Department of Pharmaceutics, University of Washington, Seattle, WA, USA.
Department of Clinical Pharmacology, University of Greifswald, Greifswald, Germany.
Biochem Pharmacol. 2018 Oct;156:32-42. doi: 10.1016/j.bcp.2018.08.003. Epub 2018 Aug 4.
Protein abundance and activity of UGT2B17, a highly variable drug- and androgen-metabolizing enzyme, were quantified in microsomes, S9 fractions, and primary cells isolated from human liver and intestine by validated LC-MS/MS methods. UGT2B17 protein abundance showed >160-fold variation (mean ± SD, 1.7 ± 2.7 pmol/mg microsomal protein) in adult human liver microsomes (n = 26) and significant correlation (r = 0.77, p < 0.001) with testosterone glucuronide (TG) formation. Primary role of UGT2B17 in TG formation compared to UGT2B15 was confirmed by performing activity assays in UGT2B17 gene deletion samples and with a selective UGT2B17 inhibitor, imatinib. Human intestinal microsomes isolated from small intestine (n = 6) showed on average significantly higher protein abundance (7.4 ± 6.6 pmol/mg microsomal protein, p = 0.016) compared to liver microsomes, with an increasing trend towards distal segments of the gastrointestinal (GI) tract. Commercially available pooled microsomes and S9 fractions confirmed greater abundance and activity of UGT2B17 in intestinal fractions compared to liver fractions. To further investigate the quantitative role of UGT2B17 in testosterone metabolism in whole cell system, a targeted metabolomics study was performed in hepatocytes (n = 5) and enterocytes (n = 16). TG was the second most abundant metabolite after androstenedione in both cell systems. Reasonable correlation between UGT2B17 abundance and activity were observed in enterocytes (r = 0.69, p = 0.003), but not in hepatocytes. These observational and mechanistic data will be useful in developing physiologically-based pharmacokinetic (PBPK) models for predicting highly-variable first-pass metabolism of testosterone and other UGT2B17 substrates.
采用经验证的 LC-MS/MS 方法，定量测定了人肝和肠来源的微粒体、S9 级分和原代细胞中 UGT2B17（一种高度可变的药物和雄激素代谢酶）的蛋白丰度和活性。UGT2B17 蛋白丰度在成人肝微粒体（n=26）中存在>160 倍的变化（平均值±标准差，1.7±2.7 pmol/mg 微粒体蛋白），与睾酮葡萄糖醛酸（TG）形成具有显著相关性（r=0.77，p<0.001）。在 UGT2B17 基因缺失样本中进行活性测定和使用选择性 UGT2B17 抑制剂伊马替尼进行实验，证实 UGT2B17 在 TG 形成中发挥主要作用，而非 UGT2B15。从小肠（n=6）分离的人肠微粒体与肝微粒体相比，平均显示出显著更高的蛋白丰度（7.4±6.6 pmol/mg 微粒体蛋白，p=0.016），并且在胃肠道（GI）的远端段呈现出上升趋势。商用混合微粒体和 S9 级分进一步证实了肠级分中 UGT2B17 的丰度和活性大于肝级分。为了进一步研究 UGT2B17 在整个细胞系统中对睾酮代谢的定量作用，在肝细胞（n=5）和肠细胞（n=16）中进行了靶向代谢组学研究。在这两个细胞系统中，TG 是除雄烯二酮之外的第二丰富的代谢物。在肠细胞中观察到 UGT2B17 丰度和活性之间存在合理的相关性（r=0.69，p=0.003），但在肝细胞中则没有。这些观察和机制数据将有助于开发用于预测睾酮和其他 UGT2B17 底物高度可变的首过代谢的基于生理学的药代动力学（PBPK）模型。
