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

J Antimicrob Chemother. 2022 Mar 31;77(4):1000-1004. doi: 10.1093/jac/dkac015.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Anti-HIV Agents / pharmacology
  • Deoxyadenosines* / pharmacology
  • Dogs
  • HIV Infections / drug therapy
  • Macaca mulatta
  • Models, Biological*
  • Rabbits
  • Rats
  • Research Design
  • Reverse Transcriptase Inhibitors* / pharmacology
  • Species Specificity
  • Swine
  • Swine, Miniature
  • Tandem Mass Spectrometry


  • Anti-HIV Agents
  • Deoxyadenosines
  • Reverse Transcriptase Inhibitors
  • islatravir