BACKGROUND MIDD0301 is an oral asthma drug candidate that binds GABAA receptors on airway smooth muscle and immune cells. OBJECTIVE The objective of this study is to identify and quantify MIDD0301 metabolites in vitro and in vivo and determine the pharmacokinetics of oral, IP, and IV administered MIDD0301. METHODS In vitro conversion of MIDD0301 was performed using liver and kidney microsomes/S9 fractions followed by quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A LC-MS/MS method was developed using synthesized standards to quantify MIDD0301 and its metabolites in urine and feces. Blood, lung, and brain were harvested from animals that received MIDD0301 by oral, IP, and IV administration, followed by LCMS/ MS quantification. Imaging mass spectrometry was used to demonstrate the presence of MIDD0301 in the lung after oral administration. RESULTS MIDD0301 is stable in the presence of liver and kidney microsomes and S9 fractions for at least two hours. MIDD0301 undergoes conversion to the corresponding glucuronide and glucoside in the presence of conjugating cofactors. For IP and IV administration, unconjugated MIDD0301 together with significant amounts of MIDD0301 glucoside and MIDD0301 taurine were found in urine and feces. Less conjugation was observed following oral administration, with MIDD0301 glucuronide being the main metabolite. Pharmacokinetic quantification of MIDD0301 in blood, lung, and brain showed very low levels of MIDD0301 in the brain after oral, IV, or IP administration. The drug half-life in these tissues ranged between 4-6 hours for IP and oral and 1-2 hours for IV administration. Imaging mass spectrometry demonstrated that orally administered MIDD0301 distributes uniformly in the lung parenchyma. CONCLUSION MIDD0301 undergoes no phase I and moderate phase II metabolism.

中文翻译：

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MIDD0301 代谢物的鉴定和定量。

背景 MIDD0301 是一种口服哮喘候选药物，可结合气道平滑肌和免疫细胞上的 GABAA 受体。目的 本研究的目的是在体外和体内鉴定和量化 MIDD0301 代谢物，并确定 MIDD0301 的口服、IP 和 IV 的药代动力学。方法 MIDD0301 的体外转化使用肝脏和肾脏微粒体/S9 级分进行，然后使用液相色谱-串联质谱 (LC-MS/MS) 进行定量。使用合成标准开发了一种 LC-MS/MS 方法，以量化尿液和粪便中的 MIDD0301 及其代谢物。通过口服、IP 和 IV 给药从接受 MIDD0301 的动物身上采集血液、肺和脑，然后进行 LCMS/MS 定量。成像质谱法用于证明口服给药后肺中存在 MIDD0301。结果 MIDD0301 在肝肾微粒体和 S9 组分存在的情况下稳定至少 2 小时。MIDD0301 在共轭辅因子存在下转化为相应的葡糖苷酸和葡糖苷。对于 IP 和 IV 给药，在尿液和粪便中发现了未结合的 MIDD0301 以及大量的 MIDD0301 葡萄糖苷和 MIDD0301 牛磺酸。口服给药后观察到较少的结合，其中 MIDD0301 葡糖苷酸是主要代谢物。MIDD0301 在血液、肺和脑中的药代动力学定量显示，在口服、IV 或 IP 给药后，脑中的 MIDD0301 水平非常低。这些组织中的药物半衰期在 IP 和口服给药的 4-6 小时和 IV 给药的 1-2 小时之间。成像质谱表明，口服 MIDD0301 在肺实质中均匀分布。结论 MIDD0301 不经历 I 期代谢和适度的 II 期代谢。