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Introduction: Pregnancy increases the clearance of CYP3A4 substrate drugs and pregnancy-related hormones (PRHs) induce hepatic CYP3A4 expression and metabolism. However, it remains unclear to what extent the magnitude of PRH-evoked changes in hepatic CYP3A metabolism varies across multiple substrates. This study quantified the impact of PRHs on CYP3A protein concentrations and buprenorphine metabolism in human hepatocytes, and compared the magnitude of these effects to nifedipine and midazolam metabolism. Methods: Sandwich-cultured human hepatocytes (SCHH) from female donors were exposed to PRHs, administered in combination across a range of physiologically relevant concentrations, for 72 h. Absolute protein concentrations of CYP3A4, CYP3A5, and CYP3A7 in SCHH membrane fractions were quantified by nanoLC-MS/MS, and norbuprenorphine (nor-BUP), dehydro-nifedipine (dehydro-NIF), and 1-hydroxy-midazolam (1-OH-MDZ) formation was evaluated. Results: Compared to control, PRH exposure increased CYP3A4, CYP3A7, and total CYP3A protein concentrations, but not CYP3A5 concentrations, and increased nor-BUP, dehydro-NIF, and 1-OH-MDZ formation in a concentration-dependent manner. The formation of nor-BUP, dehydro-NIF, and 1-OH-MDZ each positively correlated with PRH-mediated changes in total CYP3A protein concentrations. The PRH-evoked increase in nor-BUP formation was evident in all donors; however, the PRH induction of dehydro-NIF and 1-OH-MDZ formation was diminished in a hepatocyte donor with high basal CYP3A5 expression. Discussion: These findings demonstrate that PRHs increase buprenorphine, nifedipine, and midazolam metabolism in SCHH via induction of CYP3A4 and total CYP3A protein concentrations, and the magnitude of these effects vary across hepatocyte donors in a substrate-specific manner. These data provide insight into the contribution of PRH induction of CYP3A4 metabolism to increased buprenorphine clearance during pregnancy.

Pregnancy-related hormones (PRH) are recognized as important regulators of hepatic cytochrome P450 enzyme expression and function. However, the impact of PRH on the hepatic expression and function of uridine diphosphate glucuronosyltransferases (UGTs) remains unclear. Using primary human hepatocytes, we evaluated the effect of PRH exposure on mRNA levels and protein concentrations of UGT1A1, UGT2B7, and other key UGT enzymes, and on the metabolism of labetalol (a UGT1A1 and UGT2B7 substrate commonly prescribed to treat hypertensive disorders of pregnancy). Sandwich-cultured human hepatocytes (SCHH) from female donors were exposed to the PRH estradiol, estriol, estetrol, progesterone, and cortisol individually or in combination. We quantified protein concentrations of UGT1A1, UGT2B7, and four additional UGT1A isoforms in SCHH membrane fractions and evaluated the metabolism of labetalol to its glucuronide metabolites in SCHH. PRH exposure increased mRNA levels and protein concentrations of UGT1A1 and UGT1A4 in SCHH. PRH exposure also significantly increased labetalol metabolism to its UGT1A1-derived glucuronide metabolite in a concentration-dependent manner, which positively correlated with PRH-induced changes in UGT1A1 protein concentrations. In contrast, PRH did not alter UGT2B7 mRNA levels or protein concentrations in SCHH, and formation of the UGT2B7-derived labetalol glucuronide metabolite was decreased following PRH exposure. Our findings demonstrate that PRH alter expression and function of UGT proteins in an isoform-specific manner and increase UGT1A1-mediated labetalol metabolism in human hepatocytes by inducing UGT1A1 protein concentrations. These results provide mechanistic insight into the increases in labetalol clearance observed in pregnant individuals.

Intravaginal rings (IVRs) represent a well-established, woman-controlled and sustained vaginal drug delivery system suitable for a wide range of applications. Here, we sought to investigate the differences in etonogestrel (ENG) and ethinyl estradiol (EE) release from a 3D-printed IVR utilizing continuous liquid interface production (CLIP™) (referred to as CLIPLOW for low drug loading and CLIPHIGH IVRs for high drug loading) and NuvaRing, a commercially available injection molded IVR. We conducted in vitro release studies in simulated vaginal fluid to compare the release of ENG and EE from CLIPLOW IVRs and NuvaRing. CLIPLOW IVRs had a similar hormone dose to NuvaRing and exhibited slightly slower ENG release and greater EE release in vitro compared to NuvaRing. When administered to female sheep, NuvaRing demonstrated greater ENG/EE levels in plasma, vaginal tissue and vaginal fluids compared to CLIPLOW IVR despite similar drug loadings. Leveraging observed hormones levels in sheep from NuvaRing as an effective contraceptive benchmark, we developed a long-acting CLIPHIGH IVR with increased ENG and EE doses that demonstrated systemic and local hormone levels greater than the NuvaRing for 90 days in sheep. No signs of toxicity were noted regarding general health, colposcopy, or histological analysis in sheep after CLIPHIGH IVR administration. Our results provided (1) a comparison of ENG and EE release between a 3D-printed IVR and NuvaRing in vitro and in vivo, (2) a preclinical pharmacokinetic benchmark for vaginally delivered ENG and EE and (3) the generation of a 90-day CLIP IVR that will be utilized in future work to support the development of a long-acting ENG/EE IVR combined with an antiretroviral for the prevention of HIV and unplanned pregnancy.

Background/Objectives: Women and girls, particularly in sub-Saharan Africa, face high risks for both HIV and unintended pregnancy. Inconsistent condom use underscores the need for new multipurpose prevention technologies (MPTs) that combine HIV pre-exposure prophylaxis (PrEP) and contraception. Long-acting (LA) injectables are especially promising. To this end, an LA cabotegravir (CAB)/medroxyprogesterone acetate (MPA) in situ-forming implant (ISFI) has been developed. We report pharmacokinetic (PK) modeling to characterize CAB and MPA disposition and absorption to support the development of the MPT ISFI. Methods: Female BALB/c mice received single intravenous (IV) or subcutaneous (SQ) bolus doses of CAB or MPA. Sparse plasma samples were collected (~3 mice/timepoint) for PK analysis by LC-MS/MS. Noncompartmental analysis assessed SQ bioavailability. Macroparameterized compartmental PK models were fit to IV data to derive unit impulse responses (UIRs) for each drug. Results: CAB and MPA exhibited 61% and 42% bioavailability, respectively. CAB IV PK was best described by a two-compartment model with macroconstant parameters: A = 16,621 ng/mL, α = 4.52 h−1, B = 30,206 ng/mL, and β = 0.053 h−1. MPA IV PK was also best described by a two-compartment model, with A = 2506 ng/mL, α = 10.5 h−1, B = 439 ng/mL, and β = 0.65 h−1. These values define the UIR for CAB and MPA. Conclusions: Our IV PK modeling framework fully characterizes CAB/MPA disposition in mouse, enabling downstream deconvolution-based estimation of absorption from controlled-release formulations. This provides a foundation for in vitro–in vivo correlation, facilitating preclinical evaluation of long-acting formulations such as ISFIs.

HIV continues to affect millions of men and women worldwide. The development of long-acting injectables for HIV prevention can overcome adherence challenges with daily oral prevention regimens by reducing dosing frequency and stigma. We previously developed an ultra-long-acting injectable, biodegradable, and removeable in situ forming implant (ISFI) with cabotegravir (CAB) that demonstrated protection after multiple rectal SHIV challenges in female macaques. Here, we sought to further characterize CAB ISFI pharmacokinetics (PK) in mice by assessing the effect of dose and number of injections on CAB PK, time to completion of CAB release and polymer degradation, long-term genital tissue PK, and CAB PK tail after implant removal. CAB concentrations in plasma were above the benchmark for protection for 11–12 months with proportionality between dose and drug exposure. CAB ISFI exhibited high concentrations in vaginal, cervical, and rectal tissues for up to 180 days. Furthermore, depots were easily retrievable up to 180 days post-administration with up to 34% residual CAB and near complete (85%) polymer degradation quantified in depots ex vivo. After depot removal, results demonstrated a median 11-fold decline in CAB plasma concentrations across all doses. Ultimately, this study provided critical PK information for the CAB ISFI formulation that could aid in its future translation to clinical studies.

The prevention of HIV and unintended pregnancies is a public health priority. Multi-purpose prevention technologies capable of long-acting HIV and pregnancy prevention are desirable for women. Here, we utilized a preclinical macaque model to evaluate the pharmacokinetics of biodegradable ε-polycaprolactone implants delivering the antiretroviral islatravir (ISL) and the contraceptive etonogestrel (ENG). Three implants were tested: ISL-62 mg, ISL-98 mg, and ENG-33 mg. Animals received one or two ISL-eluting implants, with doses of 42, 66, or 108 µg of ISL/day with or without an additional ENG-33 mg implant (31 µg/day). Drug release increased linearly with dose with median [range] plasma ISL levels of 1.3 [1.0–2.5], 1.9 [1.2–6.3] and 2.8 [2.3–11.6], respectively. The ISL-62 and 98 mg implants demonstrated stable drug release over three months with ISL-triphosphate (ISL-TP) concentr54ations in PBMCs above levels predicted to be efficacious for PrEP. Similarly, ENG implants demonstrated sustained drug release with median [range] plasma ENG levels of 495 [229–1110] pg/mL, which suppressed progesterone within two weeks and showed no evidence of altering ISL pharmacokinetics. Two of the six ISL-98 mg implants broke during the study and induced implant-site reactions, whereas no reactions were observed with intact implants. We show that ISL and ENG biodegradable implants are safe and yield sufficient drug levels to achieve prevention targets. The evaluation of optimized implants with increased mechanical robustness is underway for improved durability and vaginal efficacy in a SHIV challenge model.

Feminizing hormone therapy (FHT) may interact with human immunodeficiency virus preexposure prophylaxis (PrEP). We found that transgender women who took FHT exhibited a 7-fold lower rectal tissue ratio of PrEP’s active metabolites vs competing deoxynucleotides compared to cisgender women and men (P = .03) that inversely correlated with estradiol (ρ = −0.79; P < .05). Thus, FHT may negatively impact PrEP efficacy.

Sparse data exist on the penetration of antiretrovirals into brain tissue. In this work, we present a framework to use efavirenz (EFV) pharmacokinetic (PK) data in plasma, cerebrospinal fluid (CSF), and brain tissue of eight rhesus macaques to predict brain tissue concentrations in HIV‐infected individuals. We then perform exposure‐response analysis with the model‐predicted EFV area under the concentration‐time curve (AUC) and neurocognitive scores collected from a group of 24 HIV‐infected participants. Adult rhesus macaques were dosed daily with 200 mg EFV (as part of a four‐drug regimen) for 10 days. Plasma was collected at 8 time points over 10 days and at necropsy, whereas CSF and brain tissue were collected at necropsy. In the clinical study, data were obtained from one paired plasma and CSF sample of participants prescribed EFV, and neuropsychological test evaluations were administered across 15 domains. PK modeling was performed using ADAPT version 5.0 Biomedical Simulation Resource, Los Angeles, CA) with the iterative two‐stage estimation method. An eight‐compartment model best described EFV distribution across the plasma, CSF, and brain tissue of rhesus macaques and humans. Model‐predicted median brain tissue concentrations in humans were 31 and 8,000 ng/mL, respectively. Model‐predicted brain tissue AUC was highly correlated with plasma AUC (γ = 0.99, P < 0.001) but not CSF AUC (γ = 0.34, P = 0.1) and did not show any relationship with neurocognitive scores (γ < 0.05, P > 0.05). This analysis provides an approach to estimate PK the brain tissue in order to perform PK/pharmacodynamic analyses at the target site.

Measurement of drug concentrations in hair provides a non-invasive approach to assess drug adherence. Here, we report on the development and validation of a method for the quantification of the antiretroviral dolutegravir (DTG) extracted from human hair. DTG is extracted from hair samples by sonication and incubation in 50:50 methanol:acetonitrile with 2% formic acid overnight at 40 °C. Following extraction, samples are analyzed by reverse-phase chromatography on a Waters Atlantis T3 (50 × 2.1 mm, 3-μm particle size) column with subsequent detection by electrospray ionization in positive ion mode on an AB Sciex API-5000 triple quadrupole mass spectrometer. The stable, isotopically labeled 13C,d5-DTG is used as an internal standard in the assay. The calibration range is 5–10,000 pg DTG/mL of extraction solvent with the ability to extract between 1 and 10 mg of hair/mL of extraction solvent. The assay was linear, accurate (inter-assay %bias within ± 6.5%), and precise (inter-assay %CV ≤ 10.3%). The assay was successfully used to analyze clinical samples from subjects on DTG regimens. Analysis of clinical samples suggested the potential presence of a degradation product, which was subsequently confirmed to occur with exposure to sunlight. The degradation of DTG could complicate absolute interpretation of clinical results, but the presence of this degradation product is easily evaluated with this assay to aid in data interpretation.

Bictegravir (BIC), an integrase inhibitor, and doravirine (DOR), a non-nucleoside reverse transcriptase inhibitor, were recently approved by the US FDA for HIV treatment and are recommended first line treatment options. Because certain clinical scenarios warrant using them in combination, we developed a fully validated LC-MS/MS method for simultaneous measurement of BIC and DOR, along with a legacy integrase inhibitor, raltegravir (RAL), in human plasma over a clinically relevant 1000-fold range for each analyte. These analytes were extracted from the plasma by protein precipitation with their stable, isotopically labeled internal standards (BIC-d5, 13C6-DOR, and RAL-d6). Following extraction, samples were analyzed by reverse phase chromatography on a Waters Atlantis T3 C18 (50×2.1mm, 3um particle size) column with subsequent detection by electrospray ionization in positive ion mode on an AB Sciex API-5000 triple quadrupole mass spectrometer. The assay was linear (R2>0.994) over the selected calibration ranges (20.0-20,000ng/mL (BIC), 3.00-3,000ng/mL (DOR), and 10.0-10,000 (RAL)). The assay was accurate (inter-assay %Bias ≤±8.5) and precise (inter-assay %CV ≤11.4). This method was validated according to FDA guidance for industry and can be used to assess the pharmacokinetics of two newly approved antiretrovirals, or to support therapeutic drug monitoring for modern antiretroviral therapy.