Division of Pharmaceutics and Biopharmaceutics, Faculty of Pharmaceutical Sciences, University of British Columbia, 2146 East Mall, Vancouver, BC, Canada V6T 1Z3.
J Pharmacol Exp Ther. 2010 Mar;332(3):755-63. doi: 10.1124/jpet.109.160614. Epub 2009 Dec 9.
Doxorubicin (DOX) and daunorubicin (DAUN) are anthracycline anticancer agents; however, considerable interpatient variability exists in their pharmacokinetics. This interpatient variability is attributed in part to altered metabolism by nonsynonymous single-nucleotide polymorphisms (ns-SNPs) in genes encoding the carbonyl reductases. This study examines the effect of seven naturally occurring ns-SNPs in the CBR3 gene on in vitro metabolism of anthracyclines to doxorubicinol and daunorubicinol. Kinetic assays measure metabolite levels by high-performance liquid chromatography separation with fluorescence detection by use of purified, histidine-tagged, human CBR3 wild type and variant enzymes. The V224M, C4Y, and V93I variants resulted in significantly reduced maximal reaction velocity (V(max)) for both anthracyclines compared with the wild-type enzyme, whereas the M235L variant had significantly reduced V(max) for DOX only. Significant increases in substrate affinity were found for the V244M variant with DAUN, as well as the C4Y and V93I variants with DOX. The catalytic efficiency values for the V244M, C4Y, and V93I variants were significantly lower than the wild type for DAUN and DOX. Furthermore, DOX was observed to be a better substrate than DAUN for the wild-type enzyme and its variants. HapMap analysis indicated that a haplotype carrying the C4Y and V244M mutations may occur in some individuals in the 11 ethnic populations studied in the HapMap project. Our preparation of the double mutant indicated a significant reduction in activity compared with the wild-type enzyme and single-mutant preparations. These findings suggest that commonly occurring ns-SNPs in human CBR3 significantly alter the in vitro metabolism of DOX and DAUN.
阿霉素（DOX）和柔红霉素（DAUN）是蒽环类抗癌药物；然而，它们的药代动力学在患者间存在相当大的变异性。这种患者间的变异性部分归因于编码羰基还原酶的基因中的非同义单核苷酸多态性（ns-SNPs）改变了代谢。本研究考察了 CBR3 基因中七个天然存在的 ns-SNPs 对蒽环类药物转化为阿霉素醇和柔红霉素醇的体外代谢的影响。通过使用纯化的、带有组氨酸标签的人 CBR3 野生型和变体酶进行高效液相色谱分离和荧光检测，来测量代谢产物水平的酶动力学测定。与野生型酶相比，V224M、C4Y 和 V93I 变体导致两种蒽环类药物的最大反应速度（V(max)）显著降低，而 M235L 变体仅使 DOX 的 V(max)显著降低。发现 V244M 变体与 DAUN 的底物亲和力显著增加，C4Y 和 V93I 变体与 DOX 的底物亲和力也显著增加。V244M、C4Y 和 V93I 变体的催化效率值明显低于野生型 DAUN 和 DOX。此外，与野生型酶及其变体相比，DOX 被观察为野生型酶及其变体的更好底物。HapMap 分析表明，在 HapMap 项目研究的 11 个人种族群体中，某些个体可能携带 C4Y 和 V244M 突变的单倍型。与野生型酶和单突变体制剂相比，我们制备的双突变体显示活性显著降低。这些发现表明，人 CBR3 中常见的 ns-SNPs 显著改变了 DOX 和 DAUN 的体外代谢。
