Establishment of in vitro methods for evaluation of induction and inhibition of human CYP450 enzymes by drugs / 药学学报

ABSTRACT

The aim of this study is to establish the in vitro methods for the study of induction and inhibition on CYP450 by drugs, and to validate the analytical method and incubation system. A method for the simultaneous determination of eight metabolites of seven subtypes of CYP450 enzymes probe substrates in human liver microsomes (HLM) was established and validated. The incubation system was optimized to confirm the incubation time and protein concentration of HLM, the enzyme activity of seven subtypes of CYP450 enzymes in HLM was determined, and the inhibition effects on each CYP450s were checked by positive controls. The method for the simultaneous determination of three metabolites of subtypes of CYP450 enzymes was established and validated in human primary cultured hepatocytes (HPCH) using the incubation medium. The enzyme activity of three subtypes of CYP450 enzymes in HPCH was determined, and the total RNA was extracted from HPCH after incubation. The expression of CYP450 enzymes were measured by Taqman fluorescence probe method. The induction effects on each CYP450s were examined using the positive controls. The established methods for the determination of metabolites of probe substrates were fully validated, and the results were conformed to the requirements of bioanalytical method validation. The induction and inhibition effects on each CYP450s were checked by positive controls. The established in vitro methods for the study of drug induction and inhibition on CYP450 were simple and reliable, which could be used in the investigation of enzyme induction or inhibition properties of new drug candidates and to evaluation the metabolic interactions of concomitant medication in clinical.