An optimised method for recovery and quantification of laboratory generated SARS-CoV-2 aerosols by plaque assay. (preprint)

We present an optimised method for the recovery of laboratory generated SARS-CoV-2 aerosols and quantification by plaque assays. This method allows easy incorporation into existing standard operating procedures of biological containment level 3 (CL3) laboratories.

Lack of antiviral activity of probenecid in Vero E6 cells and Syrian golden hamsters: a need for better understanding of inter-lab differences in preclinical assays. (preprint)

Antiviral interventions are urgently required to support vaccination programmes and reduce the global burden of COVID-19. Prior to initiation of large-scale clinical trials, robust preclinical data in support of candidate plausibility are required. The speed at which preclinical models have been developed during the pandemic are unprecedented but there is a vital need for standardisation and assessment of the Critical Quality Attributes. This work provides cross-validation for the recent report demonstrating potent antiviral activity of probenecid against SARS-CoV-2 in preclinical models (1). Vero E6 cells were pre-incubated with probenecid, across a 7-point concentration range, or control media for 2 hours before infection with SARS-CoV-2 (SARS-CoV-2/Human/Liverpool/REMRQ0001/2020, Pango B; MOI 0.05). Probenecid or control media was then reapplied and plates incubated for 48 hours. Cells were fixed with 4% v/v paraformaldehyde, stained with crystal violet and cytopathic activity quantified by spectrophotometry at 590 nm. Syrian golden hamsters (n=5 per group) were intranasally inoculated with virus (SARS-CoV-2 Delta variant B.1.617.2; 103 PFU/hamster) for 24 hours prior to treatment. Hamsters were treated with probenecid or vehicle for 4 doses. Hamsters were ethically euthanised before quantification of total and sub-genomic pulmonary viral RNAs. No inhibition of cytopathic activity was observed for probenecid at any concentration in Vero E6 cells. Furthermore, no reduction in either total or sub-genomic RNA was observed in terminal lung samples from hamsters on day 3 (P > 0.05). Body weight of uninfected hamsters remained stable throughout the course of the experiment whereas both probenecid- (6 - 9% over 3 days) and vehicle-treated (5 - 10% over 3 days) infected hamsters lost body weight which was comparable in magnitude (P > 0.5). The presented data do not support probenecid as a SARS-CoV-2 antiviral. These data do not support use of probenecid in COVID-19 and further analysis is required prior to initiation of clinical trials to investigate the potential utility of this drug.

Remdesivir-Ivermectin combination displays synergistic interaction with improved in vitro antiviral activity against SARS-CoV-2 (preprint)

A key element to the prevention and management of the COVID-19 pandemic is the development of effective therapeutics. Drug combination strategies of repurposed drugs offer a number of advantages to monotherapies including the potential to achieve greater efficacy, the potential to increase the therapeutic index of drugs and the potential to reduce the emergence of drug resistance. Combination of agents with antiviral mechanisms of action with immune-modulatory or anti-inflammatory drug is also worthy of investigation. Here, we report on the in vitro synergistic interaction between two FDA approved drugs, remdesivir (RDV) and ivermectin (IVM) resulting in enhanced antiviral activity against SARS-CoV-2, the causative pathogen of COVID-19. These findings warrant further investigations into the clinical potential of this combination, together with studies to define the underlying mechanism.

Dose prediction for repurposing nitazoxanide in SARS-CoV-2 treatment or chemoprophylaxis (preprint)

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been declared a global pandemic by the World Health Organisation and urgent treatment and prevention strategies are needed. Many clinical trials have been initiated with existing medications, but assessments of the expected plasma and lung exposures at the selected doses have not featured in the prioritisation process. Although no antiviral data is currently available for the major phenolic circulating metabolite of nitazoxanide (known as tizoxanide), the parent ester drug has been shown to exhibit in vitro activity against SARS-CoV-2. Nitazoxanide is an anthelmintic drug and its metabolite tizoxanide has been described to have broad antiviral activity against influenza and other coronaviruses. The present study used physiologically-based pharmacokinetic (PBPK) modelling to inform optimal doses of nitazoxanide capable of maintaining plasma and lung tizoxanide exposures above the reported nitazoxanide 90% effective concentration (EC90) against SARS-CoV-2. MethodsA whole-body PBPK model was constructed for oral administration of nitazoxanide and validated against available tizoxanide pharmacokinetic data for healthy individuals receiving single doses between 500 mg - 4000 mg with and without food. Additional validation against multiple-dose pharmacokinetic data when given with food was conducted. The validated model was then used to predict alternative doses expected to maintain tizoxanide plasma and lung concentrations over the reported nitazoxanide EC90 in >90% of the simulated population. Optimal design software PopDes was used to estimate an optimal sparse sampling strategy for future clinical trials. ResultsThe PBPK model was validated with AAFE values between 1.01 - 1.58 and a difference less than 2-fold between observed and simulated values for all the reported clinical doses. The model predicted optimal doses of 1200 mg QID, 1600 mg TID, 2900 mg BID in the fasted state and 700 mg QID, 900 mg TID and 1400 mg BID when given with food, to provide tizoxanide plasma and lung concentrations over the reported in vitro EC90 of nitazoxanide against SARS-CoV-2. For BID regimens an optimal sparse sampling strategy of 0.25, 1, 3 and 12h post dose was estimated. ConclusionThe PBPK model predicted that it was possible to achieve plasma and lung tizoxanide concentrations, using proven safe doses of nitazoxanide, that exceed the EC90 for SARS-CoV-2. The PBPK model describing tizoxanide plasma pharmacokinetics after oral administration of nitazoxanide was successfully validated against clinical data. This dose prediction assumes that the tizoxanide metabolite has activity against SARS-CoV-2 similar to that reported for nitazoxanide, as has been reported for other viruses. The model and the reported dosing strategies provide a rational basis for the design (optimising plasma and lung exposures) of future clinical trials of nitazoxanide in the treatment or prevention of SARS-CoV-2 infection.