Studies On Metabolic Drug-Drug Interactions Of Mailuoning Injection

1 Effect of Mailuoning injection on activities of mice CYP2E1 and CYP3AObjective:To investigate the effect of Mailuoning Injection on activities of total CYP450, CYP2E1 and CYP3A in mice.Methods:Ultraviolet-visible spectrometry(UV) has been applied to determine contents of hepatic microsomal protein at 595nm,CYP450 at 450nm and formaldehyde at 412nm.The statistical differences of liver coefficient,hepatic microsomal protein content,CYP450 content and formaldehyde formation rate between dosing group mice (normal-dose group and higher-dose group)and control group mice(physical saline group and phenobarbital group)were evaluated by One-way Analysis of Variance(One-way ANOVA).Results:Two doses of Mailuoning Injection had no significant impact on mice liver coefficient and CYP450 content(P＞0.05),but both might increase hepatic microsomal protein content;Two doses of Mailuoning Injection decreased the formation rate of aminopyrine to formaldehyde;the formation rate of erythromycin to formaldehyde has been decreased by a dose of 2 mL·kg^-1Mailuoning Injection,but not affected by a dose of 4mL·kg^-1 Mailuoning Injection.Conclusion:Both doses of Mailuoning Injection could inhibit activity of CYP2E1;Meanwhile normal dose had no significant effect on activity of CYP3A,but higher dose could inhibit activity of CYP3A.2 Pharmacokinetic drug-drug interaction of theophylline,chlorzoxazone and dapsone in ratsObjective:To investigate the possibility of pharmacokinetic drug-drug interactions of theophylline,chlorzoxazone and dapsone in rats.Methods:At first,a selective and reliable high-performance liquid chromatographic method(HPLC) was developed for simultaneous determination of theophylline,chlorzoxazone and dapsone in rat plasma,in which the plasma sample was prepared by liquid-liquid extraction with dichloromethane and evaporated to dryness under a gentle stream of nitrogen.21 rats were randomly divided into 3 groups(n=7),namely theophylline group,chlorzoxazone group and dapsone group.Each group rats underwent 2-cycle pharmacokinetic experiments.In the first cycle,3-group rats were individually given theophylline(30mg·kg^-1),chlorzoxazone(50mg·kg^-1) and dapsone(20mg·kg^-1) by gastrogavage, respectively.Blood was withdrawn by orbital bleeding at different time intervals in different time courses for 3 groups(24h for theophylline group,8h for chlorzoxazone goup and 27h for dapsone group,respectively).The plasma samples separated from whole blood by centrifugation at 5000 rpm for 10min were stored frozen at -20℃until required for analysis.After 14-day period of cleanout and recovery,3-group rats were concomitantly given theophylline, chlorzoxazone and dapsone by gastrogavage with the same doses as the first cycle.Then steps in the first cycle were repeated in the second cycle.The plasma concentrations of three probes were measured by the HPLC method developed,and DAS 1.0 software was utilized to fit the concentration-time profiles of 3 compounds and calculate their corresponding pharmacokinetic parameters,including t_1/2,AUC,CL/F and Cmax.The statistical differences exsisting within pharmacokinetic parameters of three probes in the two cycles were individually evaluated by Paired T-test and 0.05 was set as the significance level in order to see whether the pharmacokinetic interaction among 3 probes may occur.Results:In the 2-cycle pharmacokinetic experiments,no significant differences(P＞0.05) exsisted within pharmacokinetic parameters t_1/2, AUC,CL/F and Cmax for all three probes.Conclusion:When theophylline,chlorzoxazone and dapsone were concomitantly administered,their pharmacokinetic drug-drug interactions did not occur.Consequently,they can be used together as a Cocktail to simultaneously evaluate the effects of drug(s) on activities of CYP1A2,CYP2E1 and CYP3A4.3 Effects of Mailuoning Injection on activities of rat CYP1A2,CYP2E1 and CYP3A4Objective:To investigate the effects of Mailuoning Injection on activities of rat CYP1A2, CYP2E1 and CYP3A4.Methods:14 rats were divided randomly into two groups(n=7),i.e normal-dose group and higher-dose group.Each group underwent 2-cycle pharmacokinetic experiments.In the first cycle,2-group rats were concomitantly administered theophylline (30mg·kg^-1),chlorzoxazone(50mg·kg^-1) and dapsone(20 mg·kg^-1),blood was withdrawn by orbital bleeding at different time intervals within 24h.The plasma samples separated from whole blood by centrifugation at 5000 rpm for 10min were stored frozen at -20℃until required for analysis.The next day,according to clinical dosage,rats were intravenously given Mailuoning Injection(2mL·kg^-1 for normal-dose group and 4mL·kg^-1 for higher-dose group,respectively) for 14 consecutive days.On the 16th day,steps in the first cycle were repeated in the second cycle.The probe concentrations in rat plasma were quantitiated by the HPLC method developed in the above pharmacokinetic probe-probe interaction studies.DAS 1.0 software was utilized to fit the concentration-time profiles of 3 probes and calculate their corresponding pharmacokinetic parameters,including t_1/2,AUC,CL/F and Cmax.The statistical differences among the parameters in the two cycles were evaluated by Paired T-test and 0.05 was set as the significance level in order to see whether two doses of Mailuoning Injection affected the the pharmacokinetic behavior of 3 probes in rats.Results:In the 2-week dosing period,normal-dose Mailuoning Injection did not affect the pharmacokinetic parameters of 3 probes(P＞0.05);Meanwhile,after being treated with higher-dose Mailuoning Injection,there were no significant differences for theophylline's pharmacokinetics in rats,but dapsone and chlorzoxazone's average AUC0-24h increased significantly(1.27 and 1.44 times larger than before being treated,respectively) and chlorzoxazone's CL/F became smaller.Conclusion:Normal-dose Mailuoning Injection did not affect the activities of rat CYP1A2,CYP2E1 and CYP3A4;higher-dose Mailuoninh Injection could not change CYP1A2 activity,but could weakly inhibit activities of CYP2E1 and CYP3A4.4 Impact of Aspirin in combination with Mailuoning Injection on pharmacokinetics of salicylic acid in ratsObjective:To investigate whether Aspirin in combination with Mailuoning Injection could affect pharmacokinetic parameters of salicylic acid in rats.Methods:At first,a selective and reliable high-performance liquid chromatographic method(HPLC) was developed for quantitation of salicylic acid in rat plasma,in which plasma sample was deproteined with acetonitrile.16 rats were divided randomly and equally into Aspirin group(ASP group) and Combination group (CM group).According to the clinically equivalent dosage,namely 7.5mg·kg^-1 for Aspirin and 3mL·kg^-1 for Mailuoning Injection,ASP group rats were given Aspirin alone and CM group rats were given Aspirin together with Mailuoning Injection for 8 consecutive days.In the first day and eighth day,2-group rats were blood-withdrawn from orbital bleeding at different time intervals in 14h for pharmacokinetic studies.The HPLC method developed was used to quantitate plasma concentrations of salicylic acid,and DAS 1.0 software was utilized to fit the concentration-time profile of salicylic acid and calculate the corresponding pharmacokinetic parameters,including t_1/2,AUC,CL/F and Cmax.The statistical differences among the parameters between the ASP and CM group rats were evaluated by Independent Samples T-test and 0.05 was set as the significance level in order to see whether single-dose and multiple-dose Aspirin in combination with Mailuoning Injection may affect pharmacokinetics of salicylic acid in rats.Results:After single-dose administration,in contrast with ASP group,the CM group rats' pharmacokinetic parameters t_1/2,AUC_0-14h and AUC_0-∞decreased significantly,CL/F increased significantly,and Cmax changed insignificantly;but after multiple-dose administration,the parameters t_1/2,AUC_0-14h,AUC_0-∞,CL/F and Cmax exhibited insignificant differences(P＞0.05) between ASP and CM group rats.Conclusion:Single-dose Mailuoning Injection could fasten clearing of salicylic acid from the body in rats;but mulitiple-dose Mailuoning Injection failed.