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Successful development of a chemoprophylaxis against SARS-CoV-2 could provide a tool for infection prevention implementable alongside vaccination programmes. Camostat and nafamostat are serine protease inhibitors that inhibit SARS-CoV-2 viral entry in vitro but have not been characterised for chemoprophylaxis in animal models. Clinically, nafamostat is limited to intravenous delivery and while camostat is orally available, both drugs have extremely short plasma half-lives. This study sought to determine whether intranasal dosing at 5 mg/kg twice daily was able to prevent airborne transmission of SARS-CoV-2 from infected to uninfected Syrian golden hamsters. SARS-CoV-2 viral RNA was above the limits of quantification in both saline- and camostat-treated hamsters 5 days after cohabitation with a SARS-CoV-2 inoculated hamster. However, intranasal nafamostat-treated hamsters remained RNA negative for the full 7 days of cohabitation. Changes in body weight over the course of the experiment were supportive of a lack of clinical symptomology in nafamostat-treated but not saline- or camostat-treated animals. These data are strongly supportive of the utility of intranasally delivered nafamostat for prevention of SARS-CoV-2 infection and further studies are underway to confirm absence of pulmonary infection and pathological changes.

Currently nitazoxanide is being assessed as a candidate therapeutic for SARS-CoV-2. Unlike many other candidates being investigated, tizoxanide (the active metabolite of nitazoxanide) plasma concentrations achieve antiviral levels after administration of the approved dose, although higher doses are expected to be needed to maintain these concentrations across the dosing interval in the majority of patients. Here an LC-MS/MS assay is described that has been validated in accordance with Food and Drug Administration (FDA) guidelines. Fundamental parameters have been evaluated, and these included accuracy, precision and sensitivity. The assay was validated for human plasma, mouse plasma and Dulbeccos Modified Eagles Medium (DMEM) containing varying concentrations of Foetal Bovine Serum (FBS). Matrix effects are a well-documented source of concern for chromatographic analysis, with the potential to impact various stages of the analytical process, including suppression or enhancement of ionisation. Therefore, a robustly validated LC-MS/MS analytical method is presented capable of quantifying tizoxanide in multiple matrices with minimal impact of matrix effects. The validated assay presented here was linear from 15.6ng/mL to 1000ng/mL. Accuracy and precision ranged between 102.2% and 113.5%, 100.1% and 105.4%, respectively. The presented assay here has applications in both pre-clinical and clinical research and may be used to facilitate further investigations into the application of nitazoxanide against SARS-CoV-2.

Favipiravir (FAV; T-705) has been approved for use as an anti-influenza therapeutic and has reports against a wide range of viruses (e.g., Ebola virus, rabies and norovirus). Most recently FAV has been reported to demonstrate activity against SARS-CoV-2. Repurposing opportunities have been intensively studied with only limited success to date. If successful, repurposing will allow interventions to become more rapidly available than development of new chemical entities. Pre-clinical and clinical investigations of FAV require robust, reproducible and sensitive bioanalytical assay. Here, a liquid chromatography tandem mass spectrometry assay is presented which was linear from 0.78-200 ng/mL Accuracy and precision ranged between 89% and 110%, 101% and 106%, respectively. The presented assay here has applications in both pre-clinical and clinical research and may be used to facilitate further investigations into the application of FAV against SARS-CoV-2.

There is a rapidly expanding literature on thein vitroantiviral activity of drugs that may be repurposed for therapy or chemoprophylaxis against SARS-CoV-2. However, this has not been accompanied by a comprehensive evaluation of the ability of these drugs to achieve target plasma and lung concentrations following approved dosing in humans. Moreover, most publications have focussed on 50% maximum effective concentrations (EC50), which may be an insufficiently robust indicator of antiviral activity because of marked differences in the slope of the concentration-response curve between drugs. Accordingly,in vitroanti-SARS-CoV-2 activity data was digitised from all available publications up to 13thApril 2020 and used to recalculate an EC90value for each drug. EC90values were then expressed as a ratio to the achievable maximum plasma concentrations (Cmax) reported for each drug after administration of the approved dose to humans (Cmax/EC90ratio). Only 14 of the 56 analysed drugs achieved a Cmax/EC90ratio above 1 meaning that plasma Cmax concentrations exceeded those necessary to inhibit 90% of SARS-CoV-2 replication. A more in-depth assessment of the putative agents tested demonstrated that only nitazoxanide, nelfinavir, tipranavir (boosted with ritonavir) and sulfadoxine achieved plasma concentrations above their reported anti-SARS-CoV-2 activity across their entire approved dosing interval at their approved human dose. For all drugs reported, the unbound lung to plasma tissue partition coefficient (KpUlung) was also simulated and used along with reported Cmax and fraction unbound in plasma to derive a lung Cmax/EC50as a better indicator of potential human efficacy (lung Cmax/EC90ratio was also calculable for a limited number of drugs). Using this parameter hydroxychloroquine, chloroquine, mefloquine, atazanavir (boosted with ritonavir), tipranavir (boosted with ritonavir), ivermectin, azithromycin and lopinavir (boosted with ritonavir) were all predicted to achieve lung concentrations over 10-fold higher than their reported EC50. This analysis was not possible for nelfinavir because insufficient data were available to calculate KpUlungbut nitozoxanide and sulfadoxine were also predicted to exceed their reported EC50by 3.1- and 1.5-fold in lung, respectively. The antiviral activity data reported to date have been acquired under different laboratory conditions across multiple groups, applying variable levels of stringency. However, this analysis may be used to select potential candidates for further clinical testing, while deprioritising compounds which are unlikely to attain target concentrations for antiviral activity. Future studies should focus on EC90values and discuss findings in the context of achievable exposures in humans, especially within target compartments such as the lung, in order to maximise the potential for success of proposed human clinical trials.