Antiviral; SARS-CoV-2; Prodrug form of GS-441524

GS-621763 has antiviral activity against SARS-CoV-2 in cell lines and human primary cell cultures. [1]
GS-441524 is the parental adenosine nucleoside analog (Fig. 1A) of both monophosphoramidate prodrug RDV (GS-5734, Fig. 1B), and triester prodrug GS-621763 (Fig. 1C). All three molecules are metabolized to the same active nucleotide triphosphate in cells, but through different activation pathways. GS-621763 is rapidly metabolized during oral absorption to GS-441524, then intracellularly converted by cellular kinases to the analog monophosphate metabolite before further metabolism to the active nucleoside triphosphate. In contrast, the intact phosphoramidate prodrug, RDV, is broken down inside cells directly to the same monophosphate metabolite, effectively bypassing the rate-limiting first phosphorylation step of GS-441524. To determine if GS-621763 could inhibit replication of SARS-CoV-2 in cellular assays, we first evaluated its antiviral activity against a SARS-CoV-2 reporter virus expressing nanoluciferase (SARS-CoV-2 nLUC) in A549-hACE2 cells stably expressing the human entry receptor angiotensin-converting enzyme 2 (ACE2). With GS-621763, we observed a dose-dependent antiviral effect on SARS-CoV-2 nLUC replication with an average half-maximum effective concentration (EC50) of 2.8 μM (Fig. 1D, Fig.1H, and Supplementary Figure 1A). In the same assay, we measured EC50 values for the control compound RDV of 0.28 μM, similar to those reported previously in these cells and reflective of the enhanced ability of the phosphoramidate prodrug to rapidly and efficiently generate active triphosphate by bypassing the slower initial phosphorylation step (Fig. 1D, Fig. 1H, Supplementary Figure 1A). As was observed in other cell systems, the parental nucleoside, GS-441524, was less potent (EC50 = 3.3 μM) than RDV in our assay and was similar in potency to GS-621763. This suggests that the tri-isobutyryl esters of GS-621763 are efficiently cleaved in the assay to release GS-441524 (Fig. 1D, Fig. 1H, Supplementary Figure 1A). Importantly, we did not observe any measurable cytotoxicity of any of the inhibitors in A549-hACE2 cells at concentrations up to 10 μM (Figure 1F, Supplementary Figure 1B). Human primary airway epithelial (HAE) cell cultures model the cellular complexity and architecture of the human conducting airway and are often used to determine if drugs are transported and metabolized in the cells targeted by emerging CoV in vivo. In HAE cells infected with WT SARS-CoV-2 and treated with GS-621763, we observed a dose-dependent and significant reduction in infectious virus production as compared to DMSO-vehicle treated cultures (Fig. 1F). In similarly infected, control compound (i.e. RDV or GS-441524) treated cultures, a significant and dose-dependent reduction in viral titers was also observed (Fig. 1F). GS-621763, RDV, and GS-441524 inhibited reporter SARS-CoV-2 expressing Firefly luciferase (SARS-CoV-2 Fluc) replication in normal human bronchial epithelial (NHBE) cultures with EC50 values of 0.125, 0.0371, and 2.454 μM, respectively (Fig. 1G, Fig. 1H). All together, these data show that GS-621763 is transported, metabolized and potently antiviral human primary cell systems that model the tissues targeted by SARS-CoV-2 in humans.

---

Half-maximal effective concentrations (EC50) in SARS-CoV-2-infected Vero E6 cells were highly consistent, ranging from 0.11 to 0.73 μM for GS-621763 (Fig. 1b, Supplementary Table 1) and from 0.11 to 0.68 μM for GS-441524 (Fig. 1c, Supplementary Table 1). Analogous potency ranges were obtained when luciferase-expressing WA1/2020 reporter viruses were examined in dose-response assays in A549 cells stably expressing human ACE2 (A549-hACE2) (Supplementary Table 1)20. Toxicity-testing of GS-621763, remdesivir, and GS-441524 in different cell lines and primary human cells derived from different donors revealed half-maximal cytotoxic concentrations (CC50) of 40 to >100 μM (Fig. 1d, Supplementary Table 1), 36 to >100 μM (Supplementary Fig. 1, Supplementary Table 1) and >100 μM (Fig. 1e, Supplementary Table 1), respectively, corresponding to selectivity indices (SI = CC50/EC50) of GS-621763 > 137 in VeroE6 and >51 in A549-ACE2 cells. The efficacy of GS-441524 and GS-621763 was in parallel assessed on well-differentiated primary human airway epithelium cultures grown at the air-liquid interface and apically infected with VOC γ (Fig. 1f–g). Basolaterally added GS-441524 or GS-621763 displayed similar potency in this disease-relevant human tissue model, returning EC50 values of 2.83 and 3.01 µM, respectively. Parallel measurement of transepithelial electrical resistance demonstrated that epithelium integrity was fully preserved at basolateral drug concentrations ≥3 µM [2].

Dose-dependent therapeutic efficacy of GS-621763 in mouse models of COVID-19 disease.[1]
We have previously performed multiple studies describing the therapeutic efficacy of subcutaneously administered RDV in mice (Ces1c−/− C57BL/6J) genetically deleted for a secreted plasma carboxylesterase 1c (Ces1c) absent in humans but dramatically reduces drug half-life in wild-type mice.[1]
However, the prodrug GS-621763 is designed to be rapidly cleaved pre-systemically in vivo to release GS-441524 into circulation, with no or very minimal intact ester observed in plasma. Therefore, GS-621763 can be studied in wild-type mice where it should also be rapidly converted to parent GS-441524. Plasma pharmacokinetics following a single oral administration of GS-621763 at either 5 or 20 mg/kg were first determined in uninfected BALB/c mice (Fig. 2A). Doses were selected to provide high plasma exposures of GS-441524 that would support active triphosphate formation in the lung and to confirm pharmacokinetic dose proportionality needed to project exposures in efficacy studies. Previous studies had shown that parent nucleoside was at least 10-fold less efficient at generating lung triphosphate than RDV, on a molar basis, thus requiring higher plasma exposures of parental GS-441524 to account for the reduced metabolic efficiency. No exposure of intact ester prodrug, within the limit of detection, was observed in mice. GS-441524 was both rapidly absorbed and then cleared from systemic circulation, exhibiting a short plasma half-life of approximately 1 hr. Dose proportional increases in both maximal plasma concentrations (Cmax) and exposures (AUC0–24h) at the two doses were observed.[1]
To better understand the pharmacokinetic and pharmacodynamic relationship for GS-621763, we performed a series of dose-finding studies in BALB/c mice infected with mouse-adapted SARS-CoV-2 (SARS-CoV-2 MA10). In young adult BALB/c mice infected with 104 plaque forming units (PFU) SARS-CoV-2 MA10, virus replicates to high titers in the respiratory tract, mice lose 15–20% of their body weight by 4 days post-infection (dpi), and acute lung injury/loss of pulmonary function is typically observed after virus replication peaks on 2 dpi. We first defined the minimum dosage sufficient for maximal therapeutic efficacy in BALB/c mice initiating twice daily (i.e. bis in die, BID) oral treatment with either vehicle control or 3 mg/kg, 10 mg/kg, or 30 mg/kg GS-621763 beginning 8 hours post infection (hpi) with 104 PFU SARS-CoV-2 MA10 (Fig. 2B). Unlike vehicle or 3 mg/kg GS-621763 treated animals, mice receiving either 10 or 30 mg/kg GS-621763 were completely protected from weight loss thus demonstrating that early oral antiviral therapy can prevent the progression of disease (Fig. 2B). Congruent with the weight loss phenotype, both 10 and 30 mg/kg GS-621763 treated animals had significantly reduced viral lung titers as compared to both the vehicle and 3 mg/kg treated groups (Fig. 2C). To monitor the effect of drug treatment on pulmonary function, we performed daily whole-body plethysmography (WBP) with a subset of mice from each group (N=4 per treatment group). As shown with the WBP metric PenH, whose elevation is associated with airway resistance or obstruction, we observed a drug dose-dependent reduction in PenH with the maximal effect seen in the 30 mg/kg GS-621763 dose group which was completely protected from the loss of pulmonary function observed in the other treatment groups and vehicle (Fig. 2D). Mice treated with 3 and 10 mg/kg GS-621763 had impaired lung function at days 2 and 3 post infection, but lung function returned to baseline by 4 dpi for all GS-621763 treated animals (Fig. 2D). Consistent with weight loss, virus titer, and pulmonary function data, mice treated with 10 or 30 mg/kg had significantly reduced lung congestion, a gross pathologic feature characteristic of severe lung damage (Fig. 2E). We then scored lung tissue sections for the histologic features of acute lung injury (ALI) using two complementary semiquantitative tools. First, using an ALI scoring tool created by the American Thoracic Society (ATS), we blindly evaluated three diseased fields per lung section for several features of ALI including alveolar septal thickening, neutrophils in the interstitium and in air spaces, proteinaceous debris in airspaces, and the presence of hyaline membranes. Only mice treated with 30 mg/kg had significantly reduced ALI scores (Fig. 2F). Second, we used a complementary tool measuring the pathologic hallmark of ALI, diffuse alveolar damage (DAD). Mice in all treated groups showed reduced DAD scores, but only mice receiving 30 mg/kg had significantly decreased DAD in their lungs (Fig. 2G). Together, these data demonstrate that the oral delivery of the nucleoside analog GS-621763 can significantly diminish SARS-CoV-2 virus replication and associated pulmonary disease in a dose-dependent manner.

---

Extended therapeutic protection against COVID-19 disease by GS-621763 in mice[1]
To determine if the potent therapeutic efficacy of GS-621763 observed with early intervention (8 hr after infection) would extend to later times post infection, we designed a therapeutic efficacy study with six arms where we varied both time of oral therapy initiation and dose level in BALB/c mice infected with SARS-CoV-2 MA10 (Fig.3). As done previously, a control group of animals received vehicle twice daily beginning at 12 hours post infection (hpi). The next three arms of the study were dedicated to the 30 mg/kg GS-621763 dose level, with two of the three arms receiving twice daily dosing initiated at either the 12 hpi (“30 mg/kg BID 12 hr” group) or the 24 hpi (“30 mg/kg BID 24 hr” group). The third 30 mg/kg arm was designed to determine if dose frequency could be reduced to once daily (quaque die, QD) if initiated early at 12 hpi (“30 mg/kg QD 12 hr” group). In the last two arms, we wanted to evaluate if an increased dose of 60 mg/kg given QD beginning at 12 hr or 24 hr (“60 mg/kg QD 12 hr” and “60 mg/kg QD 24 hr” groups) would improve outcomes over the 30 mg/kg groups. Initiation of 30 mg/kg BID therapy at either 12 or 24 hrs offered significant protection from weight loss (Fig. 3A), extending the robust therapeutic phenotype observed for this dose level when initiated at very early times (at 8 hr) (Fig 2). Interestingly, when we decreased the frequency of 30 mg/kg treatment initiated at 12 hr to once daily (“30 mg/kg QD 12 hr” group), we also observed a significant prevention of body weight loss (Fig 3A), thus levels of drug when administered once a day and begun early (at 12 hr) in the course of infection were sufficient to prevent disease progression. Increasing the dose to 60 mg/kg QD initiated at either 12 hr or 24 hr offered similar protection from weight loss observed with vehicle treatment as the 30 mg/kg groups (Fig. 3A).

---

The therapeutic efficacy of GS-621763 is similar to molnupiravir (MPV, EIDD-2801)[1]
MPV is an oral nucleoside analog prodrug antiviral currently in Phase 3 clinical trial to treat COVID-19 with demonstrated antiviral efficacy in mice against several emerging CoV including SARS-CoV, MERS-CoV, and SARS-CoV-2. Like GS-621763, MPV is a prodrug which is metabolized in vivo into a parental nucleoside (β-D-N4-hydroxycytidine, NHC) in its metabolic progression towards the antiviral active triphosphate. To determine if GS-621763 would provide similar protection as MPV, we then designed comparative therapeutic efficacy studies in the mouse model of SARS-CoV-2 pathogenesis described above. Pre-efficacy pharmacokinetic studies in BALB/c mice (30 mg/kg or 100 mg/kg) were performed with MPV and showed dose proportional increases in NHC plasma exposures (Supplemental Fig. 2). Pharmacokinetic modeling then determined that a daily 120 mg/kg dose (given 60 mg/kg BID) would result in exposures similar to that observed in humans receiving 800 mg BID, a dose being evaluated in a human clinical trial. The comparative efficacy study included a vehicle group and 5 additional groups receiving two doses of MPV or GS-621763 per day 12 hrs apart (BID). Three arms of the study began dosing at 12 hr: 30 mg/kg GS-621763, 30 mg/kg MPV (0.5× human equivalent dose) or 60 mg/kg MPV (1× human equivalent dose). At 24 hr, we began dosing of two additional groups: 60 mg/kg GS-621763 or 60 mg/kg MPV. While SARS-CoV-2 MA10 infection caused rapid weight loss in vehicle control animals, all animals receiving either GS-621763 or MPV beginning at either 12 or 24 hr were protected from weight loss (Fig. 4A). Similarly, upon titration of lung tissues at 4 dpi for infectious virus by plaque assay, vehicle-treated animals had expectedly high levels of infectious virus which was significantly reduced in all treatment groups, independent of drug type or initiation time (Fig. 4B). When treatment was initiated at 12 hr, a moderate yet significant elevation in infectious titers was observed in the 30mg/kg MPV group, inferior to either equivalently dosed GS-621763 animals or those receiving the higher dose (60 mg/kg) of MPV (Fig. 4B). To understand the relationship between levels of infectious virus and viral RNA in lung tissue, we performed qRT-PCR on total RNA for SARS-CoV2 N RNA in parallel tissues utilized for plaque assay. The trend observed with infectious virus is mirrored in the qRT-PCR data where all groups receiving antiviral therapy had significantly reduced levels of viral RNA (Fig. 4C). In addition, animals receiving 30 mg/kg MPV (0.5× human equivalent dose) had a measurable increase in N RNA as compared to equivalently dosed GS-621763 animals. Similar to weight loss data, vehicle-treated animals had a significant loss of pulmonary function as measured by WBP on both 3 and 4 dpi which was prevented in all groups receiving antiviral treatment (Fig. 4D). We then blindly evaluated lung tissue sections for the pathological manifestations of ALI and DAD using two complementary histologic tools described above. Congruent with the above data, ALI scores in all antiviral therapy groups were significantly reduced as compared to vehicle controls (Fig. 4E). In agreement with ALI scores, the DAD histologic scores were similarly reduced in all antiviral therapy treated groups as compared to those treated with vehicle (Fig. 4F). All together, these data show that antiviral therapy with GS-621763 and MPV when initiated early or at the peak of virus replication (~24 hr) can both significantly diminish virus replication and improve disease outcomes.

---

Prophylactic efficacy in ferrets [2]
To test antiviral efficacy, we infected ferrets intranasally with 1 × 105 plaque-forming units (pfu) of WA1/2020, followed by twice daily (b.i.d.) oral treatment with GS-621763 at 20 mg/kg body weight for four days (Fig. 2b). Treatment was initiated at the time of infection, nasal lavages collected in 12-h intervals, and respiratory tissues harvested 4 days after infection. Shed SARS-CoV-2 load in nasal lavages of vehicle-treated animals reached plateau 1.5 days after infection at approximately 1 × 104 pfu/mL, whereas virus was transiently detectable in lavages of only one ferret of the GS-621763-treatment group at 12 h after infection (Fig. 2c). Clinical signs overall are minor in the ferret model6. However, only animals of the vehicle group showed elevated body temperature (Fig. 2d) and reduced weight gain (Fig. 2e). The virus was undetectable in the nasal turbinates extracted from treated animals 4 days after infection, compared to a robust load of approximately 5 × 104 pfu/g nasal turbinate of animals of the vehicle group (Fig. 2f). Viral RNA copy numbers found in lavages (Fig. 2g) and turbinates (Fig. 2h) mirrored the infectious titer results, revealing a consistent, statistically significant difference between the vehicle and treatment groups of two and three orders of magnitude, respectively. Consistent with prior studies6, no infectious virions or viral RNA were detectable in the lower respiratory tract (Fig. 2i, j).

---

Therapeutic efficacy and lowest efficacious dose [2]
To determine the lowest efficacious dose in a clinically more relevant therapeutic setting, we initiated oral treatment 12 h after infection, when the shed virus is first detectable in nasal lavages, at the 10 mg/kg and 3 mg/kg body weight levels, administered b.i.d. (Fig. 3a). EIDD-2801/molnupiravir at 5 mg/kg b.i.d. was given as reference following an identical therapeutic b.i.d. regimen6. EIDD-2801 was included as a reference compound, since at the time of study EIDD-2801 was the only nucleoside analog with demonstrated oral efficacy against SARS-CoV-2 in the ferret model. Shed virus load was significantly lower in all treated animals than in the vehicle group within 12 h of treatment onset (Fig. 3b). Virus load in nasal lavages of ferrets receiving GS-621763 at 3 mg/kg plateaued approximately one order of magnitude lower than in those from vehicle animals, while treatment with GS-621763 at 10 mg/kg or EIDD-2801/molnupiravir reduced shedding to near-detection level by day 3 after infection. Consistent with this inhibitory effect, treatment with 3 mg/kg GS-621763 reduced burden in the turbinates by one order of magnitude (Fig. 3c), while virus burden approached the limit of detection in animals of the 10 mg/kg GS-621763 and EIDD-2801/molnupiravir treatment groups. No significant differences in clinical signs were noted between vehicle animals and any of the treatment groups (Fig. 3d, e).

---

Inhibition of replication and transmission of VOC γ [2]
To probe the anti-SARS-CoV-2 indication spectrum of GS-621763, we applied the efficacious regimen, 10 mg/kg GS-621763 b.i.d. started 12 h after infection, to recently emerged VOC γ22 in a combined efficacy and transmission study (Fig. 4a). After an initial replication delay, the shed virus became detectable in vehicle-treated animals 1.5 days after infection, then rapidly reached a robust plateau of nearly 104 pfu/mL nasal lavage on day 2 after infection (Fig. 4b). Quantitation of viral RNA copies mimicked the profile of the infectious titers, although a low viral RNA load was present in lavages already on the first day after infection (Fig. 4c). Viral titers and RNA copies in nasal turbinates determined 4 days after infection were likewise high, ranging from 104 to 105 pfu/g tissue (Fig. 4d) and 108 to 1010 RNA copies/g tissue (Fig. 4e), respectively. However, no infectious VOC γ virions or viral RNA were detected in the lungs of any of these animals (Fig. 4f, g), and no clinical signs such as changes in body weight or fever emerged (Supplementary Fig. 2a, b). This presentation mimicked our previous experience with WA1/20206, indicating that VOC γ does not invade the ferret host more aggressively than WA1/2020. Treatment of VOC γ infection with oral GS-621763 was highly efficacious, reducing both shed virus burden and tissue titers to undetectable levels (Fig. 4b, d) and lowering viral RNA copies in nasal lavages and turbinates by over three orders of magnitude (Fig. 4c, e).

In vitro assays for antiviral activity [1]
A549-hACE2 cells were plated at a density of 20,000 cells/well/100 μl in black-walled clear-bottom 96-well plates 24 hrs prior to infection. Compounds GS-621763, GS-5734, GS-441524, were diluted in 100% DMSO (1:3) resulting in a 1000X dose response from 10 to 0.002 mM (10 to 0.002 μM final). All conditions were performed in triplicate. At BSL3, medium was removed, and cells were infected with 100 μl SARS-CoV-2 nLUC (MOI 0.008) for 1 h at 37 °C after which virus was removed, wells were washed (150 μl) with infection media (DMEM, 4% FBS, 1X antibiotic/antimycotic) and infection media (100 μl) containing a dose response of drug was added. Plates were incubated at 37 °C for 48 hrs. NanoGlo assay was performed 48 hpi. Sister plates were exposed to drug but not infected to gauge cytotoxicity via CellTiter-Glo assay, 48 hrs post treatment.

Cytotoxicity assays [2]
In each well of 96-well plates, 7500 cells were seeded. Cells were incubated with threefold serial dilutions of compound from a 100 µM maximum concentration. Each plate included 4 wells of positive (100 µM cycloheximide and negative (vehicle (0.2% dimethyl sulfoxide (DMSO)) controls for normalization. Plates were incubated in a humidified chamber at 37 °C and 5% CO2 for 72 h. PrestoBlue™ Cell Viability Reagent was added in each well (10 μl/well) and fluorescence recorded on a Synergy H1 multimode microplate reader after 1-h incubation (excitation 560 nm, emission 590 nm). Raw data was normalized with the formula: % cell viability = 100 × (signal sample—signal positive control)/(signal negative control—signal positive control). Fifty percentage cytotoxic concentrations (CC50) and 95% confidence intervals after nonlinear regression were determined using the inhibitor vs normalized response equation in Prism 9.1.0 for MacOS (GraphPad). For cytotoxicity assays in A549-hACE2 cells, compounds (200 nl) were spotted onto 384-well plates prior to seeding 5000 A549-hACE2 cells/well in a volume of 40 µl culture medium. The plates were incubated at 37 °C for 48 h with 5% CO2. On day 2, 40 µl of CellTiter-Glo was added and mixed 5 times. Plates were read for luminescence on an Envision and CC50 values calculated using a nonlinear four parameter regression model.

---

Virus yield reduction [2]
In 12-well plates 16  h before infection, 2 × 105 VeroE6 cells were seeded per well. Confluent monolayers were then infected with the indicated virus at a multiplicity of infection (MOI) of 0.1 pfu/cell for 1 h at 37 °C with frequent rocking. Inoculum was removed and replaced with 1 mL of DMEM with 2% FBS and the indicated concentration of compound. Cells were incubated at 37 °C and 5% CO2 for 48 h. Supernatant were harvested, aliquoted and stored at −80 °C before being analyzed by plaque assay.

---

Reporter virus assays [2]
A549-hACE2 cells (12,000 cells per well in medium containing 2% FBS) were plated into a white clear-bottomed 96-well plate at a volume of 50 µl. On the next day, compounds were added directly to cultures as 3-fold serial dilutions with a Tecan D300e digital liquid dispenser, with DMSO volumes normalized to that of the highest compound concentration (final DMSO concentration < 0.1%). The diluted compound solutions were mixed with 50 μl of SARS-CoV-2-Nluc (MOI 0.025 pfu/cell), expressing a nano luciferase reporter protein. At 48 h postinfection, 75 μl Nano luciferase substrate solution was added to each well. Luciferase signals were measured using an Envision microplate reader. The relative luciferase signals were calculated by normalizing the luciferase signals of the compound-treated groups to that of the DMSO-treated groups (set as 100%). EC50 values were calculated using a nonlinear four parameter variable slope regression model.

---

Air-liquid interface (ALI) human airway epithelial cells (HAE) shed viral titer reduction [2]
Approximately 150,000 viable “F3” cells per cm2 at passage three were seeded on Transwell 6.5 mm polyester membrane insert with 0.4 µm pore size. Upon reaching confluence (day 4 postseeding), basal media was removed and replaced with PneumaCult-Ex Plus, while apical media was removed to create an air-liquid interface. Beating ciliae, mucus production and transepithelial electrical resistance (TEER) > 300 Ohm*cm2 were noticeable ~3 weeks post ALI, confirming successful differentiation. Cells were maintained in a differentiated state with weekly apical washes of mucus with PBS for 5 months before infection. One hour prior to infection, TEER was measured with 150 µl PBS and basal media was replaced with fresh media containing indicated serial dilutions (threefold down from 10 µM) of GS-621763 or GS-441524 or vehicle (dimethylsulfoxide 0.1%). The apical side was infected with ~25,000 PFU of SARS-CoV-2 gamma isolate grown on Calu-3 cells (lineage P.1., isolate hCoV-19/Japan/TY7-503/2021 (BZ/2021; Brazil P.1) in 100 µl DMEM for 1 h at 37 °C, then the inoculum was removed and washed with PBS twice. Cells were incubated for 3 days at 37 °C before final TEER measure and fixation with 10 % neutral buffered formalin for 1 h. Shed apical viruses were harvested with 200 µl PBS for 30 min at 37 °C 48 and 72 h postinfection and viral titers were estimated by plaque assay. To determine EC50s, log viral titers were normalized using the average top plateau of viral titers to define 100% and were analyzed with a nonlinear regression with the variable slope with Prism 9.0.1 for MacOS. TEER were measured with the EVOM or EVOM3 system.

Small molecule drug synthesis and formulation [1]
Small molecules (GS-621763, RDV, Molnupiravir, and GS-441524) were solubilized in 100% DMSO for in vitro studies and in vehicle containing 2.5% DMSO; 10% Kolliphor HS-15; 10% Labrasol; 2.5% Propylene glycol; 75% Water (final formulation pH 2) (for GS-621763) and in vehicle containing 2.5% Kolliphor RH-40, 10% Polyethylene glycol 300, 87.5% Water (for MPV) for in vivo studies.

---

In vivo plasma pharmacokinetic analysis of GS-621763 and molnupiravir (MPV) [1]
Mice were orally administered either a single dose of GS-621763 (in vehicle containing 2.5% DMSO; 10% Kolliphor HS-15; 10% Labrasol; 2.5% Propylene glycol; 75% Water (final formulation pH 2) or two doses of molnupiravir (in vehicle containing 2.5% Kolliphor RH-40, 10% Polyethylene glycol 300, 87.5% Water) (BID, 12 hours apart). GS-621763 was given at either 5 or 20 mg/kg and MPV at either 30 or 100 mg/kg. Plasma was serially isolated from 4 mice at 0.25, 1, 2, 8 and 24 hrs post GS-621763 administration. Plasma was isolated from alternating groups of 4 mice per timepoint at 0.5, 2, 6, 12 (pre-second dose), 12.5, 18 and 24 hrs post MPV administration. 20 μl of plasma was added to a mixture containing 250 μl of methanol and 25 μL of internal standard solution and centrifuged. 250 μl of resulting supernatant was then transferred, filtered (Agilent Captiva 96, 0.2 μm) and dried under a stream of nitrogen at 40 °C. Following reconstitution in a mixture of 5% acetonitrile and 95% water, a 10 μl aliquot was injected onto an LC-MS/MS system. Plasma concentrations of either GS-621763 and GS-441524 or MPV and N-hydroxycytidine (NHC) were determined using 8 to 10-point calibration curves spanning at least 3 orders of magnitude with quality control samples to ensure accuracy and precision, prepared in normal mouse plasma. Analytes were separated by a 50 mm × 3.0 mm, 2.55 μm Synergi Polar-RP column (Phenomenex) using a multi-stage linear gradient from 5% to 95% acetonitrile in mobile phase A at a flow rate of 1 ml/min.

---

Quantitation of GS-441524 metabolites in the lung following oral GS-621763 administration in Balb/c mice [1]
Lungs from all mice administered GS-621763 were quickly isolated at 24 hrs post-dose and immediately snap frozen in liquid nitrogen. On dry ice, frozen lung samples were pulverized and weighed. Dry ice-cold extraction buffer containing 0.1% potassium hydroxide and 67 mM ethylenediamine tetraacetic acid (EDTA) in 70% methanol, containing 0.5μM chloro-adenosine triphosphate as internal standard was added and homogenized. After centrifugation at 20,000 × g for 20 minutes, supernatants were transferred and dried in a centrifuging evaporator. Dried samples were then reconstituted with 60 μL of mobile phase A, containing 3 mM ammonium formate (pH 5) with 10 mM dimethylhexylamine (DMH) in water, centrifuged at 20,000 × g for 20 minutes and final supernatants transferred to HPLC injection vials. An aliquot of 10 μl was subsequently injected onto an API 6500 LC/MS/MS system for analysis of GS-441524 and its phosphorylated metabolites, performed using a similar method as described previously.

---

Pharmacokinetics [2]
Female ferrets were either intravenously administered 10 mg/kg remdesivir as a 30-min infusion or orally administered 30 mg/kg GS-621763, after which plasma was isolated at 7–9 timepoints postadministration. Plasma samples underwent methanol protein precipitation followed by centrifugation. The resulting supernatants were isolated, evaporated to dryness under nitrogen and reconstituted with 5% acetonitrile for injection onto an LC-MS/MS system. Concentrations of remdesivir, GS-621763, and GS-441524 were determined using 9-point calibration curves spanning at least 3 orders of magnitude, with quality control samples to ensure accuracy and precision, prepared in normal ferret plasma. Analytes were separated by a 50 × 3.0 mm, 2.55 μm Synergi Polar-RP 30 A column using a mobile phase A consisting of 10 mM ammonium formate with 0.1% formic acid and a mobile phase B consisting of 0.1% formic acid in acetonitrile. A multi-stage linear gradient from 5% to 95% mobile phase B at a flow rate of 1 mL/min was employed for analyte separation. Pharmacokinetic parameters were calculated using Phoenix WinNonlin (version 8.2, Certara) and concentration-time profiles generated using Prism. Ferret lungs were collected at 24 h following initiation of drug administration. Whole tissues were quickly isolated and immediately placed into liquid nitrogen and stored at -80 °C until processing and LC-MS/MS analysis2. Reported values for lung total nucleosides are the sum of (GS-441524 and mono-, di-, and triphosphate (GS-443902) metabolites).

---

Ferret efficacy studies [2]
Female ferrets (6–10 months old, Mustela putorius furo) were purchased from Triple F Farms. Ferrets were rested for 7 days after arrival. Ferrets were then housed individually or in groups of 2 in ventilated negative-pressure cages in an ABSL-3 facility. Based on the previous experiments6, ferrets were randomly assigned to groups (n = 4) and used as an in vivo model to examine the efficacy of orally administered compounds against SARS-CoV-2 infection. No blinding of investigators was performed. Ferrets were anesthetized using dexmedetomidine/ketamine and infected intranasally with 1 × 105 pfu 2019-nCoV/USA-WA1/2020 in 1 mL (0.5 mL per nare). Body weight and rectal temperature were measured once daily. Nasal lavages were performed twice daily using 1 mL sterile PBS (containing Antibiotic-Antimycotic). Nasal lavage samples were stored at -80 °C until virus titration could be performed by plaque assay. Treatment (once daily (q.d.) or twice daily (b.i.d.)) was initiated at either 0 or 12 h after infection and continued until 4 days postinfection with either vehicle (2.5% dimethyl sulfoxide; 10% Kolliphor HS-15; 10% Labrasol; 2.5% propylene glycol; 75% water) or compound. Four days after infection, ferrets were euthanized, and tissues and organs were harvested and stored at -80 °C until processed.

---

Contact transmission in ferrets [2]
Eight ferrets were anesthetized and inoculated intranasally with 1 × 105 pfu of hCoV-19/Japan/TY7-503/2021. Twelve hours after infection, ferrets were split into two groups (n = 4; 2 ferrets per cage) and treated with vehicle or GS-621763 (10 mg kg−1) twice daily (b.i.d.) via oral gavage. At 54 h after infection, uninfected and untreated contact ferrets (two contacts for GS-621763; three contacts for vehicle) were co-housed with source ferrets. Co-housing was continued until 96 h after infection and source ferrets were euthanized. Contact ferrets were housed individually and monitored for an additional 4 days after separation from source ferrets and subsequently euthanized. Nasal lavages were performed on all source ferrets every 12 h and all contact ferrets every 24 h. For all ferrets, nasal turbinates and lung tissues were harvested to determine viral titers and the detection of viral RNA.

Plasma pharmacokinetics following a single oral administration of GS-621763 at either 5 or 20 mg/kg were first determined in uninfected BALB/c mice (Fig. 2A). Doses were selected to provide high plasma exposures of GS-441524 that would support active triphosphate formation in the lung and to confirm pharmacokinetic dose proportionality needed to project exposures in efficacy studies. Previous studies had shown that parent nucleoside was at least 10-fold less efficient at generating lung triphosphate than RDV, on a molar basis, thus requiring higher plasma exposures of parental GS-441524 to account for the reduced metabolic efficiency. No exposure of intact ester prodrug, within the limit of detection, was observed in mice. GS-441524 was both rapidly absorbed and then cleared from systemic circulation, exhibiting a short plasma half-life of approximately 1 hr. Dose proportional increases in both maximal plasma concentrations (Cmax) and exposures (AUC0–24h) at the two doses were observed (Fig. 2A). [1]

---

Pharmacokinetics following oral administration [2]
Assessment of GS-621763 plasma PK parameters in the ferret revealed excellent oral bioavailability (Fig. 2a), extensive cleavage presystemically to generate high exposures of GS-441524 in the blood (Supplementary Table 2), efficient distribution to soft tissues of the respiratory system (lung), and confirmed anabolism to bioactive GS-443902 (Supplementary Table 3). Following a single 30 mg/kg oral dose of GS-621763 in ferrets, the daily systemic exposure (AUC0-24h) of GS-441524 was 81 µM.h, 4.5 fold higher than the exposure following IV remdesivir at 10 mg/kg and approximately 10-fold greater than that observed following an 200/100 mg IV remdesivir dose in human. Lower levels of bioactive GS-443902 were formed from oral 30 mg/kg GS-621763 dosing compared to 10 mg/kg IV remdesivir (Supplementary Table 3), illustrating the difference in intracellular activation efficiency of the phosphoramidate prodrug remdesivir compared to systemic parent nucleoside GS-441524.

[1]. Therapeutic efficacy of an oral nucleoside analog of remdesivir against SARS-CoV-2 pathogenesis in mice. bioRxiv. 2021 Sep 17:2021.09.13.460111.

[2]. Oral prodrug of remdesivir parent GS-441524 is efficacious against SARS-CoV-2 in ferrets. Nat Commun. 2021 Nov 5;12(1):6415.

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.
In summary, we provide preclinical data demonstrating the in vitro antiviral activity and in vivo therapeutic efficacy of an orally bioavailable nucleoside analog prodrug, GS-621763. The data provided herein supports the future evaluation of orally bioavailable prodrugs of GS-441524 in humans with COVID-19. If safe and effective, this class of RdRp inhibitors could become part of the arsenal of existing oral antivirals that are desperately needed to address a global unmet need for the COVID-19 pandemic and CoV pandemics of the future. [1]

---

Remdesivir is an antiviral approved for COVID-19 treatment, but its wider use is limited by intravenous delivery. An orally bioavailable remdesivir analog may boost therapeutic benefit by facilitating early administration to non-hospitalized patients. This study characterizes the anti-SARS-CoV-2 efficacy of GS-621763, an oral prodrug of remdesivir parent nucleoside GS-441524. Both GS-621763 and GS-441524 inhibit SARS-CoV-2, including variants of concern (VOC) in cell culture and human airway epithelium organoids. Oral GS-621763 is efficiently converted to plasma metabolite GS-441524, and in lungs to the triphosphate metabolite identical to that generated by remdesivir, demonstrating a consistent mechanism of activity. Twice-daily oral administration of 10 mg/kg GS-621763 reduces SARS-CoV-2 burden to near-undetectable levels in ferrets. When dosed therapeutically against VOC P.1 gamma γ, oral GS-621763 blocks virus replication and prevents transmission to untreated contact animals. These results demonstrate therapeutic efficacy of a much-needed orally bioavailable analog of remdesivir in a relevant animal model of SARS-CoV-2 infection. [2]

C24H31N5O7

501.5322

501.222

C, 57.48; H, 6.23; N, 13.96; O, 22.33

2647442-13-3

Mindeudesivir;2647442-33-7

162625114

White to off-white solid powder

2.3

1

11

11

36

887

4

O1[C@]([H])(C([H])([H])OC(C([H])(C([H])([H])[H])C([H])([H])[H])=O)[C@]([H])([C@]([H])([C@]1(C#N)C1=C([H])C([H])=C2C(N([H])[H])=NC([H])=NN12)OC(C([H])(C([H])([H])[H])C([H])([H])[H])=O)OC(C([H])(C([H])([H])[H])C([H])([H])[H])=O

RVSSLHFYCSUAHY-JQGROFRJSA-N

InChI=1S/C24H31N5O7/c1-12(2)21(30)33-9-16-18(34-22(31)13(3)4)19(35-23(32)14(5)6)24(10-25,36-16)17-8-7-15-20(26)27-11-28-29(15)17/h7-8,11-14,16,18-19H,9H2,1-6H3,(H2,26,27,28)/t16-,18-,19-,24+/m1/s1

[(2R,3R,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-bis(2-methylpropanoyloxy)oxolan-2-yl]methyl 2-methylpropanoate

GS-621763; 2647442-13-3; GS-441524 prodrug; Remdesivir derivative; 83BU3492RP; GS621763; VV-116 free base; (2R,3R,4R,5R)-2-(4-Aminopyrrolo(2,1-f)(1,2,4)triazin-7-yl)-2-cyano-5-((isobutyryloxy)methyl)tetrahydrofuran-3,4-diyl bis(2-methylpropanoate);

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 100 mg/mL (199.39 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.98 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: 2.5 mg/mL (4.98 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (4.98 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)