Study On The Effect Of Liguzinediol On Metabolic Enzymes

Liguzinediol,named 2,5-dihydroxymethyl-3,6-dimethylpyrazine,is a water-soluble monomers derived compound of ligustrazine.The positive inotropic effect of liguzinediol with its of sarcoplasmic reticulum SR Ca2+ ATP enzyme target was identified and no side effects were found in large doses.Our group has studied the pharmacokinetics,sexual differences and metabolite of liguzinediol in rats and dogs systematically.The results turned out that the half-life of liguzinediol is very short and a significant difference between male and female rats was found.In addition,various metabolites were detected,including oxidation and glucuronide products.It suggested that liguzinediol might be metabolized by the hepatic drug-metabolizing enzymes.The cocktail probe drugs approach was initially established to evaluate the influence of liguzinediol on CYP450 isoforms in rats and incubation system that using six enzymes of CYP1A2,CYP2C9,CYP2C19,CYP2D6,CYP2E1 and CYP3A4 in liver microsomes of rat and human was set up.Each substance was determined by LC-MS/MS.Moreover,metabolites of liguzineiol in liver microsomes among different species were compared.It may provide a strong basis for the selection of appropriate animals in preclinical safety evaluation and the safety of clinical medication.The main aspects are summarized as follows:1 Single substrate assays for the effect of liguzinediol on six CYPs in rat liver microsomePhenacetin,tolbutamide,omeprazole,dextromethorphan,chlorzoxazone and midazolam were chosen as specific substrates of CYP1A2,CYP2C9,CYP2C19,CYP2D6,CYP2E1 and CYP3A4,respectively,to establish single substrate inspection system in rat liver microsomes.A novel,simple and sensitive method of LC-MS/MS was developed to evaluate the effects of liguzinediol on hepatic cytochrome P450 enzymes.The concentration of protein and incubation time were optimized in order to accurately estimate kinetic characteristics of probe drugs in rat liver microsomes mediated by cytochrome P450.First,the conditions of incubation system by the linear relationship between the metabolite yield and protein concentration or incubation time were set up,and then Michales-Menten constant(Km)of each probe substrate was determined.The inhibitory effect of liguzinediol was characterized by inhibition rate.The chromatographic separation was performed on a Shim-pack XR-ODS column(50mm×2.0mm,2.2μm)at 35℃.A gradient elution program was conducted for chromatographic separation with 0.2%formic acid in water and acetonitrile.The flow rate was 0.3 mL/min.The electrospray ionization source was operated to detect every metabolite of specific probe substrate.The verification indicated that the linearity ranges can meet the requirements for quantification and the RSD of precision and accuracy were both below 15%.The inhibition in rats was all below 15%at seven concentration levels of liguzinediol.It suggests that in a single probe substrate research in rats,the possibility of the inhibition of liguzinediol mediated by these six CYPs is very small or does not exist.2 Single substrate assays for the effect of liguzinediol on six CYPs in human liver microsomePhenacetin,tolbutamide,omeprazole,dextromethorphan,chlorzoxazone and midazolam were chosen as specific substrates of CYP1A2,CYP2C9,CYP2C19,CYP2D6,CYP2E1 and CYP3A4,respectively,to establish single substrate inspection system in human liver microsomes.The reaction was carried out in cofactor and certain time.LC-MS/MS was used to determine paracetamol,4-hydroxytolbutamide,5-hydroxy omeprazole,dextrorphan,6-hydroxychlorzoxazone,1’-hydroxymidazolam.The inhibitory effect of liguzinediol was characterized by inhibition rate.The concentration of protein and incubation time were also optimized in order to accurately estimate kinetic characteristics of probe drugs in rat liver microsomes mediated by cytochrome P450.First,the conditions of incubation system by the linear relationship between the metabolite yield and protein concentration or incubation time were set up,and then Michales-Menten constant(Km)of each probe substrate was determined.The inhibition on CYP1A2,CYP2C19,CYP2D6,CYP2E1 and CYP3A4 in human were all below 15%at seven concentration levels of liguzinediol.It indicated that in a single probe substrate research in human,there was almost no inhibition of liguzinediol on these five enzyme,but appropriate attention should be paid when combined with the drug mediated by CYP2C9.Comparing the inhibition on CYPs in human and rat,there was a little inhibition of liguzinediol on human CYP2C9 and there was no significant inhibitory effect on ray and the other five enzymes in human.It provided experimental basis for clinical safety medication.3 "N-in-One" assays for the effect of liguzinediol on six CYPs in rat liver microsomeThe method used in the first two chapters belongs to low flux method.High throughput detection method-"N-in-One" method that six metabolites can be determined at the same time was established to evaluate the effect of liguzinediol on CYPs rapidly.The inhibition ratio was calculated by comparing the generation rate of metabolites in sample group and control group.The inhibition rates of different concentrations of liguzinediol on rat CYPs were all less than 15%.The results obtained from "N-in-One" assay were consistent to the single substrate assay.4 "N-in-One" assays for the effect of liguzinediol on six CYPs in human liver microsomeHuman liver microsomal was set up and six kinds of probe substrates metabolies high-throughput detection methods,called "N-in-One" in vitro probe method,were detected simultaneously,and accord with the requirement of detecting biological samples.The result of rapid evaluating effect of liguzinediol on human liver microsomal CYP450 enzyme showed that the inhibition rate of liguzinediol on each subtype of CYP450 enzyme were all below 15%among different concentrations of liguzinediol.Some differences can be found in "N-in-One"system and single substrate system.In single substrate system,liguzinediol shows moderate inhibition toward metabolism mediated by CYP2C9,which can’t be found in "N-in-One"system.This may be concluded to the interaction effect of substrates.To sum up,no obviously inhibition acticity can be found of linguzinediol’s effect on 5 subtypes of enzyme,which indicated liguzinediol could hardly inhibit the metabolism mediated by these 5 subtypes of enzyme.But when it comes to interact with compounds mediated by CYP2C9,conditions of drug utilization of the human body needs to be attention。5 Comparation of liguzinediol metabolites in liver microsomes among different speciesAccording to previous studies in vivo,the oxidation and glucuronidase acid binding products were incubated in vitro.The metabolites of liguzinediol were various,but the drug metabolism enzyme was still not clear.There was no comparison of metabolic pathways between animals and human.LC-MS/MS method was established to determine liguzinediol and its metabolites.We simulated internal environment in liver microsome to compare metabolites of liguzinediol among different species.Hydrogen and glycosyl were provided with NADPH and UDPGA to carry out oxidation reaction and glucuronic acid conjugation,respectively.Data was analysed in BPC and IDA mode.The results showed that phase Ⅰ and phase Ⅱ metabolism of liguzinediol can be occurred in liver.In addition,the oxidation and glucuronidase acid binding products were all found in four kinds of liver microsomes.It illustrated that the metabolites of liguzinediol in liver microsomes from rats,dogs and monkeys were consistent with that in liver these from human.The conclusion can be used to select laboratory animal for safety evaluation of liguzinediol in preclinical study.Previous pharmacokinetics studies on rat revealed glutathione combination existed in bile,and sulfuric acid combination was found existed in urine from the result of pharmacokinetic studies on dogs.Metabolic-cycteine combination and metabolic-acetylcsteine combination are both characteristic metabolites,but neither of them can be obtained by ordinary incubation.However,we found the cycteine-and acetylcsteine-combinations can be transferred from glutathione-combination.In this chapter,we intented to establish LC-MS/MS methods to detect liguzinediol and glutathione-combination which research the metabolics of liguzinediol distributed in liver cytosol of rat,dog,monkey,human and etc by liver cell plasma incubation.To incubating the glutathione-combination by combining sulfate radical and glutathione,PAPS and GSH were used as provider of sulfate radical and trapping agent respectively.Subsequently,a hydrolysis procedure was applied to obtain cycteine-and acetylcsteine-combinations,and the metabolism difference among human and other species can be compaired.This makes the foundation of experiental animal selection of liguzinediol preclinical research.