Experimental Study Of Midazolam As Probe On Evaluating Activity Of Hepatic CYP3A

Objective: To study the effects of selective inhibitor of CYP3A and acute liver injury on in vivo and in vitro metabolic activity of CYP3A in rat and to estimate the activity of CYP3 A with MDZ as probe.Methods: 1. HPLC assay was established to detect the contents of midazolam(MDZ) in plasma, liver homogenate and hepatocyte supernatant samples with diazepam as internal standard. 2. Loading doses of ketoconazole(KTZ), a selective inhibitor for CYP3A, were administrated through rat sublingual vein followed by subsequently constant infusion through tail vein to obtain different steady state concentrations for inhibited groups. Different doses of carbon tetrachloride(CCL,4) were administered orally to induce different grades of liver injury models. Then midazolam, a substrate of CYP3A, was injected through sublingual vein at the 2nd hour of infusion starting for KTZ groups and 22 to 24 hours after CCU administration for model groups. The blood and liver tissue were sampled at the different time points fot MDZ detection. 3. Two-step perfusion and collagenase digestion method was applied to separate hepatocytes in rat. Several doses of KTZ and CCI4 were added to hepatocyte suspension respectively. And then MDZ was administrated in suspending cultured hepatocytes. Extracted supernatant of 1 ml of suspension sampled at the according time points were used for MDZ assay.Results: 1. For in vivo study The parameters including T1/2B CL, AUC and T1/2 and K10 for elimination phase exhibited similar tendency for both KTZ and model groups. CL for both high dose groups were significantly lower than that of control and low dose ones. T1/2 was prolonged and K10 decreased. Moreover, statistic significance existed in high dose groups compared with low dose. 2. For in vitro study T1/2B and AUC for KTZ groups were sharply increased, while CLdeclined, compared with control. And there were statistic differences for these parameters among the doses groups. K10 were reduced and there was significant difference when compared high dose with median and low doses groups. AUC was sharply increased, while CL decreased for model groups, especially of high dose group compared with low dose. K10 was significantly reduced for median dose compared with low dose. Although T1/2B were drastically prolonged, there was no statistic difference among the three dose groups. 3. There was significant correlation between CL(30,120) and CLs in vivo and in vitro. And there were clear negative correlation between CL(30,120) and KTZ concentrations or CL(30,120) and CCI4 concentrations.Conclusion: It suggested that there was concordance between hepatic CYP3A inhibition and liver injury with midazolam as probe and the clearance derived from 30 and 120min plasma concentrations of MDZ would be a valid indicator for evaluating drug-metabolizing function of hepatic CYP3A.