| Liguzinediol(LZDO)is a potential drug for the treatment of heart failure.Previous pharmacological experiments showed that LZDO had positive inotropic effects with less toxicity.Previous pharmacokinetic studies in rats and beagle dogs showed that LZDO had a short half-time and high bioavailability.In the study of excretion and disposal in rats and beagle dogs,the main metabolites of LZDO were identified by UFLC-QTOF-MS.The main metabolites were 2-NAC-LZDO,2-Cys-LZDO,oxidation product,and LZDOG.The results of in vivo experiments in rats and beagle dogs were different.In order to investigate the effects of metabolic enzymes on LZDO,an in vitro incubation method for major metabolites and an UFLC-QTOF-MS method were established to study the metabolic pathways of major metabolites and the metabolic differences of LZDO between different species.In order to investigate the effects of LZDO on metabolic enzymes,an in vitro incubation system and a LC-MS/MS method were established to evaluate the inhibitory effects of LZDO on twelve UGTs.Cocktail probe method and LC-MS/MS method were used to study the effects of LZDO on CYP450 activity in rats.1.Effect of SULT on LZDOTo elucidate the biotransformation pathway and related enzymes of 2-NAC-LZDO and 2-Cys-LZDO,an incubation system with PAPS as a cofactor and NAC as a trapping agent was established using liver cytosol.An UFLC-QTOF-MS method was used to identify the major metabolites.2-NAC-LZDO could be detected among four species(humans,monkeys,dogs,and rats)and was the dominant metabolite in HLC.The sulfotransferase(SULT)inhibitors DCNP and quercetin at a concentration of 1 μM,suppressed 2-NAC-LZDO formation in HLC by 87 and 46%,respectively.This result suggested that sulfotransferase was involved in 2-NAC-LZDO formation.The metabolism of LZDO in different species indicated that SULT activity in dogs,rats,and monkeys was higher than that in humans.Further SULT phenotyping revealed that SULT1A1 was the predominant enzyme involved in the sulfation of LZDO.The underlying mechanism for the biotransformation of LZDO was demonstrated.The potential pathway was via the sulfation of LZDO to form sulfate,and the spontaneous cleavage of the sulfate group to generate highly reactive electrophilic cations,which could bind to NAC to form the major metabolites.2.Interaction between UGT and LZDOTo elucidate the metabolic enzymes responsible for the glucuronidation of LZDO and compare metabolism differences among species,an incubation system with UDPGA as a cofactor was established using liver microsomes.LZDOG could be observed.An UFLC-QTOF-MS method was used to identify the major metabolites.These results of screening of recombinant UGTs and chemical inhibition assays demonstrated that UGT1A9 played the predominant role for the glucuronidation metabolism of LZDO.The result of species difference indicated that the hepatic metabolic activity of the glucuronidation of LZDO was in the order:dog>monkey>rat>human,which was highly consistent with pharmacokinetic results in rats and dogs.To investigate the potential inhibition of UGT activity by LZDO,an in vitro UGTs incubation system and a LC-MS/MS method were employed to study the inhibition of LZDO toward UGT isoforms with a specific probe substrate(TFP)for UGT1A4 and a nonspecific probe substrate(4-MU)for other tested UGTs.LZDO was treated as inhibitor.The result of inhibition kinetic analysis showed that LZDO exerted inhibition against UGT1A10 and UGT2B15,and induced drug-drug interaction with low possibility.3.Interaction between CYP450 and LZDOTo elucidate metabolic enzymes responsible for oxidation product of LZDO and compare metabolism differences among species,an incubation system with NADPH as a cofactor was established using liver microsomes.An UFLC-QTOF-MS method was used to identify the major metabolites.Oxidation product of LZDO could be detected among four species(humans,monkeys,dogs,and rats).The formation of oxidation product of LZDO was in the order:monkey>dog>human>rat.Chemical inhibition assays demonstrated that CYP450 was involved in the formation of oxidation product.To evaluate the effects of LZDO on the enzyme activity of CYP450 in rats,a cocktail method and a developed and validated LC-MS/MS method based on six probe drugs(tolbutamide,chlorzoxazone,caffeine,metoprolol,omeprazole and dapsone)were established.Rats were randomly divided into four groups with six rats in each group.Rats were intravenous administrated with LZDO.The administration group was given different concentrations of LZDO,while the control group was given normal saline.The plasma concentrations of probe drugs were simultaneously determined by LC-MS/MS.The corresponding pharmacokinetic parameters were calculated by the software of DAS 2.1.Statistically significant differences in the pharmacokinetic parameters between treatment groups and blank control group were assessed by t-test.The results demonstrated that compared to the control group,metabolism of caffeine in the low dosage group was evidently speeded up,while AUC of metoprolol,dapsone,and omeprazole in the medium dosage group were also increased significantly.In the high dosage groups,metabolism of metoprolol and omeprazole were evidently slowed down.There was no significant influence of pharmacokinetic parameters of tolbutamide and chlorzoxazone in LZDO pretreated rats.There was no significant difference in the metabolism of tolbutamide and chlorzoxazone among the three groups.These results indicated that a low dosage of LZDO could induce CYP1A2 activity.CYP2D6 and 2C19 activities were inhibited by a medium and high dosage of LZDO.CYP3 A4 activity was inhibited by a medium dosage of LZDO.LZDO had no significant effect on CYP2C9 and CYP2E1. |
