Study On The Influence Of Capsaicin On Rat Cytochrome P450 Isoforms By Cocktail Approach Using Probe Drugs

To study the in vitro and in vivo influence of Capsaicin on cytochrome P450 isoforms, which could provide a comprehensive understanding of the Capsaicin-drug interaction and a reference to the clinical practice.A Cocktail approach was used for the study of the in vitro and in vivo influence of Capsaicin on cytochrome P450 isoforms CYP1A2, CYP2C11, CYP2E1 and CYP3A4. In vitro study, the concentrations of each probe drug and their metabolites was determined by HPLC-DAD method, using phenacetin, tolbutamide, chlorzoxazone, testosterone as four probe drugs. A C18 analytical column and gradient elution were used for the chromatographic separation. Analytes were extracted simultaneously by SPE from rat liver microsomes. Good linearity (r≥0.9992) was observed over the concentration ranges investigated. In terms of precision, the intra-day and inter-day variation at three different concentrations in all analytes were less than 10.44%. The average relative recoveries for the analytes varied from 86.15% to 105.56%. The overall extraction recoveries for the analytes varied from 79.86% to 88.62%. The method was fast and accurate to determine the concentrations of phenacetin, tolbutamide, chlorzoxazone, testosterone and their metabolites in liver microsomes.The incubation in vitro was divided into capsaicin group and control group. Rat liver microsomes, probe drugs, Capsaicin (buffer solution in control group) and cofactors were cultured together for 20 min. When the reaction terminated, the concentrations of the metabolites were measured with HPLC to assess the enzyme activities. Then the relative activity of the four isoforms in the capsaicin group was calculated suppose the enzyme activity in control group was 100%. The result showed that the relative activity of the four isoforms decreased gradually when the concentrations of capsaicin increased. The IC₅₀ value of capsaicin on each isoform was determined to assess the inhibiton of capsaicin on the four isoforms in vitro using Graphpad prism 5.0. The result showed that the activity of the rats liver microsomes enzyme CYP450 isoforms CYP1A2, CYP2C11, CYP2E1 and CYP3A4 was inhibited by capsaicin in vitro with the IC₅₀ values of 36.21,17.19,51.64 and 18.86μmol·L^-1 respectively. While the inhibition on CYP2E1 was weak. Capsaicin and hepatic microsomess were preincubated respectively for 0, 5, 10, 15, 20, 30 min, and then the relative activity of the four isoforms at different time was calculated suppose the enzyme activity at 0 min was 100%. Whether there was mechanism-based inhibition of capsaicin on CYP450 was evaluated through preincubation of capsaicin and liver microsomes enzyme. After 30 min preincubation of capsaicin, no time-dependent manner was observed.According to the result of the in vitro study, caffeine, tolbutamide, dapsone was used as markers of CYP1A2, CYP2C11 and CYP3A4 and HPLC of simultaneous determination of three probe drugs in blood was established in vivo study. A C18 analytical column and gradient elution were used for the chromatographic separation. Analytes were extracted simultaneously using a liquid-liquid extraction from rat blood. Good linearity (r≥0.9960) was observed over the concentration ranges investigated. In terms of precision, the intra-day and inter-day variation at three different concentrations in all analytes were less than 11.54%. The average relative recoveries for the analytes varied from 92.84% to 108.93%. The overall extraction recoveries for the analytes varied from 67.59% to 84.79%. The method was fast and accurate to determine the concentrations of caffeine, tolbutamide, dapsone in blood samples.In vivo study, the male rat was divided into capsaicin group and control group. The capsaicin group were pretreated with 25 mg·kg^-1 capsaicin once a day for 7 days. The control group were given a vehicle solvent in a volume of 2 mL·kg^-1, once a day for 7 days. And the"Cocktail"probe drug was intraperitoneal injected after intragastric administration on the morning of the 7th day. Blood samples were withdrawn through tail clip at 0.17,0.5,1,1.5,2,3,4,6,9,12 and 24 h after injection of the"Cocktail"probe drugs. The concentration of the blood samples were determined by HPLC-DAD and the main pharmacokinetic parameters were calculated by DAS 2.1.1. The effects of capsaicin on CYP450 isoforms CYP1A2, CYP2C11, CYP3A4 were reflected by comparing the pharmacokinetic parameter of experimental and control groups. The result showed that T_1/2 of caffeine was shortened significantly, AUC_0-t and AUC_0-∞ of caffeine were declined significantly, CL was significantly increased. While C_max and T_max had no significance differences. Compared with control group, AUC_0-t and AUC_0-∞ of tolbutamide were significantly declined, CL was increased significantly while T_1/2, C_max and T_max had no significance differences. Whereas AUC_0-t and AUC_0-∞ of dapsone were declined significantly compared with control groups, CL was increased significantly while T_1/2, C_max and T_max had no significance differences.In conclusion, Capsaicin had inhibition on CYP1A2, CYP2C11 and CYP3A4 in rat liver microsomes in vitro and marginal effect on CYP2E1. While in vivo, capsaicin had obvious induction on CYP1A2, CYP2C11 and CYP3A4 in rats.