奈必洛尔药动学及其手性代谢机制研究

发布时间：2018-03-13 18:07

本文选题：奈必洛尔　切入点：LC-MS/MS　出处：《中南大学》2014年博士论文　论文类型：学位论文

【摘要】：目的：本研究结合奈必洛尔主要代谢酶CYP2D6的基因多态性,研究其在中国人群的药物动力学特征及其手性代谢特征和机制。方法：血浆中奈必洛尔消旋体浓度采用LC-MS/MS法测定,对映体浓度采用手性柱进行拆分后,采用LC-MS/MS法进行测定。微粒体孵化体系中药物及其代谢物浓度采用高效液相色谱仪进行测定。采用PCR-RFLP方法进行基因分型。药动学研究中,筛选16例受试者,其中CYP2D6*10突变纯合子8例,野生型纯合子3例,突变型杂合子5例。采用自身对照法进行试验,单次给药剂量为5mg、10mg；多次给药为每天给药1次,每次5mg,连续7天。在不同时间采样,测定血药浓度,计算药动学参数。体外代谢机制研究采用重组酶法和微粒体酶法,考察消旋体的酶催化机制及各对映体代谢的酶催化机制。手性药动学研究中,筛选基因分型符合规定的8例受试者(以CYP2D6*10突变位点和CYP2C19*2突变位点筛选基因型)。受试者基因型分别为CCAA、CCGG、TTAA、TTGG,各2例。单次口服给予10mg奈必洛尔后,采集血样,测定药物浓度,计算药动学参数。结果：LC-MS/MS法测定血浆中药物浓度方法学可行,符合生物样本测试的要求。采用手性柱可以有效分离血浆中奈必洛尔对映体,收集分离液后可以通过LC-MS/MS法进行测定奈必洛尔对映体浓度。在中国人群中,奈必洛尔在5mg～10mg剂量范围内符合线性动力学过程,5mg多次给药也符合线性动力学过程累加,药动学参数与国外文献报道一致。CYP2D6*10突变受试者,药物暴露有明显增加趋势,受个体差异和受试者例数等因素的影响,没有显示出统计学差异。体外试验证实,CYP2D6、CYP2C19、CYP3A4对奈必洛尔的代谢都有催化作用,CYP2D6和CYP2C19是主要催化酶,其中CYP2D6主要催化左旋体的代谢,CYP2C19主要催化右旋体的代谢。体内手性代谢研究结果显示,CYP2D6*10突变TT型具有较高的消旋体药物暴露和较高的左旋体药物暴露,CYP2C19*2突变GG型和AA型消旋体药物暴露差别不明显,但是右旋体药物暴露有差别,AA型高于GG型。结论：奈必洛尔在中国人群中药动学受CYP2D6基因多态性的影响,CYP2D6*10突变纯合子者具有较高的药物暴露；CYP2C19基因突变对奈必洛尔药动学影响较小。同时奈必洛尔各对映体受到CYP2D6和CYP2C19代谢程度不同。
[Abstract]:Aim: to study the pharmacokinetic characteristics, chiral metabolic characteristics and mechanism of CYP2D6, a major metabolic enzyme of nebiprolol, in Chinese population. Methods: the racemic concentration of nebiprolol in plasma was determined by LC-MS/MS, and the enantiomer concentration was separated by chiral column. The concentrations of drugs and metabolites in microsomal incubation system were determined by high performance liquid chromatograph (HPLC) and genotyping by PCR-RFLP method. In pharmacokinetic study, 16 subjects were selected, including 8 cases of CYP2D6*10 mutation homozygote, 3 cases of wild-type homozygote and 5 cases of mutant heterozygote. The blood concentration was measured and the pharmacokinetic parameters were calculated. In vitro metabolism mechanism was studied by recombinant enzyme method and microsomal enzymatic method. The enzymatic catalytic mechanism of racemes and enantiomers was investigated. In the chiral pharmacokinetic study, the genotypes of 8 subjects (CYP2D6*10 mutation site and CYP2C19*2 mutation site) were selected. The genotypes were CCAACCGG, TTAATTGG, 2 cases each. Blood samples were collected after single oral administration of 10 mg nebiprolol. Drug concentration was measured and pharmacokinetic parameters were calculated. Results the method of determination of plasma drug concentration by the: LC-MS / MS / MS method is feasible and meets the requirements of biological sample test. The enantiomers of nebiprolol in plasma can be effectively separated by chiral column. The enantiomer concentration of nebiprolol can be determined by LC-MS/MS after collecting the separation solution. In Chinese population, the pharmacokinetic parameters of nebiprolol were in accordance with the linear kinetic process (5mg / 10mg) and the cumulative pharmacokinetic process. The pharmacokinetic parameters were in agreement with those reported in the foreign literature. The drug exposure showed an increasing trend, but there was no statistical difference due to the individual differences and the number of subjects. In vitro experiments confirmed that CYP2D6, CYP2C19, CYP3A4 can catalyze the metabolism of nebiprolol. CYP2D6 and CYP2C19 are the main catalytic enzymes, and CYP2D6 mainly catalyzes the metabolism of levoxisome and CYP2C19 mainly catalyzes the metabolism of dextral. The results of chiral metabolism in vivo showed that there was no significant difference between CYP2D6O10 mutation TT type and CYP2C19A-2 mutation GG type and AA type racemate drug exposure. But dextral drug exposure was higher in AA type than in GG type. Conclusion: the pharmacokinetics of nebiprolol was influenced by the polymorphism of CYP2D6 gene in Chinese population. The CYP2D6C19 mutation had a higher effect on the pharmacokinetics of nebiprolol in patients with CYP2D6C10 mutation. Meanwhile, the pharmacokinetics of nebiprolol was not affected by the mutation of CYP2C19 gene, and the enantiomers of nebiprolol were enantiomeric. The body was metabolized differently by CYP2D6 and CYP2C19.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R969.1

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