Tracy T S, Rosenbluth B W, Wrighton S A, Gonzalez F J, Korzekwa K R
Department of Basic Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown 26506, USA.
Biochem Pharmacol. 1995 May 11;49(9):1269-75. doi: 10.1016/0006-2952(95)00048-5.
Flurbiprofen is a chiral non-steroidal anti-inflammatory drug used in the treatment of pain or inflammation. The primary routes of biotransformation for (R)- and (S)-flurbiprofen are oxidation (presumably cytochrome P450) and conjugation. To date, the specific cytochrome P450 (P450) involved in the oxidative metabolism of this compound (specifically 4'-hydroxylation) has not been elucidated. Experiments were conducted to characterize the kinetic parameters (Km and Vmax) for the 4'-hydroxylation of (R)- and (S)-flurbiprofen in human liver microsomes, to determine if enantiomeric interactions occur when both enantiomers are present, and to identify the specific P450 form(s) involved in this reaction. In human liver microsomes, the Km and Vmax (mean +/- SD) for (R)-4'-hydroxy-flurbiprofen formation were 3.1 +/- 0.8 microM and 305 +/- 168 pmol.min-1.mg protein)-1, respectively. In comparison, the Km and Vmax (mean +/- SD) for (S)-4'-hydroxy-flurbiprofen formation were 1.9 +/- 0.4 microM and 343 +/- 196 pmol.min-1.mg protein-1, respectively. Enantiomeric interaction studies revealed a decrease in Km and Vmax for both enantiomers and an apparent loss of stereoselectivity. Racemic-warfarin, tolbutamide, alpha-naphthoflavone and erythromycin were studied as potential inhibitors of this process. The estimated Ki values for the inhibition of (R)- and (S)-4'-hydroxy-flurbiprofen formation by racemic-warfarin were 2.2 and 4.7 microM. This reaction was also inhibited by tolbutamide. In contrast, erythromycin and alpha-naphthoflavone had no appreciable effect on 4'-hydroxy-flurbiprofen formation. cDNA-expression of individual forms was used to determine which P450 was involved in 4'-hydroxy-flurbiprofen formation. P450 2C9 and an allelic variant (R144C) readily catalyzed the formation of 4'-hydroxy-flurbiprofen. P450 1A2 was also active albeit with a turnover rate 1/140th that of P450 2C9R144C (P450s 2C8, 2E1 and 3A4 were not active toward either enantiomer). The results of these studies indicate that the enantiomers of flurbiprofen may exhibit stereoselectivity with respect to enzyme affinity but have roughly equal maximum formation velocities. Additionally, these two enantiomers may compete for the enzyme resulting in lower maximum velocities for both enantiomers. Finally, of those P450 forms examined, only P450 2C9 and an allelic variant catalyzed the 4'-hydroxylation of both (R)- and (S)-flurbiprofen.
氟比洛芬是一种手性非甾体抗炎药，用于治疗疼痛或炎症。（R）-和（S）-氟比洛芬的主要生物转化途径是氧化（可能是细胞色素P450）和结合反应。迄今为止，参与该化合物氧化代谢（特别是4'-羟基化）的具体细胞色素P450（P450）尚未阐明。进行实验以表征人肝微粒体中（R）-和（S）-氟比洛芬4'-羟基化的动力学参数（Km和Vmax），确定当两种对映体同时存在时是否发生对映体相互作用，并鉴定参与该反应的特定P450形式。在人肝微粒体中，（R）-4'-羟基氟比洛芬形成的Km和Vmax（平均值±标准差）分别为3.1±0.8μM和305±168 pmol·min-1·mg蛋白-1。相比之下，（S）-4'-羟基氟比洛芬形成的Km和Vmax（平均值±标准差）分别为1.9±0.4μM和343±196 pmol·min-1·mg蛋白-1。对映体相互作用研究表明，两种对映体的Km和Vmax均降低，并且立体选择性明显丧失。研究了消旋华法林、甲苯磺丁脲、α-萘黄酮和红霉素作为该过程的潜在抑制剂。消旋华法林抑制（R）-和（S）-4'-羟基氟比洛芬形成的估计Ki值分别为2.2和4.7μM。该反应也受到甲苯磺丁脲的抑制。相比之下，红霉素和α-萘黄酮对4'-羟基氟比洛芬的形成没有明显影响。使用单个形式的cDNA表达来确定哪种P450参与4'-羟基氟比洛芬的形成。P450 2C9和一个等位基因变体（R144C）很容易催化4'-羟基氟比洛芬的形成。P450 1A2也有活性，尽管其周转速率是P450 2C9R144C的1/140（P450 2C8、2E1和3A4对任何一种对映体均无活性）。这些研究结果表明，氟比洛芬的对映体在酶亲和力方面可能表现出立体选择性，但最大形成速度大致相等。此外，这两种对映体可能竞争酶，导致两种对映体的最大速度都降低。最后，在所检测的那些P450形式中，只有P450 2C9和一个等位基因变体催化了（R）-和（S）-氟比洛芬的4'-羟基化反应。
