Alvarez-Diez Teresa M, Zheng Jiang
Department of Pharmaceutical Sciences, School of Pharmacy, Bouvé College of Health Sciences, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts 02115, USA.
Chem Res Toxicol. 2004 Feb;17(2):150-7. doi: 10.1021/tx034143l.
Earlier phase I and II clinical studies showed that 4-ipomeanol produced selective hepatotoxicity. To investigate the mechanism of bioactivation of 4-ipomeanol, we thoroughly studied the interaction of 4-ipomeanol with human cytochrome P450 3A4 (EC 1.14.14.1). 4-Ipomeanol produced a time- and concentration-dependent inactivation of P450 3A4. More than 80% of the P450 3A4 activity was lost after its incubation with 4-ipomeanol at the concentration of 75 microM in 12 min. The inactivation was characterized by a rate of inactivation (kinact) of 0.15 min(-1) and by an inactivation potency (KI) of 20 microM. In addition, the inhibition of P450 3A4 by 4-ipomeanol was NADPH-dependent and irreversible. Glutathione, catalase, and superoxide dismutase failed to protect P450 3A4 from inactivation by 4-ipomeanol. The presence of testosterone, a substrate of P450 3A4, protected the enzyme from inactivation. The estimated partition ratio of the inactivation was approximately 257. Covalent binding studies demonstrated that reactive metabolites of 4-ipomeanol modified P450 3A4 but not P450 reductase (EC 1.6.2.4). The stoichiometry of binding between reactive metabolites of radiolabeled 4-ipomeanol and P450 3A4 was approximately 1.5:1. In addition to P450 3A4, reactive metabolites of 4-ipomeanol were found to covalently bind to other proteins. 4-Ipomeanol failed to inactivate P450 1A2 in human liver microsomes. In conclusion, 4-ipomeanol irreversibly inhibited P450 3A4, and it was characterized as a mechanism-based inactivator of P450 3A4. This finding facilitates the understanding of the mechanism of bioactivation of 4-ipomeanol by human hepatic enzymes.
早期的I期和II期临床研究表明，4-异戊烯醇具有选择性肝毒性。为了研究4-异戊烯醇的生物活化机制，我们深入研究了4-异戊烯醇与人细胞色素P450 3A4（EC 1.14.14.1）的相互作用。4-异戊烯醇对P450 3A4产生了时间和浓度依赖性的失活作用。在12分钟内，当4-异戊烯醇与P450 3A4以75 microM的浓度孵育后，超过80%的P450 3A4活性丧失。失活的特征在于失活速率（kinact）为0.15 min⁻¹，失活效力（KI）为20 microM。此外，4-异戊烯醇对P450 3A4的抑制作用是NADPH依赖性且不可逆的。谷胱甘肽、过氧化氢酶和超氧化物歧化酶均无法保护P450 3A4免受4-异戊烯醇的失活作用。P450 3A4的底物睾酮的存在可保护该酶不被失活。失活的估计分配比约为257。共价结合研究表明，4-异戊烯醇的活性代谢产物修饰了P450 3A4，但未修饰P450还原酶（EC 1.6.2.4）。放射性标记的4-异戊烯醇的活性代谢产物与P450 3A4之间的结合化学计量比约为1.5:1。除了P450 3A4外，还发现4-异戊烯醇的活性代谢产物与其他蛋白质发生共价结合。4-异戊烯醇未能使人类肝微粒体中的P450 1A2失活。总之，4-异戊烯醇不可逆地抑制了P450 3A4，其被表征为P450 3A4的基于机制的失活剂。这一发现有助于理解人类肝脏酶对4-异戊烯醇的生物活化机制。
