Background: Ronapreve demonstrated clinical application in post-exposure prophylaxis, mild/moderate disease and in the treatment of seronegative patients with severe COVID19 prior to the emergence of the Omicron variant in late 2021. Numerous reports have described loss of in vitro neutralisation activity of Ronapreve and other monoclonal antibodies for BA.1 Omicron and subsequent sub-lineages of the Omicron variant. With some exceptions, global policy makers have recommended against the use of existing monoclonal antibodies in COVID19. Gaps in knowledge regarding the mechanism of action of monoclonal antibodies are noted, and further preclinical study will help understand positioning of new monoclonal antibodies under development. Objectives: The purpose of this study was to investigate the impact of Ronapreve on compartmental viral replication as a paradigm for a monoclonal antibody combination. The study also sought to confirm absence of in vivo activity against BA.1 Omicron (B.1.1.529) relative to the Delta (B.1.617.2) variant. Methods: Virological efficacy of Ronapreve was assessed in K18-hACE2 mice inoculated with either the SARS-CoV-2 Delta or Omicron variants. Viral replication in tissues was quantified using qRT-PCR to measure sub-genomic viral RNA to the E gene (sgE) as a proxy. A histological examination in combination with staining for viral antigen served to determine viral spread and associated damage. Results: Ronapreve reduced sub-genomic viral RNA levels in lung and nasal turbinate, 4 and 6 days post infection, for the Delta variant but not the Omicron variant of SARS-CoV-2 at doses 2-fold higher than those shown to be active against previous variants of the virus. It also appeared to block brain infection which is seen with high frequency in K18-hACE2 mice after Delta variant infection. At day 6, the inflammatory response to lung infection with the Delta variant was altered to a mild multifocal granulomatous inflammation in which the virus appeared to be confined. A similar tendency was also observed in Omicron infected, Ronapreve-treated animals. Conclusions: The current study provides evidence of an altered tissue response to the SARS-CoV-2 after treatment with a monoclonal antibody combination that retains neutralization activity. These data also demonstrate that experimental designs that reflect the treatment use case are achievable in animal models for monoclonal antibodies deployed against susceptible variants. Extreme caution should be taken when interpreting prophylactic experimental designs when assessing plausibility of monoclonal antibodies for treatment use cases.
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 subgenomic 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.
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.
The SARS-CoV-2 pandemic has spread at an unprecedented rate, and 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. Niclosamide has been proposed as a candidate for repurposing for SARS-CoV-2 based upon the observation that it is amongst the most potent antiviral molecules evaluated in vitro . To investigate the pharmacokinetics of niclosamide, reliable, reproducible and sensitive bioanalytical assays are required. Here, a liquid chromatography tandem mass spectrometry assay is presented which was linear from 31.25-2000 ng/mL (high dynamic range) and 0.78-100 ng/mL (low dynamic range). Accuracy and precision ranged between 97.2% and 112.5%, 100.4% and 110.0%, respectively. The presented assay should have utility in preclinical evaluation of the exposure-response relationship and may be adapted for later evaluation of niclosamide in clinical trials.
BACKGROUND: The role of favipiravir as a treatment for COVID-19 is unclear, with discrepant activity against SARS-CoV-2 in vitro , concerns about teratogenicity and pill burden, and an unknown optimal dose. In Vero-E6 cells, high concentrations are needed to inhibit SARS-CoV-2 replication. The purpose of this analysis was to use available data to simulate intracellular pharmacokinetics of favipiravir ribofuranosyl-5â°-triphosphate (FAVI-RTP) to better understand the putative applicability as a COVID-19 intervention. METHODS: Previously published in vitro data for the intracellular production and elimination of FAVI- RTP in MDCK cells incubated with parent favipiravir was fitted with a mathematical model to describe the time course of intracellular FAVI-RTP concentrations as a function of incubation concentration of parent favipiravir. Parameter estimates from this model fitting were then combined with a previously published population PK model for the plasma exposure of parent favipiravir in Chinese patients with severe influenza (the modelled free plasma concentration of favipiravir substituting for in vitro incubation concentration) to predict the human intracellular FAVI-RTP pharmacokinetics. RESULTS: In vitro FAVI-RTP data was adequately described as a function of in vitro incubation media concentrations of parent favipiravir with an empirical model, noting that the model simplifies and consolidates various processes and is used under various assumptions and within certain limits. Parameter estimates from the fittings to in vitro data predict a flatter dynamic range of peak to trough for intracellular FAVI-RTP when driven by a predicted free plasma concentration profile. CONCLUSION: This modelling approach has several important limitations that are discussed in the main text of the manuscript. However, the simulations indicate that despite rapid clearance of the parent drug from plasma, sufficient intracellular FAVI-RTP may be maintained across the dosing interval because of its long intracellular half-life. Population average intracellular FAVI-RTP concentrations are estimated to maintain the Km for the SARS-CoV-2 polymerase for 3 days following 800 mg BID dosing and 9 days following 1200 mg BID dosing after a 1600 mg BID loading dose on day 1. Further evaluation of favipiravir as part of antiviral combinations for SARS-CoV-2 is warranted.
BACKGROUND: Severe 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 (EC 90 ) against SARS-CoV-2. METHODS: A 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 SARS-CoV-2 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 EC 90 in >90% of the simulated population. Optimal design software PopDes was used to estimate an optimal sparse sampling strategy for future clinical trials. RESULTS: The PBPK model was validated with AAFE values between 1.01 SARS-CoV-2 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 EC 90 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. CONCLUSION: The PBPK model predicted that it was possible to achieve plasma and lung tizoxanide concentrations, using proven safe doses of nitazoxanide, that exceed the EC 90 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.
