High-Throughput Assay of Cytochrome P450-Dependent Drug Demethylation Reactions and Its Use to Re-Evaluate the Pathways of Ketamine Metabolism.
High-Throughput Assay of Cytochrome P450-Dependent Drug Demethylation Reactions and Its Use to Re-Evaluate the Pathways of Ketamine Metabolism.
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在这里，我们介绍了一种可靠的，廉价的，和通用的方法，高通量的药物代谢动力学测定的基础上荧光定量甲醛（FA）形成的细胞色素P450依赖的去甲基化反应。我们描述了这种技术的自动化检测细胞色素P450依赖的药物代谢在人肝微粒体的实施。我们还报告了使用我们的新方法重新评估NMDA受体拮抗剂氯胺酮的代谢途径，氯胺酮越来越多地用作治疗酒精戒断综合征的抗抑郁药。通过对10种主要的细胞色素P450（CYP）酶的氯胺酮去甲基化动力学参数的探讨，我们证明，除了最初被认为是氯胺酮主要代谢物的CYP2B6和CYP3A酶外，CYP2C19和CYP2D6也起着重要作用。同时，在以前的报告中提出的CYP2C9的参与是微不足道的。在寻找一个可靠的，廉价的，多功能的技术，高通量的药物代谢动力学测定，我们选择重新雇用一个老派的方法的基础上，确定甲醛（FA）形成的细胞色素P450依赖性去甲基化反应。在评估了几种用于FA检测的荧光技术后，我们选择了基于Hantzsch反应与乙酰乙酰苯胺的方法作为最灵敏、最稳健且适合高通量实施的方法。在这里，我们提供了一个详细的协议，使用我们的新技术的细胞色素P450依赖性药物去甲基化的自动化检测，并讨论其适用性高通量扫描的药物代谢途径在人类肝脏。为了进一步探索我们的方法，我们将其应用于重新评估氯胺酮的代谢途径，氯胺酮是一种游离麻醉剂和强效抗抑郁剂，越来越多地用于治疗酒精戒断综合征。探索氯胺酮去甲基化的动力学参数的10个主要的细胞色素P450（CYP）酶，我们证明，除了CYP2B6和CYP3A酶，这是最初被认为是氯胺酮的主要代谢，一个重要的作用也发挥了CYP2C19和CYP2D6。同时，认为先前报告中提出的CYP2C9的参与并不重要。
Here, we introduce a reliable, inexpensive, and versatile method for high-throughput kinetic assays of drug metabolism based on fluorometric quantification of formaldehyde (FA) formed in cytochrome P450-dependent demethylation reactions. We describe the implementation of this technique for automatized assays of cytochrome P450-dependent drug metabolism in human liver microsomes. We also report the use of our new approach for re-evaluating the pathways of metabolism of the NMDA-receptor antagonist ketamine, which is increasingly used as an antidepressant in the treatment of alcohol withdrawal syndrome. Probing the kinetic parameters of ketamine demethylation by 10 major cytochrome P450 (CYP) enzymes, we demonstrate that in addition to CYP2B6 and CYP3A enzymes, which were initially recognized as the primary metabolizers of ketamine, an important role is also played by CYP2C19 and CYP2D6. At the same time, the involvement of CYP2C9 suggested in the previous reports is insignificant. In a search for a reliable, inexpensive, and versatile technique for high-throughput kinetic assays of drug metabolism, we elected to rehire an old-school approach based on the determination of formaldehyde (FA) formed in cytochrome P450-dependent demethylation reactions. After evaluating several fluorometric techniques for FA detection, we chose the method based on the Hantzsch reaction with acetoacetanilide as the most sensitive, robust, and adaptable to high-throughput implementation. Here we provide a detailed protocol for using our new technique for automatized assays of cytochrome P450-dependent drug demethylations and discuss its applicability for high-throughput scanning of drug metabolism pathways in the human liver. To probe our method further, we applied it to re-evaluating the pathways of metabolism of ketamine, a dissociative anesthetic and potent antidepressant increasingly used in the treatment of alcohol withdrawal syndrome. Probing the kinetic parameters of ketamine demethylation by ten major cytochrome P450 (CYP) enzymes, we demonstrate that in addition to CYP2B6 and CYP3A enzymes, which were initially recognized as the primary metabolizers of ketamine, an important role is also played by CYP2C19 and CYP2D6. At the same time, the involvement of CYP2C9 suggested in the previous reports was deemed insignificant.
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