Studies On The Metabolism Of PAC-1 With LC-MS Technology

This thesis describes the identification metabolites of PAC-1 in vivo and in vitro using LC-Q-TOF/MS and LC-ITMS and sums up the proposed CID pathways of parent drugs and their corresponding metabolites facilitated by accurate protonated molecules[M + H]⁺. Besides,the effects of cytochrome P450（CYP450） on the metabolism of PAC-1 in RLM and HLM were investigated,too.At last,A rapid liquid chromatography-mass spectrometry （LC/MS） method has been validated and employed routinely evaluate the potential for a new drug to modify CYP450 activities by determining the effect of the drug on in vitro probe reactions that represent activity of specific P450 enzymes.1.Metabolite identification of PAC-1 in ratIn the present study,the metabolic profile of PAC-1,a potential anticancer drug,was investigated using liquid chromatography-mass spectrometric（LC/MS） techniques.Two different types of mass spectrometers - a quadrupole time-of-flight（Q-TOF） mass spectrometer and an ion trap（IT） mass spectrometer - were employed to acquire structural information on PAC-1 metabolites.A gradient liquid chromatographic system composed of 0.2%formic acid in methanol and 0.2%formic acid in water was used for metabolite separation on an Agilent TC-C₁₈ column.A total of 14 metabolites were detected.The corresponding product ion spectra were acquired and interpreted,and structures were proposed.Accurate mass measurement using LC-Q-TOF was used to determine the elemental composition of metabolites thereby confirming the proposed structures of these metabolites.PhaseⅠmetabolic changes were predominantly observed,including debenzylation,dihydrodiol formation,hydroxylation,and dihydroxylation.The detected phaseⅡmetabolites included PAC-1 and hydroxylated PAC-1 glucuronide conjugates. Based on metabolite analysis,several PAC-1 metabolic pathways in rat were proposed.2.Metabolites characterization of PAC-1 in vitroLiver microsomes of male Wistar rats were prepared using ultracentrifuge method.The metabolism of PAC-1 in RLM and HLM were also studied using liquid chromatography-ion trap mass spectrometry and time of flight mass spectrometry with ESI sources.A total of 12 metabolites were identified including des-benzyl-PAC-1 as the major metabolites,which was same as the in vivo study.The effects of typical CYP450 inhibitors on the formation of des-benzyl-PAC-1 were investigated.In addition,there is no GSH conjugate detected by liquid chromatography-ion trap mass spectrometry in incubations of PAC-1 with NADPH and GSH-supplemented RLM or HLM.This biotransformation study of PAC-1 in vitro showed that PAC-1 is metabolized via debenzylation in RLM and HLM. The reaction is mainly catalyzed by CYP3A4/3A5.The formation of main metabolite depends on rat liver microsomes and NADPH.3.Studies on effect of PAC-1 on hepatic CYPsIn vitro drug interaction data can be used in guiding clinical interaction studies,or,the design of new candidates.To meet this need,a rapid liquid chromatography-tandem mass spectrometry（LC-MS） method has been validated and employed for routine screening of new chemical entities for inhibition of five major human cytochrome P450（CYP） isoforms. Probe substrates were used near the Michaelis-Menten constant（Km） concentration values for CYP1A2（phenacetin）,CYP2C9（tolbutamide）,CYP2D6（dextromethorphan）,CYP2E1 （chlorzoxazone）,and CYP3A4（midazolam and dextromethorphan）.Induction potential of CYP1A,CYP2C,CYP2D,CYP2Eand CYP3A by PAC-1 was examined with liver microsomes from control rats or rats treated with PAC-1 at 35 and 60 mg·kg^-1·d^-1 for 4 consecutive days or 7 consecutive days.In the induction study,there were no significant difference in the enzymatic activities between the control and PAC-1 treated rats except CYP1A（p>0.05）.