替格瑞洛的体外代谢及其与他汀类药物的相互作用研究

目的 考察替格瑞洛在大鼠肝微粒体的酶动力学及其与CYP3A的底物药物辛伐他汀、洛伐他汀及阿托伐他汀的相互作用,以期为临床上替格瑞洛与他汀类药物的合理使用提供科学依据。方法 将替格瑞洛与大鼠肝微粒体进行体外共孵育,孵育一定时间后用含有内标(地西泮,10 ng·mL^-1)的甲醇终止反应,并沉淀蛋白,14 000 r·min^-1离心10 min后取上清液进行分析,采用 LC-MS/MS测定微粒体酶孵育体系中活性代谢产物AR-C124910XX的浓度,使用 Prism 5软件计算主要的酶促动力学参数K_m,V_max与CL_int。在得到 K_m后,反应体系中底物替格瑞洛的浓度选择为1/3K_m~3K_m内的3个浓度,辛伐他汀、洛伐他汀及阿托伐他汀的浓度范围为1~100 μmol·L^-1,通过体外大鼠肝微粒体代谢实验研究其与替格瑞洛代谢性相互作用。使用 SigmaPlot 12.3 软件酶动力学模块,根据 Dixon公式计算各他汀对替格瑞洛代谢的可逆性抑制常数 K_i,并根据标准差值选择最符合的模型。结果 替格瑞洛在大鼠肝微粒体的代谢符合米氏反应动力学,其转化生成AR-C124910XX的K_m值为32.2 μmol·L^-1,V_max为149.0 pmol·min^-1·mg(pro)^-1。表观清除率CL_int为4.63 nL·min^-1·mg(protein)^-1;辛伐他汀显著抑制替格瑞洛活性代谢产物的生成,其抑制符合混合抑制模型。抑制常数K_i为0.58 μmol·L^-1,α值为7.5,β值为0.56。洛伐他汀及阿托伐他汀对替格瑞洛代谢呈现出中等强度的抑制作用,其抑制亦符合混合抑制模型,抑制常数K_i分别为2.9和7.5 μmol·L^-1,α 值分别为3和1.7,β值为0.34和0.29。 结论 替格瑞洛在大鼠肝微粒体的代谢符合米氏反应酶动力学;辛伐他汀对替格瑞洛有显著程度的代谢性抑制作用,洛伐他汀和阿托伐他汀对替格瑞洛有中等程度的抑制作用,临床上替格瑞洛与他汀类药物合用时可能需要考虑其与辛伐他汀的相互作用。

In Vitro Metabolism of Ticagrelor and Its Metabolic Interactions with Statins

OBJECTIVE To study the in vitro metabolism of ticagrelor and investigate the drug-drug interactions between ticagrelor and some commonly used statins (simvastatin, lovastatin and atorvastatin), expecting to provide some useful information for the rational use of ticagrelor in clinic. METHODS The metabolic reaction of ticagrelor was performed in rat liver microsomes. After incubation for a given time, the reactions were stopped with methanol, as well as to precipitate the protein. For drug-drug interactions study, statins (simvastatin, lovastatin and atorvastatin) were selected, the interactions between ticagrelor and these drugs were explored in vitro using rat liver microsomes. The concentrations of ticagrelor active metabolite, AR-C124910XX in the incubation system were determined by a validated LC-MS/MS method. The main enzyme kinetic parameters K_m, V_max and CL_int were calculated using Prism 5 software. After K_m was obtained, the concentrations of the substrate ticagrelor in the reaction system were selected as three concentrations in the range of 1/3K_m-3K_m, and the concentrations of simvastatin, lovastatin and atorvastatin were in the range of 1-100 μmol·L^-1. The metabolic interactions with ticagrelor were investigated by in vitro rat liver microsomal metabolism assays. The reversible inhibition constant K_i for each statin against ticagrelor metabolism was calculated according to the Dixon formula utilizing the SigmaPlot 12.3 software enzyme kinetics module, and the model that best fits was selected based on the standard deviation. RESULTS The metabolism of ticagrelor in rat liver microsomes was in accordance with Michaelis-Menten enzymatic kinetics. The K_m value of ticagrelor transformed to AR-C124910XX was 32.2 μmol·L^-1, and V_max was 149.0 pmol·min^-1·mg(pro)^-1. The apparent clearance rate, CL_int was 4.63 nL·min^-1·mg(protein)^-1. Drug-Drug interaction studies indicated that simvastatin significantly inhibited the production of ticagrelor active metabolite, and its inhibition was consistent with mixed inhibition with an inhibition constant K_i of 0.58 μmol·L^-1, α of 7.5 and β of 0.56. Lovastatin and atorvastatin showed moderate mixed inhibition of ticagrelor with inhibition constants K_i of 2.9 and 7.5 μmol·L^-1, α of 3 and 1.7, β of 0.34 and 0.29, respectively. CONCLUSION The metabolism of ticagrelor in rat liver microsomes are in accordance with Michaelis-Menten kinetics. Simvastatin inhibits the metabolism of ticagrelor significantly in a concentration dependent manner. Lovastatin and atorvastatin exhibit moderate inhibition of ticagrelor. When ticagrelor is prescribed in combination with these statin drugs, the interactions of them should not be ignored in clinic, especially for simvastatin.