FXR inhibition may protect from SARS-CoV-2 infection by reducing ACE2.

Preventing SARS-CoV-2 infection by modulating viral host receptors, such as angiotensin-converting enzyme 2 (ACE2)1, could represent a new chemoprophylactic approach for COVID-19 that complements vaccination2,3. However, the mechanisms that control the expression of ACE2 remain unclear. Here we show that the farnesoid X receptor (FXR) is a direct regulator of ACE2 transcription in several tissues affected by COVID-19, including the gastrointestinal and respiratory systems. We then use the over-the-counter compound z-guggulsterone and the off-patent drug ursodeoxycholic acid (UDCA) to reduce FXR signalling and downregulate ACE2 in human lung, cholangiocyte and intestinal organoids and in the corresponding tissues in mice and hamsters. We show that the UDCA-mediated downregulation of ACE2 reduces susceptibility to SARS-CoV-2 infection in vitro, in vivo and in human lungs and livers perfused ex situ. Furthermore, we reveal that UDCA reduces the expression of ACE2 in the nasal epithelium in humans. Finally, we identify a correlation between UDCA treatment and positive clinical outcomes after SARS-CoV-2 infection using retrospective registry data, and confirm these findings in an independent validation cohort of recipients of liver transplants. In conclusion, we show that FXR has a role in controlling ACE2 expression and provide evidence that modulation of this pathway could be beneficial for reducing SARS-CoV-2 infection, paving the way for future clinical trials.

Lack of antiviral activity of probenecid in vitro and in Syrian golden hamsters.

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.

In vitro evaluation of physicochemical-dependent effects of polymeric nanoparticles on their cellular uptake and co-localization using pulmonary calu-3 cell lines.

OBJECTIVE: The study evaluated physicochemical properties of eight different polymeric nanoparticles (NPs) and their interaction with lung barrier and their suitability for pulmonary drug delivery. METHODS: Eight physiochemically different NPs were fabricated from Poly lactic-co-glycolic acid (PLGA, PL) and Poly glycerol adipate-co-ω-pentadecalactone (PGA-co-PDL, PG) via emulsification-solvent evaporation. Pulmonary barrier integrity was investigated in vitro using Calu-3 under air-liquid interface. NPs internalization was investigated using a group of pharmacological inhibitors with subsequent microscopic visual confirmation. RESULTS: Eight NPs were successfully formulated from two polymers using emulsion-solvent evaporation; 200, 500 and 800 nm, negatively-charged and positively-charged. All different NPs did not alter tight junctions and PG NPs showed similar behavior to PL NPs, indicating its suitability for pulmonary drug delivery. Active endocytosis uptake mechanisms with physicochemical dependent manner were observed. In addition, NPs internalization and co-localization with lysosomes were visually confirmed indicating their vesicular transport. CONCLUSION: PG and PL NPs had shown no or low harmful effects on the barrier integrity, and with effective internalization and vesicular transport, thus, prospectively can be designed for pulmonary delivery applications.

Dose prediction for repurposing nitazoxanide in SARS-CoV-2 treatment or chemoprophylaxis.

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.

Predicting Drug-Drug Interactions Between Rifampicin and Long-Acting Cabotegravir and Rilpivirine Using Physiologically Based Pharmacokinetic Modeling.

BACKGROUND: Cabotegravir and rilpivirine are 2 long-acting (LA) antiretrovirals that can be administered intramuscularly; their interaction with rifampicin, a first-line antituberculosis agent, has not been investigated. The aim of this study was to simulate and predict drug-drug interactions (DDIs) between these LA antiretroviral agents and rifampicin using physiologically based pharmacokinetic (PBPK) modeling. METHODS: The designed PBPK models were qualified (according to European Medicines Agency guidelines) against observed data for oral formulations of cabotegravir, rilpivirine, and rifampicin. Induction potential of rifampicin was also qualified by comparing the DDI between oral cabotegravir and oral rilpivirine with rifampicin. Qualified PBPK models were utilized for pharmacokinetic prediction of DDIs. RESULTS: PBPK models predicted a reduction in both area under the curve (AUC0-28 days) and trough concentration (Ctrough, 28th day) of LA cabotegravir of 41%-46% for the first maintenance dose coadministered with 600 mg once-daily oral rifampicin. Rilpivirine concentrations were predicted to decrease by 82% for both AUC0-28 days and Ctrough, 28th day following the first maintenance dose when coadministered with rifampicin. CONCLUSIONS: The developed PBPK models predicted the theoretical effect of rifampicin on cabotegravir and rilpivirine LA intramuscular formulations. According to these simulations, it is likely that coadministration of rifampicin with these LA formulations will result in subtherapeutic concentrations of both drugs.

Inhibitory Effects of Commonly Used Excipients on P-Glycoprotein in Vitro.

Pharmaceutical excipients are no longer considered inert and have been shown to influence the activity of metabolic enzymes and transporters, resulting in altered pharmacokinetics of substrate drugs. In this study, the effect of 25 excipients commonly used in drug formulations were investigated for their effect on P-glycoprotein (P-gp) activity. The effect of excipients on P-gp were assessed by measuring the change in the cellular accumulation of a P-gp substrate, digoxin, in MDCK-MDR1 (Madin Darby canine kidney transfected with multidrug resistance 1 gene) cells. The cells were exposed to low (10 µM) and high (200 µM) concentrations of excipient along with 10 µM digoxin. Excipient concentrations were chosen to span the range of concentrations previously used for investigating activities in vitro. At 10 µM of excipient, an increase in the intracellular digoxin concentration was seen with d-α-tocopherol poly-(ethylene glycol) succinate (Vit-E-PEG; p = 0.002), poly(ethylene oxide)20 sorbitan monooleate (Tween 80; p = 0.001), cetyltrimethylammonium bromide (CTAB; p = 0.021), poly(ethylene oxide)35 modified castor oil (Cremophor EL; p = 0.01), polyethylene glycol15-hydroxystearate (Solutol HS 15; p = 0.006), and poly(ethylene glycol) hexadecyl ether (Brij 58; p = 0.001). At 200 µM, Vit-E-PEG ( p < 0.0001), sodium 1,4-bis (2-ethylhexoxy)-1,4-dioxobutane-2-sulfonate (AOT; p < 0.0001), Tween 80 ( p < 0.0001), CTAB ( p = 0.004), poly(ethylene oxide)20 sorbitan monolaurate (Tween 20; p < 0.0001), Cremophor EL ( p < 0.0001), Solutol HS 15 ( p < 0.0001), Brij 58 ( p < 0.0001), and sodium carboxymethyl cellulose (NaCMC; p = 0.006) increased intracellular digoxin significantly. Concentration-dependent inhibition of P-gp was then investigated for selected excipients giving an IC50 for Vit-E-PEG (12.48 µM), AOT (192.5 µM), Tween 80 (45.29 µM), CTAB (96.67 µM), Tween 20 (74.15 µM), Cremophor EL (11.92 µM), Solutol HS 15 (179.8 µM), Brij 58 (25.22 µM), and NaCMC (46.69 µM). These data add to the growing body of evidence demonstrating that not all excipients are inert and will aid excipient choice for rational formulation development.

Efavirenz Is Predicted To Accumulate in Brain Tissue: an In Silico, In Vitro, and In Vivo Investigation.

Adequate concentrations of efavirenz in the central nervous system (CNS) are necessary to suppress viral replication, but high concentrations may increase the likelihood of CNS adverse drug reactions. The aim of this investigation was to evaluate the efavirenz distribution in the cerebrospinal fluid (CSF) and the brain by using a physiologically based pharmacokinetic (PBPK) simulation for comparison with rodent and human data. The efavirenz CNS distribution was calculated using a permeability-limited model on a virtual cohort of 100 patients receiving efavirenz (600 mg once daily). Simulation data were then compared with human data from the literature and with rodent data. Wistar rats were administered efavirenz (10 mg kg of body weight-1) once daily over 5 weeks. Plasma and brain tissue were collected for analysis via liquid chromatography-tandem mass spectrometry (LC-MS/MS). The median maximum concentrations of drug (Cmax) were predicted to be 3,184 ng ml-1 (interquartile range [IQR], 2,219 to 4,851 ng ml-1), 49.9 ng ml-1 (IQR, 36.6 to 69.7 ng ml-1), and 50,343 ng ml-1 (IQR, 38,351 to 65,799 ng ml-1) in plasma, CSF, and brain tissue, respectively, giving a tissue-to-plasma ratio of 15.8. Following 5 weeks of oral dosing of efavirenz (10 mg kg-1), the median plasma and brain tissue concentrations in rats were 69.7 ng ml-1 (IQR, 44.9 to 130.6 ng ml-1) and 702.9 ng ml-1 (IQR, 475.5 to 1,018.0 ng ml-1), respectively, and the median tissue-to-plasma ratio was 9.5 (IQR, 7.0 to 10.9). Although it is useful, measurement of CSF concentrations may give an underestimation of the penetration of antiretrovirals into the brain. The limitations associated with obtaining tissue biopsy specimens and paired plasma and CSF samples from patients make PBPK modeling an attractive tool for probing drug distribution.

Measuring the operational impact of digitized hospital records: a mixed methods study.

BACKGROUND: Digitized (scanned) medical records have been seen as a means for hospitals to reduce costs and improve access to records. However, clinical usability of digitized records can potentially have negative effects on productivity. METHODS: Data were collected during follow-up outpatient consultations in two NHS hospitals by non-clinical observers using a work sampling approach in which pre-defined categories of clinician time usage were specified. Quantitative data was analysed using two-way ANOVA models and the Mann-Whitney U test. A focus group was held with clinicians to qualitatively explore their experiences using digitized medical records. The quantitative and qualitative results were synthesized. RESULTS: Four hundred six consultations were observed. Using paper records, there was a significant difference in consultation times between hospitals (p = 0.016) and a significant difference in consultation times between specialties within hospitals (p = 0.003). Using digitized records there was a significant difference in consultation times between specialties within a hospital (p = 0.001). Excluding outliers, there was no significant difference between consultation times using digitized records compared with consultations using paper records in the same hospital, either at site (p > =0.285) or specialty level (p > =0.122). With digitized records at site A, two out of three specialties showed a significant increase in time spent searching computer records (p < =0.010, Δ = 01:50-07:10) and one specialty had a corresponding reduction in time spent searching paper records (p = 0.015, Δ = -00:28). Site B showed a notable increase in direct patient care (p < 0.001, Δ = 04:20-06:00) and time spent searching computer records (p < =0.043, Δ = 00:10-01:40) and reductions in the other time categories. The focus group confirmed that the most recent clinical letter was a vital document in the patient record, often containing most of the required information. Concerns were expressed about consistency of scanning practice, causing uncertainty about what could be relied upon to exist in the digitized record. Benefits of digitized records included: access from multiple locations, better prepared ward rounds, improved inpatient handovers and an improved timeline of patient events. Limitations of digitized records included: increased complexity of creating a patient summary, display of specialised content such as hand-drawn diagrams, inability to quickly flick through the pages to find relevant content. CONCLUSIONS: Digitized medical records can be implemented without detrimental operational impact. Inherent differences between specialties can outweigh the differences between paper and digitized records. Clear and consistent operational processes are vital for the reliability and usability of digitized medical records. Divergent views about usability (such as whether patient summary information is better or worse) may reflect familiarity with features of the digitized record.

A multisystem investigation of raltegravir association with intestinal tissue: implications for pre-exposure prophylaxis and eradication.

OBJECTIVES: Recent clinical data have suggested high raltegravir concentrations in gut tissue after oral administration, with implications for treatment and prevention. We have used in silico, in vitro, ex vivo and in vivo models to further investigate the accumulation of raltegravir in gut tissue. METHODS: Affinity of raltegravir for gut tissue was assessed in silico (Poulin-Theil method), in vitro (Caco-2 accumulation) and ex vivo (rat intestine) and compared with the lipophilic drug lopinavir. Finally, raltegravir concentrations in plasma, gut contents, small intestine and large intestine were determined after oral dosing to Wistar rats 1 and 4 h post-dose. Samples were analysed using LC-MS/MS and scintillation counting. RESULTS: Gut tissue accumulation of raltegravir was less than for lopinavir in silico, in vitro and ex vivo (P < 0.05). After oral administration to rats, raltegravir concentrations 4 h post-dose were lower in plasma (0.05 µM) compared with small intestine (0.47 µM, P = 0.06) and large intestine (1.36 µM, P < 0.05). However, raltegravir concentrations in the contents of both small intestine (4.0 µM) and large intestine (40.6 µM) were also high. CONCLUSIONS: In silico, in vitro and ex vivo data suggest low raltegravir accumulation in intestinal tissue. In contrast, in vivo animal data suggest raltegravir concentrates in intestinal tissue even when plasma concentrations are minimal. However, high raltegravir concentrations in gut contents are the likely driving factor behind this observation, rather than blood-to-tissue drug distribution. The methods described can be combined with clinical investigations to provide a complete strategy for selection of drugs with high gut accumulation.

Hyaluronidase impacts exposures of long-acting injectable paliperidone palmitate in rodent models.

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.

Rilpivirine inhibits drug transporters ABCB1, SLC22A1, and SLC22A2 in vitro.

Rilpivirine is a nonnucleoside reverse transcriptase inhibitor approved for treatment of HIV-1 infection in antiretroviral-naive adult patients. Potential interactions with drug transporters have not been fully investigated. Transport by and inhibition of drug transporters by rilpivirine were analyzed to further understand the mechanisms governing rilpivirine exposure and determine the potential for transporter-mediated drug-drug interactions. The ability of rilpivirine to inhibit or be transported by ABCB1 was determined using ABCB1-overexpressing CEMVBL100 cells and Caco-2 cell monolayers. The Xenopus laevis oocyte heterologous protein expression system was used to clarify if rilpivirine was either transported by or inhibited the function of influx transporters SLCO1A2, SLCO1B1, SLCO1B3, SLC22A2, SLC22A6, and SLC22A8. The ability of rilpivirine to inhibit or be transported by SLC22A1 was determined using SLC22A1-expressing KCL22 cells. Rilpivirine showed higher accumulation in SLC22A1-overexpressing KCL22 cells than control cells (27% increase, P = 0.03) and inhibited the functionality of SLC22A1 and SLC22A2 transport with 50% inhibitory concentrations (IC50s) of 28.5 µM and 5.13 µM, respectively. Inhibition of ABCB1-mediated digoxin transport was determined for rilpivirine, which inhibited digoxin transport in the B-to-A direction with an IC50 of 4.48 µM. The maximum rilpivirine concentration in plasma in patients following a standard 25-mg dosing regimen is around 0.43 µM, lower than that necessary to substantially inhibit ABCB1, SLC22A1, or SLC22A2 in vitro. However, these data indicate that SLC22A1 may contribute to variability in rilpivirine exposure and that interactions of rilpivirine with substrates of SLC22A1, SLC22A2, or ABCB1 may be possible.

Ronapreve (REGN-CoV; casirivimab and imdevimab) reduces the viral burden and alters the pulmonary response to the SARS-CoV-2 Delta variant (B.1.617.2) in K18-hACE2 mice using an experimental design reflective of a treatment use case.

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.

Chemoprophylactic Assessment of Combined Intranasal SARS-CoV-2 Polymerase and Exonuclease Inhibition in Syrian Golden Hamsters.

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.

Quantitation of tizoxanide in multiple matrices to support cell culture, animal and human research.

Currently nitazoxanide is being assessed as a candidate therapeutic for SARS-CoV-2. Nitazoxanide is rapidly broken down to its active metabolite tizoxanide upon administration. Unlike many other candidates being investigated, tizoxanide 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 Dulbecco's 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. Herein a 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 15.6 ng/mL to 1000 ng/mL. 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.

Preclinical Evaluation of Long-Acting Emtricitabine Semi-Solid Prodrug Nanoparticle Formulations.

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.

Evaluation of Nafamostat as Chemoprophylaxis for SARS-CoV-2 Infection in Hamsters.

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.

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.

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.

Quantitation of tizoxanide in multiple matrices to support cell culture, animal and human research.

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.

Development of a highly sensitive bioanalytical assay for the quantification of favipiravir.

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.

Optimisation and validation of a sensitive bioanalytical method for niclosamide.

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.