Cyp450 Interactions. [2 - (4 - Chloro-benzoyl Hydroxamic Acid) 2 N-butyl-co-tin (dbdct) A Preliminary Study

1. BackgroundDi-n-butyl-di-(4-chlorobenzohydroxamato) tin (IV) (DBDCT) is a new antitumor compound.Our previous study has shown that DBDCT could be absorbed instantly and extensively metabolized after i.v. administration to rats. There was no parent drug of DBDCT in urine and manure; Presumably DBDCT was gradually biotransformed via stripping off ligand, alkyl and chlorine, eventually eliminated from kidney and bile through hepatic microsomal enzyme.2. AimsThe primary objectives of the present study were to characterize cytochrome P450 (CYP450) enzyme(s) involved in the metabolism of DBDCT and evaluate the effects of DBDCT on CYP450.It is useful in the further understanding of DBDCT metabolic mechanism, the prediction of pharmacokinetic drug-drug interactions and interpatient variability in drug exposure.3. MethodLiver microsomes of rats were prepared using ultracentrifuge method, followed by assays to detect changes in the levels of liver microsomel protein and CYP450.The in vitro metabolism of DBDCT was studied by incubation with rat liver microsomes and an HPLC method for the determination of DBDCT in rat liver microsomes was established.The metabolism of DBDCT was studied in rat liver microsomes using itself as substrate;To characterize the CYP450 isozymes involved in the metabolism of DBDCT, the effects of typical CYP450 inducers and of CYP450 inhibitors were investigated.The inhibitory effects of DBDCT on CYP450 were also investigated in the present study.The following CYP450 isoform specific marker reactions were measured; phenacetin(CYP1A), diclofenac sodium (CYP2C9), omeprazole(CYP2C19) and nifedipine(CYP3A4).4. ResultsLiver microsomes of rats were induced by inducer(Dex,BNF,PB),followed by assays to detect changes in the levels of liver microsomel protein and CYP450 content, the induction group compared with the control group were have significant deviation. An HPLC method for the determination of DBDCT in rat liver microsomes was established, the linear range of 3.48-174.20μmol·L-1, the lower detectable concentration was 3.48μmol·L-1,limitation of quantitation was 0.73μmol·L-1 this method proved to be convenient,fast,accurae with good reproducibility,can be applied to in vitro metabolism study of DBDCT. The kinetic parameters, Km, Vmax and liver clearance rate (Vmax/Km) were calculated by Michaelis-Menten equation and Eadie-Hofstee plot.In the metabolic system, the enzyme kinetics parameters of DBDCT were found as follows: Km=157.07μmol·L-1,Vmax=2.189μmol·(min·mg)-1 and liver clearance rate Vmax/Km=0.014L·(min·mg)-1, respectively. The results of in vitro metabolism of DBDCT in different enzyme sources suggest that:the PB, DEX group compared with the control group were have significantly deviation and the BNF group compared with the control group there have no significant deviation. DBDCT was invubated with exclusive inhibitors of various CYP450 isoforms in vitro, respectively, in order to confirm the major enzymes of mediate metabolism of DBDCT. The results showed that the ketoconazole on metabolism of DBDCT have a stronger inhibitory effect, in addition, sulfamethoxazole on DBDCT metabolism also have some inhibitory effect, and inhibition of fluvoxamine, trimethoprim, and omeprazole on DBDCT metabolism was weaker. Substrate test results:the metabolism of nifedipine in the DBDCT treated rat liver microsomes compared with the control group have significantly deviation (P <0.01, P<0.001). The metabolism of phenacetin, diclofenac sodium and omeprazole in the DBDCT treated rat liver microsomes compared with the control group was no significant difference (P> 0.05).5. ConclusionThe metabolism of DBDCT in rat liver microsomes was faster, suggesting that the liver first-pass effect of the drug may be stronger. By experiments of in vitro metabolism of DBDCT in different enzymes sources and metabolic inhibition test results showed that the leading role of CYP450 isoforms in catalytic DBDCT metabolism was CYP3A4, CYP2C9 may be partly involved, CYP1A on the metabolism of DBDCT have no significant catalytic effect; Substrate test results showed that DBDCT have a stronger inhibitory effect on CYP3A4 and no significant effect by CYP1A, CYP2C9 and CYP2C19.