Vishwanathan Karthick, Babalola Kathlene, Wang Jack, Espina Robert, Yu Linning, Adedoyin Adedayo, Talaat Rasmy, Mutlib Abdul, Scatina JoAnn
Biotransformation, Drug Safety and Metabolism, Wyeth Research, 500 Arcola Road, Collegeville, Pennsylvania 19426, USA.
Chem Res Toxicol. 2009 Feb;22(2):311-22. doi: 10.1021/tx8003328.
The recent guidance on "Safety Testing of Drug Metabolites" issued by the U.S. Food and Drug Administration, Center for Drug Evaluation and Research (CDER) has highlighted the importance of identifying and characterizing drug metabolites as early as possible in drug discovery and development. Furthermore, upon identifying significant circulating metabolites in human plasma, it has become important to demonstrate that these metabolites are present at an equal or greater exposure level (area under the curve, AUC) in any one of the preclinical species used in safety testing. Frequently, synthetic standards of metabolites are not available, and hence, obtaining their AUC values can be a challenge. In this report, we demonstrate how combinations of nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography/ultraviolet/mass spectrometry (LC/UV/MS), and plasma pooling methods were used to obtain reliable AUC values of metabolites present in the plasma of preclinical species from short-term safety studies. Plasma pooling methods were compared to the traditional approaches of obtaining quantitative information on the levels of circulating metabolites in preclinical species. The exposure values obtained via sample pooling were comparable to those obtained by traditional methods of analyzing samples individually. In the absence of synthetic chemical standards, calculations of AUC values of metabolites, using either sample pooling or traditional approaches, were achieved through the use of UV detectors. In cases where the UV properties of metabolites were significantly different from their parent compounds, NMR was used as a quantitative tool to obtain exposure values. NMR was found to be useful in quantitating biologically produced metabolites, which could subsequently be used as reference compounds for further quantitative studies. The limitations of UV detectors to obtain exposure estimates are discussed. A practical solution is presented that will enable us to obtain a quantitative assessment of metabolite exposure in humans and coverage in toxicology species, hence, circumventing the use of radiolabeled compounds or authentic chemically synthesized standards of metabolites.
美国食品药品监督管理局药品评价和研究中心（CDER）近期发布的“药物代谢物安全性测试”指南强调了在药物研发早期识别和表征药物代谢物的重要性。此外，在确定人血浆中存在显著的循环代谢物后，证明这些代谢物在安全性测试中使用的任何一种临床前物种中的暴露水平（曲线下面积，AUC）相等或更高就变得很重要。通常，代谢物的合成标准品不可用，因此，获得它们的AUC值可能是一个挑战。在本报告中，我们展示了如何结合核磁共振（NMR）光谱、液相色谱/紫外/质谱（LC/UV/MS）和血浆合并方法，从短期安全性研究中获得临床前物种血浆中存在的代谢物的可靠AUC值。将血浆合并方法与获取临床前物种中循环代谢物水平定量信息的传统方法进行了比较。通过样本合并获得的暴露值与通过单独分析样本的传统方法获得的暴露值相当。在没有合成化学标准品的情况下，使用UV检测器通过样本合并或传统方法计算代谢物的AUC值。在代谢物的UV特性与其母体化合物有显著差异的情况下，使用NMR作为定量工具来获得暴露值。发现NMR可用于定量生物产生的代谢物，随后可将其用作进一步定量研究的参考化合物。讨论了UV检测器在获得暴露估计值方面的局限性。提出了一种实际解决方案，使我们能够对人体中的代谢物暴露和毒理学物种中的覆盖范围进行定量评估，从而避免使用放射性标记化合物或代谢物的真实化学合成标准品。
