Preclinical Toxicokinetics, Excretion And Metabolism Of A Novel Caspase Inhibitor F1013

Acute liver failure is composed of hepadnavirus infection or drug substances toxic effectscaused by a group of clinical syndromes, with a higher mortality rate, in addition to livertransplantation, there is currently no effective treatment method. Pathological study on acuteliver failure that in addition to liver cell necrosis, massive hepatic apoptosis in this process hasalso played an important role in apoptosis, and the main factor is apoptotic protease （Caspase）,so the caspase inhibitors provide a possibility for the treatment of this kind of diseases.The new drug is clinically used for patients with acute liver failure in rescue andtreatment. Preliminary tests show that, the drug can selectively inhibit the different Caspaseisoforms, thereby effectively inhibit hepatocyte apoptosis, with good clinical applicationprospect. Based on the SFDA New Drug Approval, The reseach group gave a try on the drug’spreclinical toxicokinetics and excretion, metabolism studies.ObjectiveEstablish a rapid and effective LC-MS/MS method for measuring the concentration ofdrug in Beagle dog plasma and rat bile，calculating toxicokinetics parameters；identify the mainexcretion pathway of the rat, figure out the excretion rate; study the effect of a Caspase inhibitoron the total contents of CYPs enzymes in rat liver microsomes and the expression of CYP1A2,CYP2D1, CYP2E1, CYP2C11, CYP3A1mRNA.Part I Preclinical ToxicokineticsMethodsUsing high performance liquid chromatography-mass spectrometry （LC-MS/MS） toanalysis Beagle dog plasma drug concentration, and investigate the following aspects:specificity, matrix effect, lower limit of quantification, accuracy and precision, recovery rateand sample stability.ResultsEndogenous substances in dog plasma samples does not interfere with the determinationof the sample; High, medium and low concentration of plasma sample matrix effect should be98.9%¹05.1%; The average of extraction is more than88%, the RSD of intra and inter day is less than14.5%; The range of quantitative linear is10⁵0000μ g· L^-1, the lowest limit ofquantification （LLOQ） is10μ g· L^-1.ConclusionsThe injection of drug at clinically effective doses of approximately5-40times thecircumstances, the drug in Beagle dogs metabolic rule is basically consistent with the linearpharmacokinetic characteristics. The software of SPSS11.5, in low, medium and high dosagecontinuous administration of different time （1d,15d,30d） Beagle dogs mainly toxicokineticsparameters AUC0-t, AUC0-, Cmax statistically analyzed, results show that, the dogs weretreated with different doses (2,6,15mg·kg^-1) for30days, the drugs in dogs without obviousaccumulation phenomenon, no significant gender differences between male and female.Part II Excretion a of a certain new drugMethodsSamples of urine, feces and bile were collected in different time after intramuscularinjection of drug in rats. The concentrations of drug in biological samples were determined byLC-MS/MS.ResultsAfter administration of36h, the urine discharged approximately35%,95%of which isdischarged in the first6h, the discharge after12h is little. No drug was detected out from feces;drug in the bile output about20%after8h,90%of which is discharged in the first2h, thedischarge after8h is little.ConclusionsIntramuscular injection in rats after taking drug excreted mainly in prototype via theurinary and biliary, these two ways taking up about55%of the total. Among them, The newdrug prototype drugs with bile excretion is about20%after12h accumulation; while the renalroute is about35%after36h accumulation. Part Ш Metabolism of a certain new drugMethods40Wistar rats were randomly divided into the control group, induction group, low,medium and high dose group. Control group to0.9%normal saline, and inducersDexamethasone group50mg·kg^-1/d，low(1.25mg·kg^-1/d)、medium(2.5mg·kg^-1/d)、high(5.0mg·kg^-1/d)dose group, were treated by intramuscularly administration for6consecutive days. Rat liver microsomal protein concentration and CYP enzyme content wasmeasured with ultraviolet spectrophotometry. The expression of CYP1A1, CYP2D1, CYP2E1,CYP2C11, and CYP3A1mRNA was detected with Real-time quantitative RT-PCR.ResultsThe result of the new drug on CYP total enzyme in rats indicated that the new drug hadthe inducing effect on drug metabolizing enzyme at low, medium, and high doses; Theexpression levels of CYP2D1increased significantly in the low doses compared with thecontrol group （P<0.05）；The expression levels of CYP1A2and CYP2E1also both increasedsignificantly in the medium doses （P<0.05）；There is no significant difference on the expressionlevel of CYP2C11between the new drug groups and the control group.ConclusionsThe new drug has effect on inducing CYP total enzyme at1.25,2.5or5.0mg·kg^-1/d, itcan raise the expression level of CYP2D1mRNA at1.25mg·kg^-1/d, CYP1A2andCYP2E1mRNA at2.5mg·kg^-1/d. It suggests that the induced mechanism of the new drug may ascribeto its effect on the relative expression level of the CYP450mRNA.