{"title":"参与吡非尼酮主要代谢物 5-羧基吡非尼酮形成的人类醇脱氢酶 4 和醛脱氢酶 2 的特征。","authors":"Rei Sato, Tatsuki Fukami, Kazuya Shimomura, Yongjie Zhang, Masataka Nakano, Miki Nakajima","doi":"10.1124/dmd.124.001917","DOIUrl":null,"url":null,"abstract":"<p><p>Pirfenidone (PIR) is used to treatment of idiopathic pulmonary fibrosis. After oral administration, it is metabolized by cytochrome P450 1A2 to 5-hydroxylpirfenidone (5-OH PIR) and further oxidized to 5-carboxylpirfenidone (5-COOH PIR), a major metabolite excreted in the urine (90% of the dose). This study aimed to identify enzymes that catalyze the formation of 5-COOH PIR from 5-OH PIR in the human liver. 5-COOH PIR was formed from 5-OH PIR in the presence of NAD<sup>+</sup> by human liver microsomes (HLM) more than by human liver cytosol (HLC), with the concomitant formation of the aldehyde form (5-CHO PIR) as an intermediate metabolite. By purifying enzymes from HLM, alcohol dehydrogenases (ADHs) were identified as candidate enzymes catalyzing 5-CHO PIR formation, although ADHs are localized in the cytoplasm. Among constructed recombinant ADH1-5 expressed in HEK293T cells, only ADH4 efficiently catalyzed 5-CHO PIR formation from 5-OH PIR with a <i>K</i> <sub>m</sub> value (29.0 {plus minus} 4.9 µM), which was close to that by HLM (59.1 {plus minus} 4.6 µM). In contrast to commercially available HLC, in-house prepared HLC clearly showed substantial 5-CHO PIR formation, and ADH4 protein levels were significantly (<i>rs</i> = 0.772, <i>P</i> < 0.0001) correlated with 5-CHO PIR formation in 25 in-house prepared HLC samples. Some components of the commercially available HLC may inhibit ADH4 activity. Disulfiram, an inhibitor of aldehyde dehydrogenases (ALDH), decreased 5-COOH PIR formation and increased 5-CHO PIR formation from 5-OH PIR in HLM. ALDH2 knockdown in HepG2 cells by siRNA decreased 5-COOH PIR formation by 61%. <b>Significance Statement</b> This study clarified that 5-COOH PIR formation from 5-OH PIR proceeds via a two-step oxidation reaction catalyzed by ADH4 and disulfiram-sensitive enzymes, including ALDH2. Inter-individual differences in the expression levels or functions of these enzymes could cause variations in the pharmacokinetics of PIR.</p>","PeriodicalId":11309,"journal":{"name":"Drug Metabolism and Disposition","volume":null,"pages":null},"PeriodicalIF":4.4000,"publicationDate":"2024-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Characterization of human alcohol dehydrogenase 4 and aldehyde dehydrogenase 2 as enzymes involved in the formation of 5-carboxylpirfenidone, a major metabolite of pirfenidone.\",\"authors\":\"Rei Sato, Tatsuki Fukami, Kazuya Shimomura, Yongjie Zhang, Masataka Nakano, Miki Nakajima\",\"doi\":\"10.1124/dmd.124.001917\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Pirfenidone (PIR) is used to treatment of idiopathic pulmonary fibrosis. After oral administration, it is metabolized by cytochrome P450 1A2 to 5-hydroxylpirfenidone (5-OH PIR) and further oxidized to 5-carboxylpirfenidone (5-COOH PIR), a major metabolite excreted in the urine (90% of the dose). This study aimed to identify enzymes that catalyze the formation of 5-COOH PIR from 5-OH PIR in the human liver. 5-COOH PIR was formed from 5-OH PIR in the presence of NAD<sup>+</sup> by human liver microsomes (HLM) more than by human liver cytosol (HLC), with the concomitant formation of the aldehyde form (5-CHO PIR) as an intermediate metabolite. By purifying enzymes from HLM, alcohol dehydrogenases (ADHs) were identified as candidate enzymes catalyzing 5-CHO PIR formation, although ADHs are localized in the cytoplasm. Among constructed recombinant ADH1-5 expressed in HEK293T cells, only ADH4 efficiently catalyzed 5-CHO PIR formation from 5-OH PIR with a <i>K</i> <sub>m</sub> value (29.0 {plus minus} 4.9 µM), which was close to that by HLM (59.1 {plus minus} 4.6 µM). In contrast to commercially available HLC, in-house prepared HLC clearly showed substantial 5-CHO PIR formation, and ADH4 protein levels were significantly (<i>rs</i> = 0.772, <i>P</i> < 0.0001) correlated with 5-CHO PIR formation in 25 in-house prepared HLC samples. Some components of the commercially available HLC may inhibit ADH4 activity. Disulfiram, an inhibitor of aldehyde dehydrogenases (ALDH), decreased 5-COOH PIR formation and increased 5-CHO PIR formation from 5-OH PIR in HLM. ALDH2 knockdown in HepG2 cells by siRNA decreased 5-COOH PIR formation by 61%. <b>Significance Statement</b> This study clarified that 5-COOH PIR formation from 5-OH PIR proceeds via a two-step oxidation reaction catalyzed by ADH4 and disulfiram-sensitive enzymes, including ALDH2. Inter-individual differences in the expression levels or functions of these enzymes could cause variations in the pharmacokinetics of PIR.</p>\",\"PeriodicalId\":11309,\"journal\":{\"name\":\"Drug Metabolism and Disposition\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":4.4000,\"publicationDate\":\"2024-10-29\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Drug Metabolism and Disposition\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1124/dmd.124.001917\",\"RegionNum\":3,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"PHARMACOLOGY & PHARMACY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Drug Metabolism and Disposition","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1124/dmd.124.001917","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"PHARMACOLOGY & PHARMACY","Score":null,"Total":0}
引用次数: 0
摘要
吡非尼酮(PIR)用于治疗特发性肺纤维化。口服后,它通过细胞色素 P450 1A2 代谢为 5-hydroxylpirfenidone (5-OH PIR),并进一步氧化为 5-boxylpirfenidone (5-COOH PIR),这是一种通过尿液排出的主要代谢物(占剂量的 90%)。本研究旨在确定在人体肝脏中催化 5-OH PIR 形成 5-COOH PIR 的酶。在有 NAD+ 存在的情况下,人肝微粒体(HLM)比人肝细胞浆(HLC)更容易从 5-OH PIR 生成 5-COOH PIR,同时形成醛形式(5-CHO PIR)作为中间代谢物。通过纯化 HLM 中的酶,发现醇脱氢酶(ADHs)是催化 5-CHO PIR 形成的候选酶,尽管 ADHs 定位于细胞质中。在 HEK293T 细胞中表达的重组 ADH1-5 中,只有 ADH4 能有效催化 5-OH PIR 生成 5-CHO PIR,其 K m 值(29.0{正负}4.9 µM)接近 HLM 的 K m 值(59.1{正负}4.6 µM)。与市售 HLC 不同的是,内部制备的 HLC 明显显示出大量 5-CHO PIR 的形成,在 25 个内部制备的 HLC 样品中,ADH4 蛋白水平与 5-CHO PIR 的形成显著相关(rs = 0.772,P < 0.0001)。市售 HLC 的某些成分可能会抑制 ADH4 的活性。醛脱氢酶(ALDH)抑制剂双硫仑可减少 5-COOH PIR 的形成,并增加 HLM 中 5-OH PIR 形成的 5-CHO PIR。通过 siRNA 敲除 HepG2 细胞中的 ALDH2 可使 5-COOH PIR 的形成减少 61%。意义声明 本研究阐明了 5-OH PIR 经由 ADH4 和对双硫仑敏感的酶,包括 ALDH2 催化的两步氧化反应形成 5-COOH PIR。这些酶的表达水平或功能的个体差异可能会导致 PIR 药代动力学的变化。
Characterization of human alcohol dehydrogenase 4 and aldehyde dehydrogenase 2 as enzymes involved in the formation of 5-carboxylpirfenidone, a major metabolite of pirfenidone.
Pirfenidone (PIR) is used to treatment of idiopathic pulmonary fibrosis. After oral administration, it is metabolized by cytochrome P450 1A2 to 5-hydroxylpirfenidone (5-OH PIR) and further oxidized to 5-carboxylpirfenidone (5-COOH PIR), a major metabolite excreted in the urine (90% of the dose). This study aimed to identify enzymes that catalyze the formation of 5-COOH PIR from 5-OH PIR in the human liver. 5-COOH PIR was formed from 5-OH PIR in the presence of NAD+ by human liver microsomes (HLM) more than by human liver cytosol (HLC), with the concomitant formation of the aldehyde form (5-CHO PIR) as an intermediate metabolite. By purifying enzymes from HLM, alcohol dehydrogenases (ADHs) were identified as candidate enzymes catalyzing 5-CHO PIR formation, although ADHs are localized in the cytoplasm. Among constructed recombinant ADH1-5 expressed in HEK293T cells, only ADH4 efficiently catalyzed 5-CHO PIR formation from 5-OH PIR with a Km value (29.0 {plus minus} 4.9 µM), which was close to that by HLM (59.1 {plus minus} 4.6 µM). In contrast to commercially available HLC, in-house prepared HLC clearly showed substantial 5-CHO PIR formation, and ADH4 protein levels were significantly (rs = 0.772, P < 0.0001) correlated with 5-CHO PIR formation in 25 in-house prepared HLC samples. Some components of the commercially available HLC may inhibit ADH4 activity. Disulfiram, an inhibitor of aldehyde dehydrogenases (ALDH), decreased 5-COOH PIR formation and increased 5-CHO PIR formation from 5-OH PIR in HLM. ALDH2 knockdown in HepG2 cells by siRNA decreased 5-COOH PIR formation by 61%. Significance Statement This study clarified that 5-COOH PIR formation from 5-OH PIR proceeds via a two-step oxidation reaction catalyzed by ADH4 and disulfiram-sensitive enzymes, including ALDH2. Inter-individual differences in the expression levels or functions of these enzymes could cause variations in the pharmacokinetics of PIR.
期刊介绍:
An important reference for all pharmacology and toxicology departments, DMD is also a valuable resource for medicinal chemists involved in drug design and biochemists with an interest in drug metabolism, expression of drug metabolizing enzymes, and regulation of drug metabolizing enzyme gene expression. Articles provide experimental results from in vitro and in vivo systems that bring you significant and original information on metabolism and disposition of endogenous and exogenous compounds, including pharmacologic agents and environmental chemicals.