Induction Of Rat Liver CYP3A2 By ZOL And The In Vitro Metabolism Study Of MGN

In our preliminary enzyme activity and midazolam(MDZ) pharmacokinetics studies, we observed zolmitriptan(ZOL) treatment led to induction of CYP3A in male not female rats.As determined by immunoblotting and Real-time PCR,we found this gender-dependent ZOL inducibility of CYP3A is largely due to male-selective induction of CYP3A2 rather than CYP3A1.Interestingly,no changes in both genes were detected in primary cultured rat hepatocytes.Since growth hormone(GH) is known as the major mechanistic determinant of sexually-dimorphic gene expression like CYP3A2 in rat liver,the impacts of ZOL on both plasma GH levels in non MSG-treated rats and CYP3A2 expression in GH depleted MSG-treated rats were studied.ZOL was shown to partially suppress GH levels in both genders.Furthermore,CYP3A2 protein and mRNA level declined in male not female MSG-treated rats.These findings demonstrated CYP3A2 inducibility by ZOL was gender-selective and GH may play an important role in CYP3A2 induction.In order to study the possible molecular events involved in the depression of GH and gender-selective induction on rat CYP3A2 by ZOL,the mRNA and protein level (whole protein and nuclear protein) of PXR,CAR,Stat5b and HNF4αwere investigated.Nuclear accumulation of HNF4αand CAR were observed in the normal male not female rat liver tissue following ZOL treatment.However,this kind of nuclear translocation didn't occur in rat hepatocytes and MSG-treated rats.In addition,immunoblot analysis showed that the expression of PXR(whole protein and nuclear protein) in normal rats,rat hepatocytes and MSG-treated rats was not obviously altered after the treatment with ZOL though real-time PCR showed that ZOL can gender-selective induction the mRNA level of PXR.The results of part one demonstrated that this gender-dependent ZOL inducibility of CYP3A is largely due to male-selective induction of CYP3A2 rather than CYP3A1. Mitiglinide(MGN) is a new potassium channel antagonist for the treatment of type 2 diabetes mellitus.In the present study,a potential metabolic pathway of MGN,via carboxyl-linked glucuronic acid conjugation,was found.MGN carboxyl-glucuronide was isolated from a reaction mixture consisting of MGN and human liver microsomes fortified with UDP-glucuronic acid(UDPGA) and identified by a hydrolysis reaction withβ-glucuronidase and HPLC-MS/MS.With the assessment of MGN glucuronide formation across a panel of recombinant UDP-glucuronosyltransferase(UGT) isoforms(UGT1A3,UGT1A4,UGT1A6,UGT1A9,and UGT2B7),only UGT1A3 and UGT2B7 exhibited high MGN glucuronosyltransferase activity.The formation of MGN glucuronidation by human liver microsomes was effectively inhibited by quercetin(substrate for UGT1A3) and diclofenac(substrate for UGT2B7), respectively.The MGN glucuronidation activities in fifteen human liver microsomes were significantly correlated with quercetin(r~2=0.806) and diclofenac glucuronidation activities(r~2=0.704),respectively.These results demonstrate that UGT1A3 and UGT2B7 are catalytic enzymes in MGN carboxyl-glucuronidation in human liver.