key: cord-0921411-f0zvel44
authors: Prince, Tessa; Donovan-Banfield, I’ah; Goldswain, Hannah; Penrice-Randal, Rebekah; Turtle, Lance; Fletcher, Tom; Khoo, Saye; Hiscox, Julian A.
title: Antiviral activity of molnupiravir precursor NHC against SARS-CoV-2 Variants of Concern (VOCs) and its therapeutic window in a human lung cell model
date: 2021-11-29
journal: bioRxiv
DOI: 10.1101/2021.11.23.469695
sha: 393f86fd4afc22e22912929c014f7e9ea7acad83
doc_id: 921411
cord_uid: f0zvel44

Background The UK Medicines and Regulatory Healthcare Agency (MHRA) have recently licensed the anti-viral drug, molnupiravir, for use in patients with mild-moderate COVID-19 disease with one or more risk factors for serious illness. Treatment with anti-viral drugs is best initiated early to prevent progression to severe disease, although the therapeutic window for intervention has not yet been fully defined. Objectives This study aimed to determine the activity of the molnupiravir (NHC) to different SARS-CoV-2 Variants of Concern (VoCs), and to establish the therapeutic window in human lung cell model. Methods Dose response assays were performed in parallel to determine the IC50 (the concentration of drug required to inhibit virus titre by 50%) of NHC against different variants. Human ACE-2 A549 cells were treated with NHC at different time points either before, during or after infection with SARS- CoV-2. Results Here we demonstrate that ß-D-N4-hydroxycytidine (NHC), the active metabolite of molnupiravir, has equivalent activity against four variants of SARS-CoV-2 in a human lung cell line ranging 0.04-0.16µM IC50. Furthermore, we demonstrate that in-vitro activity of the drug is reduced in cells exposed to drug 48 hours after infection. Conclusions One of the main advantages of molnupiravir is that it can be administered orally, and thus given to patients in an out-patient setting. These results support giving the drug early on after diagnosis or even in prophylaxis for individuals with high risk of developing severe disease.

were performed in parallel to determine the IC50 (the concentration of drug required to 31 inhibit virus titre by 50%) of NHC against different variants. Human ACE-2 A549 cells were 32 treated with NHC at different time points either before, during or after infection with SARS-33

CoV-2. Results: Here we demonstrate that ß-D-N4-hydroxycytidine (NHC), the active 34 metabolite of molnupiravir, has equivalent activity against four variants of SARS-CoV-2 in a 35 human lung cell line ranging 0.04-0.16µM IC50. Furthermore, we demonstrate that in-vitro 36 activity of the drug is reduced in cells exposed to drug 48 hours after infection. Conclusions: 37

One of the main advantages of molnupiravir is that it can be administered orally, and thus 38 given to patients in an out-patient setting. These results support giving the drug early on after 39 diagnosis or even in prophylaxis for individuals with high risk of developing severe disease. Molnupiravir is an anti-viral pro-drug originally developed against influenza virus currently 59 undergoing clinical trials in humans for the treatment of COVID-19 4,5 . Interim phase III results 60 suggested the drug reduced the risk of hospitalisation or death by 50% with efficacy 61 unaffected by the timing of symptom onset, underlying risk factors, or variant type (gamma, 62 delta, and mu) 6 . As a result, the MHRA in the UK has granted emergency use of the drug for 

A one-way ANOVA was used to evaluate the in-vitro cytotoxicity data. The absolute IC50 159 values were calculated using GraphPad Prism 9, using a non-linear 4-parameter logistic 160 regression in a dose-response curve. 161

In order to find the appropriate concentration range of NHC in hACE2-A549 cells, to 164 investigate the effect on viral biology, Cell-titer Glo Assay (Promega) were used to measure % 165 ATP production in cells. These were treated with DMSO or different concentrations of NHC 166 diluted in hACE2-A549 culture medium compared to mock untreated cells. DMSO, at the 167 highest concentration used (0.25%) had no effect on cells (p>0.05). There was a slight increase 168 in the ATP production in cells at the lowest concentration of drug, 0.01µM (p=0.02). The only 169 concentration of drug to inhibit the ATP production was 10µM (p>0.0001) (Figure 1 variants of concern (VOCs). A non-linear regression was used to calculate the IC50 for each 191 variant (n=4). 192 infection. Further work is required to delineate the true treatment window of the drug in 237 humans with mild to moderate disease. Anti-viral drugs are best given early in infection to 238 reduce viral load. In-vivo ferret models have shown that severity is linked to the size of the 239 viral inoculum 18 . Therefore, molnupiravir likely acts to reduce disease by reducing viral load 240 in patients, and potentially subsequently reducing transmission. 241

One of the main benefits of molnupiravir as opposed to remdesivir is that it can be 242 administered orally. However, as was seen with the Influenza anti-viral, Tamiflu, resistance to 243 anti-virals can develop rapidly 19 . A thorough analysis of the potential of SARS-CoV-2 to 244 develop resistance is necessary, though it is likely that any adaptation for resistance will 245 correspond with a reduction in fitness as seen with remdesivir 20 . Use of molnupiravir would 246 likely be most beneficial if used in combination with another treatment, preferably targeting 247 a different part of the viral life cycle as has been used with success for HIV treatment. Finally, 248 molnupiravir has broad spectrum activity, shown to be effective against RSV, Influenza, and 249 seasonal coronaviruses in in-vitro models. Here we have shown that the in-vitro activity of 250 NHC is retained across a broad range of variants tested, suggesting that the drug can be 251 deployed widely, and clinical effectiveness is unlikely to be adversely impacted by these 252 different viral strains. 253

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