Study On The In Vivo/in Vitro Metabolism And Pharmacokinetics Of Harmane

Objective: To study the in vivo/in vitro metabolism and pharmacokinetics of harmane in rats after administration of harmane. Methods: 1) Using the differential centrifugation method to prepare rat liver microsomes and determine the content of protein. 2) To establish an UPLC-ESI-MS/MS method for the simultaneous determination the concentrations of nine probe metabolites, to investigate the activity and enzyme metabolic kinetics of harmane on ratLiver cytochrome P450. 3) To develop an UPLC-ESI-MS/MS method for determination the activity of phase II uridine diphosphate glucuronosyltransferase. 4) Using ultra-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry(UPLC/ESI-QTOF-MS) to analyze the metabolic profile of harmane in vitro in rats. 5) To establish an UPLC/ESI-QTOF-MS method for analyzing the metabolic profile of harmane in vivo in rats. 6) Using an UPLC-ESI-MS/MS method, to study the pharmacokinetics of harmane and its metabolites in rats after intravenous and oral administration of harmane of 1.0mg/kg and 30.0mg/kg, respectively. Results: 1) Using the differential centrifugation method, after homogenization, centrifugation and washing, ratLiver microsomes were successfully prepared. By calculation, the yield of rat liver microsomes were 32 mg microsome protein/g liver. 2) Under optimized conditions for chromatography and mass spectrometry, the concentrations of nine probe metabolites were determined, and the results shown that rat liver microsomes without the activity of CYP2A6 isoform. Furthermore, by using the GraphPad(Prism 5), the metabolic kinetic parameters(Km and Vmax) and their metabolic kinetic curves were all obtained. According to the Eadie-Hofstee plots, the hydroxylation of testosterone was catalyzed by CYP3A4 could be described by Hill equation, while the probe reactions were catalyzed by other seven isoforms could be described by Michaelis-Menten equation. 3) Under optimized conditions for chromatography and mass spectrometry, the probe metabolite of phase II UGT2B7 was observed, and the results shown that rat liver microsomes possess the activity of UGT2B7 isoform. 4) Using an UPLC/ESI-QTOF-MS method, to study the metabolic profile of harmane in rat liver microsomes and rat liver S9 in vitro in rats. The results shown that a total of 9 metabolites involved phase I and phase II were identified from the rat liver microsomes and rat liver S9 in vitro after administration of harmane, including 4 phase I metabolites and 5 phase II metabolites(3 glucuronide conjugation metabolites and 2 sulphate conjugation metabolites). 5) The results of the metabolic profile of harmane in vivo in rats shown that a total of 20 metabolites were identified from rat urine(11), feces(16), bile(16), and plasma(10) after a single oral administration of harmane using Metabolynx? and MassFragment ? software tools. It also indicated that the biliary and faecal clearance were the major excretion routes for harmane as well as its metabolites. 6) The validated method was successfully applied to a pharmacokinetic study of harmane and its ten metabolites in rats after intravenous and oral administration of harmane of 1.0mg/kg and 30.0mg/kg, respectively. As a result, ten metabolites together with harmane but no harmine were detected and identified in rat plasma collected after intravenous and oral administration, and the concentrations of ten metabolites were calculated by semi-quantification, using the calibration curve of harmane. In the present study, the absolute bioavailability(F) of harmane following an oral dose in rats was 19.41%, and the pharmacokinetic properties of harmane and its ten metabolites in rat would be addressed for the first time. Sulphate conjugation was found to be the major metabolic process of harmane in rat in vivo. Conclusion: Through the data integration and metabolic profiling analysis, we clarified the process and metabolic pathways of harmane in vitro/in vivo in rats, a total of 21 metabolites were identified including phase I and phase II, and systematically elucidated the pharmacokinetics profiles of harmane and its ten metabolites in vivo in rats.