key: cord-0729371-l43vk6p1
authors: Rasmussen, Henrik Berg; Thomsen, Ragnar; Hansen, Peter Riis
title: Nucleoside analog GS‐441524: pharmacokinetics in different species, safety, and potential effectiveness against Covid‐19
date: 2022-04-09
journal: Pharmacol Res Perspect
DOI: 10.1002/prp2.945
sha: 1c4186b7f48a12acf73a0e1f6e09276aafbdf3cf
doc_id: 729371
cord_uid: l43vk6p1

GS‐441524, the parent nucleoside of remdesivir, has been proposed to be effective against Covid‐19 based on in vitro studies and studies in animals. However, randomized clinical trials of the agent to treat Covid‐19 have not been conducted. Here, we evaluated GS‐441524 for Covid‐19 treatment based on studies reporting pharmacokinetic parameters of the agent in mice, rats, cats, dogs, monkeys, and the single individual in the first‐in‐human trial supplemented with information about its activity against severe acute respiratory syndrome coronavirus 2 and safety. A dosing interval of 8 h was considered clinically relevant and used to calculate steady‐state plasma concentrations of GS‐441524. These ranged from 0.27 to 234.41 μM, reflecting differences in species, doses, and administration routes. Fifty percent maximal inhibitory concentrations of GS‐441524 against severe acute respiratory syndrome coronavirus 2 ranged from 0.08 μM to above 10 μM with a median of 0.87 μM whereas concentrations required to produce 90% of the maximal inhibition of the virus varied from 0.18 µM to more than 20 µM with a median of 1.42 µM in the collected data. Most of these concentrations were substantially lower than the calculated steady‐state plasma concentrations of the agent. Plasma exposures to orally administered GS‐441524, calculated after normalization of doses, were larger for dogs, mice, and rats than cynomolgus monkeys and humans, probably reflecting interspecies differences in oral uptake with reported oral bioavailabilities below 8.0% in cynomolgus monkeys and values as high as 92% in dogs. Reported oral bioavailabilities in rodents ranged from 12% to 57%. Using different presumptions, we estimated human oral bioavailability of GS‐441524 at 13% and 20%. Importantly, doses of GS‐441524 lower than the 13 mg/kg dose used in the first‐in‐human trial may be effective against Covid‐19. Also, GS‐441524 appears to be well‐tolerated. In conclusion, GS‐441524 has potential for oral treatment of Covid‐19.

There remains an unmet need for easily administrable therapeutic agents with high effectiveness in the treatment of coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Drug repurposing has attracted interest to accelerate discovery and clinical development of new pharmacological Covid-19 treatments. 1 Remdesivir is a repurposed antiviral drug, which was originally designed to treat Ebola and other viral infections with a pandemic potential. This drug received approval for treatment of in USA and Europe based on the first stage of the Adaptive COVID-19 Treatment Trial known as ACTT-1. 2 However, subsequent results from the larger Solidarity trial questioned its effectiveness against Covid-19. 3 More recently, a systematic review and meta-analysis suggested that remdesivir accelerated recovery and increased rate of hospital discharge in subjects with Covid-19, but without significantly decreasing mortality or time to clinical improvement. 4 Remdesivir belongs to the group of ProTides and has the adenosine analog GS-441524 as parent nucleoside. 5, 6 Previously, GS-441524 was hypothesized to be superior to remdesivir for treatment of Covid-19. 7 Activity of GS-441524 against Covid-19 is supported by findings that it markedly inhibited SARS-CoV-2 in cell lines, 8 and possessed anti-SARS-CoV-2 activity in mouse models of Covid-19. 9 Also, GS-441524 has been found to be highly effective for treatment of feline infectious peritonitis (FIP), a coronavirus disease in cats. 10 Since GS-441524 is a nucleoside analog lacking the McGuigan moiety characteristic of ProTides, its pharmacokinetics (PK) differs markedly from that of remdesivir. Notably, cellular uptake of GS-441524 is believed to be dependent upon membrane-bound transporters since it is hydrophilic with limited ability to cross cell membranes by diffusion. 11 By contrast, uptake of remdesivir appears to be mediated by diffusion, which is facilitated by its hydrophobic prodrug moieties, thus probably to a large extent being independent of membrane-bound transporters. 5, 6, 12 Once inside the cells, the GS-441524 is phosphorylated to the active antiviral GS-441524 triphosphate metabolite, also known as GS-443902, with adenosine kinase probably being responsible for catalyzing the first and perceived rate-limiting step in the formation of this metabolite. 13 Importantly, the relatively simple chemical structure of GS-441524 may permit fast manufacture of the agent in large amounts. 7 Randomized clinical trials of GS-441524 against Covid-19 have not been conducted. Further knowledge about the pharmacokinetics, toxicity, and effectiveness against Covid-19 of GS-441524 may pave the way for the agent to reach such trials. Based on publicly available in vitro and in vivo data including data from a variety of different species, we here review the PK, anti-SARS-CoV-2 activity and safety of GS-441524 supplemented with calculations to critically evaluate the potential of this nucleoside analog for treatment of Covid-19.

PubMed, medRxiv and bioRxiv were searched on August 19, 2021 and revisited on December 19, 2021 using 'GS-441524' as search term to identify publications and preprints reporting on the PK, in vitro and in vivo anti-SARS-CoV inhibitory activities in addition to toxicity of the agent. PK data were also collected from the National Center for Advancing Translational Sciences. 14 Information about plasma protein binding of GS-441524 and PK parameters of GS-441524 triphosphate after administration of remdesivir in humans were obtained from recent publications 15 supplemented with reports assessing remdesivir for treatment of Covid-19 prepared by the European Medicines Agency 16 and the Australian Department of Health. 17 In the event that a study presented plots of time versus plasma concentration for GS-441524 but did not provide PK variables, we extracted data from the plots using WebplotDigitizer (https://autom eris.io/WebPl otDig itizer). Using the PKSolver, these data were used for calculation of PK parameters for a noncompartmental model 

GS-441524 doses and exposures following oral, intragastric, subcutaneous, intramuscular, and intravenous administration in mice, rats, cats, dogs, cynomolgus monkeys and a single human are listed (Table 1) . Since the AUC 0-24 and AUC 0-inf values were almost identical, GS-441524 does not appear to accumulate in plasma over a dosing interval of 24 h (data not shown). Therefore, we calculated average plasma concentrations of the agent at steady state (C av,ss ) using a dosing interval of 8 h and found that these concentrations ranged from 0.27 to 234.41 μM.

Assuming linear PK, the AUC 0-inf values in the animal species scaled to the only reported (n = 1) human dose of 13 mg/kg ranged from 20.23 μM · hour in cynomolgus monkey to 290,86 μM · hour in dogs ( Table 2) . Remarkably, the AUC 0-inf in cynomolgus monkey was closer to the value observed in the human of 31.08 μM · hour than it was to those reported in the other animal species.

Oral bioavailability of GS-441524 differed significantly between species, with cynomolgus monkeys displaying markedly lower ability for oral uptake than mice and dogs (Table 3) . Human oral bioavailability of GS-441524 was estimated at 13% and 20% using the scaling factors of 0.67 and 0.75, respectively. These levels of human bioavailability are consistent with previous estimates ranging from 15% to 30% obtained by comparison with acyclovir. 24 Hence, the human oral bioavailabilities calculated using different approaches suggest that oral administration of GS-441524 is feasible in humans.

Using different isolates of SARS-CoV-2, viral quantification meth- Table 4 ). Most of the calculated C av,ss values exceeded these medians (Table 1) . A study showed that GS-441524 at 3.7 μM reduced the load of SARS-CoV-2 RNA in Vero cells by more than four log 10 units to levels below lower limit of detection but did not determine the IC 50 and IC 90 values for its activity against the virus. 25 Consistent with this, exposure to GS-441524 at a concentration of 3 µM reduced the load of SARS-CoV-2 with up to four log 10 units in cultured human airway epithelial cells, which may represent a more appropriate model for studying the activity of anti-SARS-CoV-2 agents than cancer cell lines. 26 Other findings, also based on human airway epithelial cell Note: NCATS: The National Center for Advancing Translational Sciences; AUC 0-inf : area under the plasma drug concentration-time curve from time zero to infinity; C av,ss : average plasma drug concentration at steady state; NA: not applicable. a Animal doses were converted to human equivalent doses using the exponent 0.67 in body surface area-based allometric scaling. Since interspecies conversion of drug doses by allometric scaling is not supported for subcutaneous and intramuscular administration, only human equivalent doses for oral and intravenous administrations were calculated. b Calculated as AUC 0-inf /τ, where τ is the doing interval (8 h).

c For studies that presented plots of time versus drug concentrations without providing AUC 0-inf values, these values were calculated using PKSolver after extraction of data from the plots. Moreover, we recalculated areas under the plasma drug concentration-time curve from time zero to 12 or 24 h based on data extracted from plots and found that these did not deviate with more than 5% from the corresponding parameters of exposure reported by the studies in question (data not shown). 

A single intravenous injection of mice with GS-441524 at a dose of 10 mg/kg has been reported to produce wet weight concentrations above 1 µmol/kg in most organs, except for lungs and nasal mucosa, leading to the suggestion that administration of the agent at 10 mg/ kg twice daily in mice would produce concentrations above reported of treatment. 9 Given that the intracellular volume in many tissues is about 0.8 mL/g tissue, 33 intracellular concentrations of GS-441524

in μM are likely to be 25% higher than the reported wet weight Value estimated at 3.4%, which we rounded to 3%. c Calculated using the equation: Oral bioavailability = 100 · AUC Oral / AUC IV · Dose IV /Dose Oral , where AUC Oral and AUC IV are the areas under the plasma drug concentration-time curves after administration of Dose Oral and Dose IV , respectively. A human AUC oral of 31.08 uM·h measured after administration of an oral dose of 13 mg/kg was used for the calculation. Given that the AUC IV of GS-441524 has not been determined in humans, this plasma exposure in the equation was replaced with the AUC IV of 12.38 uM·h reported in cynomolgus monkeys after administration of 2 mg/kg. Furthermore, a human equivalent intravenous dose of 0.65 mg/kg, which we obtained by body surface-based allometric scaling of the monkey intravenous dose of 2 mg/kg (scaling factor = 0.67), served as intravenous dose, thus assuming that the scaled dose produces an AUC iv in humans similar to that observed in cynomolgus monkeys. Using a scaling factor of 0.75, human oral bioavailability of GS-441524 was estimated at 20%. Since conversion of a human dose of GS-441524 at 13 mg/kg by 

Reported values for the 50% cytotoxic concentration (CC 50 ) of GS-441524 ranged from 7 to above 1000 µM suggesting low generalin vitrocytotoxicity of the agent (Table 5 ). In line with this, the recommendations on compassionate use of remdesivir by the European Medicines Agency concluded that high levels of GS-441524 did not induce in vitro cytotoxic effects, although CC 50 values in the range from 9.6 to 13.9 μM affected hematopoietic stem cell proliferation. 16 However, these concentrations are 3 to 4.5-fold higher than the C max of 3.05 μM observed after administration of a single dose of GS-441524 at 13 mg/kg in the first-in-human study. 34 40 The therapeutic doses of 2, 4, 5, and 10 mg/kg, which appeared to be well-tolerated in cats, correspond to HEDs in the range from 0.82 to 4.10 mg/kg per day.

These HEDs are significantly lower than the GS-441524 dose of 13 mg/kg that was administered to a healthy human volunteer once daily for seven days and three times daily for three days, respectively, and reported to not be associated with major adverse reactions or significant alterations of key blood parameters. 34 Hence, the current evidence suggests that GS-441524 is safe in a range of species, albeit with a scarcity of human data being available at present.

Previously, we suggested that endogenous adenosine competes with GS-441524 for cellular uptake by nucleoside transporters and the perceived rate-limiting first step of its phosphorylation to GS-441524 monophosphate eventually leading to the formation of GS-441524 triphosphate. 11 Under physiological conditions, extracellular and intracellular adenosine levels are in the submicromolar range, but these levels are significantly elevated in hypoxia and critical illness, occasionally being increased by 5-to 10-fold or more, e.g. reaching a level of 8.4 μM in sepsis. [41] [42] [43] [44] Such high adenosine levels exceed most of the calculated C av,ss values of GS-441524 including that of 3.89 µM reported in the first-in-human study (Table 1) 

In aggregate, publicly available data suggest that GS-441524 has potential for oral treatment of Covid-19 although human oral bioavailability does not appear to be high. Of note, a lower oral dose than the 13 mg/kg dose administered in the so far only reported human PK and safety study (n = 1) may be effective against Covid-19. However, GS-441524 may not be equally effective in eliminating SARS-CoV-2 from all tissues and organs, potentially having lower activity against the virus in the airways than in other organs. Also, cellular uptake and intracellular phosphorylation of GS-441524 necessary for antiviral activity could be reduced by competitive inhibition due to increased adenosine levels in subjects with severe Covid-19. GS-441524 appears to be well tolerated in animal species, and although to our knowledge, new clinical studies are not currently registered in major clinical trial registries, further clinical development of the agent appears to be justified.

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guide topha rmaco logy. org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY, 46 and are permanently archived in the Concise Guide to PHARMACOLOGY 2021/22. 47, 48 

The authors declare no conflict of interest. the article. Peter Riis Hansen co-wrote the article and provided critical revision. Ragnar Thomsen provided critical revision and suggestions. All three authors interpreted the results from the calculations and approved the final manuscript.

No data available in the study.

Henrik Berg Rasmussen https://orcid.org/0000-0001-9979-7627

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