Study On The Methodology And Pharmacokinetics In Rats Of A Novel GSK-3 Inhibitor And Its Effects On Liver And Kidney

ObjectivesAlzheimer’s disease(AD)is a chronic neurodegenerative disease that occurs frequently in the elderly.In recent years,due to the rapid growth of the world’s aging population,the prevalence of AD is becoming more and more serious,which has caused a huge economic burden on society,families and other aspects.Despite the continuous development of new drugs for the treatment of AD,the existing anti ad drugs can only delay symptoms,and many newly developed anti AD drugs are not easy to be approved.In the early stage,we designed and synthesized a new type of anti-AD lead compound PIMPC,which is a new type of glycogen synthase kinase-3(Glycogen Synthase Kinase-3,GSK-3)inhibitor,which has both antioxidant and metal chelation functions.In this experiment,a high-performance liquid chromatography(High-Performance Liquid Chromatography,HPLC)method for the determination of PIMPC was established through the exploration of chromatographic conditions,and the method was evaluated to be accurate and sensitive.After intragastric administration,the content of PIMPC in rat plasma was measured at different time points to understand the pharmacokinetic(PK)process of PIMPC in rats.By measuring the relevant indicators of the liver and kidney of rats after administration,the effect of PIMPC on the liver and kidney of rats was studied,and the safety of PIMPC under the pharmacodynamic dose was evaluated.The above research provided a certain reference basis for the development and research of PIMPC as a new anti-AD drug.Methods1.Establishment of methodology for determination of PIMPC1.1 The determination of chromatographic conditionsAn ultraviolet spectrophotometer was used to scan the prepared PIMPC solution at a full wavelength in the range of 200-600 nm,and the maximum absorption wavelength of the PIMPC solution,that is,the detection wavelength,was obtained from the scan spectrum.According to the characteristics of PIMPC,the chromatographic conditions of HPLC were explored.1.2 The determination of plasma sample pretreatment methodA certain volume of PIMPC solution was added into the prepared blank plasma of rats to obtain a certain concentration of PIMPC plasma sample.Different extractants and reconstituted solvents were selected to detect the actual PIMPC content in plasma samples,and then the best pretreatment method was selected by calculating the extraction recovery.1.3 Method validationThe established detection method was verified by specificity experiment,preparation of standard curve,determination of detection limit,determination of recovery rate,precision experiment and stability experiment.2.Pharmacokinetics of PIMPC in rats2.1 The treatment of experimental animalsHealthy male Wistar rats were given PIMPC suspension by gavage at a dose of 100 mg/kg.Blood samples were collected from jugular vein at 8 time points after administration,and then the obtained blood samples were anticoagulated with heparin and centrifuged to extract the supernatant to prepare plasma samples.According to the established HPLC method for the determination of pimpc,the samples were pretreated,and then injected to determine the content of PIMPC.2.2 pharmacokinetic analysisAccording to the data of the blood concentration and time,the PK process of PIMPC in rats was analyzed by Das 2.0 software,and the PK parameters were obtained.3.The effect of PIMPC on liver and kidney in rats3.1 The treatment of experimental animalsTwenty seven healthy male Wistar rats were randomly divided into three groups:negative control group,PIMPC low-dose group and PIMPC high-dose group,with 9 rats in each group.Each group was given PIMPC suspension at doses of 0,10 and 20 mg/kg,once a day for 4 weeks.After 24 hours of fasting,the rats were decapitated and the blood was collected.The liver and kidney were quickly removed at low temperature and stored in the refrigerator at-80℃.3.2 The effect of PIMPC on liver and kidney function of normal ratsThe collected blood was centrifuged after standing for a period of time,and the supernatant was drawn to obtain the serum sample.According to the operation instructions of the kit,the content of albumin(ALB),the activities of aspartate aminotransferase(AST),alanine aminotransferase(ALT)and alkaline phosphatase(ALP),and the contents of urea nitrogen(BUN),creatinine(CR)and uric acid(UA)in serum were determined by automatic biochemical analyzer.3.3 The effect of PIMPC on antioxidant system of liver and kidney in normal ratsThe liver and kidney were made into 10%homogenate.According to the operation instructions of the kit,the activities of xanthine oxidase(XOD),glutathione peroxidase(GSH-Px),superoxide dismutase(SOD),and the content of malondialdehyde(MDA)in rat liver and kidney were determined.Results1.Establishment of methodology for determination of PIMPC1.1 The determination of chromatographic conditionsHigh performance liquid chromatography;the detector:diode array detector;the detection wavelength:246nm;the chromatographic column:C18(250mm×4.6mm,5μm);the mobile phase:acetonitrile:water=60:40;the flow rate:0.8ml/min;the column temperature:25℃;the injection volume:20μL.1.2 The determination of plasma sample pretreatment methodIn the plasma sample,ethyl acetate was added in a ratio of 3 times,then the sample was whirled for 1 min.After centrifugation at 10000r/min for 10min,the supernatant was aspirated.The obtained supernatant was blown dry with nitrogen,reconstituted with acetonitrile to 100 μL,passed through a 0.45 μm filter membrane,and then injected to determine the PIMPC content.1.3 Method validationThe established method for detecting PIMPC has good specificity,and the retention time of the sample was 9.5 min.The method had a good linear relationship(R2=0.998).The standard curve equation was Y=572.23X+393.44,and its linear range was 2～35μg/L.The lower limit of quantification was 2 μg/L,and the limit of detection was 0.034 μg/L.The recovery rate of this method was 94.04%～95.53%,and the extraction effect was great.The precision of the instrument was high,and RSD was 0.63%;the intra day precision was good,and RSD was 0.76%～1.21%;the inter day precision was good,and RSD was 0.76%～1.12%.In addition,the RSD of the short-term stability,repeated freeze-thaw sample stability,long-term stability,and the stability of the PIMPC sample to be tested after the pretreatment of the PIMPC in plasma measured by this method was 0.02%- 0.67%,and it showed that the method had high stability.2.The results of the pharmacokinetic study of PIMPC in ratsThe PK process of rats after being intragastrically administered PIMPC conformed to the two-compartment open model.Atrioventricular parameters results:the t1/2α of PIMPC in rats was 7.823min;the Ka was 0.089;the t1/2α was 14.064 min;the V1 was 1.714 L/kg;the CL was 0.012 L/min/kg;the t1/2β was 69.315 min;the AUC(0-∞)was 8076.87 mg/L*min.At the same time,statistical moment method results:the MRT(0-t)of PIMPC in rats was 178.214min;the t1/2z was521.155min;the Tmaxwas 30 min;the Cmax was 26.79mg/L;the Vz was 7.746L/kg;the CLz was 0.01 L/min/kg;the AUC(0-t)was 4740.189mg/L*min.3.Research results of the effect of PIMPC on the liver and kidney in rats3.1 The effect of PIMPC on liver function of normal ratsCompared with the negative control group,there were no significant differences in the levels of ALB and the activity of AST,ALT,ALP in the serum of rats in the low-dose PIMPC group and high-dose PIMPC group(P>0.05).3.2 The effect of PIMPC on kidney function of normal ratsThere was no significant difference in the levels of BUN,CR and UA in the serum of rats in the negative control group,PIMPC low-dose group and PIMPC high-dose group(P>0.05).3.3 The effect of PIMPC on antioxidant system of liver in normal ratsThere was no significant difference in the activity of GSH-Px,SOD,XOD and the content of MDA in the liver tissues of the negative control group,low-dose PIMPC and high-dose PIMPC groups(P>0.05).3.4 The effect of PIMPC on antioxidant system of kidney in normal ratsThere was no significant difference in the activity of GSH-Px,SOD,XOD and the content of MDA in the kidney tissues of rats in the negative control group,low-dose PIMPC and high-dose PIMPC groups(P>0.05).Conclusions1.A high performance liquid chromatography method for the determination of PIMPC has been established,and it had been evaluated that this method had the advantages of high accuracy,good sensitivity,and good repeatability.2.The PK process of PIMPC in rats showed that it conformed to the characteristics of the two-compartment model,and had fast absorption,fast and wide distribution,and fast elimination.3.Long-term administration of PIMPC at a therapeutic dose did not affect the liver and kidney function of rats,nor did it damage the liver and kidney antioxidant systems.