本文選題：奈必洛爾　切入點：LC-MS/MS　出處：《中南大學》2014年博士論文　論文類型：學位論文

【摘要】：目的：本研究結合奈必洛爾主要代謝酶CYP2D6的基因多態(tài)性,研究其在中國人群的藥物動力學特征及其手性代謝特征和機制。 方法：血漿中奈必洛爾消旋體濃度采用LC-MS/MS法測定,對映體濃度采用手性柱進行拆分后,采用LC-MS/MS法進行測定。微粒體孵化體系中藥物及其代謝物濃度采用高效液相色譜儀進行測定。采用PCR-RFLP方法進行基因分型。 藥動學研究中,篩選16例受試者,其中CYP2D6*10突變純合子8例,野生型純合子3例,突變型雜合子5例。采用自身對照法進行試驗,單次給藥劑量為5mg、10mg；多次給藥為每天給藥1次,每次5mg,連續(xù)7天。在不同時間采樣,測定血藥濃度,計算藥動學參數(shù)。 體外代謝機制研究采用重組酶法和微粒體酶法,考察消旋體的酶催化機制及各對映體代謝的酶催化機制。 手性藥動學研究中,篩選基因分型符合規(guī)定的8例受試者(以CYP2D6*10突變位點和CYP2C19*2突變位點篩選基因型)。受試者基因型分別為CCAA、CCGG、TTAA、TTGG,各2例。單次口服給予10mg奈必洛爾后,采集血樣,測定藥物濃度,計算藥動學參數(shù)。 結果：LC-MS/MS法測定血漿中藥物濃度方法學可行,符合生物樣本測試的要求。采用手性柱可以有效分離血漿中奈必洛爾對映體,收集分離液后可以通過LC-MS/MS法進行測定奈必洛爾對映體濃度。 在中國人群中,奈必洛爾在5mg～10mg劑量范圍內符合線性動力學過程,5mg多次給藥也符合線性動力學過程累加,藥動學參數(shù)與國外文獻報道一致。CYP2D6*10突變受試者,藥物暴露有明顯增加趨勢,受個體差異和受試者例數(shù)等因素的影響,沒有顯示出統(tǒng)計學差異。 體外試驗證實,CYP2D6、CYP2C19、CYP3A4對奈必洛爾的代謝都有催化作用,CYP2D6和CYP2C19是主要催化酶,其中CYP2D6主要催化左旋體的代謝,CYP2C19主要催化右旋體的代謝。 體內手性代謝研究結果顯示,CYP2D6*10突變TT型具有較高的消旋體藥物暴露和較高的左旋體藥物暴露,CYP2C19*2突變GG型和AA型消旋體藥物暴露差別不明顯,但是右旋體藥物暴露有差別,AA型高于GG型。 結論：奈必洛爾在中國人群中藥動學受CYP2D6基因多態(tài)性的影響,CYP2D6*10突變純合子者具有較高的藥物暴露；CYP2C19基因突變對奈必洛爾藥動學影響較小。同時奈必洛爾各對映體受到CYP2D6和CYP2C19代謝程度不同。
[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.
【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R969.1

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