UPLC-MS用于体外大鼠肝微粒体CYP1A2和CYP2D1酶代谢活性表征及动力学研究
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摘要
目的建立大鼠肝微粒体CYP1A2和CYP2D1酶活性的快速测定方法并考察两种CYP酶反应动力学。方法利用CYP1A2和CYP2D1选择性探针底物即非那西丁与右美沙芬进行温孵实验,采用超高压液相色谱与质谱联用(UPLC-MS)的方法建立相应代谢产物的含量测定方法。结果非那西丁代谢产物对乙酰氨基酚的线性范围为0.409~10.227μmol·L-1(r2=0.999 1),LOD和LOQ值分别为0.067和0.267μmol·L-1;右美沙芬代谢产物去甲右美沙芬为0.020~5.051μmol·L-1(R2=0.998 0),LOD和LOQ值分别为0.002和0.007μmol·L-1。方法回收率分别为95.9%~104.0%和102.1%~107.1%,精密度以RSD值表示均低于5%。CYP酶活性测试及反应动力学表明,CYP1A2和CYP2D1的米氏常数(Km值)分别为(28.4±2.7)和(13.9±1.3)μmol·L-1;其代谢活性(底物浓度为10μmol·L-1时)分别为(1.47±0.12)和(3.98±0.09)nmol·mg-1。结论本实验建立的UPLC-MS分析方法快速、灵敏、准确、可靠,适用于药物代谢研究中CYP1A2和CYP2D1体外活性测定及其反应动力学分析。
OBJECTIVE To establish a robust,fast and convenient method for in vitro assay of rat liver CYP1A2 and CYP2D1,and explore their kinetic features. METHODS Two selective substrates including phenacetin and dextromethorphan,which are probes of CYP1A2 and CYP2D1,were chosen for liver microsomes incubation,respectively; the corresponding ultra performance liquid chromatography tandem mass spectrometry( UPLC-MS) methods were developed for kinetic studies. RESULTS The fast and convenient UPLC-MS methods with high resolution and short running time( 4 ~ 5 min) were established and validated for two assays of CYP1A2 and CYP2D1 activities; both methods showed good accuracy and precision,and the values of LOQ for CYP1A2 and CYP2D1 assays could reach 0. 267 and 0. 007 μmol·L- 1,respectively. The kinetic studies showed that the Michaelis constant( Km) for CYP1A2 and CYP2D1 were( 28. 4 ± 2. 7) and( 13. 9 ± 1. 3) μmol·L- 1,respectively. Their activities were determined to be( 1. 47 ± 0. 12) and( 3. 98 ± 0. 09) nmol·mg- 1,respectively,when the substrate concentration was 10 μmol·L- 1. CONCLUSION UPLC Tandem MS technique is proved to be a rapid,convenient and efficient approach with high sensitivity and selectivity for the assays of CYP1A2 and CYP2D1 in drug metabolism.
OBJECTIVE To establish a robust,fast and convenient method for in vitro assay of rat liver CYP1A2 and CYP2D1,and explore their kinetic features. METHODS Two selective substrates including phenacetin and dextromethorphan,which are probes of CYP1A2 and CYP2D1,were chosen for liver microsomes incubation,respectively; the corresponding ultra performance liquid chromatography tandem mass spectrometry( UPLC-MS) methods were developed for kinetic studies. RESULTS The fast and convenient UPLC-MS methods with high resolution and short running time( 4 ~ 5 min) were established and validated for two assays of CYP1A2 and CYP2D1 activities; both methods showed good accuracy and precision,and the values of LOQ for CYP1A2 and CYP2D1 assays could reach 0. 267 and 0. 007 μmol·L- 1,respectively. The kinetic studies showed that the Michaelis constant( Km) for CYP1A2 and CYP2D1 were( 28. 4 ± 2. 7) and( 13. 9 ± 1. 3) μmol·L- 1,respectively. Their activities were determined to be( 1. 47 ± 0. 12) and( 3. 98 ± 0. 09) nmol·mg- 1,respectively,when the substrate concentration was 10 μmol·L- 1. CONCLUSION UPLC Tandem MS technique is proved to be a rapid,convenient and efficient approach with high sensitivity and selectivity for the assays of CYP1A2 and CYP2D1 in drug metabolism.
引文
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[2]GURLEY B J.Pharmacokinetic herb-drug interactions(Part 1):Origins,mechanisms,and the impact of botanical dietary supplements[J].Plant Med,2012,78(13):1478-1489.
[3]ZHOU S F,XUE C C,YU X Q,et al.Metabolic activation of herbal and dietary constituents and its clinical and toxicological implications:An update[J].Curr Drug Metab,2007,8(6):526-553.
[4]HU Y Z,YAO T W.Advance in cytochrome P450 1A[J].Chin Pharm J(中国药学杂志),2003,38(4):246-250.
[5]GURLEY B J,FIFER E K,GARDNER Z.Pharmacokinetic herbdrug interactions(Part 2):Drug interactions involving popular botanical dietary supplements and their clinical relevance[J].Plant Med,2012,78(13):1490-1514.
[6]LIU C X,YI X L,SI D Y,et al.Herb-drug interactions involving drug metabolizing enzymes and transporters[J].Curr Drug Metab,2011,12(9):835-849.
[7]TANG J,HATTORI M.Pyrrolizidine alkaloids-containing Chinese medicines in the Chinese pharmacopoeia and related safety concerns[J].Acta Pharm Sin(药学学报),2011,46(7):762-772.
[8]BOCHNER F,SOMOGYI A A,CHEN Z R.Dextromethorphan metabolism in rat:Interstrain differences and the fate of individually administered oxidative metabolites[J].Xenobiotica,1994,24(6):543-552.
[9]XIA D Y,ZUO J L,ZHAO D X,et al.Genetic polymorphism analysis of cytochrome CYP3A4,CYP2C9,CYP2C19,CYP2D6 in Chinese Han and Mongolian population[J].Chin Pharm J(中国药学杂志),2012,47(24):2017-2021.
[10]TANG J,AKAO T,NAKAMURA N,et al.In vitro metabolism of isoline,a pyrrolizidine alkaloid from Ligularia duciformis,by rodent liver microsomal esterase and enhanced hepatotoxicity by esterase inhibitors[J].Drug Metab Disp,2007,35(10):1832-1839.
[11]BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding[J].Anal Biochem,1976,72(1-2):248-254.
[12]HE Y Q,YANG L,LIU Y,et al.Characterization of cardamonin metabolism by P450 in different species via HPLC-ESI-ion trap and UPLC-ESI-quadrupole mass spectrometry[J].Acta Pharmacol Sin(中国药理学报),2009,30(10):1462-1470.
[13]ROBERT L W,OBACH R S.Validated assays for human cytochrome P450 activities[J].Drug Metab Disp,2004,32(6):647-660.