The Metabolism Studies Of Yonkenafil In Vivo And In Vitro

Yonkenafil is a new type of phosphodiesterase(PDE)V inhibitor,the structure of yonkenafil is cyclic Guanosine monophosphate(cGMP)five-membered and six-membered heterocyclic ketone compound.It was developed independently by Chinese scientists with independent intellectual property rights.Results from early pharmacodynamics experiments shows that yonkenafil has excellent selective inhibition to PDE V compared with the existing phosphodiesterase V inhibitor drugs vardenafil and sildenafil,it has more obvious advantages.At present,our laboratory has conducted the pharmacokinetic study of yonkenafil in rats and beagles,however,CYP450 enzymes that exist in liver microsomes are the main drug-metabolizing enzyme systems in vivo.In the preclinical pharmacokinetic study,investgating the potential interactions between target drugs and CYP450 enzymes contributes to the process of screening for new drugs,predicting clinical pharmacological effects and providing reliable data for guiding patients safe,effective and rational drug use.Therefore,based on previous studies,this research mainly focuses on the metabolism of yonkenafil in vitro and metabolite identification in vivo and in vitro.This article established a LC-MS/MS quantitative method to determine the concentration of yonkenafil and it's three metabolites in human liver microsomes.The incubation condition was optimized in regard to incubating time,concentration of liver microsomal protein and substrate to make the metabolic process be conducted in a suitable incubation condition.The final determined incubation conditions are as follows:the incubation time is set at 15 min,liver microsomal protein concentration is 0.5 mg/mL,the optimal concentration of yonkenafil is 8 ?g/mL.In the study of enzymatic reaction kinetics in human liver microsomes,the substrate yonkenafil was set up a series of concentration gradient,metabolic rate was measured under different yonkenafil concentrations,enzymatic reaction kinetics parameters Km and Vmax were calculated using Lineweaver-Burk double inverse mapping method,the result of calculated Km,Vmaxand metabolic clearance were 15.4 ?mol/L,5.56×10-3?mol/(min mg protein)-1 and 3.61×10-4 L(min mg protein)-1,respectively.By adding different specific inhibitors of CYP enzyme subtypes,the metabolic pathways of yonkenafil in liver microsome can be determined.The result shows that ketoconazole and fluconazole have obvious inhibitory effect to the metabolism of yonkenafil,which demonstrates that the metabolism of yonkenafil was mainly mediated by CYP3A4 and CYP2C9.The inbibition rate of ketoconazole to metabolites M1,M2,and M3 were all close to 100%,the inbibition rate of fluconazole were 54.7%,33.1% and 65.7% respectively,which shows that CYP2C9 plays a relatively minor role in the process of yonkenafil metabolism compared with CYP3A4.IC50 values of ketoconazole and fluconazole were determined by adding ketoconazole and fluconazole respectively to the incubation system in a series of concentration gradient and calculating the generation of metabolites,the concentration of inbibitors was set as abscissa,inhibition rate as ordinate to draw the inhibiting curve,the IC50 value can be calculated through GraphPad Prism 5.0 software,it turns out that the IC50 value of ketoconazole is 3.24?M,the IC50 value of fluconazole is 39.5?M.The result of metabolic stability study in human,monkey,rat,mouse and beagle dog liver microsomes shows that yonkenafil is metabolized in all five liver microsomes,metabolic half-lives(t1/2)in descending order is monkey > SD rat > CD mice> human > beagle dog,in which the half-life of yonkenafil in human liver microsome is similar to SD rat and CD mice liver microsomes,indicating that rat and mice are suitable species to be used as preclinical pharmacokinetic study model for yonkenafil.Mixed substrate probe(Cocktail)method was used to study the potential inhibitory effect that yonkenafil may have on different CYP enzyme subtypes,the result shows that yonkenafil has weak inhibitory effect on CYP3A4,CYP2C9,CYP2C19,CYP2D6,CYP2E1,while in normal doses,yonkenafil will not cause any severe drug-drug interactions.The unknown metabolites of yonkenafil is investigated through high resolution mass spectrometry(LC-TOF MS/MS),the result shows that a new metabolite of yonkenafil is detected in all liver microsomal incubating samples and in the collected rat bile and feces,it's molecular formula is C24H33N5O5 S,by comparing its secondary ions with parent drug,speculating that it may be a hydroxylation metabolite in piperazine ring of yonkenafil,however,NMR method is needed to further validate its structure.