In vitro characterization and physiologically based pharmacokinetic modelling for molnupiravir and its active metabolite

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In vitro characterization and physiologically based pharmacokinetic modelling for molnupiravir and its active metabolite

Mary Aladwani, Usman Arshad, Helen Cox, Paul Curley, Anthony Valentijn, Lee Tatham, Rajith Rajoli, Henry Pertinez and Andrew Owen

University of Liverpool

Introduction: Molnupiravir (MOL) is recommended by the WHO for treatment of COVID-19 patients with mild to moderate symptoms but may also have application as a broad spectrum antiviral. MOL is converted to its active metabolite NHC after absorption, which induces error catastrophe, stopping viral replication. This work aimed to develop and validate a physiologically based pharmacokinetic (PBPK) model for better prediction of MOL drug disposition, requiring assessments of apparent permeability (Papp), intrinsic clearance (Clint) and blood/plasma ratio for MOL and NHC. Validation of an LC-MS/MS method for quantifying MOL and NHC in various matrices was also undertaken.

Methods: An LC-MS/MS method was developed and validated for simultaneous quantification of MOL and NHC in transporter buffer, human plasma and human liver microsomes. Papp of MOL in Caco-2 cell monolayers was determined at 50 μM. Papp determination for NHC was unnecessary since it is formed subsequent to absorption. Clint of MOL and NHC were investigated in human liver microsomes. Blood-to-plasma ratio of NHC was determined via standard methodology. Clinical data from a published study (Khoo et al., 2021) were utilized to develop and validate a PBPK model, with simulations carried out in Teoreler (www.liverpool.ac.uk/centre-of-excellence-for-long-acting-therapeutics/teoreler), a web-based pharmacokinetic platform. A dose of 800 mg was simulated, utilizing the in vitro generated parameters and apparent clearance from clinical PK studies.

Results: The LC-MS/MS method had a runtime of 3.5 min and an analytical measuring range of 7.81–2000 ng/mL (linear regression 1/X, R² = .99) for MOL in all matrices and NHC in human plasma, and human liver microsomes. For MOL and NHC the LOD and LOQ were 1.95 ng/mL and 7.81 ng/mL. Intra- and inter-day precision and accuracy metrics were within acceptable limits and recovery rates of MOL and NHC from matrices were consistently above 70%.

MOL demonstrated a Papp of 1.46 × 10⁻⁶ cm/s. In human microsomes MOL exhibits an elimination, potentially attributable to hepatic carboxylase enzymes, whereas NHC exhibited no intrinsic clearance with a Κel values of 0. Both MOL and NHC showed a blood-to-plasma ratio of ~1.

PBPK model simulations for NHC in plasma incorporating these in vitro parameter values, combined with the necessary observed apparent clearance from an empirical fitting to the clinical data, and Kps predicted using Poulin and Theil method showed acceptable agreement with observed clinical PK exposure, with an AFE of 1.02 ± 0.24. Mean simulated vs. observed PK parameters were within an acceptable twofold range.

Conclusion: A highly sensitive, precise and accurate LC MS/MS method for the quantification of molnupiravir and NHC in diverse matrices is presented. The PBPK model using the generated in vitro data for MOL and NHC showed good performance and can be applied for forward simulation to meet the needs of future clinical use cases.