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Numerical analysis of time-dependent inhibition kinetics: comparison between rat liver microsomes and rat hepatocyte data for mechanistic model fitting.

ABSTRACT: Time-dependent inhibition (TDI) may confound drug interaction predictions. Recently, models were generated for an array of TDI kinetic schemes using numerical analysis of microsomal assays. Additionally, a distinct terminal inactivation step was identified for certain mechanism based inhibitors (MBI) following reversible metabolite intermediate complex (MIC) formation. Longer hepatocyte incubations potentially allow analysis of slow TDI and terminal inactivation. In the experiments presented here, we compared the quality of TDI parameterization by numerical analysis between hepatocyte and microsomal data. Rat liver microsomes (RLM), suspended rat hepatocytes (SRH) and sandwich-cultured rat hepatocytes (SCRH) were incubated with the prototypical CYP3A MBI troleandomycin and the substrate midazolam. Data from RLM provided a better model fit as compared to SRH. Increased CYP3A expression after dexamethasone (DEX) induction improved the fit for RLM and SRH. A novel sequential kinetic scheme, defining inhibitor metabolite production prior to MIC formation, improved the fit compared to direct MIC formation. Furthermore, terminal inactivation rate constants were parameterized for RLM and SRH samples with DEX-induced CYP3A. The low expression of CYP3A and experimental error in SCRH resulted in poor data for model fitting. Overall, RLM generated data better suited for elucidation of TDI mechanisms by numerical analysis.

SUBMITTER: Pham C

PROVIDER: S-EPMC5742070 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Developmental exposure to multiple ortho-substituted polychlorinated biphenyls (PCBs) causes adverse neurodevelopmental outcomes in laboratory animals and humans by mechanisms involving the sensitization of Ryanodine receptors (RyRs). In the case of PCB 136, the sensitization of RyR is enantiospecific, with only (-)-PCB 136 being active. However, the role of enantioselective metabolism in the developmental neurotoxicity of PCB 136 is poorly understood. The present study employed hepatic microsomes from phenobarbital (PB)-, dexamethasone (DEX)- and corn oil (VEH)-treated male Sprague-Dawley rats to investigate the hypothesis that PCB 136 atropisomers are enantioselectively metabolized by P450 enzymes to potentially neurotoxic, hydroxylated PCB 136 metabolites. The results demonstrated the time- and isoform-dependent formation of three metabolites, with 5-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-5-ol) being the major metabolite. The formation of 5-OH-PCB 136 increased with the activity of P450 2B enzymes in the microsomal preparation, which is consistent with PCB 136 metabolism by rat P450 2B1. The minor metabolite 4-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-4-ol) was produced by a currently unidentified P450 enzyme. An enantiomeric enrichment of (-)-PCB 136 was observed in microsomal incubations due to the preferential metabolism of (+)-PCB 136 to the corresponding 5-OH-PCB 136 atropisomer. 4-OH-PCB 136 displayed an enrichment of the atropisomer formed from (-)-PCB 136; however, the enrichment of this metabolite atropisomer did not affect the enantiomeric enrichment of the parent PCB because 4-OH-PCB 136 is only a minor metabolite. Although the formation of 5- and 4-OH-PCB 136 atropisomers increased with time, the enantioselective formation of the OH-PCB metabolites resulted in constant enantiomeric enrichment, especially at later incubation times. These observations not only demonstrate that the chiral signatures of PCBs and their metabolites in wildlife and humans are due to metabolism by P450 enzymes but also suggest that the enantioselective formation of neurotoxic PCB 136 metabolites, such as 4-OH-PCB 136, may play a role in the developmental neurotoxicity of PCBs.

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Numerical analysis of time-dependent inhibition kinetics: comparison between rat liver microsomes and rat hepatocyte data for mechanistic model fitting.

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