Abstract Ronapreve, a combination of two neutralising monoclonal antibodies, casirivimab and imdevimab, was amongst the authorised treatments against SARS-CoV-2 early in the COVID-19 pandemic. Ronapreve has lost some of its efficiency with the rise of new virus variants, however, it remains a valuable tool for experimental studies to gain insights into the mechanisms and effects of anti-viral drugs. In this study we combined morphological, pharmacokinetic and molecular approaches (including multiomics) to investigate the biodistribution of Ronapreve in the K18-hACE2 murine model of SARS-CoV-2 neuroinvasion, as well as possible consequences for the brain. We also investigated the effect of the treatment on the infection status. Our results showed that after intraperitoneal injection, Ronapreve accumulates in the serum and is unable to cross the blood-brain barrier, thus not reaching the brain parenchyma; treatment has only a minimal effect on the brain transcriptome, with no significant changes in the brain lipidome or metabolome. Nonetheless, post-exposure Ronapreve treatment resulted in reduced viral loads in the lung and, in particular, the brain, with markedly reduced tissue response in the brain, as shown by the transcriptomic analysis. The results suggest a peripheral mode of action of Ronapreve to block brain infection, possibly by lowering viral replication in the nasal epithelium, reducing a subsequent spread to the brain.

Immunocompromised individuals are susceptible to severe coronavirus disease 2019 and potentially contribute to the emergence of variants with altered pathogenicity due to persistent infection. This study investigated the impact of immunosuppression on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in K18-hACE2 mice and the effectiveness of antiviral treatments in this context during the first 7 days of infection. Mice were immunosuppressed using cyclophosphamide and infected with a B lineage of SARS-CoV-2. Molnupiravir and nirmatrelvir, alone and in combination, were administered, and viral load and viral sequence diversity were assessed. Treatment of infected but immunocompromised mice with both compounds either singly or in combination resulted in decreased viral loads and pathological changes compared to untreated animals. Treatment also abrogated infection of neuronal tissue. However, no consistent changes in the viral consensus sequence were observed, except for the emergence of the S:H655Y mutation. Molnupiravir, but not nirmatrelvir or immunosuppression alone, increased the transition/transversion ratio, representative of G > A and C > U mutations, and this increase was not altered by the co-administration of nirmatrelvir with molnupiravir. Notably, immunosuppression itself did not appear to promote the emergence of mutational characteristics of variants of concern (VOCs). Further investigations are warranted to fully understand the role of immunocompromised individuals in VOC development, especially by taking persistence into consideration, and to inform optimized public health strategies. It is more likely that immunodeficiency promotes viral persistence but does not necessarily lead to substantial consensus-level changes in the absence of antiviral selection pressure. Consistent with mechanisms of action, molnupiravir showed a stronger mutagenic effect than nirmatrelvir in this model. IMPORTANCE:The central aim of this study was to risk-assess the impact of administering a mutagenic antiviral compound, molnupiravir, to patients believed to already be at risk of generating increased viral diversity, which could have severe implications for antiviral resistance development. Combination therapy has a long history of mitigating antiviral resistance risk and was used in this study to demonstrate its potential usefulness in a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) context. Animals treated with molnupiravir showed an increase in transition/transversion ratios over time, consistent with the drug's mechanism of action and a recent UK-wide phase II clinical trial assessing the efficacy of molnupiravir in humans. The addition of nirmatrelvir increased viral clearance, which in turn reduces the probability of viral persistence and rapid intra-host evolution of SARS-CoV-2.

Favipiravir (FVP) and remdesivir (RDV) have demonstrable antiviral activity against SARS-CoV-2. Here, the efficacy of FVP, RDV, and FVP with RDV (FVP + RDV) in combination was assessed in Syrian golden hamsters challenged with SARS-CoV- 2 (B.1.1.7) following intraperitoneal administration. At day 4 post infection, viral RNA and viral antigen expression were significantly lower in lungs for all three treatment groups compared to the sham treatment. Similarly, viral titres in the lungs were lower in all treatment groups compared to the sham treatment. The FVP + RDV combination was the only treatment group where viral RNA in nasal turbinate and lung, virus titres in lung, and viral antigen expression (lung) were all lower than those for the sham treatment group. Moreover, lower viral titre values were observed in the FVP + RDV group compared to other treatment groups, albeit only significantly lower in comparison to those in the RDV-only-treated group. Further assessment of the potential utility of FVP in combination with RDV may be warranted. Future studies should also consider whether the combination of these two drugs may reduce the speed at which drug resistance mutations are selected.

A significant challenge in the development of long-acting injectable drug formulations, especially for anti-infective agents, is delivering an efficacious dose within a tolerable injection volume. Co-administration of the extracellular matrix-degrading enzyme hyaluronidase can increase maximum tolerable injection volumes but is untested for this benefit with long-acting injectable formulations. One concern is that hyaluronidase could potentially alter the tissue response surrounding an injection depot, a response known to be important for drug release kinetics of long-acting injectable formulations. The objective of this pilot study was to evaluate the impact of co-administration of hyaluronidase on the drug release kinetics, pharmacokinetic profiles, and injection site histopathology of the long-acting injectable paliperidone palmitate for up to four weeks following intramuscular injection in mouse and rat models. In both species, co-administration of hyaluronidase increased paliperidone plasma exposures the first week after injection but did not negate the overall long-acting release nature of the formulation. Hyaluronidase-associated modification of the injection site depot was observed in mice but not in rats. These findings suggest that further investigation of hyaluronidase with long-acting injectable agents is warranted.

Objectives: Antiviral interventions are required to complement vaccination programmes and reduce the global burden of COVID-19. Prior to initiation of large-scale clinical trials, robust preclinical data to support candidate plausibility are required. This work sought to further investigate the putative antiviral activity of probenecid against SARS-CoV-2.Methods: Vero E6 cells were preincubated with probenecid, or control media for 2 h before infection (SARS-CoV-2/Human/Liverpool/REMRQ0001/2020). Probenecid or control media was reapplied, plates reincubated and cytopathic activity quantified by spectrophotometry after 48 h. In vitro human airway epithelial cell (HAEC) assays were performed for probenecid against SARS-CoV-2-VoC-B.1.1.7 (hCoV-19/Belgium/rega-12211513/2020; EPI_ISL_791333, 2020-12-21) using an optimized cell model for antiviral testing. Syrian golden hamsters were intranasally inoculated (SARS-CoV-2 Delta B.1.617.2) 24 h prior to treatment with probenecid or vehicle for four twice-daily doses.Results: No observable antiviral activity for probenecid was evident in Vero E6 or HAEC assays. No reduction in total or subgenomic RNA was observed in terminal lung samples (P > 0.05) from hamsters. Body weight of uninfected hamsters remained stable whereas both probenecid- and vehicle-treated infected hamsters lost body weight (P > 0.5).Conclusions: These data do not support probenecid as a SARS-CoV-2 antiviral drug.

Evolving evidence underpinning musculoskeletal physiotherapy challenges the definitions of contemporary clinical practice and pre-registration curricula. This research uses an online national survey to compare clinician (272) and lecturer (61) views on the Chartered Society of Physiotherapy’s four pillars of practice (manual therapy and therapeutic handling; exercise, movement, and rehabilitation; therapeutic and diagnostics technologies; and allied approaches, including psychologically informed approaches) with the aim of establishing their clinical and educational utility. Findings indicate that close alignment exists between practising U.K. musculoskeletal physiotherapists and U.K. pre-registration physiotherapy lecturers on the ongoing relevance of exercise therapy and psychologically informed approaches to behaviour change. The paper also shows that a mismatch exists in the clinical and educational use of both manual therapy and electrotherapy between practising physiotherapists and physiotherapy lecturers. Future research should focus on the highlighted need to continue to align pre-registration musculoskeletal curricula with both the evidence base and clinical practice for the curricular areas of electrotherapy and manual therapy. This article was published open access under a CC BY licence: https://creativecommons.org/licences/by/4.0 .

Pibrentasvir (PIB) has been demonstrated to block exonuclease activity of the SARS-CoV-2 polymerase, protecting favipiravir (FVP) and remdesivir (RDV) from post-incorporation excision and eliciting antiviral synergy in vitro. The present study investigated the chemoprophylactic efficacy of PIB, FVP, RDV, FVP with PIB, or RDV with PIB dosed intranasally twice a day, using a Syrian golden hamster contact transmission model. Compared to the saline control, viral RNA levels were significantly lower in throat swabs in FVP (day 7), RDV (day 3, 5, 7), and RDV+PIB (day 3, 5) treatment groups. Similarly, findings were evident for nasal turbinate after PIB and RDV treatment, and lungs after PIB, FVP, and FVP+PIB treatment at day 7. Lung viral RNA levels after RDV and RDV+PIB treatment were only detectable in two animals per group, but the overall difference was not statistically significant. In situ examination of the lungs confirmed SARS-CoV-2 infection in all animals, except for one in each of the RDV and RDV+PIB treatment groups, which tested negative in all virus detection approaches. Overall, prevention of transmission was observed in most animals treated with RDV, while other agents reduced the viral load following contact transmission. No benefit of combining FVP or RDV with PIB was observed.

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.

The successful development of a chemoprophylaxis against SARS-CoV-2 could provide a tool for infection prevention that is implementable alongside vaccination programmes. Nafamostat is a serine protease inhibitor that inhibits SARS-CoV-2 entry in vitro, but it has not been characterised for chemoprophylaxis in animal models. Clinically, nafamostat is limited to intravenous delivery and has an extremely short plasma half-life. This study sought to determine whether intranasal dosing of nafamostat at 5 mg/kg twice daily was able to prevent the airborne transmission of SARS-CoV-2 from infected to uninfected Syrian Golden hamsters. SARS-CoV-2 RNA was detectable in the throat swabs of the water-treated control group 4 days after cohabitation with a SARS-CoV-2 inoculated hamster. However, throat swabs from the intranasal nafamostat-treated hamsters remained SARS-CoV-2 RNA negative for the full 4 days of cohabitation. Significantly lower SARS-CoV-2 RNA concentrations were seen in the nasal turbinates of the nafamostat-treated group compared to the control (p = 0.001). A plaque assay quantified a significantly lower concentration of infectious SARS-CoV-2 in the lungs of the nafamostat-treated group compared to the control (p = 0.035). When taken collectively with the pathological changes observed in the lungs and nasal mucosa, these data are strongly supportive of the utility of intranasally delivered nafamostat for the prevention of SARS-CoV-2 infection.

Long-acting injectable (LAI) formulations promise to deliver patient benefits by overcoming issues associated with non-adherence. A preclinical assessment of semi-solid prodrug nanoparticle (SSPN) LAI formulations of emtricitabine (FTC) is reported here. Pharmacokinetics over 28 days were assessed in Wistar rats, New Zealand white rabbits, and Balb/C mice following intramuscular injection. Two lead formulations were assessed for the prevention of an HIV infection in NSG-cmah(-/-) humanised mice to ensure antiviral activities were as anticipated according to the pharmacokinetics. Cmax was reached by 12, 48, and 24 h in rats, rabbits, and mice, respectively. Plasma concentrations were below the limit of detection (2 ng/mL) by 21 days in rats and rabbits, and 28 days in mice. Mice treated with SSPN formulations demonstrated undetectable viral loads (700 copies/mL detection limit), and HIV RNA remained undetectable 28 days post-infection in plasma, spleen, lung, and liver. The in vivo data presented here demonstrate that the combined prodrug/SSPN approach can provide a dramatically extended pharmacokinetic half-life across multiple preclinical species. Species differences in renal clearance of FTC mean that longer exposures are likely to be achievable in humans than in preclinical models.

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.

The Omicron variant (B.1.1.529) of SARS-CoV-2 has placed enormous strain on global healthcare systems since it was first identified by South African researchers in late 2021. Omicron has >50 mutations which mainly occur in the surface spike protein and this has led to rapid assessment of monoclonal antibodies to assess the impact on virus neutralisation. Ronapreve has shown potential application in post-exposure prophylaxis, mild/moderate disease and in seronegative patients with severe COVID19, but several early reports of loss of in vitro neutralisation activity have been documented. Here, the virological efficacy of Ronapreve was assessed in K18-hACE2 mice to provide an in vivo outcome. Ronapreve reduced sub-genomic RNA in lung and nasal turbinate 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. These data add to the growing evidence that the effectiveness of Ronapreve is compromised for the Omicron variant.

The spread of SARS-CoV-2 across the world requires the formation of a range of interventions that include immunomodulatory or antiviral therapeutics and repurposing of existing drug compounds is an important strategy to rapidly identify possible options. Niclosamide is a cheap and generic drug compound with a proven in vitro ability to inhibit viral replication of SARS-CoV-2; this drug also has known poor oral bioavailability due to very low water solubility. Here, we demonstrate the nanoparticle formulation of niclosamide using a new, cheap and scalable process to enable long-acting injectable administration and show extended circulating drug exposure in vivo over 28 days using a rodent model after depot injection.

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.

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been declared a global pandemic and urgent treatment and prevention strategies are needed. Nitazoxanide, an anthelmintic drug, has been shown to exhibit in vitro activity against SARS-CoV-2. 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 SARS-CoV-2 EC90. Methods A whole-body PBPK model was validated against available pharmacokinetic data for healthy individuals receiving single and multiple doses between 500 and 4000 mg with and without food. The validated model was used to predict doses expected to maintain tizoxanide plasma and lung concentrations above the EC90 in >90% of the simulated population. PopDes was used to estimate an optimal sparse sampling strategy for future clinical trials. Results The PBPK model was successfully validated against the reported human pharmacokinetics. The model predicted optimal doses of 1200 mg QID, 1600 mg TID and 2900 mg BID in the fasted state and 700 mg QID, 900 mg TID and 1400 mg BID when given with food. For BID regimens an optimal sparse sampling strategy of 0.25, 1, 3 and 12 hours post dose was estimated. Conclusion The PBPK model predicted tizoxanide concentrations within doses of nitazoxanide already given to humans previously. The reported dosing strategies provide a rational basis for design of clinical trials with nitazoxanide for the treatment or prevention of SARS-CoV-2 infection. A concordant higher dose of nitazoxanide is now planned for investigation in the seamless phase I/IIa AGILE trial.

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.