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

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.