Pharmacokinetics and Safety of Remdesivir in Pregnant and Non-Pregnant Women with COVID-19: Results from IMPAACT 2032.

BACKGROUND: Pregnant people with COVID-19 experience higher risk for severe disease and adverse pregnancy outcomes, but no pharmacokinetic (PK) data exist to support dosing of COVID-19 therapeutics during pregnancy. We report PK and safety data for intravenous remdesivir in pregnancy. METHODS: IMPAACT 2032 was a phase IV prospective, open-label, non-randomized opportunistic study of hospitalized pregnant and non-pregnant women receiving intravenous remdesivir as part of clinical care. Intensive PK sampling was performed on infusion days 3, 4, or 5 with collection of plasma and peripheral blood mononuclear cells (PBMCs). Safety data were recorded from first infusion through 4 weeks post-last infusion and at delivery. Geometric mean ratios (GMR) (90% confidence intervals [CI]) of PK parameters between pregnant and non-pregnant women were calculated. RESULTS: Fifty-three participants initiated remdesivir (25 pregnant; median (IQR) gestational age 27.6 (24.9, 31.0) weeks). Plasma exposures of remdesivir, its two major metabolites (GS-704277 and GS-441524), and the free remdesivir fraction were similar between pregnant and non-pregnant participants. Concentrations of the active triphosphate (GS-443902) in PBMCs increased 2.04-fold (90% CI 1.35, 3.03) with each additional infusion in non-pregnant versus pregnant participants. Three adverse events in non-pregnant participants were related to treatment (one Grade 3; two Grade 2 resulting in treatment discontinuation). There were no treatment-related adverse pregnancy outcomes or congenital anomalies detected. CONCLUSIONS: Plasma remdesivir PK parameters were comparable between pregnant and non-pregnant women, and no safety concerns were identified based on our limited data. These findings suggest no dose adjustments are indicated for intravenous remdesivir during pregnancy.

Assessment of remdesivir and its nucleoside metabolite in beagle dogs and healthy humans by liquid chromatography coupled with triple quadrupole mass spectrometry.

The aim of this study was to assess the pharmacokinetics of the existing remdesivir intravenous formulation (100 mg dose) against the newly developed oral formulation (20 mg dose) for remdesivir and its active nucleoside metabolite (GS-441524) in beagle dogs followed by healthy human volunteers. A quantification method for remdesivir and its active nucleoside metabolite (GS-441524) in beagle dog and human plasma has been developed and validated using liquid chromatography coupled to triple quadrupole mass spectrometry detection. The analytical methods for beagle dogs and human differ in the calibration curve range, plasma matrix, processing volume, reconstitution volume and injection volume; however all other parameters were same in both methods. A simple protein precipitation extraction was carried out using acetonitrile containing the internal standard remdesivir D5. Remdesivir and GS-441524 were separated on an Endurus C-18P, 100 × 4.6 mm, 3 µm column and detected using a mass spectrometer with electrospray ionization in positive ion mode. The ion transitions used were m/z 603.1 → m/z 200.0 for remdesivir, m/z 292.0 → m/z 202.2 for GS-441524 and m/z 608.2 → m/z 205.1 for remdesivir D5. The calibration curve results were linear in beagle dog plasma (2.0-2,000.8 ng/ml range for remdesivir and 2.0-1,500.4 ng/ml for GS-441524) and human plasma (30.0-4,503.9 ng/ml range for remdesivir and 2.0-200.4 ng/ml for GS-441524). The recovery was >90% in beagle dog and human plasma. These methods were successfully used to determine the pharmacokinetic parameters of the intravenous injection and subcutaneous tablets dosage forms in beagle dogs and healthy humans.

Remdesivir-induced conduction abnormalities: A molecular model-based explanation.

Purpose: Remdesivir use in COVID-19 is associated with cardiac conduction abnormalities from unclear mechanisms. A proposed mechanism is the bioaccumulation of the intermediate metabolite GS-441524 resulting in exogenous activation of cardiac adenosine A1 due to the structural similarity between adenosine and GS-441524. The prolonged half-life of GS-441524 can result in sustained activation of adenosine A1 receptors. In this study, we used molecular modeling of adenosine, GS-441524 and the adenosine A1 receptor to assess the potential mechanistic association of the proposed mechanism. Methods: Adenosine and GS-441524 structures were acquired from the PubChem database. Ligand docking was carried out using UCSF Chimera. Models were chosen based on greatest binding affinity and minimum root mean square deviation. Figures of resulting structural models were prepared using UCSF Chimera or PyMOL 2.3.5. Results: By modeling the interaction between the A1 G protein complex and both adenosine and GS-441524, we found that the proposed mechanism of exogenous A1 receptor activation is feasible based on docking compatibility. Conclusion: The proposed mechanism of exogenous cardiac A1 receptor activation from bioaccumulation of GS-441524 as a cause of observed cardiac conduction abnormalities with the use of remdesivir in COVID-19 is viable. Further studies are needed to assess causality.

Assessment of Purity, Stability, and Pharmacokinetics of NGP-1, a Novel Prodrug of GS441254 with Potential Anti-SARS-CoV-2 Activity, Using Liquid Chromatography.

SARS-CoV-2 is a highly contagious and pathogenic virus that first appeared in late December 2019 and caused a global pandemic in a short period. The virus is a single-stranded RNA virus belonging to the Coronaviridae family. Numerous treatments have been developed and tested in response to the pandemic, particularly antiviral drugs. Among them, GS441524 (GS441), a nucleoside antiviral drug, has demonstrated promising results in inhibiting SARS-CoV-2. Nevertheless, the limited oral bioavailability of GS441 restricts its application to patients with the virus. In this study, a novel prodrug of GS441 (NGP-1) with an isobutyl ester and cyclic carbonate structure was designed and synthesized. Its purity and the stability in different artificial digestive juices of NGP-1 was determined with HPLC-DAD methods. The pharmacokinetics of NGP-1 and GS441 were studied in rats via gavage administration. A new LC-MS/MS method was developed to quantitatively analyze GS441 in plasma samples. The results showed that the ka, Cmax, and MRT of converted GS441 from NGP-1 were 5.9, 3, and 2.5 times greater than those of GS441 alone. The Frel of NGP-1 was approximately four-fold that of GS441, with an AUC0-∞ of 9716.3 h·ng mL-1. As a prodrug of GS441, NGP-1 increased its lipophilicity, absorption, and bioavailability, indicating that it holds promise in improving the clinical efficacy of anti-SARS-CoV-2 medications.

Remdesivir and GS-441524 Retain Antiviral Activity against Delta, Omicron, and Other Emergent SARS-CoV-2 Variants.

Genetic variation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the emergence and rapid spread of multiple variants throughout the pandemic, of which Omicron is currently the predominant variant circulating worldwide. SARS-CoV-2 variants of concern/variants of interest (VOC/VOI) have evidence of increased viral transmission, disease severity, or decreased effectiveness of vaccines and neutralizing antibodies. Remdesivir (RDV [VEKLURY]) is a nucleoside analog prodrug and the first FDA-approved antiviral treatment of COVID-19. Here, we present a comprehensive antiviral activity assessment of RDV and its parent nucleoside, GS-441524, against 10 current and former SARS-CoV-2 VOC/VOI clinical isolates by nucleoprotein enzyme-linked immunosorbent assay (ELISA) and plaque reduction assay. Delta and Omicron variants remained susceptible to RDV and GS-441524, with 50% effective concentration (EC50) values 0.30- to 0.62-fold of those observed against the ancestral WA1 isolate. All other tested variants exhibited EC50 values ranging from 0.13- to 2.3-fold of the observed EC50 values against WA1. Analysis of nearly 6 million publicly available variant isolate sequences confirmed that Nsp12, the RNA-dependent RNA polymerase (RdRp) target of RDV and GS-441524, is highly conserved across variants, with only 2 prevalent changes (P323L and G671S). Using recombinant viruses, both RDV and GS-441524 retained potency against all viruses containing frequent variant substitutions or their combination. Taken together, these results highlight the conserved nature of SARS-CoV-2 Nsp12 and provide evidence of sustained SARS-CoV-2 antiviral activity of RDV and GS-441524 across the tested variants. The observed pan-variant activity of RDV supports its continued use for the treatment of COVID-19 regardless of the SARS-CoV-2 variant.

Synthesis and anti-SARS-CoV-2 activity of deuterated GS-441524 analogs.

The COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is continuing to spread around the world. GS-441524 is the parent nucleoside of remdesivir which is the first drug approved for the treatment of COVID-19, and demonstrates strong activity against SARS-Cov-2 in vitro and in vivo. Herein, we reported the synthesis of a series of deuterated GS-441524 analogs, which had deuterium atoms up to five at the ribose and the nucleobase moieties. Compared to GS-441524, all the deuterated compounds showed similar inhibitory activities against SARS-CoV-2 in vitro.

Why Remdesivir Failed: Preclinical Assumptions Overestimate the Clinical Efficacy of Remdesivir for COVID-19 and Ebola.

Remdesivir is a nucleoside monophosphoramidate prodrug that has been FDA approved for coronavirus disease 2019 (COVID-19). However, the clinical efficacy of remdesivir for COVID-19 remains contentious, as several trials have not found statistically significant differences in either time to clinical improvement or mortality between remdesivir-treated and control groups. Similarly, the inability of remdesivir to provide a clinically significant benefit above other investigational agents in patients with Ebola contrasts with strong, curative preclinical data generated in rhesus macaque models. For both COVID-19 and Ebola, significant discordance between the robust preclinical data and remdesivir's lackluster clinical performance have left many puzzled. Here, we critically evaluate the assumptions of the models underlying remdesivir's promising preclinical data and show that such assumptions overpredict efficacy and minimize toxicity of remdesivir in humans. Had the limitations of in vitro drug efficacy testing and species differences in drug metabolism been considered, the underwhelming clinical performance of remdesivir for both COVID-19 and Ebola would have been fully anticipated.

Validation of LC-MS/MS methods for determination of remdesivir and its metabolites GS-441524 and GS-704277 in acidified human plasma and their application in COVID-19 related clinical studies.

Remdesivir (RDV) is a phosphoramidate prodrug designed to have activity against a broad spectrum of viruses. Following IV administration, RDV is rapidly distributed into cells and tissues and simultaneously metabolized into GS-441524 and GS-704277 in plasma. LC-MS/MS methods were validated for determination of the 3 analytes in human plasma that involved two key aspects to guarantee their precision, accuracy and robustness. First, instability issues of the analytes were overcome by diluted formic acid (FA) treatment of the plasma samples. Secondly, a separate injection for each analyte was performed with different ESI modes and organic gradients to achieve sensitivity and minimize carryover. Chromatographic separation was achieved on an Acquity UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm) with a run time of 3.4 min. The calibration ranges were 4-4000, 2-2000, and 2-2000 ng/mL, respectively for RDV, GS-441524 and GS-704277. The intraday and interday precision (%CV) across validation runs at 3 QC levels for all 3 analytes was less than 6.6%, and the accuracy was within ±11.5%. The long-term storage stability in FA-treated plasma was established to be 392, 392 and 257 days at -70 °C, respectively for RDV, GS-441524 and GS-704277. The validated method was successfully applied in COVID-19 related clinical studies.

Remdesivir: A beacon of hope from Ebola virus disease to COVID-19.

Since the emergence of coronavirus disease 2019 (Covid-19), many studies have been performed to characterize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and find the optimum way to combat this virus. After suggestions and assessments of several therapeutic options, remdesivir (GS-5734), a direct-acting antiviral drug previously tested against Ebola virus disease, was found to be moderately effective and probably safe for inhibiting SARS-CoV-2 replication. Finally, on 1 May 2020, remdesivir (GS-5734) was granted emergency use authorization as an investigational drug for the treatment of Covid-19 by the Food and Drug Administration. However, without a doubt, there are challenging days ahead. Here, we provide a review of the latest findings (based on preprints, post-prints, and news releases in scientific websites) related to remdesivir efficacy and safety for the treatment of Covid-19, along with covering remdesivir history from bench-to-bedside, as well as an overview of its mechanism of action. In addition, active clinical trials, as well as challenging issues related to the future of remdesivir in Covid-19, are covered. Up to the date of writing this review (19 May 2020), there is one finished randomized clinical trial and two completed non-randomized studies, in addition to some ongoing studies, including three observational studies, two expanded access studies, and seven active clinical trials registered on the clinicaltrials.gov and isrctn.com websites. Based on these studies, it seems that remdesivir could be an effective and probably safe treatment option for Covid-19. However, more randomized controlled studies are required.

Potency and pharmacokinetics of GS-441524 derivatives against SARS-CoV-2.

The nucleoside metabolite of remdesivir, GS-441524 displays potent anti-SARS-CoV-2 efficacy, and is being evaluated in clinical as an oral antiviral therapeutic for COVID-19. However, this nucleoside has a poor oral bioavailability in non-human primates, which may affect its therapeutic efficacy. Herein, we reported a variety of GS-441524 analogs with modifications on the base or the sugar moiety, as well as some prodrug forms, including five isobutyryl esters, two l-valine esters, and one carbamate. Among the new nucleosides, only the 7-fluoro analog 3c had moderate anti-SARS-CoV-2 activity, and its phosphoramidate prodrug 7 exhibited reduced activity in Vero E6 cells. As for the prodrugs, the 3'-isobutyryl ester 5a, the 5'-isobutyryl ester 5c, and the tri-isobutyryl ester 5g hydrobromide showed excellent oral bioavailabilities (F = 71.6%, 86.6% and 98.7%, respectively) in mice, which provided good insight into the pharmacokinetic optimization of GS-441524.

Quantification of plasma remdesivir and its metabolite GS-441524 using liquid chromatography coupled to tandem mass spectrometry. Application to a Covid-19 treated patient.

Objectives: A method based on liquid chromatography coupled to triple quadrupole mass spectrometry detection using 50 µL of plasma was developed and fully validated for quantification of remdesivir and its active metabolites GS-441524. Methods: A simple protein precipitation was carried out using 75 µL of methanol containing the internal standard (IS) remdesivir-13C6 and 5 µL ZnSO4 1 M. After separation on Kinetex® 2.6 µm Polar C18 100A LC column (100 × 2.1 mm i.d.), both compounds were detected by a mass spectrometer with electrospray ionization in positive mode. The ion transitions used were m/z 603.3 â†’ m/z 200.0 and m/z 229.0 for remdesivir, m/z 292.2 â†’ m/z 173.1 and m/z 147.1 for GS-441524 and m/z 609.3 â†’ m/z 206.0 for remdesivir-13C6. Results: Calibration curves were linear in the 1-5000 µg/L range for remdesivir and 5-2500 for GS-441524, with limit of detection set at 0.5 and 2 µg/L and limit of quantification at 1 and 5 µg/L, respectively. Precisions evaluated at 2.5, 400 and 4000 µg/L for remdesivir and 12.5, 125, 2000 µg/L for GS-441524 were lower than 14.7% and accuracy was in the [89.6-110.2%] range. A slight matrix effect was observed, compensated by IS. Higher stability of remdesivir and metabolite was observed on NaF-plasma. After 200 mg IV single administration, remdesivir concentration decrease rapidly with a half-life less than 1 h while GS-441524 appeared rapidly and decreased slowly until H24 with a half-life around 12 h. Conclusions: This method would be useful for therapeutic drug monitoring of these compounds in Covid-19 pandemic.

Uroliths composed of antiviral compound GS-441524 in 2 cats undergoing treatment for feline infectious peritonitis.

Feline infectious peritonitis (FIP) historically has been a fatal disease in cats. Recent unlicensed use of antiviral medication has been shown to markedly improve survival of this infection. An 8-month-old female spayed domestic short-haired cat undergoing treatment for presumptive FIP with the antiviral nucleoside analog GS-441524 developed acute progressive azotemia. Abdominal ultrasound examination identified multifocal urolithiasis including renal, ureteral, and cystic calculi. Unilateral ureteral obstruction progressed to suspected bilateral ureteral obstruction and subcutaneous ureteral bypass (SUB) was performed along with urolith removal and submission for analysis. A 2-year-old male neutered domestic medium-haired cat undergoing treatment for confirmed FIP with GS-441524 developed dysuria (weak urine stream, urinary incontinence, and difficulty expressing the urinary bladder). This cat also was diagnosed sonographically with multifocal urolithiasis requiring temporary tube cystostomy after cystotomy and urolith removal. In both cases, initial urolith analysis showed unidentified material. Additional testing confirmed the calculi in both cats to be 98% consistent with GS-441524. Additional clinical studies are required to determine best screening practices for cats presented for urolithiasis during treatment with GS-441524.

Quality assessment and characterization of unregulated antiviral drugs for feline infectious peritonitis: implications for treatment, safety, and efficacy.

OBJECTIVE: Feline infectious peritonitis is fatal, and due to lack of approved treatments, unregulated antiviral drugs are used to treat this disease. This study set out to determine the purity of various batches of these drugs from several companies, characterize them, and note any impurities or other unusual characteristics. We also developed a method to qualitatively assess the primary components before administration. SAMPLES: We tested 30 vials from 17 brands of GS-441524 and 5 vials from 1 brand of GC376. We compared the GS-441524 to a control standard from Ambeed and the GC376 to a standard from Cayman Chemical. METHODS: We recorded physical appearance, pH, absorbance, HPLC retention times, and thin-layer chromatography retention factors for all of the samples. Some samples were used for nuclear magnetic resonance and mass spectrometric analysis. RESULTS: Some of the GS-441524 vials were 10% to 25% more concentrated than advertised, but most of the GS-441524 samples tested were similar in purity and composition, both between batches and between brands. We also tested 5 vials of GC376 and found that 1 of the 5 vials contained GS-441524 rather than GC376 and the other 4 vials contained molnupiravir. CLINICAL RELEVANCE: While all of the GS-441524 vials contained GS-441524, none of the GC376 vials tested contained GC376. GC376 is used in cats that are unresponsive to GS-441524, and use of the wrong antiviral can cause serious side effects. We provide suggested methods for distinguishing one drug from the other in new batches.

GS-441524 and molnupiravir are similarly effective for the treatment of cats with feline infectious peritonitis.

Although not registered for feline infectious peritonitis (FIP) in Japan, nucleoside analogs have shown efficacy and we have been offering them to owners of cats with FIP at our clinic since January 2020. The aim of this study was to investigate outcomes in cats with FIP who received GS-441524 or molnupiravir. Diagnosis of FIP was based on clinical signs, laboratory test results, and the presence of feline coronavirus RNA in blood or effusion aspirate. After providing verbal and written information, owners of cats with a presumptive diagnosis of FIP with a were offered antiviral treatment with commercially sourced GS-441524 from June 2020, and either GS-441524 or compounded molnupiravir from January 2022. Dosing was 12.5-25 mg/kg/day for GS-441524 and 20-40 mg/kg/day for molnupiravir, depending on the presence of effusion and neurological and/or ocular signs, and continued for 84 days. Overall, 118 cats with FIP (effusive in 76) received treatment, 59 with GS-4421524 and 59 with molnupiravir. Twenty cats died, 12/59 (20.3%) in the GS-441524 group and 8/59 (13.6%) in the molnupiravir group (p = 0.326), with most deaths within the first 10 days of starting treatment. Among survivors, neurological and ocular signs resolved in all but one cat, who had persistent seizures. Of the cats completing treatment, 48/48 in the GS-441524 group and 51/52 in the molnupiravir group achieved remission. Laboratory parameters normalized within 6 to 7 weeks of starting drug administration. Adverse events, such as primarily hepatic function abnormalities, were transient and resolved without specific intervention. Our data indicate that GS-441524 and molnupiravir show similar effects and safety in cats with FIP.

Unlicensed antiviral products used for the at-home treatment of feline infectious peritonitis contain GS-441524 at significantly different amounts than advertised.

OBJECTIVE: To analyze the content of unlicensed GS-441524-like products being used as a largely successful at-home treatment for cats suspected to have FIP. The remdesivir content and pH were also measured. SAMPLE: 127 injectable and oral samples from 30 of the most popular brands of black market producers. METHODS: Unlicensed GS-441524-like products were procured through donations and tested for GS-441524 and remdesivir content by liquid chromatography with tandem mass spectrometry. A pH meter measured the pH of injectable samples. RESULTS: Of the 87 injectable formulations, 95% contained more (on average 39% more) GS-441524 than expected based on the producer's marketed concentrations. The average pH (1.30 pH) was well below the physiologic pH conditions recommended for SC injections. The oral formulations were more variable, with 43% containing more GS-441524 (on average 75% more) than expected and 58% containing less (on average 39% less) than the expected content. There was minimal variability in GS-441524 content between replicate samples in the injectables formulations (measured by coefficient of variation). One injectable and 2 oral samples additionally contained remdesivir. CLINICAL RELEVANCE: All unlicensed products used for the at-home treatment of FIP that we tested contain GS-441524. The injectables generally contain significantly more drug than advertised at a below-physiologic pH. Unlicensed oral products vary more widely in drug content and suffer from unconventional dosing and labeling. These data should highlight the need for regulation of these products and the development of legal pathways to procure GS-441524.

First analytical confirmation of drug-induced crystal nephropathy in felines caused by GS-441524, the active metabolite of Remdesivir.

GS-441524 is an adenosine nucleoside antiviral demonstrating significant efficacy in the treatment of feline infectious peritonitis (FIP), an otherwise fatal illness, resulting from infection with feline coronavirus. However, following the emergence of COVID-19, veterinary development was halted, and Gilead pursued clinical development of a GS-441524 pro-drug, resulting in the approval of Remdesivir under an FDA emergency use authorization. Despite lack of regulatory approval, GS-441524 is available without a prescription through various unlicensed online distributors and is commonly purchased by pet owners for the treatment of FIP. Herein, we report data obtained from the analytical characterization of two feline renal calculi, demonstrating the propensity for GS-441524 to cause renal toxicity through drug-induced crystal nephropathy in vivo. As definitive diagnosis of drug-induced crystal nephropathy requires confirmation of the lithogenic material to accurately attribute a mechanism of toxicity, renal stone composition and crystalline matrix were characterized using ultra-performance liquid chromatography photodiode array detection (UPLC-PDA), ultra-performance liquid chromatography mass spectrometry (LCMS), nuclear magnetic resonance (NMR) spectroscopy, X-ray powder diffraction (XRD), and Fourier-transform infrared spectroscopy (FTIR). This work serves to provide the first analytical confirmation of GS-441524-induced crystal nephropathy in an effort to support toxicologic identification of adverse renal effects caused by administration of GS-441524 or any pro-drug thereof.