Department of Pharmaceutical Biosciences, Translational Drug Discovery and Development, Uppsala University, Uppsala, Sweden.
Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry, Uppsala University, Uppsala, Sweden.
J Pharm Biomed Anal. 2024 Oct 15;249:116348. doi: 10.1016/j.jpba.2024.116348. Epub 2024 Jul 9.
Chemically induced, targeted protein degradation with proteolysis targeting chimeras (PROTACs) has shown to be a promising pharmacological strategy to circumvent the poor "druggability" of intracellular targets. However, the favorable pharmacology comes with complex molecular properties limiting the oral bioavailability of these drugs. To foster the translation of PROTACs into the clinics it is of high importance to establish sensitive bioanalytical methods that enable the assessment of absorption, bioavailability, and disposition of PROTACs after oral dosing. In this study, two highly sensitive LC-MS/MS methods (LLOQ = 0.5 ng/mL) were developed and validated for the quantification of bavdeglutamide (ARV-110) and vepdegestrant (ARV-471) in rat plasma. Plasma samples were processed by protein precipitation and separated on a C18 column over a gradient of acetonitrile and water with 0.1 % formic acid. Selected reaction monitoring in positive ESI mode was applied to quantify ARV-110 and ARV-471. Both methods showed linearity, accuracy, and precision as well as matrix effects and carry-over within the predefined acceptance criteria. High stability of the compounds in plasma was demonstrated at long-term storage for seven weeks at -20 °C, three freeze-thaw cycles, up to 20 min at room temperature, and as extracts in the autosampler. The plasma concentration-time curves after intravenous and intraduodenal bolus single-dose administrations in rats could be successfully quantified at clinically relevant doses per body weight. The highly sensitive bioanalytical assays presented in this work enable the application of a broad spectrum of in vivo studies to elucidate the oral absorption, bioavailability, and disposition of PROTACs.
用蛋白水解靶向嵌合体（PROTAC）进行化学诱导的靶向蛋白降解已被证明是一种有前途的药理学策略，可以规避细胞内靶标的“成药性”差的问题。然而，有利的药理学伴随着复杂的分子特性，限制了这些药物的口服生物利用度。为了将 PROTAC 推向临床，建立能够评估口服给药后 PROTAC 的吸收、生物利用度和处置的灵敏的生物分析方法非常重要。在这项研究中，开发并验证了两种用于大鼠血浆中巴维昔单抗（ARV-110）和维帕西拉肽（ARV-471）定量的高灵敏度 LC-MS/MS 方法（LLOQ=0.5ng/mL）。通过蛋白沉淀处理血浆样品，并在 C18 柱上以乙腈和水的梯度洗脱，其中含有 0.1%甲酸。采用正电喷雾模式下的选择反应监测定量 ARV-110 和 ARV-471。两种方法均显示出线性、准确性、精密度以及基质效应和残留效应，均符合预定义的接受标准。在 -20°C 下长期储存 7 周、3 次冻融循环、室温下长达 20 分钟以及在自动进样器中作为提取物时，化合物在血浆中的稳定性均很高。能够成功地在大鼠体内静脉和十二指肠单次推注给药后，在临床相关的体重剂量范围内定量测定血浆浓度-时间曲线。本文介绍的高灵敏度生物分析方法能够应用于广泛的体内研究，以阐明 PROTAC 的口服吸收、生物利用度和处置。
