Prodrug Remdesivir Research Articles

Assessing in vitro stability of remdesivir (GS-5734) and conversion to GS-441524 in feline plasma and whole blood

Feline infectious peritonitis (FIP) is a potentially fatal coronavirus-driven disease of cats. Treatment with nucleoside analogue GS-441524 and or prodrug remdesivir (RDV) have produced remission in both experimentally induced and naturally occurring FIP, yet information regarding metabolism of RDV into GS-441524 in cats is scarce. This study assessed possible phase I metabolism of RDV in cats, utilising an in vitro feline microsome model with in vitro t1/2 and in vitro Clint calculated using the substrate depletion method. A previously validated high-performance liquid chromatography (HPLC) fluorescence method was utilised for detection and analysis of RDV and GS-441524. Qualitative yield of RDV and intermediate metabolite GS-441524 were determined following microsome incubation, then compared to whole blood and plasma incubations. In vitro microsome incubation resulted in rapid depletion of RDV, though it did not appear to resemble a conventional phase I-dependent reaction in cats, as it is in humans and dogs. Depletion of RDV into GS-441524 was demonstrated in whole blood in vitro, suggesting cats convert RDV to GS-441524, likely via blood esterases, as observed in mice and rats. RDV metabolism is unlikely to be impacted by impaired liver function in cats. Furthermore, as RDV depletes within minutes, whereas GS-441524 is very stable, whole blood or plasma GS-441524 concentrations, rather than plasma RDV concentrations, are more appropriate for therapeutic drug monitoring (TDM) in cats receiving RDV.

Quantification of GS-441524 concentration in feline plasma using high performance liquid chromatography with fluorescence detection

The adenosine analogue GS-441524 has demonstrated efficacy in treatment of feline infectious peritonitis (FIP). With no commercially registered formulations of GS-441524 available, global focus shifted to its pro-drug remdesivir, as it became more accessible throughout the COVID-19 pandemic. This study developed and validated a simple liquid chromatography equipped with a fluorescence detector to quantify plasma concentrations of GS-441524 applicable for routine therapeutic monitoring of remdesivir or GS-441524 therapy for FIP infected cats. A Waters X-Bridge C18, 5 µm, 150 × 4.6 mm, column was used and mixtures of 20 mM ammonium acetate (pH 4.5) with acetonitrile of 5% and 70% were prepared for gradient mobile phase. With a simple protein precipitation using methanol to clean plasma sample, GS-441524 was monitored at excitation and emission wavelengths of 250 nm and 475 nm, respectively. Using an external standard, the lowest and highest limits of quantification were 19.5 ng/mL to 10,000 ng/mL, respectively. The intra- and inter day trueness of the quality controls (QCs) were within 10% of their nominal concentrations and intra- and inter day precision of the QCs (expressed as the coefficient of variation) ranged from 1.7 to 5.7%, This assay was able to quantify plasma trough levels of GS-441524 (23.7–190.1 ng/mL) after the administration of remdesivir (9.9–15.0 mg/kg BW, IV or SC) in FIP cats (n = 12). Accordingly, this study generated an alternative and cost-effective way to quantify GS-441524 in feline biological fluids at least up to 24 hr after administrations of remdesivir.

In vitro evaluation of the impact of Covid-19 therapeutic agents on the hydrolysis of the antiviral prodrug remdesivir

Remdesivir (RDV, Veklury®) is an FDA-approved prodrug for the treatment of hospitalized patients with COVID-19. Recent in vitro studies have indicated that human carboxylesterase 1 (CES1) is the major metabolic enzyme catalyzing RDV activation. COVID-19 treatment for hospitalized patients typically also involves a number of antibiotics and anti-inflammatory drugs. Further, individuals who are carriers of a CES1 variant (polymorphism in exon 4 codon 143 [G143E]) may experience impairment in their ability to metabolize therapeutic agents which are CES1 substrates. The present study assessed the potential influence of nine therapeutic agents (hydroxychloroquine, ivermectin, erythromycin, clarithromycin, roxithromycin, trimethoprim, ciprofloxacin, vancomycin, and dexamethasone) commonly used in treating COVID-19 and 5 known CES1 inhibitors on the metabolism of RDV. Additionally, we further analyzed the mechanism of inhibition of cannabidiol (CBD), as well as the impact of the G143E polymorphism on RDV metabolism. An in vitro S9 fraction incubation method and in vitro to in vivo pharmacokinetic scaling were utilized. None of the nine therapeutic agents evaluated produced significant inhibition of RDV hydrolysis; CBD was found to inhibit RDV hydrolysis by a mixed type of competitive and noncompetitive partial inhibition mechanism. In vitro to in vivo modeling suggested a possible reduction of RDV clearance and increase of AUC when coadministration with CBD. The same scaling method also suggested a potentially lower clearance and higher AUC in the presence of the G143E variant. In conclusion, a potential CES1-mediated DDI between RDV and the nine assessed medications appears unlikely. However, a potential CES1-mediated DDI between RDV and CBD may be possible with sufficient exposure to the cannabinoid. Patients carrying the CES1 G143E variant may exhibit a slower biotransformation and clearance of RDV. Further clinical studies would be required to evaluate and characterize the clinical significance of a CBD-RDV interaction.

Eco-friendly stability-indicating HPTLC micro-determination of the first FDA approved SARS-CoV-2 antiviral prodrug Remdesivir: Study of degradation kinetics and structural elucidation of the degradants using HPTLC-MS

The worldwide spread coronavirus (covid-19) pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents a global health crisis. The world was forced to face a great challenge to control and overcome this health disaster through various containment measures including efficient vaccination side by side with effective medication. Remdesivir (RMD) is the first FDA approved antiviral agent for treatment of covid-19 pandemic and hence regarded as the first-in-class medication of this highly contagious respiratory disease. The current study represents the first stability indicating HPTLC method for the estimation of RMD in bulk form and pharmaceutical formulation. The method employed TLC silica gel aluminum plates 60 F254 as stationary phase and green mobile phase composed of ethyl acetate and ethanol (96: 4, v/v) with densitometric detection at 245 nm. Comprehensive validation of the adopted method was accomplished according to the ICH guidelines regarding linearity, ranges, detection and quantification limits, precision, accuracy and robustness. The developed method offered a neat separation of the drug in presence of pharmaceutical excipients as well as in presence of acidic, alkaline, neutral hydrolytic, oxidative and photolytic degradants. Additionally, structural elucidation of alkaline and hydrolytic oxidation degradation products was carried out using HPTLC-MS. Furthermore, for the first time the acidic and alkaline degradation kinetics of RMD were studied and its degradation rate constants and half-lives were calculated. Moreover, greenness appraisal of the developed method as well as comparison with previously published stability indicating HPLC methods were performed using analytical Eco-scale, GAPI and AGREE metrics.

SARS-CoV-2 viral load dynamics in immunocompromised critically ill patients on remdesivir treatment.

The relationship between SARS-CoV-2 quantitative viral load and risk of disease progression, morbidity such as long- COVID or mortality in immunosuppressed, remains largely undefined in COVID-19 patients. Critically ill immunosuppressed patients potentially benefit from remdesivir treatment because of the prolonged course of their infection. Four critically ill immunocompromised patients and the impact of remdesivir on viral dynamics in lower respiratory samples were studied. Bronchoalveolar lavage (BAL) samples were assessed to measure SARS-CoV-2 quantitative viral load using real-time PCR. Corresponding plasma levels of remdesivir and its metabolite GS-441524 were determined. Mean virus load of 39.74 x 107 geq/ml (±33.25 x 107 geq/ml) on day 1 dropped significantly (p<0.008) to 3.54 x 106 geq/ml (±6.93 x 106 geq/ml) on day 3 and to 1.4 x 105 geq/ml (±2.35 x 105 geq/ml) on day 5 of remdesivir treatment. Mean virus load dropped below <1% between day 1 and 5 of remdesivir treatment. Parent prodrug remdesivir and also GS441524 metabolite levels of antiviral activity in our patients were far in excess of EC 50. Our data present that remdesivir treatment potentially reduces the SARS-CoV-2 viral load in immunosuppressed critically ill patients. However, the implication of viral load reduction on morbidity and mortality needs further investigation.

Direct and Catalytic C-Glycosylation of Arenes: Expeditious Synthesis of the Remdesivir Nucleoside.

Since early 2020, scientists have strived to find an effective solution to fight SARS‐CoV‐2, in particular by developing reliable vaccines that inhibit the spread of the disease and repurposing drugs for combatting its effects on the human body. The antiviral prodrug Remdesivir is still the most widely used therapeutic during the early stages of the infection. However, the current synthetic routes rely on the use of protecting groups, air‐sensitive reagents, and cryogenic conditions, thus impeding a cost‐efficient supply to patients. We have, therefore, focused on the development of a straightforward, direct addition of (hetero)arenes to unprotected sugars. Here we report a silylium‐catalyzed and completely stereoselective C‐glycosylation that initially yields the open‐chain polyols, which can be selectively cyclized to provide either the kinetic α‐furanose or the thermodynamically favored β‐anomer. The method significantly expedites the synthesis of Remdesivir precursor GS‐441524 after a subsequent Mn‐catalyzed C−H oxidation and deoxycyanation.

Remdesivir: Mechanism of Metabolic Conversion from Prodrug to Drug.

Remdesivir (GS-5734) has emerged as a promising drug during the challenging times of COVID-19 pandemic. Being a prodrug, it undergoes several metabolic reactions before converting to its active triphosphate metabolite. It is important to establish the atomic level details and explore the energy profile of the prodrug to drug conversion process. In this work, Density Functional Theory (DFT) calculations were performed to explore the entire metabolic path. Further, the potential energy surface (PES) diagram for the conversion of prodrug remdesivir to its active metabolite was established. The role of catalytic triad of Hint1 phosphoramidase enzyme in P-N bond hydrolysis was also studied on a model system using combined molecular docking and quantum mechanics approach. The overall energy of reaction is 11.47 kcal/mol exergonic and the reaction proceeds through many steps requiring high activation energies. In the absence of a catalyst, the P-N bond breaking step requires 41.78 kcal/mol, which is reduced to 14.26 kcal/mol in a catalytic environment. The metabolic pathways of model system of remdesivir (MSR) were explored completely and potential energy surface diagrams at two levels of theory, B3LYP/6-311++G(d, p) and B3LYP/6-31+G(d), were established and compared. The results highlight the importance of an additional water molecule in the metabolic reaction. The PN bond cleavage step of the metabolic process requires the presence of an enzymatic environment.

Accelerated stability study of the ester prodrug remdesivir: Recently FDA-approved Covid-19 antiviral using reversed-phase-HPLC with fluorimetric and diode array detection.

Remdesivir (RDV) is the first antiviral drug, approved by the Food and Drug Administration, to treat severe acute respiratory syndrome coronavirus 2. RDV is a relatively new chemical entity, 'ester prodrug', with no reported stability profile. Due to the urgency of its use and thus fast production, it is important to develop a stability-indicating method for its assay. Chromatographic separation was carried out on a C18 column (250 × 4.6 mm, 5 µm) with dual detection: diode array at 240 nm and fluorescence at λex/em 245/390 nm. Isocratic elution of acetonitrile and distilled water (acidified with phosphoric acid, pH 4) in the ratio of 55:45 (v/v), respectively, was used. The linearity range using HPLC-diode array detection was 0.1-15 μg/mL, whereas that using fluorimetric detection was 0.05-15 μg/mL. As per the International Conference on Harmonization guidelines, RDV has been degraded by accelerated alkaline, acidic, neutral hydrolysis, oxidative, heat, and photolytic stress conditions. Possible degradation hypothesis of the parent molecule has been suggested and illustrated. The proposed methods have achieved selective determination of the intact drug with no peaks overlapping in all assumptions. Extensive degradation confirms threatened drug stability at thermal and basic hydrolytic stressing. The developed methods were fully validated and proved suitable for quality control routine analysis of RDV in raw material and pharmaceutical dosage forms.

Mathematical Modeling of Remdesivir to Treat COVID-19: Can Dosing Be Optimized?

The antiviral remdesivir has been approved by regulatory bodies such as the European Medicines Agency (EMA) and the US Food and Drug administration (FDA) for the treatment of COVID-19. However, its efficacy is debated and toxicity concerns might limit the therapeutic range of this drug. Computational models that aid in balancing efficacy and toxicity would be of great help. Parametrizing models is difficult because the prodrug remdesivir is metabolized to its active form (RDV-TP) upon cell entry, which complicates dose–activity relationships. Here, we employ a computational model that allows drug efficacy predictions based on the binding affinity of RDV-TP for its target polymerase in SARS-CoV-2. We identify an optimal infusion rate to maximize remdesivir efficacy. We also assess drug efficacy in suppressing both wild-type and resistant strains, and thereby describe a drug regimen that may select for resistance. Our results differ from predictions using prodrug dose–response curves (pseudo-ECs). We expect that reaching 90% inhibition (EC) is insufficient to suppress SARS-CoV-2 in the lungs. While standard dosing mildly inhibits viral polymerase and therefore likely reduces morbidity, we also expect selection for resistant mutants for most realistic parameter ranges. To increase efficacy and safeguard against resistance, we recommend more clinical trials with dosing regimens that substantially increase the levels of RDV-TP and/or pair remdesivir with companion antivirals.

Cellular Uptake and Intracellular Phosphorylation of GS-441524: Implications for Its Effectiveness against COVID-19.

GS-441524 is an adenosine analog and the parent nucleoside of the prodrug remdesivir, which has received emergency approval for treatment of COVID-19. Recently, GS-441524 has been proposed to be effective in the treatment of COVID-19, perhaps even being superior to remdesivir for treatment of this disease. Evaluation of the clinical effectiveness of GS-441524 requires understanding of its uptake and intracellular conversion to GS-441524 triphosphate, the active antiviral substance. We here discuss the potential impact of these pharmacokinetic steps of GS-441524 on the formation of its active antiviral substance and effectiveness for treatment of COVID-19. Available protein expression data suggest that several adenosine transporters are expressed at only low levels in the epithelial cells lining the alveoli in the lungs, i.e., the alveolar cells or pneumocytes from healthy lungs. This may limit uptake of GS-441524. Importantly, cellular uptake of GS-441524 may be reduced during hypoxia and inflammation due to decreased expression of adenosine transporters. Similarly, hypoxia and inflammation may lead to reduced expression of adenosine kinase, which is believed to convert GS-441524 to GS-441524 monophosphate, the perceived rate-limiting step in the intracellular formation of GS-441524 triphosphate. Moreover, increases in extracellular and intracellular levels of adenosine, which may occur during critical illnesses, has the potential to competitively decrease cellular uptake and phosphorylation of GS-441524. Taken together, tissue hypoxia and severe inflammation in COVID-19 may lead to reduced uptake and phosphorylation of GS-441524 with lowered therapeutic effectiveness as a potential outcome. Hypoxia may be particularly critical to the ability of GS-441524 to eliminate SARS-CoV-2 from tissues with low basal expression of adenosine transporters, such as alveolar cells. This knowledge may also be relevant to treatments with other antiviral adenosine analogs and anticancer adenosine analogs as well.

In vitro activity of itraconazole against SARS-CoV-2.

Although vaccination campaigns are currently being rolled out to prevent coronavirus disease (COVID‐19), antivirals will remain an important adjunct to vaccination. Antivirals against coronaviruses do not exist, hence global drug repurposing efforts have been carried out to identify agents that may provide clinical benefit to patients with COVID‐19. Itraconazole, an antifungal agent, has been reported to have activity against animal coronaviruses. Using cell‐based phenotypic assays, the in vitro antiviral activity of itraconazole and 17‐OH itraconazole was assessed against clinical isolates from a German and Belgian patient infected with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Itraconazole demonstrated antiviral activity in human Caco‐2 cells (EC50 = 2.3 µM; 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay). Similarly, its primary metabolite, 17‐OH itraconazole, showed inhibition of SARS‐CoV‐2 activity (EC50 = 3.6 µM). Remdesivir inhibited viral replication with an EC50 = 0.4 µM. Itraconazole and 17‐OH itraconazole resulted in a viral yield reduction in vitro of approximately 2‐log10 and approximately 1‐log10, as measured in both Caco‐2 cells and VeroE6‐eGFP cells, respectively. The viral yield reduction brought about by remdesivir or GS‐441524 (parent nucleoside of the antiviral prodrug remdesivir; positive control) was more pronounced, with an approximately 3‐log10 drop and >4‐log10 drop in Caco‐2 cells and VeroE6‐eGFP cells, respectively. Itraconazole and 17‐OH itraconazole exert in vitro low micromolar activity against SARS‐CoV‐2. Despite the in vitro antiviral activity, itraconazole did not result in a beneficial effect in hospitalized COVID‐19 patients in a clinical study (EudraCT Number: 2020‐001243‐15).

Current status of antivirals and druggable targets of SARS CoV-2 and other human pathogenic coronaviruses.

Coronaviridae is a peculiar viral family, with a very large RNA genome and characteristic appearance, endowed with remarkable tendency to transfer from animals to humans. Since the beginning of the 21st century, three highly transmissible and pathogenic coronaviruses have crossed the species barrier and caused deadly pneumonia, inflicting severe outbreaks and causing human health emergencies of inconceivable magnitude. Indeed, in the past two decades, two human coronaviruses emerged causing serious respiratory illness: severe acute respiratory syndrome coronavirus (SARS-CoV-1) and Middle Eastern respiratory syndrome coronavirus (MERS-CoV), causing more than 10,000 cumulative cases, with mortality rates of 10 % for SARS-CoV-1 and 34.4 % for MERS-CoV. More recently, the severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) has emerged in China and has been identified as the etiological agent of the recent COVID-19 pandemic outbreak. It has rapidly spread throughout the world, causing nearly 22 million cases and ∼ 770,000 deaths worldwide, with an estimated mortality rate of ∼3.6 %, hence posing serious challenges for adequate and effective prevention and treatment. Currently, with the exception of the nucleotide analogue prodrug remdesivir, and despite several efforts, there is no known specific, proven, pharmacological treatment capable of efficiently and rapidly inducing viral containment and clearance of SARS-CoV-2 infection as well as no broad-spectrum drug for other human pathogenic coronaviruses. Another confounding factor is the paucity of molecular information regarding the tendency of coronaviruses to acquire drug resistance, a gap that should be filled in order to optimize the efficacy of antiviral drugs.In this light, the present review provides a systematic update on the current knowledge of the marked global efforts towards the development of antiviral strategies aimed at coping with the infection sustained by SARS-CoV-2 and other human pathogenic coronaviruses, displaying drug resistance profiles. The attention has been focused on antiviral drugs mainly targeting viral protease, RNA polymerase and spike glycoprotein, that have been tested in vitro and/or in clinical trials as well as on promising compounds proven to be active against coronaviruses by an in silico drug repurposing approach. In this respect, novel insights on compounds, identified by structure-based virtual screening on the DrugBank database endowed by multi-targeting profile, are also reported. We specifically identified 14 promising compounds characterized by a good in silico binding affinity towards, at least, two of the four studied targets (viral and host proteins). Among which, ceftolozane and NADH showed the best multi-targeting profile, thus potentially reducing the emergence of resistant virus strains. We also focused on potentially novel pharmacological targets for the development of compounds with anti-pan coronavirus activity. Through the analysis of a large set of viral genomic sequences, the current review provides a comprehensive and specific map of conserved regions across human coronavirus proteins which are essential for virus replication and thus with no or very limited tendency to mutate. Hence, these represent key druggable targets for novel compounds against this virus family. In this respect, the identification of highly effective and innovative pharmacological strategies is of paramount importance for the treatment and/or prophylaxis of the current pandemic but potentially also for future and unavoidable outbreaks of human pathogenic coronaviruses.

A Chemoenzymatic Synthesis of the (RP)-Isomer of the Antiviral Prodrug Remdesivir.

The COVID-19 pandemic threatens to overwhelm healthcare systems around the world. The only current FDA-approved treatment, which directly targets the virus, is the ProTide prodrug remdesivir. In its activated form, remdesivir prevents viral replication by inhibiting the essential RNA-dependent RNA polymerase. Like other ProTide prodrugs, remdesivir contains a chiral phosphorus center. The initial selection of the (SP)-diastereomer for remdesivir was reportedly due to the difficulty in producing the pure (RP)-diastereomer of the required precursor. However, the two currently known enzymes responsible for the initial activation step of remdesivir are each stereoselective and show differential tissue distribution. Given the ability of the COVID-19 virus to infect a wide array of tissue types, inclusion of the (RP)-diastereomer may be of clinical significance. To help overcome the challenge of obtaining the pure (RP)-diastereomer of remdesivir, we have developed a novel chemoenzymatic strategy that utilizes a stereoselective variant of the phosphotriesterase from Pseudomonas diminuta to enable the facile isolation of the pure (RP)-diastereomer of the chiral precursor for the chemical synthesis of the (RP)-diastereomer of remdesivir.

Development and validation of a UHPLC-MS/MS method for quantification of the prodrug remdesivir and its metabolite GS-441524: a tool for clinical pharmacokinetics of SARS-CoV-2/COVID-19 and Ebola virus disease.

BackgroundRemdesivir has received significant attention for its potential application in the treatment of COVID-19, caused by SARS-CoV-2. Remdesivir has already been tested for Ebola virus disease treatment and found to have activity against SARS and MERS coronaviruses. The remdesivir core contains GS-441524, which interferes with RNA-dependent RNA polymerases alone. In non-human primates, following IV administration, remdesivir is rapidly distributed into PBMCs and converted within 2 h to the active nucleoside triphosphate form, while GS-441524 is detectable in plasma for up to 24 h. Nevertheless, remdesivir pharmacokinetics and pharmacodynamics in humans are still unexplored, highlighting the need for a precise analytical method for remdesivir and GS-441524 quantification.ObjectivesThe validation of a reliable UHPLC-MS/MS method for remdesivir and GS-441524 quantification in human plasma.MethodsRemdesivir and GS-441524 standards and quality controls were prepared in plasma from healthy donors. Sample preparation consisted of protein precipitation, followed by dilution and injection into the QSight 220 UHPLC-MS/MS system. Chromatographic separation was obtained through an Acquity HSS T3 1.8 μm, 2.1 × 50 mm column, with a gradient of water and acetonitrile with 0.05% formic acid. The method was validated using EMA and FDA guidelines.ResultsAnalyte stability has been evaluated and described in detail. The method successfully fulfilled the validation process and it was demonstrated that, when possible, sample thermal inactivation could be a good choice in order to improve biosafety.ConclusionsThis method represents a useful tool for studying remdesivir and GS-441524 clinical pharmacokinetics, particularly during the current COVID-19 outbreak.