小白菊内酯衍生物ACT001对P450酶的诱导作用研究
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摘要
目的使用原代培养的人肝细胞研究倍半萜烯内酯类化合物衍生物ACT001对P450酶的诱导作用,为ACT001的临床使用提供参考。方法分别将3批次的冷冻原代人肝细胞进行接种培养,使用ACT001对CYP1A2、CYP2B6和CYP3A4进行诱导,实时荧光定量PCR(q RT-PCR)测定P450酶m RNA表达水平,液相色谱-质谱联用(LC-MS/MS)法测定P450酶活力。结果 P450酶m RNA表达水平及酶活力的结果均显示模型制备成功;与对照组比较,ACT001 1、6μmol/L组细胞CYP1A2 mRNA表达水平和酶活力未见明显变化;ACT001 30μmol/L组细胞CYP1A2 mRNA表达水平显著降低,酶活力虽出现下降趋势,但不如m RNA下降明显。随着ACT001浓度增加,CYP2B6 mRNA表达水平逐渐升高,与对照组比较,ACT00130μmol/L组细胞CYP2B6 mRNA表达水平显著升高,均超过对照组的7倍,酶活力增加均>4倍,超过苯巴比妥钠诱导倍数的40%。与对照组比较,ACT001 1μmol/L组细胞CYP3A4 m RNA表达水平明显升高,超过对照组的4倍,但未达到阳性诱导剂利福平诱导倍率的40%,且随着ACT001浓度的增加,细胞内CYP3A4 mRNA表达水平逐渐降低;同时,ACT001不同浓度给药后CYP3A4酶活力未出现明显升高,均小于对照组的2倍。结论 ACT001对CYP1A2、CYP3A4没有诱导潜能,对CYP2B6存在诱导潜能,在临床联合用药时应避免与CYP2B6的底物联合使用,以减少因P450酶介导的药物-药物间相互作用(DDI)产生的不良反应。
Objective In this study, primary cultured human hepatocytes were used to study the induction of P450 enzyme by the sesquiterpene lactone derivative ACT001, which provided reference for the clinical use of ACT001. Methods Three batches of frozen primary human hepatocytes were inoculated and cultured, and CYP1 A2, CYP2 B6 and CYP3 A4 were induced by ACT001. Real-time fluorescence quantitative PCR was used to determine the mRNA expression level of P450 enzyme, and the activity of P450 enzyme was determined by LC-MS/MS method. Results The expression level of P450 enzyme mRNA and the activity of P450 enzyme showed that the P450 enzyme induction model was successfully established. Compared with the control group, the CYP1 A2 mRNA expression level and enzyme activity of ACT001 1 μmol/L and 6 μmol/L group showed no significant changes. The mRNA expression level of CYP1 A2 in ACT001 30 μmol/L group was significantly decreased, and the enzyme activity was decreased, but not as significantly as that of mRNA. With the increase of ACT001 concentration, the expression level of CYP2 B6 mRNA was gradually increased. Compared with the control group, the expression level of CYP2 B6 mRNA in the ACT001 group at 30 μmol/L was significantly increased, which was seven times higher than that in the control group, and the increase of enzyme activity was four times higher than that in the control group, which was 40% higher than that in the phenobarbital sodium induction multiple. Compared with the control group, the CYP3 A4 mRNA expression level of cells in the ACT001 1 μmol/L group was significantly increased, which was four times higher than that of the control group, but did not reach 40% of the positive inducer rifampicin, and the CYP3 A4 mRNA expression level was decreased gradually with the increase of ACT001 concentration. At the same time, there was no significant increase in CYP3 A4 enzyme activity after ACT001 administration at different concentrations, which was less than two times of that in the control group. Conclusion The data indicated that ACT001 had no induction potential for CYP1 A2 and CYP3 A4, and had potential for CYP2 B6 induction. In combination with CYP2 B6 substrates, it should be avoided in clinical combination therapy to reduce adverse reactions caused by P450-mediated drug drug interaction.
Objective In this study, primary cultured human hepatocytes were used to study the induction of P450 enzyme by the sesquiterpene lactone derivative ACT001, which provided reference for the clinical use of ACT001. Methods Three batches of frozen primary human hepatocytes were inoculated and cultured, and CYP1 A2, CYP2 B6 and CYP3 A4 were induced by ACT001. Real-time fluorescence quantitative PCR was used to determine the mRNA expression level of P450 enzyme, and the activity of P450 enzyme was determined by LC-MS/MS method. Results The expression level of P450 enzyme mRNA and the activity of P450 enzyme showed that the P450 enzyme induction model was successfully established. Compared with the control group, the CYP1 A2 mRNA expression level and enzyme activity of ACT001 1 μmol/L and 6 μmol/L group showed no significant changes. The mRNA expression level of CYP1 A2 in ACT001 30 μmol/L group was significantly decreased, and the enzyme activity was decreased, but not as significantly as that of mRNA. With the increase of ACT001 concentration, the expression level of CYP2 B6 mRNA was gradually increased. Compared with the control group, the expression level of CYP2 B6 mRNA in the ACT001 group at 30 μmol/L was significantly increased, which was seven times higher than that in the control group, and the increase of enzyme activity was four times higher than that in the control group, which was 40% higher than that in the phenobarbital sodium induction multiple. Compared with the control group, the CYP3 A4 mRNA expression level of cells in the ACT001 1 μmol/L group was significantly increased, which was four times higher than that of the control group, but did not reach 40% of the positive inducer rifampicin, and the CYP3 A4 mRNA expression level was decreased gradually with the increase of ACT001 concentration. At the same time, there was no significant increase in CYP3 A4 enzyme activity after ACT001 administration at different concentrations, which was less than two times of that in the control group. Conclusion The data indicated that ACT001 had no induction potential for CYP1 A2 and CYP3 A4, and had potential for CYP2 B6 induction. In combination with CYP2 B6 substrates, it should be avoided in clinical combination therapy to reduce adverse reactions caused by P450-mediated drug drug interaction.
引文
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[3]Lin J H.CYP induction-mediated drug interactions:In vitro assessment and clinical implications[J].Pharm Res,2006,23(6):1089-1116.
[4]Li C J,Guo S F,Shi T M.Culture supernatants of breast cancer cell line MDA-MB-231 treated with parthenolide inhibit the proliferation,migration,and lumen formation capacity of human umbilical vein endothelial cells[J].Chin Med J,2012,125(12):2195-2199.
[5]Wang W J,Meng Z L,Mo Y C,et al.Unloading the infarcted heart affect MMPs-TIMPs axis in a rat cardiac heterotopic transplantation model[J].Mol Biol Rep,2012,39(1):277-283.
[6]Czyz M,Lesiakmieczkowska K,Koprowska K,et al.Cell context-dependent activities of parthenolide in primary and metastatic melanoma cells[J].British J Pharmacol,2010,160(5):1144-1157.
[7]Holcomb B K,Yip-Schneider M T,Waters J A,et al.Dimethylamino parthenolide enhances the inhibitory effects of gemcitabine in human pancreatic cancer cells[J].J Gastrointest Surg,2012,16(7):1333-1340.
[8]Zhang Q,Lu Y,Ding Y,et al.Guaianolide sesquiterpene lactones,a source to discover agents that selectively inhibit acute myelogenous leukemia stem and progenitor cells[J].J Med Chem,2012,55(20):8757-8769.
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[10]US Department of Health and Human Services,Food and Drug Administration Center for Drug Evaluation and Research(CDER),Guidance for Industry.Drug Interaction Studies-studydesign,Data Analysis,Implications for Dosing,and Labeling Recommendations[S].2012.
[11]Sinz M,Wallace G,Sahi J.Current industrial practices in assessing CYP450 enzyme induction:Preclinical and clinical[J].Aaps J,2008,10(2):391-400.
[12]药物非临床药代动力学研究技术指导原则[S].2014.
[13]Shou M,Hayashi M,Pan Y,et al.Modeling,prediction,and in vitro in vivo correlation of CYP3A4 induction[J].Drug Metab Dispos,2008,36(11):2355-2370.
[14]慈小燕,李薇,崔涛,等.细胞色素P450酶诱导模型的建立及其应用[J].中国新药杂志,2018,27(6):688-694.
[15]Chu V,Einolf H J,Evers R,et al.In vitro and in vivo induction of cytochrome p450:A survey of the current practices and recommendations:A pharmaceutical research and manufacturers of america perspective[J].Drug Metab Dispos,2009,37(7):1339-1354.