Marathe P H, Shen D D, Nelson W L
Department of Pharmaceutics, School of Pharmacy, University of Washington.
Drug Metab Dispos. 1994 Mar-Apr;22(2):237-47.
The enantioselective formation kinetics of 4-hydroxypropranolol (4-HOP), 5-hydroxypropranolol (5-HOP), and desisopropylpropranolol (DIP) were characterized over a wide substrate concentration range (1-1000 microM) in human liver microsomes using deuterium-labeled pseudoracemic propranolol. Existing data suggest that several microsomal cytochrome P-450 enzymes are involved in the oxidative metabolism of propranolol in humans. Biphasic kinetics were observed in the formation of all three metabolites, indicating the involvement of at least two enzymes in each pathway. The R/S ratios for the formation of all three metabolites varied with respect to the substrate concentration, lending further support to the contribution of two or more enzymes with differing KM's and enantioselectivity. The high-affinity 4-hydroxylation process showed a strong R-enantioselectivity. The low-affinity component of 4-hydroxylation also exhibited a preference for R-(+)-propranolol, although to a lesser degree than the high-affinity component. A similar pattern of enantioselectivity was observed for 5-hydroxylation, except that R/S ratio showed an initial increase followed by a decrease as the propranolol concentration increased beyond 200 microM. Formation of DIP was R-enantioselective at low substrate concentrations, whereas an opposite enantioselectivity was observed at high propranolol concentrations. The metabolism of propranolol in the presence of nanomolar concentrations of quinidine (a selective inhibitor of P-450 2D6) was studied at concentrations of pseudoracemic propranolol in the high- and low-affinity regions. A significant inhibition of 4- and 5-hydroxylation was observed, whereas N-dealkylation was not affected by quinidine. The inhibition of 4-hydroxylation was slightly enantioselective toward R-enantiomer. Quinidine had no significant effect on the low-affinity component for 4-hydroxylation. Although the inhibition of 4- and 5-hydroxylation at the high-affinity site was extensive, complete inhibition was not achieved even at the highest quinidine concentration (10 microM). Data could be fitted to a mixed-type inhibition kinetics resulting from multiple high-affinity hydroxylases. Our in vitro results indicate that formation of 4-HOP and 5-HOP is mediated by more than one P-450 enzyme with major contribution from P-450 2D6, whereas the formation of DIP is catalyzed by two or more P-450 enzymes other than 2D6.
使用氘标记的伪外消旋普萘洛尔，在较宽的底物浓度范围（1 - 1000微摩尔）内，对人肝微粒体中4 - 羟基普萘洛尔（4 - HOP）、5 - 羟基普萘洛尔（5 - HOP）和去异丙基普萘洛尔（DIP）的对映体选择性形成动力学进行了表征。现有数据表明，几种微粒体细胞色素P - 450酶参与了普萘洛尔在人体内的氧化代谢。在所有三种代谢物的形成过程中均观察到双相动力学，表明每条途径中至少涉及两种酶。所有三种代谢物形成的R/S比值随底物浓度而变化，进一步支持了具有不同米氏常数（KM）和对映体选择性的两种或更多种酶的贡献。高亲和力的4 - 羟基化过程表现出强烈的R - 对映体选择性。4 - 羟基化的低亲和力组分也表现出对R -（+）-普萘洛尔的偏好，尽管程度低于高亲和力组分。5 - 羟基化观察到类似的对映体选择性模式，不同的是，当普萘洛尔浓度超过200微摩尔时，R/S比值先升高后降低。在低底物浓度下，DIP的形成具有R - 对映体选择性，而在高普萘洛尔浓度下观察到相反的对映体选择性。在高亲和力和低亲和力区域的伪外消旋普萘洛尔浓度下，研究了纳摩尔浓度的奎尼丁（P - 450 2D6的选择性抑制剂）存在时普萘洛尔的代谢。观察到4 - 和5 - 羟基化受到显著抑制，而N - 脱烷基化不受奎尼丁影响。4 - 羟基化的抑制对R - 对映体略有对映体选择性。奎尼丁对4 - 羟基化的低亲和力组分没有显著影响。尽管在高亲和力位点对4 - 和5 - 羟基化的抑制作用广泛，但即使在最高奎尼丁浓度（10微摩尔）下也未实现完全抑制。数据可拟合为由多种高亲和力羟化酶导致的混合型抑制动力学。我们的体外结果表明，4 - HOP和5 - HOP的形成由一种以上的P - 450酶介导，其中P - 450 2D6起主要作用，而DIP的形成由除2D6之外的两种或更多种P - 450酶催化。
