Kalgutkar Amit S, Nguyen Hang T, Vaz Alfin D N, Doan Anke, Dalvie Deepak K, McLeod Dale G, Murray John C
Department of Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research and Development, Groton, CT 06340, USA.
Drug Metab Dispos. 2003 Oct;31(10):1240-50. doi: 10.1124/dmd.31.10.1240.
The 3-unsubstituted isoxazole ring in the anti-inflammatory drug leflunomide undergoes a unique N-O bond cleavage to the active alpha-cyanoenol metabolite A771726, which resides in the same oxidation state as the parent. In vitro studies were conducted to characterize drug-metabolizing enzyme(s) responsible for ring opening and to gain insight into the mechanism of ring opening. Under physiological conditions, leflunomide was converted to A771726 in rat and human plasma (rat plasma,t1/2 = 36 min; human plasma, t1/2 = 12 min) and whole blood (rat blood, t1/2 = 59 min; human blood, t1/2 = 43 min). Human serum albumin also catalyzed A771726 formation, albeit at a much slower rate (t1/2 = 110 min). Rat and human liver microsomes also demonstrated NADPH-dependent A771726 formation (human liver microsomes, Vmax = 1797 pmol/min/mg and Km = 274 microM). Leflunomide metabolism in microsomes was sensitive to furafylline treatment, suggesting p4501A2 involvement. 3-Methylleflunomide, which contained a 3-methyl substituent on the isoxazole ring, was resistant to ring opening in base, plasma, blood, and liver microsomes. In microsomes, two monohydroxylated metabolites were formed, and metabolite identification studies established the 3- and the 5-methyl groups on the isoxazole ring as sites of hydroxylation. These results indicate that the C3-H in leflunomide is essential for ring opening. Although A771726 formation in human liver microsomes or recombinant p4501A2 required NADPH, its formation was greatly reduced by oxygen or carbon monoxide, suggesting that the isoxazole ring opening was catalyzed by the p450Fe(II) form of the enzyme. A mechanism for the p450-mediated ring scission is proposed in which the isoxazole ring nitrogen or oxygen coordinates to the reduced form of the heme followed by charge transfer from p450Fe(II) to the C=N bond or deprotonation of the C3-H, which results in a cleavage of the N-O bond.
抗炎药来氟米特中未被取代的异恶唑环会发生独特的N - O键断裂，生成活性α - 氰基烯醇代谢物A771726，该代谢物与母体处于相同氧化态。进行了体外研究以表征负责开环的药物代谢酶，并深入了解开环机制。在生理条件下，来氟米特在大鼠和人血浆中（大鼠血浆，t1/2 = 36分钟；人血浆，t1/2 = 12分钟）以及全血中（大鼠血液，t1/2 = 59分钟；人血液，t1/2 = 43分钟）转化为A771726。人血清白蛋白也催化A771726的形成，尽管速率要慢得多（t1/2 = 110分钟）。大鼠和人肝微粒体也显示出依赖NADPH的A771726形成（人肝微粒体，Vmax = 1797 pmol/分钟/毫克，Km = 274 microM）。微粒体中来氟米特的代谢对呋拉茶碱处理敏感，表明有p4501A2参与。3 - 甲基来氟米特在异恶唑环上含有一个3 - 甲基取代基，在碱、血浆、血液和肝微粒体中对开环具有抗性。在微粒体中，形成了两种单羟基化代谢物，代谢物鉴定研究确定异恶唑环上的3 - 和5 - 甲基为羟基化位点。这些结果表明来氟米特中的C3 - H对于开环至关重要。虽然人肝微粒体或重组p4501A2中A771726的形成需要NADPH，但其形成因氧气或一氧化碳而大大减少，这表明异恶唑环的开环是由该酶的p450Fe(II)形式催化的。提出了一种p450介导的环断裂机制，其中异恶唑环的氮或氧与血红素的还原形式配位，随后电荷从p450Fe(II)转移到C = N键或C3 - H去质子化，这导致N - O键断裂。
