(W0930-10-69) Islatravir Does Not Partition to Red Blood Cells: An In Vitro Study to Support a Total Drug Assay in Dried Blood

Purpose: Islatravir (ISL) is a nucleoside reverse transcriptase translocation inhibitor (NRTTI) targeted for HIV treatment. Intracellular triphosphate (ISL-TP) is the active moiety with observed half-life of ~ 105-265 hrs. To support the development of ISL, a “total” assay was developed for dried blood matrix to provide data on ISL and anabolites (mono-, di-, and triphosphate) exposures in clinical studies. The partitioning of ISL and its anabolites into whole blood cell fractions, namely red blood cells (RBCs) , peripheral blood mononuclear cells (PBMCs), and plasma was evaluated in ‘in vitro’ study to inform on the feasibility of this method. Uptake into RBCs , as an abundant compartment of whole blood, would allow for higher limits of quantitation; conversely if no uptake is observed in RBCs, the limit of quantitation would be lower. Uptake in RBCs would also complicate data analysis. Using the total ISL concentration as a surrogate for PBMC anabolite concentrations allows pharmacokinetic measurements in later stage studies where PBMC isolation is very challenging.

Methods: Islatravir was spiked into fresh sodium heparin whole blood from three donors and incubated overnight (20 hours) at 37°C in a 5% carbon dioxide atmosphere. Aliquots of whole blood were taken for quantitation of ISL, total ISL, and the ISL inosine metabolite (4´-ethynyl-2-fluoro-2’-deoxyinosine). Cellular fractions (RBCs and PBMCs) were isolated and counted for determination of ISL anabolites. ISL was isolated from blood by salt assisted liquid-liquid extraction with acetonitrile and separated on a Waters HSS T3 column (50x2.1mm, 2.5 mm) using acetonitrile and water with ammonium fluoride as mobile phase. ISL was detected in the positive polarity on a triple quadrupole mass spectrometer. Blood samples for the total assay (ISL plus anabolites) were generated by treating cell extracts with acid phosphatase to convert all anabolites to ISL. Anabolite samples in cellular isolates were prepared by precipitation with methanol and subsequent separation by anion exchange chromatography on a Thermo Biobasic AX (150x2.1mm, 5mm) column. Mobile phase for this separation consisted of 30/70 acetonitrile/water with 5mM ammonium acetate, 0.5% DMSO and a pH gradient to 1.5% ammonium hydroxide, and detection was in the negative polarity. Limits of quantitation for all assays was 0.5 ng/mL.

Results: Overall recovery of ISL from whole blood incubations was 70-79%, consisting of ISL, anabolites, and inosine metabolite. The inosine metabolite accounted for approximately 25% of recovered drug and was attributed to the presence of adenosine deaminase in RBC, and total ISL accounted for the remainder. No exposure of ISL or its di- or triphosphate anabolites was detected in resuspended RBCs. Concentrations of ISL-MP were detected near the limit of quantitation for the assay with significant variability in resuspended RBCs. Turnover of ISL in PBMCs was observed, where the triphosphate was the predominant species with concentrations ranging from 225-515 ng/mL. The relative ratio of ISL-TP to ISL-DP is consistent with existing clinical data.

Conclusion: Based on this in vitro data, ISL is not expected to partition into RBCs. Dried blood microsampling assays need be developed with appropriately low limits of quantitation to detect terminal phase total ISL where PBMC anabolites are the primary driver of exposure in the “total” assay.