Challenges in the Method Development and Sample Analysis for the Determination of Nucleoside Phosphates in Peripheral Blood Mononuclear Cells (PBMCs) and Tissue Samples in Support of Molnupiravir Program

Molnupiravir (MOV, MK-4482) is a nucleoside analog prodrug that has been authorized for emergency use for the treatment of mild to moderate coronavirus disease (COVID-19). MOV is rapidly converted to N-hydroxy cytidine (NHC) that is further metabolized to the bioactive moiety, NHC-triphosphate (NHC-TP). To understand the pharmacokinetic characterization of MOV, it is important to determine the correlation between plasma NHC levels and NHC-TP levels in PBMCs. To enable this, LC-MS/MS assays for nucleoside phosphates in human PBMCs were developed and qualified. These methods became the basis for the development of assays in pre-clinical (rat, dog) PBMCs, and in tissues (mouse lung, rat testes) to support pre-clinical studies with MOV. The considerations, strategies, and bioanalytical challenges in method development and sample analysis for nucleoside phosphates in atypical biological matrices will be provided in this presentation.