The Inhibition Of Danhong Injection On Liver Metabolism And Hepatic Uptake Of Atorvastatin

PurposeThe aim of this paper is to investigate the influence mechanism of pharmacokinetics of Danhong injection on atorvastatin,provide reference and guidance for Danhong injection combined with atorvastatin and other drugs.MethodsIn this research,the assays of cytochrome P450 enzymes and hepatic uptake transporters were conducted to investigate the influence mechanism of pharmacokinetics of Danhong injection on atorvastatin.?1?The pooled human liver microsomes were used as experimental model,testosterone was used as the probe substrate and ketoconazole was used as the positive inhibitor,to evaluate reliability and applicability of experimental model as well as to investigate the inhibiting effects of Danhong injection on CYP3A4 by inhibition assay of CYP450 enzymes.The variation of IC₅₀ value among three batches were also evaluated to better eliminate the influence of Danhong injection components's complexity.?2?The pooled human liver microsomes were used as the experimental model,and the experimental conditions of atorvastatin metabolism were evaluated,including incubation time,microsomal protein concentration and substrate concentration.Then,ketoconazole was used as the positive inhibitor,validate metabolic pathway of atorvastatin as well as investigate the inhibiting effects of Danhong injection on atorvastatin metabolism by inhibition assay of CYP450 enzymes,the variation of IC₅₀ value between the three batches of Danhong injection were also evaluated.?3?The hepatic uptake transport of atorvastatin were studied using OATP1B1 and OATP1B3 stably transfected HEK293 cells.?4?The OATP1B1 and OATP1B3 stably transfected HEK293 cells were used as experimental model,cyclosporin A was used as positive inhibitor,validate hepatic uptake of atorvastatin and evaluate inhibitory effect of uptake transport of Danhong injection on atorvastatin,the variation of IC₅₀value between the three batches of Danhong injection were also evaluated.Results?1?The IC₅₀ value of ketoconazole on CYP3A4 was 0.07?M,and the inhibitory rate at maximum concentration was over 95%.The inhibitory effect of Danhong injection on CYP3A4 was significantly,the IC₅₀ values were 0.60%?Lot No.:17041031?,0.50%?Lot No.:17061019?and 0.49%?Lot No.:17071008?,and no significant variability between the three batches of Danhong injection was detected.?2?The results showed that liver metabolism of atorvastatin was linear,within the incubation time of 5⁶0 minutes,within the microsomal protein concentrations of 0.0625² mg�mL^-1 and within the substrate concentration of 0.1⁵??.After a systematic consideration,the incubation time for 30min,the microsomal protein concentration at 0.25 mg�mL^-1,and substrate concentration at 0.5?M were as experimental conditions that evaluate inhibitory effect of inhibitor on atorvastatin metabolism.Ketoconazole had a significant inhibition on the metabolism of atorvastatin with an IC₅₀ value of0.05?M,and the inhibition rate at maximum concentration more than 95%.Danhong injection significantly inhibited the metabolism of atorvastatin,the IC₅₀ values were 0.43%?Lot No.:17041031?,0.35%?Lot No.:17061019?and 0.55%?Lot No.:17071008?,and the differences between three batches were not detected.?3?The experiment results showed that both transporters OATP1B1 and OATP1B3 showed significant role in hepatic uptake of atorvastatin.K_m and V_max of uptake of atorvastatin by OATP1B1 were 0.17?M and 19.79 pmol�mg protein^-1�min^-1 respectively;the K_m and V_max of uptake of atorvastatin by OATP1B3 were 0.83?M and 20.88 pmol�mg protein^-1�min^-1.?4?Cyclosporin A had significant inhibitory effect on the uptake of atorvastatin via OATP1B1 and OATP1B3 with the IC₅₀ of 0.52?M and 0.33?M,respectively,and the inhibitory rate at maximum concentration were over80%.Danhong injection significantly inhibited the uptake of atorvastatin by OATP1B1 and OATP1B3.The IC₅₀ values of Danhong injection on OATP1B1were 0.09%?Lot No.:17041031?,0.05%?Lot No.:17061019?and 0.07%?Lot No.:17071008?.The IC₅₀ values Danhong injection on OATP1B3 were0.06%?Lot No.:17041031?,0.06%?Lot No.:17061019?and 0.07%?Lot No.:17071008?.The variability between three batches was not detected.ConclusionThe enzyme metabolism experimental model and hepatic uptake transporter experimental model were developed and validated in this research.The results showed that CYP450 enzyme metabolism and OATPs stably transfected HEK293 assays were reproducible and reliable,and used for the evaluation of the inhibitory effects of Danhong injection on the liver metabolism and hepatic uptake of atorvastatin.The experimental results showed that Danhong injection could significantly inhibit atorvastatin metabolism and uptake transport process,and the inhibition of Danhong injection on uptake transport of atorvastatin was significantly greater than metabolism.No variability of three batches of Danhong injections was detected in inhibitory effected on CYP3A4,OATP1B1 and OATP1B3.Therefore,in combination of Danhong injection and atorvastatin in clinical usage,the potential risks of drug interactions should be considered.Patients should be strictly monitored the physical condition,and appropriately reduce the dosage of Danhong injection to avoid the occurrence of adverse reactions such as myopathy or rhabdomyolysis.At the same time,this research result has reference significance on Danhong injection with other drugs in combination with adverse reactions and medication safety.