{"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":" ","pages":""},"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\":\" \",\"pages\":\"\"},\"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:
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