Identification Of In Vivo And In Vitro Metabolites And Metabolic Pathway Analysis Of Two Derivatives Of Harmine

Objective:Hammine(HM)derivatives HM-Y-A and HM-Y-B are potential drugs for the treatment of echinococcosis.On the basis of previous studies,we studied the metabolism of HM-Y-A and HM-Y-B in vivo and in vitro.Methods:①Rat liver microsomes and liver S9 were prepared by differential centrifugation and determination of protein content.Metabolic enzyme activities of rat liver microsomes and liver S9 were determined by UPLC-ESI-MS/MS.②UPLC-ESI-Q-TOF-MS was used to identify the phase Ⅰ and Ⅱ metabolites of derivatives HM-Y-A and HM-Y-B in rat liver microsomes and liver S9.③An UPLC/ESI-QTOF-MS method was established and used to analyze the metabolic profile of harmane derivatives in rats.④An UPLC-ESI-Q-TOF-MS method was developed,and chemical inhibition and recombinant enzymes were used to identify the main metabolic enzyme subtypes involved in the metabolism of the derivatives HM-Y-A and HM-Y-B,separately.Results:① Rat liver microsomes and liver S9 were prepared,and the results showed that they had the activities of CYP450 and UGTs.②Identified 29 HM metabolites in rat liver microsomes and liver S9,15 in phase Ⅰ and 14 in phase Ⅱ,including 11 metabolic pathways;29 metabolites were identified by HM-Y-A,including 25 in phase Ⅰ and 4 in phase Ⅱ,including 12 metabolic pathways;HM-Y-B identified 30 metabolites,including 17 in phase Ⅰ and 13 in phase Ⅱ,including 10 metabolic pathways;and HM identified 21 metabolites were identified in human liver microsomes,including 14 in phase Ⅰ and 7 in phase Ⅱ,including 8 metabolic pathways;26 metabolites were identified by HM-Y-A,including 21 in phase Ⅰ and 5 in phase Ⅱ,including 11 metabolic pathways;HM-Y-B identified 26 metabolites,including 16 in phase Ⅰ and 10 in phase Ⅱ,including 7 metabolic pathways.③ 42 metabolites of HM were identified in rats,including 12 metabolic pathways,including 27 in plasma,16 in bile,13 in feces and 26 in urine;46 metabolites of HM-Y-A were identified,including 14 metabolic pathways,including 24 in plasma,22 in bile,29 in feces and 19 in urine;42 metabolites of HM-Y-B were identified,including 10 metabolic pathways,including 28 in plasma,25 in bile,15 in feces and 23 in urine.In addition,the results showed that HM,HM-Y-B and their metabolites were mainly excreted by the kidney and bile,while HM-Y-A and its metabolites were mainly excreted by feces.Glucoaldehyde acidification metabolites are excreted mainly by urine and bile.④ The results of chemical inhibition showed that the metabolism of the derivative HM-Y-A was mainly mediated by CYP1A2,CYP2A6,CYP2C8 and CYP2C9,and the metabolism of the derivative HM-Y-B was mainly mediated by CYP2A6,CYP2B6,CYP2C8 and CYP2C9;The results of recombinant enzyme experiments showed that the metabolism of the derivative HM-Y-A was mainly mediated by CYP1A1 and CYP1A2,and the metabolism of the derivative HM-Y-B was mainly mediated by CYP1A1,CYP1A2 and CYP3A4,Conclusion:Through data integration and metabolic profile analysis,in this study,the metabolites and pathways of derivatives HM-Y-A and HM-Y-B in vivo and in vitro of rats were systematically elucidated for the first time.In vivo and in vitro,47 metabolites of HM were identified,including 12 metabolic pathways;50 metabolites of HM-Y-A were identified,including 14 metabolic pathways;51 metabolites of HM-Y-B were identified,including 10 metabolic pathways;and major metabolic enzyme subtypes that mediate metabolism of derivatives HM-Y-A and HM-Y-B were identified.These results provide a reference for the metabolism of other β-carboline compounds in vitro and in vivo,and provide an experimental basis for the subsequent studies on the derivatives of Harmine.