Category: Preclinical Development
Purpose: The purpose of this project is to characterize the in vitro atypical kinetics and in vivo pharmacokinetics of diazepam with its metabolites by applying in vitro to in vivo correlation.
Methods: In this study, atypical kinetics of diazepam metabolites (Temazepam and Nordiazepam) formation were characterized using an in vitro enzyme assay with pooled (male Sprague Dawley) rat liver microsome (RLM), pooled (female Sprague Dawley) RLM, and purified rat cytochrome p450 (CYP450) isoforms. Rat microsome and rat plasma protein binding assays were used to determine the free fraction of the drug in the corresponding matrixes. In vitro kinetic data was modeled by Mathematica software using a numerical method. Female and male rats PK studies are being conducted to describe the atypical kinetics of diazepam in vivo.
Results: Preliminary in vitro data from pooled rat (male Sprague Dawley) liver microsome and purified CYP3A1 showed obvious sigmoidal saturation curves for Temazepam formation from Diazepam. No obvious sigmoidicity of diazepam titration curves was observed for CYP3A2 (male-specific) and CYP2C11 (male-specific) isoforms, which are other CYP450 isoforms responsible for diazepam metabolism in male rats.
Conclusion: Initial experiments show that diazepam displays atypical kinetics in vitro. The presence of sigmoidicity of Temazepam formation titration curve for the CYP3A1 isoform suggests nonlinear PK is more likely seen from female rats. In order to characterize in vitro-in vivo correlations for diazepam in rats, in vitro data with female rat liver microsomes and in vivo PK data for female and male rats are being generated and evaluated with appropriate PK models.