Department of Pharmaceutical Sciences, College of Pharmacy and Pharmaceutical Sciences, Washington State University, Spokane, Washington.
Department of Pharmaceutical Sciences, College of Pharmacy and Pharmaceutical Sciences, Washington State University, Spokane, Washington
Drug Metab Dispos. 2023 Jan;51(1):29-37. doi: 10.1124/dmd.121.000624. Epub 2022 Feb 23.
The primary mode of metabolism of nicotine is via the formation of cotinine by the enzyme CYP2A6. Cotinine undergoes further CYP2A6-mediated metabolism by hydroxylation to 3-hydroxycotinine and norcotinine, but can also form cotinine--glucuronide and cotinine--oxide (COX). The goal of this study was to investigate the enzymes that catalyze COX formation and determine whether genetic variation in these enzymes may affect this pathway. Specific inhibitors of major hepatic cytochrome P450 (P450) enzymes were used in cotinine--oxidation reactions using pooled human liver microsomes (HLMs). COX formation was monitored by ultrahigh-pressure liquid chromatography-tandem mass spectrometry and enzyme kinetic analysis was performed using microsomes from P450-overexpressing human embryonic kidney 293 (HEK293) cell lines. Genotype-phenotype analysis was performed in a panel of 113 human liver specimens. Inhibition of COX formation was only observed in HLMs when using inhibitors of CYP2A6, CYP2B6, CYP2C19, CYP2E1, and CYP3A4. Microsomes from cells overexpressing CYP2A6 or CYP2C19 exhibited similar -oxidation activity against cotinine, with maximum reaction rate over Michaelis constant values (intrinsic clearance) of 4.4 and 4.2 nL/min/mg, respectively. CYP2B6-, CYP2E1-, and CYP3A4-overexpressing microsomes were also active in COX formation. Significant associations ( < 0.05) were observed between COX formation and genetic variants in CYP2C19 (*2 and *17 alleles) in HLMs. These results demonstrate that genetic variants in CYP2C19 are associated with decreased COX formation, potentially affecting the relative levels of cotinine in the plasma or urine of smokers and ultimately affecting recommended smoking cessation therapies. SIGNIFICANCE STATEMENT: This study is the first to elucidate the enzymes responsible for cotinine--oxide formation and genetic variants that affect this biological pathway. Genetic variants in CYP2C19 have the potential to modify nicotine metabolic ratio in smokers and could affect pharmacotherapeutic decisions for smoking cessation treatments.
尼古丁的主要代谢方式是通过 CYP2A6 酶形成可替宁。可替宁进一步通过羟化作用被 CYP2A6 介导代谢为 3-羟基可替宁和去甲可替宁，但也可以形成可替宁-葡萄糖醛酸和可替宁-氧化物（COX）。本研究的目的是研究催化 COX 形成的酶，并确定这些酶的遗传变异是否可能影响该途径。使用混合人肝微粒体（HLM）在可替宁-氧化反应中使用主要肝细胞色素 P450（P450）酶的特异性抑制剂。通过超高效液相色谱-串联质谱监测 COX 形成，并使用过表达人胚肾 293（HEK293）细胞系的微粒体进行酶动力学分析。在 113 个人肝标本的基因-表型分析中进行了分析。只有在用 CYP2A6、CYP2B6、CYP2C19、CYP2E1 和 CYP3A4 的抑制剂处理 HLMs 时，才观察到 COX 形成的抑制作用。过表达 CYP2A6 或 CYP2C19 的细胞的微粒体对可替宁表现出相似的 -氧化活性，最大反应速率与米氏常数（内在清除率）值分别为 4.4 和 4.2 nL/min/mg。CYP2B6、CYP2E1 和 CYP3A4 过表达的微粒体也可在 COX 形成中发挥作用。在 HLMs 中，观察到 COX 形成与 CYP2C19 的遗传变异（2 和17 等位基因）之间存在显著关联（<0.05）。这些结果表明，CYP2C19 的遗传变异与 COX 形成减少有关，这可能会影响吸烟者血浆或尿液中可替宁的相对水平，最终影响推荐的戒烟治疗。意义：本研究首次阐明了负责可替宁-氧化物形成的酶以及影响该生物途径的遗传变异。CYP2C19 的遗传变异有可能改变吸烟者的尼古丁代谢比，并可能影响戒烟治疗的药物治疗决策。
