Sakai Noriaki, Saito Konomu, Kim Hyung-Sub, Kazusaka Akio, Ishizuka Mayumi, Funae Yoshihiko, Fujita Shoichi
Laboratory of Toxicology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, N18W9 North Ward, Sapporo 060-0818, Japan.
Drug Metab Dispos. 2005 Nov;33(11):1657-60. doi: 10.1124/dmd.105.004242. Epub 2005 Aug 4.
Diazepam was metabolized to three primary metabolites, 3-hydroxy-diazepam, N-desmethyl-diazepam, and p-hydroxy-diazepam. Our previous studies reported metabolic position-specific inter- or intrastrain differences in diazepam metabolism among Sprague-Dawley, Brown Norway, Dark Agouti, and Wistar rats. Especially, there were marked ( approximately 300 fold) inter- or intrastrain differences in diazepam p-hydroxylation activity at low concentration of substrate. In this study, we investigated the enzyme that catalyzes diazepam p-hydroxylation. The activity toward diazepam p-hydroxylation was inhibited by anti-cytochrome P450 2D (CYP2D) antibody, suggesting that this activity was catalyzed by CYP2D isoforms. Comparing the expression levels of the CYP2D subfamily in liver microsomes from various strains of rats using anti-CYP2D2 antibody, we found that there was a band of protein that was consistent with the phenotype of diazepam p-hydroxylation. N-terminal amino acid sequences of the specific protein exactly corresponded to those of CYP2D3, indicating that CYP2D3 might be involved in diazepam p-hydroxylation. Moreover, using rat CYP2D isoforms expressed in yeast, we tested CYP2Ds to catalyze diazepam p-hydroxylation. CYP2D1 and CYP2D2 practically did not participate in diazepam metabolism. On the other hand, diazepam p-hydroxylation was catalyzed by CYP2D3. CYP2D4 had high activity toward diazepam N-desmethylation, but not p-hydroxylation. In conclusion, the polymorphic expression of CYP2D3 caused the inter- or intrastrain differences in diazepam p-hydroxylation among rat strains or individuals.
地西泮被代谢为三种主要代谢产物，即3-羟基地西泮、N-去甲基地西泮和对羟基地西泮。我们之前的研究报道了在斯普拉格-道利、棕色挪威、深色刺豚鼠和Wistar大鼠中，地西泮代谢存在代谢位置特异性的种间或种内差异。特别是，在低底物浓度下地西泮对羟基化活性存在显著的（约300倍）种间或种内差异。在本研究中，我们研究了催化地西泮对羟基化的酶。抗细胞色素P450 2D（CYP2D）抗体抑制了对地西泮对羟基化的活性，这表明该活性是由CYP2D同工型催化的。使用抗CYP2D2抗体比较不同品系大鼠肝微粒体中CYP2D亚家族的表达水平，我们发现有一条蛋白带与地西泮对羟基化的表型一致。该特异性蛋白的N端氨基酸序列与CYP2D3的完全对应，表明CYP2D3可能参与地西泮的对羟基化。此外，我们使用在酵母中表达的大鼠CYP2D同工型，测试了CYP2D对地西泮对羟基化的催化作用。CYP2D1和CYP2D2实际上不参与地西泮的代谢。另一方面，地西泮的对羟基化由CYP2D3催化。CYP2D4对地西泮N-去甲基化有高活性，但对对羟基化没有活性。总之，CYP2D3的多态性表达导致了大鼠品系或个体之间地西泮对羟基化的种间或种内差异。
