The In Vitro And In Vivo Metabolism Studies Of F18 In Rat Liver Microsomes And Rats

Drug Metabolism plays an important role in drug discovery and development, which is divided into in vivo and in vitro metabolism. Cytochrome P450 (CYP 450) is a major metabolizing enzyme system for many endogenous and exogenous substances. In the early stage of new drug development, it has become an essential part of screening effective and safe new chemical entities (NCEs) by studing the metabolic characteristics under main CYP enzymes through in vitro methods, and verifying the results through in vivo experiments. To indentify the metabolites of NCEs in vivo can make clear of the transformation and excretion pathways by liquid chromatography and high resolution time-of-flight mass spectrometry, providing the basis for drug safety and effectiveness.F18, a compound with quinazolin and hydroxamate, have proved its selective inhibition for Histone Deacetylases6 (HDAC6) and significant antitumor effect through previous experiments, both in vivo and in vitro. In this paper, it is regarded as a potential small molecule antitumor drug to carry out the preliminary study on the in vivo and in vitro metabolism, including the metabolism of F18 in liver microsomes in vitro, investigating the effect of F18 on CYP3A1 and the metabolites of F18 in rats.1.In vitro metabolism of F18 in liver microsomes(1) A liquid chromatography-tandem mass spectrometry method for quantitation of F18 in liver microsomal incubation system was developed and validated. The results of method validation suggested that the specific, linearity, sensitivity, accuracy and stability all met the general criteria for biological sample analysis methods. So this method was employed to quantitative analysis of F18.(2) Metabolic stability of F18 in vitro:The metabolic stability and species difference of F18 in human, rat, dog and monkey liver microsomes in vitro was investigated. The half-life in vitro of F18 in human, rat, dog and monkey liver microsomes was 693.00,577.50,533.08 and 198.00 min, respectively. The results suggested that F18 had good metabolic stablility in liver microsomes and its metabolism rate were similar in human, rat and dog liver microsomes.(3) Metabolic phenotype of F18 in vitro:The metabolic phenotype of F18 was researched by using the specific chemical inhibitors. The results showed that CYP2C9, CYP2E1, CYP2D6 and CYP3A4 may participate in the metabolism of F18.(4) Inhibitory effect of F18 on the activities of CYP450 enzyme:The effects of F18 on CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 and CYP2E1 activities in vitro were examined using probe drugs. Comparing the concentration of metabolites with different concentrations of F18, the effects of F18 on CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 and CYP2E1 activities were obtained. F18 exhibited middle inhibitory effects on CYP3A4 activity with IC50 values of 1.63 ?M; while it exhibited weak inhibitory effect on CYP2D6, CYP2C9, CYP2C19 and CYP2E1 with IC50 value of 11.76 ?M,16.60 ?M,22.33 ?M and 37.78 ?M and no inhibitory effect on with IC50 value of larger than 100 ?M. This results suggested that F18 should be investigated the inhibition of CYP3A4 in vivo.2.Inhibitory effect of F18 on CYP3A1 in ratsThe in vivo inhibition effect of F18 on CYP3A1 activity was investigated in rats using dapsone as probe substrate. After being pretreated with total F18 (5 mg/kg) for 8 days, the rats were intragastric administrated with total dapsone (20 mg/kg) at the last F18 dosing. The plasma concentration of the substrate was determined by UPLC-MS/MS method, the plasma was obtained at different time points, for comparing their pharmacokinetic properties between the treated and untreated rats. The pharmacokinetic parameters of dapsone were calculated by DAS 2.0 program. Comparing with the control group, the main parameters such as t1/2 and AUC of F18 group increased but showed no statistically significant difference (P>0.05). The results suggested that F18 had no significant inhibition on CYP3A1. Due to the species difference between rat and human CYP enzymes, we should pay close attention to the possibility of drug drug interactions when combined use of F18 and drugs metabolized by CYP3A4 in clinical.3.The identification of the metabolites of F18 in rats.In this study, a sensitive and reliable approach based on ultra-performance liquid chromatography/quadrupole-time-of-flight-mass spectrometry(UPLC-Q-TOF-MS/MS) was applied to characterize the metabolic profile of F18 in rat plasma, urine, feces after the administration of F18. Totally 11 metabolites were discovered and tentatively identified, including 7 phase I metabolites and 4 phase II metabolites, which were divided into demethylated metabolite (Ml), deaminated and hydroxylated metabolite (M2), demethylated and deoxidated metabolite (M3), deoxidated metabolite (M4), demethylated and deaminated metabolite (M5), dehydroxylated metabolite (M6), deaminated metabolite (M7) and glucuronidated metabolites (M8-M11). The main metabolic pathways were oxidation, reduction, hydrolysis and glucuronidate. Moreover, this study provided comprehensive analysis on the metabolites in rat plasma, feces and urine, which gave better understanding of the metabolic fete and pharmacodynamic material basis of F18 in vivo.