Metabolic Characteristics Of Carvedilol Enantiomers In The Cytochrome P450 Of Human Hepatocytes Treated With Different Inducers

Chiral drugs extensively exist in the all kinds of drugs. According to the previous studies, natural and semisynthetic drugs are almost chiral drugs and many synthetic drugs, at least 40 percent, are also chiral drugs. However, 90 percent of them are used as raceme in clinical practice. In fact, the enantiomers of chiral drug have obvious stereoselectivity in vivo, which therefore make their notable different properties in the processes of absorption, distribution, metabolism and elimination. This phenomenon is regarded as the stereoselectivity in pharmacokinetics. So, it is important and necessary for the patients'health to clarify the metabolic characteristics of different enantiomers of chiral drugs.In vivo, drug and xenobiotic metabolism occur mainly in liver. While all of cytochrome P450, the large family of mixed function oxidases, are the key enzymes which involved in the phase I metabolism of a variety of chemically diverse substances ranging from endogenous compounds to xenobiotics. Cytochrome P450s consist of many members, in which CYP1A, 2A6, 2B6, 2C, 2D6, 2E1and 3A are the most important. The specific P450 enzyme(s) involved in the biotransformation of a drug can show the specific characteristic of drugs in pharmacokinetic behavior, and drug interactions can occur by competing the same P450, by which the drugs administered simultaneously are oxidated. Pharmacokinetic and clinical issues may also occur in individuals who are deficient in a specific P450 enzyme. So cytochrome P450s play central roles in pharmacology and toxicology research, and it is particularly important to clarify the profiles and variance of cytochrome P450s in the drug metabolism in human. Carvedilol is a relatively new drug withβ- andα1-receptor blocking activity and antioxidant effects recently approved for the treatment of congestive heart failure(CHF). This drug is used clinically as a racemic mixture of R(+)- and S(-)-enantiomers, but previous studies have shown that the S(-) enantiomer is metabolized faster than the R(+) enantiomer. Up to now, domestic research on pharmacokinetic of different enantiomer of carvedilol is a blank space. So we use hepatocyte culture as the tool in vitro for assessing effects of drug on metabolizing enzymes. R(+) , S(-) carvedilol are metabolized by BNF and INF, which are selective inducers of CYP1A1 and CYP2E1 respectively, induced cell. The objective of this study is to identify the metabolic difference of the R(+)- and S(-)-enantiomers in the specific enzymes for explaining and forecasting interindividual pharmacokinetic variability and helping predicting drug-drug interactions. Our research include two parts as followed.1. Analysis of carvedilol enantiomers in cell culture solution by pre-column derivatization RP-HPLCObjective: To establish a reversed-phase HPLC assay for determining the enantiomers of carvedilol in cell culture solution for the research on the metabolic stereoselctivity of enantiomers. Method: Carvedilol was extracted from cell culture solution with GITC, a pre-column chiral derivatization reagent. The derivatized products were separated on Kromasil C18 column, acetonitrile- sodium dihydrogen phosphate buffer solution(25/75, v/v) as mobile phase, flow rate at 1.0ml·min-1 , and the excitation and emission wavelength were 285nm and 340nm, respectively. Results: Carvedilol enantiomers can be separated well. The assay linear was from 4 to 20μg·mL-1 for each enantiomer. The inter-and intra-day precision was below 10%. Conclusion: The method is reliable, accurate, and suitable for the study of metabolic stereoselective of carvedilol enantiomers.2. Metabolic characteristics of carvedilol enantiomers in the cytochrome P450 of human hepatocytes treated with different inducers Objective: With the inducation of BNF and INF to the cultured hepatocytes in vitro we observed the change of particular P450 acting on the carvedilol enantiomers to clarify the metabolic stereoselectice. Method:Using 7-ER and chlorzoxazone as specific substrates to determine the activities of CYP1A1 and CYP2E1 and to define the best induced concentration. A RP-HPLC method was developed to determine the carvedilol concentration. R(+) , S(-) carvedilol were metabolized by BNF and INF induced cell. Then the substrate concentration-time curves and enzyme parameters (Km , V max) of carvedilol enantiomers were provided. Results and Conclusion:The results showed that their catalytic abilities had a stereoselectivity to S(-) carvedilol in control group and INF induced group, while there is no stereoselectivity in BNF induced group. But compared with control cell, BNF treated cell showed stronger enzymeactvities to R(+) carvedilol.