IN VITRO AND IN VIVO KINETIC MODELING OF DIAZEPAM METABOLISM

Abstract

Drug metabolism plays an important role in drug absorption and drug elimination. Therefore, it is crucial to understand the mechanism and kinetics of drug metabolism by various drug-metabolizing enzymes (DMEs). Cytochrome P450 enzymes (CYPs) are responsible for the metabolism of more than 60% of the top 200 prescribed drugs. X-ray and NMR data of CYP enzyme suggest that relatively large and flexible active sites are capable of multi-substrate binding. Due to the multiple substrate-binding, CYP reactions tend to show non-Michaelis Menten kinetics (atypical kinetics), multiple metabolite formation and sequential metabolism.To investigate the complexity of cytochrome P450 kinetics, saturation curves and intrinsic clearances (CLint) were simulated for single substrate and multi-substrate models using rate equations and numerical analysis. These models were combined with multiple product formation and sequential metabolism and simulations were performed with random error. All simulation and model fitting was performed using Mathematica. A concentration-dependent metabolite ratio plot can be observed from multi-substrate binding kinetics. Use of single substrate models to characterize multi-substrate data can result in inaccurate kinetic parameters and poor clearance predictions. It has been shown that use of different substrate concentrations may lead to highly variable in vitro CLint estimations when sigmoidal kinetics are observed. Comparing results for use of standard velocity equations with ordinary differential equations (ODEs) clearly shows that ODEs are more versatile and provide better parameter estimates. It would be difficult to derive concentration-velocity relationships for complex models, but these relationships can be easily modeled using numerical methods and ODEs. The model drug diazepam (DZP) was chosen as the probe substrate to demonstrate complex CYP kinetics with specific CYP enzyme sources, including rat liver microsome (RLM), human liver microsome (HLM), purified CYP enzyme isoforms and rat hepatocytes. All saturation curves display non-Michaelis-Menten kinetics, form multiple primary metabolites, and are sequentially metabolized to secondary metabolites. In addition, the sequential metabolism and disposition would be characterized in hepatocytes incubation under flow conditions. To provide in vivo evidence of the atypical kinetics and investigate CYP-mediated sequential metabolism, preliminary intravascular (IV) dosing PK studies with male rats was performed for DZP. In general, DZP and its metabolites were quantitated by LC/MS/MS. Numerical methods were used to solve ODEs and parameterize micro and macro rate constants for the models. It has been shown that more complex models that include explicit enzyme-product complexes can well characterize the datasets for diazepam sequential metabolism with CYP3A4. Uncommon DZP metabolite PK profiles are observed in rat PK studies. In summary, methods of in vitro data analysis are compared, new assays are developed, and new modeling approaches for complex drug and metabolite pharmacokinetics are being investigated.