Application of pharmacokinetic modelling approaches in antiviral interventions against SARS-COV-2

Abstract

This research aimed to develop and validate physiologically based pharmacokinetic (PBPK) models for (nirmatrelvir (NTV) / ritonavir (RTV)) and molnupiravir (MPV) along with its active metabolite N-hydroxycytidine (NHC). To support this objective, a series of in vitro and ex-vivo experiments were conducted encompassing bioanalytical method development, permeability profiling, metabolic stability assessment, and pharmacokinetic (PK) analysis. Highly sensitive and robust LCMS methods were developed and validated for the simultaneous quantification of NTV/RTV and MPV/NHC in transporter buffer, human plasma, and human liver microsomes. These assays exhibited excellent linearity (R² > 0.99), low limits of quantification (7.81 ng/mL), and recovery rates exceeding 95%. Permeability studies revealed that NTV undergoes significant efflux by P-glycoprotein (P-gp), with a high efflux ratio (10.09), while RTV co-administration moderately inhibited efflux, enhancing NTV absorption. MPV demonstrated a strong absorptive preference and minimal efflux activity, consistent with passive diffusion. Metabolic investigations confirmed that MPV is primarily metabolized by non-CYP-mediated hydrolysis, with rapid plasma conversion to NHC. NHC exhibited negligible microsomal clearance, highlighting a metabolism pathway dominated by intracellular phosphorylation. Blood-to-plasma distribution studies indicated dynamic changes over time, with MPV accumulating in cells and NHC demonstrating time-dependent distribution, reinforcing their suitability for PBPK modelling. Compartmental PK modelling of clinical data demonstrated that RTV significantly enhanced NTV exposure by reducing clearance and extending half-life, supporting its role as a pharmacokinetic booster. While NHC following administration of MPV showed dose-dependent tissue distribution, consistent with intracellular retention. The developed PBPK models successfully captured the absorption, distribution, metabolism, and elimination (ADME) of NTV, and MPV. For NTV, the model reproduced exposure trends across doses, with scope for refinement in representing RTV’s inhibition of P-gp. For MPV and NHC, model performance highlighted discrepancies in absorption and clearance due to differences in esterase-mediated metabolism, and tissue partitioning. Integration of intracellular retention mechanisms and CES1 metabolism is expected to improve prediction of NHC elimination