Modifying Remdesivir Prodrug Design to Enhance the Active Metabolite Accumulation in the Lung (Resubmission)

摘要 尽管瑞德韦是美国食品和药物管理局批准的少数几种治疗新冠肺炎患者的药物之一，但 很大一部分患者对治疗没有足够的反应。Remdevir形成为2- 核苷类似物GS-441524的乙基丁酯前体药物克服细胞通透性差的问题 核苷并绕过GS-441524激活所需的限速单磷酸化代谢。 雷米西韦在人体血浆中被广泛水解，这引起了人们的担忧，即只有一小部分 雷米西韦可以以其完整的前药形式进入肺部。因此，与前药设计相关的好处 对于新冠肺炎的治疗可能是达不到的。另一个限制是，redesivir仅在 静脉注射剂型由于其强烈的首过效应。由于这些限制，一些人 研究人员主张使用母体化合物GS-441524作为雷米德韦的替代治疗 新冠肺炎患者。然而，GS-441524‘S细胞通透性差如何影响细胞内仍是个未知数 药物积累和所需限速单磷酸化步骤可能影响的程度 GS-441524激活。在瑞昔维韦的开发过程中，合成了几种GS-441524酯类前药 并对其血浆稳定性和体外抗病毒活性进行了评价。雷米西韦被选为药物 是进一步开发的候选药物，因为它相对于其他前药具有更好的抗埃博拉活性 几个非肺细胞系。其中GS-441524的异丙酯类前药抗病毒作用较弱 效力，但血浆稳定性比雷米西韦高得多。鉴于所有的前药都有相同的活性 代谢物，受试前药之间抗病毒活性的差异可能是由于它们的差异。 在细胞内积累和激活。现有证据表明，羧酸酯酶1(CES1)和 组织蛋白酶A(cathepsin A，CATA)参与了酯类核苷前药的激活，CES1和CATA都是 在人的肺中高度表达。因此，异丙基-GS-441524可以在肺和 从而发挥其抗SARS-CoV-2的作用。此外，异丙基-GS-441524在水溶液中的良好稳定性 血浆、肝脏和肠道可改善肺内药物的细胞内蓄积，并使 拟开发成口服剂型的化合物。在这个项目中，我们将进行药物动力学(PK) 异丙基-GS-441524、雷米地韦和GS-3在小鼠体内的激活和处置比较研究 441524在各种组织中。我们还将测定这三种化合物在人体内的活化率。 肺和其他与PK相关的器官，并鉴定和表征负责前药的水解酶 激活。此外，我们将用体外抗SARS-CoV-2活性的方法评价三个化合物的抗SARS-CoV-2活性 模特。该项目将提供证据支持异丙基-GS-441524优于RESENDVE 治疗新冠肺炎的GS-441524。此外，组织特异性酯类前体药物激活水解酶的研究 这将为更有效的核苷前药的未来发展提供线索。

Abstract Although remdesivir is among few medications approved by the FDA for treating patients with COVID-19, a significant portion of patients did not respond adequately to the treatment. Remdesivir is formed as a 2- ethylbutyl ester prodrug of the nucleoside analog GS-441524 to overcome the poor cell permeability of nucleosides and bypass the rate-limiting mono-phosphorylation metabolism required for GS-441524 activation. Remdesivir is extensively hydrolyzed in human plasma, raising the concerns that only a small portion of remdesivir can reach the lung in its intact prodrug form. Thus, the benefits associated with the prodrug design may be unattainable for the treatment of COVID-19. Another limitation is that remdesivir is only available in an intravenous injection dosage form due to its intensive first-pass effect. Because of those limitations, some investigators advocate for using the parent compound GS-441524 as an alternative to remdesivir to treat COVID-19 patients. However, it remains unknown how GS-441524's poor cell permeability affects intracellular drug accumulation and the extent to which the required rate-limiting mono-phosphorylation step could affect GS-441524 activation. During remdesivir development, several GS-441524 ester prodrugs were synthesized and evaluated for their plasma stability and in vitro antiviral activity. Remdesivir was chosen as the drug candidate for further development because of its superior anti-Ebola activity relative to other prodrugs in several non-lung cell lines. Among those, the isopropyl ester prodrug of GS-441524 showed lower antiviral potency but much greater plasma stability than remdesivir. Given that all the prodrugs have the same active metabolite, the antiviral activity differences among the tested prodrugs are likely attributed to their differences in intracellular accumulation and activation. Existing evidence suggests that carboxylesterase 1 (CES1) and cathepsin A (CatA) are involved in the activation of ester nucleoside prodrugs, and both CES1 and CatA are highly expressed in the human lung. Thus, isopropyl-GS-441524 could be efficiently activated in the lung and consequently exert its anti-SARS-CoV-2 effect. Moreover, the excellent stability of isopropyl-GS-441524 in plasma, liver, and intestine could improve the intracellular drug accumulation in the lung and allow the compound to be developed into an oral dosage form. In this project, we will conduct a pharmacokinetics (PK) study in mice to compare the activation and disposition between isopropyl-GS-441524, remdesivir, and GS- 441524 in various tissues. We will also determine the activation rates of the three compounds in the human lung and other PK-related organs and identify and characterize the hydrolases responsible for the prodrug activation. Furthermore, we will evaluate the anti-SARS-CoV-2 activity of the three compounds using an in vitro model. The project will provide evidence supporting that isopropyl-GS-441524 is advantageous over remdesivir and GS-441524 for treating COVID-19. Moreover, studying tissue-specific ester prodrug-activating hydrolases will shed light on the future development of more effective nucleoside prodrugs.