Uncontrolled inflammation contributes to the progression of organ damage in acute conditions, such as acetaminophen-induced acute liver injury (APAP-ALI) and there are limited treatments for this condition. AT7519, a cyclic-dependent kinase inhibitor (CDKI), has been used successfully in several conditions, to resolve inflammation and return tissue homeostatic functions. AT7519 has not been assessed in APAP-ALI and its effect on APAP metabolism is unknown. Targeted chromatography and mass spectrometry can be used to assess multiple compounds simultaneously and this approach has not been applied yet to measure APAP and AT7519 in a mouse model. We show an optimised simple and sensitive LC–MS/MS method for determining concentrations of AT7519 and APAP in low volumes of mouse serum. Using positive ion mode electrospray ionisation, separation of AT7519 and APAP and their corresponding isotopically labelled internal standards [2H]8-AT16043M (d8-AT7519) and [2H]8-APAP (d4-APAP), was achieved on an Acquity UPLC BEH C18 column (100 × 2.1 mm; 1.7μm). A gradient mobile phase system of water and methanol was delivered at a flow rate of 0.5 mL/min with a run time of 9 min. Calibration curves were linear, intra-day and inter-day precision and accuracy were acceptable and the covariates of all standards and quality control replicates were less than 15%. The method was successfully applied to evaluate AT7519 and APAP levels 20 h post AT7519 (10 mg/mg) in C57Bl6J wild type mouse serum treated with either vehicle or APAP. Serum AT7519 was significantly higher in mice that had received APAP compared to control, but there was no correlation between APAP and AT7519 quantification. There was also no correlation of AT7519 and hepatic damage or proliferation markers. We optimised an LC–MS/MS method to quantify both AT7519 and APAP in mouse serum (50 µL), using labelled internal standards. Application of this method to a mouse model of APAP toxicity proved effective in accurately measuring APAP and AT7519 concentrations after i.p. dosing. AT7519 was significantly higher in mice with APAP toxicity, indicating hepatic metabolism of this CDKI, but there was no correlation with markers of hepatic damage or proliferation, demonstrating that this dose of AT7519 (10 mg/kg) does not contribute to hepatic damage or repair. This optimised method can be used for future investigations of AT7519 in APAP in mice.

中文翻译：

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通过 UPLC-MS/MS 分析 AT7519 作为对乙酰氨基酚诱导的急性炎症小鼠模型中的促消退化合物

不受控制的炎症会导致急性病症中器官损伤的进展，例如对乙酰氨基酚诱导的急性肝损伤（APAP-ALI），并且针对这种病症的治疗方法有限。AT7519 是一种循环依赖性激酶抑制剂 (CDKI)，已成功用于多种病症，以解决炎症并恢复组织稳态功能。AT7519 尚未在 APAP-ALI 中进行评估，其对 APAP 代谢的影响尚不清楚。靶向色谱法和质谱法可用于同时评估多种化合物，但该方法尚未应用于测量小鼠模型中的 APAP 和 AT7519。我们展示了一种优化的简单且灵敏的 LC-MS/MS 方法，用于测定小体积小鼠血清中 AT7519 和 APAP 的浓度。使用正离子模式电喷雾电离，在 Acquity UPLC BEH 上实现 AT7519 和 APAP 及其相应同位素标记内标 [2H]8-AT16043M (d8-AT7519) 和 [2H]8-APAP (d4-APAP) 的分离C18 色谱柱（100 × 2.1 mm；1.7μm）。水和甲醇的梯度流动相系统以 0.5 mL/min 的流速输送，运行时间为 9 分钟。校准曲线呈线性，日内和日间精密度和准确度可接受，所有标准品和质量控制重复的协变量均小于 15%。该方法成功应用于评估 AT7519 (10 mg/mg) 后 20 小时在用载体或 APAP 处理的 C57Bl6J 野生型小鼠血清中的 AT7519 和 APAP 水平。与对照组相比，接受 APAP 的小鼠血清 AT7519 显着升高，但 APAP 和 AT7519 定量之间没有相关性。AT7519 与肝损伤或增殖标志物也没有相关性。我们优化了 LC-MS/MS 方法，使用标记的内标对小鼠血清 (50 µL) 中的 AT7519 和 APAP 进行定量。将该方法应用于 APAP 毒性小鼠模型，证明可以有效准确测量 ip 给药后的 APAP 和 AT7519 浓度。AT7519 在具有 APAP 毒性的小鼠中显着较高，表明该 CDKI 的肝脏代谢，但与肝损伤或增殖标志物没有相关性，表明该剂量的 AT7519（10 mg/kg）对肝损伤或修复没有贡献。这种优化的方法可用于未来 AT7519 在小鼠 APAP 中的研究。