白术内酯II在人肝微粒体中与CYP450酶之间的相互作用研究

doi:10.5246/jcps.2021.08.052

中国药学(英文版) ›› 2021, Vol. 30 ›› Issue (8): 645-656.DOI: 10.5246/jcps.2021.08.052

白术内酯II在人肝微粒体中与CYP450酶之间的相互作用研究

陈绪龙¹^,²^,³^,^#, 廖正根¹^,^#, 李成²^,³, 黄国勇¹, 宋云燕¹, 董伟¹, Abid Naeem¹, 梁新丽¹^,^*()

1. 江西中医药大学现代中药制剂教育部重点实验室, 江西南昌 330004
2. 九江学院附属医院, 江西九江 332000
3. 九江市临床精准医学研究中心, 江西九江 332000

收稿日期:2021-01-12 修回日期:2021-03-16 接受日期:2021-05-20 出版日期:2021-08-29 发布日期:2021-08-29
通讯作者: 梁新丽
作者简介:
+ Tel.: +86-791-87118658, E-mail: paln7@163.com
基金资助:
National Natural Science Foundation of China (Grant No. 81660757); Jiangxi Provincial Academic; Technical Leader Training Program for Major Disciplines (Grant No. 20162BCB22015); The Science Foundation of Health and Family Planning Commission of Jiangxi Province (Grant No. 20181140).

In vitro metabolism and inhibitory effects of atractylenolide II on various hepatic CYPs in HLMs

Xulong Chen¹^,²^,³^,^#, Zhengge Liao¹^,^#, Cheng Li²^,³, Guoyong Huang¹, Yunyan Song¹, Wei Dong¹, Abid Naeem¹, Xinli Liang¹^,^*()

1 Key Laboratory of Modern traditional Chinese Medicine preparations, Ministry of Education, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China
2 Affiliated Hospital, Jiujiang University, Jiujiang 332000, China
3 Jiujiang Clinical Precision Medicine Research Center, Jiujiang 332000, China

Received:2021-01-12 Revised:2021-03-16 Accepted:2021-05-20 Online:2021-08-29 Published:2021-08-29
Contact: Xinli Liang
About author:
# These authors contributed equally to this work.

摘要/Abstract

摘要：

本研究旨在探讨白术内酯II在人肝微粒体中与CYP450酶之间的相互作用情况, 为预测白术内酯II在药物联用时可能产生的相互作用奠定理论基础。采用特异性抑制剂进行化学抑制实验, 阐明影响白术内酯II代谢的CYP450亚型; 以IC₅₀和K_i等相关数据为评价指标, 采用人肝微粒体系统的基于探针的测定方法, 研究白术内酯II对CYP450酶系抑制机理、动力学和抑制类型。结果显示白术内酯II代谢受CYP1A2、CYP2C9和CYP3A4抑制剂的影响, 最高抑制率分别为41.35%、41.97%和82.45%; 白术内酯II对P450五种亚型CYP2C9、CYP1A2、CYP2C19、CYP3A4和CYP2D6的IC₅₀值分别为69.7、84.3、92.4、173.8和190.1 μmol/L, K_i值分别为190.6、179.1、> 200、72.2和66.8; 白术内酯II对CYP1A2为非竞争性抑制, 对CYP2C9和3A4为竞争性抑制, 对CYP2C19和2D6为混合型抑制。研究结果表明, CYP1A2、CYP2C9和CYP3A4是参与白术内酯II代谢的CYP亚型, 白术内酯II对5种CYP450亚型具有不同的抑制机制和抑制强度。

关键词: 白术内酯II, 人肝微粒体, 代谢表型, 酶活性, 相互作用

Abstract:

In the present study, we aimed to investigate the interaction between atractylenolide II (AT-II) and CYP450 enzyme in human liver microsomes, and to lay a theoretical foundation for predicting the possible interaction of AT-II in combination with drugs. The chemical inhibition experiment was carried out with specific inhibitors to clarify the CYP450 subtypes affecting the metabolism of AT-II, and the mechanism, kinetics, and type of inhibition of CYP450 enzyme by AT-II were studied by using the probe-based determination method of human liver microsome system with the related data of IC50 and K_i as evaluation indexes. The metabolism of AT-II was affected by CYP1A2, CYP2C9 and CYP3A4 inhibitors, and the highest inhibition rates were 41.35%, 41.97% and 82.45%, respectively. The IC₅₀ values of AT-II to five subtypes of P450 CYP2C9, CYP1A2, CYP2C19, CYP3A4 and CYP2D6 were 69.7, 84.3, 92.4, 173.8 and 190.1 μmol/L, respectively. The K_i values of AT-II to five subtypes of P450 CYP2C9, CYP1A2, CYP2C19, CYP3A4 and CYP2D6 were 190.6, 179.1, > 200, 72.2 and 66.8, respectively. Among these enzymes, AT-II exhibited non-competitive inhibition on CYP1A2, showed competitive inhibition on CYP2C9 and CYP3A4, and displayed mixed AT-II inhibition on CYP2C19 and CYP2D6. CYP1A2, CYP2C9 and CYP3A4 were involved in the AT-II metabolism, and AT-II exhibited different inhibitory mechanisms and strengths for the five subtypes of CYP450.

Key words: Atractylenolide II, Human liver microsomes, Metabolic phenotype, Enzymatic activity, Drug-drug interaction

Supporting:

陈绪龙, 廖正根, 李成, 黄国勇, 宋云燕, 董伟, Abid Naeem, 梁新丽. 白术内酯II在人肝微粒体中与CYP450酶之间的相互作用研究[J]. 中国药学(英文版), 2021, 30(8): 645-656.

Xulong Chen, Zhengge Liao, Cheng Li, Guoyong Huang, Yunyan Song, Wei Dong, Abid Naeem, Xinli Liang. In vitro metabolism and inhibitory effects of atractylenolide II on various hepatic CYPs in HLMs[J]. Journal of Chinese Pharmaceutical Sciences, 2021, 30(8): 645-656.

图/表 8

Figure 1. Chemical structure of AT-II.

Figure 2. Effect of the incubation time (A) and protein concentration (B) on the elimination of AT-II in HLMs, and the plots of Michaelis-Menten and Lineweaver-Burk (C) for AT-II metabolism in HLMs.

Figure 3. Effects of speci?c inhibitors on CYP450-mediated AT-II metabolism in HLMs. (A)–(E): The metabolite rate of AT-II with the presence of ketoconazole, α-naptho?avone, nocardanone, sulfapyrazole, and quinidine. *P < 0.05, **P < 0.01, ***P < 0.0005 and ****P < 0.0001 compared with controls.

Table 1. Inhibition rate of speci?c inhibitors on CYP450-mediated metabolism of AT-II in HLMs (x ± SD, n = 3).

Figure 4. Inhibition curves of AT-II on CYP1A2 (A), CYP2C19 (B) CYP2C9 (C), CYP2D6 (D) and CYP3A4 (E) in human liver microsomes. Each data point represents the mean value ( ± SD) of triplicate.

Table 2. The Ki values and inhibition type of AT-II on CYP450 isoforms.

Figure 5. Kinetic plots for metabolites formation velocity versus substrate concentration in CYP450 enzyme systems. (A) Phenacetin; (B) S-mephenytoin; (C) Tolbutamide; (D) Dextromethorphan; (E) Testosterone.

Figure 6. Lineweaver-Burk plots and the corresponding secondary plots for the inhibition of CYPs. Primary Lineweaver-Burk plots for evaluating the types of inhibition on CYP1A2 (A), CYP2C19 (B), CYP2C9 (C), CYP2D6 (C) and CYP3A4 (E) by AT-II. Secondary plots of slopes of the Lineweaver-Burk Plots versus AT-II concentrations presented to identify inhibition potency of CYP1A2 (a), CYP2C19 (b), CYP2C9 (c), CYP2D6 (d) and CYP3A4 (e).

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白术内酯II在人肝微粒体中与CYP450酶之间的相互作用研究

In vitro metabolism and inhibitory effects of atractylenolide II on various hepatic CYPs in HLMs

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