Bsed On OAT1/3 And P-gp Exploring The Effects Of Wuzhi Capsule On The Pharmacokinetics Of Methotrexate

Part one Effects of Wuzhi Capsule on pharmacokinetics of Methotrexate in ratsObjective:To investigate the effects of different dosage of Wuzhi Capsule（WZC）on the pharmacokinetics of MTX in rats after single-and multiple-dose administration of WZC.Methods:Established a method for the determination of the concentration of Methotrexate（MTX）in rat plasma.36 male Sprague–Dawley rats were divided randomly into six groups of six rats each as follows:Group I received a single dose of 0.5%sodium carboxymethyl cellulose（CMC-Na）and 2 mg/kg MTX via oral gavage;Group II-III received a single dose of WZC（150 and 450 mg/kg for Group II and III,respectively）and 2mg/kg MTX;Group IV received 0.5%CMC-Na via oral gavage for seven consecutive days and 2 mg/kg MTX on the seventh day;Group V-VI received WZC（150 and 450 mg/kg）via oral gavage for seven consecutive days and 2mg/kg MTX on the seventh day.Blood samples were collected at different points.Methanol was used to precipitate proteins in plasma samples and the concentration of MTX in plasma was determined using UPLC-MS/MS.The pharmacokinetic parameters were calculated using the non-compartmental method with the DAS 2.1 pharmacokinetic software.Results:The calibraton curve for MTX in rat plasma samples have a good linearuty with the test concentration rangs from 1.03-515 ng/m L.The plasma methodologies met the requirements.The results of pharmacokinetic analysis showed that single-dose administration of 150 mg/kg WZC had no significant effect on the pharmacokinetic parameters of MTX.Following a single-dose administration of 450 mg/kg WZC,the AUC_0-24h and C_max of MTX increased 119%and 79%,the V_z and CL_z decreased 78%and 56%.The CL_zof MTX reduced by 34%with multiple-dose administration of 150 mg/kg WZC.After multiple doses of 450 mg/kg WZC,the AUC_0-24hand C_max of MTX increased 154%and 61%,the CL_z decreased 60%.Conclusions:A sensitive and specific UPLC-MS/MS method to quantify the concentration of MTX in rat plasma samples was established.The method can be used to determine the concentration of MTX in rat plasma and to study the effects of WZC on the pharmacokinetics of MTX.Pharmacokinetic results suggested that concomitant medication increased systemic exposure to MTX in vivo,and the possibility of drug interactions should be considered.Part two Effects of Wuzhi Capsule on the expression of transporters in ratsObjective:To investigate the effects of WZC on the m RNA and protein expression of OAT1/3 in the kidney and P-gp in the small intestine of rats and explore the role of OAT1/3 and P-gp in the interactions between WZC and MTX.Methods:Rats were anesthetized with pentobarbital,and the kidney and the small intestine were collected and stored at-80℃for later use.The m RNA and protein expression of transporters in different tissues was determined via reverse transcription quantitative PCR and Western blot.Results:Compared with control group,150 mg/kg WZC had no significant effect on the m RNA expression of OAT1/3 and P-gp.A single-dose administration of 450 mg/kg WZC inhibited the m RNA expression of OAT1in the kidney and P-gp in the small intestine by 57%and 54%.A multiple-dose administration of 450 mg/kg WZC inhibited the m RNA expression of OAT1 in the kidney and P-gp in the small intestine by 67%and64%.The m RNA expression of OAT3 was not significantly different from that of the control group.Compared with the control group,the protein expression of OAT1/3 in the single-dose administration group has no significant difference.The protein expressions of P-gp in the small intestine decreased37%and 66%with a single-dose administration of 150 mg/kg WZC and 450mg/kg WZC,respectively.The protein expression of OAT1,OAT3 and P-gp was inhibited by 64%,57%and 45%after multiple-dose treatment with 150mg/kg WZC.The protein expression of OAT1,OAT3 and P-gp was inhibited by 81%,89%and 83%after multiple-dose treatment with 450 mg/kg WZC.Conclusions:WZC can inhibit the expression of OAT1/3 in the kidney and P-gp in the small intestine.Single-and multiple-dose administration of WZC had the same effect on the trends of transporter expression.As the dosage of WZC increased,these effects became more obvious.The results implied that the expression of OAT1/3 in the kidney can be inhibited by WZC,which can lead to a reduction in the uptake of MTX in the kidney,and the inhibition of P-gp expression by WZC might cause a decrease in the non-renal clearance of MTX and an increase in the intestinal reabsorption of MTX,causing the increased systemic exposure to MTX in vivo.