Discovery of GS-5245 (Obeldesivir), an Oral Prodrug of Nucleoside GS-441524 That Exhibits Antiviral Efficacy in SARS-CoV-2-Infected African Green Monkeys.

Remdesivir 1 is an phosphoramidate prodrug that releases the monophosphate of nucleoside GS-441524 (2) into lung cells, thereby forming the bioactive triphosphate 2-NTP. 2-NTP, an analog of ATP, inhibits the SARS-CoV-2 RNA-dependent RNA polymerase replication and transcription of viral RNA. Strong clinical results for 1 have prompted interest in oral approaches to generate 2-NTP. Here, we describe the discovery of a 5'-isobutyryl ester prodrug of 2 (GS-5245, Obeldesivir, 3) that has low cellular cytotoxicity and 3-7-fold improved oral delivery of 2 in monkeys. Prodrug 3 is cleaved presystemically to provide high systemic exposures of 2 that overcome its less efficient metabolism to 2-NTP, leading to strong SARS-CoV-2 antiviral efficacy in an African green monkey infection model. Exposure-based SARS-CoV-2 efficacy relationships resulted in an estimated clinical dose of 350-400 mg twice daily. Importantly, all SARS-CoV-2 variants remain susceptible to 2, which supports development of 3 as a promising COVID-19 treatment.

Therapeutic treatment with an oral prodrug of the remdesivir parental nucleoside is protective against SARS-CoV-2 pathogenesis in mice.

The coronavirus disease 2019 (COVID-19) pandemic remains uncontrolled despite the rapid rollout of safe and effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, underscoring the need to develop highly effective antivirals. In the setting of waning immunity from infection and vaccination, breakthrough infections are becoming increasingly common and treatment options remain limited. In addition, the emergence of SARS-CoV-2 variants of concern, with their potential to escape neutralization by therapeutic monoclonal antibodies, emphasizes the need to develop second-generation oral antivirals targeting highly conserved viral proteins that can be rapidly deployed to outpatients. Here, we demonstrate the in vitro antiviral activity and in vivo therapeutic efficacy of GS-621763, an orally bioavailable prodrug of GS-441524, the parent nucleoside of remdesivir, which targets the highly conserved virus RNA-dependent RNA polymerase. GS-621763 exhibited antiviral activity against SARS-CoV-2 in lung cell lines and two different human primary lung cell culture systems. GS-621763 was also potently antiviral against a genetically unrelated emerging coronavirus, Middle East respiratory syndrome CoV (MERS-CoV). The dose-proportional pharmacokinetic profile observed after oral administration of GS-621763 translated to dose-dependent antiviral activity in mice infected with SARS-CoV-2. Therapeutic GS-621763 administration reduced viral load and lung pathology; treatment also improved pulmonary function in COVID-19 mouse model. A direct comparison of GS-621763 with molnupiravir, an oral nucleoside analog antiviral that has recently received EUA approval, proved both drugs to be similarly efficacious in mice. These data support the exploration of GS-441524 oral prodrugs for the treatment of COVID-19.

Therapeutic efficacy of an oral nucleoside analog of remdesivir against SARS-CoV-2 pathogenesis in mice.

The COVID-19 pandemic remains uncontrolled despite the rapid rollout of safe and effective SARS-CoV-2 vaccines, underscoring the need to develop highly effective antivirals. In the setting of waning immunity from infection and vaccination, breakthrough infections are becoming increasingly common and treatment options remain limited. Additionally, the emergence of SARS-CoV-2 variants of concern with their potential to escape therapeutic monoclonal antibodies emphasizes the need to develop second-generation oral antivirals targeting highly conserved viral proteins that can be rapidly deployed to outpatients. Here, we demonstrate the in vitro antiviral activity and in vivo therapeutic efficacy of GS-621763, an orally bioavailable prodrug of GS-441524, the parental nucleoside of remdesivir, which targets the highly conserved RNA-dependent RNA polymerase. GS-621763 exhibited significant antiviral activity in lung cell lines and two different human primary lung cell culture systems. The dose-proportional pharmacokinetic profile observed after oral administration of GS-621763 translated to dose-dependent antiviral activity in mice infected with SARS-CoV-2. Therapeutic GS-621763 significantly reduced viral load, lung pathology, and improved pulmonary function in COVID-19 mouse model. A direct comparison of GS-621763 with molnupiravir, an oral nucleoside analog antiviral currently in human clinical trial, proved both drugs to be similarly efficacious. These data demonstrate that therapy with oral prodrugs of remdesivir can significantly improve outcomes in SARS-CoV-2 infected mice. Thus, GS-621763 supports the exploration of GS-441524 oral prodrugs for the treatment of COVID-19 in humans.

Colloidal stability dictates drop breakup under electric fields.

Electric fields can deform drops of fluid from their equilibrium shape, and induce breakup at sufficiently large field strengths. In this work, the electric field induced breakup of a squalane drop containing a colloidal suspension of carbon black particles with polyisobutylene succinimide (OLOA 11000) surfactant is studied. The drop is suspended in silicone oil. The breakup mode of a drop containing carbon black depends strongly on the suspension stability. It is observed that a drop of a stable suspension of carbon black has the same breakup mode as a drop with surfactant alone, i.e., without added carbon black. At lower electric fields, these drops break by the formation of lobes at the two ends of the drop; and at higher fields the homogeneous lobes break in a non-axisymmetric manner. However, a drop of an unstable suspension shows a drastically different breakup mode, and undergoes breakup much faster compared to a drop with surfactant alone. These drops elongate and form asymmetric lobes that develop into fingers and eventually disintegrate in an inhomogeneous, three-dimensional fashion. As a basis for comparison, the breakup of a pure squalane drop, and a squalane drop with equivalent surfactant concentrations but no carbon black particles is examined. Axisymmetric boundary integral computations are used to elucidate the mechanism of breakup. Our work demonstrates the impact of colloidal stability on the breakup of drops under an electric field. Colloidal stability on the time scale of drop deformation leads to rich and unexplored breakup phenomena.

Microfluidic Droplet-Based Tool To Determine Phase Behavior of a Fluid System with High Composition Resolution.

The goal of this work is to develop a simple microfluidic approach to characterizing liquid-liquid phase behavior in complex aqueous mixtures of organics and salts. We take advantage of the permeability of inexpensive microfluidic devices to concentrate aqueous solutions on chip. We demonstrate a technique that allows phase boundaries to be identified with high compositional resolution and small sample volumes. Droplets of single phase samples are produced on-chip and concentrated in the device beyond the phase boundary line to map system phase behavior. Results are demonstrated on ammonium sulfate and organic (poly(ethylene oxide)) aqueous solutions and compared with macroscopic and literature results.

Droplet-based characterization of surfactant efficacy in colloidal stabilization of carbon black in nonpolar solvents.

Development of an electrostatic stabilization mechanism for colloidal suspensions in nonpolar fluids requires an improved understanding of the interactions between the inverse micelles and particles as well as the roles that steric and electrostatic effects play. A droplet-based millifluidic device is designed and used to investigate the stabilization effects of surfactants on colloidal suspensions. A system containing carbon black and the surfactant OLOA 11000 suspended in dodecane is chosen as a well-characterized system to study sedimentation quantitatively. This device takes advantage of sub-millimeter optical path lengths to characterize sedimentation at concentrations at which sedimentation is not observable in the bulk and to achieve higher resolution in composition. A simple image analysis algorithm has been developed and applied to quantify sedimentation. Conductivity measurements using electrochemical impedance spectroscopy (EIS) are coupled with the sedimentation experiments to identify the concentration ranges in which steric and electrostatic effects are dominant. A more gradual transition is observed than previously reported.