泰妙菌素对人CYP3A4、CYP1A2、CYP2E1、CYP2D6酶活性的影响

doi:10.16431/j.cnki.1671-7236.2020.04.034

摘要/Abstract

摘要： 本研究通过建立人肝微粒体体外孵育试验，考察泰妙菌素对4种CYP450亚酶的探针底物睾酮、非那西丁、氯唑沙宗、氢溴酸右美沙芬代谢的影响，以反映泰妙菌素对人CYP3A4、CYP1A2、CYP2E1、CYP2D6酶活性的作用。试验分为3组：药物试验组、阳性对照组（不含NADPH）、阴性对照组（不含CYP450抑制剂），孵育体系为100 μL，孵育试验在96孔板中进行。终止反应后使用高效液相色谱串联质谱仪（LC-MS/MS），以内标法检测96孔板中孵育液的剩余探针底物浓度。根据药物试验组与阴性对照组的探针药物代谢浓度之比，计算药物试验组的探针药物代谢率。使用Graphpad Prism 6.0软件，以药物试验组相对代谢率为纵坐标，药物浓度对数值为横坐标作图，计算试验组药物IC₅₀值。针对泰妙菌素与CYP3A4的孵育试验设置多个孵育时间点观察孵育时间对IC₅₀值的影响。试验结果显示，酮康唑对CYP3A4的IC₅₀值为0.044 μmol/L，α-萘黄酮对CYP1A2的IC₅₀值为0.030 μmol/L、4-甲基吡唑对CYP2E1的IC₅₀值为0.022 μmol/L、奎尼丁对CYP2D6的IC₅₀值为0.096 μmol/L。泰妙菌素对CYP1A2及CYP2D6的IC₅₀值均大于50 μmol/L，对CYP2E1的IC₅₀值为0.045 μmol/L，对CYP3A4的IC₅₀值为1.609 μmol/L。延长泰妙菌素与CYP3A4的孵育时间（10~50 min）后，泰妙菌素对CYP3A4的IC₅₀值由1.609 μmol/L增加至 8.657 μmol/L。本研究中4种亚酶常用抑制剂的IC₅₀值与参照值相近，表明所建立人肝微粒体体外孵育试验方法可靠。以IC₅₀值为指标显示泰妙菌素对CYP1A2和CYP2D6无抑制作用，对CYP2E1和CYP3A4存在强抑制作用，泰妙菌素可能是CYP3A4的可逆性抑制剂。

关键词: 泰妙菌素; 肝微粒体; CYP3A4; CYP2E1; CYP1A2; CYP2D6; IC₅₀值

Abstract: In this study,the in vitro CYP450 incubation assays were established using human liver microsomes (HLMs) to investigate the effects of Tiamulin on the metabolism of four substrate probes Testosterone,Phenacetin,Chlorzoxazone,and Dextromethorphan hydrobromide which reflect the effect of Tiamulin on CYP3A4,CYP1A2,CYP2E1 and CYP2D6 activity.Experiments were performed in 96-well plates with final volume of 100 μL in each well.The study consisted of three groups:a drug test group,a positive control group (without NADPH) and a negative control group (without CYP450 inhibitor).After incubation were terminated,the concentrations of probe substrates in each well were analyzed by LC-MS/MS internal standard method.The percentage of test drug metabolic was calculated from the ratio of the amount of drug metabolized of the drug test group and that of the negative control group.The percentage of test drug metabolic was plotted versus the logarithm of drugs concentration and the IC₅₀ values of test drugs were obtained from the graph using Graphpad prism 6.0.Multiple incubation time points were set to investigate the effect of the incubation time on the IC₅₀ value of Tiamulin against CYP3A4.The results showed that the IC₅₀ value of Ketoconazole against CYP3A4 was 0.044 μmol/L,the IC₅₀ value of α-Naphthoflavones against CYP1A2 was 0.030 μmol/L,the IC₅₀ value of 4-Methylpyrazole against CYP2E1 was 0.022 μmol/L and the IC₅₀ value of Quinidine against CYP2D6 was 0.096 μmol/L.The IC₅₀ values of Tiamulin against CYP1A2 and CYP2D6 were higher than 50 μmol/L.The IC₅₀ values of Tiamulin against CYP3A4 and CYP2E1 were 1.609 and 0.045 μmol/L,respectively.As the incubation time of Tiamulin and CYP3A4 prolonged from 10 min to 50 min,the IC₅₀ value of Tiamulin against CYP3A4 was increased from 1.609 μmol/L to 8.657 μmol/L.For the IC₅₀ values of the recognized inhibitors were consistent with the reference values,it could be concluded that the in vitro CYP450 incubation assays were constructed successfully.Tiamulin possessed no inhibition against CYP1A2 and CYP2D6,while existed a high inhibitory effect on CYP2E1 and CYP3A4.Tiamulin might be developed as a reversible inhibitor against CYP3A4 in the future.

Key words: Tiamulin; liver microsomes; CYP3A4; CYP2E1; CYP1A2; CYP2D6; IC₅₀ value

中图分类号:

S859.7

陈芳, 高鸿, 李博, 左香溢, 靳珍, 汤有志. 泰妙菌素对人CYP3A4、CYP1A2、CYP2E1、CYP2D6酶活性的影响[J]. 中国畜牧兽医, 2020, 47(4): 1267-1273.

CHEN Fang, GAO Hong, LI Bo, ZUO Xiangyi, JIN Zhen, TANG Youzhi. Effects of Taimulin on Human CYP3A4,CYP1A2,CYP2E1 and CYP2D6 Activity[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(4): 1267-1273.

参考文献

[1] DUIJKEREN E V,GREKO C,PRINGLE M,et al.Pleuromutilins:Use in food-producing animals in the European Union,development of resistance and impact on human and animal health[J].Journal of Antimicrobial Chemotherapy,2014,69(8):2022-2031.
[2] 王媛媛,孙淑芳,庞素芬,等.全球兽用抗菌药物使用情况[J].中国动物检疫,2018,35(4):62-65. WANG Y Y,SUN S F,PANG S F,et al.Global usage of veterinary antimicrobial agents[J].China Animal Health Inspection,2018,35(4):62-65.(in Chinese)
[3] KEMPER N.Veterinary antibiotics in the aquatic and terrestrial environment[J].Ecological Indicators,2008,8(1):10-13.
[4] MIN P,CHU L M.Fate of antibiotics in soil and their uptake by edible crops[J].Science of the Total Environment,2017,599-600:500-512.
[5] PAN M,CHU L M.Transfer of antibiotics from wastewater or animal manure to soil and edible crops[J].Environmental Pollution,2017,231:829-836.
[6] SCHL SENER M P,ARB M A V,BESTER K.Elimination of Macrolides,Tiamulin,and Salinomycin during manure storage[J].Archives of Environmental Contamination&Toxicology,2006,51(1):21-28.
[7] BEN W,QIANG Z,ADAMS C,et al.Simultaneous determination of Sulfonamides,Tetracyclines and Tiamulin in swine wastewater by solid-phase extraction and liquid chromatography-mass spectrometry[J].Journal of Chromatography A,2008,1202(2):173-180.
[8] BORAK M D,SARC L,MUGERLI D G,et al.Occupational inhalation poisoning with the veterinary antibiotic Tiamulin[J].Clinical Toxicology,2019,127:233-239.
[9] JEONG S H,KANG D J,LIM M W,et al.Risk assessment of growth hormones and antimicrobial residues in meat[J].Toxicological Research,2010,26(4):301-313.
[10] Mossad G A,El-SAYED,ASHRAF,et al.Evaluation of teratogenic potentials and tissue residues of Tiamulin in albino rats[J].Nature and Science,2014,12(1):100-105.
[11] ISVORAN A,LOUET M,VLADOIU D L,et al.Pharmacogenomics of the cytochrome P4502C family:Impacts of amino acid variations on drug metabolism[J].Drug Discovery Today,2017,22(2):366-376.
[12] MICHAELS S M,WANG M Z.The revised human liver cytochrome P450"Pie":Absolute protein quantification of CYP4F and CYP3A enzymes using targeted quantitative proteomics[J].Drug Metabolism&Disposition the Biological Fate of Chemicals,2014,42(8):1241.
[13] 舒舟,翟学佳,刘金梅,等.Cocktail探针药物法评价辣椒素对大鼠CYP450亚型的影响[J].中国药师,2014,17(10):1613-1619. SHU Z,ZHAI X J,LIU J M,et al.Effects of capsaicin on rat cytochrome P450 isoforms by cocktail probe drug method[J].China Pharmacist,2014(10):1613-1619.(in Chinese)
[14] NEBERT D W,RUSSELL D W.Clinical importance of the cytochromes P450[J].Lancet,2002,360(9340):1155-1162.
[15] STRESSER D M,BROUDY M I,HO T,et al.Highly selective inhibition of human CYP3A in vitro by azamulin and evidence that inhibition is irreversible[J].Drug Metabolism&Disposition the Biological Fate of Chemicals,2004,32(1):105.
[16] SEVRIOUKOVA I F.Structural insights into the interaction of cytochrome P4503A4 with suicide substrates:Mibefradil,Azamulin and 6',7'-Dihydroxybergamottin[J].International Journal of Molecular Sciences,2019,20(17):4245.
[17] RUOFENG S,XIUYING P,XIMING X,et al.Synthesis and biological activities of novel pleuromutilin derivatives with a substituted thiadiazole moiety as potent drug-resistant bacteria inhibitors[J].Journal of Medicinal Chemistry,2014,57(13):5664-5678.
[18] KRULL I.Handbook of analytical validation[M].Method Validation Basics,2012.
[19] YANG S,QIU Z,ZHANG Q,et al.Inhibitory effects of calf thymus DNA on metabolism activity of CYP450 enzyme in human liver microsomes[J].Drug Metabolism and Pharmacokinetics,2014,29(6):475-481.
[20] 赵秀红.创新药物对人肝微粒体CYP450酶体外抑制评价体系的建立与应用[D].天津:天津医科大学,2016. ZHAO X H.Establishment and application of an in vitro evaluation system for exploring the inhibition on CYP450 enzymes of human liver microsomes by new drugs[D].Tianjin:Tianjin Medical University,2016.(in Chinese)
[21] 徐振兴.CYP酶抑制试验中低溶解度抑制剂IC_(50)偏高问题的解决方案研究[D].苏州:苏州大学,2015. XU Z X.High IC₅₀ problem-solution of low solubility inhibitors in cytochrome P450 inhibition assays[D].Suzhou:Soochow University,2015.(in Chinese)
[22] FOOD AND DRUG ADMINISTRATION.Clinical pharmacology review[DB/OL].(2012-11-17)[2019-10-25].https://www.fda.gov/media/86818/download.
[23] YING L,JIANJIE J,CAILI Z,et al.A simplified method to determine five cytochrome p450 probe drugs by HPLC in a single run[J].Biological&Pharmaceutical Bulletin,2009,32(4):717-720.
[24] GROENE E M D,NIJMEIJER S M,HORBACH G J M J,et al.Tiamulin inhibits human CYP3A4 activity in an NIH/3T3 cell line stably expressing CYP3A4 cDNA[J].Biochemical Pharmacology,1995,50(6):771-773.
[25] HAZAI E,VERECZKEY L,MONOSTORY K.Reduction of toxic metabolite formation of acetaminophen[J].Biochemical&Biophysical Research Communications,2002,291(4):1089-1094.
[26] 王高举.氯美噻唑抑制CYP2E1预防二乙基亚硝胺诱导的大鼠肝纤维化[D].郑州:郑州大学,2017. WANG G J.Inhibitory effect of clomethiazole on CYP2E1 in the prevention of diethylnitrosamine-induced rat liver fibrosis[D].Zhengzhou:Zhengzhou University,2017.(in Chinese)
[27] STRESSER D M,BROUDY M I,HO T,et al.Highly selective inhibition of human CYP3A in vitro by azamulin and evidence that inhibition is irreversible[J].Drug Metabolism and Disposition,2004,32(1):105.
[28] BURKINA V,RASMUSSEN M K,PILIPENKO N,et al.Comparison of xenobiotic-metabolising human,porcine,rodent,and piscine cytochrome P450[J].Toxicology,2017,375:10-27.
[29] DENISOV I G,BAAS B J,GRINKOVA Y V,et al.Cooperativity in cytochrome P4503A4:Linkages in substrate binding,spin state,uncoupling,and product formation[J].Journal of Biological Chemistry,2007,282(10):7066-7076.
[30] MARIKA E,TOVE S G.Structural basis for ligand promiscuity in cytochrome P4503A4[J].Proceedings of the National Academy of Science,2006,103(37):13682-13687.
[31] FRANKLIN M R.Enhanced rates of cytochrome P450 metabolic-intermediate complex formation from nonmacrolide amines in rifampicin-treated rabbit liver microsomes[J].Drug Metabolism&Disposition,1995,23(12):1379-1382.
[32] ZWEERS-ZEILMAKER W M,MIERT A S,VAN HORBACH G J,et al.In vitro complex formation and inhibition of hepatic cytochrome P450 activity by different macrolides and tiamulin in goats and cattle[J].Research in Veterinary Science,1999,66(1):51-55.
[33] CARLETTI M,GUSSON F,ZAGHINI A,et al.In vitro formation of metabolic-intermediate cytochrome P450 complexes in rabbit liver microsomes by Tiamulin and various macrolides[J].Veterinary Research,2003,34(4):405-411.