Kim Young-Hoon, Bae Young-Ji, Kim Hyung Soo, Cha Hey-Jin, Yun Jae-Suk, Shin Ji-Soon, Seong Won-Keun, Lee Yong-Moon, Han Kyoung-Moon
Pharmacological Research Division, Toxicological and Research Department, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug safety, Cheongju 363-700.
College of Pharmacy, Chungbuk National University, Cheongju 361-763, Republic of Korea.
Biomol Ther (Seoul). 2015 Sep;23(5):486-92. doi: 10.4062/biomolther.2015.041. Epub 2015 Sep 1.
Drug metabolism mostly occurs in the liver. Cytochrome P450 (CYP) is a drug-metabolizing enzyme that is responsible for many important drug metabolism reactions. Recently, the US FDA and EU EMA have suggested that CYP enzyme induction can be measured by both enzymatic activity and mRNA expression. However, these experiments are time-consuming and their inter-assay variability can lead to misinterpretations of the results. To resolve these problems and establish a more powerful method to measure CYP induction, we determined CYP induction by using luminescent assay. Luminescent CYP assays link CYP enzyme activity to firefly luciferase luminescence technology. In this study, we measured the induction of CYP isozymes (1A2, 2B6, 2C9, and 3A4) in cryopreserved human hepatocytes (HMC424, 478, and 493) using a luminometer. We then examined the potential induction abilities (unknown so far) of mesalazine, a drug for colitis, and mosapride citrate, which is used as an antispasmodic drug. The results showed that mesalazine promotes CYP2B6 and 3A4 activities, while mosapride citrate promotes CYP1A2, 2B6, and 3A4 activities. Luminescent CYP assays offer rapid and safe advantages over LC-MS/MS and qRT-PCR methods. Furthermore, luminescent CYP assays decrease the interference between the optical properties of the test compound and the CYP substrates. Therefore, luminescent CYP assays are less labor intensive, rapid, and can be used as robust tools for high-throughput CYP screening during early drug discovery.
药物代谢大多发生在肝脏。细胞色素P450（CYP）是一种药物代谢酶，负责许多重要的药物代谢反应。最近，美国食品药品监督管理局（FDA）和欧盟药品管理局（EMA）建议可通过酶活性和mRNA表达来测定CYP酶诱导作用。然而，这些实验耗时且批间变异性可能导致结果解读错误。为解决这些问题并建立一种更有效的方法来测定CYP诱导作用，我们使用发光测定法来确定CYP诱导作用。发光CYP测定法将CYP酶活性与萤火虫荧光素酶发光技术联系起来。在本研究中，我们使用光度计测量了冷冻保存的人肝细胞（HMC424、478和493）中CYP同工酶（1A2、2B6、2C9和3A4）的诱导作用。然后，我们研究了用于治疗结肠炎的药物美沙拉嗪和用作解痉药的枸橼酸莫沙必利的潜在诱导能力（目前尚不清楚）。结果表明，美沙拉嗪可促进CYP2B6和3A4的活性，而枸橼酸莫沙必利可促进CYP1A2、2B6和3A4的活性。与液相色谱-质谱联用（LC-MS/MS）和定量逆转录聚合酶链反应（qRT-PCR）方法相比，发光CYP测定法具有快速、安全的优势。此外，发光CYP测定法减少了测试化合物光学性质与CYP底物之间的干扰。因此，发光CYP测定法劳动强度较低、速度快，可作为药物发现早期高通量CYP筛选的可靠工具。
