Department of Drug Metabolism and Pharmacokinetics, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Rd., Ridgefield, CT, USA, 06877.
Department of Drug Metabolism and Pharmacokinetics, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Rd., Ridgefield, CT, USA, 06877.
J Pharm Sci. 2024 Sep;113(9):2933-2939. doi: 10.1016/j.xphs.2024.07.019. Epub 2024 Jul 23.
In early stages of drug development, the absence of authentic metabolite standards often results in semi-quantitative measurements of metabolite formation in reaction phenotyping studies using mass spectrometry (MS), leading to inaccuracies in the determination of enzyme kinetic parameters, such as the Michaelis constant (K). Moreover, it is impossible to ascertain the maximum rate of enzyme-catalyzed reactions (k or V). The use of radiolabeled parent compounds can circumvent this problem. However, radiometric detection exhibits significantly lower sensitivity compared to MS. To address these challenges, we have developed a stepwise approach that leverages biosynthesized radiolabeled and non-radiolabeled metabolites as standards, enabling accurate determination of K, k or V without the need for authentic metabolite standards. This approach, using the carbon-14 [C] labeled metabolite to calibrate the unlabeled metabolite (C calibration method), combines radiometric with LC-MS/MS detection to generate both [C]-labeled and unlabeled metabolite standard curves to ensure that the sample concentrations measured are accurately quantitated. Two case studies were presented to demonstrate the utility of this method. We first compared the accuracy of the C calibration method to the use of authentic standards for quantitating imipramine metabolites. Next, we biosynthesized and quantitated the metabolites of BI 894416 using C calibration method and evaluated the enzyme kinetics of metabolite formation. The K values of the metabolite formation demonstrated substantially improved accuracy compared to MS semi-quantitation. Moreover, the C calibration method offers a streamlined approach to prepare multiple metabolite standards from a single biosynthesis, reducing the time required for structure elucidation and metabolite synthesis.
在药物开发的早期阶段，由于缺乏真实的代谢物标准品，使用质谱（MS）进行反应表型研究时，通常只能对半定量测量代谢物的形成，这导致酶动力学参数（如米氏常数（K））的测定不准确。此外，无法确定酶促反应的最大速率（k 或 V）。使用放射性标记的母体化合物可以避免这个问题。然而，与 MS 相比，放射性检测的灵敏度显著降低。为了解决这些挑战，我们开发了一种逐步的方法，该方法利用生物合成的放射性标记和非放射性标记代谢物作为标准品，在无需真实代谢物标准品的情况下，准确测定 K、k 或 V。这种方法使用碳-14 [C]标记的代谢物来校准非标记的代谢物（C 校准方法），将放射性与 LC-MS/MS 检测相结合，生成 [C]标记和非标记代谢物标准曲线，以确保测量的样品浓度得到准确的定量。提出了两个案例研究来证明该方法的实用性。我们首先比较了 C 校准方法与使用真实标准品定量测定丙咪嗪代谢物的准确性。接下来，我们通过 C 校准方法生物合成和定量测定 BI 894416 的代谢物，并评估了代谢物形成的酶动力学。与 MS 半定量相比，代谢物形成的 K 值的准确性有了显著提高。此外，C 校准方法提供了一种简化的方法，可从单个生物合成中制备多个代谢物标准品，减少了结构阐明和代谢物合成所需的时间。
