Study Of HZZ112’s Metabolism, Dru-enzyme Interaction And Plasma Protein Binding

HZZ112 is a chemical drug candidate for lung cancer treatment. HZZ112 blongs to prodrug and its active metabolites can inhibite keratinocyte proliferation. In this study, a non-clinical pharmacokinetic study of HZZ112 was performed for evaluating its biotransformation, metabolism pathway, and inhibitory and induction effect on cytochrome P450 (CYP) enzymes. In addition, plasma protein binding profiles of HZZ112 and its two active metabolites were also evaluated. The results of this study provide a reference for the further clinical research of HZZ112.1 Analysis of active metabolites of HZZ112Objectives To analyze HZZ112 and its two active metabolites of HZZ1018 and HZZ1005 formed in rat, and their structures by HPLC-Q-TOF/MS.Methods Plasma and urine samples of rats were collected after intragastric administration of HZZ112 and they were analyzed by HPLC-Q-TOF/MS to confirm the possible structures of activity metabolites. HZZ112 was incubated with blood cells of dog and HZZ1018 was incubated with human liver microsomes to verify their metabolites.Results Active metabolites of HZZ112 were detected in rat plasma and urine when HZZ112 was administrated to rat. HZZ1018 was found when HZZ112 was incubated in dog blood cells. HZZ1018 was transformed into HZZ1005 in human liver microsomes which indicated that carbonyl reduction not C=C double bond reduction was the reaction of HZZ1018.Conclusion HZZ1018 and HZZ1005 are the main metabolites of HZZ112 in rat. HZZ1005 is the only metabolite of HZZ1018 formed in human liver microsomes.2 Metabolic profiles of HZZ1018 in different species’s liver microsomes and cytosolObjectives To compare the metabolic profiles of HZZ1018 in different species’s liver microsomes and cytosol, such as mouse, rat, dog, mini pig, and human.Methods A simple and sensitivity HPLC method was estabolished for the determination of HZZ1005 in liver microsomes incubate. Enzyme concentration and incubation time of HZZ1018 were optimized. The enzyme kinetic paramaters of HZZ1018 were fitted by the Michaelis-Mentenequation using Graphpad Prism 5 software.Results In order to ensure the consumption of HZZ1018 is not more than 20% and avoid the non-specific adsorption of proteins, the enzyme protein concentration was set at 0.4 mg/mL and the incubation time was 40 min. The kinetics of HZZ1005 for HZZ1018 fitted the Michaelis-Menten equation. The kinetic profilesin human and pig liver microsmes were similar, and significantly different among human, mouse, rat, and dog liver microsmes. Differently, the kinetic profiles in liver cytosol of mouse, rat, dog, pig and human all showed no obvious difference. The Clint values in liver cytosol were over 100-fold higher than that in liver microsomes which indicated that liver cytosol played a major role in the metabolism process.Conclusion The kinetic profiles of HZZ1018 in human and pig liver microsmes were similar, while in liver cytosol of different species all showed no significant difference. Liver cytosol played a major role in the process of HZZ1018 transformed into HZZ1005.3 Study on enzymes invovled in metabolism of HZZ112 and HZZ1018Objectives To speculate enzymes participating ester bond cleavage of HZZ112 and carbonyl reduction of HZZ1018.Methods 1) FDA and CFDA guidances.2) the correlation experiments for metabolism and major CYPs.3) Dicumarol and wafarin were co-incubated with HZZ1018 in the cytosol to investigate the double bond reduction of HZZ1018.4) Analysis of the relation between HZZ112 and acetylcholinesterase (ACHE), butyrylcholinesterase (BCHE), carboxylesterase (CE), and paraoxonase (PON).Results 1) HZZ1018 metabolism was related to CYP enzymes not FMO, but CYPs were not the main metabolic enzymes according the kinetic results. The metabolism of HZZ1018 needs NADP-NADPH as cofactor.2) The correlation experiment showed HZZ1018 metabolism wasn’t relevent to the main CYPs.3) The metabolism of HZZ1018 wasn’t affected by coumarin and wafarin.4) HZZ112 was not matabolized in Beagle dog’s plasma (high expression of ACHE, BCHE and PON) and rat liver microsomes (carboxylesterase CE).5) Recombinant esterase enzyme rhCEl, rhCE2, rhPONl and BChE were not mediated the metabolism of HZZ112.Conclusion 1) HZZ112 was not metabolized in liver microsomes and cytosol of different species, but was metabolized in whole blood, and HZZ1018 was formed in blood cells. HZZ1018 metabolism in the cytosol was higher and faster than that in the liver microsomes.2) HZZ112 metabolism may be associated with specific esterase in blood cells, while the metabolism of HZZ1018 was likely related to the AKR1C family in the cytosol.4 The inhibition of HZZ112 and its two metabolites to CYPsObjectives To investigate the inhibitory effects HZZ112 and its two metabolites on some of CYP enzymes.Methods An HPLC-MS/MS cocktail method was estabolished for simultaneous determination of the metabolites of CYP probe substrates. HZZ112, HZZ1018 and HZZ1005 in differenct concentrations were incubated with CYP probe substrates in human liver microsomes, respectively.According to the guidelines released by CFDA, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4/5 were studied.Results The estabolished cocktail HPLC-MS/MS method succeed to simultaneously determine the concentration of the metabolites of CYPs substrates. It was found that the activities of CYP1A2,2B6,2C8,2C9,2C19,2D6, and 3A4/5 were not significantly inhibited by HZZ112 (IC50>> 100 μmol/L). However, HZZ1018 and HZZ1005 both showed strong inhibitory effect to CYP1A2,2B6,2C8,2C9,2C19,2D6 and 3A4/5.Conclusion HZZ112 exhibited no significantly inhibitory effect, but HZZ1005 and HZZ1018 showed strong inhibition on seven CYP enzymes.5 The induction effect of HZZ112 and its metabolites on CYP3A4, CYP1A2 and CYP2B6Objectives To investigate the induction effect ofHZZ112 and its metabolites on CYP3A4, CYP1A2 and CYP2B6.Methods P450 enzymes induced by drugs should be focused on human CYP3A4 and CYP1A2, CYP2B6. The induction effects of HZZ112, HZZ1018 and HZZ1005 on CYP3A4, CYP1A2 and CYP2B6 were evaluated by method of French reporter gene. And then, the levels of mRNA expression in human hepatoma cells HepG2 and homologous genes in mouse liver tissue were detected after repeated administration by RT-PCR.Results HZZ1018 and HZZ1005 exhibit no induction effection CYPs by reporter gene assay. HZZ112 exhibit weak induction effect on CYP1A2 in high-dose by reporter gene assay and RT-PCR.Conclusion HZZ1018 and HZZ1005 have no induction effect on CYP3A4, CYP1A2 and CYP2B6.6 Protein binding of HZZ112 and its metabolites in plasmaObjectives To evaluate the rate of protein binding of HZZ112 and its metabolites-HZZ1018 in human, dog, rat plasma.Methods Ultracentrifugation was used to study the rate of protein binding of three drugs. HZZ112 and HZZ1018 were dissolved in the plasma of human, dog and rat with the concentration of 2,20,200 μmol/L, respectively. After ultracentrifugation at 500,000g at 4℃ for 17 h, clear solution in middle phase was taken and then detected. The rates of protein binding of HZZ112 and HZZ1018 were calculated based on free drug concentration.Conclusion The rates of protein binding of HZZ112 and its metabolites, HZZ1018, were very high (greater than 95%) in human, dog, rat plasma.