Mathematical Modeling of In Vitro‐In Vivo Atypical Kinetics of Diazepam

Abstract

Cytochrome P450 (CYP) and uridine 5'‐diphospho‐glucuronosyltransferase (UGT) enzymes are responsible for metabolism of most marketed drugs. Due to the large binding sites of certain CYPs and UGTs, CYP and UGT substrates tend to show non ‐ Michaelis‐Menten kinetics (atypical kinetics). Although several in vitro data provide evidence for the existence of atypical kinetics, this characteristic is seldom observed with in vivo data. The purpose of this project is to characterize the atypical kinetics of diazepam and its sequential metabolism in in vitro and in vivo systems. Numerical methods will be used to obtain kinetic parameters, which will be used for in vitro‐in vivo correlations (IVIVC).Diazepam, which exhibits atypical kinetics in vitro, and its primary metabolites (temazepam, nordiazepam), and secondary metabolite (oxazepam) were characterized with in vitro studies with pooled male Sprague Dawley rat liver microsome (MRLM), pooled female Sprague Dawley (FRLM), and purified rat cyp isoforms (cyp3a1, cyp3a2, and cyp2c11) separately. Rat microsomal partition and rat plasma protein binding assays were conducted via equilibrium dialysis to determine the free fraction of the drug. Two substrate binding (ESS) models provided better fits than one substrate (ES) models, as determined from the resulting AICc values. Preliminary in vitro data from MRLM and cyp3a1 showed obvious sigmoidal saturation curves for temazepam formation. Only cyp3a1 show significant sigmoidal curves for nordiazepam formation. For cyp3a1, Km and Vmax values for temazepam and nordiazepam calculated with the EES model are significantly different from those with the ES model. The intrinsic clearance (CLint) values calculated from the MRLM and cyp3a1 ESS models is not constant within the observed range of diazepam plasma levels of rats. No obvious sigmoidicity of diazepam saturation curves was observed for cyp3a2 (male‐specific) and cyp2c11 (male‐specific) isoforms, which are other cyp isoforms responsible for diazepam metabolism in male rats. In order to characterize in vitro‐in vivo correlations for diazepam, in vivo PK studies with female and male rats are currently underway.Support or Funding InformationThis research was supported by NIGMS grants: R01GM114369 and R01GM104178.