Rilpivirine and cabotegravir trough concentrations in people with HIV on long-term treatment with long-acting injectable antiretrovirals Maria Vittoria Cossu1†, Dario Cattaneo1,2†, Davide Moschese1, Andrea Giacomelli 1, Sara Soloperto3, Antonio D’Avolio3, Spinello Antinori 1,4, Andrea Gori1,4,5, Giuliano Rizzardini1,6 and Cristina Gervasoni 1,2* 1Department of Infectious Diseases, ASST Fatebenefratelli Sacco University Hospital, Milan, Italy; 2Gestione Ambulatoriale Politerapie (GAP) Outpatient Clinic, ASST Fatebenefratelli Sacco University Hospital, Milan, Italy; 3Laboratory of Clinical Pharmacology and Pharmacogenetics, Amedeo di Savoia Hospital, Department of Medical Sciences, University of Turin, Turin, Italy; 4Dipartimento di Scienze Biomediche e Cliniche Luigi Sacco, Università degli Studi di Milano, Milan, Italy; 5Centre for Multidisciplinary Research in Health Science (MACH), Università degli Studi di Milano, Milan, Italy; 6School of Clinical Medicine, Faculty of Health Science, University of the Witwatersrand, Johannesburg, South Africa *Corresponding author. E-mail: cristina.gervasoni@asst-fbf-sacco.it †Both authors equally contributed to the study. Received 29 January 2024; accepted 6 March 2024 Objective: Large inter-individual variability in the pharmacokinetics of rilpivirine and cabotegravir has been reported in the first weeks after starting long-acting injectable (LAI) therapy. Here, we assessed the distribution of rilpivirine and cabotegravir trough concentrations in people with HIV (PWH) on long-term LAI treatment. Methods: Adult PWH treated with LAI for at least 32 weeks with an assessment of drug plasma trough concen- trations were considered. The proportion of rilpivirine and cabotegravir plasma trough concentrations below four-times the protein-adjusted concentrations required for 90% inhibition of viral replication (4×PA-IC90) was estimated. Results: Sixty-seven PWH were identified. LAI treatment duration was 216 ± 80 weeks (range 32–320 weeks). Cabotegravir concentrations were associated with lower inter-individual variability compared with rilpivirine (45% versus 84%; P < 0.05). No differences were found in rilpivirine (160 ± 118 versus 189 ± 81 ng/mL; P = 0.430) and cabotegravir (1758 ± 807 versus 1969 ± 802 ng/mL; P = 0.416) trough concentrations in males (n = 55) versus females (n = 12). A non-significant trend for lower cabotegravir concentrations was found in PWH with a body mass index >30 kg/m2 (n = 9) versus non-obese participants (1916 ± 905 versus 1606 ± 576 ng/mL; P = 0.131). Three out of the 67 PWH had at least one drug concentration <4×PA-IC90: 100% of PWH had undetectable HIV viral load. Conclusions: At steady state, optimal systemic exposure of cabotegravir and rilpivirine was found in most PWH; cabotegravir trough concentrations were associated with lower inter-individual variability compared with rilpi- virine. The study was not powered to assess the contribution of sex and/or body weight on LAI exposure due to the small number of females and obese PWH included. © The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com. Introduction Rilpivirine, a known NNRTI, and cabotegravir, a new integrase inhibitor (INI) structurally related to dolutegravir, have been re- cently approved as a long-acting injectable (LAI) antiretroviral dual regimen for the treatment of HIV infection.1–4 According to the international guidelines for HIV treatment, LAI rilpivirine and cabotegravir are now considered as a maintenance strategy for adult people with HIV (PWH) with sustained viral suppression, no history of virological failure or documented/suspected resistance to NNRTI or INI classes, and no active or occult hepatitis B virus in- fection.5,6 LAI rilpivirine and cabotegravir can be administered after an optional oral lead-in period (rilpivirine 25 mg plus cabote- gravir 30 mg once daily), or started immediately with intramuscu- lar injections given at 4- or 8-week intervals, based on the optimal efficacy and tolerability results from Phase III clinical trials.7–10 Large inter-individual variability in the pharmacokinetics of ril- pivirine and cabotegravir has been reported in the first weeks 1126 J Antimicrob Chemother 2024; 79: 1126–1132 https://doi.org/10.1093/jac/dkae080 Advance Access publication 26 March 2024 D ow nloaded from https://academ ic.oup.com /jac/article/79/5/1126/7635198 by W itw atersrand U niversity user on 06 February 2025 https://orcid.org/0000-0003-3685-4289 https://orcid.org/0000-0003-0569-9407 https://orcid.org/0000-0002-4922-6495 mailto:cristina.gervasoni@asst-fbf-sacco.it after starting LAI treatment and, most importantly, low rilpivirine and/or cabotegravir plasma trough concentrations combined with other risk factors (i.e. resistance-associated mutations, HIV subtype A6/A1 and BMI ≥ 30 kg/m2) have been associated with in- creased risk of virologic failure.11 Recent experiences with the therapeutic drug monitoring (TDM) of rilpivirine and cabotegravir plasma trough concentrations confirmed the high inter-individual variability and the potential risk of drug underexposure, mainly to rilpivirine, in the first weeks after starting LAI therapy.12,13 Here, we aimed to assess the distribution of rilpivirine and ca- botegravir trough concentrations in PWH on long-term treatment with LAI antiretrovirals (i.e. up to 320 weeks) and, eventually, identify clinical variables associated with systemic drug exposure in a real-life setting. Methods Study design and enrolment This retrospective, observational study was aimed to describe the distribution of rilpivirine and cabotegravir trough concentrations in steady-state conditions. To address this issue, we included consecutive PWH from our hospital treated with LAI rilpivirine and cabotegravir as antiretroviral regimen for at least 32 weeks with an assessment of drug plasma trough concentrations collected from March to June 2023. We considered eligible for the study all adults starting cabotegravir and rilpi- virine following the approval of the long-acting combined antiretroviral therapy (April 2022 in Italy), as well as those previously enrolled in the pivotal phase II and III studies (in these patients, the blood samples for the TDM were collected after the end of the study), regardless to lead- in phase and timing of drug injection (i.e. starting every 4 or 8 weeks), with no exclusion criteria. Demographic, clinical and pharmacologic infor- mation were extracted from the hospital database. As the primary study outcome, we estimated the proportion of rilpivirine and cabotegravir plasma trough concentrations below 4-fold the protein-adjusted concen- trations required for 90% inhibition of viral replication (4×PA-IC90) set, at 50 and 664 ng/mL for rilpivirine and cabotegravir, respectively.12,13 As secondary analyses, we attempted to identify demographic and/or clinic- al variables eventually associated with rilpivirine and cabotegravir trough concentrations. The study was approved by local Ethical Committee (IRB approval no. 11903). All data used in the study were previously anonymized, according to the requirements of the Italian Data Protection Code (leg. decree 196/2003) and by the general authorizations issued by the Data Protection Authority. All patients signed a written informed consent for medical procedures/interventions performed for routine treatment pur- poses, according to the Ethics Committee (Comitato Etico Interaziendale Area 1, Milan, Italy). Assessment of drug plasma trough concentrations Each blood trough sample for the assessment of rilpivirine and cabote- gravir trough concentration was drawn just before the scheduled injec- tion (every 8 weeks) and collected in lithium heparin vacutainer. After drawing, whole blood samples were kept at room temperature for a max- imum of 4 hours, then centrifuged (1400g, 10 min at 4°C), inactivated for HIV (35 min at 58°C), and the separated plasma was frozen at −80°C until analysis. Plasma quantification of rilpivirine and cabotegravir was per- formed using an ultrahigh-performance liquid chromatography with tan- dem mass spectrometry method, modified from the one previously published as described next.14 Fifty microlitres of plasma samples (from patients, calibrators or quality controls) were added to 100 µL of internal standard solution and 600 µL of precipitant solution (acetonitrile:metha- nol 50:50). After vortex mixing, the samples were centrifuged at 14 000 rpm for 10 min at 4°C and 300 µL of the supernatant were added to 600 µL of diluent solution (water 100%). Five microlitres were injected in the Acquity® UPLC HSS T3 column, (2.1 × 150 mm, 1.8 µm, Waters, Milan, Italy) and analysed with the chromatographic system Perkin Elmer LX-50® UHPLC system coupled with a Triple Quadrupole QSight 220® at 50°C using a column thermostat, at a flowrate of 0.4 mL/min with a gradient of two mobile phases: phase A (formic acid 0.05%) and phase B (acetonitrile + formic acid 0.05%). The instrument was settled in positive electrospray ionization mode for both drugs, a source tem- perature of 350°C, a desolvation temperature of 280°C and the chroma- tographic run lasted 15.0 minutes for each injection. Linearity ranged from 10 to 2500 ng/mL and from 40 to 10 000 ng/mL for rilpivirine and cabotegravir, respectively. All samples collected in the present study were processed in a single analytical run. Statistical analyses Continuous variables were expressed as mean ± standard deviation and compared using an unpaired t-test; categorical variables were expressed as an absolute number or as a percentage, and their frequency was com- pared using the chi-squared test. Inter-individual variability in the rilpivir- ine and cabotegravir plasma trough concentrations was calculated as the percentage of coefficient of variation (CV%). Univariate and multivariate linear regression analyses were carried out considering either rilpivirine or cabotegravir plasma trough concentrations as the dependent variable and clinical characteristics (age, sex, ethnicity, BMI, duration of LAI ther- apy, serum creatinine and transaminases) as independent variables. Only dependent variables resulting in statistical association with drug concen- trations in the univariate analyses were included in the multivariable model. Statistical significances were set at P < 0.05 (a significant difference) and P < 0.01 (a highly significant difference). Results Demographic and clinical characteristics Data on rilpivirine and cabotegravir trough concentrations were available for 67 PWH. They were mostly men (82%), with a mean age of 45 ± 13 years, a duration of viral suppression of 10 ± 5 years and had been previously treated with 1.9 ± 0.3 oral antiretroviral regimens before starting LAI therapy. No ma- jor differences in the clinical characteristics were observed be- tween females and males (Table 1). Fourteen percent of the enrolled PWH (8% females versus 15% of males; P = 0.567) had BMI >30 kg/m2. All PWH had preserved kidney and liver function and optimal immune-virologic control (100% of PWH had undetectable HIV viral load, 3% had CD4 cell count <350 cells/mm3). No viral blips were observed during the treatment period with LAI. Rilpivirine and cabotegravir dosing schemes All the patients received two injections (cabotegravir and rilpivir- ine), one into the muscle of each side of buttocks, and none of them were concomitantly treated with moderate/strong drug inducers or inhibitors. All injections were given within the ±7-day window. We used a 23G needle (0.60 × 25 mm) and a 21G (0.80 × 40 mm) in PWH with BMI < 30 and >30 kg/m2, re- spectively. Fifty-nine out of the 67 PWH (88%) had previously been enrolled in pivotal LAI studies, namely ATLAS (n = 35), FLAIR (n = 12) and SOLAR (n = 12).7–10 Sixty-one of them were treated with an oral, lead-in phase (Table 2). Forty-nine (73%) TDM of LAI 1127 D ow nloaded from https://academ ic.oup.com /jac/article/79/5/1126/7635198 by W itw atersrand U niversity user on 06 February 2025 of the PWH were initially treated with a LAI injection every 4 weeks for an average of 108 ± 56 weeks and then shifted to the 8-week frequency; the remaining (n = 18) started and main- tained LAI injections every 8 weeks (with the first two injections administered 4 weeks apart). At the time of the assessment of drug concentrations, all the PWH were on maintenance rilpivirine plus cabotegravir LAI regimens given every 8 weeks (treatment duration 216 ± 80 weeks, ranging from a minimum of 32 to a maximum of 320 weeks after starting LAI therapy). Distribution of rilpivirine and cabotegravir trough concentrations The single assessment of rilpivirine and cabotegravir trough con- centrations was performed at 216 ± 80 weeks after starting LAI treatment (range 32–320 weeks), with 88% of samples collected after a minimum of 100 weeks. Overall, rilpivirine and cabotegra- vir trough concentrations ranged from 81 to 877 ng/mL (177 ± 149 ng/mL) and from 577 to 5740 ng/mL (1792 ± 799 ng/mL), respectively (Table 2). As visualized in Figure 1, cabotegravir plas- ma trough concentrations were associated with a lower inter- individual variability over time compared with rilpivirine (CV%: 45% versus 84%; P < 0.05). A non-significant trend for lower rilpi- virine (160 ± 118 versus 189 ± 81 ng/mL, −15%; P = 0.430) and cabotegravir (1758 ± 807 versus 1969 ± 802 ng/mL, −11%; P = 0.416) trough concentrations was observed in males versus females (Figure 2). Similarly, no statistical differences were found when stratifying drug trough concentrations according to BMI (Figure 3). None of the clinical variables resulted significantly as- sociated with rilpivirine or cabotegravir trough concentrations in the univariate analysis, preventing the possibility to perform multivariate models. No significant effect was found for rilpivirine [156 ± 64 (CV% 41%) versus 153 ± 64 (CV% 42%) ng/mL, P = 0.854] and cabotegravir [1913 ± 1009 (CV% 52%) versus 1710 ± 654 (CV% 38%) ng/mL, P = 0.342] concentrations when dosing was started a 4- versus 8-week interval. None of the samples produced a result below the PA-IC90 for each drug; three rilpivirine (41, 43 and 49 ng/mL corresponding to 4.5% of all assessments) and one cabotegravir (577 ng/mL, 1.5%) trough concentrations produced a result below the target of 4×PA-IC90. The only patient with both drugs below this target was a 43-year-old Caucasian man on treatment with LAI for 132 weeks and with a BMI of 33.1 kg/m2; the other two patients with rilpivirine <50 ng/mL were both Caucasian men with BMI of 23.8 and 25.4 kg/m2, being treated with LAI, respectively, for 116 and 240 weeks. All injections were administered at the scheduled day. Two out of the 67 PWH had rilpivirine concentrations >500 ng/mL. The first was a 32-year-old man with a BMI of 21.2 kg/m2 and the second was a 62-year-old man with a BMI of 34.6 kg/m2. Neither of them was receiving enzyme inhibitor therapy; both had an ECG that showed no evidence of QT prolongation. Table 1. Demographic and clinical characteristics of the 67 PWH on long-term maintenance therapy with LAI antiretrovirals (continuous variables were expressed as mean ± standard deviation; categorical variables were expressed as absolute number or as percentage) Variable Overall Females Males Patients, n 67 12 55 Age, years 45 ± 13 47 ± 13 45 ± 14 Body weight, kg 78 ± 13 68 ± 11 80 ± 13* BMI, kg × h/m2 25.7 ± 3.9 24.4 ± 3.4 25.4 ± 3.8 Patients with BMI >30 kg × h/m2, % 14 8 15 Duration of viral suppression, years 10 ± 5 11 ± 9 10 ± 4 Previous antiretroviral regimens, n 1.9 ± 0.3 1.9 ± 0.3 1.9 ± 0.3 Previous antiretroviral regimens - bictegravir/TAF/FTC 15 5 10 - elvitegravir/c/TAF/FTC 10 2 8 - rilpivirine/TAF/FTC 12 1 11 - rilpivirine/TDF/FTC 5 1 4 - raltegravir/TDF/FTC 5 2 3 - dolutegravir/3TC 4 1 3 - others 16 0 16 Nationality, % - Italian 69 50 72 - Peruvian 14 17 13 - Brazilian 6 8 9 - Others 11 25 6 CD4, cells/mm3 707 ± 285 783 ± 357 690 ± 268 HIV RNA < 50 copies/mL, % 100 100 100 Serum creatinine, mg/dL 0.9 ± 0.2 0.8 ± 0.2 1.0 ± 0.2** Alanine aminotransferase, unit/L 23 ± 16 19 ± 8 24 ± 17 *P < 0.05 and **P < 0.01 versus females. Cossu et al. 1128 D ow nloaded from https://academ ic.oup.com /jac/article/79/5/1126/7635198 by W itw atersrand U niversity user on 06 February 2025 Discussion In this study following up PWH on long-term LAI (up to 320 weeks), we observed a lower inter-individual variability in ca- botegravir plasma trough concentrations and a lower proportion of individuals with cabotegravir and rilpivirine concentrations be- low the 4×PA-IC90 threshold compared to previous cohort studies with TDM data in the early period after LAI initiation.12,13 To the best of our knowledge, this is the first report of real-life data on the assessment of plasma trough concentrations of LAI rilpivirine and cabotegravir with an observational period largely exceeding 200 weeks. Indeed, by analysing 91 blood samples from 46 PWH, in 2022 Thoueille et al. had confirmed the consid- erable inter-individual pharmacokinetic variability previously ob- served in phase II–III trials, with rilpivirine trough concentrations that, in some instances, did not exceed twice the PA-IC90.12 However, in that study, patients were treated with LAI until the maximum of 60 weeks, and most samples were collected during the first 20 weeks: therefore, the TDM were not performed at the steady state. Both drugs are in fact characterized by a very long half-life, namely 13–28 weeks for rilpivirine and 6–12 weeks for cabotegravir, with the steady state expected to be achieved after 65–140 and 30–60 weeks, respectively (estimated as five times the half-life).4,15 More recently, the same authors published an update of the study, presenting results based on 725 TDM ob- tained from 186 PWH treated for up to 196 weeks.13 They con- firmed the significant pharmacokinetic variability of rilpivirine and cabotegravir, with a higher risk of having some drug trough concentrations below 4×PA-IC90 in the first 40–50 weeks com- pared with the second year of treatment. However, most TDM samples were collected in the first year of treatment, not at stea- dy state. Conversely, nearly 90% of our samples were collected after at least 2 years of LAI treatment, with a high probability of reaching the steady state. This may explain the reduced inter- individual variability observed compared with the Swiss study.13 As an additional finding, Thoueille et al. reported significantly lower cabotegravir trough concentrations (−30%) in males com- pared with females.13 This is only partly in agreement with our findings: thus, we observed that male PWH had, respectively, 15% and 10% lower rilpivirine and cabotegravir levels than those measured in female PWH. The different findings might reflect great differences in the population characteristics. Indeed, in the Swiss study only 24% of females were white (52% were black), whereas in our study there were 50% of white females (less than 10% were black). Therefore, the possibility that rilpivir- ine and/or cabotegravir concentrations may be differently af- fected by sex and ethnicity remains ill defined. Another clinical variable potentially associated with the pharmacokinetics of LAI and, possibly, with the response to ther- apy, is the body weight.11 Recently, Van Welzen et al., presented five cases with virologic failure on LAI rilpivirine and cabotegravir collected in different centres in the Netherlands.16 All cases dis- played low drug levels of either cabotegravir, rilpivirine or both during the treatment course that were attributed to technical problems (i.e. needle length not adjusted for increased body weight, erroneous injection during the loading phase, needle length not adjusted in an obese PWH because the body fat was Table 2. Pharmacologic data of rilpivirine and cabotegravir in the 67 PWH on long-term maintenance therapy with LAI antiretrovirals (continuous variables are expressed as mean ± standard deviation; categorical variables are expressed as an absolute number or as a percentage) Variable Data Patients with available TDM data, n 67 Patients treated with a lead-in phase, n 61 Patients starting LAI every 4 weeks, n 49 Treatment duration at 4 weeks, weeks 108 ± 96 Patients starting LAI every 8 weeks, n 18 Treatment duration at 8 weeks, weeks 108 ± 96 Patients given LAI every 8 weeks at TDM, n 67 LAI treatment duration at TDM, weeks 216 ± 80 Rilpivirine trough, ng/mL (range) 177 ± 149 (41–983) Rilpivirine inter-individual CV, % 84 Rilpivirine TDM < Pa-IC90, n (%) 0 Rilpivirine TDM <4×Pa-IC90, n (%) 3 (4.5) Cabotegravir trough, ng/mL (range) 1792 ± 799 (577–5740) Cabotegravir inter-individual CV, % 45 Cabotegravir TDM < Pa-IC90, n (%) 0 Cabotegravir TDM <4×Pa-IC90, n (%) 1 (1.5) PA-IC90, protein-adjusted 90% inhibitory concentrations (rilpivirine: 12 ng/mL, cabotegravir: 166 ng/mL). Figure 1. Time course of rilpivirine (upper panel) and cabotegravir (lower panel) plasma trough concentrations. Dashed lines represented the 4×PA-IC90 (50 ng/mL for rilpivirine and 664 ng/mL for cabotegravir). TDM of LAI 1129 D ow nloaded from https://academ ic.oup.com /jac/article/79/5/1126/7635198 by W itw atersrand U niversity user on 06 February 2025 localized in the abdominal area, prolonged dosing interval) with single injections occurring mainly in PHW with BMI >30 kg/m2. Remarkably, genotypic resistance testing revealed extensive se- lection of NNRTI-associated mutations in all and INI mutations in four cases at the time of virological failure. These cases, to- gether with pooled analyses from Phase III trials, suggest that obesity may represent an important key factor potentially asso- ciated with suboptimal exposure of rilpivirine or cabotegravir and eventually a high risk of virological failure.11,16 We explored the impact of BMI on cabotegravir and rilpivirine concentrations by stratifying data on rilpivirine and cabotegravir plasma trough concentrations according to BMI (<25 versus 25– 30 versus >30 Kg/m2) failing, however, to document statistically significant difference for both drugs in the three groups. Our re- sults agree with new findings from Elliott et al. that, by pooling together data from the FLAIR, ATLAS and ATLAS-2M trials (1245 participants, of whom 17% had a baseline BMI ≥30 kg/m2), reported that pharmacokinetic profiles of both rilpivirine and cabotegravir were comparable across BMI categories.17 The dis- cordant results on the potential association (or lack of) between obesity and the risk of suboptimal exposure to rilpivirine and ca- botegravir can be related to different factors. First, the definition of obesity as BMI >30 kg/m2 is too limited. Indeed, Berton et al. have shown that the pharmacokinetics of different antiretrovirals can also change within individual defined as obese but stratified further according to BMI sub-categories (i.e. 30–35 versus 35–40 versus 40–50 versus 50–60 kg/m2).18 Interestingly, the same authors, by performing physiologically based pharmacokinetic modelling, have recently shown that obesity may have a greater effect on cabotegravir than rilpivirine, with a decrease in cabotegravir AUC >35% for BMI >35 kg/m2 and in rilpivirine AUC >18% for BMI >40 kg/m2 at steady state.19 Second, no stan- dardized procedures are available yet for the selection of the length of the needle according to the distribution of the body fat. For instance, in one of the cases described by Van Welzen et al., the virological failure to LAI has been attributed to the underestimation of the presence of abdominal fat localization in the needle selection.16 Accordingly, it cannot be excluded that centres across the globe may adopt different procedures for LAI administration in obese versus non-obese patients, even- tually contributing to pharmacokinetic variability and discrepant TDM findings. Only three out of the 67 PWH enrolled in the present study had rilpivirine and/or cabotegravir trough concentrations below the target of 4xPA-IC90. None of them experienced virologic failure (overall, 100% of PWH at undetectable viral load both before starting LAI and at the time of TDM). We were, therefore, unable to correlate drug underexposure with clinical efficacy outcome. It must be pointed out, however, that the definition of optimal ex- posure to LAI rilpivirine and cabotegravir is presently ill defined. Indeed, some authors considered the target to be the PA-IC90 or 4×PA-IC90 (as we did), whereas others considered the cut-off to be the first quartile of the pooled trough concentrations from Phase III trials (Q1: 32 ng/mL for rilpivirine and 1120 ng/mL for cabotegravir).12,13,16 The resulting confusion makes it hard to interpret TDM results and generalize the findings. For instance, in the case series published by Van Welzen et al., one patient was considered as having an adequate concentration of rilpivirine with a trough of 38 ng/mL (because it was higher than the Q1) and a sub-therapeutic concentration of cabotegravir with a trough Figure 2. Box-plot of rilpivirine and cabotegravir plasma trough concentrations in female and male people with HIV. Dashed lines represented the 4×PA-IC90 (50 ng/mL for rilpivirine and 664 ng/mL for cabotegravir). Cossu et al. 1130 D ow nloaded from https://academ ic.oup.com /jac/article/79/5/1126/7635198 by W itw atersrand U niversity user on 06 February 2025 of 890 ng/mL (because it was lower than Q1).16 The scenario is exactly the opposite when considering as target 4×PA-IC90 (sub- therapeutic for rilpivirine and therapeutic for cabotegravir). In this regard, the correct interpretation of TDM data of LAI rilpivirine and cabotegravir concentrations may be facilitated by the guid- ance recently provided by Thoueille et al.20 Potential limitations of our study are represented in the retro- spective design and the small sample size that limits generalizabil- ity of findings. Indeed, it cannot be excluded that the reduced inter-individual variability of rilpivirine and cabotegravir concentra- tions observed in our single-centre experience compared with other multicentre studies may be related, at least in part, to the practice accumulated in more than 5 years of LAI injections (most of the patients were enrolled in the phase II–III trials) by the same nursing staff with limited turnover. Furthermore, in our study we did not assess the intra-individual variability of rilpivirine and cabotegravir concentrations. Therefore, the contribution of this additional factor to differences in trough drug concentrations cannot be ruled out. Similarly, we cannot exclude the possibility that differences in the clinical/demographic characteristics of PWH may be associated with inter-individual variability in LAI pharmacokinetics. Indeed, the lack of PWH with BMI >35 kg/m2 did not allow us to investigate in depth the contribution of morbid obesity to rilpivirine and cabotegravir pharmacokinetic variability, and the study was not powered to assess the contribution of sex and/or body weight to LAI exposure given the low number of fe- males (18%) and obese PWH (13%) included. Despite these limitations, we are confident that our study could provide preliminary evidence that, at steady state, cabote- gravir and rilpivirine are associated with optimal systemic expos- ure in most PWH, with cabotegravir trough concentrations being associated with lower inter-individual variability compared to rilpivirine. Funding This study was carried out as part of our routine work. Transparency declarations The authors declare no conflict of interest. Author contributions D.C. and C.G. supervised all the stages of the study and wrote the first draft of the manuscript. S.S. and A.D.A. performed laboratory analyses and revised the draft manuscript. M.V.C., D.M. and A.G. took care of the patients and revised the draft manuscript. S.A., A.G. and G.R. supervised the study, contributed to the interpretation of the data and revised the draft version of the manuscript. Data availability Data are available from the corresponding author upon reasonable request. References 1 Llibre JM, Kuritzkes DAR. Long-acting cabotegravir and rilpivirine: innov- ation, new challenges, and opportunities. Clin Infect Dis 2023; 76: 1655–7. https://doi.org/10.1093/cid/ciad024 2 Qazzaz H, Parganas C, Cory TJ. An evaluation of long-acting cabotegra- vir + rilpivirine for the treatment of virologically suppressed adults living with HIV. Expert Opin Pharmacother 2022; 23: 1485–95. https://doi.org/ 10.1080/14656566.2022.2126310 3 Wu YN, Yu H, Lu L et al. 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Open Forum Infect Dis. 2024; 11: ofae023. doi:10.1093/ofid/ofae023 Cossu et al. 1132 D ow nloaded from https://academ ic.oup.com /jac/article/79/5/1126/7635198 by W itw atersrand U niversity user on 06 February 2025 https://clinicalinfo.hiv.gov/sites/default/files/guidelines/documents/adult-adolescent-arv/whats-new-adult-adolescent-arv.pdf https://clinicalinfo.hiv.gov/sites/default/files/guidelines/documents/adult-adolescent-arv/whats-new-adult-adolescent-arv.pdf https://doi.org/10.1093/cid/ciad020 https://doi.org/10.1016/S2352-3018(21)00184-3 https://doi.org/10.1016/S2352-3018(21)00184-3 https://doi.org/10.1093/ofid/ofab439 https://doi.org/10.1093/ofid/ofab439 https://doi.org/10.1016/S2352-3018(23)00136-4 https://doi.org/10.1016/S2352-3018(23)00136-4 https://doi.org/10.1093/cid/ciad370 https://doi.org/10.3390/pharmaceutics14081588 https://doi.org/10.3390/pharmaceutics14081588 https://doi.org/10.1016/j.lanepe.2023.100793 https://doi.org/10.1016/j.lanepe.2023.100793 https://doi.org/10.1016/j.jpba.2017.02.002 https://doi.org/10.1007/s40262-021-01005-1 https://doi.org/10.1007/s40262-021-01005-1 https://doi.org/10.1093/cid/ciae016 https://doi.org/10.1093/infdis/jiad580 https://doi.org/10.1093/infdis/jiad580 https://doi.org/10.1093/cid/ciad495 https://doi.org/10.1093/cid/ciae060 https://doi.org/10.1093/cid/ciae060 https://doi.org/10.1093/ofid/ofae023 Rilpivirine and cabotegravir trough concentrations in people with HI Introduction Methods Study design and enrolment Assessment of drug plasma trough concentrations Statistical analyses Results Demographic and clinical characteristics Rilpivirine and cabotegravir dosing schemes Distribution of rilpivirine and cabotegravir trough concentrations Discussion Funding Transparency declarations Author contributions Data availability References