Intracellular islatravir pharmacology differs between species in an in vitro model: implications for preclinical study design

Abstract

Background Islatravir (4′-ethynyl-2-fluoro-2′-deoxyadenosine; EFdA) is a first-in-class nucleoside reverse transcriptase translocation inhibitor (NRTTI) being investigated for HIV treatment and prevention. EFdA is intracellularly phosphorylated to EFdA-triphosphate (EFdA-tp), a competitive substrate of deoxyadenosine-triphosphate (dATP). Thus, translating safety and efficacy findings from preclinical studies relies on the assumption that EFdA’s intracellular pharmacology can be extrapolated across species. Objectives We investigated how EFdA is phosphorylated across animal species commonly used for preclinical models in drug development to identify those that most closely matched humans. Methods PBMCs were isolated from whole blood of six species (human, rhesus macaque non-human primate (rmNHP), rat, minipig, dog, and rabbit) using Ficoll separation and counted on a haemocytometer by Trypan blue staining. One million live cells were cultured in media supplemented with 10 U/mL human IL-2, 10% FBS and 1% antibiotics and treated with 0, 17, 170, and 1700 nM EFdA (n = 3 replicates per concentration). After 24 h, representative cell counts were derived from untreated control wells (as above), cells were washed in PBS, and lysed with 70:30 methanol:water. EFdA-tp and dATP concentrations were quantified by HPLC-MS/MS and normalized to the representative live cell counts for each species. Results When compared to human values, EFdA-tp concentrations for each EFdA treatment concentration were lower in all species (rmNHP 1.5–2.1-fold, rat 4.5–15-fold, minipig 37–71-fold, dog and rabbit >100-fold). Additionally, rmNHP and dog PBMCs exhibited significantly higher (7–10-fold; P < 0.001) dATP when compared with human PBMCs. Conclusions Given intracellular pharmacology differences, these preclinical models may be a conservative estimate of EFdA’s intracellular pharmacokinetics and efficacy in humans.

Pharmacokinetic Modeling to Guide Preclinical Development of an Islatravir-Eluting Reservoir-Style Biodegradable Implant for Long-Acting HIV PrEP

“…We observed a significant difference in rat PBMC ISL-tp: plasma ISL ratios compared to that reported in clinical trials of an ISL-eluting implant (~2390) [ 17 ]. However, our observation of lower ISL-tp:ISL ratios in rats relative to humans is consistent with the between species differences reported in Sykes et al [ 24 ]. These findings indicate that allometric scaling of certain preclinical animal models will have limited utility in identifying clinical doses to achieve ISL’s PBMC efficacy target.…”

Pharmacokinetic Study of Islatravir and Etonogestrel Implants in Macaques

“…Compared with human studies, including the nonerodable ISL implant, we observed lower ISL-TP concentrations at equivalent plasma ISL values [ 15 , 16 , 44 , 45 ]. However, we calculated the ISL-TP:ISL ratios in other macaque studies and found a similar trend of lower ISL-TP in macaques compared to humans [ 14 , 36 , 46 , 47 ]. This could be attributed to differential rates of phosphorylation between species or possibly the sample processing method [ 36 ].…”

“…Following an established protocol, 5 million live PBMCs were resuspended in 500 µL ice-cold 80% methanol, vortexed for 1 min and immediately frozen at −70 °C [ 35 ]. ISL-TP and dATP were quantified using previously published LC-MS/MS methods [ 36 ]. The calibrated ranges were 0.05–200 and 0.005–100 ng/mL for ISL-TP and dATP, respectively.…”

Long-acting refillable nanofluidic implant confers protection against SHIV infection in nonhuman primates

“…A plausible explanation is that the nISL implant released drug at lower concentrations compared with the high C max achieved with once-monthly oral pill dosing in clinical trials. Although ISL metabolism differs between species ( 37 ), our findings motivate further investigation. Furthermore, the implant was well tolerated, and only a few mild adverse events were observed, such as implant site induration.…”