A discovery program targeting respiratory syncytial virus (RSV) identified C-nucleoside 4 (RSV A2 EC50 = 530 nM) as a phenotypic screening lead targeting the RSV RNA-dependent RNA polymerase (RdRp). Prodrug exploration resulted in the discovery of remdesivir (1, GS-5734) that is >30-fold more potent than 4 against RSV in HEp-2 and NHBE cells. Metabolism studies in vitro confirmed the rapid formation of the active triphosphate metabolite, 1-NTP, and in vivo studies in cynomolgus and African Green monkeys demonstrated a >10-fold higher lung tissue concentration of 1-NTP following molar normalized IV dosing of 1 compared to that of 4. A once daily 10 mg/kg IV administration of 1 in an African Green monkey RSV model demonstrated a >2-log10 reduction in the peak lung viral load. These early data following the discovery of 1 supported its potential as a novel treatment for RSV prior to its development for Ebola and approval for COVID-19 treatment.

Remdesivir (RDV, GS-5734), the first FDA-approved antiviral for the treatment of COVID-19, is a single diastereomer monophosphoramidate prodrug of an adenosine analogue. It is intracellularly metabolized into the active triphosphate form, which in turn acts as a potent and selective inhibitor of multiple viral RNA polymerases. RDV has broad-spectrum activity against members of the coronavirus family such as SARS-CoV-2, SARS-CoV, and MERS-CoV, as well as filoviruses and paramyxoviruses. To assess potential for off-target toxicity, RDV was evaluated in a set of cellular and biochemical assays. Cytotoxicity was evaluated in a set of relevant human cell lines and primary cells. In addition, RDV was evaluated for mitochondrial toxicity under aerobic and anaerobic metabolic conditions, and for the effects on mitochondrial DNA content, mitochondrial protein synthesis, cellular respiration, and induction of reactive oxygen species. Lastly, the active 5’-triphosphate metabolite of RDV, GS-443902, was evaluated for potential interaction with human DNA and RNA polymerases. Among all of the human cells tested under 5-14 days of continuous exposure, RDV’s CC50 values ranged from 1.7 to >20 μM, resulting in selectivity indices (SI, CC50/EC50) from >170 to 20,000, with respect to RDV anti-SARS-CoV-2 activity (EC50 of 9.9 nM in human airway epithelial cells). Overall, the cellular and biochemical assays demonstrated a low potential of RDV for off-target toxicity including mitochondria-specific toxicity, consistent with the reported clinical safety profile.

Remdesivir (RDV; GS-5734; Veklury®), the first FDA-approved antiviral to treat COVID-19, is a single diastereomer monophosphoramidate prodrug of an adenosine analogue. RDV is taken up in the target cells and metabolized in multiple steps to form the active nucleoside triphosphate (TP) (GS-443902), which in turn acts as a potent and selective inhibitor of multiple viral RNA polymerases. In this report, we profiled the key enzymes involved in the RDV metabolic pathway with multiple parallel approaches: (1) bioinformatic analysis of nucleoside/tide metabolic enzyme mRNA expression using public human tissue and lung single-cell RNAseq datasets; (2) protein and mRNA quantification of enzymes in human lung tissue and primary lung cells; (3) biochemical studies on the catalytic rate of key enzymes; (4) effects of specific enzyme inhibitors on the GS-443902 formation; and (5) the effects of these inhibitors on RDV antiviral activity against SARS-CoV-2 in cell culture. Our data collectively demonstrated that carboxylesterase 1 (CES1) and cathepsin A (CatA) are enzymes involved in hydrolyzing RDV to its alanine intermediate Met X, which is further hydrolyzed to the monophosphate form by histidine triad nucleotide-binding protein 1 (HINT1). The monophosphate is then consecutively phosphorylated to diphosphate and triphosphate by cellular phosphotransferases. Our data support the hypothesis that the unique properties of RDV prodrug not only allow lung-specific accumulation critical for the treatment of respiratory viral infection such as COVID-19, they also enable efficient intracellular metabolism of RDV and its Met X to monophosphate and successive phosphorylation to form the active TP in disease-relevant cells.

Background Accurate measurement of esophageal hiatus size is clinically important, especially when antireflux surgery is planned. We present a novel method for in vivo measurement of esophageal hiatal surface area using MDCT multiplanar reconstruction. We aimed to determine if large hiatal area is associated with hiatal hernia and gastroesophageal reflux disease. Methods We retrospectively analyzed subjects prospectively enrolled in the COPDGene® project. We created two test groups, one with hiatal hernia on chest CT, and one with GERD on medical treatment identified by history without hernia. Matched control groups were formed. We performed CT post-processing to define the double oblique plane of the esophageal hiatus, on which the hiatal surface area is manually traced. Results 48 subjects with hernia had larger mean hiatus areas than matched controls (6.9 cm2 vs. 2.5 cm2, p<0.0001), and were more likely to have GERD (42% vs. 10%, p<0.0005). Subjects with mixed (type III) hernias had larger hiatuses compared to subjects with sliding (type I) hernias, who, in turn, had larger hiatuses than subjects without hernia (p<0.0001). 55 hernia-negative subjects with GERD did not have significantly larger mean hiatal areas compared to matched controls (3.0 cm2 vs. 2.5 cm2, p=0.12). 20 measurements obtained by two radiologists showed correlation of 0.93, with mean difference of 0.5 cm2 (p=0.20). Conclusions We devised a method to measure in vivo esophageal hiatal surface area using MDCT reconstruction and established the normal size range for the first time. This methodology has the potential to guide decision-making in antireflux surgery technique pre-operatively, and assess surgical result post-operatively. Presence of hernia correlated with large hiatuses and GERD. However, hiatal area failed to identify those with GERD in the absence of hiatal hernia.

Remdesivir (RDV) is a nucleotide analog prodrug with demonstrated clinical benefit in patients with coronavirus disease 2019 (COVID-19). In October 2020, the U.S. FDA approved intravenous RDV as the first treatment for hospitalized COVID-19 patients. Furthermore, RDV has been approved or authorized for emergency use in more than 50 countries. To make RDV more convenient for nonhospitalized patients earlier in disease, alternative routes of administration are being evaluated. Here, we investigated the pharmacokinetics and efficacy of RDV administered by head dome inhalation in African green monkeys (AGM). Relative to an intravenous administration of RDV at 10 mg/kg, an about 20-fold lower dose administered by inhalation produced comparable concentrations of the pharmacologically active triphosphate in lower respiratory tract tissues. Distribution of the active triphosphate into the upper respiratory tract was also observed after inhaled RDV exposure. Inhalation RDV dosing resulted in lower systemic exposures to RDV and its metabolites as compared with intravenous RDV dosing. An efficacy study with repeated dosing of inhaled RDV in an AGM model of SARS-CoV-2 infection demonstrated reductions in viral replication in bronchoalveolar lavage fluid and respiratory tract tissues compared with placebo. Efficacy was observed with inhaled RDV administered once daily at a pulmonary deposited dose of 0.35 mg/kg beginning about 8 hours after infection. Moreover, the efficacy of inhaled RDV was similar to that of intravenous RDV administered once at 10 mg/kg followed by 5 mg/kg daily in the same study. Together, these findings support further clinical development of inhalation RDV.