A NOVEL CONTROLLED RELEASE INTRAVAGINAL BIOADHESIVE POLYMERIC DEVICE VALENCE MATHIAS KESSY NDESENDO A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfillment of the requirements for the degree of Doctor of Philosophy Supervisor: Professor Viness Pillay University of the Witwatersrand, Department of Pharmacy and Pharmacology, Johannesburg, South Africa Co-Supervisor: Professor Eckhart Buchmann Chris Hani Baragwanath Hospital, Department of Gynecology and Obstetrics, Johannesburg, South Africa 2009 ii DECLARATION I, Valence Mathias Kessy Ndesendo, declare that this thesis is my own work. It is being submitted for the degree of Doctor of Philosophy in the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg. It has not been submitted before for any degree or examination at this or any other University. ????? ? ? ? ? ? ? ? ? ? ? ? ? ? ? Signature This ???. day of ??????. . . ., 2009 iii DEDICATION This thesis is dedicated to my wife Christine, my daughter Karen and my son Collins for their love, prayers and much-needed support towards the completion of this study. I also dedicate this thesis to my father Mathias, for being a great inspiration towards the pursuance of this work, my mother Angela and my mother-in-law Bertha, for their endeavored support, prayers and love throughout my studies. iv RESEARCH OUU TPUU TS,, PATENT AND ACCOLADES A. Publications 1 . Valence M.K. Ndesendo, Viness Pillay, Yahya E. Choonara, Leith C.R. Meyer, Eckhart Buchmann and Uwe Rosin (2009). In vitro and Ex Vivo Bioadhesivity Analysis of Polymeric Intravaginal Caplets Using Physicomechanics and Computational Structural Modeling. International Journal of Pharmaceutics, 370, 151-15 9 . (Abstract in Appendix A1). 2. Valence M.K. Ndesendo, Viness Pillay, Yahya E. Choonara, Eckhart Buchmann, David N. Bayever and Leith C.R. Meyer (2008). A Review of Current Intravaginal Drug Delivery Approaches Employed for the Prophylaxis of HIV/AIDS and Prevention of Sexually Transmitted Infections. AAPS Pharmaceutical Sciences and Technology, 9, (2), 505-52 0 . (Abstract in Appendix A2). 3. Valence M.K. Ndesendo, Viness Pillay, Yahya E. Choonara, Lisa C. du Toit, Leith C.R. Meyer , Riaz A. Khan, Eckhart Buchmann, and Uwe Rosin (2009) . Investigation of the Physicochemical and Physicomechanical Properties of an Optimized Intravaginal Bioadhesive Polymeric Device in the Pig Model. AAPS Pharmaceutical Sciences and Technology, submitted. (Abstract in Appendix A3). 4. Valence M.K. Ndesendo, Viness Pillay, Yahya E. Choonara, Lisa C. du Toit, Leith C.R. Meyer , Riaz A. Khan, Eckhart Buchmann, and Uwe Rosin (2009) . In vivo Drug Content Analysis and Histopathological Evaluation of an Intravaginal Bioadhesive Polymeric Device in Pig Tissue. Submitted. (Abstract in Appendix A4). v 5 . Valence M. K. Ndesendo, Viness Pillay, Yahya E. Choonara, Lisa C. du Toit, Riaz A. Khan, Eckhart Buchmann and Uwe Rosin (2009) . Application of Artificial Neural Networks for the Elucidation of an Optimized Synergistic Polymer Combination for Effective Performance of an Intravaginal Bioadhesive Polymeric Device. Submitted. (Abstract in Appendix A5). 6. Valence M. K. Ndesendo, Viness Pillay, Yahya E. Choonara, Lisa C. du Toit, Riaz A. Khan, Eckhart Buchmann and Uwe Rosin (200 9). Preformulation Investigations for the Development of a Lead Intravaginal Bioadhesive Polymeric Device. Submitted. (Abstract in Appendix A6). B. Conference Proceedings 1 . Valence M. K. Ndesendo, Viness Pillay, Yahya E. Choonara, Lisa C. du Toit, Leith C.R. Meyer, Eckhart Buchmann and Uwe Rosin. In Vitro and Ex-vivo Evaluation of a Polystyrene Sulfonate-loaded Vaginal Polymeric Platform through Porcine Vaginal Tissue. Annual Meeting and Exposition of the American Association of Pharmaceutical Scientists (AAPS), November 8-1 2, 2009, Los Angeles Convention Center, Los Angeles, Califonia, USA. (Abstract in Appendix B1). 2 . Valence M. K. Ndesendo, Viness Pillay, Yahya E. Choonara, Lisa C. du Toit, Leith C.R. Meyer, Eckhart Buchmann and Uwe Rosin. Development of a Highly Sensitive Assay Method for the Determination of AZT in Porcine Plasma. Annual Meeting and Exposition of the American Association of Pharmaceutical Scientists (AAPS), November 8-12, 2009, Los Angeles Convention Center, Los Angeles, Califonia, USA. (Abstract in Appendix B2). vi 3 . Valence M.K. Ndesendo, Viness Pillay, Yahya E. Choonara, Lisa C. du Toit, Leith C.R. Meyer, Eckhart Buchmann and Uwe Rosin. In Vivo Morphological and Histopathological Evaluation on a Vaginal Polymeric Device in a Porcine Model. Annual Meeting and Exposition of the American Association of Pharmaceutical Scientists (AAPS), November 8-1 2, 2009, Los Angeles Convention Center, Los Angeles, Califonia, USA. (Abstract in Appendix B3). 4. Valence M.K. Ndesendo, Viness Pillay, Yahya E. Choonara, Lisa C. du Toit, Leith C.R. Meyer, Eckhart Buchmann and Uwe Rosin. In vivo Drug Content Analysis and Histopathological Evaluation of an Intravaginal Polymeric Platform in a Pig Model. Fifth International Conference of South African Pharmaceutical and Pharmacological Sciences Society (ICPPS). 23 rd-26 th September, 2009, North-West University, Potchefstroom Campus, South Africa. (Abstract in Appendix B4). 5. Valence M.K. Ndesendo, Viness Pillay, Yahya E. Choonara, Lisa C. du Toit, Leith C.R. Meyer, Eckhart Buchmann and Uwe Rosin. Development of an X-ray Detection Method for Hydroxyapatite-Loaded Matrices for Intravaginal Insertion in a Pig Model. Annual Meeting and Exposition of the American Association of Pharmaceutical Scientists (AAPS), November 16-20, 2008, Georgia World Congress Center, Atlanta GA, USA. (Abstract in Appendix B5). 6. Valence M.K. Ndesendo, Viness Pillay, Yahya E. Choonara, Lisa C. du Toit, Leith C.R. Meyer, Eckhart Buchmann and Uwe Rosin. Evaluation of the Bioadhesive Potential of A Multiconfigured Polymeric Matrix for Intr avaginal Drug Delivery. Annual Meeting and Exposition of the American Association of Pharmaceutical Scientists (AAPS), November 16-20, 2008, Georgia World Congr ess Center, Atlanta GA, USA. (Abstract in Appendix B6). vii 7. Valence M.K. Ndesendo, Viness Pillay, Yahya E. Choonara, Lisa C. du Toit and Eckhart Buchmann. Combination of Biosorbable, Relatively Low and High Compressible Polymers in Controlled Drug Delivery. Annual Meeting and Exposition of the American Association of Pharmaceutical Scientists (AAPS), November 16-20, 2008, Georgia World Congress Center, Atlanta GA, USA. (Abstract in Appendix B7). 8. Valence M.K. Ndesendo, Viness Pillay, Yahya E. Choonara, Leith C.R. Meyer, Eckhart Buchmann, Uwe Rosin, and Lisa C. du Toit. Release Rate Modulation and Ex Vivo Diffusion of Zidovudine from a Stealth Polymeric Matrix through Porcine Vaginal Tissue. 29 th Annual Conference of the Academy of Pharmaceutical Sciences of South Africa (APSSA). 22 nd to 26 th September, 2008, Magaliesberg, S. Africa. (Abstract in Appendix B8). 9. Valence M.K. Ndesendo, Viness Pillay, Yahya E. Choonara, Leith C.R. Meyer, Eckhart Buchmann, Uwe Rosin, and Lisa C. du Toit. The Influence of an Innate Moisture Protective Shell on Drug Release from a Composite Polymeric Platform. 29 th Annual Conference of the Academy of Pharmaceutical Sciences of South Africa (APSSA). 22 nd to 26 th September, 2008, Magaliesberg, South Africa. (Abstract in Appendix B9). 10. Ndesendo, V.M.K., Pillay, V., Choonara, Y.E., Buchmann, E. and Meyer, C.R.L. A Comparison between the Bioadhesivity Potential of Two Novel Polymer-Based Matrices for Intravaginal Drug Delivery. 28 thAnnual Conference of the Academy of Pharmaceutical Sciences of South Africa (APSSA), Cape Town, South Africa, 2007. (Abstract in Appendix B10). viii 1 1 . Ndesendo, V.M.K., Pillay, V., Choonara, Y.E., Buchmann, E. and Meyer, C.R.L. A Development of a Method to Investigate the Ex Vivo Bioadhesivity of a Novel Polymer- Based Platform on Freshly Exci sed Rabbit Vaginal Tissue. 28 thAnnual Conference of the Academy of Pharmaceutical Sciences of South Africa (APSSA), Cape Town, South Africa, 2007. (Abstract in Appendix B11). 12. Valence M.K. Ndesendo, Viness Pillay, Yahya E. Choonara, Leith C.R. Meyer, Eckhart Buchmann, Uwe Rosin and Lisa C. du Toit. A Resident Homeo-Prophylactic Intravaginal Polymeric Caplet for the Prevention of HIV/AIDS and STIs. Presented at the Faculty of Health Sciences Research Day. University of the Witwatersrand, 20 th August, 2008. (Abstract in Appendix B12). 13. Valence MK Ndesendo, Viness Pillay, Yahya E Choonara, Leith C.R. Meyer, Eckhart Buchmann, Uwe Rosin and Lisa C du Toit. An In vitro, Ex-vivo, In vivo Analysis as well as Histopathological and Histomorphological Evaluation of an Intravaginal Microbicidal Bioadhesive Polymeric Device for the Prevention of HIV and STIs. Presented at the Faculty of Health Sciences Research Day. University of the Witwatersrand, 15 th August, 2009. (Abstract in Appendix B13). C. Patent 1 . Valence M.K. Ndesendo, Viness Pillay and Yahya E. Choonara. A Novel Controlled Release Intravaginal Bioadhesive Polymeric Device. South Africa Patents and Trade Mark Office, 2008. Filed. ix D. Additional Outputs fr om Collaborative Research D.1 Publications 1 . Girish Modi, Viness Pillay, Yahya E. Choonara, Valence M. K. Ndesendo, Lisa C. Du- Toit and Dinesh Naidoo (2009) . A review of nanotechnological applications for the treatment of neurodegenerative disorders. Journal of Progress in Neurobiology, 88, (4), 272-28 5 . (Abstract in Appendix C1). 2. Yahya E. Choonara, Viness Pillay, Lisa C. du Toit, Girish Modi, Dinesh Naidoo, Valence M.K. Ndesendo and Sibongile R. Sibambo (2009). The Molecular Pathogenic and Clinical Therapeutic Trends in Common Neurodegenerative Disorders. International Journal of Molecular Sciences, 10, (10) , 2 5 1 0 - 2 5 5 7 . (Abstract in Appendix C2). 3. Samantha Pillay, Viness Pillay, Yahya E. Choonara, Dinesh Naidoo, Riaz A. Khan, Lisa C. du Toit, Valence M. Ndesendo, Girish Modi, Michael P. Danckwerts, Sunny E. Iyuke (2009 ) . Design, Optimization and Biomet ric Simulation of a Nano-enabled Scaffold Device for Enhanced Delivery of Dopamine to the Brain. International Journal of Pharmaceutics, 3 8 2, 277-29 0 . (Abstract in Appendix C3). 4. Y.E. Choonara, V. Pillay, N. Singh, R.A. Khan and V.M.K. Ndesendo (2008). Chemometric, physicomechanical and rheological analysis of the sol-gel dynamics and degree of crosslinking of glycosidic polymers. Biomed. Mater. 3, (2), 1-15. (Abstract in Appendix C4). 5. A Kolawole, V Pillay, Y E Choonara, L C du Toit and V M K Ndesendo (2009) . The influence of polyamide 6,10 synthesis variables on the physicochemical characteristics and drug release kinetics from a monolithic tablet matrix. Journal of Pharmaceutical Development and Technology, accepted. (Abstract in Appendix C5). x 6. Yahya E. Choonara, Viness Pillay, Valence M.K. Ndesendo, Lisa C. Du Toit, Riaz A. Khan, and Caragh S. Murphy (2009). The simultaneous in vitro characterization of poly(lactic co-glycolic acid) and poly(glucu ronide)-rich nanoparticles employing various sol-gel synthetic wet chemical processing strategies. Biomedical Materials (BMM), submitted. (Abstract in Appendix C6). 7. Valence M.K. Ndesendo, Viness Pillay, Girish Modi, Yahya E. Choonara, Lisa Du Toit. Applications of Nanotechnology in Parkinson?s and Motor neurone diseases. 2009, submitted. (Abstract in Appendix C7). 8. Pius Fasinu, Viness Pillay, Valence M.K. Ndesendo, Lisa du Toit, Yahya E. Choonara. Advances in the approaches for enhancement of oral drug delivery. 2009, submitted. (Abstract in Appendix C8). 9. Rubina Shaikh, Viness Pillay, Yahya E. Choonara, Lisa C. du Toit, Valence M.K. Ndesendo, Priya Bawa and Shivaan Cooppan. ?Smart? (stimuli-responsive) polymers: Applications in membranous drug delivery systems (and the formulation thereof). AAPS Pharmaceutical Sciences and Technology, submitted. (Abstract in Appendix C9). 10. Rubina Shaikh, Viness Pillay, Yahya E. Choonara, Lisa C. du Toit, Valence M.K. Ndesendo and Riaz A. Khan. A Modified Cro sslinked Oramucosal Lyophilized Wafer System for Gradual Drug Delivery, submitted. (Abstract in Appendix C9). 11. Ndidi Ngwuluka, Viness Pillay, Lisa C. du Toit, Valence M.K. Ndesendo, Yahya E. Choonara, Priya Bawa and Shivaan Cooppan. Progress in the Design of L-dopa-Loaded Delivery Systems for the Therapeutic Management of Parkinson?s disease. Expert Opinion on Drug Delivery, submitted. (Abstract in Appendix C11). xi 12. Ameena Wadee, Viness Pillay, Yahya E. Choonara, Lisa C. du Toit, Valence M.K. Ndesendo and Caragh S. Murphy. Recent Advances in the Design of Drug-loaded Polymeric Implants for the Treatment of Solid Tumors, submitted. (Abstract in Appendix C12). 13. Kovanya Moodley, Viness Pillay, Yahya E. Choonara, Lisa C. du Toit, Valence M.K. Ndesendo, Priya Bawa and Shivaan Cooppan. Oral Drug Delivery Systems Comprising Altered Geometric Configurations for Controlled Drug Delivery, submitted. (Abstract in Appendix C13). 14. Clare Dott, Viness Pillay, Yahya E. Choonara, Lisa C. du Toit, Valence M.K. Ndesendo and Caragh S. Murphy. Pharmaceutical Applications of Electro-Spinning, submitted. (Abstract in Appendix C14). 15. Farina Loonat, Valence M.K. Ndesendo, Viness Pillay, Yahya E. Choonara, Lisa C. du Toit and Riaz A. Khan. Design and Development of Pediatric Wafers for the Treatment of Pediatric HIV/AIDS, submitted. (Abstract in Appendix C15). 16. B Sibeko, V Pillay, YE Choonara, RA Khan, G Modi, SE Iyuke, D Naidoo and VMK Ndesendo. Fabrication, characterization and optimization of MTX-loaded PLA/MAA nanoparticles, submitted. (Abstract in Appendix C16). 17. Neha Singh, Viness Pillay, Yahya E. Choonara, Lisa C. du Toit and Valence M.K. Ndesendo. Modulation of Nicotine Release from a Gelisphere?Loaded Compressed External Polymeric Matrix. 2009, submitted. (Abstract in Appendix C17). xii D.2 Conference Proceedings 1 . Valence M. K. Ndesendo, Viness Pillay, Yahya E. Choonara and Lisa C. du Toit. The simultaneous in Vitro analysis of poly (Lactic-co-glycoli c acid) and poly (glucuronide) - rich alginate nanoparticles: A formulation approach . International Conference on Nanoscience and Nanotechnology (NanoAfrica, 2009), 1-4 February, 2009, CSIR, Pretoria, South Africa. (Abstract in Appendix C18). 2. Y.E. Choonara V. Pillay, M.P. Dankwerts, T. Carmichael, C. Wanblad, S. Naylor and V.M.K. Ndesendo. A Biodegradable Doughnut-Shaped Minitablet for Sustained Intraocular Release of Ganciclovir. Ann ual Meeting and Exposition of the American Association of Pharmaceutical Scientists (AAPS), November 16-20, 2008, Georgia World Congress Center, Atlanta GA, USA. (Abstract in Appendix C19). 3. O.A. Kolawole, V. Pillay, Y. E. Choonara L.C. du Toit and V. M. K. Ndesendo. Design and Characterization of a Pore-Regulated Matr ix for Transmucosal Drug delivery. Annual Meeting and Exposition of the American Association of Pharmaceutical Scientists (AAPS), November 16- 20, 2008, Georgia Worl d Congress Center, Atlanta GA, USA. (Abstract in Appendix C20). 4. O.A. Kolawole, V. Pillay, Y. E. Choonara L.C. du Toit and V. M. K. Ndesendo. The Influence of Pore Morphology on the Matrix Integrity, Drug Loading and Drug Release from a Porous Delivery Platform. Annual Meeting and Exposition of the American Association of Pharmaceutical Scientists (AAPS), November 16-20, 2008, Georgia World Congress Center, Atlanta GA, USA. (Abstract in Appendix C21). 5. P. Bawa , V. Pillay, Y. E. Choonara L.C. du Toit and V. M. K. Ndesendo. Formulation and Evaluation of a Buoyant Prolonged Release Drug Delivery System. Annual Meeting and Exposition of the American Association of Pharmaceutical Scientists (AAPS), xiii November 16-20, 2008, Georgia World Congres s Center, Atlanta GA, USA. (Abstract in Appendix C22). 6. D. Reddy , V. Pillay, Y. E. Choonara L.C. du Toit and V. M. K. Ndesendo. Evaluation of the Disintegration Characteristics of a Mu lti-Configured Oramucosal Drug Delivery System. Annual Meeting and Exposition of the American Association of Pharmaceutical Scientists (AAPS), November 16-2 0, 2008, Georgia World Congress Center, Atlanta GA, USA. (Abstract in Appendix C23). 7 . Z. Khan , V. Pillay, Y.E. Choonara, L.C. du Toit and V.M.K. Ndesendo. In Vitro Evaluation of the Drug Release, Swelling and Erosion Dynamics of a Layered Multi-Disk Tablet Matrix. Annual Meeting and Exposition of the American Association of Pharmaceutical Scientists (AAPS), Novemb er 16-20, 2008, Georgia World Congress Center, Atlanta GA, USA. (Abstract in Appendix C24). 8. S. Cooppan , V. Pillay, Y. E. Choonara, L.C. du Toit and V. M. K. Ndesendo . Physical Characterization of a Novel Composite Membranous System for Rate-Modulated Drug Delivery. Annual Meeting and Exposition of the American Association of Pharmaceutical Scientists (AAPS), November 16-2 0, 2008, Ge orgia World Congress Center, Atlanta GA, USA. (Abstract in Appendix C25). E. Accolades 1 . Postgraduate Merit Award (PMA), University of the Witwatersrand, South Africa, 2008 and 2009. 2 . Bradlow Postgraduate Award (BPA), University of the Witwatersrand, South Africa, 2008 and 2009. xiv 3 . University Council Postgraduate Award (U CPA), University of the Witwatersrand, South Africa, 2008 and 2009. 4. Novartis BioCamp Seminar Award, Novartis International Biotechnology Leadership Camp, Cambridge, Massachusetts, USA, 2009. 5. Novartis excellence award for the outstanding performance at Novartis BioCamp 2009, Novartis International Biotechnology Leadership Camp, Cambridge, Massachusetts, USA, 2009. F . Grants and Funding 1 . A scholarship from Norwegian Agency for Development Co-operation (NORAD)- NORWAY, August 200 6-August 2009. 2. Medical Faculty Research Endowment Fund (MFREF), University of the Witwatersrand, South Africa, 2008 and 2009. 3. International conference travelling grant, Un iversity of the Witwatersrand, South Africa, 2008 4. Academy of Pharmaceutical Scientists of South Africa (APSSA) conference grant, APSSA, South Africa, 2008. xv SUU MMARY HIV/AIDS was discovered almost a quarter of a century ago and has so far claimed the lives of more than 25 million people worldwide. Developing countries remain disproportionately affected, with sub-Saharan Africa contributing more than two-thirds of infections globally. Sexual transmission is the primary route of HIV/AIDS acquisition, and women bear the greatest burden of this pandemic. We are now at a stage where biotechnological advances are needed that can either cure HIV/AIDS, stimulate the immune system to produce anti-HIV-antibodies by vaccination, or prevent HIV infections. One of these advances has been the development of various microbicides. However, a lack of effective drug delivery systems for these agents has remained as a rate-limiting step towards successful HIV prevention. In an attempt to overcome this problem, this study aimed at designing and developing a novel intravaginal bioadhesive polymeric device (IBPD) as a delivery system to effectively deliver a microbicide {polystyrene sulfonate (PSS)} and antiretroviral (ARV) {3' -azido-3'-deoxythymidine (AZT)} combination to the vagina. The development of a successful intravaginal microbicidal drug delivery system requires the design of a formulation to deliver the microbicide-ARV combination in a safe, effective, and consistent manner. The first step therefore was to undertake extensive preliminary screening studies on various polymeric materials using a one variable at a time (OVAT) approach to find suitable polymers for developing an IBPD. Initially 18 biodegradable and biocompatible polymer s were employed to produce 62 formulations that were further screened through the OVAT approach to result in 15 lead formulations. Two major concerns of this study were the attainment of satisfactory residence time of the IBPD in the vagina as well as the ability of the IBPD to contain and release the microbicide-ARV in a controlled manner. Therefore, optimization of the IBPD was based on these two requirements for which proper matrix integrity was a pre-requisite. Artificial neural networks (ANNs), a computational technique that is able to simulate the neurological processing ability of the human brain through mathematical modeling, was employed for optimiza tion. The ANN approach confirmed that 5 of the 18 studied polymers could be suitable for the development of an optimized IBPD. To finally attain good vaginal retention for the developed delivery system, extensive bioadhesivity testing was undertaken on the optimized device. Thorough in vitro and ex vivo bioadhesivity analysis was conducted using physicomechanics and computational structural modeling. Allyl penta erythritol- crosslinked poly acrylic acid (APE-PAA) appeared to contribute most to the bioadhesivity. Apart from being employed as a matrix component, PAA was further used as a coating agent to achieve extended bioadhesivity within the posterior fornix of the vagina. Since prolonged release and suitable permeation of the microbicide-ARV across the vaginal tissue was a critical requirement of this study, the device was designed to provide a controlled and prolonged drug release. Prolonged release for up to 72 days was achieved. Furthermore, the design was constructed to ensure that the released drug could permeate into the vaginal tissue and be retained substantially. This was determined by measuring drug flux through ex-vivo permeation studies using freshly excised pig vaginal tissue in a Franz Diffusion Cell (FDC) apparatus. The ultimate aim of the study was to have the IBPD well accommodated in the vagina for successful prevention of STIs and HIV infection. Achievement of this aim was ensured by undertaking extensive in vivo studies in Large White pig model. The IBPDs were inserted under anaesthesia into the posterior fornix of the vagina, using a novel applicator. To detect the retention of the IBPDs and determine their sequential biod egradation pattern in the vagina, X-ray imaging was employed, using radio-opaque Barium Sulphate (BaSO 4 ). To demonstrate that the developed drug delivery system acted locally and was only minimally absorbed systemically, blood samples were taken from the jugular vein of each pig at pre-determined time intervals and subjected to UPLC analysis. The drug content in the vaginal ti ssue at the end of the study was also determined. Histopathological evaluation was carried out on vaginal epithelium to access the potential for toxicity of the IBPDs. The drug content analysi s revealed that greater amounts of AZT and PSS were retained in the vaginal tissue with relatively small quantities (AZT:17% ; PSS:13%) crossing into the systemic circulation. The results from the toxicity studies showed that the IBPDs were safe for use. This suggests that the developed drug delivery system (the IBPD) may be suitable for application in the prevention of STIs and HIV infections . xvi ACKNOWW LEDGEMENTS This work would not have been possible to undertake and accomplish if it was not due to endeavored support and encouragement from various people. I would therefore first and foremost like to express my deepest gratitude to my super visor, Professor Viness Pillay for his distinguished guidance, exciting sensitization towards devotion for excellence, eminent supervision, invaluable advice and support throughout this study. I am highly indebted to his vision and commitment in ensuring the successful accomplishment of this work right from the beginning to the end. He opened new highways for my scientific, academic career and the personal life. Words are very little to describe my thanks and appreciation for what he has done for me which is absolutely next to impossible to forget. I do not have anything equivalent to offer as a return but rather my constant prayers to God to enable him continue with the same pace of deliverance. I am most grateful to my co-supervisor, Prof. Eckhart Buchmann for his thorough advice and support towards the success of this work. Being a gynecologist/obstetrician, he played a leading role in bridging pharmaceutical and clinical expertise. His contribution to this thesis is unforgettable. My esteemed gratitude goes to Prof. Michael P. Danckwerts, Head and Chair: Department of Pharmacy and Pharmacology, for his invaluable encouragement, support and advice that he rendered to me as a leader which in one way or another facilitated the accomplishment of this work. His excellent leadership skills contributed subs tantially towards my success. I am grateful to Mr. David Bayever for his professional advice and assistance in legal and ethical pertaining issues and Dr. Sandy van Vuuren for her advice. I would like to extend my sincere and special thanks to Dr. Leith C.R. Meyer, Director: Central Animal Services (CAS), whose endeavored and tireless efforts made the in vivo aspect of this study possible. His contribution towards the success of this work will never ever be forgotten. I would also like to thank the rest of the CAS team including Sister Mary-Ann Costello, Patrick Selahle, Ammelia Rammekwa, Lorrain Setimo and Kershnee Chetty. Be blessed for all of your efforts towards making this work a success. xvii I would also like to extend my special thanks and appreciation to the entire management of PharmaNatura (Pty) Ltd, in particular Mr. Uwe Rosin, Director: Research and Development (R&D) Unit, and Mr. Peter Setlaelo, former Principal Technician (R&D) for making it possible to undertake the production of the IBPDs at their facilities. It will be very difficult to forget the contribution s that were made in one way or another by my PhD colleagues, Mr. Yahya E. Choonara, Mrs. Oluwatoyin Adeleke and Ms Lisa du Toit. May the Almighty reward them an equivalency in terms of academic achievement. In this regard, I also thank my other PhD colleagues, Ms Ndidi Ngwuluka, Mr. Steven Mufamadi and the entire group of Masters students, particularly Ms Samantha Pillay, Priya Bawa, Zaheeda Khan, Bongani B. Sibeko, Shivaan Cooppan, Pius Fasinu and Thiresen Govender. Their charisma, encouragement and well maintained spirit of comradely made life a bit easier for me in the Department. My special attributes go to Prof. Thirumala Govender, School of Pharmacy and Pharmacology, University of Kwazulu-Natal, who was the first person I had made contact with, regarding my intention to pursue a PhD in South Africa. I thank her for leading me to the proper channel through which I have been able to accomplish my objective . In this regard, I would also like to thank the following people who in one way or another contributed to the success of this work particularly at the initial stages: Professor Bruce Davidson, former Chairman: Animal Ethics Committee, University of the Witwatersrand; Prof. Michael Witcomb, Head: Microscopy and Microanalysis Unit, University of the Witwatersrand; Ms Caroline Lalkhan, Microscopy and Microanalysis Unit; Dr. Geoffrey Candy, Department of Surgery, University of the Witwatersrand, Prof. Josias Hamman, Tshwane University of Technology, Pretoria, Dr. Eugene Olivier, Head: Department of Pharmaceutics, Tshwane University of Technology, Pretoria, Prof. Mary Justin-Temu, Muhimbili University of Health and Allied Sciences (MUHAS), Prof. Amos Massele, MUHAS, Dr.Thecla Kohi, MUHAS, Ms Jovita Temba, MUHAS and Mr. Dennis Busuguli, Ministry of Health, Tanzania . I will be short of gratitude if I don?t remember to thank the support members of staff in our Department, particularly Ms Busi Nompumelelo Damane, Mr. Sello Ramarumo, Mrs Lehlohonolo Tebogo Chandu and the rest. They all made my studies manageable and easier in one way or another. xviii Certainly these studies would not have been possible to undertake if it were not for the sponsorship that was offered to me by the Norwegian Agency for Development Co-operation (NORAD)-NORWAY, through the University of Dar-es-Salaam (UDSM) and the logistic support from Muhimbili National Hospital (MNH) and MU HAS (particularly School of Pharmaceutical Sciences), Tanzania . I highly acknowledge and thank NORAD, MNH and the Vice chancellors of UDSM and MUHAS together with their entire administration teams in this regard. Special thanks go to Mr.Leo K. Kingalu, former Head: Staff Training and Development Unit (STDU), Directorate of Human Resource Management, UDSM, Mrs Neema G. Mshingeni, Head: STDU and Mr. Evarist F. Lyagala, STDU, UDSM, for ensuring smooth running of the NORAD scholarship. I would like to express my special gratitude to the University of the Witwatersrand not only for providing me with an opportunity to undertake these studies but also for granting me the Postgraduate Merit and Bradlow awards and the Medical Faculty Research Endowment Fund (MFREF), which apart from adding up to my inspiration towards my work, also assisted in facilitating my studies. Noteworthy, is the prestigious award that was offered to me by Novartis (South Africa) to attend the BioCamp Seminar at Massachusetts Institute of Technology (MIT), USA, October 2009, which allowed me to exchange and reap substantial knowledge related to my work. Coming to this end, I would like to express my special gratitude, thanks and gratefulness to my wife Christine, my daughter Karen and my son Collin s, for being pillars of strength during my study period, for accepting to undergo the endurance of staying without me for the whole period of my studies and for their constant love, persistent prayers, support and encouragement during this meandering arduous journey. May this achievement be a reward for their endurance. I am also highly endeavored to my Dad, Mum and my Mother-in-law for their constant prayers, wisdom, love, encouragement and support throughout my studies. I am sure that this work makes them very happy and proud. I also extend my thanks to my brothers, sisters, brother-in-laws and sister-in-law for their love and support towards this achievement. Last but certainly not the least, I would like to thank the Almighty God and his son Jesus Christ for what They have bestowed upon me. xix ANIMAL ETHICS DECLARATION I hereby confirm that the following study entitled ?A Novel Controlled Release Intravaginal Polymeric Bioadhesive Device? had received the approval from the Animal Ethics Committee of the University of the Witwatersrand with ethics clearance number 2007/2 5 / 0 5 . (Appendix D). xx TABLE OFF CONTENTS Declaration Page ii Dedication iii Research Outputs, Patent and Accolades iv Summary xv Acknowledgements xvi Animal Ethics Declaration xix List of Figures xxxvi List of Tables xLiii Chh aa ptt er Onn e Baa cc kk gg rounn dd ,, Raa tt ionn aa ll e aa nn dd Mott ivaa tt ionn ff or tt hh is Stt udd yy 1.1 Introduction 1 1.1.1 Current status of the therapeutic management of HIV/AID 6 1.1.1. 1 Combination therapy in HIV/AIDS 8 1.1.2 Anatomy and physiology of the vagina in relation to drug delivery 9 1.1.3 The vaginal mucus in relation to bioadhesion 12 1.1.4 Vagina as an application site for drug delivery 13 1.1.4 . 1 Advantages and disadvantages of vaginal drug delivery 15 1.2 Statement of the problem 17 1.3 Rationale and motivation of this study 19 1.4 Aim and objectives of this study 23 1.5 Overview of the thesis 24 1.6 Concluding remarks 27 xxi Chh aa ptt er Tww o Litt eraa tt ure Revieww off Currenn tt Inn tt raa vaa gg inn aa ll Drugg Dell iveryy Approaa cc hh es Emm pll oyy edd ff or tt hh e Prophh yy ll aa xx is off HIV// AIDS aa nn dd Prevenn tt ionn off Sexx uaa ll ll yy Traa nn smm itt tt edd Inn ff ecc tt ionn s 2.1 Introduction 28 2.2 The rationale for the development of intravaginal microbicidal delivery systems 30 2.3 Current intravaginal microbicide delivery approaches for preventing the transmission of STIs and HIV 31 2.4 Design of intravaginal microbicide delivery systems 33 2.4.1 Creams and gels 34 2.4.2 Tablets and suppositories 35 2.4.3 Vaginal rings 36 2.5 Bioadhesive intravaginal systems 37 2.6 Compounds Delivered Intravaginally as Microbicides 39 2.7 Dual-function polymer ic gel-based formulations employed in the design of intravaginal microbicide delivery systems: As excipients with potential therapeutic activity 40 2.7.1 Carageenan-based gel formulations 4 1 2.7.1 . 1 A carageenan vaginal gel formulation for HIV and Human Papilloma Virus inhibition 42 2.7.1. 2 A combinatory gel formulation of carageenan and a Non- Nucleoside Reverse Transcriptase Inhibitor 45 2.7.1 . 3 A topical non-contraceptive carageenan gel formulation 45 2.7.2 A Polyacrylic acid-based gel formulation 46 xxii 2 . 7 . 3 A Lactobacillus Crispatus soft-gel capsule formulation 4 7 2.7. 4 Thermosensitive gel formulations 4 8 2.7.5 Long-chain sulfated polysaccharides and sulfonated polymeric formulation 49 2.7.6 A carbomer, lactic acid and naphthalene sulfonate gel formulation 50 2.7.7 A micronized cellulose acetate phthalate gel formulation 51 2.7.8 A monocaprin-loaded hydrogel formulation 52 2.7.9 Polystyrene sulfonate vaginal tablets 52 2.8 Non-polymeric intr avaginal microbicide delivery systems for preventing the transmission of STIs and HIV 53 2.8.1 A cety l betaine and myristamine oxide combinatory cream formulation 53 2.8.2 A non-nucleoside reverse transcriptase inhibitor-loaded gel formulation 53 2.8.3 A nucleotide analogue-loaded gel formulation 55 2.8.4 A novel synthetically derived aryl phosphate-loaded gel formulation 55 2.8.5 A cyanovirin-N gel formulation for chemotherapeutic and immunoprophylactic prevention of HIV 56 2.8.6 Sodium lauryl sulfate as an Invisible Condom TM gel-like formulation 56 2.8.7 Dapivirine anti-HIV vaginal rings 57 2.8.8 Polyherbal anti-HIV Praneem-loaded vaginal tablets 57 2.9 Nanotechnology-based intravaginal microbicide delivery systems 58 2.9.1 A dendrimer- based microbicide formulation 58 2.9.2 Silver nanoparticles employed as microbicide delivery systems 59 xxiii 2 . 9 . 3 Polystyrene nanospheres as microbicide delivery systems 60 2.9.4 A gel-like molecular condom formulation as a barrier for STIs and HIV transmission 60 2.10 Other microbicidal compounds currently under investigation 6 1 2.10. 1 A secretory leukocyte protease inhibitor 61 2.10.2 Thrombospondin-1 as a barrier to mucosal transmission of HIV-1 62 2.10. 3 Short interfering RNA as potential liposomal microbicide delivery systems 63 2.10. 4 Thiophen-Thiourea: A non-nucleoside reverse transcriptase Inhibitor 64 2.10.5 Monoclonal antibodies as microbicides 67 2.10.6 Lime juice as a microbicide 67 2.10. 7 Yoghurt as a microbicide 68 2.11 Concluding Remarks 69 xxiv Chh aa ptt er Thh ree Preff ormm ull aa tt ionn Inn vestt igg aa tt ionn s ff or tt hh e Devell opmm enn tt off Leaa dd Inn tt raa vaa gg inn aa ll Bioaa dd hh esive Poll yy mm ericc Devicc es 3 . 1 Introduction 70 3.2 Materials and methods 74 3.2.1 Materials 74 3.2.1 . 1 Synthesis of modified polyamide 6,10 75 3.2.2 Methods 76 3.2.2 . 1 Selection of formulation components 76 3.2.2. 2 Preparation of preliminary lead intravaginal bioadhesive Polymeric devices 79 3.2.2. 3 Equilibrium swelling studies conducted on the preliminary formulations as a critical indicator of the formulation?s matrix stability 82 3.2.2. 4 Effect of changing the formulation components of the IBPD on the equilibrium swelling ratio 83 3.2.2 . 4 . 1 Effect of the elimination of carrageenan 83 3.2.2 . 4 . 2 Effect of addition of ethylcellulose 84 3.2.2 . 4 . 3 Effect of elimination of polyethylene oxide 84 3.2.2 . 4 . 4 Effect of addition of polyvinyl alcohol 85 3.2.2 . 4 . 5 Effect of addition of polyvinyl povidone 85 3.2.2 . 4 . 6 Effect of addition of the xanthan gum 86 3.2.2 . 4 . 7 Effect of the addition of guar gum 86 3,2.2.4 . 8 Effect of the addition of gelatin 87 3.2.2 . 4 . 9 Effect of the addition of beeswax 88 xxv 3 . 2 . 2 . 4 . 1 0 Effect of the addition of tragacanth 88 3.2.2 . 4 . 1 1 Effect of the addition of methyl cellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, Eudragit? S100 Eudragit ? RS100 as substitutes for gelatin 89 3.2.2 . 4 . 1 2 Effect of addition of a binary polymer combination to the formulation 90 3.2.2. 4 . 1 3 Effect of the addition of a tertiary polymer combination to the formulation 90 3.3 Results and discussions 91 3.3.1 Physical properties of mPA 6,10 and in-process validation tests 92 3.3.2 The influence of different polymeric composition on the equilibrium swelling ratio 94 3.3.2. 1 The influence of carageenan, polyethylene oxide and ethylcellulose on the matrix stability 94 3.3.2 . 2 The influence of guar gum, polyvinyl povidone, tragacanth and polyvinyl alcohol on matrix stability 94 3.3.2. 3 The influence of xanthan gum, beeswax and gelatin on the matrix stability 94 3.3.2 . 4 The influence of the addition of of methylcellulose, hydroxyethlycellulose, hydroxypropylcellulose and hydroxypropymethyllcellulose in the formulations on matrix stability 95 3.3.2 . 5 Influence of Eudragit ? S100 and Eudragit ? RS 100 on matrix swelling 95 3.3.2 . 6 Equilibrium swelling ratios of the best selected fifteen lead formulations screened through the OVAT approach 96 3.4 Concluding remarks 98 xxvi Chh aa ptt er FF our Appll icc aa tt ionn off Artt iff icc iaa ll Neuraa ll Nett ww orkk s ff or tt hh e Ell ucc idd aa tt ionn off aa nn Optt imm izz edd Syy nn ergg istt icc Poll yy mm er Comm bb inn aa tt ionn ff or Eff ff ecc tt ive Perff ormm aa nn cc e off tt hh e Inn tt raa vaa gg inn aa ll Bioaa dd hh esive Poll yy mm ericc Devicc e 4.1 Introduction 9 9 4.2 Materials and methods 101 4.2.1 Materials 1 0 1 4 . 2 . 2 Methods 101 4.2.2 . 1 Preparation of the intravaginal polymeric device 1 0 1 4 . 2 . 3 In process validation tests 1 0 2 4.2.3 . 1 Determination of the Brinell hardness number 102 4.2.4 In vitro matrix erosion of the intravaginal bioadhesive polymeric device 104 4.2.5 Optimization by ANN for the best polymer combination selection 105 4.3. Results and discussions 107 4.3.1 In process validation test 107 4.3.2 Matrix erosion evaluation 108 4.3.3 Selection of polymer combinations as per ANN optimization 110 4.4 Concluding remarks 118 xxvii Chh aa ptt er 55 In Vitro and Ex vivo Bioaa dd hh esivitt yy Ann aa ll yy sis onn tt hh e Optt imm izz edd Inn tt raa vaa gg inn aa ll Bioaa dd hh esive Poll yy mm ericc Devicc e Comm posedd off Diff ff erenn tt Crossll inn kk edd Poll yy aa cc ryy ll icc Acc idd s 5 . 1 Introduction 119 5.2 Materials and methods 121 5.2 . 1 Materials 121 5.2 . 2 Methods 121 5.2.2 . 1 Preparation of the IBPD caplets composed of allyl sucrose and allyl penta erythritol-crosslinked PAA 121 5.2.2 . 2 In-process validation tests of the IBPD caplets 122 5.2.2 . 3 In vitro and ex-vivo bioadhesivity analysis 123 5.2.2. 4 Removal of vaginal tissue from the pig model for bioadhesivity testing 123 5.2.2 . 5 Rheological analysis of hydrated AS-PAA and APE-PAA polymers 125 5.2.2 . 6 Mechanistic postulation of caplet bioadhesion employing chemometric and molecular modeling and associated interactive energy paradigms 126 5.3 Results and Discussion 127 5.3.1 Assessment of caplet bioadhesivity 127 5.3.2 Response optimization and textural analysis of the optimized AS-PAA and APE-PAA caplets 128 5.3.3 Ex-vivo bioadhesivity analysis of the optimized APE-PAA caplets on freshly excised vaginal tissue 130 xxviii 5 . 3 . 4 Rheological analysis of the hydrated AS-PAA and APE-PAA polymer solutions 131 5.3.5 Postulative Chemometric and molecular modeling of the mechanisms of caplet bioadhesion and the associated interactive energy paradigms 132 5.4 Concluding remarks 136 xxix Chh aa ptt er 66 Inn vestt igg aa tt ionn off tt hh e Phh yy sicc occ hh emm icc aa ll aa nn dd Phh yy sicc omm ecc hh aa nn icc aa ll Propertt ies off tt hh e Optt imm izz edd Inn tt raa vaa gg inn aa ll Bioaa dd hh esive Poll yy mm ericc Devicc e 6.1 Introduction 138 6.2 Materials and methods 140 6.2.1 Materials 140 6.2.2 Methods 141 6.2.2 . 1 Preparation of the intravaginal bioadhesive polymeric device 141 6.2.2 . 2 Pan coating of the intravaginal bioadhesive polymeric device 141 6.2.2 . 3 Influence of the intravaginal polymeric device on the micro- environmental pH of the vagina 142 6.2.2. 4 Thermal analysis of the polymeric composition and the coated intravaginal bioadhesive polymeric device 143 6.2.2 . 5 Ex-vivo biodhesivity testing of the coated drug-loaded intravaginal bioadhesive polymeric Device 144 6.2.2 . 6 Textural profiling analys is to determine the bioadhesivity of the intravaginal bioadhesive polymeric device 144 6.2.2 . 7 Insertion of the intravaginal bioadhesive polymeric device into the vagina of the pig 144 6.2.7 . 7 . 1 X-ray imaging of the pig for detection of the intravaginal bioadhesive polymeric device 145 6.2.2 . 8 In vitro drug release from the coated and uncoated the intravaginal bioadhesive polymeric device 145 6.2.2 . 8 . 1 Analysis of the effect of device coating on the drug release 145 xxx 6 . 2 . 2 . 8 . 2 Analysis of the drug release from the coated devices containing AZT and PSS separately and in combination 146 6.2.2 . 8 . 3 Chromatographic conditions for the analysis of AZT and PSS concentration 146 6.2.2 . 8 . 4 Preparation of standard solutions and calibration curves 147 6.2.2 . 8 . 5 The solid phase extraction procedure employed in the extraction of the drugs from simulated vaginal fluid samples for UPLC analysis 149 6.2.2 . 9 Ex-vivo drug permeation studies through pig vaginal tissue from the intravaginal bioadhesive polymeric device using the Franz Diffusion Cell apparatus 149 6.2.2 . 9 . 1 Chromatographic conditions for the analysis of AZT and PSS concentration 151 6.2.2 . 9 . 2 Preparation of standard solutions and calibration curves 151 6.2.2 . 9 . 3 The solid phase extraction procedure employed in the extraction of the drugs from simulated plasma fluid samples for UPLC analysis 152 6.2.2 . 1 0 Postulated mechanistic deduction of the drug diffusion and dissolution dynamics from the IBPD employing chemometric and molecular modeling 153 6.3 Results and discussion 153 6.3.1 Coating of the intravaginal bioadhesive polymeric device 153 6.3.2 Micro-environmental pH variation analysis within the simulated vaginal fluid 153 6.3.3 Thermal analysis of the polymer constituents of the intravaginal bioadhesive polymeric device 155 xxxi 6 . 3 . 3 . 1 Thermal characterization of the native unhydrated polymer constituents of the intravaginal bioadhesive polymeric device 156 6.3.3 . 1 . 1 Ethylcellulose 156 6.3.3 . 1 . 2 Modified Polyamide 6,10 157 6.3.3 . 1 . 3 Poly(lactic-co-glycolic acid) 158 6.3.3 . 1 . 4 Polyvinyl alcohol 159 6.3.3 . 1 . 5 Allyl pentaerythritol-crosslinked poly(acrylic acid) 1 6 0 6.3.3 . 2 Thermal characterization of the unhydrated intravaginal bioadhesive polymeric device 161 6.3.3 . 3 Thermal characterization of unhydrated and hydrated physical polymer blends as well as hydrated intravaginal bioadhesive polymeric device 162 6.3.4 Analysis of the drug release behavior from the coated and uncoated optimized drug-loaded intravaginal bioadhesive polymeric device 165 6.3.4 . 1 Chromatographic separation of 3'-azido-3'-deoxythymidine and polystyrene sulphonate with methyparaben as an internal standard 165 6.3.4 . 2 Assessment of the effect of coating on the drug release from the intravaginal bioadhesive polymeric device 167 6.3.4 . 3 Analysis of the drug release behavior from the optimized coated intravaginal bioadhesive polymeric device containing AZT and PSS separately and in combination 168 6.3.5 Assessment of drug permeation across the pig vaginal tissue 170 6.3.6 Ex-vivo bioadhesivity analysis of the intravaginal bioadhesive polymeric device 171 6.3.7 Retention of the intravaginal bioadhesive polymeric device xxxii within the pig vagina 172 6.3.8 Chemometric and molecular modeling of the intravaginal bioadhesive polymeric device drug dissolution and diffusion 174 6.3.8 . 1 Postulation of dissolution dynamics and subsequent effect on drug release 174 6.3.8 . 2 Diffusion kinetics depicting the drug flux mechanism during ex vivo studies 176 6.4 Concluding remarks 179 xxxiii Chh aa ptt er 77 In vivo Drugg Conn tt enn tt Ann aa ll yy sis aa nn dd Histt opaa tt hh oll ogg icc aa ll Evaa ll uaa tt ionn off tt hh e Inn tt raa vaa gg inn aa ll Bioaa dd hh esive Poll yy mm ericc Devicc e 7.1 Introduction 180 7.2 Materials and methods 182 7.2.1 Materials 182 7.2.2 Methods 182 7.2.2 . 1 In vivo animal studies in the pig model using the drug-loaded intravaginal bioadhesive polymeric device 182 7.2.2 . 2 Anesthesia, X-rays and blood sampling 184 7.2.2. 3 Vaginal tissue removal for drug analysis and histopathological studies 186 7.2.2 . 4 Quantitative analysis of AZT and PSS in the blood and the vaginal tissue of the pig 186 7.2.2 . 4 . 1 Preparation of standard solutions and calibration curves 186 7.2.2. 4 . 2 The drug extraction method from the plasma samples for UPLC analysis 187 7.2.2 . 4 . 3 The drug extraction met hod from the vaginal tissues for UPLC analysis 188 7.2.2 . 4 . 4 Instrumentation and chromatographic conditions for the quantitative analysis of AZT and PSS from plasma and vaginal tissue samples 189 7.2.2 . 4 . 5 Histopathological evaluation of the vaginal mucosa 190 7.3.0 Results and discussion 190 7.3.1 Chromatographic separation and retention times of AZT and xxxiv PSS from methylparaben (MP) (internal standard) 190 7.3.2 AZT and PSS contents in the blood and vaginal tissue of the pig model 192 7.3.3 Histopathological evaluation of the vaginal tissue 196 7.4 Concluding remarks 199 xxxv Chh aa ptt er 88 Conn cc ll usionn s aa nn dd Recc omm mm enn dd aa tt ionn s 8.1 Conclusions 201 8.2 Recommendations 2 0 3 Reff erenn cc es 2 0 6 Appenn dd icc es 3 0 1 Appendix A Abstracts of papers published/submitted from this thesis 302 Appendix B Abstracts of conferences proceedings 309 Appendix C Additional Outputs from Collaborative Research 323 Appendix D Animal ethics clearance certificate 349 xxxvi LIST OFF FF IGUU RES Page 1.1 A schematic of vagina vaginal wall showing 1) capillary vessels, 2) artery and 3) Vein. 11 1.2 A schematic of the upper vagina region depicting the posterior fornix as the site of IBPD application. 23 2.1 Schematic depicting the various mechanisms of preventing the transmission of sexually transmitted infections and HIV by employing microbicide delivery systems. 31 2.2 Microscopic images of gram-stai ned vaginal smears depicting different categories of vaginal microflora. a) and b), grade 1a Lactobacillus crispatus cell types; c) and d), grade 1b non-L. crispatus cell types; e) and f), grade 1ab mixtures of L . crispatus and non-L. crispatus cell types; g) and h), grade 1- l ike Gram positive rods; i) and j), grade 2 mixture of Lactobacillus cell types and bacterial vaginosis-associated bacteria cell types; k) and l), grade 3 bacterial vaginosis. 48 2.3 Schematic of the trimeric-sulphated glycoprotein Thrombospondin-1 with five distinct extracellular matrix adhesion molecules. 64 3.1 A computationally-derived model pr edicted in our laboratories depicting the polymer coiling stages a) polymer strands, b) identification of molecular sites, c) initial stages of coiling, d) secondary and tertiary coiling randomizations and patterns where (C-1)-(C-7) is non-breaded non-rope coiling and (C-8)-(C-12) is breaded rope coiling. The number of coil arm may vary in (C-1)-(C-5). 71 3.2 Synthesis of polyamide 6,10 by interfacial polymerization. 76 3.3 a) 3'-azido-3'-deoxythymidine (AZT) and b) Polystyrene Sulfonate (PSS) 79 xxxvii 3 . 4 The intravaginal bioadhesive polymeric devices (IBPDs) a) Planar view b) Side view c) Oblique view and d) Longitudinal view. 92 4.1 Typical force-distance profiles employed in the determination of matrix indentation hardness of the IBPD. 104 4.2 Constructed multilayer perceptron. 105 4.3 Network Topology depicting the hidden input and output layers. 105 4.4 A chemometric structural model developed in our laboratories for glyco- protein structure showing attachment of cellular surface protein (red) bound to the saccharide part of the glyco-protein (blue) and both (surface) protein and glyco-protein (green) bound to the polymer (here PAA) (white) for non-evenly distributed applied forc e during adhesion, with non-smooth and unevenly contacted two interacting surfaces with non-evenly distributed and non-constant, non-steadily (applied) force for the adhesion shown in a 3D optimized state with ball model. The coiling loop distortion for both protein and glycoprotein are also shown. 110 4.5 a) MSE for the primary ANN training, b) Average MSE for the primary ANN training with standard deviations for 10,000 epochs. 111 4.6 A profile depicting a) desired and b) actual network output. 113 4.7 A typical bar chart graph depicting the sensitivity coefficients of each polymer type on the matrix integr ity following the primary training. 113 4.8 a) MSE for the secondary ANN training, b) Average MSE for the secondary training with standard deviations for 10,000 epochs. 115 4.9 A typical bar chart graph depicting the sensitivity coefficients of each polymer type on the matrix integrity following the secondary training. 116 4.10 Correlation between matrix er osion and polymer quantity. 117 5.1 The dissection process to remove the vaginal tissue from the pig 124 5.2 Textural analysis method employed to generate Force-Distance profiles xxxviii for assessing the caplet bioadhesivity to freshly excised pig vaginal tissue indicating a) blue paint design b) actual process. 125 5.3 A comparison of work of adhesion (AUC FD) (J) between the AS-PAA and APE- PAA caplets. 128 5.4 A typical response optimization plot for the AS-PAA and APE-PAA caplets. 129 5.5 Typical textural profiles elucidating the peak adhesive force (PAF) (N) and work of adhesion (AUC FD) (J) for the optimized composed of: a) AS-PAA, and b) APE-PAA. 130 5.6 Typical textural profile elucidating the force (N) and work of adhesion (AUC FD) (J) for the optimized APE-PAA caplets on freshly excised vaginal tissue. 131 5.7 Rheological behavior of 2% w/ v AS-PAA and APE-PAA solutions at a shear rate between 0-500s -1 . 132 5.8 A chemometric structural model de picting caplet bioadhesion to freshly excised pig vaginal tissue with muco-epithelial cell secretions and surface bio-molecule interactions. 134 5.9 Energy estimates for the mechanism of bioadhesion. A) compartment A=interactions at physical forces levels, B=ionic interactions and ion- exchanges producing bioadhesion, C= chemical interaction involving covalent bonding between the xenobiotic polymer and cellular protein, D=interaction with surface collagen in the tissue substrate includes polymer-protein-collagen interactions. 135 6.1 Digital images depicting a) insertion of the IBPD into the vagina of the pig and b) tracking the location of the IBPD in the vagina using as a speculum. 145 6.2 a) Calibration curves for a) AZT in simulated vaginal fluid 267nm, b) xxxix PSS in simulated vaginal fluid 244nm. 148 6.3 Permeation studies of AZT and PSS ac ross pig vaginal tissue using a Franz Diffusion Cell apparatus. 151 6.4 a) Calibration curves for a) AZT in simulated plasma fluid 267nm b) PSS in simulated plasma fluid 244nm. 152 6.5 Micro-environmental pH variation in the simulated vaginal fluid containing the IBPD. 155 6.6 TMDSC thermogram for the unhydrated ethylcellulose. 1 5 7 6.7 TMDSC thermogram for t he unhydrated polyamide 6,10 . 1 5 8 6.8 TMDSC thermogram for the unhydrat ed poly (lactic-co-glycolic acid). 159 6.9 TMDSC thermogram for the unhydrated polyvinyl alcohol. 160 6.10 TMDSC thermogram for the unhy drated poly (acrylic acid) (PAA). 1 6 0 6.11 TMDSC thermogram for the unhydrated IBPD. 1 6 1 6.12 TMDSC thermogram for a) the hydr ated physical polymer blend, b) the hydrated IBPD. 163 6.13 TMDSC thermogram for a) the unhy drated physical polymer blend, b the unhydrated IBPD. 164 6.14 A typical 3D UPLC profile showing a complete separation between AZT and MP (internal standard). 166 6.15 UPLC chromatograms depicting the separation of a) AZT and MP (internal standard) and b) PSS and MP (internal standard) in simulated vaginal fluid (pH 4.5; 37?C). 167 6.16 A typical profile showing the effect of coating on the model hydrophilic drug AZT from a) uncoated IBPD and b) shellac/APE-PAA-coated IBPD in simulated vaginal fluid. 168 6.17 Drug release profiles of a) AZT (AZT-loaded IBPD), b) AZT (AZT/PSS- loaded IBPD), c) PSS (AZT/PSS-loaded IBPD) and d) PSS (PSS-loaded xl IBPD), in simulated vagina fluid (pH 4.5; 37?C). 170 6.18 Profiles showing the flux of AZT and PSS across pig vaginal tissue over a period of 24 hours. 171 6.19 Typical Force-Distance textural profiles used for computing the Peak Adhesion Force (PAF) and Work of Adhesion (AUC FD) for a) uncoated devices and b) PAA-coated devices on freshly excised pig vaginal tissue. 172 6.20 X-ray images depicting the presenc e of the intravaginal bioadhesive Polymeric device at a) day 1, b) day 14 and c) day 30 after insertion into the posterior fornix of the pig vagina. 173 6.21 Molecular model mechanistically depicting the IBPD dissolution process with lesser H-bond formation due to the excessive of simulated vaginal fluid providing more freedom to polymeric strands to disentangle. 174 6.22 A 3D model representing the IBPD matrix under dissolution along with drug associations: a) the polymer matr ix, b) fluid interactions producing hydrophilic pockets and swelling of the device, c) generation of hydrophobic areas in the matrix due to critical formulation excipients and d) the drug molecule (AZT) in associ ation at hydrophilic sites prior to release. 175 6.23 Chemometric models depicting a) dissoluted polymeric strands and b) AZT molecules (grey-colored circles) and PSS (white-colored circles) in the free float and associated forms with the loosely interacting polymeric strands in the dissolution medium. 176 6.24 Chemometric model depicting the development of a diffusion channel with a) A single polymer strand P, situated perpendicularly to a forming pore C, b) a group of strands also denoted collectively as P giving rise to the channel C which is formed perpendicular to the polymer strands backbone with F=the direction of flow fr om the starting point S to form a xli networked 3D channel and c) a polymeric strand with the generated diffusion channel. 177 6.25 3D models depicting the generation of diffusion channels with the IBPD matrix with a) polymeric strands, b) inter-strand physicochemical interactions, c) stereo-orientation within a group of associated polymeric strands generating an inter-polymeric cavity d) the cavity formed, e) associations between different polymeric strands and f) the blocked cavity due to excessive associative networking at various localized sites incorporating other components such as drug in different associative settings. 178 6.26 Qualitative status of energy paradigms and energy-time relationship for the IBPD matrix with a) stable energy status as a solid matrix, b) energy change following dissolution and c) energy transaction and changes for the drug diffusion across the pig vaginal tissue with the static energy status at equilibrium without flux also shown. 178 7.1 Summary of the in vivo study. 184 7.2 a) X-Ray imaging process and b) blood sampling procedure from the jugular vein of the pig. 185 7.3 Calibration curves for: a) AZT in blank plasma 267nm, b) PSS in blank plasma 244nm. 187 7.4 UPLC chromatograms depicting the retention times for the standard solutions of a) AZT, b) PSS and MP as an internal standard in blank plasma. 191 7.5 a) AZT and PSS plasma concentration profiles, and 28, b) AZT and PSS mean vaginal tissue and plasma concentration values on day 28. 193 7.6 A typical profile depicting the content of PSS in the vaginal tissue. 193 7.7 Correlation between a) AZT, b) PSS contents in the blood and vaginal xlii tissue. 194 7.8 A schematic modeled in our l aboratory depicting the mechanistic approach of adhesion between a polymer and the vaginal epithelium. 195 7.9 Intravaginal Bioadhesive Polymeric Device (IBPD) as observed in the excised pig vagina on the 28 th day. 196 7.10 Histological images of haematoxylin and eosin stained pig vaginal tissue samples depicting a) Epithelial hyperplasia of the vagina x 40, b) Epithelial hyperplasia, exocytosis and superficial exudate x 40, c) Lamina propria with no inflammatory cells and normal vagial epithelium x 20, d) Hyperplastic epithelium with exocytosis x 40, e) Mononuclear cell infiltrates in the lamina propria and f) Perivascular inflammation in the submucosal wall x 40. 198 xliii LIST OFF TABLES Page 2 . 1 Desirable criteria for ideal intrav aginal microbicidal delivery systems 32 2.2 Classification of the numerous intravaginal compounds delivered intravaginally 39 2.3 Chemical structures of polymeric microbicide compounds 44 2.4 Structures of non-polymer ic microbicide compounds 54 2.5 Chemical structures of ot her novel microbicide compounds 66 3.1 Polymers, properties and rationale for selection 78 3.2 Sixty two formulations obtained from different polymer combinations 81 3.3 Constituents used to prepare the simulated human vaginal and seminal fluids 83 3.4 Formulations without carrageenan 8 4 3.5 Formulations in which carrageenan was substituted with ethylcellulose 8 4 3.6 Formulations without polyethylene oxide 85 3.7 Formulations containing polyvinyl alcohol 85 3.8 Formulations containing polyvinyl povidone 86 3.9 Formulations containing xanthan gum 86 3.10 Formulations containing guar gum 87 3.11 Formulations in which xanthan gum was substituted with gelatin 87 3.12 Formulations in which gelatin was substituted with beeswax 88 3.13 Formulations containing tragacanth 88 3.14 Formulations containing methylcellulose, hydroxyethlycellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, Eudragit ? S100 and Eudragit ? RS 100 90 3.15 Formulations with a binary polymer combination among gelatin, beeswax, xanthan gum and Eudragit ? S 100 90 xliv 3 . 1 6 Formulations with a tertiary polymer combination among gelatin, beeswax, xanthan gum and Eudragit ? S 100 91 3.17 Mass, friability and diametric hardness of the sixty two caplet formulations 93 3.18 The equilibrium swelling ratios for the sixty two formulations that were subjected to screening using the OVAT approach 96 3.19 The selected fifteen lead formulations screened through the OVAT approach 98 4.1 The Polymer combinations employed in the production of the intravaginal bioadhesive polymeric devices 102 4.2 Textural settings employed for BHN value calculations 1 0 3 4.3 The input matrix erosion data that was trained by the Multilayer Perceptron network 106 4.4 Artificial neural network co nstruction parameters employing a neural builder 107 4.5 Brinell hardness number, mass and friability, of the intravaginal bioadhesive polymeric device 108 4.6 Neural network indicators char acterizing the efficiency and performance of data in the primary training as per ANN 112 4.7 Actual and desired network performance criterion data from the primary training 112 5.1 Extreme Vertices Mixture Formulation Template for caplet preparation 122 5.2 Comparative in vitro and ex-vivo bioadhesivity results for the optimized caplets 129 6.1 Parameters and settings employed for coating the intravaginal bioadhesive polymeric device 142 6.2 Temperature modulated differentia l scanning calorimetry settings xlv employed for thermal analysis of the intravaginal bioadhesive polymeric device and its formulation components 143 6.3 Chromatographic mobile phase gradient settings used for the separation of AZT 147 6.4 Constituents used to prepare the simulated plasma fluid 151 6.5 Critical thermal events evidenced by diverse temperature inflection peaks for the polymer constituents of the intravaginal bioadhesive polymeric device. 156 7.1 Parameter settings for the AZT gradient method. 190 7.2 Histopathological findings in the vaginal tissue 199 1 CHAPTER ONE B AC K G R OUND, R ATIONAL E AND MOTIV ATION F OR THIS S TUDY 1.1 Introduction Human I mmunodeficiency Virus/Acquired I mmunodeficiency Syndrome (HIV/AIDS) and other sexually transmitted infections (STIs) are a burgeoning problem globally ( Nicoll et al., 2000; Liu et al., 2005; Pusch et al., 2005; Todd et al., 2006; Becker et al., 2008; Lazarus et al., 2008; B?rtolo et al., 2009 ; Nijhawan et al., 2009) . AIDS has caused the mortality of more than 25 million people since it was first recognized in 1981 and has become one of the most destructive pandemics in history (UNAIDS/WHO, 2006 a) . With reference to the recent global estimates, more than 42 million people have contracted HIV/AIDS ( 90% of these in developing countries) , 5 million became infected in 2005 and more than 3 million deaths occurred from HIV - related diseases in 2005 (Malcolm et al., 2006; Nikolic et al., 2007) . According to a UNAIDS report, more than 6,800 people become infected with HIV daily ( UNAIDS, 2007 a and b). Furthermore, it has been estimated that heterosexual transmission of HIV accounts for over 90% of adult infections worldwide and that male- to- female transmission is eight times more likely to occur than female- to- male transmission. The biological reason is that women are more susceptible to STIs and HIV/AIDS than men due to larger mucosal exposure to seminal fluids as a result of the anatomical structure of the vagina. According to the recent UNAIDS /WHO global report, over 19 million women worldwide are currently living with HIV / AIDS, mean ing that the proportion of HIV/AIDS occuring in women is now nearly 50% ( UNAIDS /WHO, 2006b ; UNFPA, 2006; Ambrose et al., 2008) . The impact of HIV/AIDS on women continues to gain significance in terms of the number of women that become infected everyday and the implications thereof to their children and sex partners (Weeks et al., 2004; Chakraborty, 2 2008; Skoler- Karpoff et al ., 2008 ) . Once infected with an STI, many women are asymptomatic and may remain untreated, thus increasing their susceptibility to HIV infection. The effects of untreated STIs in women tend to be more serious than in men. For instance, it may result in the development of pelvic inflammatory disease (PID), which can subsequently lead to infertility or a life- threatening ectopic pregnancy. In addition, women living in low socio- economic settings are more vulnerable to STIs and HIV/AIDS than men because of their lower social status that may be related to their lower income and gender discrimination, which may render them the inability to refuse unsafe sexual practices in some cultures. In view of the above, coupled with a lack of effective protective measures, it is apparent that many women are at high risk of acquiring STIs or HIV/AIDS. Infection with HIV remains an incurable condition (Divi sion of HIV/AIDS Prevention, 2003; Frieden et al., 2005; Amet et al., 2008; Sing and Govender, 2009; Xu, 2009) . For the foreseeable future the control of HIV/AIDS will depend on the prevention of infection and alleviation of the disease process in affected persons. HIV is transmitted through direct contact of the systemic circulation or mucous membranes with bodily fluids infected with HIV, such as blood, semen, vaginal fluid, pre - seminal fluid and breast milk (San Francisco AIDS Foundation, 2008) . Transmis sion of HIV may occur during anal, vaginal or oral sex, contaminated blood transfusions, hypodermic needle pricks, exchange between mother and child during pregnancy, childbirth or breastfeeding. However, the highest rate of HIV transmission is through the exposure of the vaginal mucosal surface to HIV during sexual intercourse (San Francisco AIDS Foundation, 2008) . To date, vaginal epithelial mucosal surfaces are the major route of HIV infection worldwide ( Greenhead et al, 2000; Shattock, 2001; Wira et al. , 2004; Lackner and Veazey, 2007; Belyako and Ahlers, 2008) . This mode of transmission is increasing in prevalence more rapidly than any other (Royce et al., 1997; Wortley and Flemming, 1997). The transmission of HIV is said to be either by selective penetration or selective amplification within the infected host ( Verhofstede et al., 2003) . Interruption of cell - associated transmission requires an understanding of the 3 processes by which a virus moves from infected donor cells to cells of the recipient. The migration of HIV - infected cells and the movement of assembled virus particles out of the infected donor cell are critical to this process. Studies have shown that budding of HIV - 1 occurs selectively through lipid rafts on the epithelial cell surface (Nguyen and Hildreth, 2000; Chazal and Gerlier, 2003; Herzberg et al., 2006) . In addition, the ability of lipid rafts to act as adhesion platforms has been shown to facilitate cell- cell interactions and migration, which may be important for cell- to- cell transfer of the virus and for entry of infected cells through genital tract epithelia (Krauss and Altevogt., 1999) . Therefore , understanding of the first critical events in genital mucosal transmission of HIV infection is important in developing strategies to block or limit such transmission. The majority of deaths from HIV/AIDS have occurred in sub- Saharan Africa where the prevalence rate for sexually active adults is greater than 35% ( Pisani et al., 2003; Berger, 2004; Malcolm et al., 2006) . This culminates in c ausing the death of economically active adults with the accompanied macro- economic challenges. Much of the debates and the perpetual carnival of meetings addressing the HIV/AIDS epidemic that have taken place have ignored the truth about human sexual behav ior which includes the sexual desire of women, but rather focuses on the ?vulnerability of women? and thus is an ?incomplete? explanation for higher rates of HIV infection among women and girls (Berger, 2004) . At present, the current use of ARV treatment r educes both the mortality and morbidity associated with HIV infection, but routine access to antiretroviral medication is not available in all countries. Furthermore, the stigma accompanying HIV/AIDS is more severe than that associated with other life- threatening conditions and extends even to healthcare providers and volunteers involved with the care of people living with the disease. Although the immunopathogenetic events that culminate in HIV transmission during unprotected sex are still poorly underst ood, it is now clear that STIs enhance both HIV 4 susceptibility and secondary transmission ( Staff, 2002; Rebbapragada and Kaul, 2007 Levinson et al., 2009) . STIs may enhance HIV transmission through the following mechanisms: i) the impairment of innate muc osal defenses, including the integrity of the mucosal epithelium itself; ii) the local induction of pro - inflammatory cytokines that enhance HIV replication; iii) the recruitment of activated immune cells that might serve as targets for initial HIV infectio n and enhance local HIV replication; iv) enhanced susceptibility to other STIs and/or alterations in the normal vaginal microflora ; and v) impairment of systemic HIV immune control with an increased viral load in both blood and the genital tract. STIs affe ct men and women of all backgrounds, with approximately two- thirds of cases occurring in individuals less than 25 years of age (Brown et al., 1999; Drug Digest, 2004) . Based on a WHO report that was last revised in 2007, 340 million new cases of curable STIs (syphilis, gonorrhoea, chlamydia and trichomoniasis) occur annually worldwide in adults aged 15- 49 years (Reddy et al., 2002; Facts sheets, WHO, 2007) . The same report has indicated that STIs and their associated complications are among the top five disease categories for which adults seek health care (Facts sheets, WHO, 2007). According to WHO, STIs, excluding HIV, rank second to maternal factors as causes of disease and death in women of childbearing age in the developing countries (WHO, 2007). Practising of unsafe sex by individuals infected with HIV infection predisposes them to the risk of acquiring STIs. Such co- infection leads to both ulcerative and non- ulcerative STIs that ultimately promote HIV transmission by augmenting HIV infectivity and host susceptibility by a variety of biological mechanisms ( Royce et al., 1997). Thus, regular screening and prompt treatment of STIs should be part of the routine care of HIV - positive patients. The management of some STIs such as Neisseria gonorrhoeae, Chlamy dia trachomatis and Trichomonas vaginalis is normally the same for HIV - positive and negative individuals. However, many of the sexually transmitted pathogens result in more prolonged or severe infections in association with HIV/AIDS, and treatment may be 5 required for longer periods and may have a higher failure rate (Royce et al., 1997) . For instance, infection with herpes simplex virus may require higher doses of antiviral therapy to suppress recurrences and resistance. Infection with human papillomavirus (HPV) causes genital warts that may respond less to treatment and may recur more frequently. The incidence of HPV - associated neoplasia such as anal cancer and cervical squamous intraepithelial lesions is also increased (Cohen, 2004) . The management of syphilis in HIV - infected individuals can be a challenge (Butsashvili et al., 2001; Malow et al., 2009) . Interpretation of serological tests for syphilis can be difficult, as titers can be unusually high, low, or fluctuating. False- negative results have also occurred ( Baughn and Musher, 2005; Amy et al., 2007) . HIV - infected patients may have a higher risk of neurological complications in early syphilis, therefore making it a necessity to have a cerebrospinal fluid examination before treatment. Although such pat ients may have a higher rate of treatment failure, this is likely to be minimal (Rolfs et al., 1997), thus allowing clinicians to recommend the same treatment for primary, secondary, and early latent syphilis regardless of HIV status. However, some clinici ans prefer to treat all HIV - infected patients with the regimen recommended for neurosyphilis ( Thompson and Samuels, 2002; Goh, 2005) . In any case, regardless of the treatment regimen, careful long- term follow- up is essential. Despite the magnitude of the HIV pandemic, it is surprising that the probability of HIV transmission during a single episode of unprotected sex ranges from 0.1? 1. 0% only, irrespective of the type of sex ual practice or biological factors in each partner (Royce et al., 1997). Furthermore, studies have shown that HIV acquisition and secondary transmission is highly influenced by the presence of genital tract co- infections in either partner, particularly STIs (Cohen, 2004; Kaul et al., 2008; Sheung et al, 2008; Spinillo et al., 2008) . This may have accelerated spread of the HIV/AIDS epidemic worldwide and might serve to explain the profound regional and gender - based differences in HIV/AIDS 6 epidemics. Thus , there is a need to elucidate the immunopathogenetic events that underpin HIV sexual transmission at a mucosal level. Although studies on HIV immunopathogenesis have traditionally focused on systemic immune events, it is now evident that HIV transmission occurs mostly across the penile, oral, cervic o- vaginal or rectal mucosa (Kaul et al., 2008) . What is required is a better understanding of how HIV transmission occurs, and how it is influenced by genital co- infections and their effect on the mucosal micro- environment. This could contribute significantly in developing new technologies for HIV prevention modalities, such as HIV/STI microbicidal drug delivery systems. 1. 1. 1 Current status of the HIV/AIDS treatment modalities The treatment of HIV/AIDS was revolutionized by the introduction of highly active anti- retroviral therapy (HAART) in the 1990s (Glare, 2001; Gre enberg, 2007) . Use of HAART has resulted in a dramatic decrease in the incidence of HIV - associated morbidity and mortality (Palella et al., 1998; Farmer et al., 2001; Thirlwell et al., 2003; Batta glioli- DeNero, 2007; Marin- Muller et al., 2009 ) . However, cohort studies have found that regimens fail in many patients basically due to virological rebound or because initial suppression could not be achieved (Greub, et al., 2001, Fischer et al., 2003) . A rise in plasma viral load indicates treatment failure which may be due to several reasons including low level of adherence to the therapy, emergence of drug resistance, suboptimal drug levels, and use of less potent regimens. The c luster of differentiation 4 (CD4 ) cell count is normally used as the main determinant in deciding when to start treatment since there is an increased mortality in patients with CD4 count s below 200/mm 3 (WHO, 2006). Treatment is therefore indicated in all patients with a CD4 count < 200/mm 3 and in symptomatic patients ( those with AIDS according to the 1993 Centers for Disease Control and Prevention classification as well as patients with wasting, thrush, or unexplained fever for >2 weeks) ( Peritt et al., 1999) . However, t here is a lack of data to determine at which CD4 level antiretroviral therapies should be started for asymptomatic patients with CD4 7 counts >200/mm 3 . Quite often, an asymptomatic patient with a CD4 count >350/mm 3 presents with a high pre- treatment viral load (>50,000- 100,000), which is another predictor of clinical progression ( Bartlett, 2003; Kassaye and Katzenstein, 2003; Benson et al., 2004) . Therefore, the best option is to individualize treatment decisions by taking into account diverse factors such as the CD4 count and its rate of decline, viral load, patient interest and potential to adhere to therapy, individual risks of toxicity, drug interactions, as well as the risks and benefits of early versus late initiation of treatment. Adherence is a key determinant factor for any successful treatment regimen. To date, current treatment regimens have not been able to eradicate HIV and therefore the strategy is to: i) maximize inhibition of viral replication ; ii) restore and/or preserve immunologic function; iii) improve quality of life; and iv) reduce HIV - related morbidity and mortality. This must also consider associated problems such as expensive regimens , pill burdens, complicated dosing schedules, drug- specific toxicities, drug interactions, viral resistance, and long- term complications, especially lipodystrophy and metabolic changes. Thus far, at least 19 antiretroviral (ARV) agents have been approved for the treatment of HIV infection ( Pulido and Arribas, 2003; Marks and Gulick, 2004; Stanic and Grana, 2009), some of them available through expanded- access programs to allow a large variety of potential combinations. However, no data exists to demonstrate the superiority of one regimen over another and therefore it is considered that the first regimen may afford the best opportunity for long term control of viral replication if administered and monitored properly (Vrijens and John 2005; WHO , 2006) . Furthermore, the importance of individualization of therapy should always be emphasized. This is a complex clinical area which therefore requires proper management by experts in the field. 8 1. 1. 1. 1 Combination t herapy in HIV/AIDS It is now more than a decade since it became apparent that treatment of HIV infection with only one ARV agent is associated with rapid development of viral drug resistance (Richman et al., 1994; Brenner et al., 2002; Ma rtinez - Picado and Wai, 2007; Marconi et al. , 2008). Studies have shown that resistance is becoming more prevalent in ARV - naive patients and therefore ARV drug resistance testing is now an integral part of the management of the HIV - infected persons (Dam et al., 2001; Little et al., 2002) . C linical trials have shown that combining two antiretroviral agents leads to improved virological and immunological responses, when compared to the use of a single agent. A three- drug combination has proven to be even more effective than a two- drug combination ( Bulgheroni et al., 2004: Youle et al., 2006; Greenberg, 2007) . Therefore, ARV monotherapy is not recommended unless under special cases such as in pregnancy, to reduce perinatal transmission. Combination of two or more ARVs has now been adopted as standard treatment for maintaining viral suppression and minimizing the emergence of resistant strains, thereby reducing the risk of disease progression and death. The recommended three drug regimen of HAART usually include two Nucleoside Reverse Transcriptase Inhibit ors (NRTIs) plus a Protease I nhibitor (PI), or a Non - nucleoside Reverse Transcriptase I nhibitor (NNRT I), or three NRTIs. PI drugs may be used singly or in combination with low - dose ritonavir as a boosted regimen that increases trough levels, minimizing opportunities for viral replication and potentially allowing for activity against moderately resistant strains of virus (Stanic, 2005; Lucas, 2008). Boosting also affords a more convenient regimen in terms of pill burden, scheduling, and elimination of food restrictions. Hepatotoxicity is the commonest adverse effect of PIs, especially ritonavir (Lucas, 2008). An NNRT I - based regimen can be as effective as a PI - based regimen with the additional advantage of reserving PIs for a later date. Hepatotoxicity may be caused by both, but the risk with nevirapine (an NNRT I) , is twice that of efavirenz ( also an NNRTI ) after adjustment for PI use and hepatitis C virus 9 co- infection (Pulido and Torralba, 2002) . An advantage of efavirenz is its once- daily dosing, but central nervous system side effects can cause hyperexcitability, vivid dreams, mood disturbances, and personality changes. Both may cause severe skin rashes (Pulido and Torralba, 2002) . For triple NRTI regimens, the most promising are the ones containing abacavir (Kaufmann , and Cooper, 2000; Kessler, 2005; Dieterich, 2006). Advantages of NRTI regimens include a twice- daily schedule, low pill burden (i.e. available in a fixed- dose combination), relatively few adverse effects, and lack of undesirable pharmacokinetic interactions (Keiser, 2002; Sharma and Chakraborty, 2009) . Furthermore, there is the advantage of reserving the other two classes of drugs which may be used at a later stage. It has been observed that hypersensitivity reactions to abacavir occur in 5% of patients subjected to this regimen (Hervey and Perry, 2000; Khanlou et al., 2005). A disadvantage of NRTI regimens is that they are considered ineffective in patients with high baseline viral loads or low CD4 counts (Khanlou et al., 2005; Gallant, 2008). Studies have shown that despite the introduction of HAART, multiple treatment failures still occur ( Ledergerber et al. 1999; Lucas et al., 1999; Moyle, 2003; Flint et al., 2006; Wilcox and Saag, 2008) . Many strategies have been proposed to deal with repeated treatment failures, but with dissapointing results (Lucas et al., 1999; Idemyor, 2004; Lemiale and Korokhov, 2009) . Thus, despite the emergence of ARVs, HIV infection is still incurable. T herefore, the mainstay in the control of the pandemic remains primary prevention. 1. 1.2 A natomy and physiology of the vagina in relation to drug delivery The human vagina is a tubular, fibromuscular organ that extends from the cervix of the uterus to the vaginal vestibule with a length of about 9 cm ( das Neves and Bahia, 2006) . The vagina wall is composed of 4 layers namely the stratified squamous epithelium, lamina propria, muscular layer, and tunica adventitia (Figure 1.1). The epithelial thickness is about 200? 300?m, and it is not significantly affected by the reproductive cycle (Song et al., 2004). The epithelial layer forms a series of transverse folds called rugae which 10 dramatically increase its surface area. Vaginal ecology is normally influenced by factors such as the glycogen content of epithelial cells, glucose, pH, hormonal levels, trauma during sexual intercourse, birth- control method, age, antimicrobial treatment and child delivery ( Budtz - jorgensen, 2000; Verman and Garg, 2000; Justin - Temu et al., 2004; Valenta, 2005) . The vaginal flora is a dynamic system composed of facultative and obligate aerobes and anaerobes with Lactobacillus (D?derlein's bacilli) as the most prevalent organism (He et al., 2004) . Lactobacilli produce sufficient lactic acid to acidify vaginal secretions (including the seminal fluid) to a pH of 3 . 5 ? 5. 5 which covers the pH range that keeps the vagina healthy (4.0- 5. 0) by inactivating HIV and other pathogens . Lactobacilli maintain this pH range by converting glycogen from exfoliated epithelial cells to lactic acid (Boskey et al., 2001). Thus, Lactobacilli play a vital role in maintaining the healthy vaginal acidic environment. Normally , vaginal pH changes with age, stages of the menstrual cycle, infections and sexual arousal . T herefore, in most women, a pH gradient exist s in the vagina (Valenta, 2005). Possible causes o f alterations in vaginal pH are various bacterial infections, yeast infections particularly of the Candida species type, STIs, premature rupture of f etal membranes, hormonal imbalance, postoperative infected wounds and excessive intimate care (Vitali et al., 2007) . A disturbance in the micro - ecological balance in the vagina (e.g. dysbiosis) is often connected with an increase in the pH level ( Mastromarino et al., 2009) . Semen, menstrual flow, cervical and uterine secretions act as alkalizing agents in the vagina changing the pH from acidic to near- alkaline. ( Desphande et al., 1992; Richardson and Illum, 1992 ) . The vagina may also act as a site for drug absorption and action. Th e anterior and posterior vaginal fornixes are recesses (along with the other fornices) that exist due to the projection of the cervix to the vagina. If there is no cervix, there are no fornices. The deepest of these fornices is the posterior fornix, which is the most isolated from any activities. For instance, it is the only part of the vagina that is unlikely to be disturbed by sexual intercourse. It also happens to be out of the way of the menstrual flow, because it is behind and above the cervix. Thus, it is an ideal site for drug delivery application. Vaginal pH is an important parameter for 11 the efficacy of a drug delivery system (Ramsey et al., 2002) . For instance, it may influence drug absorption and must therefore be considered during the development of any such drug delivery system. It has been observed that changes in hormonal levels (especially estrogen) during the menstrual cycle may lead to pH changes as well as alterations in the thickness of the epithelial cell layer, width of intercellular channels and secretions ( Cajander and Rylander, 1988; Owen, 1975) . V ariations in enzyme activity (endopeptidases and aminopeptidases) with hormonal changes may further complicate the problem of achieving consistent drug delivery (Furuhjelm et al., 1980; Pscher a et al., 1989) . During the post- menopausal period, women may experience progressive atrophy of the vaginal epithelium with elevation of the pH (6.0? 7. 5), and a decrease in the quantity of vaginal secretions ( das Neves, 2006; Simon et al., 2008 ) . These fea tures may significantly impair the performance of a drug delivery system and therefore should be considered during development and evaluation of vaginal drug delivery systems. Figure 1.1: A schematic of the vaginal wall showing 1) capillary vessel s, 2) artery and 3) vein, (Adapted: das Neves, 2006) . Stratified squamous epithethelium Lamina propria Muscular layer Tunica Adventitia 1 2 3 12 1. 1. 3 The va ginal mucus in relation to bi oadhesion The t arget for the interaction of bioadhesive agents in the vagina is the mucus. Vaginal mucus is highly viscous and forms a protective coating over the lining of the vaginal epithelium in contact with external media. The vaginal mucus coating has several important physiological functions. It acts as a physical barrier to pathogenic organisms ( Eggert- Kruse et al., 2000) . It is a mixture of large glyc oproteins (mucins), water, electrolytes, epithelial cells, enzymes, bacteria, bacterial products and various other materials depending on the source and location of the mucus (Junginger, 1990) . S everal mechanisms have been proposed to explain the interacti on between a bioadhesive polymer and a biological surface, most being based on the structure of the mucin (Ch'ng et al., 1985; Lehr, 1995). The force and work of adhesion of a given bioadhesive system on a biological tissue is a good indicator of its bioadhesive capacity (Lejoyeux et al., 1989; Valenta et al., 2001). Vaginal secretions are a mixture of fluids produced from various sources (Stechever et al., 2002) . Although the vaginal epithelium is often considered to be a mucosal surface, it lacks goblet cells and therefore cannot release vaginal secretions such as mucin directly (V alenta, 2005). Vaginal mucus originates mostly from the endocervix, with small contributions from the endometrium and fallopian tubes. Vaginal discharge is comprised of a mixtur e of endocervical mucus, transudates through the epithelium, exfoliating epithelial cells, secretions of the Bartholin's glands, leukocytes, and endometrial and tubal fluids (Desphande et al., 1992) . Studies have shown that contact of synthetic polymers that have ionic characteristics with the cervical mucus may cause significant changes in the structure of mucus gels (Willits and Saltzman, 2001; Esp inosa et al., 2002; Valenta, 2005) . Mucin is negatively charged due to the presence of sialic acid residues on its carbohydrate- rich regions (Valenta, 2005) . It has been observed that anionic {e.g. poly(acrylic acid) (PAA) }, non- ionic { e.g. 13 poly(ethylene)glycol } and cationic (e.g. polyvinyl pyridine) molecules may interact with the mucin gel to form regions with higher fiber density (Valenta, 2005) . The fiber aggregation is mainly a result of electrostatic interactions between the polymer and the mucin. T he ability to modify the structure of mucus gels by adding biocompatible synthetic polymers may provide new prevention measures for pathogenic organisms that infect the vaginal mucosa such as STI and HIV pathogens (Valenta, 2005; das Neves, 2006) . Thus, the vagina has been targeted as a potential site for delivery of various drugs. The delivery of compounds is almost exclusively through the vaginal epithelium as described below. One of the most useful applications is the delivery of drugs that destroy or render dysfunctional spermatozoa and pathogens such as viruses and bacteria. 1. 1. 4 The vagina as an application site for drug delivery The vagina, much as it has been proven to be a potential route for systemic drug delivery and uterine targeting, also has huge potential for the administration of drugs intended to confer a local effect, such as microbicides (Richardson and Illum, 1992; Jim?nez - castellanos et al., 1993; Vermani and Garg, 2000; Knox, 2004; Schiffman, 2004; Hussain and Ahsan, 2005; Valenta, 2005; das Neves and Bahia, 2006; Sitruk- Ware, 2007; das Iyer et al., 2008; das Neves et al., 2008; Nelson, 2008; Lai et al., 2009 ) . The epithelium, normal flora, immune cells and acidic pH contribute to the vaginal defences which together with vaginal vascularity provide a potential route for local drug delivery ( Li et al., 2000; Vermani and Garg, 2000; Ogra et al., 2001; Valenta, 2005; Andrews et al., 2009 ) . The vaginal commensals release organic acids, inorganic compounds and antimicrobial peptides into the milieu of the vaginal fluid which includes hydrogen peroxide, lysozyme, lactoferrin, bacteriocin- like substances, biosurfactants, secretory leukocyte protease inhibitor (SLPI), human neutrophil peptides (HNP - 1, - 2, and - 3), and human ? - defensins (HBDs) which aid in killing pathogenic invaders (Wagner and Levin, 1978; Huggins and Preti, 1981; Cohen et al., 1984; B oris and Barbes, 2000; Valore et al., 2002; Venkataraman et al., 2005; Cole and Cole, 2007) . Furthermore, it has been demonstrated 14 that the anatomical position of the vagina in humans, favors secure retention of vaginal formulations (Holmes, 2000; Iyer et al., 2008) . Thus far, there has been considerable progress in the field of vaginal drug delivery in the recent years but with some challenges due to various factors ( Merabet et al., 2005 Dobaria et al., 2007) . To date, only a limited number of vaginal dosage forms are available, but increasing efforts are being made to make studies in this area of research. Apart from the novel drug- loaded inserts which are currently available on the market, there are also hydrogel drug delivery systems which may display higher retention times ( Kumar et al., 2002; Lin and Metters, 2006) . Most vaginal drug delivery systems are based on bioadhesive polymers since bioadhesitivity is an intrinsic pre- requisite for drug retention on the vaginal epithelium ( Chang et al., 2002; Francois et al., 2003; Valenta, 2005; Perioli et al., 2009) . Bi oadhesive polymers are able to swell while eroding slowly when exposed to an aqueous environment and therefore may well facilitate controlled drug release ( Gavin et al., 2002; Kumar et al., 2002; Lin and Metters, 2006; Sudhakar et al., 2006). Significant efforts have been made to improve the adhesive properties of local drug delivery systems resulting in better retention and consequently improved availability and increased therapeutic efficacy (Gavin et al., 2002; Asane et al., 2008; Madhavlal and Manordas, 2009) . Permeation of drugs through the vaginal epithelium is influenced by the physicochemical properties of the drug carrier and the tested drug, in terms of molecular weight, solubility, dissolution rates, lipophilicity, ioniz ation, chemical stability, thermal stability, and also the characteristics of the membrane (Williams, 2003, Justin- Temu et al., 2004; Sandr i et al., 2004) . In addition, physiological factors also play a role in the permeation process, for example cyclic changes in the thickness of the vaginal epithelium, fluid production volumes and composition as well as vaginal pH (Hussain and Ahsan, 2005) . For instance, an increase of vaginal fluid volume may increase the absorption rate of poorly water- 15 soluble drugs. Changes in the vaginal pH may also have an influence on the release of pH- sensitive drugs from vaginal delivery systems (Hussain and Ahsan, 2005) . Lipophilic molecules permeate faster than hydrophilic molecules (van Eyk and van der Bijl, 2005). Studies have shown that for the vaginal epithelium, the molecular weight cut- off points for permeation may be higher than those found in other epithelial surfaces (Williams, 2003, Justin- Temu et al., 2004) . In addition, ionization may affect the permeation of drugs across the vaginal epithelium ( Valenta, 2005; Nicolazzo and Finnin, 2008). D rugs that are in an unionized state penetrate epithelial membranes more freely (Williams, 2003, Justin- Temu et al., 2004) . Generally, chemical compounds cross the epithelial cell layers by two major pathways, namely the intercellular ( paracellular) pathway where compounds pass between cells and the intracellular (t ranscellular) pathway where compounds pass through the cells. In both cases, drug permeation takes place either thr ough passive diffusion, carrier- mediated transport or endocytosis (Meijer et al., 1990; Kapitza et al., 2007) . For mucosal delivery of most drugs, the intercellular route of diffusion predominates (Ghate and Edelhauser, 2006) . Furthermore, studies have shown that two types of pathways exist within the intercellular space (Wertz and Squier, 1991, Harris and Robinson, 1992; Williams, 2003) . The first one is the hydrophobic pathway through the lipid bilayers while the second is the parallel hydrophilic pathway along the narrow aqueous regions that are associated with the polar head groups of the lipids. The penetration of the compound through the epithelial barrier increases as the hydrophobicity of a permeant molecule increases (Wertz and Squier, 1991, Harris and Robinson, 1992; Williams, 2003; van Eyk and van der Bijl., 2005) . 1. 1. 4. 1 Advantages and disadvantages of v aginal d rug d elivery T he vaginal route of drug administration offers advantages over other routes such as the avoidance of hepatic first- pass metabolism, reduction in the incidence and severity of side effects within the gastrointestinal tract (GIT) , and reduction in hepatic side effects of steroids used in hormone replacement therapy or contraception (Patel, 1984; Cedars et 16 al., 1987; Vermesh et al., 1998; Dezarnaulds and Fraser, 2002) . The vaginal route overcomes the inconvenience caused by pain, tissue damage and possible infection by parenteral routes. Furthemore, it has the potential for uterine targeting of active agents such as progesterone and danazol (Cicinelli et al., 1998, 2000; Einer - Jensen et al., 2002). Compared to the oral route, the vagina might prove better for the delivery of hormonal contraceptives due to a lack of drug interactions often encountered in the GIT . Another advantage is the possibility for self- administration of single- dose drug delivery systems that may suffice in releasing drugs over a period of weeks or months and simultaneously provide optimal drug pharmacokinetic profiles (Cohen et al., 1984; Woolfson et al., 2000; Benkop- Schnurch and Hornof, 2003; Iyer et al., 2008) . Further advantages with vaginal administration include convenient access, prolonged retention of formulations, an extensive region for drug perme ation, high vascularisation and relatively low enzymatic activity. Therefore , intravaginal drug delivery has exploitable advantages compared to other routes of administration in the area of bioavailability and controlled drug delivery (Carrington et al., 1944; Goldberger et al., 1947; Mishell et al., 1972; Fried et al., 1973; Kirt on et al., 1973; Nuwayser and Williams, 1974; Johansson et al., 1975; Verman and Garg, 2000; Valenta et al., 2001; Yoo et al. , 2006; Bonferoni et al., 2007; Wang and Tang, 2008). A contributing factor to the exploitable advantages conferred by the vaginal route of drug administration is the fact that the adult vaginal cavity has an extensive surface area (100- 150cm 2 ) that is ade quately accessible for self- administration purposes (Valenta, 2005) . Furthermore, the vaginal route provides room for continuous programmed administration of drugs while simultaneously preventing the superfluous peaking of blood levels that is often seen with the use of oral drug delivery systems administered as discrete discontinuous doses (Valenta, 2005; das Neves and Bahia, 2006). 17 The vaginal route is less preferable in terms of convenience depending on the type of dosage form. The influence of the estrogen concentration on the permeability of the vaginal membrane, which can influence the pharmacokinetics of drugs designed for systemic action, is yet another disadvantage (Okada et al., 1983; Acaturk and Robinson, 1996) . The quantity of vaginal fluid in the vagina of an adult woman is in the range of 2? 3g/24h (M?ller, 1986) . This quantit y however decreases as the age increases. This volume of fluid may also affect the vaginal absorption of drugs. Furthermore, high quantity of vaginal fluid may lead to the expulsion of the dosage form. Studies have also shown that typical drug delivery systems such as foams, gels, and rings may be removed by the self cleansing action of the vaginal track ( Vermani et al., 2002; Alam et al, 2007; Bilensoy et al., 2007; Romano et al., 2008). Since drugs must be in solution before they can be absorbed, the presence of a film of moisture might be an advantage but contrary to this, the presence of thick cervical mucus may also act as a barrier to drug absorption. This may however be an advantage for drugs administered for the purpose of exerting a local effect as is the case with microbicidal agents. In either case, for successful vaginal drug delivery, factors such as changing physiology of the cervix and vagina, the menstrual cycle, coitus, as well as leakage of the formulation from the vagina may grossly affect performance of an intravaginal drug delivery system. These factors must be recognised during design and development of an intravaginal drug delivery system (Garg et al., 2003) . 1.2 Statement of the Problem Several therapeutic agents for curing HIV/AIDS have been developed, but, to date none has been proven to be ideally successful ( Norris, 2008) . Other than therapeuti c agents use, other strategies proposed and adopted over the years to prevent sexually - acquired HIV infection include the promotion of abstinence, monogamy, condom use, reduction in the number of sexual par tners and treatment of sexually - related infections. Yet, cultural, social and economic factors, particularly in the developing world, have resulted in these 18 strategies not being as successful as expected. Condom use has been the last and often preferred preventative measure advanced to combat the spread of HIV and STIs, but youth in particular do not respond appropriately to such initiatives (Fauci, 2005) . When used consistently and correctly, male condoms can reduce the risk of pregnancy and many STIs, including HIV by about 80- 90% (Weller and Davis; 2002; Hatcher,et al., 2004; Hearst and Chen, 2004; Holmes et al., 2004; Warner et al. 2004). Furthermore, condoms (including female condoms), are contraceptive methods that are substantially effective at reducing the risk of both STIs and pregnancy (USAID Health, 2009) . There is the perception that HIV and STIs only affect ?others? and despite the significant attempts to counter the belief that only certain groups are vulnerable and at risk of infection, studies show that some young men see HIV as a disease of atypical behavior associated with rape and commercial sex workers or as a result of excessive alcohol consumption (Pettifor et al., 2005). A Reproductive Health Research Unit study by Pettifor and co- workers (2005) showed an extremely high percentage of r espondents (93%), identified condom use as being an effective preventative measure but 67% did not use condoms consistently and as many as 31% had never used condoms. There is the belief that the way to protect ?innocent victims? is to stop only those who are infected from infecting others, perpetuating the view that their own conduct is unproblematic. As much of the research has failed to advance any explanation for the failure to transform knowledge into positive action, many of the crucial questions remain unasked and unanswered. In the wake of this denial, other effective female controlled measures that can protect both parties from exposure to this fatal disease are needed. The most compelling solution to HIV/AIDS is an effective vaccine. However, aft er 25 years of research, development of an effective vaccine has remained unsuccessful due to various obstacles including inadequate resources, regulatory capacity concerns, intellectual property issues and mainly the scientific challenges (Malcolm et al., 2006 ; Lau et al., 2007; Gurunathan et al., 2009; Katsnelson, 2009) . Despite major efforts and new 19 approaches, it is unlikely that an HIV vaccine will be available for human use in the near future (Web er et al., 2005; Hayden, 2009). This scenario, and the global estimate number of 5 million new cases of people suffering from HIV infection annually , coupled with the rising prevalence rate in women especially in resource- limited settings, identifies the core problem that this study seeks to address through a drug delivery perpective. 1.3 Rationale and Motivation for this Study Given the devastating effects of the HIV/AIDS epidemic and the continuing difficulties in developing an effective HIV vaccine, there i s a clear scientific rationale for developing alternative strategies to prevent STIs and the transmission of HIV infection. Th us, there is a need of searching for accessible, inexpensive and female- controlled pre- exposure prophylaxis strategies to prevent mucosal transmission of the virus. The most promising of these preventative approaches is the development of effective intravaginal microbicidal delivery systems ( Uckun and D'Cruz, 1999; Bob, 2000; Best, 2000; van Damme, 2002a and b); Moore and Shattock, 2003; Balzarini and van Damme 2004; Malcolm et al., 2004; Uckun and D'Cruz, 2004, Mantell et al., 2005; Penttinen, 2005; Weber et al., 2005; Dhawan and Mayer, 2006; Balzarini and van Damme 2007; Eaton and Kalichman, 2007 ; Holmes, 2007; Nikolic et al., 2007; Brouwers et al., 2008; Kathambi, 2008; Klasse et al., 2008; Ndesendo et al., 2008; Obioha 2008; Baloglu et al., 2009; Ndesendo et al., 2009). These are bioactive agents that when administered in the vagina prior to coitus, have the potential to either prevent or reduce HIV/AIDS and STI transmission through well established mechanisms ( D'Cruz et al., 2003b; D'Cruz and Uckun, 2004; D'Cruz et al., 2004a; Weber et al., 2005; Balzarini and Van Damme, 2007) . A microbicide as a preventive strategy may thus function as follows: i) Maintenance and mobilization of normal physiological vaginal defence mechanisms in the cervicovaginal environment. A healthy, intact vaginal mucosa is an ideal natural 20 protective barrier against infection. This barrier may be supported by gel - forming agents that provide lubrication and an additional barrier to infectious organisms. ii) Enhancement of the natural vaginal defence mechanisms by maintaining an acidic pH environment, commensal vaginal microflora, antibodies and antimicrobial peptides. iii) Direct inactivation of sexually transmitted pathogens (HIV and STIs) by disrupting the pathogens? protective membranes or envelopes. iv) Inhibition of early phases in the viral cycle of HIV and entry into the mucosal cells of the vaginal wall by specifically blocking surface proteins on the virus or by binding to receptors on human cells or by non- specifically coating the virus or human cells. v) Interruption of the viral life cycle at a post i nfection stage. ARV s already used for therapeutic use may also be employed as topical microbicides. These agents act by inhibiting viral replication or local spread of HIV infection. As opposed to the use of condoms, there may be greater potential for microbicidal drug delivery systems to empower women to protect themselves from HIV and STIs. An effective microbicide would be a valuable woman- controlled means to slow down the pace of the HIV/AIDS epidemic particularly in sub- Saharan Africa, where women aged between 15 to 24 years are three times more likely to be infected with HIV - 1 compared to the same age group of men (Fauci, 2005; Quinn and Overbaugh, 2005). To date, t he development of an ideal microbicide (i.e. safe, effective, acceptable, affordable and accessible) has not been entirely successful ( Weeks et al., 2004; Das Neves et al, 2006; Klasse et al., 2006; Balzarini and Van Damme 2007; Nuttall et al., 2007) . O nly a limited number of microbicidal drug delivery systems are available. The currently available vaginal delivery systems have several limitations which include leakage, messiness, low residence time and poor bioavailability, contributing greatly to poor compliance (Justin- Temu et al., 2004; Valenta, 2005; Biradar et al., 2009). Furthermore, the results of early clinical trials for the already developed microbicides have not been very encouraging (McCormack et al., 21 2001; Lazarus et al., 2008; Skoler- Karpoff et al., 2008) . Therefore, efforts need to be directed at the development of novel microbicidal intravaginal drug delivery systems. The ultimate success of any intravaginal drug delivery system depends on the formulation and the bioactive agent. This requires consideration of several variables including the bioactive agent, vaginal physiology and the design of the delivery system. For these drug delivery systems, the challenge is to design a delivery system that provides a high drug concentration in the vagina over a prolonged period of time (Benkorp- Schnurch et al., 2003) . For controlled, zero- order release, sustained over prolonged periods (days extending to months), solid polymeric systems may be most suitable provided they are compatible with the physicochemical nature of the drug to be delivered and the body. To date, drug release durations for intravaginal delivery systems are either too rapid which ultimately requires the drug to be applied several times daily (typical of gels and tablets) ( Gavin et al., 2002; El- Kamel et al., 2002; Wang and Lee, 2004; Bil ensoy et al., 2006) or the release period is adequate but they have been formulated for preventing HIV infection only, as is the case with vaginal rings ( van Laarhoven et al., 2002; Malcolm and Woolfson., 2006) . This research therefore proposes the development of a novel drug delivery system that will be active against both HIV and STIs with a controlled release effect of at least 1 month. Th is is a caplet- shaped device referred to as an ? Intravaginal Bioadhesive Polymeric Device ?(IBPD)? throughout this study. The delivery system will have desirable bioadhesive properties and will be capable of inducing an acidic pH environment in the vagina upon degradation, thus maintaining a wide spectrum of activity against pathogens. The main adv antages of the developed IBPD are : i) it is easier and more convenient to apply a caplet than a gel or ring; ii) t he developed bioadhesive caplet has desirable vaginal retention as opposed to poor retention conferred by gel formulations (Gavin et al., 2002); iii) controlled release of the drug will mean less frequent application and therefore enhanced patient compliance; iv) the developed IBPD is non- messy and 22 can reside in the vagina long before coitus releasing drug gradually; the IBPD will be produced at low manufacturing costs and v) the IBPD can utilize more than one preventive mechanism of action unlike existing vaginal delivery systems which utilizes single mechanism of action. The IBPD has been designed to have a dual action by releasing an ARV and microbicide simultaneou sly. A formulation that simultaneously delivers an ARV and microbicide was sought because it has been suggested that a vaginal drug delivery system combining several mechanisms of prevention would be more effective or have less side effects than one with a single mechanism of action as is the case with preparations currently being researched (D?Cruz and Uckun, 2004; Ramjee, 2006; Balzarini and Van Damme 2007; Cutler and Justman, 2008; Ndesendo et al., 2008, Gupta and Garg, 2009 ), partly due to inadequate attention to formulation, design and delivery technology development (Panttinen, 2005) . The model microbicidal agent employed was Polystyrene sulfonate ( PSS) (as a sodium salt) while the ARV agent was 3' - azido- 3' - deoxythymidine ( AZT ) . The dimensions of the I BPD were 22? 9 ? 5 mm for insertion into the posterior fornix of the vagina using an applicator (Figure 1.2) . The posterior fornix location was purposely chosen to avoid any interference with the formulation during menses and sexual intercourse ( Section 1.1. 2) . T he anterior fornix was considered to be inappropriate for this purpose because it is right in the line of entry for a male sexual organ, and the in and out movement might disrupt the adhesion of the device. The formulation was designed in such a manner that a steady- state concentration of bioactive agents could be maintained locally ( within the vaginal membrane and in the vaginal inner mucosal muscularated stratum) . 23 Figure 1.2: A schematic of the upper vagina region depicting the posterior fornix as the site of the IBPD application. 1.4 Aim and Objectives of this Study The aim of this study was to design and develop an IBPD for the potential prevention of STIs and HIV infection. In order t o achieve this aim, the following objectives have been outlined: 1. To conduct extensive pre formulation screening work on various biodegradable and biocompatible FDA- approved polymers for the selection of lead formulations that would ultimately provide an ideal bioadhesive polymeric set which is also capable of providing controlled drug delivery. 2 . To optimize the IBPD using the Artificial Neural Networks (ANN) approach. 3 . To assess the bioadhesivity of the drug - free IBPD that was developed from the blends of the selected polymers both in vitro and in vivo and optimize for bioadhesivity thereof. 4 . To perform in vitro and ex vivo analysis on the dug- loaded optimized IBPD in terms of physicomechanical and physicochemical properties, bioadhesivity, permeation and drug release characteristics. 5 . To perform computational and chemometric molecular structural modeling studies on the native polymers, drug- free IBPD and dug- loaded optimized IBPD . I Applicator Anterior fornix IBPD in the posterior fornix Cervical os Cervical os 24 6 . To conduct in vivo studies on the d rug- loaded optimized IBPD in the pig model. 1 . 5 Overview of this Thesis Chapter 1 provides the study protocol which contains the introduction, rationale, motivation for the study and objectives. The introduction provides a concise description of the current situation regarding the HIV/AIDS epidemi c, proposing the way forward for which the enaction is taken care of by the highlighted aim and objectives. A clear description on the role played by STIs in enhancing both HIV susceptibility and secondary transmission is provided. An overview of the treat ment modalities that have been employed in the management of HIV/AIDS is given. In addition, a description is provided on the anatomy and physiology of the vagina in relation to drug delivery, and the vagina as an application site for drug delivery, as well as the advantages and disadvantages of this route of adminstration. The characteristics of an ideal vaginal drug delivery system are described, and the concept of a microbicidal- Intravaginal Bioadhesive Polymeric Device is introduced. Chapter 2 provides a descriptive review of the current intravaginal drug delivery approaches that have been employed for the prophylaxis of HIV/AIDS and prevention of STIs . The current status of various microbicidal drug delivery systems, particularly in the context of thei r stage within clinical trials and their potential cervicovaginal defence successes are described. Furthermore, approaches that may lead to the successful design and development of more effective intravaginal microbicidal delivery systems for preventing the transmission of STIs and HIV are suggested. Chapter 3 explains thoroughly how various natural and synthetic biodegradable and biocompatible polymers with hydrophilic and hydrophobic properties were screened in an attempt to find suitable polymers for developing a novel intravaginal bioadhesive 25 polymeric device (i.e. IBPD). The key requirements for an intravaginal delivery system are: i) ease of manufacture; ii) low manufacturing costs; iii) ease of insertion; iv) high retention rate; v) minimal adverse effects such as damage to the vaginal epithelium; and vi) the potential for attaining controlled drug release and suitable lateral permeation (i.e. across the vaginal tissue) . To satisfy all these requirements, it was necessary that the materials used for the development of such systems should have a multitude of essential properties. These include among others, biodegradability, biocompatibility, bioadhesivity, permeation capacity, compressibility and high matrix resilience. To achieve this goal, a One Variable at a T ime (OVAT) approach was employed. Chapter 4 discusses how Artificial Neural N etworks (ANNs) have been successfully employed in the optimization of the IBPD. For successful design and development of novel drug delivery systems careful selection and optimization of several formulation and process related variables is of paramount importance. This avoids wastage of materials and time, with the subsequent benefit of reducing the production costs. I t is important to know exactly how preparation com positions determine the formulation characteristics and in particular how formulation factors and potential interactions between them affect the performance of a given delivery system. Generally, the impact of each variable can be assessed by varying one variable at a time while keeping others constant. The OVAT approach employed in Chapter 3 in the extensive investigational screening of the polymers has thus enabled the selection of the best 15 lead formulations from 62 formulations. However, the OVAT appr oach cannot take into account interactions between the formulation factors. ANNs have been shown to be uselful in dealing efficiently with this difficulty and have therefore been used in this study. Chapter 5 provides an evaluation of the bioadhesive capacities of the various polymers that have been employed in this study for developing the IBPD. For any given intravaginal drug delivery system, its retention to the vagina is an absolute requirement. 26 Good retention allows for the bioactives to be maintained at an optimal level locally and for extension of their residence time at the site of administration. To meet this requirement, an intravaginal device needs to be highly bioadhesive. Chapter 6 provides a thorough description of the evaluation of bioadhesivity, in vitro drug release, and permeation of the drugs from the IBPD. I t also seeks to explain the physicochemical and physicomechanical properties of the developed drug delivery system. One of the main objectives of this study was to design and devel op an intravaginal drug delivery system in which the formulation would initially swell in order to facilitate bioadhesion, and thereafter erode gradually to release the drug in a controlled manner, to finally permeate laterally into the vaginal tissue and provide the needed preventive effect against HIV and STIs. Chapter 7 describes the analysis of the drugs (AZT and PSS) in the vaginal tissue and plasma of a pig model admistered with an IBPD . It also provides a thorough histopathological toxicity evaluat ion of the IBPD in the pig model. This was in line with the ultimate goal of the study which required an intravaginal delivery system that could be retained in the vagina and release an ARV and microbicide simultaneously. Furthermore, the intention was that most of the released drugs would be retained in the vaginal tissue without causing toxicity . Assessment of the adherence, degradation and distribution patterns of the IBPD was also undertaken in this Chapter. Chapter 8 concludes the thesis, assimilating the significant issues addressed regarding the design and development of the novel controlled release IBPD, with recommendations for future investigations in terms of system applicability. 27 1. 6 Concluding Remarks The current status of the HIV pandemic including the successes and failures of the therapeutic approaches which therefore identified the research problem has been well addressed. The aim and objectives of this study were pragmatically selected to realize the goal of developing a bioadhesive controlled release/prolonged release microbicidal drug delivery system. The synergy between STIs and HIV which enhances the susceptibility to secondary transmission of HIV have been elucidated and emphasized as being important entities to consider for the rational development of novel HIV prevention strategies. The anatomy and physiology of the vagina in relation to drug delivery has been addressed. Although the vagina exhibits a degree of dynamism, the benefit of exploiting it as a potential route for drug delivery still holds. Thus, the vagina still has its own place as a unique site for drug administration. Vaginal drug delivery can be engineered in such a way that maximum benefit is obtained as long as factors such as cyclic changes and physiological changes during menopause, are taken into consideration. Currently, the availability of microbicides is not a challenge as several are already in existence. The greatest challenge is finding the most effective way of delivering them to the required sites. It is a nticipated that the design and methodological approach that has been undertaken in this study, will offer significant progress to this end, with the added benefit of the inclusion of an ARV into the formulation. 28 CHAPTER TWO L ITE R AT UR E R E VIE W OF C UR R E NT INTR AV AG INAL DR UG DE L IVE R Y AP P R OAC HE S E MP L OY E D F OR THE P R OP HY L AXIS OF HIV/AIDS AND P R E VE NTION OF S E XUAL L Y TR ANS MIT TE D INF E C TIONS 2.1 I ntroduction Intravaginal drug delivery systems have traditionally been used to deliver contraceptives and drugs such as antibacterials, antifungals, antiprotozoals, antivirals, labor - inducing and spermicidal agents, prostaglandins, steroids, peptides and proteins to treat vaginal infections (Katz et al., 1997; Verman and Garg, 2000; Weber et al., 2005) . Formulations have included pessaries and tablets designed after the advent of rectal suppositories. The first intravaginal controlled drug delivery system was developed in 1970, using a vaginal ring for the delivery of medroxyprogesterone acetate for contraception (Verman and Garg, 2000) . However, tablets, creams, and suppositories are now the most conventional formulations in vaginal drug delivery while vaginal rings are more commonly employed for long- term drug delivery. Recent advances have been made in the area of bioadhesive gels, microparticles and tablets, which show great promise for use as controlled intravaginal microbicide delivery systems ( Tang et al., 2005; Valenta, 2005; Bonferoni et al., 2006; Al - Tahami and Singh, 2007) . Numerous hydrophilic polymers and hydrogels have been used in a number of vaginal products ( Lew, 1994; Bogentoft and Carlsson, 1996; Robinson and Bologna, 2002; Al- Tahami and Singh, 2007). Natural hydrogels include among others, starch, collagen, various proteins, gelatin and dextrans. Swollen cellulose derivatives such as hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC) and sodium carboxy methylcellulose (NaCMC) have also been used, especially in tablet f ormulations. 29 Among the standard synthetic hydrogels, poly(ethylene oxi de) and poly(acrylic acid) have been used in vaginal microbicide delivery formulations (Lee et al., 2000) . The thick cervical mucus in the vagina may assist in the bioadhesion of various delivery systems, at the same time serving as a diffusion barrier to subsequent microbicidal delivery (Lard- Whiteford et al., 2004) . V aginal gels such as carageenan and PRO 2000 ? Gel ( Indevus Pharmaceuticals, Inc. Lexington, MA, USA) formulations when employed for the prevention of HIV and herpes virus transmission may be e ffective for up to one hour after sexual intercourse (Phillips and Maguire, 2002; Cadena, 2006; Rando et al., 2006; Brache et al., 2009) . Thus, there is a need to develop affordable and easy to apply microbicidal delivery systems. Among potential intravaginal delivery systems about which researchers at the WHO, UN AIDS, and other organizations have expressed hope in terms of safety and effectiveness, are vaginal gel formulations such as BuffergelTM and CarraguardTM ( Maguire, 2001; Thom, 2007) . It is antici pated that future research will focus on the development of combination products capable of delivering microbicides that violate the HIV life - cycle at multiple steps, have increased efficacy and have limited cross- resistance and which can minimize microbic ide- induced host toxicity (Penttinen, 2005) . In the absence of an effective prophylactic anti- HIV vaccine or therapy, current efforts are aimed at developing topical intravaginal formulations of anti- HIV agents or microbicides to reduce vaginal and perinatal virus transmission and cure and prevent STIs which may enhance HIV transmission. Therefore the purpose of this Chapter is to provide an overview and description of recent developments in intravaginal delivery systems for microbicides employed for the prophylaxis of HIV/AIDS and prevention of STIs. 30 2.2 The Rationale for the Development of Intravaginal Microbicidal Delivery Systems Currently, much research on intravaginal drug delivery focuses on the prevention of HIV infection and STIs. Development of vaccines for the prevention of HIV transmission is still a challenge (Bourinbaiar et al., 2006 ; Lau et al., 2007; Hayden, 2009; Katsnelson, 2009 ) . Microbicides circumvent many of the immunological difficulties associated with HIV vaccine development and make topical formulations such as gels a more realistic goal, especially in the short term. On the other hand, male condoms are not always accepted and correctly used. Unlike condoms, microbicidal del ivery systems may be controlled by women, and do not require the cooperation, consent or even knowledge of the partner. Microbicidal delivery systems can provide an option to thousands of women who are at risk of contracting HIV and other STIs but are unable to negotiate condom use because of gender inequality, illiteracy, cultural resistance and poverty in many developing countries (Joshi et al., 2005) . Thus, microbicidal delivery systems may be a solution to the prevention of STIs and sexual transmission of HIV infection (Moore et al., 2003; Malcolm et al., 2005 ) . The potential for developing controlled release formulations for long- term intravaginal delivery of microbicides has recently gained momentum and may overcome challenges relating to compliance, acceptability and efficacy associated with current gel- based formulations (Woolfson et al., 2000; Moore et al., 2003; Nikolic et al., 2007; Klasse et al., 2008; Obioha 2008; Baloglu et al., 2009 ) . Furt hermore, the emergence of resistant HIV strains and toxic side- effects of current ARVs require intravaginal delivery systems with superior safety profiles and potential for improved patient compliance. An intravaginal microbicidal delivery system combining several mechanisms of preventing the transmission of STIs and HIV would need to possess added effectiveness and less side- effects than a delivery system having a single mechanism (Penttinen, 2005) . Figure 2.1 depicts various mechanisms of preventing the transmission of sexually transmitted 31 infections and HIV by employing microbicide delivery systems. The challenge is to design a delivery system that is able to provide high concentrations of the microbicidal compound in the vagina over a prolonged period of time (Benkorp- Schnurch et al., 2003) . Studies have indicated that dosages in first- generation microbicides remain effective only for a few hours and therefore necessitate administration shortly before coitus ( Kiser , 2006a ) . Figure 2.1: Schemati c depicting the various mechanisms of preventing the transmission of sexually transmitted infections and HIV by employing microbicide delivery systems, (Adapted: Stone, 2002) . 2.3 C urrent Intravaginal Microbicide Delivery Approaches for Preventing the Transmission of STIs AND HIV The majority of microbicidal delivery systems for intravaginal administration that have been developed and evaluated in ongoing clinical trials are conventional semi- solid aqueous gels and vaginal ring formulations. These have been designed to provide a single dose of a microbicidal agent (Di Fabio et al., 2003; Herrewege et al., 2004; Alliance for Microbicide Development, 2005; Mitchel, 2006) . Prior to widespread use, an intravaginal microbicide delivery system should meet several criteria (Table 2. 1) in that the microbicide employed should be effective against a diversity of HIV strains. Gels or creams form physical barriers or lubrication Maintenance of normal vaginal microflora Prevention of other transmitted diseases Fusion/absorpt ion inhibition onto cell co- receptors Inhibition by non- nucleoside reverse transcriptase Inhibition of HIV uptake by dendritic cells Viral disruption prevents entry Vaginal Epithelium Stoma Layer 32 Table 2.1: Desirable criteria for ideal intravaginal microbicidal delivery systems i) Simple to manufacture, cost effective and easy to apply thus facilitating patient compliance. ii) Non - irritative and free from producing any physical discomfort. iii) Provide immediate and sustained protection by releasing the microbicide in a controlled manner over a prolonged period of time. iv) Have suitable vaginal retention and distribution. v) Be versatile against various pathogens encompassing STIs and HIV. Currently, the maximum duration of drug release for intravaginal microbicidal delivery systems is as follows: i) Vaginal gels (6 hours) ( Wang and Lee, 2004; Bilenso y et al., 2006), ii) vaginal tablets (8 hours) (El - Kamel et al., 2002; Gavin et al., 2002), and iii) vaginal rings (71 days) (van Laarhoven et al., 2002; Malcolm and Woolfson., 2006) . Vaginal gels and tablets have rapid release rates which, for effective use, ultimately require administration several times a day. Vaginal rings have adequate release rates but have only been formulated for preventing the transmission of HIV and as a contraceptive. Several intravaginal delivery systems for microbicides have been formulated and traditionally include a large variety of pharmaceutical dosage forms such as semi- solids, tablets, capsules, pessaries, liquid preparations, vaginal films, vaginal rings, foams, and tampons. The most widely used semi - solid preparations for vaginal drug delivery include creams, ointments, and gels (Verman and Garg, 2000) . These systems could be used to reverse the rising rates of STIs such as external genital warts, gonorrhea, chlamydia, and syphilis, which significantly predispose individuals to acquiring HIV ( Penttinen. 2005; Kiser, 2006a ) Over the past decade, there have been major advances in the field of microbicidal delivery with diverse types of delivery systems in various stages of development (Harrison, 2003) . The ultimate success of an intravaginal microbicide delivery system requires consideration of variables including the microbicidal agent, vaginal physiology and the design of the delivery system. Targeting HIV entry into the body is a favored 33 preventative approach as it is the initial step in the process of infection. Several readily available anionic polymers interfere with the entry processes of HIV, and hence these polymers qualify as primary candidates for designing various microbicidal delivery formulations (Bernkop - Schnurch et al., 2003) . However, few of these formulations have been tested in Phase I/II clinical trials, in HIV - uninfected, and HIV - infected populations (D?Cruz and Uckun, 2004) . Current multi - center Phase I/II safety and Phase II/I II efficacy studies are being conducted or planned in different geographical locations, by various groups which aim to design systems for rapid clinical applications ( Bentley et al., 2002; Morin et al., 2008) . 2.4 Design of Intravaginal Microbicide Delivery Systems Intra vaginal delivery may be formulated for administration of microbicides by using an applicator or specifically designed systems for intravaginal administration. In general, based on the delivery system or the microbicide used, drug absorption, distribution and residence time in the vagina may vary. Early studies by Johnson and Masters (1962) showed that the microbicide distribution in the vaginal tissue varies considerably with the nature of the delivery system. Solutions, suspensions and foams displayed greater superiority over oral dosage forms. Ideally, a vaginal delivery system that is intended for localized microbicidal delivery should distribute uniformly throughout the vaginal cavity. Thus, for a local effect to occur in the vagina, semi - solid, or fast dissolving solid systems are required. Bioadhesive delivery systems or intravaginal ring systems are more suited for topical effects. However, it has been difficult to measure the distribution of a microbicide after intravaginal administration (Di Fabio et al., 2003; Hussain and Ahsan, 2005; Scorpio et al., 2008; Hendrix et al ., 2009) . Currently there is no data available regarding the bioavailability of most intravaginal microbicide delivery systems after extended vaginal exposure (D?Cruz and Uckun, 2004; Cutler and Justman, 2008; Hendrix et al., 2009). Furthermore , most of the intravaginal drug delivery systems for contraception and the prophylaxis against HIV lack efficacy (Katz et al., 1997) . 34 Engineering and materials science may provide critical and new information to the process of designing and developing superior intravaginal microbicide delivery systems. First- generation microbicidal compounds that are currently under investigation are expected to be available within a f ew years. These systems may only be 50- 60% effective in delivering microbicides (Kiser, 2006a ), but even with this efficacy if they are used by only 20% of women, in 73 low - income countries, they may still lead to the prevention of 2.5 million new infections during a three- year period (Penttinen. 2005; Kiser, 2006a ). 2.4.1 Creams and gels Although creams and gels are commonly used for the topical intravaginal delivery of microbicides ( Bernkop- Schn?rch and Hornof, 2003; Hardy et al., 2007) , these systems are messy, uncomfortable and may not provide an exact dose due to non - uniform distribution and their leakage (DuBouchet, et al., 1998; Hussain and Ahsan, 2005). To evaluate the efficacy of a 3- day course of clindamycin vaginal cream in the treatment of bacterial vaginosis, some researchers performed a randomized, placebo controlled trial in pregnant women (Broumas and Basara, 2000; Lamont et al., 2003) . They found that the clindamycin cream was well tolerated and more efficacious than placebo (Broumas and Basara, 2000; Lamont et al., 2003) . Vaginal creams and gels are based on the principle of emulsion or hydrogel- based drug delivery. During the past few years, considerable work has been done on the development of hydrogel controlled release microbicide delivery systems (Owen, et al., 1999; Verman and Garg, 2000; Alvarez - Lorenzo and Concheiro, 2002; Tang et al., 2005; Valenta, 2005; Bonferoni et al., 2006; das Neves and Bahia, 2006; Al- Tahami and Singh 2007; Alvarez - Lorenzo and Concheiro, 2008 ) . For example, a 3% alginate gel of nonoxynol - 9 has been investigated for intravagi nal spermicidal activity (Owen et al., 1999). The study found that the spermicidal activity and the diffusion of the agent changed with the pH and osmolarity of the formulation. Recently, a gel microemulsion- based spermicide formulation, phenyl phosphate derivative of zidovudine, with anti- HIV effect, has been developed (Yiv, 2006; D?Cruz and Uckun, 2006a and b; 35 D?Cruz and Uckun, 2007) . Multiple intravaginal applications of this drug as a microemulsion gel formulation did not cause any damage in the vaginal epithelium in the pig model (D?Cruz, 2003b; D?Cruz et al., 2004b; D?Cruz and Uckun, 2006a; Yiv, 2006; D?Cruz and Uckun, 2007 ) . 2.4.2 Tablets and suppositories Some authors use the terms pessaries and suppositories interchangeably and consider vaginal tablets as a separate dosage form. These f ormulations are designed to melt in the vaginal cavity and release the microbicide over several hours. Suppositories are most commonly used to administer drugs for cervical ripening prior to child birth and for local delivery of various drugs. Vaginal tablets may contain binders, disintegrants and other excipients that are used to prepar e conventional oral tablets. Bi oadhesive polymers are sometimes used in tablet formulations to increase the vaginal residence time of the microbicide been delivered. The presence of hydrophobic and release retarding materials may decrease the absorption of microbicides from a vaginal delivery system. Highly hydrophobic microbicides may not be suitable for vaginal tablets. The presence of penetration enhancers such as surfactants or bile salts can also significantly enhance systemic absorption if required. Other vaginal tablet- like formulations are extrapolations of silicone- based vaginal rings. Research groups have studied the release of microbicides from silicone matrices (K atz and Dunmire, 1993; Klavinskis, 1999) . Release studies were performed in vitro for up to 1 year and in vivo in pigs for up to 52 days. Both in vitro and in vivo studies showed consistent release profiles over time, showing that microbicide delivery is controlled by diffusion from the silicone delivery device and was not limited by absorption through the vaginal epithelium. Further analysis of this diffusion process was performed by the development of a composite physical model. This involved the simultaneous receding boundary release of drug from a cylindrical silicone matrix delivery system, diffusion across an aqueous layer (vaginal fluid) and tran sport across the vaginal membrane by way of parallel lipoidal and aqueous pore pathways. The important 36 parameters in the analysis of this simulation are the solubility of the drugs in the silicone matrix, the solubility of the drugs in water, diffusion coe fficients, silicone/water partition coefficients, and the permeability coefficient of the vaginal membrane to the microbicide. 2.4.3 Vaginal r ings Vaginal rings are circular ring- type drug delivery devices designed to release microbicides in a controlled manner after insertion, to prevent the transmission of STIs and HIV (Malcom et al., 2004 and 2006; Woolfson et al., 2006) . The advantages of such a device are that they are controlled by the user; they do not interfere with coitus and they allow for the continuous delivery of microbicidal compounds. Vaginal rings are approximately 5.5cm in diameter with a circular cross section diameter of 4? 9mm. In simple vaginal rings, the microbicide is homogeneously dispersed within a polymeric ring with the surface of the ring releasing the microbicide faster than the inner layers. The key challenge in development of these systems is finding the optimum dose that will deliver the least amount of microbicide necessary to ensure protection. Advances have been made on the original two- layer ring system by adding a third, outer, rate controlling drug- free elastomer layer ( Washington, et al., 2001) . This formulation design minimizes the drug concentration spike which was often observed during the first treatment cycle using a new ring. The rings are usually designed so that a new device is re- inserted at a time interval which is dependent upon the precise design of the delivery system and the microbicide being delivered. To obtain constant microbicide release from the vaginal ring, sandwich or reservoir- type rings have also been developed. Sandwich type devices consist of a narrow microbicide- loaded layer located below the surface of the ring and positioned between a central core and an outer band. In reservoir type ring systems, microbicides are dispersed in a centralized core, which is then encapsulated by a polymeric layer. In a single ring, it is possible to have several cores of different microbicidal compounds, thereby allowing administration of several microbicidal compounds from the same device. The rate of drug release can be modified by changing 37 the core diameter or thickness of the polymeric coating. Much of the vaginal ring literature relates to the commonly used polymer, poly(dimethylsiloxane) or silicone devices, although other elastomeric polymers such as ethylene vinyl acetate and styrene butandiene block copolymers have been tested (Roumen and Dieben, 1999; and van Laarhoven, et al., 2002; Airey et al., 2008; Hamid et al., 2008) . The addition of vinyl acetate units in the polyethylene provides increased flexibility and greater adhesion. In a study by Roumen and Dieben (1999), evaluating the tolerability of ethylene vinyl acetate placebo vaginal rings of diameter 54 mm, there was 91% acceptability among subjects involved in the study. The ring remained inserted for 21 consecutive days after insertion, with temporary removal during coitus (Roumen and Dieben, 1999). Most women judged the ring easy to insert and remove, and no side- effects were experienced among the tes t group during the study period (Roumen and Dieben, 1999; Ballagh, 2001; Harwood and Mishell, 2001; Dezarnaulds and Fraser, 2002; Nov?k, et al., 2003). 2.5 Bioadhesive Intravaginal Systems Most conventional intravaginal f ormulations however are associated with disadvantages of low retention to the vaginal epithelium, leakage and messiness, thereby causing poor patient compliance. To circumvent these challenges, bioadhesive microbicidal deliv ery systems are being promoted. A bioadhesive polycarbophil gel, Replens ? (One Nil Trade Ltd, Swanbridge, Caerphilly, UK), is currently available on the market, which is used to retain moisture and lubricate the vagina. The formulation remains in the vagina for 2? 3 days and maintains the vagina at a healthy acidic pH ( Richardson and Armstrong, 1999). Bioadhesive polymers that have been used for intravaginal formulations include among others polycarbophil, hydroxyprop ylcellulose and PAA (Saettone et al., 2002; Hussain and Ahsan, 2005; Andrews et al., 2009). The first bioadhesive systems for vaginal drug delivery were in the form of tablets which could be placed on a diseased area for delivery of bleomycin, an anti- cancer agent ( Hwang, et al., 1977; Brannon - Peppas, 1992) . These tablets contained hydroxypropylcellulose and PAA (Carbopol ? 934) . Release rates in vitro 38 increased with increasing quantities of hydroxypropylcellulose and water absorption increased with increasing quantities of PAA. Studies in vivo showed that after 5 days of treatment with a total of 150mg of bleomycin, in some cases, cancerous lesions had necrosed and the normal mucosa was unaffected ( Hwang, et al., 1977; Brannon- Peppas, 1992) . Bioadhesive tablets have been studied for the delivery of zinc sulphate as a model drug from compressed tablets of PAA and sodium carboxymethylcel lulose, as bioadhesive polymers and cellulose (Avicel ? PH102) (FMC BioPolymer , Philadelphia, USA) as a diluent ( Sanders and Matthews, 1990) . Experiments were performed in situ using bovine vaginal tissue. The force necessary to det ach the tablets from bovine tissue was measured and correlated with the tablet composition. It was found that tablets containing only polyacrylic acid and zinc sulphate exhibited the optimal integrity. The presence of Avicel? in the formulations increased the tablet swelling in all cases but did not substantially decrease the force of detachment nor alter the drug release behavior. Increasing the quantity of polyacrylic acid increased the bioadhesion of the tablets to the vaginal epithelium ( Woodley, 2001; Knuth, et al., 1993; Ceschel, et al., 2001) . Attempts have also been made to deliver microbicides using bioadhesive microparticulate vaginal systems ( Vasir et al., 2003; Hussain and Ahsan, 2005) . These systems may be a multi- phase liquid or a semi- solid, but they have been designed so as not to seep from the vagina like pessary formulations. The system is designed to provide controlled microbicidal delivery for at least 3 hours. The units of the release system have a lipoidal external phase and a non- lipoidal internal phase, providing bioadhesion when applied. A bioadhesive controlled release delivery system for nonoxynol - 9 was reported by Lee and co- workers (1996) consisting of varying levels of nonoxynol - 9 and ethylenediamine tetra - acetic acid ( EDTA ) , a chelating agent, formulated using the polymer Carbopol? 934P. Carbopol? 934P provided a high burst release of nonoxynol - 9 in the first 2 minutes and controlled release for up to 6 hours (Lee et al., 1996) . Ceschel and co - workers (2001) developed a new bioadhesive vaginal dosage form by incorporating bioadhesive polymers 39 namely polycarbophyl, hydroxypropylmethylcellulose and hyaluronic sodium salt, into pessaries made of semi- synthetic solid triglycerides. The authors of this study argued that these polymers hold the pessaries in the vagina for longer periods of time, without side- effects, thereby prolonging the permanence of the drug on the vaginal epithelium. The presence of bioadhesive polymers largely modulated the behavior of pessaries in terms of adhesive force, liquefaction time and permanence of the drug in the simulated application site. However, their presence did not alter the release of the drug. The developed formulations showed good technological and adhesion properties and the ability to hold the delivery system at the application site. 2.6 Compounds Delivered Intravaginall y as Microbicides T he numerous intravaginal compounds delivered intravaginally as microbicides are listed in Table 2.2 for which a concis e description is provided hereafter. Table 2.2: Classification of the numerous intravaginal compounds delivered intravaginally. Classification Compound Polymeric Compounds: Carageenan, Monocaprin, Polyacrylic Acid, Lact obacillus, Cellulose Acetate Phthalate, Cellulose Sulfate, Polystyrene Sulfonate, Naphthalene Sulfonate, Sulfated Polyvinyl Alcohol and Lactic Acid Non - Polymeric Compounds: Cetyl Betaine, Myristamine Oxide, Stampidine, Cyanovirin- N, Monoclonal Antibodies, Lyposomes, Thrombospondin - 1, Lime Juice, Yoghurt, Tenofovir and Zidovudine Nano- Structured Compounds: Dendrimers, Thiourea, Silver, Polystyrene and Sodium Lauryl Sulfate 40 2.7 Dual -function Polymeric Gel -based Formulations Employed in the Design of In travaginal Microbicide Delivery Systems: As Excipients with Potential Therapeutic Activity Microbicidal gel f ormulations are likely to be only partially effective, but can produce a significant difference in places such as Africa, where most gels can be produced at reasonable costs. The properties of the gel in which microbicides are included are known to affect the epithelial permeation rate of microbicides and their microbicidal properties (Wang and Lee, 2002; Geonnotti and Katz, 2006; Hendrix et al., 2009 ) . A formulation comprising Carbopol? 974 ( Noveon Inc, Cleveland, OH, USA ) 1.0? 1. 5% w/ w and HPMC 1.0? 1. 5% w/ w displayed a desirable gel- base intravaginal microbicide delivery system (Garg et al., 2001) . Furthermore, the development of a ?universal? placebo gel is required in order to assure that the placebo formulation does not distort either safety or efficacy assessments of microbicides. Recently, a hydroxyethylcellulose (HEC) placebo gel has been proposed as an adequate ?universal? placebo, which is safe and sufficiently inactive for use in the clinical study of investigational microbicides (Wang and Lee, 2004a ). Patient compliance is one of the main attributes that excipients should provide to gel - based vaginal delivery systems for microbicides. Classes of excipients usually added to vaginal gels include gelling agents, humectants, preservatives and vehicles (Garg et al., 2004b ; Oshlack et al., 2009) . Various polymers are also commonly used as gelling agents in the formulation of hydrophilic vaginal gels. By definition, excipients are usually chosen from among materials that are very nearly pharmaco- toxicologically inert, and they usually have no therapeutic activity. However, some sulphated polymers, such as the carageenans, that are usually used as gelling agents; present themselves as one of the most promising classes of potential microbicidal compounds ( Perotti, et al 2003; Smit, 2004; Buck et al., 2006; das Neves and Bahia, 2006; Blakemore et al., 2008; Andrews et al., 2009 ) . 41 2.7.1 Carageenan- based gel formulations Carageenan is a mixture of highly sulfated polysaccharides, comprised mainly of polygalactans such as gums, agar and fruit pectins. It is a hydrocolloid obtained from various members of the Gigartinaceae or Solieriaceae families or the red seaweed, Rhodophyceae. It is a yellow to white powder and is odorless and tasteless. The extracted product consists of three types of carageenan, known as iota , kappa or lambda ( Table 2.3) . Carageenan formulations may be effective against the spread of HIV infection because they appear to prevent the trafficking of HIV - infected mononuclear cells from vaginal fluids into the epithelium. These polymers form a neg atively charged coat around HIV - infected mononuclear cells preventing their adhesion to the vaginal epithelial surface (Perotti et al., 2003) . Data from a Phase I preliminary safety study indicates that a Carbopol? 974 G el containing carageenans as microbicidal agents do not cause significant irritation to the female reproductive tract ( Spencer et al., 2004 ) . This may be an advantage over other microbicides or formulation excipients that are irritant to the vagina ( Spencer et al., 2004). Before usage, excipients that are used to formulate vaginal drug delivery systems must prove their biocompatibility. These tests are usually performed in cell culture models. Biocompatibility can be determined by cell growth/cell viability assays, cell proliferation assays, cytotoxicity assays, nitrite assays, and by protein assays and identification. Carageenan has been classified as "Generally Recognized as Safe" (GRAS) material by the United States Food and Drug Administration (US FDA). Further extensive oral pharmacokinetic studies have been conducted in various animal models. These have shown that the breakdown of carageenans in the gastrointestinal tract is minimal and that absorption is virtually non- existent (Blakemore et al., 2008) . Sulfated polysaccharides that are useful in inhibiting cell- to- cell transmission of HIV and thus the sexual transmission of HIV as well as STIs include iota carageenan, dextran sulfate, kappa carageenan, lambda carageenan, heparin mimetics, heparin sulfate, pentosan polysulfate, chondrotin sulfate, 42 lentinan sulfate, curdlan sulfate, de- N - sulfated heparin and fucoidan (Smit, 2004) . It has been shown that certain carageenans, or permutations of various carageenans, possess specific physicochemical properties which enable them to provide a prolonged antimicrobial effect and inhibit or reduce the possibility of STIs. Carageenan is one of the most active microbicides among the sulfated polysaccharides (Pearce - Pratt and Phillips, 1996) . Iota carageenan is the most efficacious of the commercially available sulfated carageenans, in preventing HIV infection and in blocking chlamydia infection i n vitro and in vivo (Blakemore et al., 2008). 2.7.1. 1 A carageenan vaginal gel f ormulation for HIV and H PV inhibition Carraguard? (The Population Council Co., New York, USA) is a gel- like delivery system obtained from lambda and kappa carageenan that acts as an absorption inhibitor by coating the vagina. This may prevent HIV from entering the vaginal epithelium (Forbes and Harrison, 2000) (Table 2.3) . The system forms a three- dimensional polymeric matrix with a high degree of physicochemical reticulation. Long, disordered chains are connected at specific points, but the connections are reversible. The molecular mechanisms of gelation are poorly understood, but researchers are attempting to design and enhance molecules with these properties. Carraguard? has several advantages over other vaginal microbicide delivery systems such as a higher bioavailability, safety, versatility, and economic saving (Justin- Temu et al., 2004). There appears to be no effect on sperm motility (Coetzee et al., 2006; Fernandez - Romero et al., 2007) . Furthermore, carageenan has shown the potential of being a potent inhibitor of sexually transmitted external genital warts. Certain sexually transmitted HPV types are causally associated with the development of cervical cancer (Perotti, et al., 2003; Smit, 2004; Buck et al., 2006) . Recent development of high - titer HPV pseudoviruses has made it possible to perform high- throughput in vitro screening to identify HPV infection inhibitors. Comparison of a variety of compounds has revealed that carageenan is an extremely potent infection inhibitor for a broad range of sexually transmitted HPVs and it acts primarily by preventing 43 the binding of HPV virions to cells (Buck et al., 2006). C linical trials revealed that carageenan- based intravaginal microbicide delivery systems are effective against genital warts ( Hartmann et al., 2006) . However, phase 3 clinical trial s did not show that Carraguard? is effective in preventing HIV t ransmission during vaginal sex (Population council, 2009). 44 Table 2.3: Chemical structures of polymeric microbicide compounds Compound Structure Key Function Reference Kappa Carageenan Absorption inhibitor ( Forbes and Harrison, 2000 ) Carbomer Interruption of HIV ( D?Cruz and Uckun, 2004 ) cell binding Cellulose Acetate Inactivates HIV and HSV ( Manson et al., 2000 ) Phthalate CapricAcid Inactivates HIV and HSV ( Neyts et al., 2000 ) Polystyrene Sulfonate Activity against HIV ( Simoes et al., 2002 ) and HSV 45 2.7.1.2 A combinatory gel f ormulation of carageenan and a non -nucleoside reverse transcriptase i nhibitor PC- 815 gel is an intravaginal delivery system combining a microbicide containing carageenan with MIV - 150. MIV - 150 is a Non- Nucleoside Reverse Transcriptase Inhibitor (NNRT I) which binds to reverse transcriptase enzymes that in turn prevents HIV - infected cells from replicating. The safety and ac tivity of carageenan against STIs has already been proven (Finley et al., 2006; Fernandez - Romero et al., 2007) . Pre - clinical tests of MIV - 150 have shown a significant increase in activity against HIV - 1 primary isolates. Extensive pharmacological and toxico logical profiles of MIV - 150 have shown that the compound is non- toxic in vitro and in vivo (Fernandez - Romero et al., 2007) . However, it has a low oral bioavailability which makes it an ideal compound for intravaginal delivery, since the chances of causing systemic side- effects become diminished (Fernandez - Romero et al., 2007) . In vitro pharmacological studies have indicated that the PC- 815 delivery system has significantly higher activity against HIV than that of the Carraguard? system (Fernandez - Romero et al., 2007) . MIV - 150 and carageenan have different mechanisms of action, which when combined, have a synergistic effect. The anti - HIV activity of MIV - 150 is not affected by the presence of seminal fluid or vaginal sec retions and the activity of carageenan is not affected by the addition of MIV - 150 to the formulation. 2.7.1. 3 A topical non- contraceptive carageenan gel f ormulation PC- 515 gel is a topical gel f ormulation containing 3% w/ w carageenan, benzyl alcohol as a preservative, and hydrochloric acid as pH adjust er. It is under development as a non- contraceptive microbicidal delivery system that may offer disease protection while allowing women to conceive. Zacharopoulos and Phillips ( 1997) measured the protective properties of PC- 515 in the vagina of mice inoculated with Herpes Simplex Virus (HSV), a standard model used to test microbicidal formulations. It was shown that PC - 515 protected against HSV, with an effect superior to many other microbicidal delivery 46 systems. Even at 100 times the lethal dose of HSV, 40% of mice were protected from infection. The protective effect was seen across a wide range of pH levels and lasted up to 18 hours (Zacharopoulos and Phillips, 1997) . PC - 515 has undergone developmental trials in humans to ascertain the overall performance of the formulation (Maria - Elisa et al., 2003; Hart and Evans- Strickfaden, 2007; Ramjee, 2007) . 2.7 .2 A polyacryl ic acid -based g el f ormulation A PAA polymeric gel (BufferGel?, ReProtect, LLC, Baltimore, MD, USA), was subjected to Phase II clinical trials as an intravaginal spermicidal delivery system for contraception and prevention of HIV in 2006 (Malcolm et al., 2006) . Its principal mechanism of act ion involves maintaining the natural acidity of the vagina, even in the presence of semen, by buffering the vagina to a pH of less than 5 due to the presence of PAA which ensures the survival of lactobacilli (Boskey et al., 2000) . Ideally vaginal delivery systems should not be detrimental to lactobacilli because they are responsible for producing lactic acid and hydrogen peroxide which inactivate many pathogens that cause STIs (Boskey et al., 2000; Quayle, 2002; Lohr, 2007). The pH of a healthy human vagina ranges from 4- 5 (~pH 4.5) , an environment too acidic for STI pathogens and HIV to survive (Forbes and Harrison 2000). However, semen is alkaline and therefore during intercourse the vaginal pH increases, allowing the survival of STI pathogens and HIV (For bes and Harrison 2000) . The BufferGel? system acidifies semen, thereby destroying sperm cells, HIV and a wide range of STI pathogens and keeps the vagina acidic even during sexual intercourse and thus creates a physical barrier that inhibits the passage of pathogens into the vaginal and cervical epithelium (Forbes and Harrison, 2000; Boskey et al., 2000 and 2001; van de Wijgert, 2001; D?Cruz and Uckun, 2004) . A similar system includes Acidform? gel (CONRAD/CDC, Atlanta, Georgia, USA) , an acid buffer anti- HI V vaginal gel formulation which is currently under Phase III clinical trials (Amarl et al., 2004; O'Brien, 2006; Mumper et al., 2009). However, recent clinical trials have revealed that BufferGel? has no effect on HIV prevention (PENN Medicine News, 2009) . 47 2.7.3 A Lactobacillus crispatus soft -gel c apsule f ormulation A Lactobacillus crispatus ( LC) microbicidal agent ( Lactin Vaginal Capsules?, Gynelogix, Louisville, CO, USA) which acts by re- colonizing the vagina with hydrogen peroxide producing lactobacillus once released from the soft- gel capsule has been developed (Harrison et al., 2003) . LC (Figure 2.2) is a normal commensal bacteria found in healthy vaginas (a sister species of lactobaccilli is found in yoghurt). LC assists in keeping the vagina free from infection by producing hydrogen peroxide, a substance that is weakly acidic. Normally, upon disturbing the ecology of the vagina either through infection, douching, or poor hygiene is when the vaginal pH rises above 4.5. This leads to the loss of lactobacilli resulting in the overgrowth of other bacteria and the proliferation of bacterial vaginosis which is a significant risk for pre- term labor, amnionitis, PID and HIV acquisition (Paavonen, 1983; Boskey et al., 2000; Forbes and Harrison, 2000 ) . Maggi a nd co- workes (2000) conducted a study on ten strains of lactobacilli by evaluating their viability in restoring the normal indigenous flora for the treatment of urogenital tract infections (UTI) in women. The microorganisms were formulated in single- and double- layer vaginal tablets. Each layer was characterized by different release properties. The first one was an effervescent composition that ensured a rapid and complete distribution of the active ingredient over the whole vaginal surface while the second one was a sustained release composition capable of releasing the lactobacilli over a longer period of time (Maggi et al., 2000) . Three different retarding polymers were tested and all formulations were evaluated in terms of technological processability, bacterial viability and stability, and cell adhesion properties of the microorganisms. The results indicated that three out of ten strains appeared to be suitable for the application in the treatment of UTI. 48 . Figure 2.2: Microscopic i mages of gram- stained vaginal smears depicting different categories of vaginal microflora. a) and b), grade 1a Lactobacillus crispatus cell types; c) and d), grade 1b non - L. crispatus cell types; e) and f), grade 1ab mixtures of L. crispatus and non- L. cri spatus cell types; g) and h), grade 1- like Gram positive rods; i) and j), grade 2 mixture of Lactobacillus cell types and bacterial vaginosis- associated bacteria cell types; k) and l), grade 3 bacterial vaginosis. (Source: Verhelst et al., 2005) . 2.7.4 Thermosensitive gel f ormulations Thermosensitive gels are systems that alter their physical characteristics with exposure to environmental changes within the vagina. This may be advantageous for intravaginal drug delivery due to the dynamic changes continually occurring in the vagina. These systems are capable of gelling in response to temperature changes, generally from ambient temperature to body temperature. Usually, the gelation temperatures have been considered suitable if they are in the range of 25? 3 7? C (Chang et al., 2002) . The thermo - gelation mechanisms include partial crystallization, coil - to- helix transition, hydrophobic association, and micelle packing, which serve as reversible physical crosslinking points to form a gel (Jeong et al., 2002). The thermo - reversible properties of gels such as carageenan can be evaluated by rheological parameters such as the shear stress changes upon shear rates, sol? gel transition temperature, and viscoelasticity moduli 49 (Chang et al., 2002 ) . Commonly used thermosensitive polymers employed for intravaginal microbicide delivery include the polysaccharides, N - isopropylacrilamide copolymers, poloxamers and its copolymers, poly (ethylene oxide)/poly(D,L- lactic acid- co- glycolic acid), and a few liposome- based systems. Poloxamer hydrogels perhaps represent the most extensively studied thermosensitive polymeric systems, while polysaccharides usually demonstrate good biocompatibility and/or biodegradability, with their solutions being thermosensitive at low polymeric concentrations (Ruel - Gariepy and Leroux, 2004). Chang and co- workers (2002) managed to produce thermosensitive vaginal gels containing clotrimazole. Results indicated that the gels were good potential candidates for safe, convenient, and effective intravaginal treatment. Thes e gels, which also presented bioadhesive behavior, were prepared with mixtures of poloxamers and polycarbophil (Chang et al., 2002 ) . 2.7.5 Long- chain sulfated polysaccharides and sulfonated polymeric formulations A microbicidal delivery system that is a byproduct of non- cytotoxic p oly(sodium 4- styrene sulfonate) (T - PSS) and cellulose sulfate (Ushercell?, Polydex Pharmaceuticals, Toronto, Canada) have reached Phase I and III clinical trials respectively ( Bourne et al., 2003; Rohan and Sassi, 2009) . Ushercell TM is a long- chain sulfated polysaccharide ( 1,900 kDa) and T - PSS is a long- chain sulfonated polymer (Table 2. 3) . Both have broad- spectrum antimicrobial activity and act as adsorption inhibitors when released (Simoes et al., 2002) . They are known to inhibit multiplication or activity of HIV, HPV, HSV - 1, HSV - 2, Chlamydia trachomatis, and Neisseria gonorrhea in vitro ( Anderson et al., 2000; Herol d et al., 2000; Christensen et al., 2001; Anderson et al., 2002; Simoes et al., 2002; Zanevelde et al., 2 002) . T - PSS gel formulations have shown promising in vivo activity as topical microbicidal delivery systems. Gel formulations tested contained 5? 10% w/ w T - PSS, hydroxyethylcellulose, glycerine, propylene glycol, benzoic acid, methylparaben, and sodium hydroxide (Bourne et al., 1999). These systems do not inhibit Lactobacilli and have been proven safe in a variety of animal studies (Anderson et al., 2000; Anderson et 50 al., 2002; Zanevelde et al., 2002 ) . As a result, the US FDA approved an Investigational New Drug (IND) application for both products. However, recently the Contraceptive Research and Development Program (CONRAD) reported that from interim data, from a trial where more than 1300 women in South Africa, Benin, Uganda and India that received UshercellTM , showed a greater prevalence of HIV infection compared to those who received the placebo gel (Highleyman, 2007; Thom, 2007). The Independent Data Mo nitoring Committee (IDMC, Rockville, MA, USA) overseeing this trial has agreed to conduct a detailed review of the data so as to better understand the findings and help determine their implications. Thus, these clinical trials have been halted pending the outcome of these investigations. Other, novel synthetic sulfated polymers, namely, sulfated polyvinyl alcohol (PVAS) and sulfated copolymers of acrylic acid with vinyl alcohol (PAVAS) have proved to be potent and selective inhibitors of HIV - 1 and HIV - 2 in vitro (Baba et al., 1990; Balzarini and van Damme, 2007). These polymers have been found to completely inhibit HIV - 1 - induced cytopathogenicity and HIV - 1 antigen expression at a concentration of 0.8?g/mL and equally effective against HIV - 2 replication (B aba et al., 1990) . As is the case wit h other sulfated polysaccharide formulations, the mechanism of action of PAVAS and PVAS resides in the inhibition of virus entry into vaginal epithelial cells once released from the delivery system (Baba et al., 1990; D e Clercq, 2004) . 2. 7 .6 A carbomer, lactic acid and naphthalene sulfonate gel formulation A water- based gel comprising a synthetic carbomer, a lactic- acid/lactate buffer system (pH 4.5) (Table 2.3), common preservatives and naphthalene sulfonate as an anti viral agent ( PRO 2000? Gel, Indevus Pharmaceuticals, Inc. Lexington, MA, USA) is an intravaginal microbicide delivery system currently under development for the prevention of STIs and HIV (Morrow et al., 2003; Dhawan and Mayer, 2006; Joshi et al., 2006). T he gel is clear, with a slight yellow tint and has a viscosity twice that of KY ? Jelly (Johnson and 51 Johnson, New Brunswick, NJ, USA). The mechanism of action of the microbicide released from the PRO 2000 T M Gel formulation is by binding to the HIV virus, thereby disrupting any interaction of the virus with target cells. Similarly it is able to prevent chlamydia and HSV- 2 infections (Forbes and Harrison, 2000) . Results from in vivo animal studies have shown that PRO 2000? G el is safe and well tolerated (Ruscnis et al., 1996; Bourne et al., 1999; van Damme et al., 2000; Morrow et al., 2003; Smita et al., 2006). Currently it is under Phase III clinical trials where it has displayed promising results in low - risk women (Mayer et al., 2003 and Smita et al., 2006; MT N microbicide trials network, 2009) . A recent clinical trial that involved more than 3,000 women in Southern Africa and USA has demonstrated for the first time the potential of PRO 2000? gel for preventing HIV infect ion in women. According to the findings, PRO 2000? G el was found to be 30% effective (MT N microbicide trials network, 2009) . However, additional evidence is required to thoroughly ensure the effectiveness of PRO 2000? G el. PRO 2000? G el is also compatible with the use of condoms, and therefore its further development may even prove to provide more benefits for women worldwide (Cadena, 2006). 2.7.7 A micronized cellulose acetate phthal ate gel formulation Cellulose Acetate Phthalate (CAP) has been used for several decades in the pharmaceutical industry for enteric coating of oral tablets and capsules (Table 2. 3). Micronized CAP, is available commercially as an aquateric system, and contains excipients required for micronization and tablet coating. It has shown to adsorb and inactivate HIV - 1, HSV and other STIs (Manson et al., 2000). S tudies have indicated that a gel formulation of micronized CAP has the potential to be used for topical intravaginal delivery of microbicides for prevention of STIs and HIV (Neurath et al., 2003; Fichora et al., 2005). It is converted into a gel and therefore does not have to be removed following its application (Neurath et al., 2003). CAP is one of the potential anti - HIV vaginal gel formulations that are under Phase II clinical trials (Neurath et al., 2003; Fichora et al., 2005) . 52 2.7.8 A m onocaprin- loaded hydrogel formulation Hydrogel formulations containing a 1- monoglyceride of capric acid (monocaprin) possess potent in vitro microbicidal activity against HIV, HSV, Chlamydia tr achomatis and Neisseria gonorrhea (Thormar et al., 1999; Neyts et al., 2000) (Table 2. 3). In vitro studies have shown that gels containing monocaprin, formulated using gel- forming agents such as either sodium carboxymethylcellulose and polyvinylpyrrolidone (pH 7.0) or carbomer and hydroxypropylmethylcellulose (pH 5.0), are highly virucidal to HSV - 1 and less cytotoxic than nonoxynol - 9 (Krist mundsdottir et al., 2000) . In addition, the gels had spermicidal properties, and thus they may have potential contraceptive activity (Thormar et al., 1999) . In vivo studies in mice showed that these gels were non- irritant and non- toxic in the vagina (Neyts et al., 2000). The results from these studies also indicated that 20mM monocaprin hydrogel could prevent HSV- 2 infecti on and mortality in mice (Neyts et al., 2000) . Hydrogel formulations of monocaprin could be further pursued in the development of intravaginal microbicide delivery systems for the prevention of STIs and HIV. 2.7.9 Polystyrene sulfonate vaginal tablets Polystyrene Sulfonate (PSS) is also a microbicide that has been developed as an intravaginal tablet formulation ( Garg et al., 2004a ; Romano et al., 2008) (Table 2. 3) . When PSS intravaginal tablets were used as a microbicide delivery system they showed that they provided superior antimicrobial activity against HIV and HSV (Garg et al., 2004a ). This formulation did not immobilize sperm, was not cytotoxic and did not inhibit normal vaginal microflora. This demonstrates its potential as a safe and effective vaginal microbicide delivery system for the prevention STIs and HIV (Garg et al., 2004a; Romano et al., 2008) . 53 2.8 Non -polymeric Intravaginal Microbicide Delivery Systems for Preventing the Transmission of STIs and HIV 2.8.1 A c etyl betaine and myristamine ox ide combinatory cream formulation C31G cream (Savvy?, Cellegy Pharmaceuticals, Inc, San Francisco, CA, USA) comprises a broad- spectrum antimicrobial and spermicidal agent with two surface- active compounds, cetyl betaine (a C 16 alkyl betaine) and myristamine oxide (a C 14 alkyl amino oxide) (Table 2.4) . The f ormulation is an equimolar mixture of the two amphoteric, surface- active compounds and displays activity against gram- positive and gram- negative bacteria, fungi, yeasts, and enveloped viruses (Krebs et al ., 2000). Savvy? has shown activity against numerous micro- organisms that cause STIs, including chlamydia, HSV - 1, HSV- 2 and gonorrhea. These effects occur once the microbicidal agent diffuses through the cream, which is formulated with HEC and also acts as a surface- active microbicide. In Phase III clinical trials, the delivery system has shown the ability to prevent HIV - 1 and HIV - 2 transmission and also has a desirable contraceptive activity (van Damme et al., 2002) . Other studies have shown that Savvy? , when used alone, may also be approximately 85% effective in the prevention of pregnancy (van Damme et al., 2002; Dhawan and Mayer, 2006) . 2.8.2 A non- nucleoside reverse transcriptase inhibitor -loaded gel formulation UC781 (Cellegy Pharmaceuticals, Inc South San Fransisco, CA, USA) is a tight- binding Non - Nucleoside Reverse Transcriptase Inhibitor (NNRT I) that is incorporated into gel formulations (Table 2.4). It has significant potential for use as an anti - HIV microbicide. Short pre- treatment of both isolated cells and human cervical tissue explants, with low concentrations of UC781 provided a strong barrier to subsequent virus infection by cell - free or cell- associated HIV - 1 (Gadi and Parniak, 2001; Kerr, 2007) . UC781 is readily formulated as an appropriate carrier for vaginal application in the form of Replens ? Gel (Anglian Pharma, Hertfordshire, UK). Furthemore, UC781 has shown to have a f avorable 54 toxicity profile (Gadi and Parniak, 2001) . UC781 warrants further clinical assessment for its use as a topical intravaginal delivery system for the prevention of STIs and HIV. Table 2.4: Structures of non- polymeric microbicide compounds Compound Structure Key Function Reference Cetyl Betaine Broad- spectrum antimicrobial ( Krebs et al., 2000 ) Myristamine Spermicidal agent ( Krebs et al., 2000 ) UC781 Tight binding NNRTI ( Kerr, 2007 ) Tenofovir Blocks HIV replication ( De Clercq, 2006 ) Stampidine Broad- spectrum anti- HIV agent ( D?Cruz, 2003a ) Dapirivine Potent NNRT I Malcolm and Woolfson, ( 2006 ) 55 2.8.3 A nucleotide analogue- loaded gel formulation PMPA Gel (Tenofovir?, Gilead Sciences, Foster City, CA, USA) is a microbicide delivery system that blocks HIV from replicating once it has entered cells (De Clercq, 2006). The hope is that Tenofovir ? could be absorbed by the vaginal epithelium to prohibit the replication and penetration of the virus in the outer cells of the vaginal wall (Table 2.4). Numerous ARVs initially explored for potential AID S therapy were later abandoned due to inadequate delivery systems that provided poor systemic absorption. These may be suitable for re- formulation into newer effective intravaginal microbicide delivery systems (Forbes and Harrison, 2000) . Preliminary resul ts from studies of Tenofovir ? are encouraging and indicate that it may be a new microbicide delivery system to consider as part of a potent anti- HIV regimen. Tenofovir ? may require less frequent dosing than currently available therapies (only once daily) and appears to be active agai nst resistant strains of HIV, including protease resistance (Forbes and Harrison, 2000). 2.8.4 A novel synthetical l y derived aryl phosphate- loaded gel formulation Stampidine is a novel aryl phosphate derivative of stavudine (Table 2.4) . It is a potent, broad- spectrum anti- HIV agent with potential to be used as a newer class of non - spermicidal microbicides (D?Cruz, 2003a). A vaginal gel formulation of stampidine was tested for its potential to cause vaginal mucosal toxicity in New Zealand white pigs (D?Cru z, 2003a; D?Cruz et al., 2003a ). Application of 0.5- 2. 0% w/ w of this formulation, produced only minimal- to- mild vaginal irritation, of which is in an acceptable range for a clinical trial (total score <4 out of a possible 16). Repeated intravaginal treatmen t with stampidine gel was shown to cause no inflammation or hyperplasia in the vaginal epithelium and sub- epithelial tissue (D?Cruz et al., 2003a ). Stampidine and its major metabolites were not detectable in the plasma of pigs after they received a 2% w/ w stampidine- containing gel intravaginally. Thus, this finding coupled with the favorable toxicity profile of intravaginally administered stampidine- containing gel may provide the 56 foundation for its clinical development as a safe and effective broad- spectrum anti- HIV microbicide delivery system. 2.8.5 A cyanovirin -N gel formulation for chemotherapeutic and immunoprophylactic prevention of HIV Cyanovirin- N (CV - N) is a novel protein, originally isolated from cultures of the cyanobacterium (blue- green algae) called Nostoc ellipsosporum that has generated interest as a natural product for the chemotherapy of HIV infection (Boyd et al., 1997; De Clercq, 2000) . A possible combined chemotherapeutic and immunoprophylactic approach for preventing HIV may be based upon the concept that continuous exposure of HIV to gp120 - binding agents from a gel formulation may diminish glycosylation sites, thus triggering the production of specific neutralizing antibodies to previously hidden gp120 epitopes (Boyd et al., 1997; De Cl ercq, 2000) . Both natural and recombinant CV - N are highly potent as virucidal agents (Boyd et al., 1997; De Clercq, 2000; Tsai et al., 2003; Balzarini, 2005; Xiaowen et al., 2006). CV - N inhibits the in vitro fusion of HIV - infected and non- infected cells as well as cell- to- cell transmission of HIV - 1 infection. These effects make CV- N a promising candidate as a microbicide gel f ormulation for intravaginal delivery ( Boyd et al., 1997; O? Keefe, 2001). In cell cultures it is non- toxic, it is remarkably resistant to physical degradation and it is water- soluble and stable in solution with a shelf life of at least 6 months (Boyd et al., 1997). 2.8.6 Sodium lauryl sulfate as an Invisible Condom TM gel -like formulation Sodium lauryl sulfate (SLS) which is an anionic surfactant that denatures membrane proteins of pathogens is currently under Phase II clinical trials and is classed as an entry fusion inhibitor (Malcolm et al., 2006). It is also called an Invisible Condom TM (Invisible CondomTM , Laval University, Infecti ous Diseases Research Center, Quebec City, QC, Canada). Ward and Ashley (1979) were the first to demonstrate that SLS at low concentrations is a potent inactivator of rotavirus and poliovirus. Previous in vitro studies 57 have demonstrated the ability of SLS to inhibit the infective capacity of different enveloped viruses such as HSV- 1, HSV - 2 and HIV - 1 (Roy et al., 1999; Piret et al., 2000) . SLS has also been reported to inhibit the infective capacity of non- enveloped bovine HIV and HPV (Howett et al., 1999). This suggested that SLS could be a candidate for use as an intravaginal microbicide gel formulation to prevent the sexual transmission of STIs and HIV. As previously mentioned, thermosensitive gels are recognized to allow advantageous application of active substances in the vagina. A thermoreversible gel composed of polyoxypropylene and polyoxyethylene in citrate buffer (pH 4.0) presents a phase transition temperature of 28?C. This showed to be an ideal vehicle to deliver microbicidal substances, such as SLS or n- lauroylsarcosine, when applied in a murine model of HSV- 2 (Roy et al., 1999) . 2.8.7 Dapivirine a nti -HIV vaginal rings As mentioned earlier vaginal rings are suitable for the delivery of microbicides to prevent the transmission of STIs and HIV ( Malc olm and Woolfson, 2006). While most research on microbicides has focused on single- dose and semi- solid topical gels, ring- based delivery systems could reduce the burden of patient compliance. These can provide long - term, controlled release of microbicides that may confer continuous protection against STIs and heterosexually transmitted HIV, and rule out the need for application of the intravaginal microbicide delivery system near the moment of sexual intercourse (Smith, 2006; Woolfson et al., 2006) . Dapivir ine, which is also known as TMC120, is a potent non - nucleoside reverse transcriptase inhibitor and is the only vaginal ring system used as an intravaginal microbicide delivery system for preventing the transmission of STIs and HIV ( Malcolm and Woolfson, 2006; Smith, 2006; Nutall et al., 2007) (Table 2.4). 2.8.8 Polyherbal anti -HIV praneem -loaded vaginal t ablets Praneem has been developed as an anti- HIV vaginal tablet formulation. Praneem contains purified extracts of Azadirachta indica also known as the Ne em tree. Praneem 58 has preventative activity against STIs in vitro and therefore it has been developed as a possible intravaginal microbicide delivery system. Phase I safety studies on Praneem tablets, revealed that the formulation is safe for once daily intravaginal use, for 14 consecutive days, in sexually active uninfected women (Joshi et al., 2005). 2.9 Nanotechnology -based Intravaginal Microbicide Delivery Systems 2.9.1 A Dendrimer -based microbicide formulation SPL7013 (VivaGel?, Starpharma Ltd., Melbourne, V ictoria, Australia) is a dendrimer with a polyanionic outer surface that allows multiple interactions with target sites. It is the most advanced dendrimer- based microbicide delivery system in which the dendrimer is not a carrier but an active ingredient (Short, 2007). These substances are large highly branched macromolecules synthesized from a polyfunctional core (Bourne et al., 1999) . SPL7013 emerged as the most promising dendrimer after pre- clinical studies, and which was formulated as a gel that has been undergoing clinical trials ( Jiang, et al., 2005; Nikolic et al., 2007 ) . Bernstein and co- workers (2003) showed that a 5% w/ w Carbopol? 974 G el (pH 4.5) can also be a suitable vehicle for the intravag inal administration of SPL7013. SPL7013 binds and blocks HIV - 1, and chimeric simian/HIV - 1 (SHIV) replication, thereby preventing STIs, including HIV and genital herpes ( Okada, 1991; Highleyman, 2007; Short, 2007) . It works by making use of its branched structure to bind viral surface proteins thereby preventing attachment of the virus to human cells. Gels containing different concentrations of SPL7013 were formerly used as intravaginal topical microbicide systems in female pigtailed macaques to study their ability to prevent vaginal transmission of SHIV. All untreated macaques (8/8) and seven (out of eight) macaques treated with a placebo gel formulation were infected with SHI V within 2 weeks after exposure. T hey showed a low plasma viremia and dramatic CD4 + cell decline within 4 weeks (Short, 2007). Extr apolation from these results suggests that VivaGel? may be a promising anti- HIV microbicide f ormulation. Phase I trials have shown that VivaGel? is safe and well- tolerated ( Rupp et al., 2007; Short, 2007; Fairley, 2009a ). Further trials are 59 being conducted to test its efficacy against genital herpes and HIV ( Highleyman, 2007; Short, 2007 ; Fairley, 2009b). 2.9.2 Silver nanoparticles employed as microbicide delivery systems Various studies are currently investigating the production of a novel class of nanomaterials called protein- conjugated noble metal nanoparticles (Cho et al., 2005; Yacaman et al., 2006; Pal et al., 2007). The production involves direct conjugation of noble- metal nanoparticles with proteins that may be used in numerous forms including antiviral and antibacterial delivery systems. The process includes one or more targeting molecules (e.g. cytokines) that bind specifically to target cells expressing the cognate receptor for the cytokine. Researchers initially developed a method for synthesizing water- soluble noble metal nanoparticles, grown within and directly functionalized by bovine serum albumin (BSA), a globular protein, without the use of additional protecting or linking agents. Both gold and silver nanoparticles have been produced using t his method, and the approach can easily be extended to other noble metals, such as the platinum group (Yacaman et al., 2006) . Other globular proteins such as human serum albumin (HSA) may also be substituted for BSA and include, immunoglobulins, cytokines, receptors, lectins, glycoproteins, lipoproteins, toxins, toxoids, collagen, bacterial proteins, viral proteins, parasitic proteins, and fusion proteins (Richardson and Illum, 1992) . The method of synthesis includes chemical reduction of an ionic metal precursor at room temperature in aqueous solution in the presence of a globular protein. Under certain pH conditions, disulfide bonds of the protein are available for direct bonding with the noble metal nanoparticles. The polypeptide backbone of the protein r emains intact and the method does not affect the functional groups of the constituent amino acid residues. Silver nanoparticles take advantage of the microbicide properties of silver with different materials to produce effective microbicide delivery systems for preventing STIs and HIV transmission (Cho et al., 2005; Barbara et al., 2006; Yacaman et al., 2006). 60 2.9.3 Polystyrene nanospheres as microbicide delivery systems Mucosal secretory IgA may have an important role in the prevention of HIV - 1 transmiss ion during sexual intercourse. Therefore, substances that induce HIV - 1 - specific IgA antibodies in the genital tract have shown promise for use as prophylactic vaccines against HIV - 1 infection. It has been reported that Concanavalin A - immobilized Polystyrene Nanospheres (Con A - NS) could efficiently capture HIV - 1 particles and gp120 antigens on their surface and that intravaginal immunization with these inactivated HIV - 1 - capturing nanospheres (HIV - NS) induce vaginal anti- HIV - 1 IgA antibodies in mice (Agaki et al., 2003) . Thus, application of HIV - NS by intravaginal delivery is a practical approach to promote HIV - 1 - specific IgA responses as an effective immunization strategy . Likewise, lectin- immobilized polystyrene nanospheres have been synthesized and examined for their HIV - 1 capturing abilities (Hayakawa et al., 1998) . When Con- A is immobilized on the surface of polystyrene nanospheres (400nm in diameter) with polymethacrylic acid branches and incubated with HIV - 1 suspensions at room temperature for 60min, the Con A- NS achieves greater reduction in viral infectivity of HIV - 1 (II I B strain) than does Con A - free nanospheres (Hayakawa et al., 1998) . The combination of Con A - NS treatment followed by in vitro filtration with a microporous membrane efficiently removes virion- free gp120 as well as infectious viral particles from HIV - 1 suspensions. Electron microscopic examination has demonstrated that HIV - 1 virions may be entrapped on the surface of Con A- NS (Hayakawa et al., 1998; Carmen and Angel, 2003 ) . Thus, Con A - N S is able to capture HIV - 1 virions and gp120 with a high affinity, and may potentially be an effective nanotechnology- based delivery system for the prevention of HIV - 1 transmission (Hayakawa et al., 1998; Carmen and Angel, 2003 ) . 2.9.4 A g el -like molecula r condom formulation as a barrier for STIs and HIV transmission The molecular condom is a recently developed anti - HIV vaginal gel (Kiser, 2006a and b). It is a polymeric construct designed, to incorporate controlled release characteristics, to 61 allow for enhanced tissue coating, to reduce viral transport and to allow for triggered release of anti- HIV bioactives by interacting with semen. The term ?molecular condom? arises from the concept that the polymer construct is liquid at room temperature and, when applied intravaginally, spreads and converts to a gel that effectively coats the vaginal wall (Kiser , 2006 a and b). The gel is designed to release the anti - HIV bioactives upon contact with semen during sexual intercourse. It is an aqueous - based gel or "hydrog el" sensitive to body temperature and pH, and serves as a "smart semen- triggered vaginal microbicide delivery vehicle." This system is nanotechnology - based and explores the use of bioresponsive drug delivery by tailoring the physiological and mechanical requirements essential for intravaginal application. This is a type of a prophylactic agent that women may use without relying on their partners. It is of particular interest in areas such as Sub- Saharan Africa and South Asia where HIV - infection rates are substantially high while use of condoms is not well accepted. It is believed that the bio- distribution and bioavailability of antiviral bioactives can be modulated to increase potency and allow for burst release of free HIV inhibitors at the initial onslaught of viral exposure. Thus far, in vitro studies have demonstrated that the substance converts from liquid to gel at body temperature and a vaginal pH of 4 - 5 and then reverts to liquid and releases bioactives, such as microbicides, when exposed to semen, wi th a pH of 7.5 (Kiser, 2006a and b ). The ultimate hope for this technology is to protect women and their unborn or nursing children from HIV. It is estimated that if all goes well, clinical trials in humans will start in three to five years, and the gel is expected to reach the market in several more years (Kiser, 2006a and b). The future plan is to incorporate an ARV into the gel so that it can cater both for blocking HIV movement and prevent ing the virus from replicating. 2.10 Other Microbicidal Compounds Currently under Investigation 2.10. 1 A secretory leukocyte protease inhibitor Secretory leukocyte protease inhibitor (SLPI) is an endogenous antimicrobial agent found in mucosal tissue and saliva, most prominently in mucosal sites open to the external 62 environment. Its original function is believed to be an anti - protease defense against neutrophils at the mucosal surfaces ( Bob, 2000). In vitro laboratory studies have shown that SLPI can inhibit HIV entry into CD4 + cells by interacting with targeted cells and blocking access as a barrier function. This may provide a reason as to the low rate of HIV infection through oral sex (San Francisco AIDS Foundation, 2008) due to the presence of SLPI in saliva. Data from studies of various mucosal tissues such as the mouth, gut, rectum and endocervix, suggest an inverse relationship between infectious HI V and the presence of SLPI (Bob, 2000; Zuckerman et al., 2003; Campo et al., 2006; Odaka and Ding, 2009 ) . In breast milk, levels of SLPI rise dramatically at birth and begin to drop two to three weeks later, to a point where they are not sufficient to inhibit HIV infections in infants (Bob , 2000; Cheshenko et al., 2004). Further studies of SLPI as "a natural endogenous product" need to be performed since it has been sho wn to have the potential to function as an effective microbicide (Bob, 2000; Cheshenko et al., 2004; Keller et al., 2005) . 2.10.2 Thrombospondin- 1 as a barrier to mucosal transmission of HIV -1 During an investigation of the physiology of HIV inhibition by human saliva, laboratory studies identified thrombospondin- 1 (TSP1) to have potential as a barrier agent against local mucosal transmission of HIV - 1 (Crombie et al., 1998, 2000, 2001) . TSP1 is a trimeric- sulfated glycoprotein that belongs to a family of five distinct extracellular matrix adhesion molecules ( Bornstein, 1995; Adams, 1997; Crombie, 2000). Unlike other multifunctional extracellular matrix glycoproteins, TSP1 is found in lower concentrations in plasma and most body fluids (Mosher, 1990) (Figur e 2.3) but not in saliva (Crombie et al., 1998) . Since TSP1 is a high molecular mass compound, difficult to purify, and labile in the presence of tissue proteases, it is an impractical candidate to be formulated as a localized intravaginal delivery system. Investigations have therefore focused on the activity of peptides prepared from different regions of this molecule for which the attention has been on type 1 properdin/malaria- like repeat sequence (Crombie et al., 1998, 2000, 2001). 63 Characterization of al ternative TSP1 peptide modifications currently under design may identify conformations that are more potent and stable and thus better for localized intravaginal therapy (Crombie et al., 2001) . 2.10.3 Short interfering RNA as potential liposomal microbici de delivery systems Short interfering RNA (siRNA) is a type of microbicide based on RNA interference (RNAi), a natural selective process for turning off genes, which holds great potential in its ability to treat disease at the genetic level ( Dykxhoorn and Lieberman, 2006; Palliser et al., 2006; Soifer et al., 2007) . In contrast to other classes of therapeutics, such as proteins, antibodies and small molecules, RNAi do not require either "drugable" domains on the molecular target, or specific locations at the cell membrane nor an extracellular site for their effective action. RNAi is triggered by siRNA molecules that engage a group of cellular proteins, known as RNA - induced silencing complexes (RISC). RISC guides the siRNA to its target mRNA (messenger RNA) b y complementary base pairing and splits it in a selective fashion, thus halting protein expression or viral replication (Roumeliotis, 2006) . Currently, in vitro vaginal tissue culture systems have been investigated and work continues with in vivo vaginal x enograft models to determine the optimum dose required for maximum down- regulation of virus replication. In vitro tests utilizing fluorescent tagged siRNA in liposomal delivery systems indicate that siRNAs are absorbed throughout the vaginal tissue (Cristo faro and Ramratnam, 2005 ; Holmes, 2007 ) . 64 Figure 2.3: Schematic of the trimeric - sulphated glycoprotein Thrombospondin- 1 with five distinct extracellular matrix adhesion molecules , (Adapted: Mosher, 1990). 2.10.4 Thiophen- Th iourea: A non- nucleoside reverse transcriptase inhibitor PHI - 443 ( N ?- [ 2 - ( 2 - thiophene) ethyl] - N ?- [ 2 - ( 5 - bromopyridyl)] thiourea) is a rationally designed novel thiophene- thiourea NNRTI with potent activity against the wild- type and drug- resistant primary clinical HIV - 1 isolates ( D?cruz et al., 2004a ; D?cruz and Uckun, 2005c ) (Table 2.5) . Its potential as a non- spermicidal microbicide for preventing the sexual transmission of HIV has been examined by testing its effect on in vitro sperm function where it has shown to inactivate viruses in human cells. It completely prevents the vaginal transmission of a genotypically and phenotypically drug- resistant HIV - 1 isolate in the humanized severe combined immunodeficient mouse model of sexually transmitted HIV (D?cruz e t al., 2004 a). Exposure of human sperm to PHI - 443 at doses 30,000 times greater than those that yield effective concentrations against HIV has shown that PHI - 443 has no effects on sperm motility, kinematics, cervical mucus penetrability, or the viability of vaginal and cervical epithelial cells. Exposure of pig semen to PHI - 443 either ex vivo or in vivo resulted in no side- effects on the in vivo fertilizing ability. Repeated intravaginal administration of 0.5? 2% w/ w of PHI - 443 as a gel has been found to be safe in pig s CD3 TGF W R C C C C W W R 65 (D?cruz et al., 2004a ). Thus, PHI- 443 has potential as a prophylactic broad- spectrum anti- HIV microbicide without contraceptive activity. 66 Table 2.5: Chemical structures of other novel microbicide compounds Compound Structure Key Function Reference Thiophen - Thiourea NNRT I against drug resistant HIV (D?Cruz et al., 2004a) Monoclonal Antibodies Passive immunization (Cairns, 2007) Lime Juice Retards HIV transmission ( Hemmerling et al., 2007) Yoghurt Inhibits transmission of H IV (Turpin, 2006; Ramratnam, 2006 ) O OH OH OH OH OH n-1 67 2.10.5 Monoclonal antibodies as microbicides Monoclonal antibodies are Y - shaped molecules synthesized by the immune system as ?mirror images? of foreign substances (Cairns, 2007) . A long- range goal in vaginal protection is to develop monoclonal human antibodies in a microbicidal gel for protecting genital skin and epithelia against infections by topical passive immunization. Mucus secretions contain large quantities of antibodies that are highly specific and potent agents for preventing the entry of pathogens (Workman, 2003) (Table 2.5). Secreted mucosal antibodies also help maintain healthy bacterial flora on mucosal surfaces. Mucosal antibodies are polyvalent and hence bind with high affinity to pathogens. Monoclonal synthetic antibodies against sperm, HIV, and other STI pathogens can be applied directly to genital skin and epithelia for protection (Cone and Whaley, 1994; Sherwood et al., 1996) . This may closely mimic the normal function of antibodies in the mucosal immune system, e.g. anti bodies in breast milk help protect the surfaces of the mouth, nose, eyes, and digestive tract of the baby. To date, the results of studies performed indicate that monoclonal antibodies delivered to the vagina may help prevent pregnancy as well as sexual tr ansmission of genital herpes and HIV (Castle et al., 1997; Zeitlin, et al., 1999; Castle 2002; Perotti et al., 2003; Whaley and Zeitlin, 2005) . Topical application of these synthetic antibodies protects mice from HSV infection (Briggs and Zeitlin, 2000; Yi m et al., 2005; Pedras- Vasconcelos et al., 2006). The end objective is to develop a protective topical gel containing these synthetic antibodies for human use (Briggs and Zeitlin, 2000) . 2.10.6 Lime juice as a microbicide Lime juice has been known for long in history as a contraceptive and vaginal douche ( Thaineua et al 2003; Clarke et al., 2006; Hemmerling et al., 2007) (Table 2.5). In vitro studies that were conducted by Potts and co- workers (2004) demonstrated that lemon/lime juice is an effective micr obicide and in in vivo primate studies, repeated use of lime juice did not damage the vagina. Hemmerling and co - workers (2007) further conducted a study on lime juice that involved twenty five sexually abstinent women. Tests 68 for genital infections, measurement of inflammatory biomarkers and a colposcopy were conducted, before and after treatment. Results indicated that no participant showed severe vaginal irritation. The reported side effects were mostly singular, mild and transient events. Thus, lime juice up to 20% concentration proved to have an acceptable safety profile for vaginal use ( Potts et al., 2004; Hemmerling et al., 2007). Recent in vitro research has shown that, for effective prevention of HIV transmission the concentration of lime juice should be at least 50% which is associated with some toxicity uncertainties (Hemmerling et al., 2007) . Therefore, t here is an urgent need for a systematic study to determine the best ways of delivering lime juice effectively without any toxicity risks. If the use of lime juice can retard HIV transmission, then lime juice could prove to be a life- saving microbicide. If the effect is modest, lime juice might still prove to be a 'gold standard' microbicide with which artificial microbicides could be compared, shor t circuiting the need for time- consuming expensive placebo controlled trials (Potts et al., 2004). However, thorough research still needs to be conducted on the efficacy of lime juice as an inexpensive and ubiq uitous intravaginal microbicide drug delivery system. 2.10.7 Yoghurt as a microbicide It has been shown that yoghurt may soon be enlisted in the battle against HIV/AIDS (Turpin, 2006) (Table 2.5) . Lactobacillus which is a harmless bacterium that helps to turn milk into yoghurt has been engineered to produce HIV - fighting microbicides (Turpin, 2006) . Consuming yoghurt containing Lactobacillus may provide a way for women to fend off HIV if no other means are available (Turpin, 2006) . Apart from being present in yoghurt, lactobacillus is a natural inhabitant of the human vagina. It survives the passage through the gastrointestinal tract and can easily cross from the anus to the vagina and colonize there. Once in the vagina, the bioengineered bacteria can produce compounds that inhibit transmission of HIV and maintain a normal vaginal pH, thus helping prevent HIV and STIs infection (Turpin, 2006) . Similarly, Ramratnam (2006) showed that it was possible to genetically manipulate bacteria present in yoghurt to release an anti- HIV drug. 69 This could successfully alter the genetic constitution of Lactobacillus lactis to enable it to release cyanovirin which has already been found to confer protection against HIV infection in monkeys and human cells. Although successful results (followed by clinical trials) are yet to be obtained, there is optimism that yoghurt may eventually pave a way for the development of cost- effective microbicide delivery systems ( Ramratnam, 2006). . 2.11 Concluding Remarks Intravaginal microbicide delivery systems are providing a new opti on for preventing the transmission of STIs and HIV. This could have considerable public health and economic impact especially in resource- poor countries. As described in this Chapter, numerous safe and effective anti- HIV intravaginal microbicide delivery systems are currently being evaluated at various stages in clinical trials that include the dual- function polymeric compounds, non- polymeric compounds and nano- structured compounds. To date, none of the delivery systems described, which comprise mainly gel formulations, have been found to be fully effective at preventing the transmission of STIs and HIV. Several clinical trials have demonstrated that intravaginal microbicide delivery systems have the ability to protect against the transmission of STIs and HI V but numerous challenges still remain before these systems are used commercially. These challenges include: i) the need for the development of newer and innovative intravaginal microbicide delivery technologies that can completely prevent the transmission of STIs and HIV; ii) securing a cost - effective and efficient production and distribution of these compounds and systems; iii ) securing wide access to these compounds and systems that takes into account cultural sensitivities of the most affected countries; and iv) securing extensive education in the use of the intravaginal microbicidal delivery system. It is thus anticipated that future research will focus on the design and development of more effective intravaginal microbicidal delivery systems for preventing the transmission of STIs and HIV employing a wide variety of dosage forms with multiple mechanisms of action and microbicidal delivery. 70 CHAPTER THREE P R E F OR MUL ATION INVE S TIG AT IONS F OR THE DE VE L OP ME NT OF L E AD INTR AV AG INAL B IOADHE S IVE P OL Y ME R IC DE VIC E S 3.1 Introduction Major efforts have been directed towards designing intravaginal drug delivery systems that can deliver drugs to the vagina at a desired rate to maximize drug efficacy while minimizing side effects (Garg and Tambwekar, 2003; das Neves and Bahia, 2006; D'Cruz and Uckun, 2006a ; Andrews et al., 2009) . However, successful design of intravaginal drug delivery systems still poses numerous challenges ( Justin- Temu et al., 2004; Ndesendo et al., 2008; Rohan and Sassi, 2009) . It is imperative that the type and properties of the drug and the drug carrier (e.g. polymer) be thoroughly understood if successful intravaginal delivery is to be achieved. In the case of a polymer as the carrier agent, appropriate selection is of paramount importance. The polymer employed can be either degradable or non- degradable. Biodegradable polymers that are biocompatible to the body are most frequently chosen for the design of intravaginal drug delivery systems. Their value is attributed to the fact that they do not require to be removed from the body after application. The type, physicochemical and physicomechanical properties, proportions of the polymers in the formulation, as well as the degree of swelling and /or erosion for the polymers employed in formulating an intravaginal delivery system, play an important role in matrix integrity and retention capacity. These are the determinant factors for bioadhesion and release characteristics of a given system (Vueba et al., 2004; Ravi et al., 2008). Figure 3.1 depicts a biometric simulation undertaken in our laboratories on ACD/I - Lab, V5.11 (Add- on) software (Advanced Chemistry Development Inc., Toronto, Canada, 2000), indicating the different levels of coiling that polymers may undergo to produce the highest degree of matrix integrity. A polymer?s physicochemical and 71 physicomechanical properties contribute substantially to diffusion, swelling, degradation and erosion dynamics, which play an important role in terms of drug release kinetics ( Siepmann and Peppas, 2001; Avgoustakis, 2004; Arifin et al., 2006). Other phenomena such as osmotic, magnetic or electric effects may also be involved. The chemical nature of polymers commonly used for pharmaceutical purposes differs significantly. Some are freely water- soluble, while others are completely water- insoluble. Thus, they vary in the rate and extent of swelli ng, bioerosion/biodegradation and their potential to interact with the drug. Figure 3.1: A computationally- derived model predicted in our laboratories depicting the polymer coiling stages a) polymer strands, b) identification of molecular sites, c) initial stages of coiling, d) secondary and tertiary coiling randomizations and patterns where (C - 1) - (C - 7) is non - breaded non- rope coiling and (C - 8) - (C - 12) is breaded rope coiling. The number of coil arm may vary in (C - 1) - (C - 5). R 1 , R 2 and R 3 are functional groups. The lines represent polymeric strains while the blocks represent polymer strain interactions. . . . . ... . .. . . .. . ... . . .. . . .. . . ... . . . ... ... .. ... .... .. .. .. . .. .. . . .. . . . . . .. . .. . . .. .. . .. . . . ... . . . . .. (R 1 ) n +2(R 1 ) n + 1(R 1 ) n + 1(R 1 ) n + 1 or or or (R 1 ) n +2 (R 1 ) n +2 (R 1 ) n + 1or (R 1 ) n +2 Mixed Randomiz ations/Patterns of or (R 2) n + 1 or or or (R 2) n + 1 (R 2) n +2 (R 2) n +2 or or (R x ) n + y . .. . ... ..... ( A) ( B) ( C -1 ) ( C-2 ) ( C-3 ) ( C-4 ) ( C-5 ) ( C-6 ) ( C-7 ) ( C-8 )( C-9 ) . ... . . ( C-10 ) Coiling Extension StagesSecondary & Tertiary Coiling Randomizations/ Patterns . .. . .. ( C-11 ) (R 3 ) n + 3 or (R 3 ) n +2 or (R 3 ) n + 1 (R x ) n + y .. ( C-12 ) (R x n)n n + y n Super -breaded Rope Coiling Breaded Rope Coiling Non-Breaded Non-Rope Coiling (D ) (D ) 72 In the current study, both natural and synthetic biodegradable and biocompatible polymers have been employed, some with hydrophilic properties, others with hydrophobic properties and yet others with bioadhesion properties. Hydrophilic polymers can control drug release because of their compressibility and swelling properties, as well as the ability to accommodate high levels of drugs (Pinto et al., 2004; Hiremath and Saha, 2008; Jin et al., 2008). However, hydrophilic matrix syste ms generally result in nearly first- order release profiles ( Hassan et al., 2003; Chopra et al., 2007; Rahman et al., 2009). For this reason, use of both hydrophilic and hydrophobic polymers have been considered to be an absolute necessity in this study since the ultimate aim was to achieve zero- order delivery ( Einmahl et al., 2001; Breton et al., 2008; Barakat et al., 2009) . Studies have shown that an increase in hydrophobicity may substantially alter the release kinetics of a drug delivery system (Nirmal, 2006; Khoee et al., 2007; Wu and Jin, 2008; Smith et al., 2009). This is attributable to its ability to hinder the penetration of the solvent molecules into the polymer matrix thereby leading to a reduction in the drug release rate (Sanchez - Lafuente et al. , 2002; Sant et al., 2008; Yu et al., 2009) . Furthermore, an increase in hydrophobicity often assists to control the rate of polymer disentanglement (Khoee et al., 2007; Smith et al., 2009) . This occurs through various mechanisms, but mostly through a decr eased tendency for the polymer to undergo hydrolysis due to decreased exposure to water (Liu et al., 2001; Khoee et al., 2007; Wu and Jin, 2008; Smith et al., 2009 ). In addition, hydrophobilic polymers may also provide several advantages, ranging from differing stability at varying pH and moisture levels, to well- established safe applications (Tiwari et al., 2003) . In this study a novel caplet - shaped device was considered to be the most appropriate based on the route of application. Conventional vaginal delivery systems including solutions, semi- solids (ointments, creams, gels), tablets and pessaries suffer in one way or another from the problems of retention, spreadability and drug release control (Garg and Tambwekar, 2003; das Neves and Bahia, 2006; Garg et al., 2007 ). Semi- solids in 73 particular are perceived as messy in use and prone to leakage ( Justin- Temu et al., 2004; Valenta, 2005). Furthermore, the conventional vagi nal drug delivery systems do not provide sufficient design flexibility in the control of drug release over periods extending from days to months. In this regard, there is then a great need for designing more specific formulations that can circumvent the problems associated with the conventional vaginal drug delivery systems. Polymer swelling is one of the factors that determines the matrix stability and subsequently the drug release kinetics from polymer matrices ( Patel a nd Patel, 2007; Barakat et al., 2008; Sibeko et al., 2009) . Normally in swellable polymer matrices, pol ymer relaxation and erosion mechanisms may coexist ( Bettini et al., 2001). Swelling and erosion front domains specifically define the gel layer thickness, which is considered to be the key factor in drug release kinetics and in most cases occur simultaneously (Christ et al., 1998; Bettini et al., 2001; Al - Taani and Tashtoush, 2003) . Thus, the extent of polymer swelling, relative mobilities of dissolution medium and drug, and matrix erosion dictate the kinetics as well as mechanism of drug release from polymeric matrices (Dahlberg et al., 2007) . Generally, swelling ? controlled release systems consist of a drug, which is molecularly dispersed in a polymeric matrix. Swelling occurs with subsequent formation of a thin gel layer as soon as water begins to penetrate the polymer matrix. In addition, it has been shown that the porosity of a polymeric system is another dominant factor that may control the swelling behavior ( Mehta et al., 2000; Vlachou et al., 2001) . Increased porosity leads to fast initial rates of water uptake and therefore high extent of swelling equilibrium. For a better outcome in terms of matrix stability and therefore optimal drug release kinetics, low swelling and slow erosion are the most desirable features. The aim of this Chapter was therefor e to screen various polymers through an extensive preformulation investigation so as to ultimately obtain polymer combinations for the design of lead Intravaginal Bioadhesive Polymeric Devices (IBPDs). A one- dimensional 74 search with successive variation in variables i.e. OVAT approach was employed. Although it is practically impossible for a one- dimensional search to accomplish an appropriate optimum in a finite number of experiments (Haltrich et a., 1994; He et al., 2005), it does successfully provide a lead formulation/s upon which mathematical optimization can be used for further refinement. 3.2 Materials and Methods 3.2.1 Materials The following polymers were employed in this study: Modified polyamide 6,10 ( mPA 6,10) was synthesized using hexamethylenediamine, sebacoyl chloride, anhydrous n- hexane and cyclohexane, all purchased from Sigma- Aldrich Chemie (Sigma- Aldrich Chemie, Steinheim, Germany). The remainder of the polymers employed were commercially available. These were poly(acrylic acid) (Carbopol ? 734 and 974) (Noveon Inc Cleveland, OH, USA), carageenan (Type one- kappa and alpha), ethylcellulose (Ethocel - 10), xanthan gum, tragacanth, bovine serum albumin (BSA) (Sigma - Aldrich Chemie, Steinheim, Germany); poly(ethylene oxide) (Union Carbide Corporati on, Danbury, CT, USA); poly(lactic - co- glycolic) acid (Resomer ? RG504; Boehringer Ingelheim, Ingelheim, Germany); poly(vinyl alcohol), polyvinyl povidone (Merck - Schuchardt, Hohenbrunn, Germany); gelatin, beeswax (Saarchem (Pty) Ltd., Krugersdorp, South Afri ca); methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylymethylce- llulose (Merck - Schuchardt? , Hohenbrunn, Germany); poly(ethyl acrylate, methyl - methacrylate, and chlorotrimethyl- ammoniumethylmethacrilate) S 100 (Eudragit ? S- 100) and poly(ethyl acrylate, methyl - methacrylate, and chlorotrimethyl- ammoniumethylmetha- crilate) RS 100 (Eudragit ? RS - 100) (Rohm & Co., Pharma Polymers, Darmstadt, Germany); calcium hydroxide, glycerol, acetic acid (Associated Chemical Enterprises (Pty) Ltd. , Southdale, South Africa). All other reagents used were of analytical grade and employed as received. 75 3.2.1.1 Synthesis of modified polyamide 6,10 The modified polyamide 6,10 ( mPA 6,10) was synthesized in our laboratory using a method developed by Kolawole and co- workers (2007) employing hexamethylenediamine (HMD), sebacoyl chloride (SC), hexane (HXN), cyclohexane (C - HXN), sodium hydroxide (NaOH) and deionized water (DW). The overall chemical reaction is illustrated in Figure 3.2. These researchers focused on exploring the effect of volume ratio, stoichiometric variations and the addition of solvent phase modifiers such as sodium hydroxide and cyclohexane on the physicochemical and physicomechanical properties of the mPA 6,10. In the present study, it is only the optimized mPA 6,10 that was synthesized. Briefly two sets of solutions were prepared. The first solution comprised SC dissolved in a mixture of HXN and C - HXN while the second solution comprised of specific quantities HMD and NaOH dissolved in DW. T he optimized formulation comprised of HMD (1.5g), SC (0.63), HXN 40 (mL ), C - HXN (40mL ), NaOH (0.1g) and DW 10 (mL ). The first solution was gradually added to the second to form two immiscible phases which resulted in a polymeric film being formed at the interface (i.e. by an interfacial polymerization process). The polymeric film was collected as a mass by slowly rotating a glass rod at the interface. Upon collection of the polymeric mass, it was thoroughly washed, with HXN to remove any unreacted SC and t hen with DW (3?300mL) to remove any un- reacted NaOH. The polymeric mass was then lightly rolled on filter paper (diameter 110mm, pore size 20?m) to remove any excess solvent and dried to a constant weight at 40?C over 48 hours. 76 Figure 3.2: Synthesis of modified polyamide 6,10 by interfacial polymerization. 3.2.2 Methods 3.2.2.1 Selection of formulation components In polymeric drug delivery syst ems, controlled drug delivery occurs when a polymer, whether natural or synthetic, is combined with a drug or other bioactive agent in such a manner that the bioactive agent is released from the polymeric material in a pre- determined manner. Initially 18 polymers were investigated. These were modified polyamide 6,10 ( mPA 6,10), poly(lactic - co- glycolic acid) (PLGA), polyethylene oxide ( PEO), poly(acrylic acid) (PAA), carrageenan (CG), ethycellulose (EC), polyvinylalcohol ( PVA), polyvinyly polidone (PVP), tragacanth (TG), xanthan gum (XG), gelatin (GL), methylcellulose (MC), Hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), hydroxypropymethylcellulose (HPMC), beeswax (BWX), poly(ethyl acrylate, methyl - methacrylate, and chlorotrimethyl- ammoniumethylmethacrilate) S 100 ( Eudragit ? S100) ( ED- S100) and poly(ethyl acrylate, methyl - methacrylate, and chlorotrimethyl- ammoniumethylmethacrilate) RS 100 ( Eudragit ? RS 100) ( ED- RS100) . Out of the 18 polymers, 5 of them namely modified polyamide 6,10 ( mPA 6,10) , poly(lactic - co- glycolic acid) (PLGA), ethycellulose (EC), polyvinylalcohol (PVA) and poly(acrylic acid) (PAA) N N C C O H H O C Cl O C O Cl N N H H H H + Sebacoyl chloride dispersed in the non- polar phase Hexamethylenediamine dispersed in the polar phase Interfacial polymerization - 2HCl Polyamide 6,10 n + NaoH + NaCl + H 2 0 77 have finally been employed to encapsulate PSS and AZT (Figure 3.3) with the purpose of attaining controlled release within the vagina (see Chapter 6 l ater). These polymers have their own inherent properties when used alone. However, this study has aimed at using their combination, a move that is expected to provide superior physicochemical and physicomechanical properties suitable for development of the IBPD. Furthermore, these polymeric materials are FDA approved and are biocompatible and biodegradable. Table 3.1 depicts the polymer type, properties and the rationale for selection of each polymer. 78 Table 3.1: Polymers, properties and rationale for selection Polymer Properties Rationale for selection mPA 6,10 ? High matrix resilience ? Abrasion resistance ? Chemical inertness ? High modulus ? Thermoplasticity ? To control drug release ? To control permeation ? To intensify the robustness of the IBPD PLGA ? Highly hydrophobic ? Degrades into two acidic units namely lactic acid and glycolic acid ? To produce an acidic pH environment within the vagina upon degrading into lactic and glycolic acid. ? Acidic pH maintains the normal vaginal ecology by favoring the growth of Lactobacilli - containing microflora which prevents bacterial vaginosis. ? To control drug release EC ? Highly hydrophobic ? Easily compressible ? To control drug release ? To control permeation PVA ? Hydrophilic ? Compressible ? Mildly bioadhesive ? To control drug release ? To foster bioadhesivity PAA ? Hydrophilic ? Possesses hydrogen bonds and strong anionic/ cationic charges ? High molecular mass and chain flexibility ? Surface energy interactions favoring spreading onto mucus ? Confers bioadhesiveness to the IBPD, thus favoring extension of drug residence time in the vagina ? To control drug r elease ? To control permeation For an intravaginal drug delivery system to be effective as an anti- HIV microbicidal agent, the drug employed should be able to inactivate HIV replication in lymphocytes, epithelial cells and sperm cells (D?Cruz and Uckun, 2002). Given the fact that the passage of HIV - infected mononuclear cells in semen contributes to the sexual transmission of HIV (Zhang et al., 1998; D?Cruz and Uckun, 2002), the anti - HIV microbicide should be metabolized 79 with equal efficiency by both the seminal cells and the epithelial cells of the cervico- vaginal region (D?Cruz and Uckun, 2002) . AZT (Figure 3.3a) is one such a compound and therefore the reason for being employed in this study. It undergoes intracellular hydrolysis yielding monophosphate derivatives which further become phosphorylated by thymidylate kinase to produce a bioactive triphosphate derivative. PSS (Figure 3.3b) is a microbicidal polymer which is also homogenous and viscous, and forms bioadhesive dispersion itself, thus facilitating its retention in the vagina for prolonged periods of time (Garg et al., 2004a ). Furthermore, It has been FDA approved as an alternative vaginal topical microbicide following the drawbacks found with the Nonoxynolol - 9 when it was once used as a spermicidal and contraceptive (i.e. it potentiated bacterial vaginosis as well as HIV transmission) (Richardson et al., 1998; Rosenstein et al., 1998; Anderson et al., 2000; Simoes et al., 2002). In addition, PSS has been shown to possess some ARV effect (Simoes et al., 2002), and it has gelling properties which may help in preventing HIV transmission by acting as a physical barrier Figure 3.3: a) 3' - azido- 3' - deoxythymidine (AZT), molecular weight 267. 24 and a solubility 20.1 mg/mL, b) Polystyrene Sulfonate (PSS) molecular weight 70,000, water soluble. 3.2.2.2 Preparation of preliminary lead intravaginal bioadhesive polymeric devices In the search for a suitable combination of polymers which will finally have the suitable bioadhesivity, matrix integrity and controlled release effect for the development of a novel controlled release intravaginal bioadhesive device, vast in vitro preformulation work was HN N O N 3 H2 CHO O O CH3 a) SO3 - n b) 80 carried out on 18 different polymers as mentioned above (Section 3.2. 2. 1). From these polymers, 62 different formulations weighing 800mg each were obtained through different polymer combinations at varying quantities of each polymer (Table 3.2) . All polymer combinations were thoroughly blended using an Erweka cube blender (Erweka Apparatebau, Heusenstamm, Germany) and then directly compressed into caplet devices using a Carver Press (Carver Inc. Hydraulic Laboratory Press, Wabash, IN, USA) at a force of 5 tons. Each device was subjected to i n- process validation tests that involved longitudinal crushing force, friability and mass uniformity analysis to ensure manufacturing reproducibility. A sample of 10 devices from each formulation was used for each test. Longitudinal crushing force test was conducted on a Hardness Tester (Pharma Test, Hainburg, Germany) while friability was conducted on a Friabilator (Erweka D - 63150, Heusenstamm, Germany) at 25rpm for 4 minutes with 1% set as the upper limit of acceptability. The weight of each device was determined using an electronic balance (Mettler, Model AE 240, Griefensee, Switzerland) with readings recorded to 2 decimal places. 81 Table 3.2: The sixty two formulations obtained from different polymer combinations F : Formulatio number; P 6: Modified polyamide 6,10; P L : Poly(lactic -co-glycolic acid); PE : Polyethylene oxide P A : Polyacrylic acid; CG: Carrageenan; EC: Ethycellulose; PV : Polyvinylalcohol; XG: Xanthan gum; GL : gelatin; ML: Methylcellulose; HE: Hydroxyethylcellulose; HP : Hydroxypropylcellulose; BW : beeswax; ES: Eudragit S100; ER: Eudragit RS 100; MA, MB, MC, MD, ME, MF: Mixture of two polymers among gelatin, beeswax, xanthan gum and Eudragit S 100 25mg each; MG, MH, MI: Mixture of three polymers among gelatin, beeswax, xanthan gum and Eudragit S 100 25m g each; WT : Weight Polymeric Composition (mg) F P6 PL PE PA CG EC PV XG GL ML HE HP BW ES ER MA MB MC MD ME MF MG MH MI WT 1 150 150 100 200 200 - - - - - - - - - - - - - - - - - - - 800 2 100 150 150 200 100 - - - - - - - - - - - - - - - - - - - 800 3 100 100 200 250 15 0 - - - - - - - - - - - - - - - - - - - 800 4 130 155 170 205 140 - - - - - - - - - - - - - - - - - - - 800 5 120 180 160 210 130 - - - - - - - - - - - - - - - - - - - 800 6 200 150 150 200 100 - - - - - - - - - - - - - - - - - - - 800 7 105 155 105 20 5 195 - - - - - - - - - - - - - - - - - - - 800 8 110 165 120 230 175 - - - - - - - - - - - - - - - - - - - 800 9 140 160 110 240 150 - - - - - - - - - - - - - - - - - - - 800 10 230 120 100 110 240 - - - - - - - - - - - - - - - - - - - 800 11 210 105 130 205 250 - - - - - - - - - - - - - - - - - - - 800 12 205 150 145 200 100 - - - - - - - - - - - - - - - - - - - 800 13 150 300 150 200 - - - - - - - - - - - - - - - - - - - - 800 14 250 200 150 200 - - - - - - - - - - - - - - - - - - - - 800 15 50 3 00 125 125 - 200 - - - - - - - - - - - - - - - - - - 800 16 50 200 125 125 - 300 - - - - - - - - - - - - - - - - - - 800 17 50 150 125 125 - 350 - - - - - - - - - - - - - - - - - - 800 18 50 100 125 125 - 400 - - - - - - - - - - - - - - - - - - 800 19 150 250 - 125 - 275 - - - - - - - - - - - - - - - - - - 800 20 150 225 - 175 - 250 - - - - - - - - - - - - - - - - - - 800 21 150 200 - 225 - 225 - - - - - - - - - - - - - - - - - - 800 22 160 250 - 190 - 200 - - - - - - - - - - - - - - - - - - 800 23 170 250 - 205 - 175 - - - - - - - - - - - - - - - - - - 800 24 180 250 - 220 - 150 - - - - - - - - - - - - - - - - - - 800 25 190 250 - 235 - 125 - - - - - - - - - - - - - - - - - - 800 26 195 250 - 225 - 100 - - - - - - - - - - - - - - - - - - 800 27 200 250 - 25 - 300 25 - - - - - - - - - - - - - - - - - 800 28 175 250 - 25 - 300 50 - - - - - - - - - - - - - - - - - 800 29 150 250 - 25 - 300 75 - - - - - - - - - - - - - - - - - 800 30 125 250 - 25 - 300 100 - - - - - - - - - - - - - - - - - 800 31 100 250 - 25 - 300 125 - - - - - - - - - - - - - - - - - 800 32 220 275 - 25 - 250 25 5 - - - - - - - - - - - - - - - - 800 33 215 275 - 25 - 250 25 10 - - - - - - - - - - - - - - - - 800 34 210 275 - 25 - 250 25 15 - - - - - - - - - - - - - - - - 800 35 205 275 - 25 - 250 25 20 - - - - - - - - - - - - - - - - 800 36 200 275 - 25 - 250 25 25 - - - - - - - - - - - - - - - - 800 37 195 275 - 25 - 250 25 30 - - - - - - - - - - - - - - - - 800 38 190 275 - 25 - 250 25 35 - - - - - - - - - - - - - - - - 800 39 185 275 - 25 - 250 25 40 - - - - - - - - - - - - - - - - 800 40 180 275 - 25 - 250 25 45 - - - - - - - - - - - - - - - - 800 41 175 275 - 25 - 250 25 50 - - - - - - - - - - - - - - - - 800 42 195 300 - 25 - 250 25 - 5 - - - - - - - - - - - - - - - 800 43 185 300 - 25 - 250 25 - 15 - - - - - - - - - - - - - - - 800 44 175 300 - 25 - 250 25 - 25 - - - - - - - - - - - - - - - 800 45 165 300 - 25 - 250 25 - 35 - - - - - - - - - - - - - - - 800 46 155 300 - 25 - 250 25 - 45 - - - - - - - - - - - - - - - 800 47 175 300 - 25 - 250 25 - - 25 - - - - - - - - - - - - - - 800 48 175 300 - 25 - 250 25 - - - 25 - - - - - - - - - - - - - 800 49 175 300 - 25 - 250 25 - - - - 25 - - - - - - - - - - - - 800 50 175 300 - 25 - 250 25 - - - - - 25 - - - - - - - - - - - 800 51 175 300 - 25 - 250 25 - - - - - - 25 - - - - - - - - - - 800 52 175 300 - 25 - 250 25 - - - - - - - 25 - - - - - - - - - 800 53 150 400 - 25 - 150 25 - - - - - - - - 50 - - - - - - - - 800 54 150 400 - 25 - 150 25 - - - - - - - - - 50 - - - - - - - 800 55 150 400 - 25 - 150 25 - - - - - - - - - - 50 - - - - - - 800 56 150 400 - 25 - 150 25 - - - - - - - - - - - 50 - - - - - 800 57 150 400 - 25 - 150 25 - - - - - - - - - - - - 50 - - - - 800 58 150 400 - 25 - 150 25 - - - - - - - - - - - - - 50 - - - 800 59 75 400 - 25 - 200 25 - - - - - - - - - - - - - - 75 - - 800 60 75 400 - 25 - 200 25 - - - - - - - - - - - - - - - 75 - 800 61 75 400 - 25 - 200 25 - - - - - - - - - - - - - - - - 75 800 62 150 400 - 25 - 200 25 - - - - - - - - - - - - - - - - - 800 82 For the mixture of two polymers, MA was GL+ES; MB: GL+BW; M C: ES+BW; MD: ES+XG; ME: BW+XG and MF: GL+XG while for the mixture of three polymers, MG was GL+ES+BW; MH: GL+BW+XG and MI: ES+BW+XG. 3.2.2.3 Equilibrium swelling studies conducted on the preliminary formulations as a critical indicator of the formulation?s matrix stability A polymer combination selected as the working formulation onto which the OVAT approach was applied in an attempt to find lead IBPDs was: mPA 6,10 (50mg), PLGA (300mg), PEO (125mg), PAA (125mg) and CG (200mg). A series of swelling tests were conducted on various sets of the formulations shown in Table 3.2 to investigate which device would have the optimal swelling at both human and pig vaginal pH (i.e. ~4. 5) and human/pig seminal pH (i.e. ~7. 4) by employing the OVAT approach. Due to the physiological similarity between the human and pig ( Buddhikot et al. 1999; Qui ntanar- Guerrero et al., 2001; Sandri et al., 2004; D?cruz et al., 2005b), simulated human vaginal and seminal fluids (Owen and Katz, 1999, 2005) prepared as indicated in Table 3.3, were employed for the tests. Each of the tested devices was weighed, immers ed into both simulated fluids and then placed in an orbital shaking incubator (MRC Laboratory Instruments Ltd., Hahistadrut, Holon, Israel), maintained at 20rpm and a temperature of 37 ? C for 24 hours. After 24 hours each device was removed from the orbital shaking incubator, gently blotted on a filter paper and then re- weighed. The swelling behavior was determined in terms of the equilibrium swelling ratio (ESR) which was calculated using Equation 3.1. The ESR (after 24 hours) which was the critical indicat or of the formulation?s matrix stability (i.e. the degree of matrix robustness), was used as a screening parameter for each formulation. W WWESR o ot ? = Equation 3.1 83 where ESR is the equilibrium swelling ratio, Wo is the initial weight of the device and Wt is the weight of the device after 24 hours. Table 3.3: Constituents used to prepare the simulated human vaginal and seminal fluids SHVF 1 SHSF 2 Component Quantity( g/L) Compon ent Quantity( g/L) NaCl 3.510 NaH 2 PO4?H2O 16.974 KOH 1.400 Na 2 HPO4 17. 466 Ca(OH) 2 0.222 Na 3 C3 H5 O(CO 2 ) 3 8.130 Bovine serum albumin 0.018 KCl 0.908 Lactic acid 2.000 KOH 0.881 Acetic acid 1.000 CaCl2 1.010 Glycerol 0.160 MgCl 2 0.920 Urea 0.400 ZnCl 2 0.344 Glucose 5.000 Glucose 1.020 Fructose 2.720 Urea 0.450 Lactic acid 0.620 Bovine serum albumin 50.400 1 Simulated human vaginal fluid according to Owen and Katz, (199 9) 2 Simulated human seminal fluid according to Ow en and Katz, (200 5) 3.2.2.4 Effect of changing the formulation components of the IBPD on the equilibrium swelling ratio The formulations components of the IBPD were changed sequentially (changes highlighted in grey color) to see the effect on equilibrium swelling ratio. 3.2.2.4.1 Effect of the elimination of carrageenan Carrageenan is a highly hydrophilic compound which is substantially water- soluble with the capacity to transform into a gel that presents with a high degree of swelling in an alkaline pH (Campo et al., 2009) . It was thus eliminated to see the effect in terms of the composite swelling of the devices. After elimination of the carrageenan an adjustment of the quantities of the remaining polymers was undertaken to maintain a constant weight of 800mg (Table 3.4) . 84 Table 3.4: Formulations without carrageenan Polymeric Composition (mg) F # m PA 6,10 PLGA PAA PEO 1 50. 0 400. 0 250. 0 100 2 100. 0 350. 0 250. 0 100 3 200. 0 250. 0 250. 0 100 4 250. 0 200. 0 250. 0 100 5 300. 0 150. 0 250. 0 100 6 350 100. 0 250. 0 100 3.2.2.4.2 Effect of the addition of ethyl cellulose Due to its hydrophobic nature and high compressibility, EC was employed to replace CG. The quantities of PAA and PEO were substantially reduced as they enhanced matrix swelling, due to their high degree of hydrophilicity. The quantities of EC employed ranged from 240mg - 460mg as shown in Table 3.5. Table 3.5: Formulations in which carrageenan was substituted with ethylcellulose Polymeric Composition (mg) F # m PA 6,10 PLGA PEO PAA EC 1 9 0. 0 340. 0 65 65. 0 240. 0 2 90. 0 240. 0 65 65. 0 340. 0 3 90. 0 190. 0 65 65. 0 390. 0 4 90. 0 140. 0 65 65. 0 440. 0 5 90. 0 130. 0 65 65. 0 450. 0 6 90. 0 120. 0 65 65. 0 460. 0 3.2.2.4.3 Effect of the elimination of polyethylene oxide PEO is a hydrophilic polymer with a high tendency to undergoing swelling ( Varma et al., 2004) . PEO as a bioadhesive and a rate- controlling polymer was removed to reduce the number of hydrophilic polymers so as to reduce the extent of swelling of the devices. However, PAA remained included throughout all formulations due to its substantial bioadhesive imparting (Table 3.6). 85 Table 3.6: Formulations without polyethylene oxide Polymeric Composition (mg) F # m PA 6,10 PLGA PAA EC 1 100 300. 0 75. 0 325 2 100 300. 0 100 300 3 100 300. 0 150 250 4 100 300. 0 200. 0 200 5 100 300. 0 250. 0 150 6 100 300. 0 300. 0 100 3.2.2.4.4. Effect of the addition of polyvinyl alcohol PVA has high tensile strength and flexibility, as well as adhesive and high oxygen barrier properties ( Yan et al., 2006; Yang et al., 2008; Mukherjee, 2009). It is also an atactic material but exhibits crystallinity as the hydroxyl groups are small enough to fit into the lattice without disrupting it. It was therefore incorporated into the formulation to enhance the control of drug release as well as the bioadhesivity of the devices. Quantities ranging from 25mg - 150mg were employed in formulations to produce a total weight of 800mg/device while keeping the quantities of PLGA, EC and PAA constant (Table 3.7) . Table 3.7: Formulations containing polyvinyl alcohol Polymeric Composition (mg) F# m PA 6,10 PLGA EC PAA PVA 1 200. 0 250. 0 300. 0 25. 0 25. 0 2 175. 0 250. 0 300. 0 25. 0 50. 0 3 150. 0 250. 0 300. 0 25. 0 75. 0 4 125. 0 250. 0 300. 0 25. 0 100. 0 5 100. 0 250. 0 300. 0 25. 0 125. 0 6 75. 0 250. 0 300. 0 25. 0 150. 0 3.2.2.4 .5 Effect of the addition of polyvinyl povidone PVP is a water- soluble material with a Newtonian- type viscosity. When dry it is a light flaky powder, capable of readily absorbing up to 40% of its weight in atmospheric water. In solution, it has excellent w etting properties and readily forms films which make it a good coating agent. It was therefore added to the formulations ranging from 25 - 150mg with the intention of fostering the bioadhesivity of the devices. The quantities of PLGA, EC and PAA were kept constant as shown in Table 3.8. 86 Table 3.8: Formulations containing polyvinyl povidone Polymeric Composition (mg) F # m PA 6,10 PLGA EC PAA PVP 1 200. 0 250. 0 300. 0 25. 0 25. 0 2 175. 0 250. 0 300. 0 25. 0 50. 0 3 150. 0 250. 0 300. 0 25. 0 75. 0 4 125. 0 250. 0 300. 0 25. 0 100. 0 5 100. 0 250. 0 300. 0 25. 0 125. 0 6 50. 0 250. 0 300. 0 25. 0 150. 0 3.2.2.4.6 Effect of the addition of xant han gum XG is a water - soluble polysaccharide produced by Xanthomonas campestris . It forms a single or double- stranded helix in dilute solut ion that transforms into molecular assemblages which show liquid crystallinity in the presence of water (Rodd et al., 2000; Iseki et al., 2001) . XG can form hydrogels upon annealing its solution in the sol state and then cooling (Quinn et al., 1994; Song e t al., 2006) . XG was therefore added to the formulations due to its gel- forming and viscosity- enhancing properties in an attempt to facilitate adhesion of the device to the vaginal epithelium. The quantities added ranged from 5 - 50mg. The quantities of PLGA , EC, PAA and PVA were kept constant (Table 3.9) . Table 3.9: Formulations containing xanthan gum Polymeric Composition (mg) F # m PA 6,10 PLGA EC PAA PVA XG 1 220. 0 275. 0 250. 0 25. 0 25. 0 5.0 2 210. 0 275. 0 250. 0 25. 0 25. 0 15. 0 3 200. 0 275. 0 250. 0 25. 0 2 5. 0 25. 0 4 190. 0 275. 0 250. 0 25. 0 25. 0 35. 0 5 180. 0 275. 0 250. 0 25. 0 25. 0 45. 0 6 175. 0 275. 0 250. 0 25. 0 25. 0 50. 0 3.2.2.4.7 Effect of the addition of guar gum GG is a polysaccharide comprising galactose and mannose. In water it is nonionic and hydrocolloidal and remains stable in solution over a pH range of 5 - 7. Guar gum presents with high low- shear viscosity but is strongly shear- thinning. It is very thixotropic above 1% w/ v with a low degree of thixotropy below a concentration of 0.3% w/ v ( Soukoulis et al., 2007) . It shows viscosity synergy with xanthan gum. Guar gum has almost eight times 87 the water- thickening potency of other thickening agents such as starch, and therefore can be used in the preparation of various multi- phase formulations (Billa et a l., 2000) . It was therefore added to the current formulations so as to enhance the bioadhesivity. The quantities added ranged from 5 - 50mg (Table 3.10) . Table 3.10: Formulations containing guar gum Polymeric Composition (mg) F # m PA 6,10 PLGA EC PAA PVA GG 1 220. 0 275. 0 250. 0 25. 0 25. 0 5.0 2 210. 0 275. 0 250. 0 25. 0 25. 0 15. 0 3 200. 0 275. 0 250. 0 25. 0 25. 0 25. 0 4 190. 0 275. 0 250. 0 25. 0 25. 0 35. 0 5 180. 0 275. 0 250. 0 25. 0 25. 0 45. 0 6 175. 0 275. 0 250. 0 25. 0 25. 0 50. 0 3.2.2.4.8 Effect of the addition of gelatin GL is a translucent, brittle, solid substance derived from the collagen obtained from skin and bones of animals. It forms a solution of high viscosity in water, which sets to a gel on cooling. Gelatin solutions present with viscoelastic flow and st reaming birefringence (Ward and Courts, 1977) . It qualifies as a binder, and therefore was used to substitute xanthan gum in an attempt to improve the bioadhesivity of the devices. The quantities added ranged from 5- 50mg while the quantities of the other polymers remained constant (Table 3.11) . Table 3.11: Formulations in which xanthan gum was substituted with gelatin Polymeric Composition (mg) F # m PA 6,10 PLGA EC PAA PVA GL 1 195. 0 300. 0 250. 0 25. 0 25. 0 5 2 185. 0 300. 0 250. 0 25. 0 25. 0 15 3 175. 0 300. 0 250. 0 25. 0 25. 0 25 4 165. 0 300. 0 250. 0 25. 0 25. 0 35 5 155. 0 300. 0 250. 0 25. 0 25. 0 45 6 150. 0 300. 0 250. 0 25. 0 25. 0 50 88 3.2.2.4.9 Effect of the addition of beeswax Beeswax is comprised of a mixture of many organic compounds, including hydrocarbons, wax esters, and fatty acids . It has emollient, soothing and softening properties. Furthermore, it is very stable over prolonged periods. It is resistant to hydrolysis and natural oxidization and is completely insoluble in water. Thus, beeswax was added to the formulations ranging from 5- 50mg (Table 3.12) so as to increase the matrix stability of the devices. Table 3.12: Formulations in which gelatin was substituted with beeswax Polymeric Composition (mg) F # m PA 6,10 PLGA EC PAA PVA BW 1 195. 0 300. 0 250 . 0 25. 0 25. 0 5 2 185. 0 300. 0 250. 0 25. 0 25. 0 15 3 175. 0 300. 0 250. 0 25. 0 25. 0 25 4 165. 0 300. 0 250. 0 25. 0 25. 0 35 5 155. 0 300. 0 250. 0 25. 0 25. 0 45 6 150. 0 300. 0 250. 0 25. 0 25. 0 50 3.2.2.4.10 Effect of the addition of tragacant h Tragacanth is a translucent and horny fracture short material. It swells in water forming smooth nearly uniform stiff opalescent mucilage that is free of cellular fragments. It imparts great viscidity to water, a property that renders it useful for the suspension of heavy insoluble powders. Pharmaceutically, it is has good applications particularly in imparting consistence to troches and emulsions. It was added to the formulations for its effect on the matrix stability. The quantities added ranged from 5- 50mg (Table 3.13). Ta ble 3.13: Formulations containing tragacanth Polymeric Composition (mg) F # m PA 6,10 PLGA EC PAA PVA TG 1 195. 0 300. 0 250. 0 25. 0 25. 0 5 2 185. 0 300. 0 250. 0 25. 0 25. 0 15 3 175. 0 300. 0 250. 0 25. 0 25. 0 25 4 165. 0 300. 0 250. 0 25. 0 25. 0 35 5 155. 0 300. 0 2 50. 0 25. 0 25. 0 45 6 150. 0 300. 0 250. 0 25. 0 25. 0 50 89 3.2.2.4.11 Effect of the addition of methyl cellulose, hydroxyethyl cellulose, hydroxyl - propylcellulose, hydroxypropylmethyl cellulose, Eudragit ? S100 and Eudragit ? RS 100 as substitutes for gelatin MC, HEC, HPC and HPMC are cellulose ethers that differ in their type and degree of substitution. These polymers are hydrophilic in nature with pharmaceutical attributable properties that favor their use in the preparation of controlled release formulations. They are also used as bulking agents in the tabletting process. When exposed to aqueous media, they swell forming a gel layer around the tablet core (Baumgartner et al., 2002) . Chain dissolution may take place at the gel surface depending on the type of cellulose ester. These polymers were added to the formulations to investigate their effect on the equilibrium swelling ratio of the devices with the intention of finally employing them to control drug release. ED- S100 and ED - RS100 are poly(ethyl acrylate, methyl - methacrylate, and chlorotrimethyl- ammoniumethylmethacrilate) co - polymers. They are insoluble at physiological pH but undergo swelling in water (Pignatello et al., 2002). ED - S100 and ED - RS100 are commonly employed for the enteric coating of tablets and i n the preparation of controlled release formulations. Both are good materials for the dispersion of drugs and have been used successfully to obtain appropriate controlled release matrix formulations (Ndesendo et al., 1996; Wong et al., 1999; Trapani et al., 2007). They were therefore added to the formulations for the purpose of modulating the release kinetics. The quantity of each polymer added to the formulations was 25mg. The quantities of the other polymers were kept constant (Table 3.14). 90 Table 3.14: Formulations containing methylcellulose, hydroxyethlycellulose, hydroxyl - propylcellulose, hydroxypropylmethylcellulose, Eudragit ? S100 and Eudragit ? RS100 Polymeric Composition (mg) F # m PA 6,10 PLGA EC PAA PVA Additional polymer 1 175. 0 250. 0 300. 0 25. 0 25. 0 25 (MC) 2 175. 0 250. 0 300. 0 25. 0 25. 0 25 (HEC) 3 175. 0 250. 0 300. 0 25. 0 25. 0 25 (HPC) 4 175. 0 250. 0 300. 0 25. 0 25. 0 25 (HPMC) 5 175. 0 250. 0 300. 0 25. 0 25. 0 25(ED - S100) 6 175. 0 250. 0 300. 0 25. 0 25. 0 25 (ED - RS100) 3.2.4.12 Effect of the addition of a binary polymer combination to the formulation Combinations of 2 polymers in equal quantities of 25mg each (1:1), from among gelatin, beeswax, xanthan gum and ED - S100 were added to the formulations to investigate their effect on the matrix stabil ity. The quantities of the other polymers were kept constant (Table 3.15) . Table 3.15: Formulations with a binary polymer combination among gelatin, beeswax, xanthan gum and Eudragit ? S100 Polymeric Composition (mg) F # m PA 6,10 PLGA EC PAA PVA Combinati on of 2 polymers 1 150. 0 400. 0 150. 0 25. 0 25. 0 50 (GL+ED - S100) 2 150. 0 400. 0 150. 0 25. 0 25. 0 50 (GL+BWX) 3 150. 0 400. 0 150. 0 25. 0 25. 0 50 (ED - S100+BWX) 4 150. 0 400. 0 150. 0 25. 0 25. 0 50 (ED - S100+XG) 5 150. 0 400. 0 150. 0 25. 0 25. 0 50 (BWX+XG) 6 150. 0 400. 0 150. 0 25. 0 25. 0 50 (GL+XG) 3.2.2.4.13 Effect of the addition of a tertiary polymer combination to the formulation Combinations of 3 polymers in equal quantities of 25mg each (1:1: 1), from among gelatin, beeswax, xanthan gum and ED - S100 were added to the formulations to investigate the effect on the matrix stability of the devices. The quantities of the other polymers were kept constant (Table 3.16) . 91 Table 3.16: Formulations with a tertiary polymer combination among gelatin, beeswax, xanthan gum and Eudragit? S100 Polymeric Composition (mg) F # m PA 6,10 PLGA EC PAA PVA Combination of 3 polymers 1 75. 0 400. 0 200. 0 25. 0 25. 0 75(GL+ED - S100+BWX ) 2 75. 0 400. 0 200. 0 25. 0 25. 0 75 (GL+BWX+XG) 3 75. 0 400. 0 200. 0 25. 0 25. 0 75 (ED - S100+BWX+XG) 3.3 Results and Discussion 3.3. 1 Physical properties of m PA 6,10 and in -process validation tests The mPA 6,10 product synthesized presented as a firm white crystalline spherical compact. The unrefined mPA 6,10 powder was subsequently sieved through an aperture size of 1mm in keeping with the other polymeric components of the caplet matrix to obtain a particle size ranging from 0.8 - 1. 2mm which facilitated desirable powder compression properties. The compressed caplets (Figure 3.4) were sufficiently strong and robust (i.e. sturdy and with high resilience) with an average longitudinal crushing force of 286? 0. 03N, a uniformity mass of 800? 0. 48mg and average friability of 0.31? 0. 04% which was within the set limits (Table 3.17) . 92 Figure: 3.4: The intravaginal bioadhesive polymeric devices (IBPDs) a) Planar view, b) Side view, c) Oblique view and d) Longitudinal view. a b c d 93 Table 3.17: Mass, friability and longitudinal crushing force of the sixty two caplet formulations F # Mass (mg) ?SD % Friability ?SD Longitudinal Crush ing Force (N) ?SD 1 789. 22 3. 633 0. 39 0. 018 284. 81 1. 311 2 803. 65 4. 109 0. 41 0. 019 285. 76 1. 317 3 788. 11 2. 99 8 0. 44 0. 020 284. 28 1. 309 4 799. 06 3. 678 0. 42 0. 019 284. 82 1. 313 5 800. 09 4. 00 3 0. 40 0. 018 284. 92 1. 315 6 801. 89 4. 006 0. 40 0. 018 285. 61 1. 318 7 798. 09 3. 593 0. 42 0. 019 285. 90 1. 319 8 788. 72 3. 701 0. 41 0. 020 285. 82 1. 317 9 804. 29 4. 015 0. 39 0. 017 285. 09 1. 312 10 803. 22 4. 012 0. 39 0. 015 284. 41 1. 308 11 804. 21 4. 018 0. 46 0. 024 2 80. 63 1. 309 12 790. 32 3. 456 0. 37 0. 013 285. 71 1. 326 13 800. 33 4. 009 0. 35 0. 011 285. 92 1. 328 14 803. 43 4. 013 0. 36 0. 013 286. 03 1. 330 15 805. 21 4. 127 0. 33 0. 010 286. 41 1. 338 16 802. 44 4. 018 0. 32 0. 010 286. 65 1. 339 17 801. 29 4. 003 0. 32 0. 012 286. 91 1. 40 0 18 801. 29 4. 003 0. 30 0. 009 287. 25 1. 403 19 803. 64 4. 012 0. 29 0. 009 287. 07 1. 401 20 789. 08 3. 987 0. 28 0. 007 287. 38 1. 328 21 789. 00 3. 967 0. 26 0. 005 286. 82 1. 330 22 805. 78 4. 134 0. 27 0. 006 286. 71 1. 338 23 788. 99 3. 556 0. 31 0. 011 286. 52 1. 339 24 801. 98 4. 007 0. 33 0. 013 286. 33 1. 400 25 800. 37 4. 001 0. 32 0. 013 287. 18 1. 403 26 789. 98 3. 897 0. 30 0. 010 288. 97 1. 401 27 810. 02 5. 004 0. 24 0. 005 289. 36 1. 428 28 801. 99 4. 120 0. 29 0. 009 287. 66 1. 429 29 788. 89 3. 898 0. 30 0. 010 287. 94 1. 407 30 802. 77 4. 108 0 . 31 0. 010 286. 84 1. 330 31 807. 22 4. 130 0. 32 0. 012 286. 62 1. 338 32 789. 69 3. 789 0. 34 0. 012 285. 51 1. 339 33 803. 11 4. 015 0. 37 0. 015 285. 42 1. 400 34 801. 25 4. 001 0. 35 0. 014 285. 08 1. 403 35 800. 66 4. 002 0,33 0.013 284. 93 1. 381 36 798. 76 3. 972 0. 30 0. 009 287. 19 1. 328 37 792. 53 3. 864 0. 32 0. 010 287. 28 1. 330 38 801. 48 3. 999 0. 33 0. 013 287. 39 1. 338 39 790. 98 3. 870 0. 26 0. 010 288. 14 1. 429 40 808. 79 4. 137 0. 29 0. 010 287. 66 1. 400 41 789. 98 2. 987 0. 27 0. 012 288. 49 1. 403 42 803. 44 4. 121 0. 35 0. 009 285. 65 1. 4 01 43 802. 00 4. 002 0. 36 0. 009 285. 23 1. 428 44 788. 89 2. 998 0. 29 0. 007 287. 09 1. 429 45 801. 66 4. 006 0. 33 0. 005 285. 25 1. 407 46 787. 98 2. 999 0. 37 0. 006 285. 73 1. 326 47 799. 86 3. 003 0. 39 0. 011 284. 93 1. 328 48 808. 00 4. 985 0,36 0.013 285. 06 1. 330 49 803 . 33 4. 006 0. 38 0. 013 285. 34 1. 338 50 800. 30 4. 000 0. 31 0. 010 286. 78 1. 339 51 803. 01 3. 998 0. 24 0. 005 289. 02 1. 4 23 52 808. 22 5. 000 0. 25 0. 005 288. 77 1. 328 53 789. 56 3. 979 0. 32 0. 012 285. 88 1. 330 54 809. 29 5. 001 0. 29 0. 010 286. 23 1. 338 55 804. 44 4. 110 0. 33 0. 010 285. 56 1. 429 56 789. 79 3. 961 0. 30 0. 012 285. 68 1. 400 57 800. 67 3. 998 0. 30 0. 012 286. 05 1. 403 58 804. 58 4. 029 0. 34 0. 015 285. 83 1. 301 59 798. 98 4. 001 0. 37 0. 014 285. 07 1. 328 60 802. 44 4. 008 0. 32 0. 013 285. 41 1. 329 61 789. 88 3. 989 0. 31 0. 009 285. 90 1. 307 62 800. 45 4. 007 0. 23 0. 006 289. 69 1. 4 25 94 3. 3.2 The influence of different polymeric composition on the equilibrium swelling ratio 3.3.2.1 The influence of carageenan, polyethylene oxide and ethyl cellulose on the matrix stability The formulations prepared with CG and PEO (F1 - F12) (Table 3.2) presented with the highest level of matrix swelling as shown by their ESR values (0.55- 0. 76) (Table 3.18). The highest ESR was observed in F11 (ESR=0. 76; Table 3.18) . This was most likely due to the hydrophilic nature of CG and PEO. T hese polymers, being highly hydrophilic, rapidly absorbed water which led to a high degree of swelling. A similar trend was apparent in F3 (ESR=0.71) though to a lesser extent, due to relatively lower quantity of CG. The elimination of PEO and substitution of CG with EC resulted in a marked negative impact on matrix swelling in that a formulation with an ESR as low as 0.12 (F 19; Table 3.18) was obtained. This was due to the hydrophobic nature of EC. 3. 3.2.2 The influence of guar gum, polyvinyl povidone, t ragacant h and polyvinyl alcohol on matrix stability GG produced matrix swelling to a large extent after 12 hours. PVP and TG were found to induce initial swelling and subsequent erosion of the formulation after 24 hours. PVA resulted in formulations with minimal matrix swelling (Table 3.18; F27; ESR=0.02) and therefore was considered to have the most favorable matrix stability among the entire group. 3. 3.2.3 The influence of xant han gum , beeswax and gelatin on the matrix stability Xanthan gum provided substantially swollen formulations which remained intact (no evidence of erosion), in addition to being highly bioadhesive. Overall, XG , BWX and GL at low quantities (25mg each) (Table 3.18) resulted in formulations with minimal ESR (0.02- 0. 16). This may be due to the ability of XG, BWX and GL to form gel - like microstructure (Gimeno et al., 2003) which interspersed itself throughout. 95 3. 3.2.4 The influence of the addition of methyl cellulose, hydroxethyl cellulose, hydroxypropylcellulose and hydroxylpropylmethyl cellulose in the formulations, on matrix stability The MC - containing formulations underwent initial massive swelling and thereafter erosion after 24 hours (formulations completely collapsed). HEC, HPC and HPMC - containing formulations remained stable for at least 72 hours. This finding may be attributed to the fact that these polymers are non- ionic and hydrophilic with water retention properties. 3.3.2.5 The influence of Eudragit ? S100 and Eudragit ? RS 100 on matrix stability ED- S100 and ED - RS100 are random copolymers of methacrylic acid and ethyl acrylate, which are insoluble at acidic pH but become increasingly soluble in a neutral to weakly alkaline solution by forming salts (Guoqiang et al., 1995; Arasaratnam et al., 2000). Thus, the ED- S100- and ED- RS100- containing formulations presented with minimal swelling tendency (ESR=0.02 and 0.03) in simulated vaginal fluid (pH 4.5) (Table 3.18; F51 and F52). 96 Table 3.18: The equilibrium swelling ratios for the sixty two formulations that were subjected to screening using the OVAT approach F# ESR ?SD 32 0 .13 0.006 33 0.14 0.006 3 4 0.15 0.007 35 0.16 0.007 36 0.06 0.003 37 0.08 0.004 38 0.08 0.004 39 0.04 0.002 40 0.05 0.002 41 0.04 0.002 42 0.14 0.0 0 6 43 0.16 0.007 44 0.06 0.003 45 0.13 0.006 46 0.15 0.007 47 0.53 0.025 48 0.49 0.023 49 0.56 0.026 50 0.07 0.003 51 0.02 0.001 52 0.03 0.001 53 0.12 0. 00 6 54 0.09 0.004 55 0.14 0.0 0 6 56 0.12 0.006 57 0.11 0.005 58 0. 13 0.006 59 0.15 0.007 60 0.14 0.0 0 6 61 0.12 0.006 62 0.01 0.001 F# ESR ?SD 1 0.63 0.029 2 0.58 0.027 3 0.71 0.033 4 0.65 0.030 5 0.6 0 0.028 6 0.58 0.027 7 0.6 0 0.028 8 0.61 0.028 9 0.59 0.027 10 0.68 0.032 11 0.76 0.035 12 0.55 0.026 13 0.53 0.025 14 0.50 0.023 15 0.47 0.022 16 0.45 0.021 17 0.42 0.020 18 0.11 0.005 19 0.12 0. 00 6 20 0.11 0.005 21 0.08 0.004 22 0.09 0.004 23 0.10 0.005 24 0.12 0. 00 6 25 0.08 0.004 26 0.06 0.003 27 0.02 0.001 28 0.05 0.002 29 0.07 0.003 30 0.09 0.004 31 0.08 0.004 3.3.2.6 Equilibrium swelling ratios of the selected fifteen lead formulations screened through the OVAT approach The ESRs for the selected 15 lead formulations are summarized in Table 3.19. Depending on the polymer combination and hydrophilic/hydrophobic proportions, all devices demonstrated a different swelling equilibrium as depicted by their ESR values (Table 3.19) . Swelling ratio describes the amount of water that was contained within the device at equilibrium and is a function of the proportion between hydrophilicity and hydrophobicity in the device network structure. Ionization of the polymer functional groups, crosslinking density, charge density, and simulated vaginal fluid ionic strength, 97 may have played a role as well in this regard. The higher the hydrophobicity the lower the ESR and the higher the hydrophilicity the higher the ESR. The opposite holds true in both cases. Low ESR is an indication of low swelling rate and therefore high matrix stability and vice versa (Baumgartner et al., 2000; Girish et al, 2008; Wen et al., 2008) . Among the 15 lead formulations, F62 presented with the lowest ESR (0.011). These findings may be associated with the presence of a high quantity of PLGA (400mg) in the formulation which prevented the influx of water into the IBPD matrix due to its high hydrophobicity. The presence of EC in the formulation at a relatively high quantity (200mg) may have as well attributed to the obtained results since EC is also a polymer with high degree of hydrophobicity. Most certainly, minimal quantities of the hydrophilic polymers PAA and PVA coupled the superior matrix resilience of mPA 6,10 contributed as well to the small ESR value of F62. ED - S100 and ED - RS100 are both pH - dependent polymer materials that are only soluble at pH above 6.0 ( Ohmura et al., 1991) . Thus, at the simulated vaginal fluid pH (4.5), these polymers were insoluble indicating that they may have played a role in preventing the entrance of water molecules to the IBPD matrix, therefore leading to the low ESR values obtained in F51 (ESR=0.023) and F52 (ESR=0. 033) respectively (Table 3.19) . The high tendency GL, BWX and XG to form a non- collapsible networked- structure may have improved the veracity of the IBPD matrix and there fore the relatively low values of ESR values obtained in the formulations containin g these polymers (Table 3.19).The ESR for the best 15 formulations was in the following order F62>F27>F51>F52>F41>F39> F28 >F40>F44> F36>F29>F50> F37> F38>F31 (i.e. lowest t o the highest) (Table 3.19) . Overall, the high degree of hydrophobicity in the polymers constituting these formulations, prevented rapid penetration of water molecules into the IBPD matrix resulting in a low swelling tendency (Table 3.19) and therefore low er ESR values when compared to the rest of the tested formulations (Table 3.18) . 98 Table 3.19: The selected fifteen lead formulations screened through the OVAT approach Polymeric Composition (mg) F# P6 PL PA EC PV XG GL BW ES ER ESR 62 150 400 25 200 25 - - - - - 0.011 27 200 250 25 300 25 - - - - - 0.020 51 175 300 25 250 25 - - - 25 - 0.023 52 175 300 25 250 25 - - - - 25 0.033 41 175 300 25 250 25 - 25 - - - 0.041 39 185 275 25 250 25 40 - - - - 0.044 28 175 250 25 300 50 - - - - - 0.050 40 180 2 75 25 250 25 45 - - - - 0.053 44 175 275 25 250 25 50 - - - - 0.060 36 200 275 25 250 25 25 - - - - 0.064 29 150 250 25 300 100 - - - - - 0.071 50 175 300 25 250 25 - - 25 - - 0.073 37 195 275 25 250 25 30 - - - - 0.080 38 190 275 25 250 25 35 - - - - 0.082 31 100 250 25 300 125 - - - - - 0.084 P6: Modified polyamide 6,10; PL : Poly(lactic -co-glycolic acid); PE: Polyethylene oxide PA : Polyacrylic acid; C G : Carrageenan; EC: Ethycellulose; PV: Polyvinylalcohol; X G : Xanthan gum; G L : gelatin; B W: beeswax; ES: Eudragit S100; ER: Eudragit RS 100; ESR: Equilibrium swelling ratio 3.4 Concluding Remarks Through extensive screening of the developmental formulations using an OVAT approach, robust caplets with substantial matrix integrity were produced, as evi denced by the Equilibrium Swelling Ratios. Fifteen lead formulations with minimal swelling tendencies were obtained. F62 which is composed of mPA 6,10 (150mg), PLGA (400mg), EC (200mg), PVA ( 25mg) and PAA (25mg) appeared to be the most suited in terms of minimal swelling capacity and highest matrix stability. Optimization was further conducted on the fifteen lead formulation employing ANN in Chapter 4. 99 CHAPTER FOUR AP P L IC AT ION OF AR TIF IC IAL NE UR AL NE TWOR K S F OR THE E L UC IDAT ION OF AN OP TIMIZE D S Y NE R G IS TIC P OL Y ME R C OMB INATION F OR E F F E C TIVE P E R F OR MANC E OF THE INTR AV AG INAL B IOADHE S IVE P OL Y ME R IC DE VIC E 4. 1 I ntroduction Successful delivery of drugs to the human vagina is mainly dependent on the residence time as well as the ability of the delivery system to contain and release the drug in a controlled manner ( Justin- Temu et a., 2004; Grassi et al., 2005; Andrews et al., 2009; Rohan and Sassi, 2009; Serra et al., 2009). Therefore, the selection of polymers to achieve good matrix integrit y that will subsequently lead to the control of drug release is a vital goal in formulation optimization of an intravaginal drug delivery system. C onducting a comparative analysis of polymers for developing a specific drug delivery system is of paramount importance if the appropriate matrix integrity and controlled release effect is to be achieved. Comparative studies of polymers would also facilitate the process of formulation optimization. In addition, matrix integrity evaluation may enable identification of the physicochemical and physicomechanical properties of a polymeric delivery system which may be used to predict the in vivo behavior and therefore its suitability for intravaginal drug delivery. R eproducibility of experimental procedures during formulation development is frequently hindered by numerous factors. In solving such problems , several techniques have been employed in attempting to discover the rules, patterns and relationships in a given data set. Software - based computational techniques namely Artificial Neural N etworks (ANNs) are able to simulate the neurological processing ability of the human brain through mathematical modeling of its functional unit (Hopfied and Tank 1985; Achanta et al., 100 1995; Arulsudar et al., 2005; Paroj?i? et al., 2007; Patel et al., 2007; Hassanien et al., 2008; Lee et al., 2008; Munasur et al., 2008; Lancashire et al., 2009). Essentially ANNs are networks of adaptable nodes, which store experimental knowledge through the learning process from the given samples (Yang et al., 2005; Lancashire et al., 2009). They can also be applied in establishing a non- linear relationship between causal factors and the required efficacy (Chen et al., 2002; Sun et al., 2003; Rizkalla and Hildgen 2005; Agatonovic- Kustrin et al., 2008; Najah et al., 2009) . Thus , an ANN may be considered as an advanced non- linear regression model that can be used to assess the association of variables by means of iterative training of data obtained from a designed experiment (Ta kayama et al., 1999). Thus far, ANNs have successful ly been used to solve various problems in pharmaceutical research, including pharmaceutical process optimization (Takahara et al., 1997; Peh et al., 2000), product development ( Leane et al., 2003; Paroj?i? et al., 2007), predicting drug release patterns (Brier et al., 1995; Peh et a., 2000; Sun et al., 2003; Reis et al., 2004; Mendyk and Jachowicz, 2005; Fogel, 2008), predicting diffusion dynamics (Jha et al., 1995; Yuzgec et al., 2008; Lee et al., 2008 ), predicting the mechanism of action of drugs (Nair et al., 1994; Leane et al., 2003; Gunther et al. 2003; Ghaffari et al., 2006) , predicting pharmacokinetic parameters, and pharmaceutical product coefficients ( Weinstein et al., 1992; Huss ain et al., 1993; Brier et al., 1995; Smith and Brier, 1996; Peh et al., 2000; Fu et al., 2003; Leane et al., 2003). Furthermore, ANNs are more accurate than conventional linear correlation techniques in identifying specific input variables such as the type of polymers that may substantially affect the performance of a formulation (Bourquin et al., 1998; Chen et al, 2002; Caputo and Pelagagge 2008 ) . ANNs are also capable of generating non- linear input- output mappings for use in producing accurate confirmation or predictions of previous results within shortest periods and with highest computational precision ( Agatonovic- Kustrin and Beresford 2000; Lee et al., 2008) . 101 The aim of this Chapter was therefore to assess the matrix integrity of the initial selected 15 lead formulations derived from the OVAT - based developmental screening approach (Chapter 3 ) , in terms of matrix erosion. A NN was used to optimize the erosion kinetics of the 15 lead formulations to generate an optimized formulation with the best erosion properties. Furthermore, to achieve ideal matrix erosion, ANN was employed to undertake sensitivity testing on the polymers that constituted the 15 lead formulations ( Chapter 3; Section 3.3. 2. 6; Table 3.19) so as to optimize a selection of polymers that would ultimately lead to the achievement of this objective. 4.2. M aterials and M ethods 4.2.1 Materials Materials employed are the same as in Chapter 3; Section 3.2. 1. 4.2.2 Methods 4.2.2.1 Preparation of the intravaginal bioadhesive polymeric device The polym ers employed in this study were modified polyamide 6,10 ( mPA 6,10 ), poly(lactic - co- glycolic acid) (PLGA), polyacrylic acid (PAA 974), polyvinly alcohol (PVA), ethylcellulose (EC), polyethlene oxide (PEO), carageenan (CG) gelatin (GL), xanthan gum (XG), beesw ax (BW X), Eudragit S - 100 (E S100) and Eudragit RS - 100 (E RS 100) . Different combinations and quantities of these polymers ( Table 4.1) , derived from the preliminary screening experiments that were conducted in Chapter 3 , were blended by using a cube blender (Erweka ? GmbH, Heusenstamm, Germany). The powder blends were then compressed at a pressure of 5 tons into caplet - shaped intravaginal bioadhesive polymeric devices (IBPDs) on a Manesty D3B 16 station tableting p ress equipped with D3B oblong tooling of 22 ?9 mm in dimension (Manesty D3B L249Q, Liverpool, England) . 102 Table 4.1: The Polymer combinations employed in the production of the intravaginal bioadhesive polymeric devices Polymeric Composition (mg) F# P6 PG PA EC PV XG GL BW ES ER WT 62 150 400 25 200 25 - - - - - 800 27 200 250 25 300 25 - - - - - 800 51 175 300 25 250 25 - - - 25 - 800 52 175 300 25 250 25 - - - - 25 800 41 175 300 25 250 25 - 25 - - - 800 39 185 275 25 250 25 40 - - - - 800 28 175 250 25 300 50 - - - - - 800 40 180 275 25 250 25 45 - - - - 800 44 175 275 25 250 25 50 - - - - 800 36 200 275 25 250 25 25 - - - - 800 29 150 250 25 300 100 - - - - - 800 50 175 300 25 250 25 - - 25 - - 800 37 195 275 25 250 25 30 - - - - 800 38 190 275 25 250 25 35 - - - - 800 31 100 250 25 300 125 - - - - - 800 P6: Modified polyamide 6,10; PL : Poly(lactic -co-glycolic acid); PE: Polyethylene oxide PA : Polyacrylic acid; C G : Carrageenan; EC: Ethycellulose; PV: Polyvinylalcohol; X G : Xanthan gum; G L : gelatin; B W: beeswax; ES: Eudragit S100 ; ER: Eudragit RS 100; WT : Weight 4.2.3. In -process validation tests In - process validation tests involving indentation hardness, mass uniformity and friability analysis were performed to ensure the reproducibility of the matrices. Indentation hardness was represented by the Brinell Hardness Number (BHN) derived through the textural profiling analysis. A sample of 10 caplets from each formulation batch was tested for mass uniformity and friability using the same instruments, parameters and limits as described in Chapter 3, Section 3.2. 2. 2. 4.2.3.1 Determination of the Brinell hardness number The BHN (N/mm 2 ) was computed for each set of devices using the ball probe approach. A calibrated Texture Analyzer (TA. XTplus , Stable Micro Systems, Surrey, UK) fixed with a ball probe indenter of diameter 3.125mm, indentation depth set at 1.5625mm and 5kg load cell for all readings was employed. The textural settings used to calculate the BHN values are depicted in Table 4.2. The typical force- distance profiles generated for the 103 calculation of the BHN value are illustrated in Figure 4.1. Each analysis was conducted in triplicate. The indentation hardness was represented by a conversion to the Brinell Hardness Number (BHN) (Equation 4. 1). Equation 4.1 where F= force generated from indentation (N), D= diameter of ball probe indenter (3.125mm) and d= indentation depth (1.5625mm). Table 4.2: Textural settings employed for BHN v alue calculations Parameters Settings Pre- test speed 1.000 mm/sec Test speed 0.500 mm/sec Post- test speed 1.000 mm/sec Compressive distance 1.563 mm Trigger force 0.050 N ? ? ? ? ? ? ? ? 2 d- 2 D-DD? 2F =BHN 104 Figure 4.1: Typical force - distance profiles employed in the determination of matrix indentation hardness of the IBPD. 4.2. 4 In vitro matrix erosion of the intravaginal bioadhesive polymeric device Each formulation was subjected to in vitro matrix erosion studies. Formulations were immersed in 100mL of simulated vaginal fluid (pH 4.5; 37?C) using a sealable glass vessel (150mL) and then placed in an orbital shaking incubator (LM - 530- 2, MRC Laboratory Instruments Ltd., Hahistadrut, Holon, Israel) maintained at 20rpm. After 24 hours, each formulation was removed from the medium, blotted on filter paper (diameter 110mm and pore size 20?m) and dried to constant weight at 40?0. 5?C in an oven. All determinations were conducted in triplicate. The mathematical expression stated in Equation 4.2 was employed to determine the percentage matrix erosion (ME) (i.e. % w/ w) of each formulation. 1 0 0xOM RMOM(%)ME ?= Equation 4.2 where ME is the matrix erosion, OM original mass, and RM the residual mass. 105 4.2.5 Optimization by ANN for the best polymer combination selection Optimization was conducted by employing the feedback Multilayer Perceptron (M L P) neural network to train the empirical input matrix erosion data with static back propagation. Fig ure 4.2 illustrates the typical construction of the MLP network while Table 4.3 shows the input matrix erosion data (obtained from the matrix erosion studies) that was trained. The main advantage of these networks is that they can approximate any input/output map. Figure 4. 2: Constructed multilayer perceptron network. A genetic algorithm with a Sigmoid Axon transfer function and Conjugated Gradient learning rule was employed for the hidden input and output layers. Figure 4.4 shows the network topology for the hidden input and output layers. Figure 4.3: Network topology depicting the hidden input and output layers. 106 Table 4.3: The input matrix erosion data that was trained by the Multilayer Perc eption Network Polymeric Composition F# P6 PG PA EC PV XG GL BW ES ER ME% 62 150 400 25 200 25 - - - - - 1.21 27 200 250 25 300 25 - - - - - 1.43 51 175 300 25 250 25 - - - 25 - 2.89 52 175 300 25 250 25 - - - - 25 3.2 6 41 175 300 25 250 25 - 25 - - - 3.58 39 185 275 25 250 25 40 - - - - 3.81 28 175 250 25 300 50 - - - - - 3.83 40 180 275 25 250 25 45 - - - - 4.37 44 175 275 25 250 25 50 - - - - 4.45 36 200 275 25 250 25 25 - - - - 5.57 29 150 250 25 3 00 100 - - - - - 6.31 50 175 300 25 250 25 - - 25 - - 6.48 37 195 275 25 250 25 30 - - - - 7.20 38 190 275 25 250 25 35 - - - - 7.53 31 100 250 25 300 125 - - - - - 7.68 P6: Modified polyamide 6,10; PL : Poly(lactic -co-glycolic acid); PE: Polyethylene oxide PA : Polyacrylic acid; C G : Carrageenan; EC: Ethycellulose; PV: Polyvinylalcohol; X G : Xanthan gum; G L : gelatin; B W: beeswax; ES: Eudragit S100; ER: Eudragit RS 100; ME% : Matrix erosion percentage A maximum of 10,000 epochs were run on NeuroSolutions Version 5.0 (NeuroDimension Inc., Gainsville, Florida) for ensuring optimal training of data. Sensitivity analysis was used for extracting the cause and effect relationship between the inputs and the outputs of the network. This could provide feedback pert aining to the input variable that was the most significant by testing the network with regard to its sensitivity about the matrix erosion for each formulation, thus elucidating the polymer combination type that was most significant. The parameters used to construct the neural network are as shown in Table 4.4 107 Table 4.4: Artificial neural network construction parameters employing a neural builder Parameter Setting Hidden layer 2 Exemplars 17 Output Processing element 1 Transfer function SigmoidAxon :sigmoid (0/1) Learning rule ConjugateGradient: second order method for gradient Maximum Epochs 10,000: Supervised Learning Control Termination at Mean Square Error; Load Best Weights Approach Probe Configuration Quantitative - MatrixViewer, MatrixEdito r; Qualitative - MegaScope, Hinton 4.3. Results and Discussion 4.3. 1 In -process validation tests The devices were uniform in mass (800? 0. 48mg). Textural analysis confirmed BHN values ranging from 2.687- 4. 981N/mm 2 while friability was at an average of 0. 21% (Table 4.5), demonstrating desirable matrix compatibility and compressibility. F15 presented with the highest BHN value (4.981N/mm 2 ) while F1 presented with the lowest BHN value (2.687N/mm 2 ). The high compressibility of PLGA and EC coupled with the abi lity of GL, BWX and XG to form a gel - like macrostructure may have contributed to the good friability and BHN values obtained. In addition, GL, BWX and XG resulted in non- collapsible formulations with a swelling tendency which improved the veracity of the matrix networked- structure, which contributed to the highest matrix integrity and thus the high BHN values obtained. 108 Table 4.5: Brinell hardness number, mass, and friability of the intravaginal bioadhesive polymeric device F# BHN (N/mm 2) ?SD Mass (mg) ?S D % Friability ?SD 1 2.687 0.109 801. 23 4.459 0.369 0.015 2 2.745 0.110 800. 22 4.013 0.350 0.024 3 2.778 0.100 799. 45 4.127 0.312 0.013 4 2.884 0.115 802. 02 4.018 0.298 0.011 5 2.922 0.121 798. 79 3.933 0.274 0.013 6 2.989 0.120 800. 98 4.003 0.231 0.01 2 7 3.063 0.132 802. 00 4.012 0.227 0.012 8 3.340 0.138 799. 32 3.987 0.217 0.010 9 3.521 0.139 801. 79 3.967 0.201 0.009 10 3.894 0.141 800. 38 4.134 0.131 0.014 11 4.367 0.140 797. 97 3.556 0.108 0.007 12 3.708 0.129 800. 33 4.009 0.138 0.017 13 4.703 0 .148 800. 58 4.013 0.103 0.006 14 3.988 0.140 797. 98 4.018 0.123 0.011 15 3.897 0.138 800. 23 4.127 0.326 0.017 4.3.2 Matrix erosion evaluation Matrix erosion, polymer swelling and drug dissolution are the important factors that determine drug release from polymer matrices ( Colombo et al., 1996; Gao and Meury, 1996) . Disolution systems can provide a means of overcoming the well- known advantage of a purely diffusion control system ( Kim and Fassihi, 2000; D?rig and Fassihi, 2002; Al - Taani and Tashtoush, 2 003; Lopes et al., 2007; Sriamornsak et al., 2007; Efentakis and Peponaki, 2008; Malik et al., 2008 ) . Furthermore, synchronization between erosion and diffusion fronts may produce zero- order drug release kinetics (Lee, 1980; Pillay and Fassihi, 2000; Pillay and Danckwerts, 2002; Pillay et al., 2005; Al- Taani and Tashtoush, 2003) . Normally in polymer matrices , polymer relaxation and erosion mechanisms may coexist (Christ et al., 1998; Bettini et al., 2001 ) . The results of the matrix integrity as determined in terms of the matrix erosion percentage for the fifteen lead formulations are as shown in Table 4.3. Depending on the polymer combination and proportions of hydrophilic/hydrophobic polymer, all devices demonstrated different degrees of erosion as depicted in Table 4.3. F1 presented with the least matrix erosion (1.21%) while Formulation F3 1 presented with the highest matrix erosion (7.68%) (Table 4.3). Generally, the rate of matrix erosion from the IBPDs appeared to depend mainly on the networking 109 level of their hydrophilic and hydrophobic polymer- based microstructure. Matrix erosion in a polymer matrix is usually determined by the rate at which the polymer undergoes hydration and swelling ( Roy et al., 2002; Khamanga and Walker, 2006; Sriamornsak et al., 2007; Choonara et al., 2008) . Therefore, matrix erosion from the IBPDs depended on the relative magnitude of polymer hydration at the moving rubbery/glassy front within the IBPDs . F1 contained the highest content of PLGA (400mg) and a relativey high quantity of EC (200mg) ( Table 4.3). These polymers are highly hydrophobic with high compressibility properties. The presence of relatively low quantities of the hydrophilic polymers in this formulation ( mPA 6,10: 150mg; PAA: 25mg; PVA 25mg) coupled with the hydrophobic nature of PLGA and EC are certainly the reasons for the minim al matrix erosion observed. The same reason holds for the rest of the formulations which apparent ly were the best formulations from the OVAT - based screening that was undertaken in Chapter 3. Thus, the high degree of hydrophobicity prevented rapid penetration of water molecules into the polymer matrices, leading to less hydration, swelling and erosion and therefore the lowest degree of matrix erosion. PAA has played the leading role in fostering the bioadhesivity of the IBPD (see Chapter 5 later), but it had to be applied in minimal quantities due its high swelling properties. PAA in water swells up to 1000 times its original volume (Bonacucina et al., 2004) , which permits the ionization of its carboxylic groups, that facilitate the formation of adhesive bonds with either the protein or glycoprotein of the vaginal epithelium (Figure 4.4). A chemometric structural model developed in our laboratories showed that the protein and polymer bonding at the cell surface of the vaginal epithelium, via the embedded glyco- protein and other surface proteins plays a key role in linking the incoming polymer or other smaller units. The saccharide part in the glycol - protein binds to the inherent surface protein and available functional groups in the protein and to some extent the g lyco- proteins saccharide units are extended in binding to the polymer (i.e. PAA) (Figure 4. 4 ) . 110 Figure 4.4 : A chemometric structural model developed in our laboratories for a glyco- protein structure showing attachment of cellular surface protein (red) bound to the saccharide part of the glyco- protein (blue) and both (surface) protein and glyco- protein (green) bound to the polymer (here PAA) (white) for non- evenly distributed applied force during adhesion, with non- smooth and unevenly contacted two interacting surfaces with non- evenly distributed and non- constant, non- steadily (applied) force for the adhesion shown in a 3D optimized state with ball model. The coiling loop distortions for both protein and glycoprotein are also shown. 4. 3. 3 Selection of polymer combinations as per ANN optimization For the 12 polymers that were employed in the study, the M L P network was able to accurately confirm that mPA 6,10 , PLGA, EC, PVA and PAA were the most significant input variables in terms of matrix integrity of the IBPD based on the empirical data. The approach followed in this work required prior assumption for the selection of a mathematical model before applying the ANN models so as to be able to confirm the sensitivity coefficients of the various polymer types as input variables that significantly contributed to characterizing the matrix integrity. In order to obtain accuracy and maximum degree of precision, the training was done twice ( i.e. primary and secondary training). For the primary training, the three run outcomes are as shown in Figure 4.5a. Run # 2 gave the lowest level of the mean square error ( MSE ) . The gradual leveling of the MSE with standard deviation (SD) boundaries for the training runs indicated a sequen tial PAA Glyco- protein Surface protein Surface protein and glycol- protein 111 improvement of data modeling as illustrated in Figure 4.5b. Table 4. 6 depicts the average of the MSE values for the three runs of the primary training, the best network run out 10,000 epochs, and the overall efficiency and performance of the neural network during the data training. Figure 4. 5 : a) MSE for the primary ANN tr aining, b) Average MSE for the primary ANN trains with standard deviations for 10,000 epochs. Epoch 0 2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0 1 0 0 0 0 MS E 0 2 4 6 8 1 0 Run # 1 Run # 2 Run # 3 (a) Epoch 0 2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0 1 0 0 0 0 Av erage M SE -2 0 2 4 6 8 1 0 Training + 1 Standard Deviation -1 Standard Deviation (b) 112 Table 4.6: Neural network indicators characterizing the efficiency and performance of data in the primary training as per ANN Best Network Training Performance ME ( % ) Epoch # 10,000 MSE 0.768 Minimum MSE 0.001 NMSE 0.010 Final MSE 0.002 MAE 0.519 - - Min Abs Error 0.002 - - Max Abs Error 2.512 R 2 - - 0.99 MSE: Mean square error NMSE: Normal ized mean square error MAE: Mean absolute error Min Abs Error: Mi nimum absolute error Max Abs Error: Maximum absolute error Basing on the obtained results, it is evident that the employed training model was highly efficient (MSE= 0.001) . Results revealed a highly satisfactory fit for the input variables ( R 2 = 0.99) . T he performance criterion employed to assess the closeness and correlation between the desired and the actual network output for matrix erosion of each formulation is listed in Table 4.7 and illustrated in Figure 4.6. The sensitivity coefficient of each polymer type (input variables) is depicted in Figure 4.7 . Table 4.7: Actual and desired network performance criterion data from the primary training Desired output : ME ( %) Actual network output ( %) 1.21 1.19 905 708 1.43 1.73 679 249 2.89 3.27 070 479 3.26 3. 27 070 479 3.58 3.27 070 479 3.81 4.75 966 199 3.83 3.48 203 039 4.37 4.02 684 657 4.45 4.48 380 486 5.57 5.98 735 234 6.31 6.08 292 899 6.48 6.48 950 2 81 7.2 7.01 026 921 7.53 6.66 716 927 7.68 7.91 356 107 113 Figure 4.6: A profile depicting the a) desired and b) actual network output. Figure 4. 7 : A typical bar chart graph depicting the sensitivity coefficients of each polymer type against the matrix erosion following the primary training. From the primary ANN training it was apparent that 6 among 10 polymer types had the highest sensitivity against matrix erosion. These were mPA 6,10 , PLGA, EC, PEO PVA and PAA (Figure 4.7 ) . Thus, a second (secondary) ANN training was run to explore the potential of these polymers to constitute an optimal formulation for developing the IBPD. The MSE for the secondary training is shown in Figure 4.8a. As can be noted, the secondary training resulted in highly reduced MSE values. For the primary training the Polymer Type 114 MSE rang ed between 0.02- 9. 02 (Figure 4.5a) while for the secondary training the MSE ranged between 0.01 - 0.92 (i.e. les s than 1) (Figure 4.8a). The gradual level ing of the MSE with standard deviation (SD) boundaries for the secondary training exhibited a sequential improvement of data modeling in that the average MSE decreased from values that were almost close to 10 (for pr imary training) (Figure 4.5) to values les s than 1 as shown in Figure 4.8b. Overall these results depict a high degree of sensitivity for this set of polymers when compared to the rest. 115 Figure 4.8 : a) MSE for the secondary ANN training, b) Average MSE for the secondary training with standard deviations for 10,000 epochs . As was the case with the primary training, the MSE for the seco ndary training was 0.001 with an excellent fit for the input variables as well (i.e. R 2 = 0.99) . The sensitivity Epoch 0 2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0 1 0 0 0 0 MS E 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 Run # 1 Run # 2 Run # 3 Epoch 0 2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0 1 0 0 0 0 Av erage M SE 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 Training + 1 Standard Deviation - 1 Standard Deviation (b) (a) 116 coefficient of each polymer type against matrix erosion is as shown in Figure 4.9. EC presented with the highest sensitivity against matrix erosion followed by PLGA (Figure 4.9) . This depicts their high capacity in controlling matrix erosion. This behavior may be attributed to their high degree of hydrophobicity. The sequence of the other remainng polymers in terms of sensivity against matrix erosion was PVA, PAA and mPA 6,10. The results observed with PVA is a reflection of the results obtained earlier in matrix swelling tests where it had presented with minimal swelling (Chapter 3; Section 3.3. 2. 2 ). The low sensitivity against matrix erosion shown by PAA and PA 6,10 (Fig ure 4. 9 ) , may be associated with their hydrophilic nature. Figure 4.9: A typical bar chart graph showing the sensitivity coefficients of each polymer type against the matrix erosion following the secondary training. The desirable matrix integrity exhibited by formulations with markedly reduced quantities of PAA is reflected in the formulation containing mPA 6,10 (150mg), PLGA (400mg), EC (200mg), PVA (25mg) and PAA (25mg). This formulation was able to provide a matrix erosion value which was as low as 1.21% (Section 4.2. 5; Table 4.3) . These results are supported by the relationship between the matrix erosion and the varied input polymer quantity (Figure 4.10). PAA, which was mainly employed due to its bioadhesivi ty potential, showed a markedly high influence on matrix erosion in that even at quantities as low as 25mg , it could result in a static impact on matrix erosion (Figure 10). This is certainly due Polymer Type 117 to its high degree of hydrophilicity. PVA which was mainly used for the purpose of enhancing bioadhesivity and controlled release to the IBPD, portrayed a substantially high positive sensitivity for matrix erosion (Figure 10) and therefore the reason why it was also used in minimal quantities (25mg) . mPA 6,10 which was employed for the purpose of achieving desirable controlled drug release, presented with a relatively low sensivity to matrix erosion compared to the other hydrophilic polymers i.e PAA and PVA. PLGA and EC which are highly hydrophobic polymers, presented with a markedly high degree of negative sensitivity for matrix erosion (Figure 4.10) . The substantially high matrix integrity of this formulation, may therefore be attributed to the high degree of hydrophobicity in PLGA and EC, good matrix resilience of mPA 6,10 , and the hydrophilic nature of PAA and PVA which allowed formation of the networked microstructure in the IBPD polymer matrix. The bioadhesivity capacity of the optimized formulation is assessed later in Chapter 5. Figure 4.10: Correlation between matrix erosion and polymer quantity. Varied Input Polymer Quantitity (mg) 0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 3 5 0 Matr ix E ro sio n (%) 0 2 4 6 8 1 0 PVA EC PAA PLGA PA 6,10 118 4.4. Concluding Remarks A comprehensive analysis of several polymers for the development of the IBPD was conducted in terms of matrix erosion. The results of this study have highlighted polymeric effects on the matrix erosion of the IBPD. The high sensitivity against matrix erosion shown by the best 5 polymers (Figure 4.7 and 4.9) in both primary and seconadary ANN training was an indication of their suitability in formulating a stable IBPD. The ANN approach confirmed that mPA 6,10 (150mg), PLGA (400m g), EC (200mg), PVA (25mg) and PAA (25mg) could lead to the development of an optimized intravaginal bioadhesive polymeric drug delivery device. 119 CHAPTER FIVE INN VITT RR OO AND EXX VIVOO BIOADHESIVITY ANALYSIS ON THE OPTIMIZZ ED INTRAVAGINAL BIOADHESIVE POLYMERIC DEVICE COMPOSED OFF DIFF FF ERENT CROSSLINKED POLYACRYLIC ACIDS 5.1 I ntroduction The development of bioadhesive intravaginal drug delivery systems allows for therapeutic levels of bioactive to be maintained locally, extends the bioactive residence time within the vagina and reduces the dosing frequency and quantity of bioactive administered ( Mathowitz and Jacob, 1999; Ugwoke et al., 1999; Ceschel et al., 2001) . Thus, the therapeutic efficacy of locally- acting bioactives may be improved due to the increased availability at the vaginal epithelium afforded through bioadhesion (Elson et al., 2000; Genc et al., 2000; Mandal, 2000) . Currently, the majority of bioadhesive polymers include polyacrylic acid and cellulose derivatives (Repka and McGinity 2001) , poly(v inylpyrrolidone) (PVP), poly(vinylalcohol) (PVA), chitosan and various gum - based polymers such as xanthan gum (XG) (Tambwekar et al., 2002) . Multifunctional polymers exhibiting bioadhesive and/or gelling properties such as the thiolated polyacrylates and poloxamers represent a useful framework for the design of intravaginal drug delivery systems (Bogentoft et al., 1996; Remu??n - L?pez et al., 1998; Lee et al., 2000; Lehr, 2000;Valenta et al., 2001; Kast et al., 2002; Robinson and Bologna, 2002; Valenta et al ., 2002; Tang et al., 2005 ; Bonferoni et al., 2006; Al- Tahami and Singh, 2007; Trapani et al., 2009 ) . Bioadhesivity is essentially defined as the interfacial force between the polymeric drug delivery system and the mucus layer coating an epithelium (Peppas and Buri, 1985). Bioadhesion may occur in three distinct phases namely wetting, interpenetration and 120 subsequent mechanical interlocking between mucin and the polymer (Longer and Robinson, 1986; Jast et al., 2003). Bioadhesion is thus thought to be a result of the presence of hydrogen bonding groups, strong anionic/cationic charges, high molecular mass, chain flexibility and surface energy interactions which favor spreading onto the vaginal tissue. Basic theories such as electronic, adsorption, wetting and diffusion phenomena have been described and associated with the mechanisms by which bioadhesion occurs (Helfand and Tagami, 1972; Kaelble, 1977; Deryaguin et al., 1997). Each of the steps involved in bioadhesion may be facilitated by the polymeric composition of the drug delivery system and the mode of application (Hussain, 2000) . Several techniques for the in vitro assessment of bioadhesion have been explored and include adhesion mass methods (Smart and Kellaway, 1982), fluorescent probe methods (Park and Robinson, 1984), shear stress testing (Smart et al., 1984), flow channel techniques (Mikos and Peppas, 1986), tensile testing (Park and Robinson, 1987) , colloidal gold staining methods (Park, 1989) , averted sac technique and the CAHN microbalance (Santos, 1999) . However, these techniques lack sufficient sensitivity. Therefore the aim of this Chapter was to investigate the use of a comprehensive multi- dimensional approach including chemometrical molecular modeling and pertinent mathematical descriptors to assess the polymeric bioadhesivity to vaginal tissue. The Area Under the Force- Distance Curve (AUC FD) under a global locale was computed for all data points iterated during bioadhesion testing of optimized polyacrylic acid (PAA) - based intravaginal matrices generated through an Extreme Vertices Mixture Experimental Design template. Bioadhesivity testing was conducted on both simulated vaginal membrane and freshly excised pig vaginal tissue. The ideal polymers obtained through the optmization by ANN approac h i.e. modified polyamide 6,10, ( mPA 6,10 ), poly ( lactic co- glycolic acid (PLGA), e thylcelloluse (EC), p olyvinyl alcohol (PVA) and polyacrylic acid (PAA) were employed for the study. Apart from the ensured matrix integrity, these polymers also have the potential for controlling drug release, ensuring desirable 121 erodability, homeostatic control of the vaginal environment and bioadhesive potential. Studies have shown that PAA is a highly bioadhesive polymer ( Semalty et al., 2006; Semalty and Semalty, 2008) . Therefore , for the purpose of achieving optimal bioadhesivity, this study explored two combinatory matrices comprising PAA , namely allyl sucrose- crosslinked PAA (AS- PAA) and allyl penta erythritol - crosslinked PAA (APE- PAA) . 5. 2 Materials and M ethods 5.2.1 Materials The simulated vaginal membranes employed were dialysis flat sheet membranes of M w=12,000- 14,000g/mol purchased from Spectrum Laboratories Inc. (Rancho Dominguez, CA, USA ). Allyl sucrose- crosslinked PAA (AS- PAA) and allyl penta erythritol - crosslinked PAA (APE- PAA) were purchased from Noveon Inc Cleveland, OH, USA. Pig vaginal tissue was excised from euthanized p igs obtained from the University of the Witwatersrand Central Animal Services Unit. The modified PA 6,10 was synthesized as per the method described in Chapter 3, Section 3.2. 1. 1 . T he rest of the materials used were as described in Chapter 3. 5.2.2 Methods 5.2.2 .1 Preparation of the intravaginal bioadhesive polymeric device caplets composed of all yl sucrose and all yl penta eryt hritol -crosslinked PAA An Extreme Vertices Mixture Desi gn template was generated employing Minitab ? V15 (Minitab ? Inc., PA, USA) statistical software to produce various caplet formulations comprising 11 polymer combinations (Table 5.1 ) . Each formulation had an equivalent mass of 8 00mg. Formulation response optimization was performed using an inherent D - optimal technique by combining mixture components and processing factors to converge to pre- optimal settings prior to achieving a global optimized solution with the desirable polymeric proportions for the AS- PAA and APE- PAA caplets that were subsequently prepared and further tested. 122 Table 5.1: Extreme Vertices Mixture Formulation Template for caplet preparation F # m PA 6,10 (mg) PLGA(mg) EC (mg) PVA (mg) PAAa ( m g) PAAb (mg) 1 150 50 100 100 200 200 2 100 50 100 150 250 200 3 100 50 100 100 205 250 4 130 55 105 105 205 205 5 105 80 105 105 200 205 6 100 100 100 100 205 200 7 105 55 130 105 230 205 8 105 55 105 105 210 230 9 110 60 110 110 200 210 10 100 50 150 100 205 200 11 105 55 105 130 200 205 aAS-PAA: allyl sucrose-crosslinked PAA bAPE-PAA: allyl penta erythritol -crosslinked PAA PLGA, mPA 6,10, EC, PVA and the two crosslinked PAA variants were weighed in triplicate and subsequently blended using a cube blender (Erweka Apparatebau, Heusenstamm, Germany). Two sets of blends were prepared in each case, one containing AS- PAA and the other APE- PAA. The polymer blends were then compressed into IBPD caplets using a Manesty D3B 16 station tableting press (Manesty D3B L249LQ, Liverpool, England) equipped with D3B oblong tooling of 22 ?9 mm in dimension at a compression pressure of 5 tons. 5.2.2.2 In -process validation tests Diametric hardness, mass uniformity and friability analysis were conducted on the IBPD caplets to ensure adequate manufacturing reproducibility. A sample of 10 caplets from each formulation batch was tested for diametric hardness, mass uniformity and friability using the same instruments, parameters and limits as described in Chapter 3, Section 3.2. 2 . 123 5.2.2.3 In vitro and ex vivo bioadhesivity anal ysis Bioadhesivity testing was performed on all experimental formulation batches and both sets of optimized caplets (i.e. the AS - PAA and APE- PAA caplet formulations) using a highly sensitive Texture Analyzer (TA.XT plus, Stable Microsystems, UK) . This approach was employed to principally measure the bioadhesiveness using a cylindrical probe that was robotically guided to make contact and induce a test force onto the non- deformed substrate surface comprising either a simulated vaginal membrane or freshly excised pig vaginal tissue and thereafter disengage in order to simultaneously measure the force required to detach the probe containing the vaginal tissue from the hydrated caplet. The maximum force required to detach the simulated vaginal tissue attached to the cylindrical probe from the secured caplet on the platen was obtained from the Peak Adhesive Force (PAF) and also computed from the Area Under Force- Distance Curves (AUC FD.) representing the Work of Adhesion. In each case, the simulated vagi nal membrane and the caplets were hydrated for 30 minutes in simulated vaginal fluid (pH 4.5; 37?C) ( Chapter 3, Section 3.2. 2. 3, Table 3.3) prior to testing (N=3). For further method and formulation validation bioadhesivity testing was also conducted using freshly excised vaginal tissue obtained from a pig model on the APE- PAA and AS- PAA caplets. The textural analysis par ameter settings employed were pre- test and test speeds = 2.0mms - 1 , a post test speed = 10mms - 1 , a trigger force = 0.5N and a contact time of 5s. 5.2. 2. 4 Removal of vaginal tissue from the pig model for bioadhesivity testing A Large White female pig (84kg) was euthanized with 40mL of sodium pentobarbitone (200mg/mL) administered intravenously. The pelvic canal of the pig was opened by dissecting through the symphysis pubis and then exposing the intra- abdominal vaginal tract. The external vaginal tract was carefully dissected from the surrounding tissues before removing the vaginal tissue (Figure 5.1) . An incision was made through the vaginal canal to expose the inner lining of the tissue, which was then placed in an airtight specimen jar and immediately subjected to bioadhesivity testing. 124 Figure 5.1: The dissection process to remove the vaginal tissue from the pig . The freshly excised vaginal tissue was secured onto a cylindrical textural probe of a Texture Analyzer (TA.XT plus, Stable Microsystems, UK). An optimized APE - PAA caplet was fixed onto the textural platen after exposure to simulated vaginal fluid (pH 4.5; 37?C) for 30 m inutes. Bioadhesive testing was then conducted immediately by measuring the maximum force ( N) required to detach the vaginal tissue from the hydrated APE- PAA caplet matrix. Data was represented as the Peak Adhesion Force (PAF) and the work of adhesion (AUC FD.) (Figure 5.2). The work of adhesion per unit area ( w A ???), was characterized by the work executed on the matrices when the two contact phases i.e. the vaginal tissue ( ?) and the caplet matrix ( ?) , formed an interface of unit area which were subsequently separated reversibly to form unit areas of each of the ?? - and ??- interfaces. This relationship is mathematically described by Equation 1. w A??? = ? ? ? + ? ? ? ? ? ?? Equation 5. 1 Where, ? ?? , ? ?? and ? ?? are the surface tensions between the two bulk phases comprising the vaginal tissue and the APE- PAA IBPD caplets , ?, ? ; ?, ? and ? , ? respectively. 125 Figure 5.2: T extural analysis method employed to generate Force - Distance profiles for assessing the caplet bioadhesivity to freshly excised pig vaginal tissue indicating a) blue paint design, b) actual process. 5.2. 2. 5 Rheological anal ysis of hydrated AS-PAA and APE -PAA polymers The ability of PAA to par tially hydrate within the IBPD matrix ultimately results in wetting and volumetric distension of the system which facilitates the intimate contact and interaction with the simulated vaginal membrane and freshly excised pig vaginal tissue. Rotational rheological analysis was therefore performed to generate flow curves for F reshl y excised pig vaginal tissue Test speed=2mm/sec Hydrated caplet matrix secured on platen a) Textural anal ysis probe 15mm in diameter IBPD secured on the platen Textural analysis probe ( 15mm diameter) Freshly excised vaginal tissue from the pig model b ) 126 analyzing relationships between the viscosity and shear stress as a function of shear rate using a Modular Advanced Rheometer system (ThermoHaake MARS Rheometer, Thermo Fischer Scientific, Karlsuhe, Germany) equipped with a T - type spindle sensor (C35/1 oTi) set at a sample gap of 1mm and a Thermocontr oller (UTC- MARS II). The AS - PAA and APE- PAA polymers were dissolved in simulated vaginal fluid at a concentration of 2% w/ v, vortexed for 2 minutes, and then assessed for their rheological behavior at 25?1. 0 oC with a test time of 200s and a controlled rate starting at 5.00s - 1 decreasing to 0.001s - 1 and a shear rate ranging from 0 to 500s - 1 . 5.2. 2. 6 Mechanistic postulation of caplet bioadhesion employing chemometric and molecular modeling and associated interactive energy paradigms Cellular adhesion will play a critical role toward the mechanisms of achieving superior bioadhesion. While a number of computational models and experimental studies have addressed the issue of cell adhesion to surfaces, no model or theory has adequately addressed cell adhesion at the molecular level employing freshly excised pig vaginal tissue as the bioadhesive substrate ( King, 2001; Yang and Zaman, 2007). Computational and molecular structural modeling was performed in our laboratories to deduce the transient mechanisms of bioadhesivity, chemical interactions and inter- polymeric interfacing during caplet adhesion to the freshly excised pig vaginal tissue as the adhesive substrate. This approach led to predictive findings solely based on the chemical interactions underlying the bioadhesion of the caplet to the vaginal tissue substrate. Semi - empirical quantum mechanics was used to generate molecular interactions and computational energy paradigms of the caplet components based on inherent interfacial phenomena underlying the mechanisms of bioadhesion provided by the inter- polymeric blended caplet. Models and graphics suppor ted on the step- wise molecular caplet- tissue interaction, polymeric interconversion and bioadhesion as envisioned by the molecular behavior and stability of the hydrated caplet network were generated on ACD/I - Lab, V5.11 (Add- on) software (Advanced Chemist ry Development Inc., Toronto, Canada, 127 2000) . The energy distribution curves assumed a componential progression of bioadhesion stability, a spatially uniform distribution of polymers within the caplet, and an irreversible gelation process ( Schroeder et al., 1998; Yu et al., 1998; Picout et al., 2000a and b; Kalugin et al., 2001; Gilsenan et al., 2003a , b, c). A comprehensive chemometric molecular understanding of the mechanisms by which certain macromolecules adhere to the vaginal tissue epithelium is not yet available (Mikos and Peppas, 1990; Valenta, 2005) . T herefore a thermodynamic model that addresses receptor- mediated cell adhesion at the molecular level was explored. By incorporating the entropic, conformational, solvation, and long- and short- range interactive components of receptors and the extracellular matrix molecules, adhesive free energy as a function of a number of key variables such as surface coverage, interaction distance, molecule size, and solvent conditions were predicted. This allows the computation of the free energy of adhesion in a multi- component system in order to simultaneously elucidate adhesion receptors and ligands of different sizes, chemical identities, and conformational properties. This approach not only provides a fundamental understanding of adhesion at the molecular level but may also aid in identifying possible strategies for designing novel biomaterials to improve bioadhesion of the caplet to the pig vaginal tissue substrate. 5. 3 Results and Discussion 5.3. 1 Assessment of caplet bioadhesivity Work of adhesion values for the APE- PAA caplets were significantly higher (P< 0.05) than those of AS- PAA caplets. The difference in work o f adhesion was 94.1% (Figure 5.3 ). This clearly indicated that the APE - PAA caplets had superior bioadhesivity than the AS- PAA caplets. The difference in bioadhesiv ity may be due to the difference in percentage crosslinking of allyl sucrose and allyl penta erythritol to PAA. Allyl sucrose is 1.5 % crosslinked ( Corring, 1992) while penta erythritol is 2.0 % crosslinked ( Wilber et al., 2003) . 128 T his slightly higher density of crosslinking in APE- PAA may therefore have translated into an intense hydrated network capable of facilitating superior bioadhesion. Figure 5.3 : A comparison of work of adhesion (AUC FD) (J) between the AS - PAA and APE- PAA caplets ( N=3; SD<0.03) . 5. 3.2 Response optimization via textural analysis outputs from the optimized AS- PAA and APE -PAA caplets Response optimization of the AS - PAA and APE- PAA caplets using Minitab ? V15 ( Minitab ? Inc., PA, USA) software revealed the optimum level for each polymer ( mPA 6,10 , PEO, PLGA, CG and PAA) within the caplets in order to provide the most desirable relative bioadhesivity (Figure 5.4 ) . The bioadhesivity values for the in vitro test were significantly higher compared to the in vivo test (P<0.01) (Table 5.2) . The reason is mainly based on the nature of the surfaces with which the caplets made contact with to establish the bioadhesive force. The in vitro test employed artificial simulated vaginal membrane whereas the in vivo test employed freshly excised vaginal membrane. The mucus present on the surface of the freshly excised vaginal membrane underwent a constant turn- over with the old layer being replaced by a new one. This process int errupted the establishment of hydrogen bonds and van der Waal forces between the caplet and the 129 vaginal membrane thereby resulting in low bioadhesion. With articial membrane, there was no turn- over effect and therefore once a bond formed, it remained intact (i.e. the interaction holds) which may thus be the reason for the higher bioadhesivity observed. Furthermore, mucus degrades while artificial membranes do not and therefore the biodhesive forces established in the later, remained intact and undisturbed. The difference in bioadhesivity between the AS- PAA and APE- PAA caplets was 46.7% (Figure 5.5). The desirability (D) values were 0.98 and 0.92 respectively indicating the sufficient convergence of data toward an optimized global solution . Figure 5.3: A typical response optimization plot for the AS - PAA and APE- PAA caplets Figure 5.4: A typical response optimization plot for the AS - PAA and APE- PAA caplets. Table 5.2: Comparative in vitro and ex vivo bioadhesivity results for the optimized caplets Textural parameters Predicted (x10 -3 ) Experimental (x10 -3 ) In vitro anal ysis of optimized AS -PAA and APE -PAA caplets Force of adhesion (N) AS- PAA caplet 1 300 . 00 1 168 . 10 ? 93 . 27 0 APE- PAA caplet 2 000 . 00 1 671 . 60 ? 23 . 221 Work of adhesion (J) AS- PAA caplet 0. 50 0.32 ? 0. 13 APE- PAA caplet 0. 90 0.60 ? 0. 21 Ex vivo anal ysis of the optimized APE -PAA caplet Force of adhesion (N) 1 000 . 00 883 . 00 ? 82 . 73 Work of adhesion (J) 0. 35 0. 30 ? 0 . 035 130 5. 3. 3 Ex vivo bioadhesivity anal ysis of the optimized APE-PAA caplets on freshly excised vaginal tissue The PAF value obtained was 0.883? 0. 08273N while the work of adhesion (AUC FD) was 0.0003? 3. 53553 X10 - 5 J. The results portrayed a direct correlation between the PAF and the work of adhesion (Table 5.2 and Figure 5. 6 ). The optimized caplet matrices with desirability (D) values close to 1 depicted excellent correlation (R 2 =0. 99) between the predicted and the experimental results (Table 5.2) . Figure 5. 5 : Typical textural profiles elucidating the peak adhesive force (PAF) (N) and work of adhesion (AUC FD) (J) for the optimized composed of: a) AS - PAA, and b) APE - PAA. a ) b) Force (N) Force (N) Distance (m) Distance (m) 2 2 131 Figure 5. 6 : Typical textural profile elucidating the force (N) and work of adhesion (AUC FD) (J) for the optimized APE - PAA caplets on freshly excised vaginal tissue. 5. 3. 4 Rheological anal ysis of the h ydr ated AS -PAA and APE -PAA polymer solutions AS- PAA and APE- PAA are polymers that are able to produce mucilages with short rheological flow properties that can be associated with the high degree of crosslinking in both polymers. In terms of rheological properties, APE - PAA was found to be highly viscous (900x10 - 3 mPas) in comparison to AS - PAA (80x 10 - 3 mPas) with a subsequent higher shear stress (180Pa vs. 58Pa) (Figure 5.6) . Justifiably, this rheological observation may explain the superior bioadhesivity of the APE- PAA caplets over the AS- PAA caplets. In both cases the viscosity decreased as the shear rate increased (Figure 5.7 ) . Force (N) Distance (m) 132 Figure 5.7: Rheological behavior of 2% w/ v AS- PAA and APE- PAA solutions at a shear rate between 0- 500s - 1 . The bioadhesivit y behavior of the caplets to the freshly excised pig vaginal tissue is a typical reflection of the fact that the polymer combination employed for matrix forma tion typically possesses different structural deformation mechanisms due to their backbone, molecular mass, polymeric concentration, chain flexibility, spacial conformation, pH of volumetric distention at the polymer- substrate interface, the associated forces, the initial contact time and the extent of mucin turn- over. This may be a result of their variation in moduli and rheological deformation energies. Hence deformation or flow at the polymer - vaginal tissue substrate interface occurred within the polymeric matrix with subsequent failure at the interface to induce and control the degree of bioadhesion between the caplet and the freshly excised vaginal tissue. 5. 3. 5 Postulative chemometric and molecular modeling of the mechanisms of caplet bioadhesion and the associated interactiv e energy paradigms Results obtained from chemometric and computational analysis of the associated energy paradigms have revealed that the caplet bioadhesivity to the freshly excised pig vaginal tissue substrate may be explained in terms of the Differential Adhesion Hypothesis ( DAH) 0 20 40 60 80 100 120 140 160 180 0 100 200 300 400 500 0200 400600 800 She ar S tres s (P a) Shear Rate (1/s) Viscosity (X10 -3 mPa) AS-PAA matrix APE-PAA matrix 133 theory of cell adhesion ( Steinberg, 1996; Brodland, 2003; Foty and Steinberg, 2005; Steinberg, 2007; Katsamba et al., 2009) . The pig vaginal tissue substrate acted as a viscoelastic flexible semi - solid with a tissue surface tension corresponding to a mutual classification behavior occupying an internal position relative to the gelled caplet with a lower surface tension during interaction. Quantit ative energy variations in homo- and hetero- typic bioadhesion were sufficient to account for the phenomenon without the need to postulate cell- type specific bioadhesion. The caplet bioadhesion occurred in a stepwise mechanism involving weak inter- atomic and inter- molecular physical forces that interplay at the outset (Figure 5.8). During initial contact of the caplet with the freshly excised pig vaginal tissue substrate mechanical bioadhesion occurred with all surface voids between the caplet and tissue substrate filled and therefore resulted in surface interlocking. As the caplet hydrated a more unyielding chemically adhesive interaction progressed between the hydrated inter- polymeric blend of the caplet to form a bioadhesive compound at the interface either through ionic or covalent bonding prior to caplet dispersion. In the dispersive phase of bioadhesion the hydrated caplet and vaginal tissue substrate were bound by van der Waals forces as a result of polymeric ionic species generated due to disentanglement and volumetric distention of the constituent polymers. In the simplest case, molec ules during this phase of bioadhesion were polar with respect to the average charge density and may be due to Keesom forces or a transient effect of London forces . Once the caplet and vaginal tissue substrate diffusively merged, both interfaces were miscible through a sintering- like mechanism. The strength of bioadhesion depended on a hybrid between the dispersive and diffusive phases as well as the contact surface area. Polymers such as the AS- PAA and APE- PAA were able to significantly wet during sol- gel interconversion and therefore provided a reduced contact angle facilitating bioadhesion. 134 Figure 5.8: A chemometric structural model developed in our laboratories depicting caplet bioadhesion to freshly excised pig vaginal tissue with muco- epithelial cell secretions and surface bio- molecule interactions. Figure 5.9a - d depicts the energy estimates for bioadhesion representing the physical forces, ionic and chemical interactions as well as ion- exchanges during bioadhesion. This involved electron donations and sharing of covalent bonds between the xenobiotic polymers of the caplet and cellular proteins of the vaginal tissue substrate. Interactions with collagen in the substrate surface with polymer- protein interactions were also present. The extent of hydration also played a significant role in hydration of the caplet resulting in ionic interaction pockets that contributed towards bioadhesion. Structural deformities of the caplet and proteins occurred with proteins transitioning into coiling at primary, secondary, tertiary and quaternary structure levels that participated in bonding to the polymers and facilitated bioadhesion. The energy barrier between the compartmental interactions determined the extent of bioadhesion as a factor of energy (Figure 5.9a - d). The protein and polymer bonding at the cell surface via the embedded glyco- protein and other surface proteins played a role in linking the polymer. The saccharide component of the glyco- proteins bonded to the inherent surface protein and available functional groups and to some extent also bonded to the AS- PAA and APE- PAA polymers within the caplet. The energy differences and 135 level played a rate- limiting factor in the proposed bioadhesion phenomenon. Helical structural transitions and distortions within the polymer were predicted by molecular modeling and also affected by stereo- orientation though no effects on the coiling or helical turns of the protein were observed. The embedded glyco- proteins were least affected and provided the anchoring groups for polymer- substrate bioadhesion. Figure 5.9 : Energy estimates for the mechanism of bioadhesion developed in our laboratories: a) compartment A=interactions at physical forces levels, B=ionic interactions and ion- exchanges producing bioadhesion, C= chemical interaction involving covalent bonding between the xenobiotic polymer and cellular protein, D=interaction with surface collagen in the tissue substrate includes polymer- protein- collagen interactions. Hydration also played a role and facilitated polymeric gelling and induction of ionic interaction pockets that contributed towards bioadhesion. Furthermore, collagen was dissolved from the fibrils by proteases and structurally adjusted to the requirements of bioadhesion as a result of polymer- protein and polymer- collagen interactions. Generally, the barriers to break weak physical bonds do not usually exceed 10? 20 times the thermal 136 energy, kT (where k is the Boltzmann constant and T is the absolute temperature). The lifetime of these bonds can vary from microseconds to seconds. Hence, polymers form and break these weak physical bonds numerous times during the course of bioadhesion. The caplets comprised two types of polymeric chain molecules arising from the AS - PAA and APE- PAA within the matrices. In order to predict the mechanisms of cell - matrix bioadhesion it was assumed that both surfaces (i. e. the caplet and vaginal tissue substrate) were two dimensional parallel planar impenetrable surfaces. In addition to polymeric chain molecules, the residual voids between the two surfaces were assumed to be filled with simulated vaginal fluid. To simulat e protein interactions with the polymers, ?generic? amino acids were employed and conformations chosen randomly from sterically allowable regions using dihedral angles. The polymer molecules were simulated with a similar strategy. Bioadhesive mechanisms were modeled to capture the essential features of polymer- protein interactions. Increasing the number of sampled conformations did not significantly alter the results and for simplicity only interactions between polymer- protein, collagen- polymer and polymer- protein- collagen were considered with self- interaction terms ignored. All interaction terms contained attractive and repulsive phases with an incompressibility condition or volume- filling constraint introduced to account for repulsive interactions. Under t his assumption, no two segments could occupy the same volume which was the result of repulsive interactions. 5.4 Concluding Remarks The polymer - based caplets adhered to the simulated vaginal membranes and the freshly excised pig vaginal tissue revealing the potential for application in intravaginal drug delivery. C hemometrical and molecular structural modeling deduced that the potential mechanisms of bioadhesivity are a combination of mechanical, chemical, dispersive and diffusive bioadhesive phenomena. Rheological analysis revealed that AS - PAA and APE- PAA displayed varying rheological behavior contributing to bioadhesion to the in vitro and 137 ex vivo substrates evaluated. Furthermore, the results demonstrated that APE - PAA caplets were more bioadhesive than the AS- PAA caplets and therefore capable of providing an optimized IBPD formulation. The bioadhesivity testing approach employed using freshly excised pig vaginal tissue suggests that the method developed may be useful for measuring the bioadhesivity of other intravaginal drug delivery systems on vaginal tissue substrates. Rational design of future bioadhesive intravaginal drug delivery systems should focus on the polymer properties that conform to vaginal retention to target bioactives more appropriately. 138 CHAPTER SIX INVE S TIG AT ION OF THE P HY S IC OC HE MIC AL AND P HY S IC OME C HANIC AL P R OP E R TIE S OF THE OP TIMIZE D INTR AV AG INAL B OADHE S IVE P OL Y ME R IC DE VIC E 6.1 I ntroduction The thermodynamic behavior of polymers is known to have a significant impact on the physicomechanical properties and therefore their final performance ( Pillay and Fassihi, 1999; Liu et al., 2003; Parija, et al., 2004) . Therefore, accurate thermal analysis of polymer blends employing temperature modulated differential scanning calorimetry (TMDSC) is critical during formulation design ( Ribeiro and Grolier, 1999) . TMDSC provides a clearer interpretation of thermal transitions on analyzed samples due to the improved sensitivity and resolution, coupled with the ability to separate reversible glass transitions that have diminutive transitions in heat capacity from overlapping non- reversible relaxation endotherms. TMDSC is also used to reveal thermal transitions at sub- zero temperatures that cannot be detected by conventional DSC due to poor equilibrium of the heat flow baseline below 0?C ( Pijpers et al., 2000) . Thus, the development of multi- polymeric intravaginal drug delivery systems requires a comprehensive understanding of the thermodynamic stability of the polymeric constituents used in designing the system. The natural and most important protection against potential infections in the vagina is the presence of a normal, healthy vaginal flora (Vitali et al., 2007; Obiero et al., 2008; Mastromarino et al., 2009) . An acidic vaginal pH value ranging from 4 - 5 is a crucial requirement for the optimal functioning of the body's natural protective system in the vagina. Thus, maintenance of this pH range in the vagina is important as it can effectively 139 avoid colonization by pathogens which may lead to gynecological problems since most of the pathogens cannot thrive under acidic conditions (Boskey et al., 2001; Ramsey 2002; Mastromarino et al., 2009) . There are numerous factors that regulate the composition and dynamics of the vaginal microbial ecosystem and that maintain the natural protective functions of the vagina. The most important one, however, is the breakdown of glycogen (released from the upper vaginal epithelium), which is then metabolised to glucose, and finally lactic acid by Lactobacilli (Boskey et al., 2001) . This results in an ideal acidic environment in the vagina, thus providing effective protection against infection. Generally, drug permeation rates correlate with partition coefficients and are inversely proportional to the molecular mass of the drug ( Iyer et al., 2008) . Thus, successful development of an intravaginal drug delivery system should consider permeation characteristics as a result of the formulation design, the therapeutic agent and vaginal physiology ( Verman and Garg, 2000; Iyer et al., 2008; Ndesendo et al., 2008 ) . Therefore the aim of this Chapter was to design, develop and evaluate the bioadhesivity, permeability, thermal stability and the in vitro drug release of a novel drug- loaded intravaginal bioadhesive polymeric device (IBPD) for the prevention of HIV and other STIs employing model drugs AZT and PSS. The device should provide controlled drug release over an experimental period of at least one month when inserted into the posterior fornix of the vaginal cavity. Pig vaginal tissue was employed for permeation analysis due to the fact that the pig vaginal mucosa is very similar in many respects to human vaginal mucosa (Kremer et al., 2001; Thomson et al., 2001; Davis et al., 2003; Squier et al., 2008) . Both ty pes of mucosa consist of stratified squamous epithelium that is supported by connective tissue and lamina propria with lipid compositions that are comparable (van Kremer et al., 2001; Thomson et al., 2001). The specific polymers expressly selected in formulating the device included combinations of modified polyamide 6,10 ( mPA 6,10 ), poly(lactide- co- glycolide) (PLGA), allyl penta erythritol - crosslinked PAA (APE- PAA), polyvinyl alcohol (PVA) and ethylcellulose (EC) that were primarily employed for 140 controlling the release of AZT and PSS from the IBPD. PSS played a dual role as a polymer matrix and a bioactive (Garg et al., 2004a ; Chu et al., 2007) ( Chapter 3, Section 3.2. 1. 1) . PAA was used as both polymer matrix constituent as well as a coating agent for the purpose of achieving extended bi oadhesivity within the posterior fornix of the vagina (Bonacucina et al., 2006; Ndesendo et al., 2009) . The acidic environment in the vagina may be influenced by the changes in pH and ionic concentrations as a result of PLGA degradation into lactic and glycolic acid through cleavage by enzymatic or non- enzymatic hydrolysis ( Park et al., 2005; Yasukawa et al., 2006; Kulkarni et al., 2007) . Thus, since PLGA was one of the polymers selected for formulating the IBPD, it was impor tant to determine the changes in micro- environmental pH the simulated vaginal fluid when exposed to the degradating constituents of the IBPD . A specific X - ray imaging approach employing radiopaque barium sulfate ( BaSO4 ) was developed for detecting and determining the retention and sequential biodegradation pattern of the IBPD device within the vagina of the Large White pig. In addition to having radiopaque properties, BaSO4 ( which is biocompatible) was employed as a polymer matrix stabilizer for the purpose of enhancing matrix integrity ( Bailey and Swett, 2007) . Furthemore, since chemometric and molecular modeling approaches can precisely explicate various interactive mechanis ms of drug release and permeation dynamics that occur from a drug delivery system, a model was developed in our laboratories to elucidate these mechanisms at a molecular level. 6.2 M aterials and M ethods 6.2.1 Materials The two model drugs were AZT (Evershine Ind., Naejar Malad, Mumbai, India) and PSS (Omega (Pty) Ltd., Montreal, Canada). M ethylparaben (Merck (Pty) Ltd., Darmstadt, Germany), shellac (Roeper GmbH, Hamburg, Germany), castor oil (Jayant Oils and Derivatives Ltd., Mumbai, India) , barium sulphate (BaSO 4 ) (Merck - Schuchardt, 141 Hohenbrunn, Germany), pentobarbitone, ketamine (Bayer (Pty) Ltd, Wrenchwerg, Isando, South Africa), midazolam (Roche Products (Pty) Ltd, Isando, Gauteng, South Africa) and isoflurane (Safe Line Pharmaceuticals (Pt y) Ltd, Florida, South Africa) were utilized to facilitate the in vivo studies. Th e mobile phase solvents comprising acetonitrile (99. 9% ) and methanol (99. 9% ) were purchased from Romil - SpS? (Cambridge, UK) . All other materials employed were the same as in Chapter 3. 6.2.2 Methods 6.2.2.1 Preparation of the intravaginal bioadhesive polymeric device Biodegradable and biocompatible polymers namely mPA 6,10 (150mg), PLGA (400mg), APE- PAA (25mg), PVA (25mg) and EC (200mg) were blended with model drugs AZT and PSS (separately and in combination) as well as radio- opaque barium sulfate (BaSO 4 ), using a cube blender (Erweka ? GmbH, Heusenstamm, Germany), and then compressed into robust devices on a tableting press. One set of devices was coated with 2% w/ v APE- PAA while another set remained uncoated. In process validation tests were performed to ensure that the IBPD device had desirable quality attributes in terms of diametric hardness, uniformity in mass and friability. 6.2. 2.2 Pan coating of the intravaginal bioadhesive polymeric device A dual coating process using the Thai C oater? (Pharmaceutical and Medical Supply Limited Partinership, Yannawa, Bankok, Thailand) was employed with a protective undercoat comprising shellac and thereafter a mixture of XG and APE- PAA as an overcoat in order to prevent any irritation to the vaginal tissue during device insertion. The addition of APE- PAA was to facilitate bioadhesion of the IBPD to the posterior fornix of the vagina. The process involved firstly undercoating the IBPD with a combination of shellac (4mg/device), cold pressed castor oil (3mg/device) and ethanol (96%). This was followed by an overcoat of XG (2% w/ v) and APE- PAA (2% w/ v). XG was used for its viscoelastic non- collapsible swellability in order to facilitate bioadhesion of the IBPD in 142 conjunction with APE- PAA that was employed as a biodhesive polymer ( Iseki et al., 2001; Gimeno et al., 20003; Verhoeven et al., 2006) . The processing conditions utilized for effective coating of the IBPDs are listed in Table 6. 1. A non- coating period of 30 minutes was allowed after each coating phase to effect a reduction in pan temperature and avoid sticking or fracture of the undercoat or overcoat seal. The increase i n weight after coating the IBPD was determined using an electronic balance (Mettler, Model AE 240, Griefensee, Switzerland) while the increase in thickness was determined using a digital vernier caliper (Taizhou hangyu tools gauge and blades Co., Ltd, Wenqi ao, Zhejiang, China) with a sensitivity of 0.01. Table 6.1: Parameters and settings employed for coating the intravaginal bioadhesive polymeric device Parameter Settings Temperature 50 - 56?C Relative humidity 23 - 28% Warming- up period 10min Pan rotation 2 - 3rpm Spray rate 4g/min Volume of coating 2L Number of caplets coated 2000 Undercoating duration 30min Over- coating duration 60min 6.2.2.3 Influence of the intravaginal bioadhesive polymeric device on the micro- environmental pH of the vagina The changes in micro- environmental pH within simulated vaginal fluid due to the presence of the IBPD were assessed by incubation of 3mL simulated vaginal fluid (containing the IBPD) in a Multi - Purpose T itrator (MPT - 2) equipped with a rapid response, liquid filled glass pH micro- electrode supported on a vertical puller (Malvern Instruments Ltd., Worcestershire, UK ) . The changes in pH were evaluated from a pH- time profile over 30 days. The electrode calibration standards were adjusted to cover the buffer range from pH 3.5- 5. 5 with a linear Nernstian response maintained. 143 6.2.2.4 Thermal anal ysis of the polymeric composition and the coated intravaginal bioadhesive polymeric device T MDSC was performed on the constituent polymers ( mPA 6,10 , PLGA, APE- PAA, PVA, and EC) as well as the unhydrated and hydrated physical mixtures of the polymers and the IBPD (Mettler Toledo, DSC1, STAR e System, Schwerzenback, Switzerland) . The thermal events were explicated in terms of the glass transition (T g) measured as the reversible heat flow (?H) due to changes in the magnitude of the C p- complex values ( ?Cp), melting (T m) and crystallization (T c) temperature peaks which are consequences of irreversible and reversible ?H values corresponding to the total heat flow. The temperature calibration was accomplished with the melting transition of indium. The transitions of the individual polymers and their physical mixtures were compared with the transition of the composite IBPD matrix. Samples were weighed (5mg) on perforated 40?L aluminum pans, crimped and then ramped from - 35- 230?C under a nitrogen (Afrox, Germiston, Gauteng, South Africa) atmosphere in order to diminish oxidation at a rate of 1?C/min. The instrument parameter s and settings employed are listed in Table 6.2. Table 6. 2: Temperature modulated differential scanning calorimetry settings employed for thermal analysis of the intravaginal bioadhesive polymeric device and its formulation components Segment Type Parameter Setting SINE PHASEa Start - 35?C Heating rate 1?C/min Amplitude 0.8?C Period LOOP PHASE b To segment Increment End Count 0.8?C 1 0.8?C 230?C 436 aSinusoidal oscillations bOscillation period 144 6.2.2.5 Ex vivo bioadhesivity testing of the coated drug -loaded intravaginal bioadhesive polymeric device The excision of vaginal tissue from the pig model for bioadhesivity testing was undertaken following the method described in (Chapter 5, Section 5.2. 2. 4 ) . 6.2. 2. 6 Textural profiling anal ysis to determine the bioadhesivity of the intravaginal bioadhesive polymeric device Bioadhesivity of the IBPD was determined using a method described in one of our recent publications ( Ndesendo et al., 2009 ) . Briefly, the freshly excised pig vaginal tis sue was secured on the textur al probe and the IBPD was fixed onto the heated textural platen after exposure to simulated vaginal fluid (pH 4.5, 37?C) ( Chapter 3, Section 3.2. 2. 3, Table 3.3) for 30 minutes . Testing was then conducted by measuring the maximum force (N) required to detach the vaginal tissue from the fixe d device. This was determined by measuring the Peak Adhesive Force (PAF) or the Work of Adhesion that was computed as the area under the curve of a Force- Distance textural profile (AUC FD). 6. 2. 2. 7 Insertion of the intravaginal bioadhesive polymeric device into the vagina of the pig Three Large White pigs each weighing 35kg were anaesthetized with midazolam (0.3mg/kg I.M. ) and ketamine (11mg/kg I.M. ) . 2% isoflurane in 100% oxygen was administered via a face mask to maintain anesthesia. The IBPD was then deeply inserted into the posterior fornix of the vagina of each pig with the aid of an applicator and a speculum as shown in Figure 6.1a and b. 145 Figure 6.1 : Digital images depicting a) insertion of the IBPD into the vagina of the pig and b) tracking the location of the IBPD in the vagina using a speculum. 6.2.2.7 . 1 X -ray imaging of the pig for detection of the intravaginal bioadhesive polymeric device To detect the presence and position of the IBPD in the pig vagina after insertion, animals were X - rayed (Siemens AG, Medical Engineering Group, Erlangen, Germany) directly after device insertion and thereafter three times weekly for 2 weeks, then twice weekly for a further 2 weeks to confirm the retention of the IBPD in the vagina and to qualitatively analyze its swellability and bioerosion dynamics. 6.2.2.8 In vitro drug release from the coated and uncoated intravaginal bioadhesive polymeric device In vitro drug release evaluation was conducted on the APE- PAA coated and uncoated AZT - loaded IBPDs and coated AZT - and PSS- loaded IBPD s 6.2. 2. 8.1 Anal ysis of the effect of device coating on the drug release To assess the effect of coating on drug release, analysis was conducted on IBPDs (c oated and uncoated) containing AZT as a representative drug model due to its hydrophilicity. An IBPD was immersed in a 100mL (Umamaheshwari et al., 2004; Charde et al., 2008) simulated vaginal fluid (pH 4.5; 37?C) (Chapter 3, Section 3.2. 2. 3, Table 3.3) using a sealable glass vessel (150mL) and placed in an orbital shaking incubator (LM - 530- 2, MRC Laboratory Instruments Ltd., Hahistadrut, Holon, Israel) maintained at 20rpm and a temperature of 37 ? C. For the determination of AZT concentration, 3 mL samples a) b) 146 were withdrawn at predetermined time intervals over a period of 30 days and subjected to Ultra Performance Liquid Chromatography (UPLC) analysis. An equivalent volume of drug- free simulated vaginal fluid was replaced into the release medium to maintain sink conditions. The analysi s was conducted in triplicate. A correction factor was appropriately applied in all cases where dilution of samples was required. 6.2. 2.8.2 Anal ysis of the drug release from the coated devices containing AZT and PSS separately and in combination For the analysis of the drug release from the coated devices containing AZT and PSS separately and in combination, the same procedure as in Section 6.2. 2. 8.1 was employed the only difference being that in this case samples for analysis were withdrawn over a period of 72 days. 6.2. 2. 8.3 Chromatographic conditions for the anal ysis of AZT and PSS concentration Quantitative analysis was performed using a Waters ? Acquity Ultra Performance Liquid Chromatographic (UPLC) system (Waters Corp., Milf ord, MA, USA), equipped with a photodiode array ( PDA) detector and interchangeable columns, namely, a UPLC ? BEH phenyl column (1.7?m; 2.1? 50mm) for AZT separation, and a UPLC ? BEH C18 column (1.7?m; 2.1? 100mm) for PSS separation. The binary mobile phases w ere composed of water/acetonitrile (60: 40 v/ v) and methanol/water (50: 50 v/ v) for AZT and PSS respectively. All solutions were filtered using a 0.22?m membrane filter (Millipore Corp., Bedford, Massachusetts , USA) prior to injection onto the UPLC column. A gradient assay method was used for AZT separation with a column temperature set at 25 ?C, injection volume of 2?L and a UV detection wavelength of 267nm. The gradient settings for the ass ay method are shown in Table 6.3. An isocratic assay method was used for PSS separation employing methanol/water (50: 50 v/ v) as the mobile phase, a flow rate of 0.2mL/min, a 147 column temperature of 25?C, an injection volume of 1.7 ?L and a UV detection wavelength of 244nm. Table 6.3: Chromatographic mobile phase gradient settings used for the separation of AZT Separation events Time (min) Flow Rate (mL/min) % A (Water) % B (Acetonitrile) 1 0.00 0.500 60. 0 40. 0 2 1.00 0.500 5. 0 95. 0 3 2.60 0.500 5. 0 95. 0 4 3.50 0.500 60. 0 40. 0 6.2. 2. 8 . 4 Preparation of standard soluti ons and calibration curves The internal standard employed for both model drugs was methylparaben (MP). Standard solutions of AZT, PSS and MP (internal standard) were separately prepared by mixing specific quantities in water/acetonitrile (60: 40 v/ v) for AZT and methanol/water (50: 50 v/ v) for PSS to yield a concentration of 0.1mg/mL in each case. The standard solutions employed in preparing the calibration curve of the test drug and internal standard were obtained by further serial dilutions with a final concentration range of 25- 1 0,000ng/mL. The internal standard solution was prepared at a concentration of 5000ng/mL and was added to all samples prepared for UPLC analysis. Calibration curves were developed using blank simulated vaginal fluid (pH 4.5) and comput ed as a ratio of the Area Under the Curve (AUC) of AZT and PSS chromatographic peaks to that of the internal standard MP against the corresponding standard concentrations of AZT and PSS (Figure 6. 2 a and b) . 148 Figur e 6.2 : a) Calibration curves for: a) AZT in simulated vaginal fluid 267nm (N=3, SD<0.001 in all cases); b) PSS in simulated vaginal fluid 244nm (N=3, SD<0.003 in all cases). Y=0.1601+0.0029x R2=0.99 Concentration of AZT (ng/mL) 0 2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0 1 0 0 0 0 Ar ea (P DA : 2 67nm A ZT) 0 5 1 0 1 5 2 0 2 5 3 0 AZT Linear Regression Curve 95% Confidence Band 95% Prediction Band a) Y=0.2457+0.0039x R2=0.99 Concentration of PSS (ng/mL) 0 2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0 1 0 0 0 0 Ar ea (P DA : 24 4 PSS ) 0 1 0 2 0 3 0 4 0 PSS Linear Regression Curve 95% Confidence Band 95% Prediction Band b) 149 6.2. 2. 8 .5 The solid phase extraction procedure employed in the extraction of t he drugs from simulated vaginal fluid samples for UPLC anal ysis This was carried out by using single use Water Oasis? HLB 3cc (60mg) extraction cartridge (Waters Corporation, Milford, Massachusetts, USA) adapting a method developed by Notari and co- workers (2006). The Solid Phase Extraction (SPE) cartridge was conditioned with 1.0mL methanol followed by 1.0mL water Milli - Q. For AZT sample preparation, 1mL of sample was mixed with 1mL of acetonitrile vortexed for 1 minute and centrifuged (Nison Instrument (S hangai) Limited, Shangai, China) at 13,000rpm for 6 minutes at 24 oC. 650?L of the supernatant was diluted by adding water Milli - Q (1mL) and loaded in the cartridge. Thereafter, the cartridges were washed with 1.0mL of 5% v/ v methanol in water Milli - Q. Analytes were eluted by washing cartridges with 550?L 0.01M KH 2 PO4 followed by 2mL methanol. The eluate was evaporated to dryness in a slow stream of high purity nitrogen gas (Afrox, Germiston, Gauteng, South Africa). The extracted sample was re- constituted with 100?L absolute methanol, mixed with 400?L of MP, and then filtered into the injection vials using 0.22?m syringe- driven filter units (Millipore Corporation, Bedford, Massachusetts , USA) for UPLC analysis. The same procedure was followed for PSS samples. However, methanol was used as the mobile phase. 6.2. 2. 9 Ex vivo drug permeation studies through pig vaginal tissue from the intravaginal bioadhesive polymeric device using the Franz Diffusion Cell Apparatus The perm eation studies to assess the extent of drug perm eation across pig vaginal tissue were performed using a Franz Diffusion Cell (FDC) apparatus (PermeGear Inc. Bethlehem, PA, USA) equipped with a 12mL receptor compartment, clamp and stirrer - bar. Freshly excised pig vaginal tissue obtained from the euthanized Large White pig described above was placed between the donor and receptor compartments of the FDC apparatus (Figure 6.3 ) . 10mL simulated plasma (pH 7.4; 37?C) (Giannola et al., 2007 ) (Table 6.4) was used in the receptor compartment and continuously agitated. IBPD device 150 containing AZT and PSS was dissolved in 50m L simulated vaginal fluid (pH 4.5; 37?C) (Chapter 3; Section 3.2.2. 3; Table 3.3) , by crashing and triturating it into a powder form and then solubilising the powder by the aid of a sonicator ( SB 5200D, Ultrasonic, Ningbo Scientists Biotechnology Co Ltd, Ni ngbo, China) . The solutions were then assessed for the permeation of the drug across the pig vaginal tissue (thickness=1.5? 0. 06 mm; permeation area=2.0? 0. 01cm 2 ) and into the simulated plasma in the receptor compartment. Samples (2 mL) were withdrawn from the receptor compartment, at predetermined intervals over a period of 24 hours, and subjected to quantitative drug analysis using UPLC. An equivalent volume of drug- free simulated plasma was replaced into the receptor compartment to maintain sink conditions throughout the permeation study. The analyses were conducted in triplicate. A correction factor was appropriately applied in all cases where dilution of samples was required. The extent of permeation of AZT and PSS across the pig vaginal tissue was determi ned in terms of drug flux. The flux (mg.cm ?2.h r?1) of drug across the vaginal tissue was calculated at steady- state per unit area by linear regression analysis of permeation data using Equation 6.1. tA rQsJ ?= Equation 6.1 where, Js is the flux (mg.cm ?2.h rg?1), Q r (mg) is the quantity of AZT or PSS that diffused through the pig vaginal tissue into the receptor compartment, A ( cm?2) is the effective cross- sectional area available for drug permeationand t (h) is the time of drug exposure to the vaginal tissue. 151 Figure 6. 3 : Permeation studies of AZT and PSS across pig vaginal tissue using a Franz Diffusion Cell apparatus. Table 6.4: Constituents used to prepare the simulated plasma fluid Simulated Plasma Component Quantity (g/L) KH 2 PO4 0.144 Na 2 HPO4 0.795 NaCl 9.000 ( Giannola et al., 2007) 6.2. 2. 9.1 C hromatographic conditions for the anal ysis of AZT and PSS concentration The c hromatographic conditions for the analysis of AZT and PSS concentration in the simulated plasma fluid were the same as described in Section 6.2. 2. 8. 3 . 6.2. 2. 9.2 Preparation of standard solutions and calibration curves This was conducted in the same way as described in Section 6.2. 2. 8. 4 , the only difference being that calibration curves were developed using blank simulated plasma fluid (pH 4.5) (Section 6.2. 2. 9, Table 6.4) instead of simulated vaginal fluid. The constructed calibration curves are as shown in Figure 6. 4a and b. Donor compartment Vaginal tissue Receptor compartment 152 Figure 6.4: a) Calibration curves for: a) AZT in simulated plasma fluid 267nm (N=3, SD<0.001 in all cases); b) PSS in simulated plasma fluid 244nm (N=3, SD<0.003 in all cases). 6.2. 2. 9.3 The solid phase extraction procedure employed in the extraction of the drugs from simulated plasma fluid samples for UPLC anal ysis The solid phase extraction procedure employed in the extraction of the drugs from simulated plasma fluid samples was the same as described in Section 6.2. 2. 8. 5 . Y=0.1226+0.0030x R2=0.99 Concentration of AZT (ng/mL) 0 2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0 1 0 0 0 0 Ar ea (P DA : 2 67nm AZT ) 0 5 1 0 1 5 2 0 2 5 3 0 AZT Linear Regression Curve 95% Confidence Band 95% Prediction Band a) Y=0.0862+0.0038x R2=0.99 Concentration of PSS (ng/mL) 0 2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0 1 0 0 0 0 Ar ea (P DA : 24 4nm PSS ) 0 1 0 2 0 3 0 4 0 PSS Linear Regression Curve 95% Confidence Band 95% Prediction Band b) 153 6.2.2 .10 Postulated mechanism of the drug permeation and di ssolution dynamics from the intravaginal bioadhesive polymeric device employing chemometric and molecular modeling Chemometric and molecular structural modeling was used to deduce the transient mechanisms of diffusion and dissolution, chemical interactions and inter- polymeric interfacing during the dissolution of the IBPD device and the permeation of AZT and PSS across the vaginal tissue. This approach allowed us to make predictive findings based on the chemical and physical interactions underlying the dissolution of the IBPD and the diffusion of AZT/PSS from the IBPD (contained in the simulated vaginal fluid) and finally the permeation of these drugs to simulated plasma fluid across the pig vaginal tissue. In addition, semi- empirical quantum mechanics were employed to generate molecular interactions and computational energy paradigms of the IBPD components based on inherent interfacial phenomena underlying the mechanisms of dissolution and diffusion as provided by the inter- polymeric blended IBPD. Models and graphics supported on the step- wise molecular IBPD - simulated fluids and IBPD - tissue interactions, polymeric interconversion, dissolution and diffusion as envisioned by the molecular behavior and stability of the gelled IBPD network were generated on ACD/I - Lab, V5.11 (Add- on) software (Advanced Chemistry Development Inc., Toronto, Canada, 2000). 6. 3 R esults and D iscussion 6.3. 1 Coating of the intravaginal bioadhesive polymeric device Uniformly coated IBPD s were produced. The final mass for the coated caplets was 1278 ? 0. 04mg while the uncoated caplets weighed 1200? 0. 01 mg. Thus, the increase in weight was 6.5? 0. 02 % w/ w for which the thickness of the coat was 0.520? 0.005mm. 6. 3.2 Micro -environmental pH variation anal ysis within the simulated vaginal fluid It was generally observed that the superficial simulated vaginal fluid immediately adjacent to the immersed IBPD exhibited higher pH values than the simulat ed vaginal fluid 154 immediately surrounding the device. The initial pH, measured as close as possible to the device, upon insertion of the IBPD into the titration system (MPT - 2) was 4.5? 0. 01 (N= 3). The pH electrode was inserted using a Narashige micro - manipulator and was submerged towards the IBPD by careful hydraulic micro- movements to avoid creating any unnecessary turbulent hydrodynamic flow. A slight drop in pH was routinely recorded as the electrode passed in proximity to the IBPD with a slightly more aci d pH than the entire simulated vaginal fluid (pH 4.48? 0. 02) around a superficial surface diameter of 5mm. The pH, measured away from the superficial layer was 4.58? 0. 03 (N= 3) . The relatively higher pH at the superficial layer may have been due to the extruding OH- ions from mPA 6,10 , EC, PAA or PVA. T he relatively lower pH observed at the IBPD proximity (micro - environmental pH) could most certainly have been due to the break down of PLGA into lactic and glycolic acids. This biphasic response in pH was obser ved over an experimental period of 30 days. A profile depicting this sequence is shown in Figure 6.5. Attempts were made to perturb the inherent pH of the simulated vaginal fluid by adding incremental volumes of simulated seminal fluid of pH 7.4 (a signif icantly > volume than would be produced on degradation of the PLGA - based IBPD in the simulated vaginal fluid) . After the addition of simulated seminal fluid, the pH recorded within the simulated vaginal fluid was 4.58+ 0. 02 (N= 3) and not significantly diff erent from the resting pH of 4.50 (P<0.01) . Upon further addition of simulated seminal fluid into the M PT - 2 , the inherent pH was reduced to 4.52? 0. 03 (N= 3) indicating that the simulated vaginal fluid was able to maintain its internal pH close to a value of 4.50 in the presence of a more alkaline medium (e.g. seminal fluid). Overall the results portrayed the potential of the IBPD to control and buffer the pH range of 3.5 - 5. 5 in the simulated vaginal fluid by the virtue of the degradation of PLGA to lactic and glycolic acids. This pH range is the same as that produced by Lactobacilli species (Boskey et al., 2001) which plays a vital role in keeping the vagina healthy. It also covers the average pH range of a normal healthy 155 human vagina which is 4 - 5 ( Smith, 1993; Ferris et al., 2006; WebMD, 2008) indicating the suitability of the IBPD for the intended purpose. Time (Days) 0 5 10 15 20 25 30 Mi cro- Env iro nmental C hange i n pH 0.0 1.5 3.0 4.5 6.0 7.5 9.0 Baseline Micro-Environmental pH IBPD device Upper Limit Lower Limit Figure 6.5: Micro - environmental pH variation in the simulated vaginal fluid containing the IBPD (N=3) . 6. 3. 3 Thermal anal ysis of the polymer constituents of the intravaginal bioadhesive polymeric device The thermal stability of the constituent polymers as well as the composite unhydrated IBPD was investigated by T MDSC at a temperature range from - 35- 230?C. The corresponding TMDSC thermograms are shown in Figures 6.6 to 6.13 . The polymers displayed multi- transitional thermal behaviors with multiple T g, T m and T c values (Table 6.5) that were attributed to the existence of reversing and non- reversing endothermic signals arising from the transient melting of molecules within each polymer. 156 Table 6.5: Critical thermal events evidenced by diverse temperature inflection peaks for the polymer constituents of the intravaginal bioadhesive polymeric device Sample Anal yzed Critical temperature transition points (?C) Tg Tc Tm mPA 6,10 163 120; 200 65; 140 PLGA 45 - 55; 210 230 22; 220 APE- PAA 90 60 30; 130 - 170 PVA 22; 115 163; 215 30; 180 EC 100 130 170 Hydrated polymer blend 70; 160 210 22; 163; 200 Unhydrated polymer blend 170 220 200 Hydrated IBPD 83; 163 160 - 10; 38; 85 Unhydrated IBPD 150 140; 220 222 Tg=Glass transition temperature Tc=Crystallization temperature Tm=M elting temperature 6.3. 3 . 1 Thermal characterization of the native unhydrated polymer constituents of the intravaginal bi oadhesive polymeric device 6.3. 3 . 1. 1 Ethyl cellulose (EC) EC was characterized by a T g at 100?C and an exothermic T c at 130? C (Figure 6. 6 ) . The T m of EC occurred at 170?C which was relatively high compared to the other polymers employed. This revealed the superior thermodynamic stability of the polysaccharide subunits of EC. 157 Figure 6. 6 : T MDSC thermogram for the unhydrated ethylcellulose. 6.3. 3 . 1.2 Modified polyamide 6,10 ( m PA 6,10) The mPA 6,10 had a distinct T g at 163 oC and two endothermic T m values at 65 ?C and 140?C. In addition, the exothermic T c peaks observed at 120?C and 200 ?C followed this melting phases. This suggested partial decomposition of mPA 6,10 that may be related to matrix erosion (Figure 6. 7 ) and the fact that the aliphatic polyamides inherently have a high heat of fusion and low entropy of fusion dependent on the collective dissociation energy of intramolecular H- bonds before any macroscopic dimensional changes can be realized ( de Candia et al., 1998; Murthy, 2006 ) . a ) Standard DSC Curve Total heat flow Non - reversing curve Reversing curve C C w R 158 Figure 6. 7 : T MDSC thermogram for the unhydrated polyamide 6,10 ( PA 6,10 ). 6.3. 3 . 1. 3 Poly(l actic -co- glycolic acid) PLGA was characterized by two T g values (Figure 6. 18). The first T g peak appeared during the initial heating scan in which an enthalpy of relaxation peak superimposed the T g between 20- 30?C. The second T g value at 210?C was also noted as PLGA is a quench cooled amorphous material ( Teixeira et al., 2005; Mainardes et al., 2006; Rouse et al., 2007) . Furthermore, therm ograms representing the total heat flow depicted a transient endothermic T m peak at 22?C which was a recovery of heat loss. As heating proceeded, the T g and the temperature at the apex of the overheating peak shifted to higher temperatures ( T g=210?C; T m=220?C) due to the formation of a percolated network structure resulting from the quench cooling properties of PLGA . b) Standard DSC Curve Total heat flow Non - reversing curve Reversing curve C C w Non-reversing curve R 159 Figure 6.8: T MDSC thermogram for the unhydrated poly (lactic - co- glycolic acid). 6.3. 3 . 1. 4 Polyvinyl alcohol PVA is a semi- crystalline polymer in which high physical polymeric chain interactions exist due to H- bonding between the OH- groups. Thermograms for PVA revealed two T g values at 22?C and 115?C and two endothermic T m peaks at 30?C and 180?C (Figu re 6.9 ) due to the thermal decomposition of the ordered PVA elements. Physical interactions between the polymer chains such as H- bonding contributed to the high enthalpy of fusion and T m values observed for PVA. Thermodynamic analysis of PVA isotherms and crystallite growth rates has shown that crystallization of PVA (163?C; 215?C) is one- dimensional ( Peppas and Hansen, 2003) . Thus, due to the absence of water or other swelling agents, kinetic hindrances predominated as a result of interactions between the OH- groups of PVA. c ) Standard DSC Curve Total heat flow Non - reversing curve Reversing curve C C w R 160 Figure 6. 9: T MDSC thermogram for the unhydrated polyvinyl alcohol. 6. 3. 3 . 1. 5 Allyl penta erythritol -crosslinked poly(acrylic acid) The salient thermal events for APE- PAA are shown in Figure 6.10 and Table 6.5. The broad endothermic peak ( 130- 170?C) resulted from conformational changes in the macromolecules of APE- PAA related to the magnitude of crosslinking interactions between the proton- donating pendent COOH- groups and polar OH- groups of APE- PAA during synthesis ( Sugama et al., 2007). The presence of COOH- groups favored the bioadhesiveness of APE- PAA within the pig vaginal tissue when used as a coating agent for the IBPD device. Figure 6. 1 0: T MDSC thermogram for the unhydrated allyl penta erythritol- crosslinked poly(acry lic acid). e) Standard DSC Curve Total heat flow Non - reversing curve Reversing curve C C w R d) Standard DSC Curve Total heat flow Non - reversing curve Reversing c urve C C w R urve 161 6. 3 . 3 .2 Thermal characterization of the u n hydrated intravaginal bioadhesive polymeric device Thermal analysis of the unhydrated IBPD revealed a T g at 150?C, two T c peaks at 140?C and 220?C and a T m peak at 220?C (Figure 6.11). The presence o f transient T m peaks in the total TMDSC signals for the unhydrated IBPD indicated that the polymers were dispersed within the device matrix. In addition, diminutive exothermic events were observed at the corresponding T c ranges for the constituent polymers indicating a high degree of crystallinity within the matrix structure. The deconvolution of the total TMDSC signals for the unhydrated IBPD in terms of reversing and non- reversing events reflected the average of the equivalent signals for each polymer. However, for the unhydrated IBPD the T m appeared to be predominantly reversing due to the concurrent re- crystallization and melting phenomena that offset one another. This indicated that solid- solid phase transitions may have occurred within the unhydrated I BPD due to polymeric compression, and subsequently contributed to the prolongation and control of drug from the device. Thus, the thermodynamic stability of polymer blends may affect the drug release process and can therefore be used to predict the drug release behavior based on unequivocally defined thermodynamic events. Figure 6. 11: T MDSC thermogram for the unhydrated IBPD . Standard DSC Curve Total heat flow Non - reversing curve Reversing curve 162 6. 3. 3 . 3 Thermal characterization of unhydrated and hydrated physical polymer blends as well as hydrated intravaginal bioadhesive polymeric device T MDSC analysis was also performed on hydrated and unhydrated physical blends of the constituent polymers of the IBPD as well as the hydrated IBPD in order to determine the effect of compression on the polymer blend. Thermograms obtained on the hydrated and unhydrated physical polymer blends as well as the hydrated and unhydrated IBPD are depicted in Figures 6. 1 2 and 6.13 and in Table 6.5. Overall, there was a distinct similarity between thermal events of the hydrated physical polymer blend and the hydrated IBPD (Figures 6. 1 6 a and b). Thermograms presented with regions associated with very low temperatures ( - 10?C and - 15?C ) for the hydrated samples of the physical polymer blend and the IBPD while dehydration was complete at 200?C (Figure 6. 16a and b) . For the hydrated physical polymer blend (Figure 6. 15 a), the onset of the low- temperature endothermic T m peak ( 22?C) was attributed to the high moisture content in the physical polymer blend while the apparent T m peaks (163?C and 200?C ) resulted from the loss of residual water as heating proceeded. The T m endotherms were distinctly separated from the total heat flow in the non- reversing signal. 163 Figure 6. 1 2: T MDSC thermogram for a) the hydrated physical polymer blend, b) the hydrated IBPD. T he thermal behavior for the unhydrated physical polymer blend was markedly different from that of the unhydrated IBPD (Figure 6. 1 3 a and b). This was attributed primarily to the effect of polymer compression on the physical polymer blend to produce the device. Contrary to the hydrated physical polymer blend, the unhydrated polymer blend showed fewer thermal events ( Figure 6.13a ). A single T g at 170?C and a T m peak at 200?C were observed (Figure 6. 13a). Furthermore, the T g and T m peaks that appeared for the Non-reversi curve Reversin curve Standar DS Curve Total heat flow Non-reversing curve Reversing curve Standard DSC Curve Non-reversing curve Reversing curve Standard DSC Curve Total heat flow Non-reversing curve Reversing curve a ) Standard DSC Curve Total heat flow Non-reversing curve Reversing curve b) 16 4 hydrated physical polymer blend prior to 170?C, were absent in the unhydrated polymer blend (Figure 6.1 3 a). This may be associated with a baseline transition at ?170?C in the reversing heat flow signal. Overall, the hydrated physical polymer blend presented with lower T m peaks ( 22?C and 163?C). These observations were consistent with previous results reported by Frushour (2004), where upon hydrating a physical polymer blend, the T m peak was reduced well below the onset temperature. Figure 6. 1 3 : T MDSC thermogram for a) the unhydrated physical polymer blend, b) the unhydrated IBPD . Standard DSC Curve Total heat flow Non-reversing curve a ) Standard DSC Curve Total heat flow Non-reversing curve b ) Standard DSC Curve Total heat flow Non-reversing curve c ) d ) Reversing curve b) Standard DSC Curve Total heat flow Non-reversing curve Reversing curve Standard DSC Curve Total heat flow Non-reversing curve Reversing curve a) 165 6. 3. 4 Anal ysis of the drug release behavior from the coated and uncoated optimized drug- loaded intravaginal bioadhesive polymeric device 6.3. 4. 1 Chromatographic separation of 3' -azido -3' -deoxyth ymidine and polystyrene sulphonate with methyparaben as an internal standard A UPLC assay method was used for quantifying the concentration of AZT and PSS released from the IBPD. The 3 dimensional chromatographic analysis of blank simulated vaginal fluid revealed complete separation with no interfering peaks at the retention times within the UV wavelength range of 200- 400 (AZT/PSS and MP) as shown by the typical representative 3D UPLC profile in Figure 6.14 . Chromatograms depicting the retention times for MP (internal standard), AZT and PSS in simulated vaginal f luid are as shown in Figure 6. 1 5 a and b. 166 Figure 6.14: A typical 3D UPLC profile showing a complete separation between AZT and MP (internal standard). 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 AU 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 Minutes 300.00 400.00 AZ T MP ? 167 Figure 6. 15: UPLC chromatograms depicting the separation of a) AZT and MP (internal standard) and b) PSS and MP (internal standard) in simulated vaginal fluid (pH 4.5; 37?C). 6. 3. 4.2 Assessment of the effect of coating on the drug release from the intravaginal bioadhesive polymeric device The shellac/APE - PAA- coated IBPD containing only one of the model hydrophilic drugs, AZT, demonstrated extended drug release when compared to the uncoated devices (28 vs 20 days) (Figure 6.16a and b). This was due to the shielding effect of the initial shellac undercoat and APE- PAA overcoat applied. Once the APE - PAA coating was hydrated the shellac gradually solubilised in a manner that diffusion channels formed within the coating layer. This facilitated the drug diffusion from the IBPD. Furthermore, shellac (used as an undercoat) shielded the device against the i ngress of release medium due to its wax - like 244nm 267nm a) AU 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 Minutes 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 AZ T (R t=3. 73min) MP (R t= 1. 48min) 0.660 1.438 AU 0.001 0.002 0.003 0.004 0.005 0.006 0.007 0.008 Minutes 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 P SS (R t= 1. 44 min) MP (R t= 0. 66min) b ) 244nm 168 properties (Sinha, 2003 ) . This may be attributed to its inherent moisture protecting properties, as well as its ability to act as a plasticizer. Figure 6.16: A typical profile showing the effect of coating on the model hydrophilic drug AZT from a) uncoated IBPD and b) shellac/APE - PAA- coated IBPD in simulated vaginal fluid (pH 4.5; 37?C) (N=3; SD<0.18 in all cases). 6. 3. 4. 3 Anal ysis of the drug release behavior from the optimized coated intrav aginal bioadhesive polymeric device containg AZT and PSS separately and in combination The substantial matrix integrity imparted by the polymers used to formulate the IBPD resulted in the minimization of the rate of matrix disentanglement and consequently prolonged and controlled the release of AZT and PSS from the IBPD. Controlled drug release representing zero- order was realized consistently over 40 days for AZT and 72 days for PSS (Figure 6.17 ) . These results can be attributed to the hydrophobic nature and high compressibility of EC, PLGA and PSS, coupled with the superior matrix resilience of mPA 6,10. The electrolytic nature of BaSO4 may have also contributed to the prolongation and control of drug release. The PSS- loaded IBPD achieved superior drug rel ease behavior with consistent and controlled release over a period of 72 days (Figure 6.17d ). For the IBPD loaded with both PSS and AZT, the release of PSS occurred over 56 days Time (Days) 0 4 8 1 2 1 6 2 0 2 4 2 8 Fractio nal Dr ug R ele ase 0 .0 0 .2 0 .4 0 .6 0 .8 1 .0 Un coated IBPD Coated IBPD 169 compared to 40 days for AZT (Figure 6.17b and c) . However this was still dimin utive in comparison to the 72 days achieved from the PSS - only loaded IBPD (Fig ure 6.17d ). Conversely, the period of AZT release from the IBPD was more prolonged than from the AZT - only loaded IBPD device (i.e. 40 v s 28 days) ( Figure 6.17a and b ). This was c lear that the inclusion of PSS had a significant role in controlling the release of AZT from the IBPD due to the hydrophobicity of PSS and electrostatic properties (as a sodium salt) arising from its polymeric segmental charge density ( Singh et al., 1995 ) . Studies have shown that charge density of polyelectrolytes such as PSS enhance binding interactions and favors oppositely charged compounds, culminating in the lowering of the rate of desorption and diffusion thereby slowing the drug release rate from the polyelectrolyte compound ( Singh et al., 1995; Vishalakshi, 1995 , Pu et al., 2004). The hydrophobicity of PSS results from the presence of strong electrostatic charges and internal linkages (H - and S- bonds) in the residual un- sulfonated aromatic moieties of the PSS molecule (Jiang et al., 2003; Bonifazi et al., 2007). In addition, previous studies conducted on polyelectrolytes such as PSS have shown that the higher the osmotic coefficient and radius of gyration, the greater the ability to control the rate of drug release ( Alvarez - Lorenzo et al., 1999; Griffiths et al., 2004; Le Cer et al., 2004; Sen et al., 2007; Thapa et al., 2009; Virid?n et al., 2009) . 170 Time (Days) 0 12 24 36 48 60 72 Fractio nal D ru g R ele ase 0.0 0.2 0.4 0.6 0.8 1.0 a) b) c) d) Figure 6.17: Drug release profiles of a) AZT (AZT- loaded IBPD), b ) AZT (AZT/PSS- loaded IBPD), c) PSS (AZT/PSS- loaded IBPD) and d) PSS (PSS - loaded IBPD), in simulated vagina fluid (pH 4.5; 37?C) (N=3; SD<0.38 in all cases). 6. 3. 5 Assessment of drug permeation across t he pig vaginal tissue The flux of AZT and PSS acros s the pig vaginal tissue over time is shown in Figure 6.18. A relatively constant increase in the rate of flux occurred over the initial 12 hours and thereafter a steady- state was achieved up to 24 hours (Figure 6. 18 ) . This suggests that the mechanism of passive drug transport across the pig vaginal tissue became saturated with drug. The lower flux of PSS reflects its high degree of hydrophobicity coupled with the presence of strong intermolecular charges in the polymer (Knudsen et al., 2004; Pu et al., 2004; Chu et al., 2007) . This may therefore have contributed to controlling the permeation of the drug across the vaginal tissue. Overall, it can therefore be proposed that most of the drug was retained within the vaginal tissue. 171 Figure 6. 18 : Profiles showing the flux of AZT and PSS across pig vaginal tissue over a period of 24 hours (N=3 , SD<0.23 in all cases ). 6. 3. 6 Ex vivo bi oadhesivity anal ysis of the intravaginal bioadhesive polymeric device The devices that were produced were strongly bioadhesive. Textural profile analysis indicated that the uncoated devices had the lowest bioadhesivity (PAF=1.1976? 0. 150N; AUCFD=0.0019? 0. 0001J) compared to the PAA - coated devices (PAF=3.699? 0. 0. 464N; AUCFD=0.0098? 0. 0004J) (Figure 6.19a and b). This indicates the superiority of APE- PAA as a bioadhesive coating as may be attributed to its hydrophilicity, H- bonding capacity, the high molecular mass and the surface tension properties of the polymer. APE- PAA controlled the extent of interpenetration betw een the polymer and the vaginal mucosal/epithelial surface. The high hydrophilicity of APE- PAA enabled the formation of strong bioadhesive bonds due to the high water content within the mucosal layer of the pig vaginal tissue. The presence of OH - and COOH- groups in APE- PAA may have favored the formation of H- bonds between the entangled APE- PAA chains and the pig vaginal tissue that ultimately resulted in bioadhesion (Chapter 4; Section 4.3. 2; Figure 4.4) . In addition, t he desirable surface tension of PAA facilitated spreading over the epithelial surface of the vaginal mucosal layer thereby enhancing bioadhesion. Time (Hours) 0 4 8 12 16 20 24 Mas s Flu x (mg.cm -2 .h- 1 ) 0.0 0.2 0.4 0.6 0.8 1.0 AZT PSS 172 Figure 6. 19 : Typical Force- Distance textural profiles used for computing the Peak Adhesion Force (PAF) and Work of Adhesion (AUC FD) for a) uncoated devices and b) PAA- coated devices on freshly excised pig vaginal tissue. 6.3. 7 Retention of the intravaginal bioadhesive polymeric device within the pig vagina Analysis of X - ray images (Figure 6.2 0 ) revealed that the devices were maintained in the posterior fornix of the pig vagina for the experimental period up to 30 days. The devices underwent swelling and gradually eroded over time as shown in Figures 6.20 a, b and c which is in accordance with the stipulated design in which the formulation is expected to initially swell in order to facilitate bioadhesion and thereafter gradually erode and release the drug over the vaginal tissue for the required clinical preventative effect. Distance (mm ) Distance (mm ) a a) b) 173 Figure 6.20: X - ray images depicting the presence of the intravaginal bioadhesive polymeric device at a) day 1, b) day 14, and c) day 30 after insertion into the posterior fornix of the pig vagina. a ) b) c) 174 6. 3. 8 Chemometric and molecular modeling of the intravaginal bioadhesive polymeric device drug dissolution and diffusion 6 . 3 . 8 . 1 Postulation of d issolution dynamics and subsequent effect on drug release Chemometric and computational analysis conducted in our laboratories revealed that polymer- polymer and polymer- drug ratios, as well as the ratio between the coating polymers and components of the dissolution medium contributed substantially to drug dissolution kinetics obtained. Figure 6.21 depicts a step- wise model of the IBPD undergoing dissolution. inner core shellac & castor oil layeringouter PAA layer dissolution AZT PSS Polymer strands Medium & its ingredients Hydrophilic areas Hydrophobic areas Hydrogen bondings & other ionic and molecular associations Water intakeCaplet ruptures Figure 6.2 1 : Molecular model mechanistically depicting the IBPD dissolution process with lesser H- bond formation due to the excessive of simulated vaginal fluid providing more freedom to polymeric strands to disentangle. Regarding the physicochemical associations of the polymers, AZT and PSS, all components were homogenized to produce a characteristic even distribution within the IBPD matrix. The hydrophile- hydrophobe properties, molecular interactive sites and functional groups in the mass and stoichiometry of the matrix was in a 2:3 ratio as depicted in Figures 6.2 2 a- c for a typical IBPD matrix. The core of the IBPD was a heterogenous matrix medium in the active solid- state and molecular properties of the polymers and excipients were in a dormant soli d- state. The dissolution medium provided the induction phase for the dormant properties to become activated. With the presence of excess dissolution medium and subsequent complete dissolution of the IBPD, this 175 weakened the interactions and physicochemical associations with no further channel formations that may not be conclusive for channelized diffusion of drug. The hydrophilic and hydrophobic areas of certain polymeric components within the matrix and their association provided the flexible hydration sites. The hydrophilic sites were located within the outer regions of the matrix and the hydrophobic sites were confined to fewer interactive regions at the center of the IBPD matrix. AZT was confined near hydrophilic regions of the matrix while PSS consolidat ed the inner core as well as areas associated with other hydrophobic polymeric interactions such as PLGA and EC. This segregated hydrophile- hydrophobe clusters within the IBPD matrix was primarily responsible for modulating the diffusion path of drug molecules through the matrix and subsequently controlling drug release. Figure 6.22 depicts the IBPD matrix in a solid and heterogeneous form as well as the inter- polymeric entanglements that involved the polymer- polymer, polymer- drug associations and other componential interactions. A B C D Figure 6.22: A 3D model representing the IBPD matrix under dissolution along with drug associations: a) the polymer matrix, b) fluid interactions producing hydrophilic pockets and swelling of the device, c) generation of hydrophobic areas in the matrix due to critical formulation excipients and d) the drug molecule (AZT) in association at hydrophilic sites prior to release. The chemometric and molecular modeling revealed that, remarkably, the rate of transport for the hydrophilic drug AZT compared to the hydrophobic PSS was found to be in a ratio of 3:2 indicat ing that AZT diffused at a rate that was 1.5 times faster than PSS. This explains the longer controlled release effect obtained when PSS was incorporated into the IBPD device. Through chemometric modeling, it was also ascertained that the difference in molecular mass between AZT and PSS was not solely responsible for this behavior and 176 that the high degree of charge density in PSS, the ionic interactions between the cationic PSS and the polymer constituents as well as the high osmotic coefficient and radius of gyration of PSS, were the primary contributing factors. Figure 6.23 depicts the polymeric strands in the dissolution medium, the free floating AZT in the simulated vaginal fluid and PSS occupying the inner core regions of the matrix, in comparison to the interactions/entanglement in the solid/semi - solid/gel state at the onset of the dissolution process when the IBPD device was not fully dissolved. Figure 6.2 3 : Chemometric models depicting a) dissoluted polymeric strands and b) AZT molecules (grey - colored circles) and PSS (white- colored circles) in the free float and associated forms with the loosely interacting polymeric strands in the dissolution medium. 6.3. 8 .2 Diffusion kinetics depicting the drug flux mechanism during ex vivo studies The presence of excess simulated vaginal fluid, led to complete dissolution of the caplet in the donor compartment of the Franz Diffusion Cell apparatus during the ex vivo vaginal tissue permeation studies weakening the interactions and physicochemical associations. It was observed that approximately 21% of AZT and 14% of PSS permeated across the pig vaginal tissue in 24 hours. Thus, the actual transport of the drugs (considering that 16.67% of both drugs i.e. 200m g each in a 1200mg IBPD matrix) from the donor compartment to the receptor compartment was 42mg and 28m g for AZT and PSS respectively. This finding substantiates the results obtained in Section 6.3. 5 for which it N O O N R H H H H H H H H H H H H H H H H H H H H HH H H HH H H HH H H HH RH H O O O O O H R H H H H H H2- O O O OH O CH3 O O CH3 O CH3 O OHOH O R R O O O O O R HH H H H H HH H H H H H H H R H H H H H H H O O O O O O O O R H H H H H H H H H H H H H H H HH RHH H a ) N O O N R H H H H H H H H H H H H H H H H H H H H HH H H HH H H HH H H HH RH H O O O O O H R H H H H H H2- O O O OH O CH3 O O CH3 O CH3 O OHOH O R R O O O O O R HH H H H H HH H H H H H H H R H H H H H H H O O O O O O O O R H H H H H H H H H H H H H H H HH RHH H b) 177 was proposed that most of the drug remained in the vaginal tissue. A chemometric model depicting the step- wise process of generating diffusion/transport channels perpendicularly to a polymer- strand localized in the IBPD is depicted in Figure 6.24 . A B C Figure 6. 24 : Chemometric model depicting the development of a diffusion channel with a) a single polymer strand P, situated perpendicularly to a forming pore C, b) a group of strands also denoted collectively as P giving rise to the channel C which is formed perpendicular to the polymer strands backbone with F=the direction of flow from the starting point S to form a a networked 3D channel and c) a polymeric s trand with the generated diffusion channel. With regard to the diffusion kinetics of the IBPD device, the pH of the simulated vaginal fluid had a contributory effect on the transport of drug across the pig vaginal tissue. An osmotic gradient mediated transport across the vaginal tissue based on the higher concentration of drugs, ions and other molecular entities in the 2mL donor compartment of the Franz Diffusion Cells. The permeation of drug was also inversely proportional to the molecular mass and almost all components i.e. ions, drugs, polymeric strands, protein, salts and acids as well as urea from the donor cell permeated though in varying concentrations. Figure 6.25 shows the actual events that occurred during the generation of diffusion/transport cha nnels within the IBPD matrix. 178 A B C D E F Figure 6.2 5 : 3D models depicting the generation of diffus ion channels with the IBPD matrix with a) polymeric strands, b) inter - strand physicochemical interactions, c) stereo- orientation within a group of associated polymeric strands generating an inter- polymeric cavity d) the cavity formed, e) associations between different polymeric strands and f) the blocked cavity due to excessive associative networking at various localized sites incorporating other components such as drug in different associative settings. The considerable energy paradigms were governed by the solvation of the dissolution medium and osmotic flux. The qualitative status of energy paradigms and energy - time relationships for the IBPD mat rix is as shown in Figure 6.26a - c. A simple energy status is depicted as a qualitative energy- time relationship for the IBPD matrix as clearly portrayed in Figure 6.26b. X-axis X-axis X-axis Y-axis Y-axis Y-axis T T T E E E 0 0 0 a) b) c) Figure 6.26: Qualitative status of ener gy paradigms and energy- time relationship for the IBPD matrix with a) stable energy status as a solid matrix, b) energy change following dissolution and c) energy transaction and changes for the drug permeation across the pig vaginal tissue with the static energy status at equilibrium without flux also shown. 6.4 Concluding remarks Robust polymeric devices were produced that confirmed the ability of the polymer blend selected ( PA 6,10 , PLGA, PVA, PAA and EC) to control the release of the model drugs AZ T and PSS over a period of 72 days (PSS) . The thermodynamic stability of the native 179 polymers and device was substantiated by TMDSC thermograms. Ex vivo bioadhesion and permeation studies revealed that the APE- PAA- coated devices were desirably bioadhesive and a relatively substantial fraction of the drug- load was confined within the vaginal tissue. The produced IBPDs showed the potential of maintaining the acidic microenvironmental pH of the simulated vaginal fluid upon degrading which is a desirable feature in the vagina. The chemometric and molecular structural modeling approach qualitatively supported the deduction of the IBPD rate of dissolution and has shown that the drug release rate was dependant on the stoichiometric parameters between the polymers, drugs and the simulated vaginal fluid. Furthermore, it was mechanistically deduced that the permeation of drug across the pig vaginal tissue during ex vivo studies was based on an osmotic gradient and depended on the degree of ionization as well as the size and molecular mass of the drug molecules. Thus, the developed IBPD may be suitable for use as a localized intravaginal drug delivery system for the potential treatment and prevention of HIV infection and STIs. 180 CHAPTER SEVEN INN VIVOO DRUU G CONTENT ANALYSIS AND HISTOPATHOLOGICAL EVALUU ATION OFF THE INTRAVAGINAL BIOADHESIVE POLYMERIC DEVICE 7.1 I ntroduction Prevention of sexual transmission of HIV at the vaginal mucosal epithelial level is a critical global health priority because of the devastating impacts of HIV/AIDS on women, young adults and infants (Keller et al., 2005). Studies using cervical organ culture as a model of primary infection, have demonstrated that HIV - infected cells mostly reside within subepithelial mucosa ( Miller et al., 19 92; Nuovo et al., 1993; Padian et al., 1997). This indicates that the establishment of HIV infection requires transepithelial penetration and therefore blocking this step may assist to prevent the HIV infection. HIV pathogens cross the mucosal surface either by: a) direct HIV infection of epithelial cells; b) transcytosis of HIV through epithelial cells; c) epithelial transmigration of HIV - infected donor cells; d) uptake of HIV by intraepithelial dendritic cells (e.g. Langerhans cells) and e) circumvention of the epithelial barrier through physical breaches ( Ibata et al., 1997; Shattock, 2001, Sa?di et al., 2009) . Furthermore, it has been shown that penetration of the mucosal surface by HIV to reach the submucosal target cells (macrophages, lymphocytes and dendritic cells) may be modulated by: a) supraepithelial factors, such as seminal complement components (opsonized HIV); b) epithelial factors released in the submucosal microenvironment, such as antimicrobial soluble factors, cytokines and chemokines; and c) by the potent intraepithelial and submucosal innate immunity ( Kozlowski and Neutra, 2003; Sa?di et al., 2009) . Following the translocation across the mucosal epithelium, HIV - 1 finds potential target cells in the dense lymphocyte and macrophage populations of the lamina propria. Cervical 181 mucosa, lamina propria and CD4 + T lymphocytes are early target cells for HIV - 1 and Simian Immunodeficiency Virus (SIV) in humans and support viral replication (Spira et al., 1996; Zhang et al., 1999; Collins et al., 2000; Greenhead et al, 2000; Gupta et al., 2002; Moore et al., 2004; Shen et al., 2009). Thus, the trajectory of anti - HIV microbicide delivery system development approach should preferably be towards agents that can block specific virus? host cell interactions at the vaginal mucosal epithelial level. Targeting HIV entry has been a favored approach because it is the first step in the process of HIV infection (D?Cruz and Uckun, 2004; Cutler and Justman, 2008) . An anti- HIV microbicide delivery system that can block transmission of HIV at the vaginal mucosal epithelial level may provide a realistic method of intervention worldwide. The s trong epidemiological association of inflammatory ulcerative venereal disease with HIV transmission and the observation that mucos al SIV transmission may be enhanced following thinning of vaginal epithelium by steroids such as progesterone implants suggests an important barrier role of the genital epithelium ( Marx et al., 1996; Royce et al., 1997 ) . Thus, f ollowing any study in the vagina that involves administration of a foreign body (tablet, caplet, ring etc), i t is imperative to undertake histopathological and histomorphological evaluation on the vaginal epithelium since it plays a vital role, both as a physical and as an immunological barrier, in preventing entry of bacterial and viral pathogens into host tissues ( Catalone et al., 2005; Hirbod and Broliden 2007; Rebbapragada and Kaul, 2007; Levinson et al., 2009) . Disruption of this barrier and the presence of genital tract inflammation are risk factors for HIV infection as well as other STIs (Coom bs et al., 2003; Catalone et al., 2005; Dhawan and Mayer, 2006; Sundstrom et al., 2007; Brenchley and Douek 2008; Kaul et al., 2008; Sheung et al., 2008; Broliden et al., 2009; Hendrix et al, 2009; Shafir et al., 2009) . Therefore, it is important for a vaginal delivery system to not only target STI pathogen s but also to have negligible or no toxic effects on the vaginal epithelium ( Levine et al., 1998; Stafford et al., 1998; Greenhead et al, 2000; Miller and Shattock, 2003; Shen and Iwasaki, 2006; Iijima et al., 2008 ) . 182 The aim of this Chapter therefore, was to report on the in vivo study that was conducted in a pig model administered with AZT and PSS - loaded IBPD with the following purposes: i) to analyze the dru g content contained in plasma (if any) as well in the vaginal tissues after sacrificing the animals; and ii) to conduct a thorough toxicity study that involved histopathological evaluation on the vaginal tissues exposed to drug - containing IBPDs and placebo in comparison to a control. In each case, the IBPDs (properly coated to avoid irritation to the vaginal tissue as well as to facilitate easy insertion) were inserted into the vagina using a special applicator designed in our laboratories. A pig model was selected because of many similarities with the human vagina, particularly in genital tract physiology and histology. (Pond and Houpt, 1988; D?Cruz et al., 2005b ; Wang et al., 2009 ) . 7.2 Materia ls and Methods 7.2.1 Materials For anesthesia, ketamine was purchased from Bayer (Pty) Ltd ( Bayer Pty Ltd, Isando, Gauteng, South Africa., m idazolam from Roche products (Pty) Ltd ( Roche products (Pty) Ltd, Isando, Gauteng, South Africa) and isoflurane from Safeline Pharmaceuticals (Pty) Ltd (Safeline Pharmaceuticals (Pty) Ltd, Florida, South Africa) . The digestive enzyme for the vaginal tissues was subtilisin which was purchased from Merck Co. Ltd (Merck - Schuchardt? , Hohenbrunn, Germany). The rest of the materials employed were the same as in Chapter 6. 7.2.2 Methods 7.2.2.1 In vivo studies in the pig model using the drug- loaded intravaginal bioadhesive polymeric device The study involved 20 female, healthy Large White pigs (35kg) divided into 4 groups of 5 each. G roup 1 was a control group in which no IBPDs were inserted. Groups 2 and 3 were used for testing the model drugs, and therefore IBPDs with drugs were inserted. Group 4 was used as a placebo group in which IBPDs with no drug ( i.e. formulated with 183 native polymers) were inserted. The study period for each group was 1 month spanning across a total of 4 months in staggered manner. In Group 2 and Group 3, IBPDs containing AZT and PSS respectively were inserted after anaesthetizing the pigs. X - ray imaging was done three times a week (1 st, 3 rd and 5 th day) for two weeks and then twice a week (1 st and 5 th day) for another two weeks , each time under anesthesia. Furthermore, blood samples (10mL) were withdrawn from the jugular vein of each pig on day s 1, 3, 5 , 7, 14 and 28 while the pigs were still under anesthesia. In G roup 4 , placebo IBPDs were inserted and then subjected to the same procedure as above. G roup 1 was subjected to blood sampling procedures on the scheduled days. On the 28 th day, each pig was euthanized, followed by dissection of the vaginal tissue for drug content analysis using UPLC, and toxicity studies. The toxic ity studies involved histological examination for inflammation, exocytosis, hyperplasia, hypoplasia, epithelial exudate, ulceration, polymorphonuclear infiltration and any evidence of infection. A summary of the in vivo study is shown in Figure 7. 1 . 184 Figure 7.1: Summary of the in vivo study. 7.2.2.2 Ane sthesia, X -ray imaging and blood sampling Each group of pigs was anesthetiz ed with ketamine (11mg/kg I.M. ) and midazolam, (0.3mg/kg I.M. ) . The pigs were then intubated and anesthesia was maintained with 2% isoflurane in 100% oxygen . X - ray imaging of the pelvic region was performed (Figure 7.2a), and blood sam ples were taken following the procedure stipulated in Section 7.2. 2. 1 (Figure 7.2b). All blood samples were immediately transferred into heparinised vacutainers (BD Vacutainer ? , Plymouth, BD Beliver Industrial State, UK) and then stored 20 Large White Pigs 5 Pigs Controls with no IBPDs intravaginally administered 5 Pigs for IBPDs with PSS adminstration 5 Pigs for IBPD s with AZT adminstration 5 Pigs for IBPDs with no drug (placebo) adminstration 5 pigs no t anaesthesized Anesthesia to 5 pigs for intravaginal administration Anesthesia and sampling of 10ml blood from the jugular vein at 1 , 3, 5, 7, 14, 21, 28 days for analysis by UPLC Anesthesia for X - ray and sampling of 10ml blood from the jugular vein at 1 , 3, 5, 7, 14, 21, 28 days for drug content analysis by UPLC Euthanasia and vaginal tissue removal for UPLC analysis Euthanasia and vaginal tissue removal for UPLC analysis of AZT and PSS Toxicity studies Toxicity studies 0 0 185 in a refrigerator at 4 oC for one hour. The blood was then centrifuged (Nison Instrument (Shangai) Limited, Shangai, China) at 15,000rpm for 10 minutes and the supernatant was pipetted as blank plasma using an adjustable volume micropipette (Boeco Gmbh, Hamburg, Germany). The blank plasma was stored in a refrigerator at - 70 oC for the quantitative drug analysis using UPLC. Figure 7.2: a) X - Ray imaging process and b) blood sampling procedure from the jugular vein of the pig. a ) b) 186 7.2.2.3 Vaginal tissue removal for drug anal ysis and histopathological studies On 28 th day, each pig (weighing between 80- 85kg) was euthanized and then dissecting out the vaginal tract following the procedure described in Chapter 5, Section 5.2. 2. 4 , Figure 5.1. Transverse cuts of tissues (at the anterior, middle and posterior part of the vagina) with a cross- sectional size 1.5? 3. 0cm were made through the vaginal wall. Half of the tissues were kept in 100mL of 10% formalin and then subjected to histopathological studies (including t oxicity evaluation). The remaining tissue was immediately frozen using liquid nitrogen, and then stored in a refrigerator at - 70 oC for quantitative drug analysis. 7.2.2.4 Quantitative anal ysis of AZT and PSS in the blood and the vaginal tissue of the pig. 7.2.2 . 4. 1 Preparation of standard solutions and calibration curves The same procedure as previously described under Chapter 6, Section 6.2. 2. 8. 4 , was used except that in this case calibration curves were developed using blank pig plasma instead of simulated vaginal fluid. The constructed calibration curves are as shown in Figures 7.3a and b . 187 Figure 7.3: a) Calibration curves for: a) AZT in blank plasma 267nm (N=5, SD<0.001 in all cases); b) PSS in blank plasma 244nm (N=5 , SD< 0.009 in all cases). 7.2.2 . 4. 2 The drug extraction method from the plasma samples for UPLC anal ysis This was conducted employing a single use Water Oasis? HLB 3cc (60mg) extraction cartridge (Waters Corporation, Milford, Massachusetts, USA) adapting a method Y=0.0291+0.0029x R2=0.99 Concentration of AZT (ng/mL) 0 2000 4000 6000 8000 10000 Ar ea (P DA : 2 67nm AZT ) 0 5 10 15 20 25 30 AZT Linear Regression Curve 95% Confidence Band 95% Prediction Band a) Y=0.1437+0.0039x R2=0.99 Concentration of PSS (ng/mL) 0 2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0 1 0 0 0 0 Ar ea (P DA : 24 4nm PSS ) 0 1 0 2 0 3 0 4 0 PSS Linear Regression Curve 95% Confidence Band 95% Prediction Band b) 188 developed by Notari and co- workers (2006). The Solid Phase Extraction (SPE) cartridge was conditioned with 1.0mL methanol followed by 1.0mL ultra pure water Milli - Q. 1mL of acetonitrile (for AZT) was added to 600?L of pig plasma. The solution was vortexed for 1 minute and then centrifuged ( Nison Instrument (Shangai) Limited, Shangai, China) at 15,000 rpm for 6 minutes at 24 oC. 650?L of the supernatant was diluted by adding ultra pure water Milli- Q (1mL) a nd loaded in the cartridge. The reafter, the cartridges were washed with 1.0mL of 5% v/ v methanol in ultra pure water Milli- Q. Analytes were eluted by washing cartridges with 550?L 0.01M KH 2 PO4 followed by 2mL methanol. The eluate was evaporated to dryness in a slow stream of high purity nitrogen gas (Afrox, Germiston, Gauteng, South Africa). The extracted sample was re- constituted with 100?L absolute methanol, mixed with 400?L of MP, and then filtered into the injection vials using 0.22?m syringe- driven filter units (Millipore Cor poration, Bedford, Massachusetts , USA) for UPLC analysis. The same procedure was followed for PSS but substituting acetonitrile with methanol as a mobile phase. 7.2.2 . 4. 3 Drug extraction from the vaginal tissue for UPLC anal ysis To extract the drug from the tissues, a method developed by Wang and co- workers (2009) was adapted but with some modifications. Briefly, each tissue sample (8g each) was homogenized in 16 mL of simulated plasma (pH 7.4) . The homogenized sample was digested by incubating it with 10 mg of subtilisin (Merck - Schuchardt? , Hohenbrunn, Germany), vortexed for 1 minute, and then placed in a thermostatic bath for 1 hour at 56?C while mixing after every 10 minutes to ensure complete degradation of the tissue. After enzymatic digestion, the sam ple was centrifuged at 10,000rpm for 15 minutes . For the AZT sample preparation, 1mL of the supernatant was withdrawn, mixed with 1mL of acetonitrile and then pipetted into the centrifuge tubes into which 550?L of 0.01M KH 2 PO4 was added as an extracting medium. The tubes were vortexed for 1 minute and then centrifuged at 13,000 rpm for 6 minutes at 24 oC. 650?L of the supernatant was diluted by adding ultra pure water Milli- Q (1mL) followed by the addition of 2mL absolute methanol. 189 The supernatant was placed into sample vials and then evaporated to dryness in a slow stream of high purity nitrogen gas (Afrox, Germiston, Gauteng, South Africa). The samples were re- constituted with 100?L absolute methanol mixed with 400?L of MP, then filtered into the injection vials using 0.22?m syringe- driven filter units (Millipore Corporation, Bedford, Massachusetts , USA). The same procedure was followed for the PSS sample preparation but substituting acetonitrile with methanol as the mobile phase. 7.2. 2.4 .4 Instrumentation and chromatographic conditions for the quantitative anal ysis of AZT and PSS from plasma and vaginal tissue samples Quantitative analysis was performed using the Waters Acquity Ultra Perf ormance Liquid Chromatographic (UPLC) system (Waters Corporation, Milford, Massachusetts , USA), equipped with the Acquity Photodiode Array ( PDA) and Evaporative Light Scattering (ELS) detec tors. The columns employed were Acquity UPLC ? BEH Phenyl 1.7?m , 2.1? 50mm column and Acquity UPLC ? BEH C18 , 1.7 ?m , 2.1? 100mm for AZT and PSS analysis respectively. The mobile phases were composed of water/acetonitrile (60/ 40 v/ v) and methanol/water (50/50 v/ v) for AZT and PSS respectively. The wash solutions used, namely strong and weak washes were composed of 90/10 acetonitrile/water, 10/90 water/acetonitrile, and 100% ultra pure water Milli - Q , respectively. All prepared solutions were filtered using 0.22?m membrane filters (Millipore Corporation, Bedford, Massachusetts, USA) under vacuum and degassed before their use. For AZT analysis, a gradient method was used at a column temperature of 25?C, injection volume was 2?L and UV detection wavelength of 267nm. The employed gradient settings were as shown in Table 7.1. For PSS analysis, an isocratic method was employed using methanol/water (50/ 50 v/ v) as a mobile phase, flow rate 0.2mL/minute, column temperature 25?C, total run time of analysis was 3 minutes, injection volume was 1.7?L and UV detection wavelength of 244nm. 190 Table 7.1: Parameter settings for the AZT gradient method. Separation events Time (min) Flow (mL/min) % A (Water) % B (Acetonitrile) 1 0.00 0.500 60. 0 40. 0 2 1.00 0.500 5. 0 95. 0 3 2.60 0.500 5. 0 95. 0 4 3.50 0.500 60. 0 40. 0 7.2. 2. 4. 5 Histopathological evaluation of the vaginal mucosa The vaginal tissue specimens obtained were cut into three tissue blocks containing the anterior, middle and posterior sections. These blocks were processed with routine histological methodology in an automated tissue processor. They were then sectioned at 5?m, placed on slides and stained with haematoxylin and eosin in an automated stainer ( Rankin Biomedical Corporation, Michigan, USA) and finally subjected to thorough histological evaluation. The evaluation was divided into three parts. The first was an evaluation on epithelial histological lesions; the second one was an assessment of the lamina propria and the third was an evaluation on the subepithelial tissues and vaginal wall. The evaluation of epithelial histological lesio ns encompassed: i) hyperplasia which is an increase in epithelial cell layers which finally results in acanthosis and thickening of the epithelium; ii) exocytosis which refers to transepithelial leukocyte migration of inflammatory cells; iii) exudate on the epithelial surface; and iv) ulceration. In the lamina propria, mononuclear inflammation, polymorphonuclear infiltration and foreign body inflammation were assessed. The evaluation of subepethelial tissues and vaginal wall was mainly concerned with a search for perivascular inflammation. 7. 3. Results and D iscussion 7.3. 1 Chromatographic separation and retention times of AZT and PSS from m ethylparaben (MP) (Internal S tandard) AZT/PSS and MP were eluted at 1.701? 0. 43 and 1.184? 0. 18 for AZT and 0.583? 0. 15 and 1.275? 0. 13 for PSS after extract ion from pig plasma (Figures 7.4a and b). Assay method 191 validation analysis revealed that the intra- and inter- day precision and accuracy were satisfactory (R 2 =0. 9987 for AZT; R 2 =0. 9998 for PSS). The signal to noise ratio at the lower limit of quantification (25ng/mL) was >10. 8 in both cases. For the validation testing of the linear regression model, the weighting factors were selected to be proportional to the reciprocal of the standard deviations which was optimal under the least squares estimation with R 2 >0. 99. The three dimensional chromatographic analysis of blank plasma revealed no interfering peaks at the retention times within the UV wavelength range of 200 - 400 (AZT/PSS and MP). Figure 7.4: UPLC chromatograms depicting the retention times for the standard solutions of a) AZT, b) PSS and MP as an internal standard in blank plasma. 1.184 1.701 1.184 1.701MP a) AZ T 0.583 1.275 AU 0.002 0.004 0.006 0.008 0.010 0.012 Minutes 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 MP PSS b) 192 7. 3.2 AZT and PSS c oncentrations in the blood and vaginal tissue of the pig model The drug concentration in the plasma from day 1- 28 ranged between 0.012- 0. 332mg/mL (AZT) and 0.009- 0. 256 mg/mL (PSS) (N=5) (Figure 7.5 a) . This indicates that only a minimal quantity of the AZT and PSS crossed into the systemic circulation. This may be attributed to the hydrophobicity and high matrix resilience of the poly mers employed. A steady- state concentration of AZT/PSS was observed from day 14 throug h day 28 indicating that no further drug crossed into systemic circulation. The lower concentration value of PSS may be due to the presence of strong intermolecular forces in the polymer that reduced the rate of permeation of the drug into blood compartment ( Pu et al., 2004; Chu et al., 2007). The mean drug concentration in the vaginal tissue at day 28 was 1.2148? 0. 062mg/mL (N=5) for AZT and 1.4004? 0. 071mg/mL (N=5) for PSS , while the plasma concentration was 0.332? 0. 014mg/mL (N= 5) for AZT and 0.256? 0. 013mg/mL (N=5) for PSS (7.5b). This is an indication that the majority of the drug was retained in the vaginal tissue. For each pig, the v aginal tissue drug concentration at three vaginal sites (namely anterior, middle and posterior) was approximately the same (P>0.05) . On average, PSS presented with the highest vaginal tissue drug concentration at the 28 th day (Figure 7.5b). This indicates that PSS was better retained in the vaginal tissue than AZT. This may be attributed to the high degree of hydrophobicity coupled with the presence of strong intermolecular charges in PSS. Figure 7.6 depicts a typical profile for the PSS content in the vaginal tissue. The finding corresponds well with the earlier results of drug flux analysis (Chapter 6, Section 6.3. 5) whereby PSS had lower flux values, indicating that its permeation rate across the vaginal tissue was slower compared to AZT. Likewise, the results of the analysis of drug concentration in the plasma revealed that a lower concentration of PSS crossed into the blood compared to AZT. Overall, the results portrayed a direct correlation (R 2 = 0.99) between the drug concentration in the blood and in the vaginal tissue (Figures 7. 7 a and b). 193 Figure 7.5: a) AZT and PSS plasma concentration profiles (N=5, SD<0.01), b) AZT and PSS mean vaginal tissue and plasma concentration values on day 28 (N=5, SD< 0.014 in all cases) . Figure 7.6: A typical profile depicting the content of PSS in the vaginal tissue. Time (Days) 0 4 8 1 2 1 6 2 0 2 4 2 8 Plasma Co ncentratio n (mg/mL) 0 .0 0 .1 0 .2 0 .3 0 .4 PSS AZT a ) AZT PSS Co ncentrati on (mg/mL ) 0 .0 0 .2 0 .4 0 .6 0 .8 1 .0 1 .2 1 .4 1 .6 Vaginal Tissue Plasma b ) 0.607 1.038 AU 0.002 0.004 0.006 0.008 0.010 0.012 Minutes 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 MP PSS 194 Figure 7. 7 : Correlation between a) AZT, b) PSS concentrations in the blood plasma and vaginal tissue for the 5 pigs tes ted at the 28 th day. On the 28 th day, the IBPD was still found retained in the vagina. The adherence o f the IBPD to the vagina was mechanistically deduced (in our laboratories) to be a result of the role played by the components forming the vaginal epithelium and the polymers of the IBPD as depicted in Figure 7. 8 . However, the IBPD was found to have broken into several parts (close to the cervix) and most of it had eroded (Figure 7. 9 ). A gel - like soft matrix was also observed at the vaginal epithelial lining. This is an indication that the IBPD was Y=0.0024+3.2796x R2=0.99 AZT Content in Plasma (mg/ mL) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 AZT Co ntent in Vaginal Tis su e (mg/mL ) 0.0 0.2 0.4 0.6 0.8 1.0 AZT Linear Regression Curve 95% Confidence Band 95% Prediction Band a) Y=0.0019+3.2179x R2=0.99 PSS Content in Plasma (mg/ mL) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 PS S C onte nt in V aginal T iss ue (mg/mL) 0.0 0.2 0.4 0.6 0.8 1.0 PSS Linear Regression Curve 95% Confidence Band 95% Prediction Band b) 195 degrading slowly into a gel- like matrix that adhered to the vaginal walls. These findings correlate well with the objectives of the study whereby the IBPDs were supposed to degrade, adhere to the vaginal walls and release the ARV and microbicide simultaneously. Figure 7.8: A schematic modeled in our laboratories depicting the mechanistic approach of adhesion between a polymer and the vaginal epithelium. Figure generated on ACD/ I - Lab, V5.11 (Add - on) software (Advanced Chemistry Development Inc., Toronto, Canada, 2000) . . . . . . . . . . . . . . . Epithelial cells Collagen Binding Area Non - Frle- helix Collag en Protein Binding Area Deformed Protein Polymer Binding Area Polymer Chain Distorted Polymer Backbone Polymers' Functional & End Groups Ionic Interaction Sites Water, Sugar & other Mucosal Materials 196 Figure 7.9 : Intravaginal Bioadhesive Polymeric Device (IBPD) as observed in the excised pig vagina on the 28 th day. 7.3. 3 Histopathological evaluation of the vaginal tissue In this study, negative to mild epithelial hyperplasia was observed (Figure 7. 10a) except for pig number 3, 9 and 14 which presented with moderate to severe hyperplasia (Table 7.2). This was noted from an increase in the epithelial rete ridges (Figure 7.10b). The controls showed no epithelial hyperplasia thus showing normal vaginal epithelium (Figure 7.10c) (Table 7.2) . The reason for the moderate to severe hyperplasia observed in pig number 3, 9 and 14 may have been due to the small vaginas of these pigs. In these pigs, the insertion of the IBPD was achieved with some di fficulty resulting in a relatively higher degree of inflammation. Some irritation from the polymeric device was most likely responsible for the hyperplastic epithelial response. Regarding exocytosis, neutrophils were detected in intercellular spaces in the vaginal epithelium, in the process of migrating to the vaginal lumen. The exocytosis varied among the different test groups as well as the placebo, but was not found in the control animals. The exudate attached to the muc osal surface followed the transepithelial migration and exocytosis of neutrophils into the lumen. This was found in only one of the animals 197 receiving PSS and two that received AZT (Figure 7.10d ). However it was not observed in the placebo and control pigs. Ulceration in the vagina refers to the complete loss of superficial epithelium due to an underlying submucosal inflammatory process and was confirmed only in a few animals from the AZT group (Table 7.2), and more so in pig number 9 which again may have been caused by the epithelial damage or necrosis during the insertion process due to the small size of the vagina. M ononuclear inflammatory cells (lymphocytes and plasma cells) were observed in the lamina propria and stromal tissue directly underneath the vaginal epithelium (Figure 7.10e). As shown in Table 7.2, (Figure 7. 10e), moderate mononuclear inflammation was recorded in PSS (N =1) and AZT- containing IBPDs (N =2) as well as placebo animals (N =1) , but was not visible in the controls. P olymorphonuclear cells ( eosinophils and neutrophils) , were found in the submucosa as well as in the superficial exudate. These cells originated from the circulation and migrated into the tissues from the blood vessels and also by exocytosis into the lumen of the vagina. The mononuclear and polymorphonuclear inflammation indicates that there was mild to moderate chemotaxis of polymorphonuclear cells as well as an antigenic stimulation of mononuclear leukocytes arising from the polymeric devices. This is normal for any foreign object introduced onto an epithelium. In pig number 9, a foreign body granuloma and inflammation was observed in the focal area of the vaginal epithelium. The foreign material appeared as homogenous eosinophilic staining and was surrounded by numerous macrophages, epithelioid cells and multinucleated foreign body giant cells. This reaction was also associated with ulceration in the epithelium. Possibly the foreign body reaction was related to the traumatic insertion process in this pig. The perivascular inflammatory infiltrates consisted mainly of mononuclear cells with few polymorphonuclear infiltrates dispersed among the mononuclear cells in the submucosa and the wall of the vagina (Figure 7.10 f) ( Table 7.2). It was negative to mild and could be demonstrated in just few individual pigs (PSS: N =1; AZT : N=2; placebo: N =1) . 198 Figure 7.10: Histological images of haematoxylin and eosin stained pig vaginal tissue samples depicting a) Epithelial hyperplasia of the vagina x 40, b) Epithelial hyperplasia, exocytosis and superficial exudate x 40, c) Lamina propria with no inflammatory cells and normal vagial epithelium x 20, d) Hyperplastic epithelium with exocytosis x 40, e) Mononuclear cell infiltrates in the lamina propria and f) Perivascular inflammation in the submucosal wall x 40. a b c d e f a b c d f a ) b) c ) d) ) f) 199 Table 7.2: Histo pathological findings in the vaginal tissue 7.4 C oncluding R emarks The results of the drug content analysis in t he blood and vaginal tissue revealed that only 0.332mg/mL (16. 78 % ) of AZT and 0.256mg/mL (12. 94 % ) of PSS crossed into the systemic circulation, indicating that the majority of the drugs was not absorbed and was thus in the vaginal lumen and possibly the tissues. This shows the superior ability of the employed polymers in retaining the drugs within the vaginal tissue. The observed gradual erosion of the IBPD and the subsequent adherence of its byproducts to the vagina is a clear indication that the developed IBPDs had the required matrix integrity attributes and were also substantially bioadhesive. The microscopical examination of the vaginal tissue following the termination of the pigs revealed that the histopathological changes (e.g. --------1 +-1 +1 +_2 +---1 +--Perivascular inflammation Submucosa and vaginal w all -----------3 +--------Foreign body inflammation 1+1+1 +1 +1 +1 +2 +-1 +1 +2 +1 +1 +1 +2 +-1 +Polymorphonucle ar infiltration -----1 +1 +2 +1 +1 +2 +2 +1 +1 +1 +1 +_2 +1 +-Mononuclear inflammation Lamina propria -----------2 +--1 +-----Ulceration ----------1 +--1 +2 +--2 +1 +-Exudate on epithelial surface -----1 +2 +2 +1 +1 +1 +2 +1 +1 +2 +1 +1 +2 +-1 +Exocytosis -----1 +2 +1 +1 +1 +1 +3 +1 +1 +-1 +1 +2 +1 +1 +Hyperplasia Epithelium Histopathologi cal findings 2019181716151413121110987654321Animal No. Negative controlPlaceboIBPD with AZT IBPD with PSS Tested item --------1 +-1 +1 +_2 +---1 +-- bm c a and ag nal w all -----------3 +-------- 1 +1 +1 +1 +1 +1 +2 +-1 +1 +2 +1 +1 +1 +2 +-1 + -----1 +1 +2 +1 +1 +2 +2 +1 +1 +1 +1 +_2 +1 +-M am na a -----------2 +--1 +----- ----------1 +--1 +2 +--2 +1 +-x -----1 +2 +2 +1 +1 +1 +2 +1 +1 +2 +1 +1 +2 +-1 +x -----1 +2 +1 +1 +1 +1 +3 +1 +1 +-1 +1 +2 +1 +1 + t h l m t at h l g cal f nd ng 118171615141311118765431n m al . gat c nt ll ac bw th Zw tht d t m - Negative 1+ Mild 2+ Moderate 3+ Severe 200 inflammation, ulceration) were either negative, mild or moderate, which was pathologically or clinically a sign of non- toxicity. The developed microbicide- ARV delivery system meets most of the essential requirements of an ideal microbicidal delivery system with characteristics that are defined as desirable in that they have shown substantial retention to the vaginal epithelium with very low systemic absorption and high permeation of the drug within the vaginal tissue. 201 CHAPTER EIGHT C ONC L US IONS AND R E C OMME NDAT IONS 8.1 C onclusions An effective anti- HIV intravaginal microbicide delivery system represents a practical and accessible means of reducing male to female sexual transmission of HIV. However, successful design and development of such a system brings with it a multitude of challenges, evidenced by a need for efficacy, acceptability, accessibility and affordability. Thus, exhaustive efforts have been made during the design and development of an IBPD in this study to address these challenges, leading to the successful achievements of the aim and objectives. The c urrent status of the treatment modalities of HIV/AIDS has been well described. Factors such as vaginal physiology, vaginal pH, the type and the intrinsic properties of the polymer vehicles as well as the drugs employed have been well addressed. N umerous safe and effective HIV vaginal microbicide delivery systems are still being evaluated at various stages in clinical trials, meaning that this research has been conducted at a period when the successful development of an effective anti- HIV intravaginal microbicide delivery system is still an ongoing agenda. Polymer ic biomaterials appear to have great potential for intravaginal drug delivery development because of their peculiar physicochemical and physicomechanical properties which allow delivery and maximization of the drug concentration at the required site, ultimately resulting in improved efficacy of drug therapy. Striking a balance between the hydrophobicity and hydrophilicity was an essential criterion upon which polymer selection was based using the equilibrium swelling ratio. The polymeric materials employed after undergoing a 202 thorough screening have resulted in the development of a delivery system that demonstrated the potential for providing controlled release over a period of 72 days . This is a significant achievement in the current research. The major prerequisite for the developed IBPD was proper matrix integrity whic h would eventually ensure highly controlled drug release and substantial bioadhesion ( retention) to the vaginal epithelium. To attain these goals, this research utilized multifunctional polymers with intrinsic properties that could lead to the development of a delivery system with a high degree of matrix inte grity and subsequently controlled drug release. Successful optimization mainly based on polymer matrix erosion as a measure of matrix integrity, and was carried out using the most recent highly- enabled, mathematically- based technology of Artificial N eural N etworks. Extensive bioadhesiv ity testing on the employed polymers on both a simulated artificial membrane and on freshly exercised pig vaginal tissue under conditions simulating the vaginal fluid pH demonstrated the bioadhesivity capacity of the developed intravaginal drug delivery system. Deduction of the transient mechanisms of bioadhesivity and the associated interactive energy paradigms during caplet adhesion to the freshly excised pig vaginal tissue was successfully carried out using computational and structural modeling techniques. Economic and social conditions as well as patient?s preference are prime factors that needed to be considered during the design and development of the IBPD . Acceptability depends on affordability, ease of use and minimal side- effects. Failure in this regard would result in non- compliance and non- adherence, amounting to no progress with the intended mission. Thus, it was absolutely important to conduct a thorough scientific evaluation of the physicochemical and physicomechanical properties of the IBPDs. The benefit of this evaluation, attested to by the good matrix hardness, matrix integrity , 203 thermal stability and acceptable friability of the IBPD , also ensured safety, functionality and efficacy and therefore its suitability as a microbicide- ARV delivery system. Furthermore, the highly controlled release pattern shown by the IBPD , the permeation dynamics obtained and the fact that the IBPD could adhere to the vaginal epithelium, satisfied the formulation requirements. This entailed, among other things, the ability of the IBPD to maintain a steady- state concentration of the bioactives within the vaginal tissue. Numerous challenges are currently faced by the pharmaceutical industry. There fore, there is a pressing need for new and innovative technology in drug delivery, for preventing HIV/AIDS and STIs . For this reason, novel techniques and appropriate materials were employed in this study which produced a robust IBPD with the following important attributes: a) the IBPD could remain stable in simulated human vaginal fluid (pH 4.5) and in simulated human seminal fluid (pH 7.4) for more than 30 days; b) the IBPD was substantially vaginally bioadhesive both ex vivo and in vivo; c) the IBPD could provide controlled release rate of the microbicide- ARV ensuring the potential for prolonged protection in women; and d) the IBPD was non- toxic. Thus, the novel developed controlled release microbicide- ARV loaded IBPD may significantly contribute in solving the problems currently faced in drug delivery technology development and may lead to the following: i) provide an al ternative preventive measure in the transmission of HIV/AIDS; i i) reduce the burden of HIV/AIDS; iii) improve the quality of life with subsequent macroeconomic benefits; iv ) reduce the cost of medication; v) develop products locally; v i) create intellectual property; vi i) overcome the challenges of current intravaginal drug delivery systems; and vi ii) a versatile IBPD may be suitable for the treatment of other diseases burdening developed and developing countries. 8.2 R ecommendations Although prevention of HIV infection is an overwhelming task, a human, female- controlled anti- HIV intravaginal microbicide delivery sys tem points to promising future for success in 204 the fight against HIV - 1 with the eventual eradication of the HIV pandemic. The development of an ideal anti- HIV intravaginal microbicide delivery system that will pass through all clinical trial phases will be achieved through focused and endeavored scientific efforts. The work presented in this thesis provides a significant contribution towards this goal. Local delivery of an anti - HIV intravaginal microbicide to the vaginal compartment at the correct time and concentration is a key requirement, and this presents a formidable challenge to overcome if the potential for microbicides in preventing HIV infection is to be realized. This r esearch has identified priority challenges and opportunities for pre- competitive research in intravaginal anti- HIV microbicide delivery for the prevention of STIs and HIV infection. Much needed further research should gain greater interactive- based understanding of the physicochemical and physicomechanical properties of biomaterials ( in addition to polymers), microbicidal agents and anti - HIV drugs . Such studies will discover how these properties can be modified within the human vaginal biological environment so as to determine how optimal efficiency towards prevention of STIs and HIV i nfection can be achieved. It will also be of great importance to conduct in vivo human- oriented clinical trials to validate and compare the stability, viability and efficacy of the IBPD . T he developed IBPD may be applied not only for preventin g STIs and H IV but also in other areas like hormone replacement therapy, contraception, infections, infertility, and other female- related conditions, as an alternative to oral or parenteral administration. One such example is the everlasting problem that has been incurred so far in the treatment of Candidiasis (from Candida albicans ) due to resistance complications. The great stability of the IBPD in the vagina, its high bi oadhesivity and therefore unquestionable substantial retention, and the superior ability to control drug release rate over a prolonged period may make it a gold standard in this endeavor. Needed at this point in time is to streaml ine the 205 developed IBPD by undertaking more directed studies aimed at realizing its best clinical applicability. Such attempts should involve a study on irritation potential of this device which goes beyond the histology work but also involves looking at the liberation of any cytokines like IL- 1, alpha IL - 6 and IL - 8 in the skin tissue. Furthermore, it may be necessary to look for pharmacodynamic disease models to which this device can be applied in order to finally assess the benefit of this approach which will be a very good preclinical model. Finally, this study does not only provide encouraging results for the delivery of PSS and AZT but also provides a novel systematic approach for the development of delivery systems for other microbicide- ARVs. It is therefore highly recommended that t he approach employed in this study for the development of a microbicide- ARV delivery system for the prevention of STIs and HIV infections be adopt ed as a design tool for other similar bioactive systems. 206 REFERENCES Acaturk F. and. Robinson, J.R., (1996). Effect of the spermicide, nonoxynol 9, on vaginal permeability in normal and ovariectomized pig s, Pharmaceutical Research, 13 , 950? 951. Achanta, A.S. , Kowalski , J.G. , James K.W. and Rhodes, C.T., (1995). Artificial neural network: implications for pharmaceutical sciences, Drug Development and Industrial Pharmacy, 21, 119 ? 155. Adams, J.C., (1997) . Thrombospondin- 1. The International Journal of Biochemistry & Cell Biology, 29, 861 - 865. Agatonovic- Kustrin, S. and Beresford, R., (2000) . Basic concepts of artificial neural network (ANN) modeling and its application in pharmaceutical research. Journal of Pharmaceutical and Biomedical Analysis, 22, 717 - 727. Agatonovic- Kustrin, S . Turner, J.V. and Glass, B.D., (2008). Molecular structural characteristics as determinants of estrogen receptor selectivity. Journal of Pharmaceutical and Biomedical Analysis, 48, 369- 375. Airey, G.D., Mohammed, M.H. and Fichter C., (2008) . Rheological characteristics of synthetic road binders. Fuel , 87, 1763- 1775. Akagi, T., Kawamura, M., Ueno, M., Hiraishi, K., Adachi, M., Serizawa, T., Akashi M. and Baba, M., (2003) . Mucosal immunization with inactivated HIV - 1 - capturing nanospheres induces a significant HIV - 1 - specific vaginal antibody response in mice. Journal of Medical Virology, 69, 163- 172. 207 Alam, M.A., Ahmad, F.J., Khan, Z.I., Khar, R.K. and Ali, M., (2007) . Development and Evaluation of Acid- buffering Bioadhesive Vaginal Tablet for Mixed Vaginal Infections. AAPS Pharmaceutical Sciences and Technology . 8, 229- 236. Alliance for Microbicide Development . M icrobicide Research and Development Database, 2005.http://secure.microbicide.org/NetReports/ClinicalTrialsOngoingByProduct.aspx [ Accessed July 21, 2007 ] . Al- Taani, B.M. and Tashtoush, B.M., (2003). Effect of Microenvironment pH of Swellable and Erodable Buffered Matrices on the Release Characteristics of Diclofenac Sodium. AAPS Pharmaceutical Sciences and Technology, 4, 1 - 6. Al- Tahami, K. and Singh, J., (2007) . Smart polymer based delivery systems for peptides and proteins. Recent Patents on Drug Delivery and Formulation, 1, 65- 71. Alvarez- Lorenzo C, Gomez - Amoza JL., Martinez - Pacheco, R. and Souto, C., (1999). Microviscosity of hydroxypropylcellulose gel as a basis for prediction of drug diffusion rates. International Journal of Pharmaceutics, 180, 91 - 103. Alvarez- Lorenzo, C., Concheiro, A., Gomez - Amoza, J.L., Souto C. and Martinez - Pacheco, R., (2002). Effect of microcrystalline cellulose grade and process variables on pellets prepared by extrusion- spheronisation, Drug Development and Industrial Pharmacy, 28 , 451 ? 456. Alvarez- Lorenzo, C. and Concheiro, A., (2008) . Intelligent Drug Delivery Systems: Polymeric Micelles and Hydrogels. Mini Reviews in Medicinal Chemistry , 8, 1065 - 1074. 208 Amarl, E., Perdigao, A., Souza, M.H., Mauck, C., Waller, D., Zaneveld, L. and Faundes, A., (2004). Postcoital testing after the use of acid buffering gel (ACIDFORM) and a 2% nonoxynol - 9 product. Contraception, 70, 492- 7. Ambrose, Z., Compton , L. and Piatak Jr., M., (2008). Incomplete Protection against Simian Immunodeficiency Virus Vaginal Transmission in Rhesus Macaques by a Topical Antiviral Agent R evealed by Repeat Challenges. Journal of Virology, 82, 6591- 6599. Amet, T., Nonaka, M., Dewan M., Saitoh, Y., Xi, X., Ichinose, S., Yamamoto, N and Yamaoka, S., (2008) . Statin- induced inhibition of HIV - 1 release from latently infected U1 cells reveals a critical role for protein prenylation in HIV - 1 replication. Microbes and Infection, 10, 471- 480. Amy, E. , Rachel, L. and Amy, M. , (2007). HIV Revisited: The Global Impact of the HIV/AIDS Epidemic. SKINmed: Dermatology for the Clinician, 3, 149 ? 156. Anderson, R.A., Feathergill X., Diao M., Cooper, R., Kirkipatrick, P., Spear, D.P., Waller, C., Chany, G.F., Doncel, F., Herold, B. and Zaneveld, L.J.D., (2000). Evaluation of poly (st yrene- 4 - sulfonate) as a preventive agent for conception and sexually transmitted diseases, Journal of Andrology, 21, 862 - 875. Anderson, R.A., Feathergill, K.A., Diao, X.H., Cooper, M.D., Kirkpatrick, R., Rencher, W.F., Waller, D.P., Chany, C.J., Doncel, G.F., Herold, B.C. and Zaneveld, L.J.D., (2002). Preclinical evaluation of sodium cellulose sulfate (Ushercell) as a contraceptive antimicrobial agent. Journal of Andrology, 23, 426 - 438. 209 Andrews, G.P., Laverty, T.P. and Jones D.S., (2009) . B ioadhesive polymeric platforms for controlled Drug Delivery. European Journal of Pharmaceutics and Biopharmaceutics, 71, 505- 518. Arasaratnam, V., Galaev I. Y., and Mattiasson, B., (2000) . Reversibly soluble biocatalyst: optimization of trypsin coupling to Eudragit S - 100 and biocatalyst activity in soluble and precipitated forms. Enzyme and Microbial Technology , 27, 254 - 263. Arifin, D.Y., Lee L.Y. and Wang C., (2006). Mathematical modeling and simulation of drug release from microspheres: Implications to Drug Delivery systems. Advanced Drug Delivery Rev iews , 58 , 1274- 1325. Arulsudar, N., Subramanian N. and Murthy , R.S.R., (2005) . Comparison of artificial neural network and multiple linear regression in the optimization of formulation parameters of leuprolide acetate loaded liposomes Journal of Pharmacy and Pharmaceutical Sciences, 8, 243 - 258. Asane, G.S., Nirmal, S. A. and Rasal, K. B. Naik, A.A. Mahadik, M.S., and Rao Y.M., (2008). Polymers for Mucoadhesive Drug Delivery System: A Current Status. Drug Development and Industrial Pharmacy, 34, 1246 ? 1266. Avgoustakis, K., (2004) . Pegylated poly(lactide) and poly (lactide- co- glycolide) nanoparticles: Preparation, properties and possible applications in Drug Delivery. Current Drug Delivery, 1, 321- 333. Baba, M., Schols, D., De Clercq, E. Pauwels, R., Nagy, M. , Gy?rgyi - Edel?nyi, J., L?w, M. and G?r?g, S., (1990). Novel sulfated polymers as highly potent and selective inhibitors of human immunodeficiency virus replication and giant cell formation. Antimicrobial Agents and Chemotherapy, 34, 134- 138. 210 Bailey, M.L and Swett, J.E., (2007). Radiopaque composi tions, articles and methods of making and using same. US Patent 20070270691. http://www.freshpatents.com/Radiopaque- compositions- articles- and- methods- of- making- and- using- same- dt20071122ptan20070270691.php [Accessed 05 April 2009] . Ballagh, S.A., (2001). Vaginal ring hormone delivery systems in contraception and menopause, Clinical Obstetrics and Gynecology , 44, 106? 113. Baloglu E., Senyigit, Z.A., Karavana, S.Y., and Bernkop- Schn?rch, A., (2009) . Strategies to Prolong the Intravaginal Residence Time of Drug Delivery Systems. Journal of Pharmaceutical Sciences, 12, 312 ? 336. Balzarini, J. and van Damme, L., (2007). Microbicide drug candidates to prevent HIV infection. Lancet , 369, 787 - 797. Balzarini, J., Hatse, S., Vermeire, K., Princen, K., Aquaro, S., Perno, C.F. De Clercq, E., Egberink, H., Mooter, G.V., Peumans, W., van Damme, E. and Schols D., ( 2004). Mannose- specific plant lectins from the Amaryllidaceae family qualify as efficient microbicides for prevention of human immunodeficiency virus infection. Antimicrobial Agents and Chemotherapy, 48, 3858 - 3870. Balzarini, J., (2005) . Targeting the glycans of gp120: a novel approach aimed at the achilles heel of HIV. Lancet infectious diseases, 5, 726 - 731. Barakat, N.S. Elbagory, I.M. and Almurshedi A.S., (2009) . Controlled- Release Carbamazepine Matrix Granules and Tablets Comprising Lipophilic and Hydrophilic Components. Drug Delivery, 16, 57? 65. 211 Barakat, N.S. Elbagory, I . M. and Almurshedi, A.S., ( 2008). Controlled- Release Carbamazepine Granules and Tablets Comprising Lipophilic and Hydrophilic Matrix Components. AAPS Pharmaceutical Sciences and Technology, 9, 1054- 1062. Barbara, E., Valeria, F., Fabrizio, E., (2006) . Candidate HIV - 1 tat vaccine development frombasic science to clinical trials. AIDS, 20, 2245 - 2261. Bartlett, J.G., (2003). The Hopkins HIV report. The Johns Hopkins University AIDS Service, 15 , 1 - 16. B?rtolo, I., Rocha, C., Bartolomeu J. Gama, A., Marcelino, R ., Fonseca, M ., Mendes, A ., Epalanga, M , Silva, P.C. and Taveira, N ., (2009) . Highly divergent subtypes and new recombinant forms prevail in the HIV/AIDS epidemic in Angola: New insights into the origins of the AIDS pandemic. Infection, Genetics and Evolution , 9, 672- 682. Battaglioli- DeNero , A., (2007) . Strategies for Improving Patient Adherence to Therapy and Long- Term Patient Outcomes. Journal of the Association of Nurses in AIDS care, 18, S17 - S22. Baughn, R.E. and Musher D.M.(2005). Secondary Syphilitic Lesions. Clinical Microbiology Reviews, 18, 205- 216. Baumgartner, S., Kristl, J. and Peppas, N.A., (2002) . Network structure of cellulose ethers used in pharmaceutical applications during swelling and at equilibrium. Pharmaceutical Research, 19, 1084- 1090. 212 Baumgartner, S., Kristl J., Vrecer F., Vodopivec, P. and Zorko, B., (2000) . Optimisation of floating matrix tablets and evaluation of their gastric residence time. International Journal of Pharmaceutics. 195, 125 - 135. Becker, J.U., Theodosis, C., and Kulkarini, R., (2008) . HIV/AIDS, conflict and security in Africa: rethinking relationships. Journal of the International AIDS Society, 11, 1 - 7. Belyakov, I.M. and Ahlers J.D., (2008). Functional CD8 + CTLs in mucosal sites and HIV infection: moving forward toward a mucosal AIDS vaccine. Trends in Immunology, 29, 574- 585. Benson, C.A., Kaplan, J.E. and Masur, H., (2004) . Tr eating opportunistic infections among HIV - infected adults and adolescents. Centers for Disease Control and Prevention, 53, 1 - 112. Bentley, M.E., Marrow, K.M., Fullem A., Chesney M., Horton D. S., Zosenberg Z. and Mayer H. K., (2002). Acceptability of a novel vaginal microbicide during safety trial among low- risk women, Family Planning Perspective, 32, 184. Berger, J., (2004). Re - sexualizing the Epidemic: Desire, risk and HIV prevention. Development Update, 5, 45- 67. Bernkop- Schnurch, A., (2005) . Mucoadhesive Polymers: Strategies, Achievements and Future Challenge. Advanced Drug Delivery Review s, 57, 1553 - 1555. Bernkop- Schn?rch, A. and Hornof, M., (2003) . Intravaginal Drug Delivery sys tems: Design, challenges, and solutions. American Journal of Drug Delivery, 1, 241 - 254. 213 Bernstein, D.I., Stanberry, L.R., Sacks, S. Ayisi, N. K., Gong, Y. H., Ireland, J., Mumper, R. J., Holan, G., Matthews, B., McCarthy, T. and Bourne, N., (2003). Evaluations of unformulated and formulated dendrimer- based microbicide candidates in mouse and guinea pig models of genital herpes. Antimicrobial Agents and Chemotherapy , 47, 3784? 3788. Best, K., (2000) . Microbicide products enter human trials. FHI?s Prevention Trials Network. 20, 1 - 12. Bettini, R., Catellani, P. L., Santi, P., Massimo, G, Peppas, N.A. and Colombo, P., (2001) . Translocation of drug particles in HPMC matrix gel layer: effect of drug solubility and influence on release rate. Journal of Controlled Release, 70, 383 - 391. Bilensoy, E., Cirpanli, Y. and Sen, M., (2007) . Thermosensitive mucoadheisive gel formulation loaded with 5- Fu: cyclodextrin complex for HPV - induced cervical cancer. Journal of Inclusion Phenomena and Macrocyclic Chemistry, DOI 10.1007/s 10847- 006 - 9259- y, 1 - 8. Bilensoy, E., Rouf, A.M, Vural, I., Sen, M. and Hincal, A.A., (2006). Mucoadhesive, thermosensitive, prolonged- release vaginal gel for clotrimazole cyclodextrin complex. AAPS Pharmaceutical Sciences and Technology, 7, 1 - 13. Billa, N. and Yuen, K.H., (2000). Formulation Variables Affecting Drug Release From Xanthan Gum Matrices at Laboratory Scale and Pilot Scale. AAPS Pharmaceutical Sciences and Technology, 1, 1 - 8. Biradar,S.V., Dhumal, R.S., Shah, M.H. , Paradkar, A.R. and Yamamura , S., (2009). Preparation of Multiparticulate Vaginal Tablet Using Glyceryl Monooleate for Sustained Progesterone Delivery. Pharmaceutical Development and Technology , 14, 41- 52 214 Blakemore, W.R., Ball ard, A.D, Sewall, C.J., Modliszewski, J.J. and Bubnis, W.A., (2008) . Carageenan based antimicrobial compositions. US Patent 0161551 . http://www.freshpatents.com/Carrageenan - based- antimicrobial- compositions- dt20080703ptan20080161551.php [ Accessed June 23, 2009] . Bob, R., (2000). Microbicide. Fashioning new tools to deter HIV transmission International Association of Physicians in AIDS Care, 6, 157. Bogentoft, C. and Carlsson., A., (1996). Gel forming liquid carrier composition. US Patent 5492937. http://www.patentstorm.us/patents/5492937/ claims.html [Accessed June 2 , 2007] . Bonacucina, G., Martelli, S. and Palmieri G.F., (2004). Rheological, mucoadhesive and release properties of Carbopol gels in hydrophilic cosolvents. International Journal of Pharmaceutics, 282, 115 - 130. Bonacucina, G., Cespi, M., Misici- Falzi, M. and Palmieri, G.F., (2006) . Rheological, adhesive and release characterisation of semisolid Carbopol/tetraglycol systems . International Journal of Pharmaceutics, 307, 129 - 140. Bonferoni, M.C., Sandri, G., Ros si, S., Ferrari, F., Gibin, S. and Caramella, C., (2007) . Chitosan citrate as multifunctional polymer for vaginal delivery: Evaluation of penetration enhancement and peptidase inhibition properties. European Journal of Pharmaceutical Sciences, 33, 166 - 176. Bonferoni, M.C . , Giunchedi, P., Scalia, S. Rossi, S., Sandri, G. and Caramella, C., ( 2006). Chitosan gels for the vaginal delivery of lactic acid: Relevance of formulation parameters 215 to mucoadhesion and release mechanisms. AAPS Pharmaceutical Sciences and Technology, 7, E1 - E8. Boris, S. and Barb?s, C., (2000). Role played by lactobacilli in controlling the population of vaginal pathogens, Microbes Infect. 2, 543? 546. Bonifazi, D., Enger, O and Diederich F., (2007) . Supramolecular [60]fullerene chemistry on surfaces, Chemical Society Reviews , 36, 390- 414. Bornstein, P., (1995). Diversity of function is inherent in matricellular proteins: an appraisal of thrombospondin- 1. Journal of Cell Biology, 130, 503 - 506. Boskey, E. A., Jansen, M., Merski, I.K., Whaley, I.T ., Moench, T. and Cone, R., (2000) . BufferGelTM favors in vitro growth of lactobacilli while inhibiting BV- associated organisms Johns Hopkins University and ReProtect, Inc. Abstract A07. http://curezone.com/blogs/fm.asp?i =974816 [ Accessed November 18, 2007] . Boskey, E.R. and Cone, R.A., (2001) . Whaley KJ and Moench TR. Origin of vaginal acidity: High D/L lactate ratio is consistent with bacteria being the primary source. Human Reproduction, 16, 1809- 1813. Bourinbaiar, A.S., Root - Bernstein, R.S., Abulafia- Lapid, R., Rytik, P.G., Kanev, A.N., Jirathitikal, V. and Orlovsky, V.G., (2006) . Therapeutic AIDS vaccines. Current Pharmaceutical Design, 12, 2017 - 2030. Bourne, N., Bernstein, D.L., Ireland, J., Sonderfan, A.J., Prof y, A.T. and Stanberry, L.R., ( 1999). The topical microbicides PRO 2000 protects against genital herpes infection in a mouse model. Journal of infectious Diseases, 180, 203 - 205. 216 Bourne, N., Zanevelde, L.J.D., Ward, J.A., Ireland, J.P. and Stanberry, L.R., ( 2003). Poly (sodium 4- sulfonate): Evaluation of a topical microbicide gel against herpes simplex virus type 2 and Chlamydia trachomatis infection in mice. Clinical Microbiology Infections, 9, 816- 822. Bourquin, J., Schmidli, H., van Hoogevest, P. and Leuenberger, H., (1998). Advantages of artificial neural networks (ANN) as alternative modelling technique for data sets showing non- linear relationship using data from a galenical study on a solid dosage form. European Journal of Pharmaceutical Sciences, 7, 5 - 16. Boyd, M.R., Gustfson, K.R., Mc Mahon, J.B. Shoemaker, R.H., O'Keefe, B.R., Mori, T., Gulakowski, R.J., Wu, L., Rivera, M.I., Laurencot, C.M., Currens, M.J., Cardellina, J.H., Buckheit, R.W., Nara, P.L., Pannell, L.K., Sowder, R.C. and Henderson, L.E., (1997). Discovery of cyanovirin- N, a novel human immunodeficiency virus- inactivating protein that binds viral surface envelope glycoprotein gp 120: potential application to microbicide development. Antimicrobial Agents and Chemotherapy, 41, 1521- 1530. Brache, V., Croxatto, H., Kumar, N. , Sitruk- Ware, R., Coch?n, L., Schiappacasse, V., Sivin, I., Mu?oz, C., Maguire, R. and Faundes A., (2009). Effect of sexual intercourse on the absorption of levonorgestrel after vaginal administration of 0.75 mg in Car raguard? gel: a randomized, cross - over, pharmacokinetic study. Contraception , 79, 150- 154. Brannon- Peppas, L., (1992) . Novel vaginal drug release applications. Advanced Drug Delivery Reviews, 11 , 169 ? 177. Brenchley, J.M. and Douek D.C., (2008). HIV infec tion and the gastrointestinal immune system. Mucosal Immunology, 1, 23? 30. 217 Brenner, B.G., Wainberg, M.A . and Turner , D., (2002) . HIV - 1 drug resistance: can we overcome? Expert Opinion on Biological Therapy, 2, 751 - 761. Breton, P., Larras V., Roy, D., Segodira, S., Limal, D., Bonnafous, D., Colin, N, Bru, N, Fattal, E. and Couvreur, P., (2008) . Biocompatible poly(methylidene malonate) - made materials for pharmaceutical and biomedical applications. European Journal of Pharmaceutics and Biopharmaceutics, 68, 479 - 495. Brier, M.E. Zurada J.M. and Aronoff, G.R. (1995) . Neural network predicted peak and trough gentamicin concentrations, Pharmaceutical Research, 12, 406? 412. Briggs, K. and Zeitlin, L., (2000). An anti - HSV antibody produced in transgenic rice plants prevents HSV- 2 infecti on in mice. AIDS, 15 , S19 - S20. Brodland, G.W., (2003). New information from cell aggregate compression tests and its implications for theories of cell sorting. Biorheology, 40, 1 - 3, 273- 277. Broliden, K., Haase, A. T., Ahuja, S. K., Shearer, G.M. and Andersson, J., (2009). Back to basics: mucosal immunity and novel HIV vaccine concepts. Journal of Internal Medicine, 265, 5 - 17. Broumas, A.C. and Basara, L.A., (2000) . Potential patient preference for three day treatment of bacterial vaginosis: Responses to a new suppository form of clindamycin, Advances in Urethrane Science and Technology, 17, 159 - 166. Brouwers, J., Vermeire, K., Schols, D. and Augustijns P ., (2008). Development and in vitro evaluation of chloroquine gels as microbicides against HIV - 1 infection. Virology, 378, 306- 310. 218 Brown, T.J., Yen - Moore, A. and Trying, S.K., (1999) . An overview of sexually transmitted diseases. Part 1. Journal of American Academy of Dermatology, 41 , 511- 532. Buck, B.C., Thompson, C.D., Roberts, J.N. , M?ller, M., Lowy D.R. and Schiller J.T., (2006). Carageenan is a potent inhibitor of papillomavirus infection. PloS Pathogens, 2, 0671- 0680. Buddhikot, M., Falkenstein, E., Wehling M. and Meizel, S., (1999) . Recognition of a human sperm surface protein involved in the progesterone- initiated acrosome reaction by antisera against an endomembrane progesterone binding protein from porcine liver. Molecular and Cellular Endocrinology . 158 , 187 - 193 Budtz - jorgensen, E., (2000) . Ecology of candida- associated denture stomatitis. Mic robial Ecology in Health and Disease, 12, 170- 185. Bulgheroni, E., C itterio, P., Croce, F. Cicero, M.L., Vigan?, O., Soster, F., Chou, T., Galli M. and Rusconi S., ( 2004) . Analysis of protease inhibitor combinations in vitro: activity of lopinavir, amprenavir and tipranavir against HIV type 1 wild- type and drug- resistant isolates. Journal of Antimicrobial Chemother apy, 53, 464- 468. Butsashvili, M., Tsertsvadze, T., McNutt, L.A. Kamkamidze, G., Gvetadze, R. and Badridze N., (2001) . Prevalence of hepatitis B, hepatitis C, syphilis and HIV in Georgian blood donors. European Journal of Epidemiology , 17, 693? 695. Cadena, C., A., (2006) . HIV Vaginal Gel: An overview of PRO. http://www.associatedcont ent.com /article/92443/HIV_vaginal_gel_an_overview_of_pro.html [ Accessed March 24, 2007] . 219 Cairns, G., ( 2007). Microbicides trials get London start. Prevention. http://www.guscairns.com/ [ Accessed July 23, 2007] . Cajander, S.S. and Rylander, E., (1988). Morphometric characteristics of the vaginal epithelium during the menstrual cycle. Gynecologic and Obstetric Investigation, 26, 136? 144. Campo, J., Perea, M.A., del Romero, J., (2006) . Oral transmission of HIV, reality or fiction? An update. Oral diseases, 12, 3219 ? 228. Campo, V. L., Daniel F?bio Kawano, D.F., da Silva D.B. and Carvalho I., (2009).Carrageenans: Biological properties, chemical modifications and structural analysis ? A review. Carbohydrate Polymers , 77, 167- 180. Caputo, A.C. and Pelagagge, P.M., (2008). Parametric and neural methods for cost estimation of process vessels. International Journal of Production Economics. 112, 934 - 954. Carmen, A. and Angel, C., (2003). Effects of surfactants on gel Behavior: Design implications for Drug Delivery systems. American Journal of Drug Delivery, 1 , 77 - 101. Carrington, G.L, Rohrer, T., Jones, E., Moore, P., (1944). Sulfanilamide absorption via the rectum and vagina. Surgery, Gynecology and Obstetrics, 78, 333- 334. Castle, P.E., Whaley, K.J., Hoen, T.E., Moench, T.R. and Cone, R.A., (1997). Contraceptive effect of sperm- agglutinating monoclonal antibodies in pigs. Biolology of Reproduction, 56, 53- 159. 220 Castle, P.E., (2002). Human monoclonal antibody stability and activity at vaginal pH Journal of Reproductive Immunology , 56, 61 - 76. Catalone, B.J., Kish- Catalone, T.M., Neely, E.B., Budgeon, L.R. Ferguson, M.L., Stiller, C., Miller, S.R., Malamud D., Krebs, F .C., Howett, M.K. and Wigdahl B., ( 2005). Comparative Safety Evaluation of the Candidate Vaginal Microbicide C31G. Antimicrobial Agents and Chemotherapy, 49 , 1509? 1520. Cavalcanti, O.A., Petenuci, B., Bedin, A.C. , Pineda, E.A.G and Hechenleitner, A.A.W., ( 2004). Characterisation of ethylcellulose films containing natural polysaccharides by thermal analysis and FTIR spectroscopy . Latin American Journal of Pharmacy, 23, 53 - 57. Cedars, M.I. and Judd, H.L. (1987). Nonoral routes of estrogen administration. Obstetrics & Gynecology Clinics of North America, 14, 269? 298. Ceschel, G.C ., Maffei, P ., Lombardi, B.S ., Ronchi, C ., and Rossi, S ., 2001. Development of a mucoadhesive dosage form for vaginal administration. Drug Development and Industrial Pharmacy, 6 , 541 - 547. Chakraborty, R., (2008). Update on HIV - 1 infection in children. Paediatrics and Child Health , 18 , 496 - 501. Chang, J.Y., Oh, Y. and Choi, H. Kim, Y.B . Kim, C.K ., (2002) . Rheological evaluation of thermosensitive and mucoadhesive vaginal gels in physiological conditions. International Journal of Pharmaceutics. 241, 155 - 163. Charde, S., Mudgal, M., Kumar, L. and Saha, R., (2008) . Development and Evaluation of Buccoadhesive Controlled Release Tablets of Lercanidipine. AAPS Pharmaceutical Sciences and Technology, 9, 182 - 190. 221 Chazal, N. and Gerlier, D., (2003). Virus Entry, Assembly, Budding, and Membrane Rafts. Microbiology and Molecular Biology Reviews, 67, 226? 237. Chen, X., Galeski, A. and Michler, G.H., (2006). Morphological alteration and strength of polyamide 6 subjected to high plane? strain compression. Polymer, 47, 3171- 3185. Chen, Y. Jiao, T. McCall, T.W. Baichwal A.R. and Meyer, M.C., (2002). Comparison of four artificial neural network software programs used to predict the in vitro dissolution of controlled- release tablets. Pharmaceutical Development and Technology, 7 , 373? 379. Cheshenko, N., Keller, M.J. and MasCasullo, V., (2004) . Candidate topical microbicides bind herpes simplex virus B and prevent viral entry and cell - to- cell spread. Antimicrobial Agents and Chemotherapy , 48, 2025 - 2036. Ch'ng, H.S. Park, H. Kelly, P. and Robinson, R., (1985) . Bioadhesive polymers as platforms for oral controlled drug delivery: II. Synthesis and evaluation of some swelling water insoluble bioadhesive polymers. Journal of Pharmaceutical Sciences, 74, 399? 405. Cho, K., Park, J., Osaka, T. and Park, S., (2005) . The study of antimicrobial activity and preservative effects of nanosilver ingredient. Electrochimica Acta, 51, 956 - 960. Choonara, Y.E., Pillay , V., Singh, N., Khan, R.A. and Ndesendo, V.M.K., (2008) . Chemometric, physicomechanical and rheological analysis of the sol- gel dynamics and degree of crosslinking of glycosidic poymers. Biomedical Materials, 3, 1 - 15. 222 Chopra, S., Patil, G.V. and Motwani, S.K., (2007) . Release modulating hydrophilic matrix systems of losartan potassium: Optimization of formulation using statistical experimental design. European Journal of Pharmaceutics and Biopharmaceutics, 66, 73 - 82. Choudhry, M. and Choudhry, V. P., (2003). Prevention and control of HIV/AIDS. Indian Journal of Pediatrics, 70, 975- 981. Christ, K.A., Zessin, G. and Cobet, U., (1998) . The use of ultrasound and penentrometer to characterize the advancement of swelling and eroding fronts in HPMC matrices. International Journal of Pharmaceutics, 163, 123? 131. Christensen, N.D., Reed, C. A., Culp, T.D., Hermonat, P.L., Howett, M.K., Anderson, R.A. and Zaneveld L.J.D., (2001) . Papillomavirus microbicidal activities of high- molecular- weight cellulose sulfate, dextran sulfate, and polystyrene sulfonate. Antimicrobial Agents and Chemotherapy , 45, 3427- 3432. Chu, H., Yeo, Y., Chuang, K.S., (2007) . Entry in emulsion polymerization using a mixture of sodium polystyrene sulfonate and sodium dodecyl sulfate as the surfactant. Polymer, 48, 2298 - 2305. Cicinelli, E. , Cignarelli, M., Sabatelli, S. Romano, F., Schonauer, L..M., Padovano, R. and Einer- jensen, N., (1998) . Plasma concentrations of progesterone are higher in the uterine artery than in the radial artery after vaginal administration of micronized progesterone in an oil- based solution to postmenopausal women. Fertility and Sterility, 69, 471? 473. Cicinelli, E., de Ziegler D., Bulletti, C., Matteo, M.G., Schonauer L.M. and Galantino P., (2000). Direct transport of pro gesterone from vagina to uterus. Obstetrics and Gynecology , 95 , 403? 406. 223 Clarke, G., McCoombe, S. and Short, R., (2006). Sperm immobilizing properties of lemon juice. Fertility and Sterility , 85, 1529 - 1530. Coetzee, N., Hoosen, A., Blanchard, K., de Kock, A., Sebda, H., Friedland, B., Ellerton, C., Nkompela, N., Altin, L., Ndlovu, G. and Tweedy, K., (2006). Safety of lambda carageenan microbicides ( Carraguard? ) in South Africa. University of Cape Town, Medical University of Southern Africa, Population Council and Family Health International. http://www.itg.be/micro2002/downloads/presentations/2Monday_May_13 _2002/Track_B_ sessions/Nicol_Coetzee.pdf [ Accessed October 17, 2007 ] . Cohen, M.S. (2004) . HIV and sexually transmitted diseases: lethal synergy, Topics in HIV Medicine, 12 104? 107. Cohen, M.S., Black J.R., Proctor R.A., Sparling P.F., (1984). Host defences and the vaginal mucosa: a re - evaluation. Scandinavian Journal of U rology and Nephrology, 86, (Suppl.) , 13 - 22. Cole, A.M. and Cole. A.L., (2007). Antimicrobial polypeptides are key anti - HIV - 1 effector molecules of cervicovaginal host defense. American Journal of Reproductive Immunology, 59, 27? 34. Collins, K. B., Patterson, B. K., Naus, G. J., Landers, D. V. and Gupta, P., ( 2000) . Development of an in vitro organ culture model to study transmission of HIV - 1 in the female genital tract. Nature Medicine, 6, 475- 479. Colombo, P., Bettini, R., Santi, P., De Ascentiis A. and Peppas, N.A., (1996) . Analysis of the swelling and release mechanisms from Drug Delivery systems with emphasis on drug solubility and water transport. Journal of Controlled Release, 39, 231? 237. 224 Cone, R.A. and Whaley, K.J., (1994) . Monoclonal antibodies for reproductive health: Preventing sexual transmission of disease and pregnancy with topically applied antibodies. American Journal of Reproductive Immunology , 32, 114- 131. Coombs, R.W.,Reichelderfer, P.S. anf Landay, A.L., (2003) . Recent observations on HI V type- 1 infection in the genital tract of men and women. AIDS, 17, 455- 480. Corring, R., (1992) . Gel detergent compositions containing a clay and a cross- linked polycarboxylic polymer. US Patent 5160448. http://ww w.freepatentsonline.com/5160448 [ Accessed August 28, 2000] . Cristofaro, P. and Ramratnam, B., (2005). Prevention Strategies: Vaccines and Microbicides. 12 th conference on retrovirus and opportunistic infections, Boston, USA. 22- 25. Crombie, R. and Silverstein, R.L., (1998). Lysosomal integral membrane protein LIMP II binds thrombospondin- 1: structure - function homology with the cell adhesion molecule CD36 defines a conserved recognition motif. Journal of Biologic al Chemistr y, 273, 4855- 66. Crombie, R., (2000). Mechanism of thrombospondin- 1 anti - HIV - 1 activity. AIDS Patient Care and STDs, 14, 211 - 214. Crombie, R., Kawasaki, K., Hojo, K. and Laurence, J., (2001). Peptides derived from salivary thrombospondin- 1 replicate its anti - HIV effect: Potential role in microbicide development. Journal of Acquired Immune Deficiency Syndromes, 27, 91 - 93. 225 Cutler, B. and Justman, J., (2008) . Vaginal microbicides and the prevention of HIV transmission. Lancet Infectious Diseases, 8, 685? 697. Dahlberg, C. , Fureby, A., Schuleit, M., Dvinskikh, S.V. and Furo, I., (2007). Polymer mobilizat ion and drug release during tablet swelling. A 1 H NMR and NMR microimaging study. Journal of Controlled Release, 122, 199 - 205. Dam, E., Clavel, F., Calvex, V. Salmon, D., Pellegri n, J.L., Mamet, J.P., Antoun, Z. and Vauthier, J.M., (2001) . Comparison of HIV - 1 resistance phenotypes obtained by two different assay systems. Antiviral Therapy, 6 , 122. das Neves, J. and Bahia, M.F., (2006). Gels as vaginal drug delivery systems. International Journal of Pharmaceutics, 318, 1 - 14. das Neves, J., Santos, B., Teixeira, B. Dias, G., Cunha, T . and Brochado, J., (2008) . Vaginal drug administration in the hospital setting. American Journal of Health- System Pharmacy, 65, 254 - 259. Davis, C.C. Kremer, M.J. Schlievert P.M. and Squier, C.A., (2003). Penetration of toxic shock syndrome toxin - 1 across porcine vaginal mucosa ex vivo: permeability characteristics, toxin distribution, and tissue damage, Am erican Journal of Obstetrics and Gynecol ogy. 189, 1785? 1791. D?Cruz, O.J . and Uckun, F.M . , (2002). Pre- clinical safety evaluation of novel nucleoside analogue- based dual- function microbicides ( WHI - 05 and WHI - 07 ) . Journal of Antimicrobial Chemotherapy, 50 , 793 - 803. D?Cruz, O.J., (2003a). Stampidine is a potential nonspermicidal broad- spectrum anti- human immunodeficiency virus microbicides. Fertility and Sterility, 81, 831 - 841. 226 D?Cruz, O.J., (2003b). Contraceptive activity of a spermicidal aryl phosphate derivative of bromo- methoxy - zidovudine (compound WHI - 07) in pigs. Fertility and Sterility , 79, 864- 872. D'Cruz, O.J., Samuel, P., Waurzyniak, B. and Uckun, F.M., (2003a). In vivo evaluation of a gel formulation of stampidine, a novel non- spermicidal broad- spectrum anti- HIV microbicide. American Journal of Drug Delivery, 1, 275 - 285. D'Cruz, O.J. Samuel, P., Waurzyniak, B., and Uckun F.M., (2003b). Development and evaluation of a thermoreversible ovule formulation of stampidine. a novel nonspermicidal broad- spectrum anti- human immunodeficiency virus microbicide. Biology of Reproduction, 69, 1843? 1851. D?Cruz, O.J. and UcKun, F.M., (2004). Clinical development of microbicides for the prevention of HIV infection. Current Pharmaceutical Design, 10, 315 - 335. D'Cruz, O.J., Samuel, P. and Uckun, F.M., (2004a). PHI - 443: A novel noncontraceptive broad- spectrum anti- human immunodeficiency virus microbicide, Biology of Reproduction, 71, 2037 - 2047. D'Cr uz, O.J., Samuel, P. and Uckun, F.M., (2004b). Antiretroviral Spermicide WHI - 07 Prevents Vaginal and Rectal Transmission of Feline Immunodeficiency Virus in Domestic Cats. Antimicrobial Agents and Chemotherapy, 48, 1082 - 1088. D'Cruz, O.J., Samuel, P. and Uckun, F.M., (2005a). Conceival, a novel non- contraceptive vaginal vehicle for lipophilic microbicides. AAPS Pharmaceutical Sciences and Technology, 6, E56 - E64. 227 D?cruz, O.J., Erbeck, D. and Uckun F.M., (2005b). A Study of the potential of the pig as a model for the vaginal irritancy of benzalkonium chloride in comparison to the nonirritant microbicide PHI - 443 and the spermicide vanadocene dithiocarbamate. Toxicologic Pathology, 33, 465 - 476. D'Cruz, O.J. and Uckun, F.M., (2005c). Discovery of 2, 5 - dimethox y- substituted 5- bromopyridyl thiourea (PHI - 236) as a potent broad- spectrum anti- human immunodeficiency virus microbicide. Molecular Human Reproduction, 11, 767- 777. D?Cruz, O.J. and Uckun, F.M., (2006a). Influence of long- term stability conditions on microbicidal nucleoside prodrug (WHI - 07) - loaded gel- microemulsion. AAPS Pharmaceutical Sciences and Technology, 7, 1530- 9932. D'Cruz, O.J.D. and Uckun, F.M., (2006b). Dawn of non- nucleoside inhibitor- based anti- HIV microbicides. Journal of Antimicrobial Chemotherapy , 57, 411- 423. D'Cruz, O.J. and Fatih, Uckun, F.M., (2007). Preclinical Evaluation of a Dual- Acting Microbicidal Prodrug WHI - 07 in Combination with Vanadocene Dithiocarbamate in the Female Reproductive Tract of Pig , Pig, and Cat. Toxicologic Pathology, 35, 910 - 927. De Clercq, E., (2000). Current lead natural products for the chemotherapy of human immunodeficiency virus (HIV) infection. Medical Care Research and Review, 20, 323- 349. De Clercq, E., (2004) . New anti - HIV agents in preclinical and clinical development. Frontiers in Medicinal Chemistry, 1, 543- 579. 228 De Clercq, E., (2006) . The role of tenofovir in the prevention of HIV infections. AIDS, 20, 1990- 1991. de Candia, F, Maglio, G, Palumbo, R and Sirletti, M., (1998). Synthesis and physical behavior of polyamide 6,10- poly(butadiene- co- acrylonitrile) segmented block copolymers. Colloid and Polymer Science, 267, 9 - 15. Deryaguin, B.V ., Toporov , Y.P., Mueler, V.M. and Aleinikova, I.N., (1997). On the relationship between the electrostatic and molecular component of the adhesion of elastic particles to a solid surface, Journal of. Colloid and Interface Science, 58 , 528 - 533. Desphande, A., Rhodes , C.T., and Danish M., (1992). Intravaginal drug delivery. Drug Development and Industrial Pharmacy, 18, 1225- 1279. Dezarnaulds, G., and Fraser, I.S., (2002) . Vaginal ring delivery of hormone replacement therapy?a review. Expert Opinion on Pharmacotherapy, 4 , 201? 212. Dhawan, D. and Mayer, K.H., (2006) . Microbicides to Prevent HIV Transmission: Overcoming Obstacles to Chemical Barrier Protection. The Journal of Infectious Diseases, 193, 36? 44. Di Fabio, S., Van Roey, J., Giannini, G. van den Mooter, G., Spada, M., Binelli, A ., Pirillo, M. F., Germinario, E., Belardelli, F., de Bethune, M. and Vella, S., (2003) . Inhibition of vaginal transmission of HIV - 1 in hu - SCID mice by the non- nucleoside reverse transcriptase inhibitor TMC120 in a gel formulation. AIDS, 17 , 1597? 1604. 229 Dieterich, D.T., (2006) . Disease management? constructing optimal NRT I - based combinations: Past, Present, and Future. Medscape General Medicine, 8, 16. Division of HIV/AIDS Prevention, (2003). HIV and its transmission. Center for Disease Control and Prevention (CDC). https://www.cdc.gov/hiv/pubs/facts/transmission.htm [ Accessed May 23, 2006 ] . Dobaria, N., Mashru, R. and Vadia, N.H., (2007) . Vaginal drug delivery systems: A Review Current Status. East and Central African Journal of Pharmaceutical Studies , 10, 3 - 13. Drug Digest, (2004). Sexually transmitted infections (STIs). http : //www.drugdigest.org/DD/ Printab- lePages/HealthCondit ions/1,20041,550367,00.html [ Accessed April 20, 2007 ] . DuBouchet, L, McGregor, J.A., Ismail, M. and McCormack, W.M., (1998). A pilot study of metronidazole vaginal gel versus oral metronidazole for the treatment of trichomonas vaginalis vaginitis. Sexually Transmitted Diseases, 25, 176? 179. D? rig, T. and Fassihi, R., (2002). Guar- based monolithic matrix systems: effect of ionizable and non- ionizable substances and excipients on gel dynamics and release kinetics. Journal of Con trolled Release, 80, 45- 56 Dykxhoorn, D.M. and Lieberman, J., (2006) . Silencing Viral Infection. PLoS Medicine, 3, e242. 230 Eaton L.A. a nd Kalichman S.C. , (2007). Risk compensation in HIV prevention: Implications for vaccines, microbicides, and other biomedical HIV prevention technologies. Current HIV/AIDS Reports , 4, 165 - 172. Efentakis M. and Peponaki C., (2008). Formulation Study and Evaluation of Matrix and Three- layer Tablet Sustained Drug Delivery Systems Based on Carbopols with Isosorbite Mononitrate. AAPS Pharmaceutical Sciences and Technology, 9, 917- 923. Eggert- Kruse, W., Botz, I., Pohl, S., Rohr, G. a nd Thom as Strowitzki, T., (2000) . Antimicrobial activity of human cervical mucus. Hum an Reproduction, 15, 778- 784 Einer- Jensen, N., Cicinelli, E. , Galantino, P., Pinto V. and Barba B., (2002) . Uterine first pass effect in postmenopausal women, Human Reproduction, 17, 3060? 3064. Einmahl, S., Capancioni, S. Schwach - Abdellaoui, K. , Moleller, M., Behar - Cohen, F. and Gurny, R., (2001) . Therapeutic applications of viscous and injectable poly(ortho esters). Advanced Drug Delivery Reviews , 53, 45 - 73. El- Kamel A., Soker M., Naggar V. and Al Gamal S., (2002) . Chitosan and sodium alginate- based bioadhesive vaginal tablets, AAPS Pharmaceutical Sciences and Technology, 4, 1 - 8. Elson, C., Milne, A., Curran, D. and Kydonieus, A. N., (2000) . O- Carboxymethylchitosan as a mucoadhesive for vaginal delivery of levonorgestrel, Proceedings of International Symposium on Controlled Release of Bioactive Materials, 27, 7201 - 7202. 231 Espinosa, M. No?, G., Troncoso, C. Ho, S.B. and Villal?n M., (2002) . Acidic pH and increasing [Ca 2+ ] reduce the swelling of mucins in primary cultures of human cervical cells. Human Reproduction, 17, 1964 - 1972. Fairley, J., (2009a ). Completes Patient Testing In Retention Of VivaGel Antiviral Activity Study. Starpharma Holdi ngs Limited (ASX:SPL). http://www.ecplaza.net/news/0/22998/ starpharma _holdings_limited.html [ Accessed July 5 , 2009] . Fairley, J., (2009b ). VivaGel Ne w Clinical Trial Commences. Starpharma Holdings Limited (ASX:SPL). http://www.femail.com.au/vivagel - new- clinical- trial- commences.htm [Accessed August 20, 2009] . Farmer, P., Le? andre, F., Mukherjee, J. , Gupta, R ., Tarter, L . and Kim, J.Y ., (2001). Community- based treatment of advanced HIVdisease: introducing DOT - HAART (directly observedtherapy with highly active antiretroviral therapy). Bulletin of the World Health Organization, 79, 1145? 1151. Fauci, A.S., (2005) . International Trial Of Two Microbicides Begins, Science Daily. http://www.sciencedaily.com/releases/2005/ 02/ 050213135251.htm [Accessed May 22, 2009] . Ferris, D.G., Francis, S.L. , Dickman, E.D . Miler - Miles, K., Waller , J.L . and McClendon N ., (2006). Variability of Vaginal pH Determination by Patients and Clinicians. The Journal of the American Board of Family Medicine, 19, 368- 373. Fern?ndez - Romero, J. A. , Thorn, M ., Turville, S.G ., Titchen, K ., Sudol, K ., Li, J., Miller, T ., Robbiani, M ., Maguire, R.A ., Buckheit, R.W. Jr , Hartman, T.L. and Phillips, D,M ., (2007). 232 Carrageenan/MIV - 150 (PC - 815), a combination microbicide. Sexually Transmitted Diseases, 34, 9 ? 14. Fichora, R.N., Zhou, F., Ratnan, V., Atanass ova, V., Giang, S., Strick, N. and Neurath, A.R., (2005). Anti - human immunodeficiency virus type I microbicides cellulose acetate 1,2- benzene dicarbxylate in a human in vitro model of vaginal inflammation. Antimicrobial Agents and chemotherapy, 49 , 323 - 325. Finley, B.M., Plescia, C.J., Harrison, P.F. and Vignes, F.N., (2006) . An analytical overview of the microbicide preclinical and clinical pipeline. AIDS Conference. Toronto Canada; Abstract No. TuPE0438 [ Accessed November 18, 2007] . Fischer, M., Hafner, R., Schneider, C. , Trkola, A., Joos, B., Joller, H., Hirschel, B., Weber, R. and Gunthard, H., (2003) . HIV RNA in plasma rebounds within days during structured treatment interruptions. AIDS, 17, 195 - 199. Flint, S.R., Tappuni, A., Leigh, J. , Schmidt- Westhausen, A.M. and MacPhail L., ( 2006). (B3) Markers of Immunodeficiency and Mechanisms of HAART Therapy on Oral Lesions . Advances in Dental Research. 19, 146- 151. Fogel, G.B., (2008). Computational intelligence approaches for pattern discovery in biological systems. Briefings in Bioinformatics, 9, 307- 316. Forbes, A. and Harrison, P., (2000). Microbicides . Aids Community Research Initiative of America. http://www.thebody.com/c ontent/art14112.html [Accessed November 15 , 2007 ] . Foty R.A. and Steinberg M.S., (2005) . The differential adhesion hypothesis: a direct evaluation. Developmental Biology, 278, 255 - 263. 233 Francois, M., Snoeckx , E., Putteman, P. et al., (2003).A Mucoadhesive, cyclodextrin- based vaginal cream formulation of itraconazole. American Association of Pharmaceutical Scientists, 5, 1 - 5. Fried, N.D., Tredway, D.R. and Mishell, D.R., (1973) . Ter mination of early pregnancy with prostaglandin E2 vaginal suppositories. Contraception. 8, 255 - 263. Frieden, T.R., Das- Douglas, M., Kellerman, S.E. and Henning, K.J., (2005) . Applying Public Health Principles to the HIV Epidemic. The New England Journal of Medicine, 353, 2397- 2402. Frushour, B.G., (2004) . A new thermal analytical technique for acrylic polymers. Polymer Bulletin, 4, 305 - 314. Fu, X. C., Wang, G.P., Gao, J. Q. Zhan, S.Y. and Liang W.Q., ( 2003). Prediction of plasma protein binding of cephalosporins using an artificial neural network. Pharmazie, 62, 157- 158. Furuhjelm, M., Karlgren, C. and Carlstrom, K., (1980). Intravaginal administration of conjugated estrogens in postmenopausal women. International Journal of Gynecology and Obstetrics, 17, 335 - 339. Gadi, B. and Parniak, M.A., (2001) . Anti - HIV - 1 microbicide potential of the tight - binding non- nucleoside reverse transcriptase inhibitor UC781. AIDScience, 1, 12. Galeski, A., (2003). Strength and toughness of crystalline polymer systems. Progress in Polymer Science, 28, 1643- 1699. 234 Gallant, J.E., (2008) . Initial regimen. Johns Hopkins poc -IT Center. http://www.hopkins - hivguide.org/management/antiretroviral_therapy/full_initial_regimen.html [Accessed August 15 , 2009 ] . Gao, P. a nd Meury, R.H., (1996). Swelling of hydroxypropyl methylcellulose matrix tablets. 1. Characterisation of swelling using a novel optical imaging method. Journal of Pharmaceutical Sciences, 85 , 725? 731. Garg, S., Kandarapu, R., Vermani, K., (2003) . Development Pharmaceutics of Microbicide Formulations. Part I: Preformulati on Considerations and Challenges. AIDS Patient Care and STDs, 17, 17- 32. Garg, S. and Tambwekar, K.R., (2003). Development pharmaceutics of microbicide formulations Part II. Formulation, evaluation and challenges. AIDS Patient Care and STDs, 17, 377- 399. Garg, S., Verman, K., Anderson, R.A. and Zaneveld, L.J., (2004a). Rapidly disintegrating novel bioadhesive vaginal tablets of polystyrene sulfonate (PSS), a potential microbicide formulation. International Conference of AIDS. Abstract No. TuPeB4656. http://gateway.nlm.nih.gov/MeetingAbstracts/ma?f=102282478.html [Accessed November 12, 2007] . Garg, S., Zaneveld, L.J.D., Anderson, J.R.A. and Waller, D.P., (2004b). Compositions and methods for trapping and inactivating pathogenic microbes and spermatozoa. United States Patent 6706276. http://www.freepatentsonline.com/6706276.html [Accessed June 19, 2 009] . 235 Garg, S., Jambu L. and Vermani K., (2007). Development of Novel Sustained Release Bioadhesive Vaginal Tablets of Povidone Iodine. Drug Development and Industrial Pharmacy, 33, 1340- 1349. Gavin, E., Sanna, V., Juliano, C., B enfero, C.M. and Giunchedi, P., (2002) . Mucoadhesive vaginal tablets as veterinary system for the controlled release of an antimicrobial drug, acriflavine. AAPS Pharmaceutical Sciences and Technology, 3, 1 - 7. Genc, L., Oguzlar, C. and G?ler, E., 2000. Stu dies on vaginal bioadhesive tablets of acyclovir. Pharmazie, 55, 297 - 299. Geonnotti, A. and Katz, D., (2006). Dynamics of HIV neutralization by a microbicide formulation layer: Biophysical fundamentals and transport theory. Biophysical Journal, 91, 2121- 2130. Ghaffari, A., Abdollahi, H., and Khoshayand, M.R., (2006) . Performance comparison of neural network training algorithms in modelling of biomodal drug delivery. International Journal of Pharmaceutics, 327, 126 - 138. Ghate, D. and Edelhauser, H.F., (2006). Ocular drug delivery. Pharmaceutical Developmemt and Technology, 3, 275 - 287. Giannola, L.I., De Caro, V., Giandalia, G., Siragusa, M.G., Tripodo., Florena, A.M., Campisi, G., (2007). Release of naltrexone on bucal mucosa: Permeation studies, histological aspects and matrix system design. European Journal of Pharmaceutics and Biopharmaceutics, 67, 425 - 433. 236 Gilsenan, P.M., Richardson, R.K., and Morris, E.R., (2003a). Associative and seggregative interactions between gelation and low methoxy pectin: Part I. Associative interactions in the absence of Ca+ , Food H ydrocolloids, 17, 723- 737. Gilsenan, P.M., Richardson, R.K., and Morris, E.R., (2003b). Associative and seggregative interactions between gelation and low methoxy pectin: Part II. Co- gelation in the presence of Ca+ , Food Hydrocolloids, 17, 739 - 749. Gilsenan, P.M., Richardson, R.K., and Morris, E.R., (2003c). Associative and seggregative interactions between gelation and low methoxy pectin: Part III. Quantitative analysis of co- gel moduli, Food Hydrocolloids, 17, 751- 761. Gimeno, E., Moraru, C.I, Kokini , J.L., (2003) Effects of Xanthan gum and CMC on the structure and texture of corn flour pellets expanded by microwave heating. Cereal Chemistry, 81, 100- 107. Girish, K.L. aand Dhiren P.S., (2008). Evaluation of Mucilage of Hibiscus rosasinensis Linn as Rate Controlling Matrix for Sustained Release of Diclofenac. Drug Development and Industrial Pharmacy, 34, 807- 816. Glare, P.A., (2001). Pain in patients with HIV infection: issues for the new millennium European Journal of Pain, 5, 43 - 48 Goh, B., (2005). Syphilis. Medicine, 33, 48- 51. Goldberger MA, Walter RI and Lapid LS. (1947). Absorpton of penicillin from the vagina. American Journal of Obstetrics & Gynecology , 53, 529- 531. 237 Grassi, G., Farra, R., Caliceti, P., Guarnieri, G., Salmaso, S., Carenza, M. and Grassi, M., (2005). Temperature - Sensitive Hydrogels: Potential Therapeutic Applications. American Journal of Drug Delivery, 3, 239- 251. Greenberg, B., Mccorkle, R., Vlahov, D. and Selwyn P.A., (2000) . Palliative care for HIV disease in the era of highly active antiretroviral therapy. Journal of Urban Health, 77, 150- 165. Greenberg M.L., (2007) . Enfuvirtide: Basic science to FDA approval Entry Inhibitors in HIV Therapy Book. 161 - 177. Greenhead, P., Hayes, P., Watts, P.S. Laing, L.G. Griffin, G .E. and Shattock R.J., ( 2000). Parameters of Human Immunode ficiency Virus Infection of Human Cervical Tissue and Inhibition by Vaginal Virucides. Journal of Virology, 74, 5577? 5586. Greub, G., Cozzi, L.A., Ledergerber, B. , Staszewski, S., Perrin, L., Miller, V., Francioli, P., Furrer, H., Battegay, M., Vernazza, P., Bernasconi, E., Gunthard, H.F., Hirschel, B., Phillips, A.N. and Telenti, A., (2001). Low - level HIV viral rebound and blips in patients receiving potent antiretroviral therapy. http://gateway.nlm.nih.gov/MeetingAbstracts/ma?f=102244305.html [ Accessed August 15, 2009] . Griffiths, P.C., Paul, A., Khayat, Z Wan, K., King, S.M., Grillo, I., Schweins, R., Ferruti, P., Franchini J. and Duncan R., (2004). Understanding the Mechanism of Action of Poly(amidoamine)s as Endosomolytic Polymers: Correlation of Physicochemical and Biological Properties. Biomacromolecules, 5 , 1422? 1427. 238 Gunther, E.C., Stone, D.J., Gerwien, R.W., Bento, P. and Heyes, M.R., (2003) . Prediction of clinical drug efficacy by classification of drug- induced genomic expression profiles in vitro. Proceedings of the National Academy of Sciences, U S A., 100, 9608 ? 9613. Guoqiang, D., Batra, R . , Kaul , R., Gupta, M.N. and Mattiasson, B., (1995) . Alternative modes of precipitation of Eudragit S 100: a potential ligand carrier for affinity precipi tation of protein. Bioseparation, 5 , 339? 350. Gupta, P., Collins, K. B., Ratner, D., Watkins, S., Naus, G. J., Landers, D. V. and Patterson, B. K., (2002) . Memory CD4 + T cells are the earliest detectable human immunodeficiency virus type 1 (HIV - 1) - infected cells in the female genital mucosal tissue during HIV - 1 transmission in an organ culture system. Journal of Virology, 76, 9868 - 9876. Gupta, V. and Garg. R., (2009). Probiotics. Indian Journal of Medical Microbiology, 27, 202 - 209. Gurunathan, S., El Habib, R., Baglyos, L., (2009) . Use of predictive markers of HIV disease progression in vaccine trials. Vaccine, 27, 1997 - 2015. Haltrich, D. Lavssamayer B. and Steiner, W., (1994) . Xylanase formation by Sclerotium rolfsii effect of growth substrates and development of a culture medium using statistical designed experiments, Applied Microbiology and Biotechnology, 42 , 522? 530. Hamid, B., Hassan, Z. and Shams, N., (2008) . The use of polymer modification of bitumen for Durant hot asphalt mixtures. Journal of Applied Sciences Research, 4, 96- 102. Hardy, E., Hebling, E., Sousa, M., Al meida, A. and Amaral E., (2007) . Delivery of microbicides to the vagina: difficulties reported with the use of three devices, adherence to use and preferences. Contraception, 76, 126 - 131. 239 Harris, D. and Robinson, J.R. (1992) . Drug delivery via the mucous membranes of the oral cavity, Journal of Pharmaceutical Sciences, 81, 1 ? 10 . Harrison, P.F., Rosenberg, Z. and Bowcut, J., (2003) . Topical microbicides for disease prevention: Status and challenges. Clinical Infectious Diseases, 26, 1290- 1294. Hart C.E. and Evans- Strickfaden T., (2007) . HIV - 1 entry inhibitors as microbicides. Entry Inhibitors in HIV Therapy Book, 99- 117 Hartmann, S.U., Berlin, C.M. and Howett, M.K., (2006) . Alterna tive modified infant- feeding practices to prevent postnatal transimission of human immunodeficiency virus type 1 through breast milk: Past, present and future. Journal of Human Lactation, 22, 75- 88. Harwood, B., and Mishell, D.R., (2001) . Contraceptive vaginal ring. Seminars in Reproductive Medicine, 19, 381? 390. Hasan, E.I., Amro, B.I., Arafat, T. and Badwan A.A., (2003). Assessment of a controlled release hydrophilic matrix f ormulation for metoclopramide HCl. European Journal of Pharmaceutics and Biopharmaceutics, 55, 339 - 344. Hassanien, A., Milanova, M.G., Smolinski, T.G. et al., (2008). Computational Intelligence in Solving Bioinformatics Problems: Reviews, Perspectives, and Challenges. Computational Intelligence in Biomedicine and Bioinformatics , 151, 3 - 47. Hatcher, R., Trussell, J., Stewart F. et al., (2004). Contraceptiv e Technology . 18th revised edition. New York: Ardent Media, Inc. http://www.amazon.com/Contraceptive- Technology - 18th- Revised - 2004/dp/0966490266 [Accessed May 25, 2007] . 240 Hayakawa, T., Kawamura, M., Okamoto, M., Baba, M. et al., (1998) . Concanavalin A- immobilized polystyrene nanospheres capture HIV - 1 virions and gp120: Potential approach towards prevention of viral transmission. Journal of General Virology, 56, 327- 331. Hayden, E.C., (2009) . Mystery of HIV vaccine failure deepens. Nature news. doi:10. 1038. http://www.nature.com/news/2009/ 090720/full/news.2009. 707.html [Accessed September 28, 2009] He, G.Q., Chen, Q.H., Ju, X.J. and Shi, N.D., (2004) . Improved elastase production by Bacillus sp. EL31410- further optimization and kinetics studies of culture medium for batch fermentation, Journal of Zhejiang University Sc ience, 5 , 149? 156. Hearst, N. and Chen, S., (2004) . Condom promotion for AIDS prevention in the developing world: is it working? Studies in Family Planning. 35, 39 - 47. Helfand, E., and Tagami, Y., 1972. Theory of the interface between immiscible polymers, Journal of Chemical Physics, 57, 1812 - 1815. Hemmerling, A., Potts, M ., Walsh, J., (2007). Lime juice as a candidate microbicide? An open- label safety trial of 10% and 20% lime juice used vaginally. Journal of Womens Health, 16, 1041 - 1 051. Hendrix , C.W., Cao , Y.J. and Fuchs, E.J., (2009) . Topical microbicides to prevent HIV: Clinical drug development challenges. Annual Review of Pharmacology and Toxicology , 49, 349 - 375. 241 Herold, B.C., Bourne, N., Marcellino, D., Kirkpatrick, R., Strauss, D.M., and Zaneveld, L.J.D., Waller, D.P., Anderson, R.A., Chany, C.J., Barham, B.J., Stanberry, L.R. and Cooper, M.D., (2000) . Poly (sodium 4- styrene sulfonate): an effective candidate topical antimicrobial for the prevention of sexually transmitted diseases. Journal of infectious Diseases, 181, 770 - 773. Herrewege, Y.V., Michiels, J., Van Roey, J., Fransen, J.K., Kestens, L., Balzari ni, J., Lewi, P., Vanham, G. and Janssen, P., (2004). In vitro evaluation of non- nucleoside reverse transcriptase inhibitors UC- 781 and TMC120- R147681 as human immunode ficiency virus microbicides. Antimicrobial Agents and Chemotherapy , 48, 337? 339. Hervey, P. S. and Perry , C. M. , ( 2000). Abacavir : A review of its clinical potential in patients with HIV infection. Drugs, 60, 447- 479. Herzberg, M.C., Weinberg, A. and Wahl, S.M., (2006) . (C3) The oral epithelial cell and first encounters with HIV - 1. Advances in Dental Research, 19, 158 - 166. Highleyman, L., (2007) . UsherCell microbicides provides no protection against HIV transmission but VivaGel yields promising early data. 4 th International Conference on HIV Treatment, Pathogenesis and Prevention, Sydney, Australia. http://www.hivandhepatitis.com/recent/ 2007/ 020607_a.html [ Accessed November 20, 2007] . Hirbod, T. and Broliden, K., (2007). Mucosal immune responses in the genital tract of HIV - 1 - exposed uninfected women. Journal of Internal Medicine, 262, 1 44 ? 58. 242 Hiremath, P.S, and Saha, R.N., (2008). Oral matrix tablet formulations for concomitant controlled release of anti- tubercular drugs: Design and in vitro evaluations. International Journal of Pharmaceutics, 362, 118 - 125. Holmes, L., (2000). The Internal Triangle: New Theories of Female Development. Modern Psychoanalysis, 25, 207 - 226. Holmes, K.K., Levine, R., Weaver, M., (2004) . Effectiveness of condoms in preventing sexually transmitted infections . Bulletin of the World Health Organization, 82, 454 - 461. Holmes, V., Ramratnam, B., and Hartmann., (2007) . Developing a microbicide containing CCR5 siRNA for preventing the transmission of human immunodeficiency virus type 1 (HIV - 1). Alliance for Microbicides Development, 8, 1 - 21. Hopfield, J.J., and Tank, D.W., (1985). ?Neural? computation of decisions in optimization problems. Biological Cybernetics, 55, 141? 146. Howett, M.K., Neely, E.B., Christensen, N.D., Wigdahl, B., Krebs, F.C., Malamud, D,, Patrick, S.D., Pickel, M.D., Welsh, P.A., Reed, C.A., Ward, M.G., Budgeon, L.R. and Kreider, J.W., (1999). A broad- spectrum microbicide with virucidal activity against sexually transmitted viruses. Antimicrobial Agents and Chemotherapy , 43, 314? 321. Huggins G. R., Preti G., (1981). Vaginal odors and secretions. Clinical Obstetrics and Gynecology , 24, 355 - 377. Hussain, A. and Ahsan, F., (2005). The vagina as a route for systemic drug delivery. Journal of Controlled Release, 1 03, 301 - 313. 243 Hussain, N., ( 2000) . Bioadhesive Drug Delivery systems: Fundamentals, novel approaches and development, International Journal of Pharmaceutics, 205, 201 - 202. Hussain, A.S., Johnson, R.D., Vachh arajan , N. N. and Ritschell, W.A., (1993) . Feasibility of developing a neural network for prediction of human pharmacokinetic parameters from animal data. Pharmaceutical Research, 10, 466- 469. Hwang, S., Wada, E.O., Yotsuanagi , T., Suhardja, I., Ho, N.F.H., Flynn, G.L. and Higuchi, W.I., (1977) . Systems approach to vaginal delivery of drugs: II. In situ vaginal absorption of unbranched aliphatic alcohols. Journal of Pharmaceutical Sciences, 65, 1574? 1578. Ibata, B., Parr, E., King, N. and M. Parr, M., ( 1997) . Migration of foreign lymphocytes from the mouse vagina into the cervicovaginal mucosa and to the iliac lymph nodes. Biology of Reproduction, 56, 537- 543. Idemyor, V., (2004) . The concept of structured treatment interruptions in the management of patients with Human Immunodeficiency Virus (HIV) disease: Where Are We Currently? HIV Clinical Trials , 4, 79- 83 Iijima, N., Line han, M.M., Zamora, Me., Butkus, D. , Butkus, D., Dunn , R., Kehry , M.R., Laufer T.M. and Iwasaki A., (2008). Dendritic cells and B cells maximize mucosal Th1 memory response to herpes simplex virus Isek i, T., Takahashi, M., Hattori, H., Hatakeyama, T. and Hatakeyama, H., ( 2001). Viscoelastic properties of xanthan gum hydrogels annealed in the sol state. Food and Hydrocolloids, 15, 503- 506. . The Journal of Experimental Medicine, 205, 3041- 3052. 244 Iyer, V., Bendgude, N. and Poddar, S.S., (2008) Vaginal Drug De livery. Express Pharma. http://www.expresspharmaonline.com/20080715/research02.shtml [Accessed 19 March 2009] . Jast, B., Li, X. and Cleary, G., (2003) . Recent advances in mucoadhesive drug delivery systems. Drug Delivery Polymers, 1, 194 - 196. Jeong, B., Kim S.W. and Bae Y.H., (2002) . Thermosensitive sol ? gel reversible hydrogels. Advanced Drug Delivery Reviews , 54, 37 - 51. Jha, B.K., Tambe, S.S. and Kulkarni, B.D., (1995) . Estimating diffusion coefficients of micellar system using an ANN, Colloid and Interface Science, 170, 392? 398. Jiang, C., Li, H., Tripp, C. P., (2003) . Infrared Method for In Situ Studies of Polymer/Surfactant Adsorption on Silica Powders from Aqueous Solution, Appllied. Spectroscopy, 57, 1419- 1424. Jiang, Y., Emau, P., Cairns, J.S. , Flanary, L ., Morton, W.R ., McCarthy, T.D . and Tsai, C.C. (2005). SPL7013 gel as a topical microbicide for prevention of vaginal transmission of SHIV in macaques. AIDS Research and Human Retroviruses , 21, 207 - 213. Jim?nez - castellanos, M.R., Zia, H. and Rhodes C. T., (1993). Mucoadhesive Drug Delivery systems. Drug Development and Industrial Pharmacy , 19, 143- 194. Jin, x., Zhang, y., Xiao , l. and Zhao , z., (2008) . Optimization of Extended Zero- order Release Gliclazide Tablets Using D- optimal Mixture Design. Yakugaku Zasshi , 128, 1475- 1483. 245 Johansson, E.D.B., Luukkainen, T., Vartiainen, E., Victor , A., (1975) . The effect of progestin R 2323 released from vaginal rings on ovarian function. Contraception, 12, 299 - 307. Johnson, V.E. and Masters, W.H., (1962). Intravaginal contraceptive study: Phase I. Anatomy. Western Journal of Surgery, Obstetrics and Gynecology , 70, 2 02 ? 207. Joshi, S.N., Katti, U., Godbole, S., Bharucha, K., Kulkarini, S., Risbud, A. and Mahendale, S., (2005) . Phase I safety study of praneem polyherbral vaginal tablet use among HIV - uninfected women in Pune, India. Transactions of t he Royal Society of Tropical Medicine and Hygiene, 99, 769 - 774. Joshi, S.N., Dutta, S., Bell, B., et al., (2006) . Phase I safety study of 0.5% PRO 2000 vaginal gel among HIV un- infected women in Pune, India. AIDS Research and Therapy, 3, 2 - 6. Junginger, H.E., (1990) . Bioadhesive polymer systems for peptide delivery, Acta Pharmaceutica Technologica, 36, 110? 126. Justin- Temu, M., Damian, F., Kinget, R. and Van Den Mooter, G., (2004) . Intravaginal gels as Drug Delivery systems. Journal of Women?s Health, 3 1, 834- 843. Kaelble, D.H., 1977. A surface energy analysis of bioadhesion, Polymer, 18 , 475 - 482. Kalugin, O.N., Volobuev, M.N., Ischenko, A.V., and Adya, A.K., 2001. Structure and dynamics of Na + and Cl? salvation shells in liquid DMSO: molecular dynamic s simulations. Journal of Molecular Liquids, 91, 135- 148. 246 Kapitza, S.B., Michel, B.R., van Hoogevest, P. , Leigh, M.L.S. and Imanidis, G., (2007). Absorption of poorly water soluble drugs subject to apical efflux using phospholipids as solubilizers in the Caco - 2 cell model. Intentional Journal of Pharmaceutics, 66, 146 - 158. Kassaye S.G. and Katzenstein D., (2003) . HIV/AIDS care and treatment in sub- Saharan Africa. AIDS Revision, 5, 195- 204. Kast, C.E., Valenta, C. , Leopold, M., and Bernkop- Schn?rch, A., ( 2002) . Design and in vitro evaluation of a novel bioadhesive vaginal Drug Delivery system for clotrimazole, Journal of Controlled Release, 81, 347- 354. Kathambi, K., (2008). Microbicides: Women?s weapon in the war against HIV/AIDS, Association for Women?s Rights in Development (AWID). http://www.awid.org/eng/Issues - and- Analysis/Library/Microbicides - A- weapon- for- women- in- the- war- against- HIV - AI DS2 [Accessed June 15 , 2009] . Katsamba , P., Carroll, K. Ahlsen, G., Bahna, F., Vendome, J., Posy, S., Rajebhosale, M., Price, S., Jessell, T. M. Ben - Shaul, A., Shapiro, L. and Honig , B.H. Link ing molecular affinity and cellular specificity in cadherin- mediated adhesion. Proceedings of the National Academy of Sciences. 106, 1194 - 11599. Katsnelson, A., (2009) . New wrinkle for HIV vaccine. The Scientist.com. http://www.the - scientist.com/blog/display/55478/ [Accessed September 28, 2009] . Katz, D.F., Dunmire, E.N., Henderson, M.H., Owen, D.H. and Plenys, A.M., (1997). Applications of biomedical engineering in reproductive biomedicine: sensing and Drug Delivery to the lower female reproductive tract. Engineering in Medicine and Biology Society, 6 , 2656 ? 2658. 247 Katz, D.F. and Dunmire, E.N., (1993). Cervical mucus. Problems and opportunities for Drug Delivery via the vagina and cervix. A dvanced Drug Delivery Reviews, 11 , 385? 401. Kaufmann, G.R. and Cooper D.A., (2000) . Antiretroviral therapy of HIV - 1 infection: established treatment strategies and new therapeutic options. Current Opinion in Microbiology, 3, 508- 514. Kaul, R., Pettengell, C., Sheth, P., Sunderji , S. , Biringer, A. , MacDonald, K . , Walmsley, S. and Rebbapragada , A., (2008) . The genital tract immune milieu: an important determinant of HIV susceptibility and secondary transmission. Journal of Reproductive Immunology , 77, 32 - 40. Keiser, P., (2002) . Role of Sequencing in Therapy Selection. Journal of Acquired Immune Deficiency Syndromes, 29, S19 - S27. Keller, M.J. , Tuyam, A., Carlucc i, M.J. and Herold, B.C., (2005). Topical microbicides for the prevention of genital herpes infection. Antimicrobial Agents and Chemotherapy, 55, 420- 423. Kerr, M., (2007) . NNRTI shows safety as a topical anti - HIV microbicide in animal tests. Antimicrobial Agents and Chemotherapy, 51, 1608- 1615. Kessler, H.A., (2005) . Triple - Nucleoside Analog Antiretroviral Therapy: Is There Still a Role in Clinical Practice? A Review. Medscape General Medicine, 7, 70. Khamanga, S.M. and Walker R.B., ( 2006). Evaluation of Rate of Swelling and Erosion of Verapamil (VRP) Sustained- Release Matrix Tablets . Drug Development and Industrial Pharmacy, 32, 1139 ? 1148. 248 Khanlou H., Yeh, V ., Guyer, B. and Farthing, C., (2005) . Early virologic failure in a pilot study evaluating the efficacy of therapy containing once- daily Abacavir, Lamivudine, and Tenofovir DF in treatment of Na?ve HIV - infected patients. AIDS Patient Care and STDs, 19, 135 - 140. Khoee, S., Hassanzadeh, S. and Goliaie, B., (2007) . Effects of hydrophobic drug - polyesteric core interactions on drug loading and release properties of poly (ethylene glycol) triblock core shell nanoparticles. Nanotechnology, 18, 1 - 9. Kim, H. and Fassihi, R., (2000). Application of a binary polymer system in drug release rate modulation. 1. Characterization of release mechanism. Journal of Pharmaceutical Sciences, 86, 316 ? 322 . King, M.R., (2001) . Multiparticle Adhesive Dynamics. Interactions between Stably Rolling Cells Biophysical Journal, 81, 799 - 813 Kirton, K.T., Roseman, T.J., Forber, A.D., (1973) . Ev aluation of progesterone- containing silicone vaginal devices in rhesus monkeys. Contraception, 8, 561- 568. Kiser, P ., (2006a ). A molecular condom against AIDS. Science Daily. http://www.sciencedaily.com/releases/2006/ 12/ 061212091837.htm [ Accessed March 26, 2007] . Kiser, P ., (2006b ). HIV - blocking gel for women: New 'Molecular Condom' meant to prevent AIDS. http://www.sciencedaily.com/releases/2009/ 08/ 090810024837.htm [Accessed August 20, 2009] . 249 Klasse, P.J. Shattock R. and Moore, J.P., (2008). Antiretroviral drug- based microbicides to prevent HIV - 1 sexual transmission. Annual Revi ew of Medicine, 59, 455 ? 471. Klavinskis, L.S., Daheshia, M., Karem, K., Manickan, E. and Rouse, B.T., (1999) . Intranasal immunization with plasmid DNA - lipid complexes elicits mucosal immunity in the female genital and rectal and rectal tracts. Journal of Immunology, 1, 254? 262. Knox, P., (2004). Knox, P., (2004) . Drug Delivery device for insertion into the vagina, rectum or nasal cavity. European Patent EP1200151. http://www.freepatentsonline.com/ EP1200151. html [ Accessed May 20, 2009] . Knudsen, K.D., Lauten, R.A., Kj?niksen, A., Nystr?m, B., (2004). Rheological and structural properties of aqueous solutions of a hydrophobically modified polyelectrolyte and its unmodified analogue. European Polymer Journal, 40 , 721 - 733. Knuth, K., Amiji, M. and Robinson, J.R., (1993). Hydrogel delivery systems for vaginal and oral applications: Formulation and biological consideration. Advanced Drug Delivery Reviews, 11, 137? 164. Kolawole, O.A., Pillay, V. and Choonara, Y.E., (2007). Novel polyamide 6,10 variants synthesized by modified interfacial polymerization for application as a rate- modulated monolithic Drug Delivery System. Journal of Bioactive and Compatible Polymers, 22, 281- 313. Kozlowski, P.A. and Neutra, M.R., (2003) . The Role of mucosal immunity in prevention of HIV transmission. Current Molecular Medicine, 3, 217- 228. 250 Krauss, K. and Altevogt, P., (1999) . Integrin leukocyte function- associated antigen- 1 - mediated cell binding can be activated by clustering of membrane rafts. Journal of Biological Chemistry, 274 , 36921 - 36927. Krebs, F.C., Miller, R.S., Catalone, B.J., Welsh, P.A. Malamud, D., Howett, M.K. and Wigdahl, B., ( 2000). Sodium dodecyl sulfate and C31G as microbicidal alternatives to nonoxynol 9: Comparative sensitivity of primary human vaginal keratinocytes. Antimicrobial Agents and Chemotherapy 44, 1954 - 1960. Kremer, M.J., Wertz, P.W. and Squier, C.A., (2001) . Permeability and barrier function of three porcine non- keratinized mucosae, Journal of Dental Res earch, 80, 851. Kristmundsd?ttir, T., ?rnad?ttir, S.G., Bergsson, G. and Thormar, H., (2000) . Development and evaluation of microbicidal hydrogels containing monoglyceride as the active ingredient. Journal of Pharmaceutical Sciences, 88, 1011 - 1015. Kumar, M.N. V.R., Kumar, N., Dom b, A. J. and Arora M., (2002) . Pharmaceutical Polymeric Controlled Drug Delivery Systems. Advances in Polymer Science, 160, 47- 117. Kulkarni, A., Reiche J . , Lendlein, A., (2007) . Hydrolytic degradation of poly( rac- lactide) and poly[( rac- lactide) - co- glycolide] at the air - water interface. Surface and Interface Analysis, 39, 740- 746. Lackner, A.A. and Veazey, R.S., (2007) . Current concepts in AIDS pathogenesis: Insights from the SIV/Macaque model. Annual Review of Medicine, 58, 461 - 467. 251 Lai S.K., Wang Y. and Hanes J., (2009) . Mucus - penetrating nanoparticles for drug and gene delivery to mucosal tissues. Advanced Drug Delivery Reviews , 61, 158 - 171. Lamont, R.F., Jones, B.M., Mandal, D., Hay, P. E. and Sheehan, M., (2003) . The efficacy of vaginal clindamycin for the treatment of abnormal genital tract flora in pregnancy. Infectious Diseases in Obstetrics and Gynecology , 11, 181? 189. Lancashire, L.J., Lemetre C. and Ball, G.R., (2009) . An introduction to artificial neural networks in bioinformatics?application to complex microarray and mass spectrometry datasets in cancer studies. Briefings in Bioinformatics, 10, 315- 329. Lard- Whiteford, S.L., Matecka, D., O'Rear, J.J., Yuen, I.S ., Litterst, C . and Reichelderfer, P., (2004) . Recommendations for the Nonclinical Development of Topical Microbicides for Prevention of HIV Trans mission: An Update. JAIDS Journal of Acquired Immune Deficiency Syndromes, 36, 541 - 552. Lau, C., Velasco P.P. and Johnston, M. I ., (2007). A new era in HIV vaccine development. Expert Review of Anti -infective Therapy, 5, 205- 215. Lazaridou, A. and Biliaderis, C.G., (2004) . Cryogelation of cereal ? - glucans: structure and molecular size effects. Food Hydrocolloids , 18, 933 - 947. Lazarus, J.V., H?ggblom, A., Kirkegaard, L., Lundgren, J.D. and Matic S., (2008). The elusive search for an HIV vaccine?and what to do meanwhile. Vaccine, 26, 6491 - 6493. Le Cer, R.D., Picton, L., Argillier, J.F., Muller, G., (2004) . Entrapment and release of sodium polystyrene sulfonate (SPS) from calcium alginate gel beads. European Polymer Journal, 40 , 2709- 2715. 252 Leane, M.M., Cumming, I., Corrigan, O.I., (2003) . The Use of Arti ficial Neural Networks for the selection of the most appropriate formulation and processing variables in order to predict the in itro dissolution of sustained release minitablets. AAPS Pharmaceutical Sciences and Technology, 4, 1 - 12. Ledergerber, B., Egger, M., Opravil, M. , Telenti, A ., Hirschel, B., Battegay, M ., Vernazza, P., Sudre, P., Flepp, M ., Furrer, H., Francioli, P. and Weber R ., (1999). Clinical progression and virological failure on highly active antiretroviral therapy in HIV - 1 patients: a prospective cohort study. Swiss HIV Cohort Study. Lancet, 353, 863 - 868. Lee, C.H., Bagdon, R. and Chien, Y.W., (1996). Comparative in vitro spermicidal activity and synergistic effect of chelating agents with nonoxynol - 9 on human sperm functionality. Journal of Pharmaceutical Sciences, 85, 91? 95. Lee, P.I., (1980). Diffusional release of a solute from a polymeric matrix ?approximate analytical solutions. Journal of Membrane Science, 7, 255 - 275. Lee, W.J., Park, J.H. and Robinson, J.R., (2000). Bioadhesive- based dosage forms: The next generation. Journal of Pharmaceutical Sciences, 89, 850- 866. Lee, Y., Khemka, A., Yoo J. and Lee C.H., (2008) . Assessment of diffusion coefficient from mucoadhesive barrier devices using artificial neural networks. International Journal of Pharmaceutics, 351, 119 - 26. Lehr, C.M., (2000). Lectin- mediated Drug Delivery: The second generation of bioadhesive, Journal of Cont rolled. Release, 65, 19- 29. 253 Lehr, C.M., (1995) . Bioadhesion technologies for the controlled delivery of peptide and protein drugs to the gastrointestinal tract. Critical Reviews in Therapeutic Drug Carrier Systems, 11, 177? 218. Lejoyeux, F., Ponchel, G ., Wouessidjewe, D., Peppas, N.A. and Duchene, D., (1989) . Bioadhesive tablets?influence of the testing medium composition on bioadhesion, Drug Dev. Ind. Pharm. 15, 2037? 2048. Lemiale, F. and Korokhov, N., (2009) . Lentiviral vectors for HIV disease prevention and treatment. Vaccine, 27, 3443- 3449. Levine, W. C., Pope, V., Bhoomkar, A., Tambe, P. , Lewis, J.S ., Zaidi, A.A ., Farshy, C.E ., Mitchell, S . and Talkington, D.F., (1998) . Increase in endocervical CD4 lymphoc ytes among women with nonulcerative sexually transmitted diseases. Journal of infectious Diseases, 177 , 167 - 174. Levinson, P., Kaul, R., Kimani, J. Ngugi, E ., Moses, S ., MacDonald, K.S ., Broliden, K . and Hirbod, T ., Kibera HIV Study Group. , (2009). Levels of innate immune factors in genital fluids: association of alpha defensins and LL- 37 with genital infections and increased HIV acquisition. AIDS, 23, 309 - 317. Lew, C.W., (1994). Controlled release pH sensitive capsule and adhesive sy stem and method. US Patent 5364634. http://www.freepatentsonline.com/5364634.html [Accessed June 2, 2007] . Li, X., Kolltveit, K.M., Tronstad , L . and Olsen I., (2000) . Systemic Diseases Caused by Oral Infection. Clinical Microbiology Reviews, 13, 547? 558. 254 Lin C.C. and Metters A.T., (2006). Hydrogels in controlled release Formulations: Network design and mathematical modeling. Advanced Drug Delivery Reviews , 58, 1379- 1408. Little, S.J., Holte, S., Routy, J.P ., Daar E.S., Markowitz, M., Collier, A.C., Richard A. Koup, R.A., Mellors, J.W. , Connick, E., Conway, B., Kilby, M., Wang, L., Whitcomb, J.M., Hellmann, N.S. and Richman, D.D., (2002). Antiretroviral - drug resistance among patients recently infected with HIV. New England Journal of Medicine, 347, 385 - 394. Liu, J., Zhang, F. and McGinity, J.W., (2001). Properties of lipophilic matrix tablets containing phenylpropanolamine hydrochloride prepared by hot- melt extrusion. European Journal of Pharmaceutics and Biopharmaceutics, 52, 181- 190. Liu, T.X ., Liu, Z.H., Ma, K.X., Shen, L., Zeng, K.Y., He, C.B., (2003) . Morphology, thermal and mechanical behavior of polyamide 6/layered- silicate nanocomposites. Composites Science and Technology, 63, 4 331 - 337. Liu, H., Li, X., Stanton, B. Liu, H., Liang, G., Chen, X., Yang, H. and Hong Y., (2005) . Risk Factors for Sexually Transmitted Disease Among Rural - to- Urban Migrants in China: Implications for HIV/Sexually Transmitted Disease Prevention. AIDS Patient Care STDS, 19, 49 - 57. Lohr, P.A., (2007). The Future Role of Vaccines and Microbicides. Sexually Transmitted Diseases Book . 321- 344. Longer, M.A., and Robinson, J.R., (1986) . Fundamental aspects of bioadhesion, Pharmacy International, 7 , 114 - 117. 255 Lopes, C.M., Lobo, J.M.S., Pinto, J.F. and Costa, P.C., (2007). Compressed Matrix Core Tablet as a Quick/Slow Dual - Component Delivery System Containing Ibuprofen. AAPS Pharmaceutical Sciences and Technology, 8 , E1 - E8. Lozinsky, V.I. and Damshkaln, L.G., (2001). Study of cryostructuration of polymer systems. XX. Foamed poly(vinyl alcohol) cryogels . Journal of Applied Polymer Science, 82, 1609 ? 1619. Lucas, G.M., (2008) . Antiretroviral adherence, drug resistance, viral fitness and HIV disease progression: a tangled web is woven. Journal of Antimicrobial Chemotherapy, doi:10. 1093/jac/dki042, 1 - 4. Lucas, G.M., Chaisson, R.E. and Moore, R.D., (1999) . Highly active antiretroviral therapy in a large urban clinic: risk factors for virologic failure and adverse drug reactions. Annals of Internal Medicine, 131, 81 - 87. Madhavlal, P.G. and Manordas, P.M., (2009) . Design and In Vitro Evaluation of a Novel Vaginal Drug Delivery System Based on Gelucire. Current Drug Delivery , 6, 159 - 165. Maggi, L., Mastromarino, P., Macchia, S., Brigidi, P., Pirovano, F., Matteuzzi, D. and Conte, U., (2000) . Technological and biological evaluation of tablets containing different strains of lactobacilli for vaginal administration. European Journal of Pharmaceutics and Biopharmaceutics, 50, 389 ? 395. Maguire, R.A., Bergman, N. and Phillips, D.M., (2001). Comparison of Microbicides for Efficacy in Protecting Mice Against Vaginal Challenge With Herpes Simplex Virus Type 2, Cytotoxicity, Antibacterial Properties, and Sperm Immobilization. Sexually Transmitted Diseases, 28, 259 - 265. 256 Mainardes, R.M., Gremi ao, M.P.D. and Evangelistica, R.C., (2006) . Thermoanalytical study of praziquantel - loaded PLGA nanoparticles. Brazilian. Journal of Pharmaceutical Sciences, 42, 523 - 530. Malcolm, K.R. and Woolfson, D.A., (2006). Delivery hope on HIV - Vaginal rings for controlled release of microbicides. Journal of Controlled Release, 74, 52- 55. Malcolm, K.R., Woolfson, D.A., Toner, C., Morrow R.J. and McCullagh, S.D., ( 2005) . Long- term, controlled release of the HIV microbicide TMC120 from silicone elastomer vaginal rings. Journal of Antimicrobial Chemo therapy, 56, 954 - 956. Malik, R., Tondwal, S., Venkatesh, K.S. and Misra, A., (2008) . Nanoscaffold matrices for size - controlled pulsatile transdermal testosterone delivery: nanosize effects on the time dimensions. Nanotechnology, 19, 1 - 6. Malow, R.M., Rosenberg, R. and Devieux, J.G., (2009) . Cognitive- behavioral stress management interventions for ethnic- minority HIV - positive alcohol/drug abuses in resource limited and cultural diverse communities. American Journal of Infectious Diseases, 5, 48 - 59. Mandal, T.K., ( 2000) . Swelling - controlled release system for the vaginal delivery of miconazole, European Journal of Pharmaceutics and Biopharmaceutics, 50 , 337 - 343. Manson, K.H., Wyand, M.S., Miller, C. and Neurath, A.R., (2000) . Effect of cellulose acetate phthalate topical cream on vaginal transmission of simian immunodeficiency virus in rhesus monkeys. Antimicrobial Agents and chemotherapy , 44, 3199 - 3302. 257 Mastromarino, P., Macchia, S., Meggiorini, L. , Trinchieri, V., Mosca, L ., Perluigi, M. and Midulla C., ( 2009). Effectiveness of Lactobacillus - containing vaginal tablets in the treatment of symptomatic bacterial vaginosis. Clinical Microbiology and Infection, 15, 67- 74. Mantell, J.E., Myer, L., Carballo- Di?guez, A., (2005) . Microbicide acceptability research: current approaches and future directions. Social Science & Medicine, 60, 319 - 330. Marconi, V.C., Sunpath, H. and Lu, Z., (2008). Prevalence of HIV - 1 Drug Resistance after Failure of a First Highly Active Antiretroviral Regimen in KwaZulu Natal, South Africa. Clinical Infectious Diseases , 46, 1589? 1597. Maria - Elisa, P., Pirovano, A. and Phillips, D.M., (2003) . Carageenan formulation prevents macrophage trafficking from vagina: Implications for microbicide development. Biology of Reproduction, 69, 933? 939. M arks, K. and Gulick, R.M., (2004) . New antiretroviral agents for the treatment of HIV infection Current Infectious Disease Reports , 6, 333 - 339. Marin - Mulle r, C., Li, M., Chen, C., and Yao. O., (2009) . Current Understanding and Potential Immunotherapy for HIV - Associated Squamous Cell Carcinoma of the Anus (SCCA) . World Journal of Surgery , 33, 653 - 660. Martinez - Picado, J. and Wai, Y.T . L., (2007). Risk of selecting resistance mutations during treatment interruption. Current Opinion in HIV and AIDS, 2, 6 - 13. Marx, P., Spira, A. Gettie, A. (1996). Progesterone implants enhance SIV vaginal transmission and early virus load. Nature Medicine, 2, 1084 - 1089. 258 Mathowitz, E.C.D. and Jacob, S.J., (1999). Bioadhesive Drug Delivery systems. In Encyclopaedia of Controlled Drug Delivery 1 ; John Wiley and Sons: New York, USA . Mayer, K.H., Karim, S.A., Kelly, C., Maslankowski,L., Rees, H., Profy, A.T., Day, J., Welch. J. and Rosenberg Z., (2003). The safety and tolerability of a novel vagi nal microbicides, PRO 2000/ 5Gel in sexually active HIV - uninfected and abstinent HIV - infected women. AIDS, 17, 321 - 329. McCormack , S., Hayes, R., Lacey , C.J.N. and Johnson, A.M., (2001). Microbicides in HIV prevention. British Medical Journal , 322, 410- 413. Mehta, K.A., Kisla lioglu, M.S., Phuapradit, W., Malick A.W. and Shah, N.H. (2000) . Effect of formulation and process variables on porosity parameters and release rates from a multi unit erosion matrix, Journal of Controlled Release, 63, 201 - 211. Mendyk, A. and Jachowicz, R., (2005). Neural network as a decision support system in the development of pharmaceutical formulation? focus on solid dispersions. Expert Systems with Applications , 28, 285 - 294. Meijer, D.K.F., Mol, W.E.M., M?ller , M. and Kurz, G., (1990). Carrier - mediated transport in the hepatic distribution and elimination of drugs, with special reference to the category of organic cations. Journal of Pharmacokinetics and Pharmacodynamics , 18, 35- 70. Merabet , J., Thompson, D. and Levinson, R.S., (2005) . Advancing vaginal drug delivery. Expert Opinion on Drug Delivery, 2, 769 - 777. 259 Mikos, A.G., and Peppas, N.A., 1986. Proceedings of International Symposium on Controlled Release of Bioactive Materials, 13, 97. Mikos, A.G., and Peppas, N.A., ( 1990) . Scaling concepts and molecular theories of the adhesion of synthetic polymers to glycoproteins, In: Lenaerts, V., and Gurny, R. (Ed.), Bioadhesive Drug Delivery systems. Boca Raton. Fl., 25 - 42. Miller, C.J., Vogel, P., Alexander, N., Sutjipto, N., Hendrickx, A. G. and Marx, P. A., (1992). Localization of SIV in the genital tract of chronically infected female rhesus macaques. American Journal of Pathology, 141, 655- 660. Miller, C.J. and Shattock R.J., (2003) . Target cells in vaginal HIV transmission. Microbes and Infection, 5, 59 - 67. Mishell, D.R., Lumkin, M.and Stone, S., (1972) . Inhibition of ovulation with cyclic use progesterone- impregnanted devices. American Journal of Obstetrics and Gynecology, 13, 927 - 932. Mitchel, D., (2006). Focus renewed on HIV microbicides, International Health Conference ,Canada. https://www.ipm - microbicides.org/news _room/English/feature_room/ English/feature _ stories_ full.htm [Accessed June 15, 2009] . Moore, J.P. and Shattock, R.J., ( 2003). Preventing HIV - 1 sexual transmission?not sexy enough science, or no benefit to the bottom line? Journal of Antimicrobial Chemotherapy, 52, 890? 892. Moore, J.P., Kitchen S.G., Pugach, P. and Zack , J. A., ( 2004) . The CCR5 and CXCR4 coreceptos- central to understanding the transmission and pathogenesis of human 260 immunodeficiency virus type 1 infection. Aids Research and Human Retroviruses, 20, 111- 126. M orin, S.F., Morfit, S., Majorana, A. , Amamrattana, A., Goicochea, P., Mutsambi, J.M., Robins J.l. and Richards, T.A., (2008). Building community partnerships: case studies of Community Advisory Boards at research sites in Peru, Zimbabwe, and Thailand. Clinical Trials, 5, 147. Morrow, K., Rosen, R., Ritcher, L., Emans, A., Forbes, A., Day, J., Morar, N., Maslankowski. L., Profy, A.T., Kelly, C., Abdool Karim, S.S. and Mayer, K.H., (2003). The acceptability of an investigational vaginal microbicide, PRO 2000 Gel, among women in a phase I clinical trial. Journal of Women?s Health, 12, 655 - 666. Mosher, D.F. , (1990) . Physiology of thrombospondin. Annual Review of Medicine, 41, 85 - 89. Moyle, G., (2003). Stopping HIV fusion with enfuvirtide: the first step to extracellular HAART Journal of Antimicrobial Chemothe rapy, 51, 213 - 217. MT N microbicide trials networ k (2009). Trial finds microbicide promising as HIV prevention method for women. http://www.mtnstopshiv.org/node/765 [Accessed August 20, 2009 ] . Mukherjee, G.S., (2009). Calorimetric c haracterization of membrane materials based on polyvinyl alcohol. Journal of Thermal Analysis and Calorimetry , 96, 21 - 25. M?ller, B.W., (1986). Factors which are influencing the drug liberation as well as topical effects, Suppositoria Wissenschaftl , Verlagsges, Stuttgart, 272? 275. 261 Mumper R.J., Bell, M.A. and Worthen, D.R. , Cone, R.A., Lewis, G.R., Paull, J.R.A., Moench, T.R., (2009). Formulating a Sulfonated Antiviral Dendrimer in a Vaginal Microbicidal Gel Having Dual Mechanisms of Action. Drug Development and Industrial Pharmacy, 35, 515 - 524. Munasur, A.P., Pillay, V., Choonara, Y.E., Mackraj, I.and Govender, T., (2008) . Comparing the mucoadhesivity and drug release mechanisms of various polymer- containing propranolo buccal tablets. Drug Development and Industrial Pharmacy , 34, 189- 198. Murthy, N.S., (2006). Hydrogen bonding, mobility, and structural transitions in aliphatic polyamides. Journal of Polymer Science Part B: Polymer Phys ics, 44, 1763 ? 1782 . Nair, T.M., Tambe S.S. and Kulkarni., (1994) . B.D. Application of artificial neural networks for prokaryotic transcription terminator prediction, FEBS Letters, 346, 273? 277. Najah, A., Elshafie, A., Karim O.A. and Jaffar, O., (2009). Prediction of Johor river water quality parameters using artificial neural networks. European Journal of Scientific Rese arch, 28, 422- 435. Ndesendo, V.M.K., Meixner, W., Korsatko, W., Kortsatko - Wabnegg, B., (1996). Microencapsulation of chloroquine diphosphate by Eudragit RS 100. Journal of Microencapsulation, 13, 1 - 8. Ndesendo, V.M.K., Pillay, V., Choonara, Y.E., Buchmann, E., Bayever, D.N., Meyer, L.C.R., (2008). Current intravaginal Drug Delivery approaches employed for the 262 prophylaxis of HIV/AIDS and prevention of sexually transmitted infections. AAPS Pharmaceutical Sciences and Technology, 9, 505 - 520. Ndesendo, V.M .K., Pillay, V., Choonara, Y.E., Khan, R.A., Meyer, L., Buchmann, E., Rosin, U., (2009) . In vitro and ex vivo bioadhesivity analysis of polymeric intravaginal caplets using physicomechanics and computational structural modeling. International Journal of Pharmaceutics, 370, 151 - 159. Nelson, A.L., 2008. The Vagina: New options for the administration of medications. http://www.medscape.com/ viewarticle/504375_6 [ Accessed, March 10] . Neurath, A.R., Srick, N. and Li, Y., (2003). Water dispersible microbicidal cellulose acetate phthalate film. BMC Infectious Diseases, 3, 27. Neyts, K.T.J., De Clercq, E., and Thormar, H., (2000) . Hydrogels containing monocaprin prevent intravaginal and intracutaneous infections with HSV- 2 in mice: Impact on the search for vaginal microbicides. Journal of Medical Virology, 61, 107 - 110. Nguyen, D.H., Hildreth, J.E., (2000) . Evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid- enriched membrane lipid rafts. Journal of Virology, 2000. 74, 3264 - 3272. Nicoll, A., Gill, O., Peckh am C. S., Ades A.E., Parry, J., Mortimer P., Goldberg D., Noone A., Bennett D. and Catchpole M., (2000) . The public hea lth applications of unlinked anonymous seroprevalence monitoring for HIV in the United Kingdom . International Journal of Epidemiology, 29, 1 - 10. 263 Nicolazzo, J.A. and Finnin, B.C., (2008) . In Vivo and In Vitro Models for Assessing Drug Absorption Across the Buccal Mucosa. Biotechnology: Pharmaceutical Aspects , VII, 89- 111. Nijhawan, A., Zaller, N., Cohen, D. and Rich J.D., (2009) . Interventions with incarcerated persons. HIV Prevention, Doi:10. 1016/B978- 0 - 12- 374235- 3. 00016 - 9, 444- 471. Nikolic, D.S., Garcia, E. and Piguet, V., (2007). Microbicides and other topical agents in the prevention of HIV and sexually transmitted infection. Expert Review of Anti-infective Therapy, 5 , 77- 88. Nirmal, M., (2006). Control release formulation containing a hydrophobic material as the sustained release agent. US Patent, 7052706 . http://www.patentstorm.us/patents/70527 06/ description .html [ Accessed June 9, 2009] . Norris, S., (2008). HIV/AIDS - Past, Present and Future. http://www.parl.gc.ca/informa tion/ http://www.parl.gc.ca/information/library/PRBpubs/prb0208 - e.htm [ Accessed June 16, 2009] . Nov?k, C., de la Loge, L. and van der Meulen, E.A., (2003) . The combined contraceptive vaginal ring, NuvaRing: an international study of user acceptability, Contraception, 67, 187? 194. Nuovo, G., Forde, A., MacConnell, P. and Fahrenwald, R., ( 1993) . In situ detection pf PCR- amplified HIV - 1 nucleic acids and tumor necrosis factor cDNA in cervical tissues. American Journal of Pathology, 143, 40 - 48. 264 Nuttall, J., Romano, J., Douville, K. Galbreath, C. and Nel, A., (2007) . The future of HIV prevention: Prospects for an effective anti - HIV microbicide. Infectious Disease Clinics of North America, 21, 219 - 239. Nuwayser, E.S. and Williams, D.L., (1974). Development of delivery system for prostaglandins. Advances in Experimental Medicine, 47, 45 - 164. Obiero, J.A., Mburu, M.N., Ndung'u B.M. , Waititu, K.K., Mulei, I., Farah, I.O. and Mwet hera, P.G., (2008) . UniPron is A Fully Effective Non- hormonal Reversible Contraceptive in Baboon Model ( Papio Anubis ) . Journal of Reproduction and Contraception , 19, 107 - 118 Obioha, E.E., (2008) . Exploring the cultural context of HIV/AIDS pandemic in a Nigerian community: Implications of cultural specific prevention programmes. Anthropology , 10, 269- 276. O'Brien, R.F., (2006) . Vaginal Microbicides. MD Consult Preview , 17, http://www.mdconsult.com/php/about/155168591 - 2/pm_SubOps.html [Accessed August 19, 2009 ] . Odaka, C. and Ding, A., (2009). Secretory Leukocyte Protease Inhibitor: More than Just A Protease Inhibitor. Current Immunology Reviews , 5, 135- 142. Ogra, P.L., Faden, H. and Welliver R.C., (2001) . Vaccination Strategies for Mucosal Immune Responses. Clinical Microbiology Reviews, 14, 430 - 445. Okada, H., Yashiki, T. and Mima, H., (1983). Vaginal absorption of a potent luteinizing hormone- releasing hormone analog (leuprolide) in rats: III. Effect of es trous cycle on 265 vaginal absorption of hydrophilic model compounds, Journal of Pharmaceutical. Sciences, 72, 173? 176. Okada, H., (1991). Vaginal route of peptide and protein delivery . In: Lee VHL. ed. Peptide and protein Drug Delivery, 633 - 666. O? Keef, B.R., (2001) . Biologically active proteins from natural product extracts. Journal of Natural Products, 64, 1373- 1381. Ohmura, T., Fukui, M., Sugi ura, H., Yoneya, S., Hosono, T. and Kajiyama, A., (1991). Drug- release controlling coating material for long acting formulations US Patent 5028664 . http://www.freepatentsonline.com/5028664.html [Accessed October 1, 2009) . Oshlack, B., Wright C. and Breder C., (2009) . Pharmaceutical composition containing gelling agent. US Patent 424456 . http://www.faqs.org/patents/app/20090081287 [Accessed June 2 , 2009 ] . Owen, J.A.J., (1975). Physiology of the menstrual cycle, Clinical Nutrition, 28, 333? 338. Owen, D.H. and Kartz, D.F., (1999). A vagina fluid simulant, Contraception, 59, 91 - 95. Owen, D.H., Dunmire, E.N., Planys, A.M. and Katz, D.F., (1999) . Factors influencing nonoxynol - 9 permeation and bioactivity in cervical mucus. Journal of Controlled Release, 60, 23? 34. Owen D.H. and Katz D.F., (2005). A Review of the Physical and Chemical Properties of Human Semen and the formulation of a Semen Simulant. Journal of Andrology, 26, 459- 266 469. Paavonen, J., (1983). Physiology and ecology of the vagina. Scandinavian Journal of Infectious Diseases, 40 , 31? 35. Padian, N., Shiboski, S., Glass S. and Vittinghoff. E. , (1997) . Heterosexual transmission of human immunodeficiency viruss (HIV) in northern California: results of a ten - year study. American Journal of Epidemiology, 146, 350 - 357. Pal, S., Tak, Y.K. and Song, J.M., (2007). Does the antibacterial activity of silver nanoparticles depend on the shape of the nanoparticle? A study of the gram - negative bacterium Escherichia coli. Applied and Environmental Microbiology, 73, 1712- 172. Palella, F.J.Jr, Delaney, K.M., Moorman, A.C. Lovel ess, M.O., Fuhrer, J., Satten, G.A., Aschman, J.A. and Holmberg, S.D., (1998). Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. HIV Outpatient Study Investigators. New England Journal of Medicine, 338, 853 - 860. Palliser, D., Chowdhury, D., Wang, Q.Y ., (2006). An siRNA - based microbicide protects mice from lethal herpes simplex vir us 2 infection. Nature , 439, 89- 94. Panttinen, P., (2005) Microbicides as an option for HIV prevention, A Report for the International Task Force on Global Public Goods . http://www.gpgtaskfo rce.org/uploads/ files/183.pdf [ Accessed June 15, 2006] . Parija, S., Nayak, S.K. , Verma, S.K. and Tripathy, S.S., (2004) . Studies on physico- mechanical properties and thermal characteristics of polypropylene/layered silicate nanocomposites. Polymer composites, 25 , 646 ? 652. 267 Park, H., and Robinson, J.R., ( 1987 ) . Mechanisms of mucoadhesion of poly( acrylic acid) hydrogels. Pharmaceutical Research, 4, 457- 464. Park, J.H., Ye, M. and Park, K., (2005). Biodegradable Polymers for Microencapsulation of Drugs. Molecules, 10 , 146- 161. Park, K., ( 1989) . A new approach to study mucoadhesion: Colloidal gold staining. International Journal of Pharmaceutics, 53, 209 - 217. Park, K., and Robinson, J.R., ( 1984) . Bioadhesive polymers as platforms for oral controlled Drug Delivery: Method to study bioadhesion, International Journal of Pharmaceutics. 19, 107- 127. Paroj?i?, J., Ibri?, S., Djuri? Z. et al., (2007). An investigation into the usefulness of generalized regression neural network analysis in the development of level A in vitro? in vivo correlation. European Journal of Pharmaceutical Sciences, 30, 264- 272. Patel, L.G. (1984) . Propanolol concentration in plasma after insertion into the vagina, British Medical Journal, 287, 1247? 1248. Patel, R., Pillay, V. and Choonara, Y.E., (2007) . A novel cellulose- based hydrophilic wafer matrix for rapid bioactive delivery. Journal of Bioactive and Compatible Polymers, 22, 119- 142. Patel, V.F. and Patel N.M., ( 2007) . Statistical Evaluation of Influence of Xanthan Gum and Guar Gum Blends on Dipyridamole Release from Floating Matrix Tablets. Drug Development and Industrial Pharmacy, 33, 3, 327 - 334. 268 Pearce- Pratt, R. and Phillips, D.M., (1996) . Sulfated polysaccharides inhibit lymphocyte- to- epithelial transmission of human immunodeficiency virus- 1. Biology of Reproduction, 54, 173 - 182. Pedras- Vasconcelos, J.A., Goucher, D., Puig, M. Tonelli, L.H. , Wang V., Ito S. and Verthelyi D., (2006) . CpG Oligodeoxynucleotides Protect Newborn Mice from a Lethal Challenge with the Neurotropic Tacaribe Arenavirus. The Journal of Immunology, 176, 4940- 4949. Peh, K.K., Lim C.P., Quek S.S. and Khoh, K.H. , (2000). Use of Artificial Neural Networks to Predict Drug Dissolution Profiles and Evaluation of Network Performance Using Similarity Factor. Pharmaceutical Research , 17, 1384- 1389. PENN Medicine News, (2009). Anti- HIV Gel Shows Promise in Large - Scale Study. http://www.health.upenn.edu/news/News_Releases/2009/ 02/anti - hiv- gel.html [Accessed August 19 , 2009 ] . Penttinen, P., (2005). Microbicides as an option for HIV prevention, A Report for the International Task Force on Global Public Goods. http://www.gpgtaskforce.org/show_file. aspx?file_id= 43 [ Accessed February 2, 2007 ] . Peppas, N.A., Hansen, P.J., (2003). Crystallization kinetics of poly(vinyl alcohol. Journal of Applied Polymer Science, 27 , 4787 ? 4797. Peppas, N.A., and Buri, P.A., (1985) . Surface, interfacial and molecular aspects of polymer bioadhesion on soft tissues, Journal of Controlled Release, 2 , 257 - 275. 269 P?rez, O.E, Wargon, V. and Pilosof, A.M.R., (2006). Gelation and structural characteristics of incompatible whey proteins/hydroxypropylmethylcellulose mixtures. Food Hydrocolloids , 20, 966- 974. Perioli, L., Ambrogi, V., Venezia, L. Giovagnoli, S., Pagano, C. and Rossi, C., (2009) . Formulation studies of benzydamine mucoadhesive f ormulations for vaginal administration. Drug Development and Industrial Pharmacy, 35, 769 - 779 . Perotti, M., Pirovano, A. and Phillips, D.M., (2003) . Carageenan formulation prevents macrophage trafficking from vagina: Implications for microbicide development. Bi ology of Reproduction, 69, 933- 939. Peritt, D., Sesok- Pizzini, D.A., Schretzenmair , R ., Macgregor, R.R., Valiante N.M., Xin Tu , X., Trinchieri G. and Kamoun M., ( 1999). C1.7 Antigen Expression on CD8 + T Cells Is Activation Dependent: Increased Proportion o f C1.7 + CD8 + T Cells in HIV - 1 - Infected Patients with Progressing Disease. The Journal of Immunology, 162, 7563- 7568. Pettifor, A.E., Rees, H.V., Steffenson, A., Hlongwa - Madikizela, L., MacPhail, C., Vermaak, K. and Kleinschmidt, I., (2005) . HIV and sexual behavior among young South Africans: a national survey of 15- 24year olds. R eproductive Health Research Unit, University of the Witwatersrand. http://www.rhruco.za/images/Docs/national%20survey%20RHRU.pdf [Acce ssed November, 2007] . Phillips, D.M. and Maguire R.A., (2002) . The development of microbicides for clinical use to prevent sexually transmitted diseases. Current Infectious Disease Reports , 4, 135 - 140. 270 Picout, D.R., Richardson, R.K., and Morris, E.R., (2000a ). Co - gelation of calcium pectinate with potato maltodextrin: Part I. Network formation on cooling, carbohydrate polymer, 43, 1 33 - 141. Picout, D.R., Richardson, R.K., Rolin C., Abeysekera, R.M., and Morris, E.R., (2000b ). Ca++ induced gelation of low methoxy pectin in the presence of oxidized starch: Part I. Collapse of network structure. C arbohydrate polymer, 43, 113 - 1 22. Pignatello, R., Bucolo, C., Ferrara, P. , Maltese, A ., Puleo, A., Puglisi, G., (2002) . Eudragit RS 100 nanosuspensions for the ophtalmic c ontrolled delivery of ibuprofen. European Journal of Pharmaceutical Sciences, 16, 53? 61. Pijpers, T.F.J., Mathot VBF, Goderis B, et al., (2000) . High - speed calorimetry for the study of the kinetics of (d e)vitrification, crystallization, and melting of macromolecules. Macromolecules, 35, 3601? 3613. Pillay, V. and Fassihi, R., (1999) . In vitro release modulation from crosslinked pellets for site- specific Drug Delivery to the gastrointestinal tract: I. Comparison of pH - responsive drug release and associated kinetics, Journal of Controlled Release, 59, 229- 242. Pillay, V. and Fassihi, R., (2000). Electrolyte- induced compositional heterogeneity: A novel approach for rate- controlled oral drug delivery. Journal of Pharmaceutical Sciences, 88, 1140? 1 1 48 . Pillay, V. and Danckwerts, M.P., (2002). Textural profiling and statistical optimization of crosslinked calcium- alginate- pectinate- cellulose acetophthalate gelisphere matrices, Journal of Pharmaceutical Sciences, 91, 2559 - 2570. 271 Pillay, V., Danckwerts, M.P., Muhidinov, Z. and Fassihi, R., (2005) . Novel modulation of Drug Delivery using binary zinc - alginate- pectinate polyspheres for zero- order kinetics over several days: Experimental strategy to elucidate the crosslinking mechanism. Drug Development and Industrial Pharmacy, 31, 191- 207. Pinto, J.F., Wunder, K.F., Okoloekwe, A., (2004) . Evaluation of the Potential Use of Poly(ethylene oxide) as Tablet - and Extrudate - Forming Material. A APS Pharmaceutical Sciences and Technology, 6, 1 - 10. Piret, J., Lamontagne, J., Bestman - Smith, J., Roy, S., Gourde, P., D?sormeaux, A., Omar, R.F., Juh?sz, J. and Bergeron, M.G., (2000) . In vitro and in vivo evaluations of sodium lauryl sulfate and dextran sulfate as microbicides against herpes simplex and human immunodeficiency viruses. Journal of Clinical Microbiology, 38, 110 ? 119. Pisani, E., Garnett, G.P., Grassly, N.C., Brown, T., Stover, J., Hankins, C., Walker, N. and Ghys, P.D., ( 2003). Back to basics in HIV prevention: focus on exposure. British Medical Journal, 326, 1384- 1387. Pond, W.G., and Houpt, K.A., (1988) . Reproductive physiology. The Biology of the Pig, 129- 180. New York: Cornell University Press. Population council, (2009) . The Population Council, HIV and AIDS, and Microbicides. http://www.popcouncil.org/microbicides/more_info.html#Phase%203%20trial [ Accessed August 19, 2009 ] . Potts, M., Perlman, D., Mandara, M., Prata, N., Campbell, M., (2004) . Is lime/lemon juice an effective microbicide? International Conference of AIDS, University of California, Berkeley, CA, United States, Abstract No. C11663. http://gateway.nlm.nih.gov/Meeting/ Abstracts/ma?f =102277391.html [ Accessed November 21, 2007] . 272 Pschera, H., Hjerpe, A. and Carlstroem, K., (1989) . Influence of the maturity of the vaginal epithelium upon the absorption of vaginally administered 17b - estradiol and progesterone in postmenopausal women, Gynecol ogic and Obstetrics Investigation, 27, 204? 207. Pu, Q., Ng, S., Mok, V. and Chen, S.B., (2004) . Ion Bridging Effects on the Electroviscosity of Flexible Polyelectrolytes. Journal of Physical Chemistry B, 108, 14124? 14129. Pulido, F. and Arribas J.R., (2003). Treatment of advanced HIV infection. Journal of Antimicrobial Chemo therapy, 51 , 225 - 227. Pulido, F. and Torralba M., (2002). NNRT I hepatotoxicity: efavirenz versus nevirapine. Journal of HIV Therapy, 7, S3 - S16. Pusch, O., Boden, D., Hannify, S., Lee, F., Tucker, L.D ., Boyd, M.R ., Wells, J.M . and Ramratnam, B ., (2005). Bioengineering lactic acid bacteria to secrete the HIV - 1 virucide cyanovirin. Journal of Acquired Immune Deficiency Syndromes, 40 , 512 - 520. Quayle, A.J., ( 2002). The innate and early immune response to pathogen challenge in the female genital tract and the pivotal role of epithelial cells. Journal of Reproductive Immunology, 57, 61. Quinn, F.X., Hatakeyama, T., Takahashi, M. and Hatakeyama, H., (1994) . The effect of annealing on the conformational properties of xanthan hydrogels. Polymer, 35 , 1248? 1252. Quinn, T.C. and Overbaugh , J., (2005). HIV/AIDS in women: an expanding epidemic. Science, 308, 1582? 1583. 273 Quintanar - Guerrero, D., Villalobos - Garc?a, R., Alvarez - Col?n, E. and Cornejo- Bravo, J. M., (2001). In vitro evaluation of the bioadhesive properties of hydrophobic polybasic gels containing N,N- dimethylaminoethyl methacrylate- co- methyl methacrylate. Biomaterials, 22, 957- 961. Rahman, B.M., Islam, M.A., Wahed, M.I.I ., Ahmed, M., Islam, R., Barman, R.K., Anisuzzaman, A.S.M. and Khondkar P., ( 2009) . In vitro studies of pentoxifylline controlled- release from hydrophilic matrices. Journal of Applied Sciences Research, 1, 353- 362. Ramjee, R., Shattock, R., Delany, S., McGowan, I. , Morar, N., Gottemoeller, M., ( 2006). Microbicides. AIDS Research and Therapy, 3, 25. Ramjee, G., (2007). Microbicides in HIV prevention. Future HIV Therapy, 1, 161 - 170. Ramratnam, B ., (2006). Bacteria in yogurt can combat HIV infection. /Bacteria- In - http://www.bio - medicine.org/medicine- news/Bacteria- In - Yogurt --Can - Combat- HIV - Infection- 7111- 1/ [ Accessed June 23, 2009] . Ramsey, P.S. , Ogburn, P.L., Harris, D.Y., Heise, R.H. and Ramin, K .D., (2002) . Effect of vaginal pH on efficacy of the dinoprostone gel for cervical ripening/labor induction, American Journal of Obstetrics and Gynecology, 187, 843? 846. Rando, R.F., Obara, S., Osterling, M.C., (2006) . Critical Design Features of Phenyl Carboxylate- Containing Polymer Microbicides. Antimicrobial Agents and Chemotherapy, 50, 3081? 3089. 274 Ravi, P.R., Kotreka, U.K. and Saha, R.N., (2008) . Contolled release matrix tablets of zidovudine: Effect of formulation variables on the in vivo drug release kinetics. AAPS Pharmaceutical Sciences and Technology, 9, 302 - 313. Rebbapragada, A. and Kaul, R., ( 2007) . More than their sum in your parts: the mechanisms that underpin the mutually advantageous relationship between HIV and sexually transmitted infectio ns. Drug Discovery Today: Disease Mechanisms , 4, 237- 246. Reddy, S.P., Swart. D., Jinabhai, C.C., Amosun, S.L., James, S., Monyeki, K.D., Stevens, G., Morejele, N., Kambaram, N. S., Omardien, R.G., and Van den Borne, H.W., (2002) . The 1 st South African National Youth Risk Behavior Survey 2002 . http://www.info.gov.za/ otherdocs/ 2003/youth/part1.p df [ Accessed May 20, 2007 ] . Reis, M.A.A., Sinisterra, R.D. and Belchior, J.C., (2004) . An alternative approach based on artificial neural networks to study controlled drug release. Journal of Pharmaceutical Sciences, 93, 418 - 430. R epka, M.A., and McGini ty, J.W., (2001). Bioadhesive properties of hydroxyproplycellulose topical films produced by hot - melt extrusion, Journal of Controlled Release, 70, 341- 351. Ribeiro, M., Grolier, J.P.E., (1999) Temperature Modulated DSC for the Investigation of Polymer Materials: A brief account of recent studies. Journal of Thermal Analysis and Calorimetry, 57, 253- 263. Richardson, B.A., Martin, H.L., Stevens, C.E., Hillier, A. K., Mwatha, B.H., Chohan, M.P., Nyange, M., Mandaliya, K., Ndinya- Achola, J. and Kreiss, J.K., (1998). Use of 275 nonoxynolol - 9 and changes in vaginal lactobacilli, Journal of Infectious Diseases, 178, 441- 445. Richardson, J.L. and Armstrong, T.I., (1999). Vaginal delivery of calcitonin by hyaluronic acid formulations. In: Mathiowitz E, Chickering DE and Lehr CM. eds. Bioadhesive Drug Delivery systems: fundamentals, novel approaches and development. Marcel Dekker, New York, 563? 599. Richardson, J.L. and Illum, L., (1992) . The vaginal route of peptide and protein drug delivery. Advanced Drug Delivery R eviews, 8, 341 - 366. Richman D.D., Havlir, D., Corbeil, J. , Looney, D., Ignacio, C., Spector, S.A., Sullivan, J., Cheeseman, S., Barringer K. and Pauletti D., ( 1994) . Nevirapine resistance mutations of human immunodeficiency virus type 1 selected during t herapy. Journal of Virology, 68, 1660? 1666. Rizkalla N. and Hildgen P., (2005). Artificial Neural Networks: Co mparison of Two Programs for Modeling a Process of Nanoparticle Preparation. Drug Development and Industrial Pharmacy, 31, 1019- 1033. Robinson, J.R. and Bologna, W.J., (2002). Use of polycarboxy lic acid polymers to treat vaginal infections. US Patent 6017521. http://www.freepatentsonline.com/6017521.html [Accessed March 15, 2008] . Rodd, A.B., Dunstan, D.E. and Boger, D.V., (2000) . Characterisation of xanthan gum solutions using dynamic light scattering and rheology. Carbohydrate Polymers, 42, 159 ? 174. 276 Rohan L.C. and Sassi A.B., (2009). Vaginal Drug Delivery Systems for HIV Prevention. American Association of Pharmaceutical Scientist Journal, 11, 78 - 87. Rolfs, T.R., Joesoef, M.R., Hendershot, E.F. Rompalo, A.M., Augenbraun, M.H., Chiu, M.,Bolan, G., Johnson, S.C., French, P., Steen, E., Radolf, J.D., Larsen, S., Brady, W.E., Wagner, K.F., D'Aquilante, D.A. for The Sy philis and HIV Study Group, (1997). A randomized trial of enhanced therapy for early syphilis in patients with and without human immunodeficiency virus infection. New England Journal of Medicine. 337, 307- 314. Romano, J., Malcolm, R.K., Garg, S., Rohan, L.C., Kaptur, P.E., (2008) . Microbicide delivery: formulation technologies and strategies. Current Opinion in HIV and AIDS , 3, 558- 566. Rosenstein, I.J., Stafford, M.K., Kitchen, V.S., Ward, H., Weber, J.N. and Taylor - Robinson, D., (1998) . Effect on normal vaginal flora of three intravaginal microbicidal agents potentially active against human immunodeficiency virus type 1, Journal of Infectious Diseases, 177, 1386 - 1390. Roumeliotis, G., (2006) . SR Pharma claims formulation triumph in siRNA drugs. Allian ce for Microbicide Development, 73, 542 - 547. Roumen, F.J.M.E. and Dieben, T.O.M., (1999). Clinical acceptability of an ethylene- vinyl- acetate non- medicated vaginal ring. Contraception, 59, 59? 62. Rouse, J.J., Mohamed, F. and van der Walle, C.F., (2007) . Physical ageing and thermal analysis of PLGA microspheres encapsulating protein or DNA. International Journal of Pharmaceutics, 339, 112 - 120. 277 Roy, D.S. and Rohera B.D., (2002) . Comparative evaluation of rate of hydration and matrix erosion of HEC and HPC and study of drug release from their matrices. European Journal of Pharmaceutical Sciences, 16, 193 - 199. Roy, S., Piret, J., Gagne, N. Desormeaux, A., Omar, R.F., Tremblay, M.J., Juhasz, J., Bergeron, M.G., (1999). Thermoreversible gel containing sodium lauryl sulfate as a potential topical microbicide for the prevention of STDs/HIV. National HIV Prevention Conference Atlanta Georgia, USA. Abstract No. 419. http://gateway.nlm.nih. gov/MeetingAb- stracts/ma?f=102187614.html [ Accessed November 21, 2007] . Royce, R.A. Se?a, A., Cates, W. and Cohen, M.S., (1997). Sexual transmission of HIV. The New England Journal of Medicine. 336 , 1072? 1078. Ruel - Gariepy, E. and Leroux, J., (2004). In situ- forming hydrogels- review of temperature- sensitive systems. European journal of Pharmaceutics , 58, 2, 409- 426. Rupp, R., Rosenthal, S.L. and Stanberry, L.R., (2007) . VivaGel ? (SPL7013 Gel): A candidate dendrimer ? microbicide for the prevention of HIV and HSV infection. International Journal of Nanomedicine, 2, 561? 566. Ruscnis, S, Moonis, M., Merril, D.P. Pallai, P.V., Neidhardt, E.A., S K Singh, S.K., Willis, K.J., Osburne , M.S., Profy, A.T., Jenson, J.C., and Hirsch, M.S., (1996) . Naphtalene sulfonate polymers with CD4- blocking and anti- human immunodeficiency virus type 1 activities. Antimicrobial Agents and Chemotherapy, 40, 234 - 236. Saettone, M.F., Panichi, L., Giannacc ini, B. et al., (2002) . Bioadhesive complexes of polycarbophil and azole antifungal or antiprotozoal drugs. United States Patent 6423307. http://www.freepatentsonline.com/6423307.html [Accessed June 19, 2009 ] . 278 Sa?di, H. , Jenabian M. and B?lec, L., (2009). Early events in vaginal HIV transmission: implications in microbicide development, Future Virology , 4, 259 - 269. San Francisco AIDS Foundation (2008) . How HIV is spread. http://www.sfaf.org/aids101 transmi- ssion.html [ Accessed June 16, 2007] . Sanchez - Lafuente, C. , Teresa, F.M., Fernandez - Arevalo, M., Alvarez - Fuentes, J., Rabasco, A.M., Mura, P., (2002) . Development of sustained release matrix tablets of didanosine containing methacrylic and ethylcellulose polymers. International Journal of Pharmaceutics, 234, 213 - 221. Sanders, J.M. and Matthews, H.B., (1990). Vaginal absorption of polyvinyl alcohol in Fischer 344 rats, Human and Experimental Toxicology, 9, 71? 77. Sandri, G., Rossi, S., Ferrari, F . , Bonferoni M.C., Muzzarelli, C. and Caramella C., (2004). Assessment of chitosan derivatives as buccal and vaginal penetration enhancers. European Journal of Pharmaceutical Sciences, 21, 51 - 59. Sant, S., Thommes, M., and Hildgen, P., (2008). Microporous Structure and Drug Release Kinetics of Polymeric Nanoparticles. Langmuir , 24 , 280? 287. Santos, C.A., Jacob, J.S., Hertzog, B.A. Freedman, B.D., Press, D.L., Harnpicharnchai, P. and Mathiowitz, E. (1999) . Correlation of two bioadhesive assays: The averted sac technique and the CAHN microbalance. Journal of Controlled Release, 61, 113- 122. 279 Schroeder, G., Leska, B., and Brzezinski, B., (1998) . Solvent effect for proton transfer reaction from dimethyl(4- nitrophenyl)malonate to cis 1,2 - bis(dimethylaminomethyl) cyclohexane. Journal of Molecular Structure, 446, 235 - 239. Scorpio, D.G., Ruben D.S., Liao, Z., (2008). Cervicovaginal evaluation in macaques used as a model for topical microbicide safety studies. Journal of Medical Primatology , 37, 65 ? 73. Semalty, A., and Semalty, M., 2008. Mucoadhesive Polymers - A Review. http://www.pharmainfo. net/revie ws/mucoadhesive- polymers- review [ Accessed, February 28] . Semalty, A., Bhojwani, M., Bhatt, G. Gupta, G.D. and Shrivastav, A.K., (2006). Mucoadhesive Polymers. Latest Reviews , 4, 1 - 5. Sen, A.K., Roy, S., Juvekar, V.A., (2007) . Effect of structure on solution and interfacial properties of sodium polystyrene sulfonate (NaPSS). Polymer International, 56, 167- 174. Serra, L., Dom?nech, J. and Peppas N.A., (2009) . Engineering design and molecular dynamics of mucoadhesive Drug Delivery systems as targeting agents. European Journal of Pharmace- utics and Biopharmaceutics, 71, 519 - 528. Shafir, S.C., Sorvillo, F.J. and Smith L., (2009) . Current Issues and Considerations Regarding Trichomoniasis and Human Immunodeficiency Virus in Afric an- Americans. Clinical Microbiology Reviews, 22, 37- 45. Sharma, D. and Chakraborty R., (2009) . Managing HIV infection in infants, children and adolescents with HAART. HIV Therapy , 3, 391- 404. 280 Shattock, R.J., (2001). HIV infection of human cervical tissue in vitro and the effects of vaginal virucides. AIDS , 15, S39. Shen, H. and Iwasaki A., (2006) . A crucial role for plasmacytoid dendritic cells in antiviral protection by CpG ODN ? based vaginal microbicide. The Journal of Clinical Investigation, 116, 2237 - 2243. Shen, R., Richter, H.E. , Clements, R.H., Novak, L., Huff, K., Bimczok, D., Sankaran - Walters, S., Dandekar, S. , Clapham, Paul R., Smythies, L.E., and Smith P.D., (2009). Macrophages in Vaginal but Not Intestinal Mucosa Are Monocyte- Like and Permissive to Human Immunodeficiency Virus Type 1 Infection. Journal of Virology, 83, 3258 - 3267. Sherwood, J.K., Zeitlin, L., Whaley, K.J, Cone, R.A. and Saltzman, W.M., (1996). Controlled release of antibodies for long- term topical passive immunoprotection of female mice against genital herpes. Nature Biotechnology , 14, 468 - 471. Sheung, A., Rebbapragada, A., Shin, L.Y. Y., and Dobson- Belaire, W., (2008). Mucosal Neisseria gonorrhoeae coinfection during HIV acq uisition is associated with enhanced systemic HIV - specific CD8 T- cell responses. AIDS, 22, 1729 - 1737. Schiffman, R.M., (2004). A method for treating uterine disorders, including hyperplasic, hypertonic, cystic and/or neoplastic uterine gland tissue by loc al administration of a botulinum toxin to or to the vicinity of the afflicted uterine tissue. US Patent No. 20040175399. http://www.freepatentsonline.com/y2004/ 0175399.html [Accessed September 12, 2009] Short, I., (2007). Molecular trees bear fruit. Chemistry World. http://www.rsc.org/chemistry world /restricted/2007/ May/MolecularTreesBearFruit.asp [ Accessed June 9, 2007 ] . 281 Sibambo, S.R., Pillay, V. and Choonara, Y.E., (2008). A novel salted - out and subsequently crosslinked poly (Lactic - co- glycolic acid) polymeric scaffold applied to monolithic Drug Delivery. Journal of Bioactive and Compatible polymers , 23, 132 - 153. Sibanda, W., Pillay, V., Danckwerts, M.P., Viljoen, A.M., Van Vuuren, S. and Khan, R.A., (2004). Experimental design for the f ormulation and optimization of crossinked oilispheres developed for in vitro site- specific release of Mentha piperita oil, AAPS Pharmaceutical Sciences and Technology, 5, 1 - 14. Sibeko, B., Pillay, V., Choonara, Y.E., Khan, R.A., Modi, G., Iyuke, S.E., Naidoo, D and Danckwerts, M.P., (2009) . Computational molecular modeling and structural rationalization for the design of a drug loaded PLLA/PVA biopolymeric membrane. Biomedical Materials, 4, 1 - 11. Siepmann, J. and Peppas, N. A., (2001) . Modeling of drug release from delivery systems based on hydroxypropyl methylcellulose (HPMC). Advanced Drug Delivery Reviews , 48, 139- 157. Simoes, J.A., Citron, D.M., Aroutcheva, A., Anderson, R.A., Chany, C.J., Waller, D.P., Faro, S. and Zaneveld, L.J.D., (2002). Two novel vaginal microbicides (Polystyrene sulfonate and cellulose sulfate) inhibit Gardnerella vaginalis and anaerobes commonly associated with bacterial vaginosis. Antimicrobial Agents and chemotherapy, 46, 2692 - 2695. Simon, J., Nachtigall, L. and Gut, R., (2008) . Effective Treatment of Vaginal Atrophy with an Ultra- Low - Dose Estradiol Vaginal Tablet. Obstetrics & Gynecology, 112, 1053 - 1060. 282 Singh, M.P., Lumpkin, J.A., Rosenblatt, J., (1995) . Effect of electrostatic interactions on polylysine release rates from collagen matrices and comparison with model predictions. Journal of Controlled Release, 35 , 165 - 179. Sing, Y. and Govender D., (2009). Infections in the HIV - infected child. Diagnostic Histopathology, 15, 251- 263. Sinha, V.R. , Kumria, R., (2003). Coating polymers for colon specific Drug Delivery. A comparative in vitro evaluation. Acta Pharmaceutica, 53, 41- 47. Sitruk- Ware, R. (2007) . New hormonal therapies and regimens in the postmenopause: routes of administration and timing of initiation. Climacteric , 10, 358 - 370. Skoler- Karpoff, S., Ramjee G., Ahmed, K., Altini, L ., Plagianos, M.G ., Friedland, B., Govender, S., De Kock, A ., Cassim, N ., Palanee, T., Dozier, G ., Maguire, R . and Lahteenmaki, P ., (2008) . Efficacy of Carraguard for prevention of HIV infection in women in South Africa: a randomised, double- blind, placebo- controlled trial. Lanc et, 372, 1977- 1987. Smart, J.D., and Kellaway, I.W., 1982. In vitro techniques for measuring mucoadhesion, Journal of Pharmacy and Pharmacology, 34, 70- 70. Smart, J.D., Kellaway, I.W., and Worthington, H.E.C., 1984. An in vitro investigation of muco- adhesive materials for use in controlled delivery, Journal of Pharmacy and Pharmacology, 36 , 295 - 299. Smit, A.J., (2004). Medicinal and pharmaceutical uses of seaweed natural products: A review. Journal of Applied Psychology, 16, 245- 262. 283 Smita, J., So ma, D., Beverly, B., Albert, P. , JoAnn, K., Fang, G., Missy, C., Lydia, S., Anjali, P., Arun, R., Sanjay, M., Steven R., J, and the HIV Prevention Trial Network (HPTN) 047 Protocol Tea, (2006) . Phase I safety study of 0.5% PRO 2000 vaginal gel among HIV un- infected women in Pune, India. AIDS Research and Therapy, 3, 4. Smith, K.P.B., (1993) . Estrogens and the urogenital tract. Studies on steroid hormone receptors and a clinical study on a new estradiol releasing vaginal ring. Acta Obstetricia et Gynecologi ca Scandinavica, 72, S1 - 26. Smith, B.P. and Brier, M.E., (1996) . Statistical approach to neural network model building for gentamicin peak predictions, Journal of Pharmaceutical Sciences, 85, 65 ? 69. Smith, M., (2006) . TMC 120 vaginal ring promising as m icrobicides carrier. XVI International Conference, Toronto Canada . http://www.natap.org/2006/IAS/IAS_65.htm [Accessed November 21 , 2007 ] . Smith, R.C., Leung, A., Kim, B. and H ammond, P.T., (2009). Hydrophobic Effects in the Critical Destabilization and Release Dynamics of Degradable Multilayer Films. Chemistry of Materials, 21 , 1108? 1115. Soifer, H.S., Rossi, J.J. and S?trom, P., (2007) . MicroRNAs in Disease and Potential Ther apeutic Applications. Molecular Therapy , 15 , 2070? 2079. Song, K., Kim, Y. and Chang, G., (2006). Rheology of concentrated xanthan gum solutions: Steady shear flow behavior. Fibers and Polymers , 7, 129- 138. 284 Soukoulis, C., Panagiotidis, P. , Koureli , R. and Tzia, C., (2007). Industrial Yogurt Manufacture: Monitoring of Fermentation Process and Improvement of Final Product Quality. Journal of Dairy Science, 90, 2641 - 2654. Spencer, S.E., Valentin - Bon, I.E., Whaley, K. and Jerse A.E., (2004) . Inhibition of Neisseria gonorrhoeae Genital Tract Infection by Leading - Candidate Topical Microbicides in a Mouse Model. Journal of infectious diseases, 189, 410- 419. Spinillo, A., Gardella, B. , Zanchi , S. Roccio, M., Preti and E., (2008) . Determinants of Genital Shedding of Human Immunodeficiency Virus: A Review. Current Women's Health Reviews , 4, 118- 123. Spira, A. I., Marx, P. A., Patterson, K. B ., Mahoney, J ., Koup, R.A ., Wolinsky, S.M ., Ho, D.D., (1996) . Cellular targets of infection and route of viral dissemination after an intravaginal inoculation of simian immunodeficiency virus into rhesus macaques. The Journal of Experimental Medicine, 183, 215- 25. Squier, C.A. , Mantz , M.J., Schlievert , P.M. and Catherine C. Davis, C.C., (2008). Porcine vagina Ex Vivo as a model for studying permeability and pathogenesis in mucosa. Journal of Pharmaceutical Sciences, 97, 9 - 21. Sriamornsak, P., Thirawong, N., Weerapol, Y., Nunthanid, J. and Sungthongjeen S., (2007). Swelling and erosion of pectin matrix tablets and their impact on drug release behaviour. European Journal of Pharmaceutics and Biopharmaceutics, 67, 211 - 219. Staff, P., (2002). Strategies f or interrupting the transmission of genital and neonatal HSV infection. The journal of the IHMF, 11, 1470- 1537. 285 Stafford, M. K., Ward, H., Flanagan, A., Rosenstein, I. J., Taylor - Robinson, D ., Smith, J.R ., Weber, J., Kitchen, V.S ., (1998). Safety study of nonoxynol - 9 as a vaginal microbicide: evidence of adverse effects. Journal of acquired immune deficiency syndromes and human retrovirology, 17 , 327 - 331. Stanic, A. and Grana J.C., (2009) . Review of antiretroviral agents for the treatment of HIV infection. Formulary, 44, 47? 54. Stanic, A. and Schneider, T.K., (2005) . Overview of antiretroviral agents in 2005. Journal of Pharmacy Practice, 18, 228. Steinberg, M.S., (1996) . Adhesion in development: An historical overview, Developmental Biology, 180, 377? 388. Steinberg, M.S., (2007). Differential adhesion in morphogenesis: a modern view. Current Opinion in Genetics & Development , 17, 4 , 281 - 286 Stone, A., (2002) . MICROBICIDES: A new approach to preventing HIV and other sexually transmitted infections. Nat ure Reviews Drug Discovery, 1, 977- 985. Sudhakar Y., Kuotsu K. and Bandyopadhya A.K., (2006) . Buccal bioadhesive drug delivery ? A promising option for orally less efficient drugs. Journal of Controlled Release , 114, 5 - 40. Sugama, T., Kukacka, L.E., Carciello, N., (1984) . Nature of interfacial interaction mechanisms between polyacrylic acid macromolecules and oxide metal surfaces. Journal of Materials Science, 19, 4045- 4056. 286 Sun, Y., Peng, Y., Chen, Y., Chen, Y. and Shukla, A.J., (2003). Application of artificial neural networks in the design of controlled release Drug Delivery systems. Advanced Drug Delivery Reviews, 55, 1201 - 1215. Sundstrom, J.B., Ellis, J.E., Hair, G.A., Kirshenbaum A.S. et al, (2007). Human tissue mast cells are an inducible reservoir of persistent HIV infection. Blood, 109, 5293 - 5300. Takahara, J., Takay ama K. and Nagai T., (1997). Multi - objective simulataneous otpimization besed on artificial neural networks in sustained release f ormulation. Journal of Controlled Release, 49, 11 - 20. Takayama, K., Takahara, J., Fujikawa, M. , Ichikawa, H . and Nagai, T ., (1999) . Formula optimization on artificial neural networks in transdermal Drug Delivery. Journal of Controlled Release, 6, 161 - 170. Tambwekar, K. R., Gunjan, Verman, K., Kandarapu, R., Zaneveld, L.J.D. and Garg, S., (2002). Effect of different bioadhesive polymers on performance characteristics of vaginal tablets, National Institute of Pharmaceutical Education and Research (NIPER), S.A.S. Nagar, Punjab- 160062, India. Tan, X. and Philips, D., (1996) . Cell - mediated infection of cervix derived epithelial cells with primary isolates of human immunodeficiency virus. Archives of Virology. 141, 1177- 1189. Tang, C., Yin, C., Pen, Y. Zhang M. nd Lifang Wu, L., (2005). rporous hydrogels composites based on aqueous Carbopol? solution (SPHCcs): Synthesis, characterization and in vitro bioadhesive force studies. European Polymer Journal, 41, 557 - 562. 287 Teix eira, M., Alonso M.J., Pinto, M.M. M. and Barbosa C.M., (2005). Development and characterization of PLGA nanospheres and nanocapsules containing xanthone and 3- methoxyxanthone. European Journal of Pharmaceutics and Biopharmaceutics 59, 491- 500. Thaineua, V., Viravaidya, M., Short, R., (2003). AIDS Information Services. Lemons & AIDS Project History http://www.aids.net.au/lemons - news- thai- aust- bg.htm [Accessed June 22, 2009] . Thapa, P., Stevens, H.N.E., Baillie, A.J., (2009) . In vitro drug release studies from a novel lyophilized nasal dosage form. Kathmandu University Journal of Engineering, Sc ience and Technology, 5, 71- 86. Thirlwell, C., Debashis Sarker, D., Stebbing, J. , Bower M., (2003) . Acquired Immunodeficiency Syndrome? Related Lymphoma in the Era of Highl y Active Antiretroviral Therapy. Clinical Lymphoma & Myeloma, 4, 86- 92 Thom, A., (2007) . South Africa: Microbicide trial failure. http://allafrica.com/stories/200702010864 htm [ Accessed February 2, 2007 ] . Thompson, I.O.C. , van der Bijl, P. , van Wyk, C.W. and van Eyk, A.D., (2001) . A comparative light- microscopic, electron- microscopic and chemical study of human vaginal and buccal epithelium, Archives of Oral Biology, 46, 1091? 1098. Thompson, M.M. and Samuels S.C., (2002). Neurosyphilis: is it still a clinically relevant form of dementia? Expert Review of Neurotherapeutics , 2, 665- 668. 288 Thormar, H., Bergsson, G., Gunnarsson, E. , Georgsson, G., Witvrouw, M. Steingrimsson, O. De Clercq, E. and Kristmundsdottir, T. (1999). Hydrogels containing monocaprin have potent microbicidal activities against sexually transmitted viruses and bact eria in vitro. Sexually Transmitted Infections, 75, 181- 185. Tiwari, S.B., Murthy, T.K., Pai, M.R., Mehta, P.R., Chowdary, P.B., (2003) . Controlled release formulation of tramadol hydrochloride using hydrophilic and hydrophobic matrix system. AAPS Pharmaceutical Sciences and Technology, 4, 1 - 6. Todd, C., Alibayeva, G., Sanchez, J. Bautista, C., Carr, J. and Earhart, K. (2006). Utilization of contraception and abortion and its relationship to HIV infection among female sex workers in Tashkent, Uzbekistan. Contraception, 74, 318 - 323. Trapani, A., Laquintana V., Denora, N. and Lopedota, A., (2007). Eudragit RS 100 microparticles containing 2- hydroxypropyl - ? - cyclodextrin and glutathione: Physicochemical characterization, drug release and transport studies. European Journal of Pharmaceutical Sciences, 30, 64- 74. Tsai, C.C., Emau, P., Jiang, Y., Tian, B., Morton, W.R., Gustafson, K.R . and Boyd M.R ., (2003). Cyanovirin- N gel as a topical microbicide prevents rectal transmission of SHIV89.6P in macaques. AIDS Research and Human Retroviruses , 19, 535 - 541. Turpin, J., (2006). Yoghurt could help fend off HIV. New Scientist Magazine, 17, 2572. Uckun F.M. and D'Cruz O.J., (1999). Prophylactic contraceptives for HIV/AIDS. Human Reproduction Update, 5, 506- 514. Uckun F.M. and D'Cruz O.J., (2004). Clinical development of microbicides for the prevention of HIV infection. Current Pharmaceutical Design, 10, 315 - 336. 289 Ugwoke, M.I., Exaud, S., Van Den Mooter, G., Verbeke, N., and Kinget, R., (1999) . Bioavailability of apomorphine following intranasal administration of mucoadhesive Drug Delivery systems in pigs. European Journal of Pharmaceutical Sciences, 9, 213 - 219. Umamaheshwari, R.B., Ramteke, S., Jain, N.K., (2004). Anti ? Helicobacter Pylori Effect of Mucoadhesive Nanoparticles Bearing Amoxicillin in Experimental Gerbils Model. AAPS Pharmaceutical Sciences and Technology, 5, 1 - 9. UNAIDS, (2007) . AIDS Epidemic Update. http://www.globalhealth.org/hiv_aids/global_ view/ [ Accessed June 24, 2009 ] . UNAIDS, (2007) . UNAIDS Revises Global HIV/AIDS Estimates. The Foundation for AIDS Research. http://www.amfar.org/hill/article.aspx?id=882 [ Accessed 23 May, 2009] . UNAIDS/WHO, (2006 a). Overview of the global AIDS epidemic. Report on the global AIDS epidemic. http:// www.cmaj.ca/cg i/content/full/176/ 12/ 172 [ Accessed June 8, 2006] . UNAIDS/WHO, ( 2006b) . Number of People Worldwide Living With HIV/AIDS Increases in All Regions; Nearly 40 Million People Have Virus. http://www.thebody.com/content/ art38849. html [ Accessed June 24 , 2009 ] . UNFPA, (2006) . Top Level Push to Tackle Priorities in Sexual and Reproductive Health. http://www.unfpa.org/news/news .cfm?ID=812 [ Accessed June 24, 2009 ] . USAID Health, (2009) . Condom Use: How It Relates to HIV and STI Prevention. http://www.usa id.gov/our_work/global_health/aids/TechAreas/prevention/condomfactshee t.html [Accessed September 13 , 2009 ] . 290 Valenta, C., Constantia, E., Kast, C.E., Harich, I., Bernkop - Schn?rch, A., (2001). Development and in vitro evaluation of a mucoadhesive vaginal delivery system for progesterone. Journal of Controlled Release, 77, 323- 332. Valenta, C., (2005). The use of mucoadhesive polymers in vaginal delivery . Advanced Drug Delivery Reviews. 57, 1692 - 1712. Valenta, C., Kast, C.E., Harich, I., and Bernkop- Schn?rch, A., 2001. Development and in vitro evaluation of a mucoadhesive vaginal delivery system for progesterone, Journal of Controlled Release, 77, 323- 332. Valenta, C., Marsch?tz, M., Egyed, C. and Bernkop- Schn?rch, A., 2002. Evaluation of the inhibitory effect of thiolated poly(acrylates) on vaginal membrane bound aminopeptidase, Journal of Pharmacy and Pharmacology, 54, 603- 610. Valore, E. V., Park C. H., Igreti S. L., Ganz T., (2002). Antimicrobial components of vaginal fluid. American Journal of Obstetrics and Gynecology, 187, 561 - 568. van Damme, L., Wright, A. and Depraetene, K., (2000). A phase I study of a novel potential intravaginal microbicides, PRO 2000, in healthy sexually inactive women. Sexually Transmitted Infections, 76, 126. van Damme, L., Adriens, E. and Ramjee, G., (2002) . The evaluation of the local tolerance of vaginal formulations with or without nonxynolol - 9 using the slug mucosal irritation test. Contraception, 66, 369 - 375. van Damme L., (2002 a). Special report: Microbicides. Heatlh and Sexuality, 7, 5 - 9. 291 van Damme, L., (2002b ) Alliance for microbicide development. Health and Sexuality Microbicides. Special report, 1 - 8. https://www.arhp.org/healthcar eproviders/onlinepublicati ons/healthand sexuality/micro [ Accessed October 10 , 2006 ] . van der Wijgert, J., Fullem, A., Kelly, G. , Mehendale, S., Kumwenda, N., Rugpao, S., Joshi, S., Taha, T., Nelson, K. and Padian, N., ( 2001). Phase I trial of the topical microbicide buffer gel: Safety results from four international sites. Journal of Acquired Immune Deficiency Syndromes, 26, 21 - 27. van Eyk, A.D. and van der Bijl, P., (2005) . Porcine vaginal mucosa as an in vitro permeability model for human vaginal mucosa. International Journal of Pharmaceutics, 305, 105 - 111. van Laarhoven, J.A.H., Kruft, M.A.B., Vromans, H., (2002). In vitro release properties of etonogestrol and ethinyl estradiol from a contraceptive vaginal ring. International Journal of Pharmaceutics, 232, 163 - 173. Varma, M., Singla, A. K. and. Dhawan, S., (2004) . Release of Diltiazem Hydrochloride from Hydrophilic Matrices of Polyethylene Oxide and Carbopol. Drug Development and Industrial Pharmacy, 30, 545- 553. Vasir, J.K., Tambwekar, K. and Garg, S., (2003) . Bioadhesive microspheres as a controlled Drug Delivery system. International Journal of Pharmaceutics, 255, 13 - 32. Venkataraman, N., Cole, A.L., Svoboda, P., Pohl, J. and Cole A.M., (2005) . Cationic Polypeptides Are Required for Anti - HIV - 1 Activity of Human Vaginal Fluid. The Journal of Immunology, 175, 7560- 7567. 29 2 Verhelst, R., Verstraelen, H., Claeys, G. Verschraegen, G., Van Simaey, L., De Ganck, C., De Backer, E., Temmerman, M. and Vaneechoutte M., (2005). Comparison between Gram stain and culture for the characterization of vaginal microflora: defination of distinct gade that resembles grade I microflora and revised categorization of grade I microflora. BMC Microbiology, 5, doi:10. 1186/ 1471- 2180 - 5 - 61. Verhoeven, E., Vervaet, C. and Remon, J.P., (2006) . Xanthan gum to tailor drug release of sustained- release ethylcellulose mini- matrices prepared via hot- melt extrusion: in vitro and in vivo evaluation. European Journal of Pharmaceutics and Biopharmaceutics, 63, 320- 330. Verhofstede, C., Demecheleer, E., De Cabooter, N., Gaillard, P., Mwanyumba, F., Claeys, P., Chohan, V., Mandaliya, K., Temmerman, M., and Plum J., (2003) . Diversity of the Human Immunodeficiency Virus Type 1 (HIV - 1) env Sequence after Vertical Transmission in Mother - Child Pairs Infected with HIV - 1 Subtype A. Journal of Virology, 77, 3050? 3057. Verman., K. and Garg, S., (2000). The scope and potential of vaginal drug delivery, Pharmaceutical Sciences Technology Today, 3, 359- 364. Vermani, K., Garg S. and. Zaneveld L.J.D., (2002) . Assemblies for In Vitro Measurement of Bioadhesive Strength and Retention Characteristics in Simulated Vaginal Environment. Drug Development and Industrial Pharmacy, 28, 1133- 1146. Vermesh, M. Fossum, G.T. and Klet zky, O.A., (1998). Vaginal bromocriptine: pharmacology and effect on serum prolactin in normal women, Obstetrics and. Gynecology , 72 , 693? 698. 293 Virid?n, A., Wittgren, B., Larsson, A., (2009) . Investigation of critical polymer properties for polymer release and swelling of HPMC matrix tablets. European Journal of Pharmaceutical Sciences, 36 , 297 - 309. Vishalakshi, B., (1995) . The effect of the charge density and structure of the pol ymer on the dye- binding characteristics of some cationic polyelectrolytes. Journal of Polymer Science Part A: Polymer Chemistry, 33, 365- 371. Vitali, B., Pugliese, C., Biagi, E. Candela, M., Turroni, S., Bellen, G., Gilbert G. G. Donders, G.G.G. and Brig idi P., (2007) . Dynamics of Vaginal Bacterial Communities in Women Developing Bacterial Vaginosis, Candidiasis, or No Infection, Analyzed by PCR - Denaturing Gradient Gel Electrophoresis and Real - Time PCR. Applied and Environmental Microbiology, 73, 5731? 5741. Vlachou, M., Naseef, H., Efentakis, M., Tarantili P.A. and Andreopoulos, A.G., (2001). Swelling Properties of Various Polymers Used in Controlled Release Systems. Journal of Biomaterials Applications, 15, 293 - 306. Voravuthikunchai, S.P., Bilasoi, S. and Supamala, O., (2006). Antagonistic activity against pathogenic bacteria by human vaginal lactobacilli. Anaerobe, 12, 221? 226. Vrijens B. and Urquhart J., (2005). Patient adherence to prescribed antimicrobial drug dosing regimens. Journal of Antimicrobial Chemo therapy, 55, 616- 627. Wagner, G., R. and Levin J., (1978) . Vaginal fluid. E. S. Hafez, and T. N. Evans, eds. In The Human Vagina, 2, 121 - 137. 294 Vueba, M.L., Batista de Carvalho, L.A.E., Veiga, F., Sousa, J.J. and Pina M.E., (2004). Influence of cellulose ether polymers on ketoprofen release from hydrophilic matrix tablets. European Journal of Pharmaceutics, 58, 51- 59. Wang, Y. and Lee C., (2002). Characterization of a female controlled Drug Delivery system for microbicides. Contraception, 66, 2 81 - 287. Wang, Y. and Lee, H.C., (2004) . Effects of intrinsic variables on release of sodium dodecyl sulfate from a female controlled Drug Delivery system. International journal of Pharmaceutics, 282, 173 - 181. Wang, L. and Tang, X., (2008). A novel ketoconazole bioadhesive effervescent tablet for vaginal delivery: Design, in vitro and ?in vivo? evaluation. International Journal of Pharmaceutics, 350, 181 - 187. Wang, Z., Li, X., Su, D., Li , Y., Wu L., Wang, Y. and Wu, W., (2009). Residue depletion of Imido carb in swine tissue. Journal of Agricultural and. Food Chemistry, 57 , 2324? 2328. Ward, R.L., Ashley, C.S., (1979) . pH modification of the effects of detergents on the stability of enteric viruses. Applied and Environmental Microbiology, 38 , 314 ? 322. Ward, G. and Courts, A. (1977) . The science and technology of gelatin. London ; New York : Academic Press, 1977. http://catalogue.nla.gov.au/Record/828694 [Accessed August 24 , 2009 ] . Warner, L, Hatcher, R.A., Steiner, M.J., (2004) . Male Condoms. In: Hatcher RA, Trussel J, Stewart F, et al, editors. Contraceptive Technology. New York: Ardent Media Inc . 331- 353. 295 Washington, N., Washington, C. and Wilson, C.G., (2001) . Vaginal and int rauterine Drug Delivery . In: Washington N, Washington C and Wilson CG. Eds. Physiological pharmaceutics: barriers to drug absorption: Taylor and Francis, London, 271? 281. Weber, J., Desai, K. and Darbyshire, J., (2005). The development of vaginal microbic ides for the prevention of HIV transmission, PLo Medicine, 2, e142. WebMD, (2008). Vaginal Wet Mount. Women Health. http://women.webmd.com/vaginal - wet- mount [Accessed Sept ember 9 , 2009 ] . Weeks, M.R., Mosack, K.E. Abbott, M ., Sylla, L.N., Valdes, B., and Prince M., (2004) . Microbicide Acceptability Among High - Risk Urban U.S. Women: Experiences and Perceptions of Sexually Transmitted HIV Prevention. Sexually Transmitted Diseases, 31, 682? 690. Weinstein, J.N., Kohn, K.W., Grever, M.R. Viswanadhan, V. N. , Rubinstein, L.V. , Monks, A. P. , Scudiero, D.A. , Welch, L., Koutsoukos, A.D. , Chiausa, A.J. , Paull, K.D. , (1992). Neural computing in cancer drug development: predicting mechanism of action. Science, 258, 447? 451. Weller, S. and Davis, K., (2002) . Condom effectiveness in reducing heterosexual HIV transmission. Cochrane Database Systematic Review. 1, CD003255. Wen, X., Wang T., Wang, Z., Li, L., and Zhao C., (2008). Preparation of konjac glucomannan hydrogels as DNA - controlled release matrix. International Journal of Biological Macromolecules, 42, 256 - 263 296 Wertz, P.W. and Squier, C.A., (1991). C ellular and molecular basis of barrier function in oral epithelium, Critical Reviews in Therapeutic Drug Carrier Systems . 8, 237 ? 269. Whaley, K. J. and Zeitlin, L., (2005) . Preventing transmission: plant - derived microbicides and mucosal vaccines for reproductive health. Vaccine , 23, 1819 - 1822. WHO, (2006) . Antiretroviral therapy for HIV infection in adults and adolescents. http://www.who.int/hiv/pub/guidelines/artadultguidelines.pdf [ Accessed August 17, 2009] . WHO, (2007). Sexually transmitted infections. People and planet.net. People and reproductive health. http://www.peopleandplanet.net/doc.php?id=211 [Accessed June 17, 2009] . WHO Facts sheets, (2007) . Sexually transmitted infecti ons (STIs). http://www.who.int/mediacent - re/factsheets /fs110/en/ [ Accessed June 17, 2009 ] . Wilcox, C.M. and Saag, M.S., (2008). Gastrointestinal complications of HIV infection: changing priorities in the HAART era. An International Journal of Gastroenterology and Hepatology , 57 , 861 - 870. Williams, A., (2003). Theoretical aspects of transdermal drug delivery. In: A. Williams, Editor, Transdermal and Topical Drug Delivery, Pharmaceutical Press, London, 35? 40. Willits, R.K. and Saltzman, W.M., (2001) . Synthetic polymers alter the structure of cervical mucus. Biomaterials, 22, 445? 452. Wilber, W.R., Guo, J.and Greenberg , E.S., (2003) . Directly compressed solid dosage particles. European Patent EP1276466, http://www.freepatentsonline.com/ EP1276466. 297 html [ Accessed August 28 , 2009 ] . Wira C.R. and Fahey J.V., (2004). The innate immune sy stem: gatekeeper to the female reproductive tract. Immunology, 111, 13? 15. Wong, C.F., Yuen, K.H., and Peh, K.K., 1999. Formulation and evaluation of controlled release Eudragit? buccal patches, International Journal of Pharmaceutics, 178, 1 1 - 22. Woodley, J., (2001) . Bioadhesion: new possibilities for drug adhesion? Clinical Pharmacokinetics , 40, 77 ? 84. Woolfson, A.D., Malcolm, R.K., Gallagher, R., (2000). Drug Delivery by the intravaginal route. Critical Reviews in Therapeutic Drug Carrier Systems , 17, 509? 55. Woolfson, A.D., Malcom, R.K., Morrow, R.J., Tonner, C.F. and Mc Cullag, S.D., (2006). Intravaginal ring delivery of the reverse transcriptase inhibitor TMC 120 as an HIV microbicides. International Journal of Pharmaceutics, 325, 82 - 89. Workman, M ., (2003). The cellular basis of bacterial infection. Critical Care Nursing Clinics of North America , 15, 1 - 11. Wortley, P. and. Flemming, P., (1997) . AIDS in women in the United States. Journal of the American Medical Association, 278, 911- 916 Wu, F. a nd Jin, T., (2008) . Polymer - Based Sustained- Release Dosage Forms for Protein Drugs, Challenges, and Recent Advances. AAPS Pharmaceutical Sciences and Technology, 9, 1218 - 1229. 298 Xiaowen, L., Lagenaur, L.A. and Simpson, D.A., (2006) . Engineered vaginal Lactobacillus strain for mucosal delivery of the human immunodeficiency virus inhibitor cyanovirin- N. Antimicrobial Agents and chemotherapy, 50, 3250 - 3259. Xu, X., Patel, D.A. Vanessa, K. et al., (2009) . Can Routine Neonatal Circumcision Help Prevent Human Im munodeficiency Virus Transmission in the United States? American Journal of Men's Health, 3, 79 - 84. Yacaman, M.J., Elechiquerra, J.L., Lara, H.H. and Burt, J.L., (2006). Protein- noble metal nanoparticles. WO/2006/ 053225. http://www.wipo.int/pctdb/en/wo.jsp?wo=2006053225 [ Accessed June 5, 2007 ] . Yan, C., Wu, G. and Lin, S., (2006) . Alkaline blend polymer electrolytes based on polyvinyl alcohol (PVA)/tetraethyl ammonium chloride (TEAC). Journ al of Applied Electrochemistry, 36, 655 - 661. Yang, Y., Tai, N.L and Yu, W.Y., (2005) . ART artificial neural networks based adaptive phase selector. Electric Power Systems Research, 76, 115- 120. Yang, T . and Zaman, M.H ., (2007). Free energy landscape of receptor - mediated cell adhesion. Journal of Chemical Physics , 126, 045103. Yang Y., Liu C. and Wu, H., (2008) . Preparation and properties of poly(vinyl alcohol)/exfoliated ?- zirconium phosphate nanocomposite films. Polymer Testing, 28, 371- 377. Yasukawa, T. , Ogura Y., Kimura H. et al., (2006) . Drug delivery from ocular implants . Expert Opinion on Drug Delivery, 3, 261 - 273. 299 Yim, K.C., Carroll, C.J., Tuyama, A., (2005) . The Cotton Rat Provides a Novel Model To Study Genital Herpes Infection and To Evaluate Preventive Strategies. Journal of Virology, 79, 14632? 14639. Yiv, S., Li, M., D?Cruz, O. and Uckun, F.M., (2006) . Gel - microencapsulation formulations. USP Patent 7064114. http://www.patentstorm.us/patents/7064114.html [Accessed October 15 , 2007] . Yoo, J., Dharmala K. and Lee, C.H., (2006) . The physicodynamic properties of mucoadhesive polymeric films developed as female controlled Drug Delivery system. International Journal of Pharmaceutics, 309, 139 - 145. Youle, M., Staszweski, S., Clotet, B., (2006) . Concomitant Use of an Active Boosted Protease Inhibitor with Enfuvirtide in Treatment - Experienced, HIV - Infected Individuals: Recent Data and Consensus Recommendations. HIV Clinical Trials , 7, 86 - 96. Yu, D., Branford - White, C., Ma, Z., Zhu, L., Li, X. and Yang, X.L., (2009) . Novel Drug Delivery devices for providing linear release profiles fabricated by 3DP. International Journal of Pharmaceutics, 370, 160 - 166. Yu, Z., and Quinn, P.J., 1998. Solvation effects of dimethyl sulfoxide on the structure of phospholipids bi- layers, Biophysical. Chemistry, 70, 3 5 - 39. Yuzgec, U., Becerikli, Y and Turker, M., (2008) . Dynamic neural - networks- based- model- predictive control of an industrial baker?s yeast drying process. IEEE Transactions on Neural Networks, 19, 1231- 1241. 300 Zacharopoulos, V.R. and Phillips, D.M., (1997). Vaginal formulations of carrageenan protect mice from herpes simplex virus infection. Clinical and diagnostic laboratory immunology, 4, 465? 468. Zaneveld, L.J.D., Waller, D.P., Anderson, R.A., Chany, C., Rencher, W.F., Feathergill, K., Diao, K., Doncel, G.F., Herold, B. and Cooper, M., (2002). Efficacy and safety of a new vaginal contraceptive antimicrobial formulation containing high molecular weight poly (sodium 4 - styrenesulfonate). Biology of Reproduction, 66, 886- 894. Zang, H., Dornadula, G., Beumount, M. et al., (1998). Human immunodeficiency virus type 1 in semen of men receiving highly active antiretroviral therapy. New England Journal of Medicine, 339, 1803 - 1809. Zhang, Z., Schuler,T., Zupancic, M., Wietgrefe, S., Staskus, K. A., Reimann, K. A. Reinhart, T. A., Rogan, M., Cavert, W., Mi ller, C. J., Veazey, R. S., Notermans, D., Little, S., Danner, S. A., Richman, D. D., Havlir, D., Wong, J., Jordan, H. L., Schacker, T. W., Racz, P., Tenner - Racz, K., Letvin, N. L., Wolinsky, S. and Haase, A. T., ( 1999) . Sexual transmission and pr opagation of SIV and HIV in resting and activated CD4 + T cells. Science, 286, 1353 - 1357. Zeitlin, L., Cone, R.A. and Whaley, K.J., (1999). Monoclonal antibodies for preventing mucosal transmission of epidemic infectious diseases. Emerging Infectious Diseases, 5, 54- 64. Zuckerman, R. A., Whittington, W.L.H., Celum C.L. et al., (2003). Factors Associated with Oropharyngeal Human Immunodeficiency Virus Shedding. The Journal of Infectious Diseases, 188, 142? 145. 301 APPENDICES 302 APPENDIX A ABSTRACTS OF PAPERS PUBLISHED/SUBMITTED FROM THIS THESIS 303 APPENDIX A1 APPENDIX A2 304 APPENDIX A2 305 APPENDIX A3 Submitted Investigation of the Physicochemical and Physicomechanical Properties of an Optimized Intravaginal Bioadhesive Polymeric Device in the Pig Model Valence M. K. Ndesendo ?, Viness Pillay?*, Yahya E. Choonara 1 , Lisa C. du Toit 1 , Leith Meyer 1 , Riaz A. Khan 2 , Eckhart Buchmann3 , and Uwe Rosin 4 1 University of the Witwatersrand, 7 York Road, Parktown, 2193, Johannesburg, South Africa 2 Integral University, Department of Industrial Chemistry, Lucknow, 226026, India 3 Chris Hani Baragwanath Hospital, Department of Gynaecology and Obstetrics, Bertsham, 2013, Johannesburg, South Africa 4 PharmaNatura (Pty) Ltd., Research and Development Unit, Sandton, 2012, South Africa *Corresponding Author: viness.pillay@wits.ac.za Abstract The purpose of this study was to develop and evaluate the bioadhesivity, diffusivity, thermal stability and the in vitro drug release an Intravaginal Bioadhesive Polymeric Device (IBPD) loaded with 3' - azido- 3 ' - deoxythymidine (AZT) and polystyrene sulfonate (PSS). Biodegradable and biocompatible polymers namely, modified polyamide 6,10, poly(lactic - co- glycolic acid), polyacrylic acid, polyvinyl alcohol and ethylcellulose were blended with model drugs AZT and PS S (separately and in combination) as well as radio- opaque barium sulfate (BaSO 4 ) and then compressed into robust caplet devices on a tableting press at a pressure of 5 tons. One set of devices was coated with 2% w/ v PAA while the other remained uncoated. Thermal analysis was performed on the constituent polymers as well the IBPD employing temperature modulated differential scanning calorimetry. The micro - environmental pH changes within simulated vaginal fluid due to the degrading constituents of the IBPD wer e monitored using a Multi - Purpose Titrator (MPT - 2) instrument. Textural profile analysis indicated that the mucoadhesivity of the PAA - coated devices (3.699?0. 464N; 0.0098? 0. 0004J) was higher than that of the uncoated devices (1.198? 0. 150N; 0.0019? 0. 0001J) . In addition, BaSO 4 facilitated x - ray imaging revealed that the IBPD adhered to pig vaginal tissue over the experimental period of 30 days in vivo. Controlled drug release kinetics was obtained over 72 days, and this was attributed to the superior physicomechanical properties of the polymer blend selected. A characteristic bi- phasic response in pH was within a range of 3.5 - 5. 5 was maintained indicating that the IBPD did not significantly alter the pH within the simulated vaginal fluid. This was certainly enabled by PLGA upon breaking down into its constituent units, i.e. lactic and glycolic acids. During a 24 - hour permeation study, an increase in drug flux for both AZT (0. 84 mg.cm ?2.h ?1) and PSS ( 0. 72 mg.cm ?2.h ?1) was realized up to 12 hours and thereafter a steady- state was reached. This indicated that a large proportion of AZT and PSS were retained within the vaginal tissue beyond steady- state. The permeation and dissolution dynamics were mechanistically deduced based on a chemometric and molecular structure modeling approach. Overall, r esults suggested that the IBPD may be sufficiently bioadhesive with desirable physicochemical and physicomechanical stability for use as a prolonged intravaginal drug delivery device. Keywords: Intravaginal drug delivery, Microbicidal polymeric device, Mucoadhesivity, Controlled release, Physicochemical and physicomechanical characterization 306 APPENDIX A4 Submitted In Vivo Drug Content A nal ysis and Histopathological Evaluation of an Intravaginal Bio adhesive Polymeric Dev ice in Pig Tissue Valence M. K. Ndesendo ?, Viness Pillay?*, Yahya E. Choonara 1 , Lisa C. du Toit 1 , Leith Meyer 1 , Riaz A. Khan 2 , Eckhart Buchmann3 , and Uwe Rosin 4 1 University of the Witwatersrand, 7 York Road, Parktown, 2193, Johannesburg, South Africa 2 Int egral University, Department of Industrial Chemistry, Lucknow, 226026, India 3 Chris Hani Baragwanath Hospital, Department of Gynaecology and Obstetrics, Bertsham, 2013, Johannesburg, South Africa 4 PharmaNatura (Pty) Ltd., Research and Development Unit, Sandton, 2012, South Africa *Corresponding Author: viness.pillay@wits.ac.za Abstract The purpose of this study was to determine the concentration of drug in plasma and vaginal tissues from a pig model administered with a 3' - azido- 3' - deoxythymidine (AZT) and Polystyrene Sulfonate (PSS) - loaded Intravaginal Bioadhesive Polymeric Device (IBPD), intended to be used as homeo- prophylactic agent against HIV and STIs. In addition, histopathological evaluation was undertaken on the vaginal epithelium to assess the potential for toxicity. Biocompatible polymers namely modified polyamide 6,10, poly(lactic - co- glycolic acid), polyacrylic acid, polyvinylalcohol and ethylcellulose were blended with model drugs AZT and PSS, c ompressed into caplet shaped IBPD s and then intravaginally administered into the pig model. The study employed twenty pigs which were divided into four groups of five each (i.e. control, placebo, AZT- containing and the PSS- containing group). The IBPDs were deeply inserted into the posterior fornix of the vagina of group 2, 3 and 4 pigs using a novel applicator under anesthesia. Plasma samples (10mL) were taken from the jugular vein of each pig at day 0, 3, 7, 14, 21 and 28. AZT and PSS were extracted via Solid Phase Extraction (SPE) and subjected to Ultra Performance Liquid Chromatography (UPLC) analysis. At day 28 each pig was euthanized to remove the vaginal tissue which was digested with subtilisin, extracted and subjected to AZT and PSS content analysis. Histopathological evaluation was conducted on vaginal epithelium, lamina propria as well as submucosa and vaginal wall. The mean drug concentration in the vaginal tissue at day 28 was 1.2148? 0. 062mg/mL (N=5) for AZT and 1.4004? 0. 071mg/mL (N=5) for PSS, wh ile the plasma concentration was 0.332? 0. 014mg/mL (N=5) for AZT and 0.256?0. 013mg/mL (N=5) for PSS. This is an indication that the majority of the drug was retained in the vaginal tissue. For each pig, the vaginal tissue drug concentration at three vaginal sites (namely anterior, middle and posterior) was approximately the same (P>0.05). On average, PSS presented with the highest vaginal tissue drug concentration at the 28 th day. These results may be attributed to the hydrophobicity and high matrix resilience of the polymers employed coupled with the presence of strong intermolecular forces in PSS. This findings correlated well (R 2 = 0. 99) with the results obtained from AZT/ PSS content analysis in blood samples from day 1 - 2 8 where AZT ranged between 0.012- 0. 332mg/mL and PSS between 0.009- 0. 256 (N= 5). The histopathological events were negative to mild levels in most cases. AZT and PSS concentration in the pig blood and vaginal tissue and the histopathological findings suggests that the developed caplets may be suitable for intravaginal drug delivery that may be useful for the prevention of female- related disease conditions. Keywords: Histopathological evaluation, HIV/AIDS, STIs, Microbicidal drug delivery system, UPLC analysi s. 307 APPENDIX A5 Submitted Application of Artificial Neural Networks for the Elucidation of an Optimized Synergistic Polymer Combination for Effective Performance of an Intravaginal Bioadhesive Polymeric Device. Valence M. K. Ndesendo ?, Viness Pillay?*, Yahya E. Choonara 1 , Lisa C. du Toit 1 , Leith Meyer 1 , Riaz A. Khan 2 , Eckhart Buchmann3 , and Uwe Rosin 4 1 University of the Witwatersrand, 7 York Road, Parktown, 2193, Johannesburg, South Africa 2 Integral University, Department of Industrial Chemistry, Lucknow, 226026, India 3 Chris Hani Baragwanath Hospital, Department of Gynaecology and Obstetrics, Bertsham, 2013, Johannesburg, South Africa 4 PharmaNatura (Pty) Ltd., Research and Development Unit, Sandton, 2012, South Africa *Corresponding Author: viness.pillay@wits.ac.za Abstract This study aimed at elucidating the synergistic polymer combination that govern the matrix integrity, controlled release kinetics and the release mechanisms of the loaded drugs in an Intravaginal Bioadhesive Polymer ic Device (IBPD), using Artificial Neural Networks (ANN) approach. Fifteen formulations derived from hydrophilic and hydrophobic polymer combinations were blended with the model drugs 3' - azido- 3' - deoxythymidine (AZT) and Polystyrene Sulfonate (PSS) and then compressed into caplet shaped devices on a Manesty D3B 16 station tableting press (Manesty D3B L249LQ, Liverpool, England) at a compression pressure of 5 tons. In process validation tests were performed to ensure that the IBPDs had desirable quality attr ibutes in terms of matrix hardness, uniformity in mass and friability. Matrix erosion test was conducted to the formulations at pH 4.5 to assess their matrix integrity. In vitro drug release evaluation was conducted on the AZT and PSS- loaded IBPDs in simul ated vaginal fluid (pH 4.5; 37?C) and samples were analysed by UV spectroscopy (Cecil Instruments, Cambridge, England). Artificial Neural Networks (ANN) was used to enable the selection of the appropriate polymers for developing the IBPD. Robust devices wi th mass uniformity=800? 0. 48mg, matrix hardness of BHN values ranging from 2.687- 4. 981N/mm 2 and an average friability of 0.21% were produced. The tested formulations presented with good matrix integrity with matrix erosion percentage ranging from 1.21- 7. 68. Zero order release kinetics was obtained over a prolonged period ranging from 24 hours to 72 days. Polymers employed were polyamide 6,10, poly(lactic - co- glycolic acid), polyethylene oxide, polyacrylic acid, carrageenan, ethycellulose, polyvinylalcohol, xanthan gum, gelatin, beeswax, Eudragit? S100 and Eudragit? RS 100. ANNs elucidated that among these polymers a combination comprised of modified polyamide 6,10, poly(lactic - co- glycolic acid), polyvinylalcohol, poly(acrylic acid) and ethycellulose was the most appropriate polymer combination for formulation optimization of the IBPD. This combination presented with the highest degree of matrix integrity that contributed highly to the good drug release kinetics obtained. Results revealed a highly satisfactory f it for the input variables (R 2 = 0.99) for matrix erosion. A linear correlation between the matrix loss and drug release was also realized (R 2 = 0.99). Keywords: Artificial neural networks, Controlled drug release, Micro- environmental pH, Matrix integrity, Matrix erosion, Optimization. 308 APPENDIX A6 Submitted Preformulation Investigations for the Development of a Lead Intravaginal Bioadhesive Polymeric Device Valence M. K. Ndesendo ?, Viness Pillay?*, Yahya E. Choonara 1 , Lisa C. du Toit 1 , Leith Meyer 1 , Riaz A. Khan 2 , Eckhart Buchmann3 , and Uwe Rosin 4 1 University of the Witwatersrand, 7 York Road, Parktown, 2193, Johannesburg, South Africa 2 Integral University, Department of Industrial Chemistry, Lucknow, 226026, India 3 Chris Hani Baragwanath Hospital, Department of Gynaecology and Obstetrics, Bertsham, 2013, Johannesburg, South Africa 4 PharmaNatura (Pty) Ltd., Research and Development Unit, Sandton, 2012, South Africa *Corresponding Author: viness.pillay@ wits.ac.za Abstract The purpose of this study was to screen various polymers through extensive preformulation investigations to ultimately obtain a lead polymer combination that can be employed in designing desirable I ntravaginal Bioadhesive Polymeric Device (IBPD). Hydrophilic and hydrophobic polymers at different combinations were blended and compressed into caplet shaped devices at a force of 5 tons. One of the hydrophilic polymers was a modified synthetic product of polyamide 6,10 ( mPA 6,10). The devi ces were subjected to i n- process validation tests and thereafter to preformulation investigational screening using a One Variable at a Time (OVAT) approach. A series of swelling tests were conducted on 62 formulations that were derived from 18 polymers, to investigate which formulation had the optimal swelling at both simulated and seminal vaginal fluids. Each formulation was weighed, immersed into both vaginal and semen fluids and then placed in an orbital shaking incubator, maintained at 20rpm (37 ? C) for 24 hours. After 24 hours each device was removed from the orbital shaking incubator, gently blotted on filter paper and re- weighed. The swelling behavior was determined in terms of the equilibrium swelling ratio (ESR) which was the critical indicator of th e formulation?s matrix stability (i.e. the degree of matrix robustness), and was used as a screening parameter for each formulation. The developed devices were sufficiently s trong and robust with a mean hardness of 286? 0. 01N, mean weight 600? 0. 48mg and a mean friability of 0.31? 0.04% which was within the limits. Through OVAT approach, 15 suitable formulations (out of 62) with ESRs ranging from 0.011- 0. 084. Through extensive screening of the developmental formulations using an OVAT approach, robust and stron g devices with substantial matrix integrity were produced, as evidenced by the swelling equilibrium ratios. Fifteen lead formulations with minimal swelling tendencies were obtained. F62 which is comprised of mPA 6,10, (150mg), PLGA (400mg), EC (200mg), PVA ( 25mg) and PAA (25mg) appeared to be the most suited in terms of minimal swelling capacity and highest stability. Overall, the results suggested that PA 6,10, PLGA, EC, PVA and PAA at an appropriate weight ratio may be suitable for development of an IBPD . Keywords: OVAT, Intravaginal Bioadhesive Polymeric Device (IBPD), Developmental design, Differential scanning calorimetry, Matrix swelling. 309 APPENDIX B ABSTRACTS OF CONFERENCE PROCEEDINGS 310 APPENDIX B1 311 APPENDIX B 2 312 APPENDIX B3 313 APPENDIX B4 In vivo Drug Content Anal ysis and Histopathological Evaluation of an Intravaginal Polymeric Platform in a Pig Mode l Valence M. K. Ndesendo ?, Viness Pillay?*, Yahya E. Choonara 1 , Lisa C. du Toit 1 , Leith Meyer 1 , Riaz A. Khan 2 , Eckhart Buchmann3 , and Uwe Rosin 4 1 University of the Witwatersrand, 7 York Road, Parktown, 2193, Johannesburg, South Africa 2 Integral Universit y, Department of Industrial Chemistry, Lucknow, 226026, India 3 Chris Hani Baragwanath Hospital, Department of Gynaecology and Obstetrics, Bertsham, 2013, Johannesburg, South Africa 4 PharmaNatura (Pty) Ltd., Research and Development Unit, Sandton, 2012, South Africa *Corresponding Author: viness.pillay@wits.ac.za Purpose To determine the drug concentration in both plasma and vaginal tissue in a pig model inserted with a zidovudine (AZT) - loaded Intravaginal Polymeric Platform (IPP) for the prophylaxis and/or prevention of HIV and STIs. In addition, histopathological and morphological evaluation was undertaken on the vaginal mucosa to assess the potential for toxicity. Methods Biocompatible polymers namely PLGA, ethylcellulose (EC) and PAA were blended with model drug AZT, compressed into caplet shaped IPPs and then intravaginally inserted into the pig model. Fifteen pigs divided into 3 groups (control, placebo and AZT- loaded IPPs) of 5 pigs each were employed for this study. The IPPs were inserted into the posterior fornix of the vagina (group 2 and 3) using a novel applicator under anesthesia. Plasma samples (10mL) were taken from the jugular vein of each pig at day 0, 3, 7, 14, 21 and 28. AZT was extracted v ia Solid Phase Extraction (SPE) and subjected to Ultra Performance Liquid Chromatography (UPLC) analysis using a phenyl column (1.7 ?m; 2.1? 50mm). A gradient method was used with a binary mobile phase (water/acetonitrile) varying from 60:40 at t o, 5:95 at t 1 - 2.6min, and 60:40 at t 3.5- 3 .6min with an injection volume of 2?L, a flow rate=0.5mL/min and UV detection set at 267nm. At day 28 each pig was euthanized to remove the vaginal tissue which was digested with subtilisin, extracted and then subjected to AZT content analysis using UPLC. Histopathological and morphological evaluation involved assessment of the epithelial histological lesions, the lamina propria, as well as submucosa and vaginal wall. Results and Discussion AZT content in the porcine vaginal tissue from day 0- 28 ranged between 56- 67% (N= 5) indicating that AZT was substantially retained within the vaginal tissue. This was attributed to the high matrix integrity of the IPP that was imposed by the networked microstructure that was formed by the hydrophobic and hydrophilic polymers employed. This finding correlates (R 2 =0. 99) with the results obtained from AZT content analysis in blood samples where AZT content was minimal and ranged between 22- 37% (N= 5). Histopathological and morphological evaluation revealed that hyperplasia, exocytosis, exudates on the mucosal surface, ulceration, polymorphonuclear infiltration and perivascular ranged from negative to mild- moderate levels. Conclusions AZT concentration in the plasma and vaginal tissue as well as the histopathological and morphological findings suggests that the developed IPP may be suitable for intravaginal drug delivery. 314 APPENDIX B5 315 APPENDIX B6 316 APPENDIX B7 317 APPENDIX B8 318 APPENDIX B9 319 APPENDIX B10 320 APPENDIX B11 321 APPENDIX B12 322 APPENDIX B13 An In vitro, Ex vivo, In vivo Anal ysis as well as Histopathological and Histomorphological Evaluation of an Intravaginal Microbicidal Bioadhesive Polymeric Device for the Prevention of HIV and STIs. Valence M. K. Ndesendo?, Viness Pillay?*, Lisa C. du Toit 1 , Yahya E. Choonara?, Leith Meyer 2 , Riaz A. Khan 3 , Eckhart Buchmann4 , and Uwe Rosin 5 1 University of the Witwatersrand, Department of Pharmacy and Pharmacology, 7 York Road, Parktown, 2193, Johannesburg, South Africa 2 University of the Witwatersrand, Central Animal Services and Brain Function Research Group, School of Physiology, 7 York Road, Parktown, 2193, Johannesburg, South Africa 3 Integral University, Department of Industrial Chemistry, Lucknow, 226026, India 4 Chris Hani Baragwanath Hospital, Department of Gynaecology and Obstetrics, Bertsham, 2013, Johannesburg, South A frica 5 PharmaNatura (Pty) Ltd., Research and Development Unit, Sandton, 2012, South Africa. Introduction: The global HIV/AIDS epidemic calls for the need to search ideal microbicides for curbing transmucosal transmission of HIV and STI pathogens. The aim of this study therefore was to develop an Intravaginal Bioadhesive Polymeric Device (IBPD) loaded with zidovudine (AZT) and polystyrene sulfonate (PSS) for the prevention of HIV and STIs. Methods: Suitable polymers were blended with model drugs AZT, PSS, barium sulfate (BaSO 4 ) then compressed into IBDPs, and thereafter subjected to in vitro drug release analysis, ex vivo drug flux analysis and ex vivo and in vivo bioadhesivity testing. Furthermore, the IBPDS were also subjected to in vivo drug content analysis as well toxicity evaluation in the pig model Results: Controlled drug release was obtained over a period of 72 days. A suitable ex vivo drug flux and ex vivo bioadhesivity was obtained. The IBPDs could adhere to the vaginal tissue over 30 days. Most of the drug was retained in the vaginal tissue (77%) with only little quantity crossing to the blood (22%). Histopathological and histomorphological evaluation indicated that the IBPDs were non- toxic. Conclusions: Results suggest that the developed IBPDs may be suitable for use as a localized intravaginal drug delivery system for the potential prevention of HIV infection and STIs. Ethical clearance No. 2007/ 25/ 05. 323 APPENDIX C ADDITIONAL OUTPUT S FROM COLLABORATIVE RESEARCH 324 APPENDIX C1 325 APPENDIX C2 APPENDIX C2 326 APPENDIX C3 327 APPENDIX C4 328 APPENDIX C 5 The inf l u en ce of polyami d e 6,10 syn th es i s vari ab l es on the physi coch emi cal characte ri s ti cs and dru g releas e kin eti cs fro m a mon ol i th i c tab l et ma t ri x O A Kol awol e , V Pill a y * De pa rt me nt of Pharm a cy and Pha rm ac ology, Un i ve rs ity of the Wit wat er s rand, 7 York Roa d, Par ktown 2193, Johanne sburg, South Afr ic a , Y E Choonara, L C Du toit and V M K Ndes e ndo Corres ponding aut hor: vi nes .pi l l ay@wi t s .ac.za APPENDIX C6 329 APPENDIX C 6 The sim ult ane ous in vitro charact e ri zat ion of pol y( lac t ic co - gl yc ol ic aci d) a nd pol y( gl uc ur oni de ) - ri c h nanopar ti cl e s empl oyi ng vari ous sol - gel synt he ti c wet chemi c al proc e ss i ng strate gie s Ya hya E. Choona ra 1 , Vi ness Pi ll a y *1 , Val e nc e M.K. Ndes e ndo 1 , Li sa C. du Toi t 1 , Ri az A. Kha n 2 , and Cara gh S. Murphy 1 1 Uni ve r s it y of the Wit wat ers r a nd, Depar t me nt of Pharma c y and Pha r ma c ol ogy, 7 Yor k Road, Par kt own, 2193, J oha nnes bur g, South Afr ic a 2 Int e gr a l Uni ve r si t y, Depa rt me nt of Indust ri al Chemi s tr y, Luc know, 226026, Indi a *Corr e s pondi ng Aut hor: vines s .pi ll a y@wi t s .ac .za Te l : +2711 717- 2274 F ax : + 2711 642 4355 Abst r a c t T his stud y fo cu sse d on t he fo r mu la tio n a nd eval ua tio n of po lyme r ic na no p a r tic le s ad o p ting var io u s prep a r a tio n ap pr o a c he s and atte mp ts to e xp lic a te t he me cha ni s ms of na no p a r tic le fo r ma tio n e mp lo yin g mo le c ula r mo d e li n g and chemo me tr ic a l co mp uta tio ns. Na no p a r tic le s wer e fo r mula te d usin g thr e e dif fe r e n t ap p r o a c he s suc h as an E mu lsi fic a tio n/So l ve n t Evap o r a tio n (ESE ) , E mul si fic a tio n /Sur fa c ta n t/So l ve n t Evap o r a tio n (ESSE ) a nd Io nic Geli fic a tio n (IG) ap p r o a c he s. T he ESE ap p r o a c h co mp r is e d the e mu ls ific a tio n of an aq ue o u s a nd orga nic so lutio n, wh ile so r b ita n mo no o le a te was ad d e d as a sur fa c ta n t dur in g t he E SSE ap p r o a c h. Catio n - i nd uc e d cro ssli n kin g of hyd r o p hil ic algi n a te wa s e mp lo ye d fo r the IG ap p r o a c h. Fo ur ie r T ransfo r m Infr a r e d (FT - I R) anal ysi s wa s per fo r me d to eluc id a te an y cha nge s in the str u c tur a l backb o ne of the native po l yme r s due to na no p a r tic le fo r ma t io n. T he size and mo r p ho lo g y of na no p a r tic le s wer e anal yse d by Zeta siz e anal ys is a nd Sca nni n g Elec tr o n Micr o sc o p y ( SE M ) wit h pho to m ic r o gr a p h s take n at se ve r a l mag ni fic a t io ns. Step - wise mo le c ula r si mu la tio n mo d e l s rev e a le d the mecha ni s ms of na n o p a r tic le fo r ma tio n to occur via so lva tio n, sur fa c e i nte r a c tio n s, cro ssli n ki ng/p r e c ip ita tio n i nitia tio n a nd sur fa c e - vo lu me mi ni miz a tio n wi th sp h e r ic a l iz a tio n and in te r la c e d net wo r k fo r ma tio n. T he s ize distr ib utio n o f the nano p a r tic le s wer e manip ula te d by the sur fa c ta nt in tr o d uc tio n T he ad d itio n o f so r b ita n mo no o le a te preve nte d co ale sc e nc e of par tic le s, resul tin g in stab le na no - e mul sio n s wit h disti nc t par tic le mo r p ho lo gie s and par tic le size s a nd zeta po te ntia l val u e s in the re gio n o f 20 0 n m a nd - 4 0 mV , resp e c tive l y. T he IG ap p r o a c h also pro d uc e d stab le nano - e mu lsio n wit h a hig he r yie ld of na no p a r tic le s wit h sup e r io r size and stab ilit y co ntr o l of co nfi ne d na no p a r tic le s. Bo th the ESSE a nd IG ap p r o a c he s wer e fo u nd to be sui ta b le fo r pro d uc ing stab le nano - e mul sio n s tha t ma y po te ntia ll y be e mp lo ye d fo r the no ve l delive r y of var io us dru g mo le c ule s. Key wo r ds: Polyme r ic na no p a r tic le s, algi na te , po l y( la c t id e - c o - gl yc o lid e ) , po l yme r ic char a c te r iz a tio n, sur fa c ta n t, cro sslin ki n g, e muls ific a tio n, drug deli ve r y 330 APPENDIX C7 Applications of Nanotechnology in Parkinson?s and Motor neurone diseases Valence M.K. Ndesendo 1 , Viness Pillay1 *, Girish Modi 2 , Yahya E. Choonara1 , Lisa Du Toit 1 1 University of the Witwatersrand, Department of Pharmacy and Pharmacology, 7 York Road, Parktown, 2193, South Africa 2 University of the Witwatersrand, Department of Neurology, Division of Neurosciences, 7 York Road, Parktown, 2193, Johannesburg, South Africa Corresponding author: vines.pillay@wits.ac.za Abstract Parkinson's and motor neurone diseases are ruthless nerve disorders. While the etiology of these diseases is not yet fully known the drugs so far available for treating these conditions are still not as effective and often have serious side- effects. The greatest challenge in treating these conditions lies in the difficulties involved in delivering drugs to the brain which is due to the resistance imposed by arachnoid tight junctions, choroid plexus and blood brain barrier (BBB). Treatment strategies, such as use of alternative drugs (e.g. Levo dopa instead of dopamine), viral vectors, gene, and lipid therapy have not been as successful, due to either the inability to cross the BBB, or development of side effects or triggering of immune responses. Nanotechnology poses to be a likely solution to these challenges. Thus far, nanotechnology has enabled the creation of smart nanomaterials and microdevices that can deliver drug molecules (e.g. dopamine) to the nervous system conveniently. This review article addresses various avenues in which nanotechnology has been involved in the management of Parkinson?s and Motor neurone disease to date, with specific emphasis on nano- based therapeutic modalities that can limit and reverse neuropathological disease states, support and promote functional regeneration of damaged neurons, provide neuroprotection, as well as facilitate the delivery of drugs and small molecules across BBB. Nano- surgery as well as the diagnosis of Parkinson?s and motor neurone diseases through nano- based techniques has also been discussed. Keywords: Parkinson's disease, Motor neurone disease, Dopamine, Brain delivery, Nano- based drug delivery systems. 331 APPENDIX C8 Review: Advances in the Approaches for Enhancement of Oral Drug Bioavailability Pius Fasinu, Viness Pillay, Valence M.K. Ndesendo, Lisa du Toit, Yahya E. Choonara University of the Witwatersrand, Department of Pharmacy and Pharmacology, 7 York Road, Parktow n, 2193, Johannesburg, South Africa *Corresponding Author: viness.pillay@wits.ac.za Abstract In conscious and co - operating patients, oral drug delivery remains the preferable route of drug administration. H owever, not all drugs possess the desirable physicochemical and pharmacokinetic properties which favour oral administration mainly due to poor bioavailability. This has in some cases led to the choice of other routes of administration, which may compromise the convenience and increase the risk of non- compliance. Poor bioavailability has necessitated the administration of higher than normally required oral doses which often leads to economic wastages, risk of toxicity, erratic and unpredictable responses. The challenge over the years has been to design techniques that will allow oral administration of most drugs, irrespective of their properties and yet achieve a therapeutic systemic availability. This will be a worthy achievement since over 90% of therapeutic compounds are known to possess oral bioavailability limitations. In this review, an attempt has been made to explore various approaches that have been used in the recent years to improve oral drug bioavailability, including physical and chemical means. D esign of prodrugs to bypass metabolism or enhance solubility as well as modification of formulation techniques such as use of additives, permeation enhancers, solubilizers, emulsifiers and non aqueous vehicles have been discussed. Pharmaceutical application of nanotechnology, which is an emerging area in drug delivery, has also been addressed. This review sought to assess each method aimed at enhancing the oral bioavailability of drugs in terms of the purpose, scientific basis, limitations, commercial application as well as the areas for further research improvement. Keywords: Absorption, Bioavailability, Cytochrome P450, Efflux transporters, Enzymes, Inhibitors, Permeation enhancers, P - glycoproteins, Pre- systemic metabolism 332 APPENDIX C 9 Rubina P. Shaik h, Viness Pillay*, Yahya E. Choonara, Lisa C. du Toit, Valence M.K. Ndesendo, Priya Bawa and Shivaan Coopan University of the Witwatersrand, 7 York Road, Parktown, 2193, Johannesburg, South Africa *Corresponding Author: viness.pillay@wits.ac.za Abstract: Membrane technology is broadly applied in the medical field. The ability of membranous systems to effectively control the movement of chemical entities is pivotal to their significant potential for use in both drug delivery and surgical/medical applications. An alteration in the physical properties of a polymer in response to a change in environmental conditions is a behavior that can be utilized to prepare 'smart' drug delivery systems. Stimuli- responsive or 'smart' polymers are polymers that upon exposure to small changes in the environment undergo rapid changes in their microstructure. A stimulus, such as a change in pH or temperature, thus serves as a trigger for the release of drug from membranous drug delivery systems that are formulated from stimuli- responsive polymers. This article has sought to review the use of stimuli - responsive polymers that have found application in membranous drug delivery systems. Polymers responsive to pH and temperature have been extensively addressed in this review since they are considered the most important stimuli that may be exploited for use in drug delivery, and biomedical applications such as in tissue engineering. In addition, dual - responsive and glucose- responsive membranes have also been addressed as membranes responsive to diverse stimuli. Keywords: Membranous drug delivery systems, stimuli - responsive polymers, dual responsive membranes, glucose responsive membranes, pH, temperature, "On - off" gating mechanisms. 333 APPENDIX C10 A Modified Crosslinked Oramucosal Lyophilized Wafer System for Gradual Drug Delivery Rubina Shaikh 1 , Viness Pillay1 *, Yahya E. Choonara 1 , Lisa C. du Toit 1 , Valence M. K. Ndesendo 1 and Riaz A. Khan 2 1 University of the Witwatersrand, Department of Pharmacy and Pharmacology, 7 York Road, Parktown, 2193, Johannesburg, South Africa 2 * Corresponding Author: Integral University, Department of Industrial Chemistry, Lucknow, 226026, India viness.pilla y@wits.ac.za Abstract The aim of this study was to develop a polymeric wafer system targeted for gradual oramucosal drug delivery. The mucoadhesive polymer pectin was crosslinked with various salts such as calcium chloride, barium chloride and zinc sulphate for the production of the wafers and subjected to three methods of lyophilization. A simultaneous qualitative and quantitative analysis of the wafer surface morphology and porosity was undertaken. In addition, vibrational transitions, the thermal behav ior as predicted by temperature modulated differential scanning calorimetry, textural attributes, and in vitro drug release behavior of the highly water soluble model drug, diphenhydramine HCl, from the various wafer systems were systematically characteriz ed. The techniques employed yielded wafers of varying porosities. Crosslinked pectin wafers generally demonstrated a smaller pore size (having a mean diameter of 107. 63? compared to 180. 53? for non- crosslinked pectin wafers); due to the highly interconnect ed matrix structure afforded by the formation of inter- and intra- molecular crosslinks between the pectin chains. The drug release capabilities of the wafers varied markedly with the approach employed in the production of the wafers. Non- crosslinked wafers released 82.5% of drug within 30 minutes. Wafers that included a crosslinking step in their formulation design showed a more gradual release of drug. Furthermore, wafers crosslinked after the lyophilization process controlled drug release more efficiently than those that were crosslinked before lyophilization. Vibrational and thermal analysis confirmed the crosslinking effect and resultant drug release capabilities of the wafer systems. There was a strong negative correlation between the normalized pore si ze of the wafers and their resilience (R 2 =0. 99) . It was established that ?), possessed the propensity to form a number of ionic bridges between pectin monomers in intra- and inter- molecular associations producing an interconnected wafer matrix, culminating in wafers of high porosity (0.048cm 2 /g) on lyophilization. Keywords: Oramucosal, wafer matrix, mucoadhesive polymer, gradual drug release, lyophilization, crosslinking, porosity, physicochemical, physicomechanical 334 APPENDIX C 11 Levodopa Delivery Systems: Advancements in Delivery of the Gold Standard Ndidi Ngwuluka , Viness Pillay, Lisa C. du Toit, Valence M.K. Ndesendo, Yahya E. Choonara, Priya Bawa and Shivaan Cooppan University of the Witwatersrand, 7 York Road, Parktown, 2193, Johannesburg, South Africa *Corresponding Author: viness.pillay@wits.ac.za Abstract Background: Despite the fact that Parkinson?s disease (PD ) was discovered almost 200 years ago, its treatment and management remains an immense challenge since progressive loss of dopaminergic nigral neurons, motor complications experienced by the patients as the disease progresses and drawbacks of pharmacotherapeutic management still persists. Various therapeutic agents have been employed in the management of PD, including levodopa (L- dopa), selegiline, amantadine, bromocriptine, entacapone, pramipexole dihydrochloride and more recently istradefylline and rasagiline. Of all agents, L- dopa though the oldest, remains the most effective. L- dopa is easier to administer, better tolerated, less expensive and is required by almost all PD patients. However, L- dopa?s efficacy in advanced PD is significantly reduced due to metabolism, subsequent low bioavailability and irregular fluctuations in its plasma levels. To date, significant strides have been made to improve the delivery of L- dopa in order to enhance its bioavailability and reduce plasma fluctuations as well as motor complications experienced by patients purportedly due to pulsatile stimulation of the striatal dopamine receptors. Objective: The ultimate aim was to critically assess the attempts made thus far directed at improving L - dopa absorption, bioavailability and maintenance of constant plasma concentrations, including the drug delivery technologies implicated. Methods: Drug delivery systems that have been instituted for the delivery of L- dopa include immediate release formulations, liquid formulations, dispersible tablets, controlled release formulations, dual release formulations, microspheres, infusion and transdermal delivery, among others. In this review, the L- dopa loaded drug delivery systems developed over the past three decades were elaborated on. Conclusion: This review highlights the fact that neuropharmaceutics is at a precipice, which is expected to spur investigators to take that leap to enable the generation of innovative delivery systems for the effective management of PD. Keywords: Bioavailability, Conventional dosage forms, Drug delivery systems, Levodopa, Microspheres, Motor complications, Neuropharmaceutics, Nanotechnology, Parkinson?s disease, Pulmonary delivery, Transdermal delivery. 335 APPENDIX C 12 Review Article: Recent Advances in the Design of Drug- loaded Polymeric Implants for the Treatment of Solid Tumors Ameena Wadee1 , Viness Pillay* 1 , Yahya E. Choonara 1 , Lisa C. du Toit 1 , Clement Penny2 , Valence Ndesendo 1 , and Caragh Murphy 1 1 University of the Witwatersrand, Department of Pharmacy and Pharmacology,7 York Road, Parktown, 2193, Johannesburg, South Africa. 2 University of the Witwatersrand, Department of Oncology, 7 York Road, Parktown, 2193, Johannesburg, South Africa. *Corresponding Author: viness.pillay@wits.ac.za Abstract The effective treatment of solid tumors still continues to be a great challenge to clinicians despite the development of novel drugs. In order to improve the clinical effic acy of the existing chemotherapeutics agents, researchers have considered the possibility of local treatment at the site of the solid tumor. The greatest advantage of this localised delivery is the significantly fewer side effects experienced by the patient. In the past, the peri - or intra- tumoral delivery of chemotherapeutics agents was mainly based on implants that used to be inserted surgically into the affected region. In the recent years, in situ forming implants have attracted considerable interest. These are polymeric systems which are injected as solutions into the tumor site using commercially available syringes and needles. The injected solution forms an implant at the tumor site as a result of local environmental stimuli and hence removes the need for surgical implantation. However, while these implants have been shown to improve the treatment of various solid tumors, the ideal implant is yet to be formulated. To date, it is only a few implants that are biodegradable and able to deliver the chemotherapeutic agent over a prolonged period of time. Many of these implants also have an undesirable initial burst release effect. This review summarises the attempts that have so far been made in the development of polymeric implants for the treatment of solid tumors. Keywords: Chemotherapy, Environmental stimuli, Implant, Polymeric systems, Solid tumors. 336 APPENDIX C 13 A review on oral drug delivery systems comprising altered geometric configurations for controlled drug delivery Kovanya Moodley , Viness Pillay*, Yahya E. Choonara, Lisa C. du Toit, Valence M.K. Ndesendo, Shivaan Cooppan and Priya Bawa University of the Witwatersrand, Department of Pharmacy and Pharmacology, 7 York Road, Parktown, 2193, Johannesburg, South Africa *Corresponding Author: viness.pillay@wits.ac.za Abstract Recent pharmaceutical research has focused on controlled drug delivery as an advantage over conventional methods. Adequate controlled plasma drug levels, reduced side effects as well as improved patient compliance are some of the benefits that these systems may offer. Controlled delivery systems that can provide zero- order drug delivery have the potential for maximizing efficacy while minimizing dose frequency and toxicity. Thus, zero - order drug release is ideal in a large area of drug delivery which has therefore led into the development of various technologies with such drug release patterns. Systems such as multilayered tablets and other geometrically altered devices have been created to perform this function. One of the principles of multilayered tablets involves creating a constant surface area for release. Polymeric materials play an important role in the functioning of these systems. Technologies so far developed include among others, Geomatrix? multilayered tablets which utilizes specific polymers that may act as barriers to control drug release; Procise? which has a core with an aperture that can be modified to achieve various types of drug release, core- in- cup tablets where the core matrix is coated on one surface while the circumference forms a cup around it, donut- shaped devices which have a hole in the middle, Dome Matrix? and ?release modules assemblage? which can offer alternating drug release patterns. This review discusses the novel altered geometric system technologies that have been developed to provide controlled drug release, also focusing on polymers that have been employed in such developments. Keywords: Controlled drug delivery, geometrically altered devices, multilayered tablets, Polymeric materials, ?Release modules assemblage?. 337 APPENDIX C14 Pharmaceutical Applications of Electro- Spinning Clare Dott, Viness Pillay*, Yahya E. Choonara, Lisa C. du Toit, Valence M.K. Ndesendo and and Caragh S. Murphy University of the Witwatersrand, Department of Pharmacy and Pharmacology, 7 York Road, Parktown, 2193, Johannesburg, South Africa *Corresponding Author: viness.pillay@wits.ac.za Abstract Electro- spinning of polymers is a unique technology which produces very fine fibres using electrostatic repulsive forces obtained from applying an electrical potential to a liquid. The resultant fibres have much smaller diameters in comparison with fibres obtained using different methods. Electro- spun fibres have found application in many and varied fields such as drug and gene delivery, tissue engineering, wound dressing, electronics, filtration as well as absorption and adsorption. This review will cover the various process ing parameters which play a role in electro- spinning, including the applied voltage, solution flow rate, solution viscosity, solvents, solution conductivity, capillary- to- collector distances and the influence of surfactants. A main focus will be on the application of electro- spun fibres in drug delivery, where researchers have already investigated the use of these fibres in transdermal delivery systems, long- term implants and grafts. Different methods of drug incorporation will be discussed as well as numerous polymers which have been successfully utilised in this field, including cellulose acetate, poly(? - caprolactone), poly(ethylene oxide), poly(vinyl alcohol), gelatin, poly(d,l - lactide- co- glycolide), poly(lactic acid), poly(l - lactic acid), polyurethane, poly(ethylene- co- vinyl acetate), poly(ethylene glycol), and poly(acrylic acid). Keywords: Electro- spinning, drug delivery, electrospun fibres, electrospun scaffolds, parameters, 338 APPENDIX C 15 Design and Development of Pediatric Wafers for the Treatm ent of Pediatric HIV/AIDS. Farina Loonat 1 , Valence M.K. Ndesendo 1 , Viness Pillay* 1 , Yahya E. Choonara 1 , Lisa C. du Toit 1 and Riaz A. Khan 2 1 University of the Witwatersrand, Department of Pharmacy and Pharmacology, 7 York Road, Parktown, 2193, Johannesbur g, South Africa 2 Integral University, Department of Industrial Chemistry, Lucknow, 226026, India Corresponding author: vines s.pillay@wits.ac.za Abstract The purpose of this study was to fabricate pediatric wafers containing 3' - azido- 3' - deoxythymidine (AZT) as an antiretroviral (ARV) drug for the treatment of HIV/AIDS in pediatric patients. Oramucosal drug delivery systems have many advantages over the other routes mainly because of the ability to circumvent the pitfalls of traditional drug delivery systems by directly entering the systemic circulation via the internal jugular vein. A 5% hydroxypropylcellulose solution and a separate 1% carbopol solution were allowed to homogenize, these solutions were then combined in a ratio of 1:1. Approximately 200mg of A ZT needed to be delivered to each cylindrical well of the wafer mould. Other excipients employed included mannitol and lactose as bulking agents, sodium starch glycolate as a disintegrant and glycine as an anti- collapsing agent. Cylindrical wells were lubricated with a mineral oil. 1.5mL of the final solution was transferred into each well under continuous stirring. The formulation was then frozen for a period of 24 hours at negative 60?C. Thereafter, the formulation was lyophilized for a period of 48 hours. Extensive in vitro and ex-vivo studies including disintegration test, determination of DLC and DEE, drug release analysis and bioadhesivity testing were conducted on the wafer systems. An optimal formulation consisting of a ratio of 1:2 (HPC/C934), gly cine (0.03g) as an anti- collapsing agent and sodium starch glycolate (4%w/w) as a disintegrant was obtained. This optimal formulation had a disintegration time as short as 6.7 seconds, a high DLC ( 190. 4mg) and a desirable DEE ( 95. 175% ). A zero order drug r elease kinetics was realized (optimal formulation) and almost the whole quantity of drug was released within 10 seconds. The developed wafers were substantially bioadhesive. The Peak Adhesion Force and the work of adhesion (AUC FD) for the optimal wafer sys tem were PAF=0.193? 0. 004N and AUC FD=0.104? 0. 006J respectively. The developed pediatric a wafers may be suitable for the treatment of pediatric HIV/AIDS. Keywords: Bioadhesivity, HIV/AIDS, Drug loading capacity, Drug entrapment efficiency, Disentergration time. 339 APPENDIX C16 Fab ri ca ti on, ch aracte ri zati on and opti mi zati on of MTX - l oad ed PLA/ MAA nan op arti cl es B Si be ko 1 , V Pil l a y* 1 , YE Choonar a 1 , RA Khan 1 , G Modi 2 , SE Iyuke 3 , D Nai doo 4 and VMK Ndes e ndo 1 1 Uni ve r s it y of the Wit wat ers r a nd, Depar t me n t of Pharma c y and Pha r ma c ol ogy, 7 Yor k Road, Par kt own, 2193, J oha nnes bur g, South Afr ic a 2 Uni ve r s it y of the Wit wat ers r a nd Depar t me nt of Neur ology, Di vi s ion of Neuros ci enc e s , 3 Uni ve r s it y of the Wit wat ers r a nd School of Chemi c a l and Met al l ur gi c al Engi ne er ing, 4 Uni ve r s it y of the Wit wat ers r a nd Depar t me nt of Neur os ur ge r y, Di vi s i on of Neur os c i enc es *Cor r es ponda nce : vi nes s .pil l a y@wi t s .a c .za Abst r a c t The pur p o se of thi s rese a r c h was to deve lo p pol y ( D L - la c t id e ) (P LA) /E ud r a g it S 10 0 (ES 100 ) nano p a r tic le s (NP ) wit h t he po te ntia l to ser ve as car r ie r syst e ms fo r me tho tr e xa te (MT X) an an tic a nc e r dru g . T he nano p a r tic le s wer e prepa r e d by do ub le e mu lsio n so lve n t evap o r a tio n tech niq ue . A 3 - Fa c to r Bo x Behn ke n desi gn was ap p lie d fo r prep a r ing var io u s nano p a r tic le fo r mu la tio n s. T he size o f t he na no p a r tic le s var ie d fro m 21 1 nm to 37 8 . 3 nm and t he na no p a r tic le reco ve r y var ie d fro m 36 . 8 mg to 86 . 2 mg. Mo s t fo r mula tio n s sho we d a po l y - d isp e r sit y ind e x (P d I ) value l ess t ha n 0.5 ind ic a t i n g t hat t he nano p a r tic le siz e distr ib utio n was ho mo ge no us wit hi n t he fo r mula tio ns. T he drug lo ad in g cap a c it ie s ( DLC) o f t he nano p a r tic le s wer e ver y lo w wit h t he maxi mu m bein g 12 % for the op ti miz e d fo r mula tio n. I n ord e r to i mp r o ve the dru g lo ad in g cap a c it y of t he op ti miz e d fo r mu la t io n, we ad so r b e d the drug ont o the nano p a r tic le s matr i x b y inc ub a ti ng t he na no p a r tic le s i n a conc e ntr a te d MT X so lutio n. B y so do in g, we succ e ss f ull y i mp r o ve d drug lo ad ing to 98 %. Fo ur ie r - T r a ns fo r m I nfr a r e d (FT I R) stud ie s sho we d that MT X wa s prese nt i n the nano p a r tic le s. T he na no p a r tic le s sho we d an in itia l b ur st rele a se wit h 50 % o f MT X rele a se d in 24 ho ur s, ther e a fte r t he rele a se wa s pro lo nge d fo r 84 ho ur s. Scann in g elec tr o n micr o gr a p hs reve a le d almo s t sp he r ic a l so lid na no p a r tic le s wit h a smo o th sur fa c e a nd tran s mis sio n elec tr o n micr o gr a p hs sho we d the prese nc e MT X wit hi n t he na no p a r tic le s. Key wo r ds: P o ly ( D L - la c tid e ) (P L A) , Eud r a gi t S 10 0 (ES 10 0 ) , Metho tr e xa te (MT X) and Dru g lo ad in g cap a c it y ( DLC) 340 APPENDIX C 17 Modulation of Nicotine Release from a Gelisphere? Loaded Compressed External Polymeric Matrix Neha Singh, Viness Pillay* Yahya E. Choonara and Valence M.K. Ndesendo University of the Witwatersrand, Department of Pharmacy and Pharmacology, 7 York Road, Parktown 2193, Johannesburg, South Africa *Cor r es ponda nce : vi nes s .pil l a y@wi t s .a c .za Abstract The purpose of this study was to develop a prolonged- release drug delivery device for site- specific delivery of a newly researched neuroprotective agent, namely nicotine. The device was formulated as a novel reinforced crosslinked composite polymeric system with the potential for implantation into the substantia nigra pars compacta of the brain to provide site- specific drug delivery for the treatment of Parkinson?s disease. Polymers with biocompatible and bioerodible characteristics were selected to incorporate nicotine within electrolyte- crosslinked alginate- hydroxyethylcellulose gelispheres compressed within a release- rate modulating external polymeric matrix, comprising either hydroxypropylcellulose, polyethylene oxide and poly(lactic - co- glycolic) acid to further attenuate nicotine release. The degradation and erosional behavior of the discs following exposure to simulated CSF are also conducted to elucidate their influence on drug release. Kinetic modeling of drug release data was performed to understand the predominant mechanisms involved in controlling nicotine release from the device. Zero- order drug release was observed for a period of 50 days in simulated cerebrospinal fluid from the device comprising poly(lactic - co- glycolic) acid as the external matrix. Keywords: Alginate gelispheres, textural analysis, crosslinked matrices, kinetic modeling, PLGA discs, prolonged release, BH N, powder flow properties 341 APPENDIX C 18 APPENDIX C 19 342 APPENDIX C 19 343 APPENDIX C 20 344 APPENDIX C 21 345 APPENDIX C 22 346 APPENDIX C23 347 APPENDIX C24 348 APPENDIX C25 349 APPENDIX D ANIMAL ETHICS CLEARANCE CERTIFICATE The rabbit model was changed to pig model due the pig vagina more closely resembling that of the human (see Modifications and Extensions to Experiments approval next page). 350