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.

Relating CYP2B6 genotype and efavirenz resistance among post-partum women living with HIV with high viremia in Uganda: a nested cross-sectional study.

BACKGROUND: We investigated the association between CYP2B6 polymorphisms and efavirenz drug resistance among women living with HIV who started on antiretroviral therapy during pregnancy and with high viremia during post-partum. METHODS: This was a cross-sectional study of women with viral loads greater than 1000 copies/ml who were at least 6 weeks postpartum. Sanger sequencing was used to detect resistant mutations, as well as host genotyping, and efavirenz resistance was compared among the metabolizer genotypes. RESULTS: Over the course of one year (July 2017-July 2018), 322 women were screened, with 110 (34.2%) having viral loads of 1000 copies/ml and 62 having whole blood available for genotyping. Fifty-nine of these women had both viral resistance and human host genotypic results. Efavirenz resistance according to metabolizer genotype was; 47% in slow, 34% in extensive and 28% in intermediate metabolizers, but the difference was not statistically significant due to the small sample size. CONCLUSIONS: There was no statistically significant difference in EFV resistance between EFV metabolizer genotypes in women who started antiretroviral therapy during pregnancy and had high viremia in the postpartum period. However, a numerical trend was discovered, which calls for confirmation in a large, well-designed, statistically powered study.

A Holistic Review of the Preclinical Landscape for Long-Acting Anti-infective Drugs Using HIV as a Paradigm.

Lack of predictive preclinical models is a key contributor to the steep attrition rate in drug development. Successful clinical translation may be higher for new chemical entities or existing approved drugs reformulated for long-acting (LA) administration if preclinical studies designed to identify any new uncertainties are predictive of human exposure and response. In this review, we present an overview of standard preclinical assessments deployed for LA formulations and delivery systems, using human immunodeficiency virus LA therapeutics preclinical development as a paradigm. Key progress in the preclinical development of novel LA antiretrovirals formulations and delivery systems are summarized, including bispecific broadly neutralizing monoclonal antibody and small molecule technologies for codelivery of multiple drugs with disparate solubility properties. There are new opportunities to take advantage of recent developments in tissue engineering and 3-dimensional in vitro modeling to advance preclinical modeling of anti-infective activity, developmental and reproductive toxicity assessment, and to apply quantitative modeling and simulation strategies. These developments are likely to drive the progression of more LA anti-infective drugs and multipurpose technologies into clinical development in the coming years.

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.

Genetic influence of ABCG2, UGT1A1 and NR1I2 on dolutegravir plasma pharmacokinetics.

OBJECTIVES: Dolutegravir has replaced efavirenz as first-line treatment in universal HIV guidelines. We sought to ascertain the contributory effect of SNPs in four key genes linked to dolutegravir disposition (UGT1A1, ABCG2, CYP3A and NR1I2) on plasma dolutegravir pharmacokinetics. METHODS: Paired pharmacogenetic/pharmacokinetic data from 93 subjects were analysed for association using multivariate linear regression. RESULTS: Co-occurring UGT1*28 and NR1I2 c.63396C>T homozygosity was associated with a 79% increase in AUC0-24 (P = 0.001; 27% if analysed individually), whilst combined ABCG2 c.421C>A and NR1I2 c.63396C>T variants were associated with a 43% increase in Cmax (P = 0.002) and a 39% increase in AUC0-24 (P = 0.002). When analysed individually, homozygosity for the NR1I2 c.63396C>T variant alleles was associated with a 28% increase in Cmax (P = 0.033) and homozygosity for the ABCG2 c.421C>A variant alleles was associated with a 28% increase in Cmax (P = 0.047). The UGT1A1*28 (rs8175347) poor metabolizer status (*28/*28; *28/*37; *37/*37) was individually associated with a 27% increase in AUC0-24 (P = 0.020). The combination of UGT1A1*28 poor metabolizer and UGT1A1*6 intermediate metabolizer statuses correlated with a 43% increase in AUC0-24 (P = 0.023). CONCLUSIONS: This study showed a pharmacogenetic association between dolutegravir pharmacokinetics and variants in the ABCG2, UGT1A1 and NR1I2 genes, particularly when combined. Further research is warranted to confirm these associations in population-specific studies and to investigate their putative relationship with dolutegravir pharmacodynamics.

Pharmacokinetics of Efavirenz 400 mg Once Daily Coadministered With Isoniazid and Rifampicin in Human Immunodeficiency Virus-Infected Individuals.

Background: The World Health Organization recommends efavirenz 400 mg (EFV400) as first-line antiretroviral therapy, with a disclaimer that no data with anti-tuberculosis (TB) treatment exist. Many people living with human immunodeficiency virus (PLWH) require TB treatment with isoniazid (INH) and rifampicin (RIF), which affect cytochrome P450 and antiretroviral exposure. Methods: PLWH receiving tenofovir disoproxil fumarate (TDF)/emtricitabine (FTC)/EFV 600 mg with a viral load (VL) <50 copies/mL switched to TDF/FTC/EFV400. Genetic polymorphisms and pharmacokinetic (PK) parameters of EFV400 without (PK1) and with INH/RIF following 4 (PK2) and 12 (PK3) weeks of coadministration were evaluated. Results: Twenty-six PLWH were enrolled; 22 completed PK2. All maintained VL <50 copies/mL throughout the study. Geometric mean ratio (GMR) PK2/PK1 of EFV400 maximum plasma concentration (Cmax), area under the curve (AUC), and concentration at 24 hours postdose (C24h) were 0.91 (90% confidence interval [CI], .83-.99), 0.91 (90% CI, .79-1.05), and 0.85 (90% CI, .72-.99), respectively. GMRs (90% CI) of PK3/PK2 and PK3/PK1 Cmax, AUC, and C24h were 0.95 (.86-1.05) and 0.92 (.83-1.01), 0.88 (.75-1.03) and 0.84 (.75-.93), and 0.84 (.72-.99) and 0.75 (.62-.92), respectively. Eleven of 22 participants carried polymorphisms in the CYP2B6 gene associated with slow EFV metabolism. Conclusions: INH/RIF coadministration was associated with limited changes in EFV400 AUC (<25%), and EFV400 concentrations were maintained within ranges of those measured in PLWH in the ENCORE-1 study, irrespective of CYP2B6 genotype. The coadministration of EFV400 with anti-TB treatment can be considered and this is being confirmed in PLWH with TB. Clinical Trials Registration: NCT02832778.

Meta-analysis of the effect of CYP2B6, CYP2A6, UGT2B7 and CAR polymorphisms on efavirenz plasma concentrations.

BACKGROUND: Efavirenz primary metabolism is catalysed by CYP2B6 with minor involvement of CYP2A6. Subsequently, phase I metabolites are conjugated by UGT2B7, and constitutive androstane receptor (CAR) has been shown to transcriptionally regulate many relevant enzymes and transporters. Several polymorphisms occurring in the genes coding for these proteins have been shown to impact efavirenz pharmacokinetics in some but not all studies. OBJECTIVES: A meta-analysis was performed to assess the overall effect of CYP2B6 rs3745274, CYP2A6 (rs28399454, rs8192726 and rs28399433), UGT2B7 (rs28365062 and rs7439366) and NR1I3 (rs2307424 and rs3003596) polymorphisms on mid-dose efavirenz plasma concentrations. METHODS: Following a literature review, pharmacokinetic parameters were compiled and a meta-analysis for these variants was performed using Review Manager and OpenMetaAnalyst. A total of 28 studies were included. RESULTS: Unsurprisingly, the analysis confirmed that individuals homozygous for the T allele for CYP2B6 rs3745274 had significantly higher efavirenz concentrations than those homozygous for the G allele [weighted standard mean difference (WSMD) = 2.98; 95% CI 2.19-3.76; P < 0.00001]. A subgroup analysis confirmed ethnic differences in frequency but with a similar effect size in each ethnic group (P = 0.96). Associations with CYP2A6 and UGT2B7 variants were not statistically significant, but T homozygosity for CAR rs2307424 was associated with significantly lower efavirenz concentrations than in C homozygotes (WSMD = -0.32; 95% CI -0.59 to -0.06; P = 0.02). CONCLUSIONS: This meta-analysis provides the overall effect size for the impact of CYP2B6 rs3745274 and NR1I3 rs2307424 on efavirenz pharmacokinetics. The analysis also indicates that some previous associations were not significant when interrogated across studies.

Effect of patient genetics on etonogestrel pharmacokinetics when combined with efavirenz or nevirapine ART.

BACKGROUND: We previously demonstrated that etonogestrel concentrations were 82% lower in women using etonogestrel contraceptive implants plus efavirenz-based ART compared with women not receiving ART. OBJECTIVES: To investigate the genetic contribution to this previously observed drug-drug interaction through studying SNPs in genes known to be involved in efavirenz, nevirapine or etonogestrel metabolism in the same group of women. PATIENTS AND METHODS: Here, we present a secondary analysis evaluating SNPs involved in efavirenz, nevirapine and etonogestrel metabolism and associated etonogestrel pharmacokinetics among 57 women, 19 not receiving ART (control group), 19 receiving efavirenz- (600 mg daily) based ART and 19 receiving nevirapine- (200 mg twice daily) based ART. Associations between patient genotype and etonogestrel pharmacokinetic parameters were determined through univariate and multivariate linear regression. This study was registered at clinicaltrials.gov (NCT02082652). RESULTS: Within the control group, CYP2B6 983 T>C was associated with 27% higher etonogestrel Cmax and 28% higher AUC0-24weeks. In the efavirenz group CYP2B6 516 G>T was associated with 43% lower etonogestrel Cmin and 34% lower AUC0-24weeks. For participants receiving nevirapine, NR1I2 63396 C>T was associated with 39% lower etonogestrel Cmin and 37% lower AUC0-24weeks. CONCLUSIONS: This study demonstrates the influence of pharmacogenetics on the extent of drug-drug interactions between etonogestrel and efavirenz- or nevirapine-based ART. Efavirenz plus the etonogestrel contraceptive implant results in a detrimental drug-drug interaction irrespective of patient genetics, which is worsened in women possessing variant alleles for these CYP2B6 SNPs.

Rifampicin effect on intracellular and plasma pharmacokinetics of tenofovir alafenamide.

OBJECTIVES: Tenofovir alafenamide produces lower plasma tenofovir and higher intracellular tenofovir diphosphate (DP) concentrations than tenofovir disoproxil fumarate but it is likely a victim of interactions with rifampicin. We aimed to investigate the pharmacokinetics of tenofovir alafenamide/emtricitabine with rifampicin. PATIENTS AND METHODS: Healthy volunteers received tenofovir alafenamide/emtricitabine at 25/200 mg once daily, followed by tenofovir alafenamide/emtricitabine + rifampicin daily followed by tenofovir disoproxil fumarate. Plasma tenofovir alafenamide, tenofovir, emtricitabine and intracellular tenofovir-DP and emtricitabine triphosphate pharmacokinetics and genetic polymorphisms were assessed. RESULTS: Tenofovir alafenamide exposure decreased when tenofovir alafenamide/emtricitabine + rifampicin was used compared with tenofovir alafenamide/emtricitabine [geometric mean ratio (GMR) (90% CI): 0.45 (0.33-0.60)]. Plasma tenofovir and intracellular tenofovir-DP concentrations decreased with rifampicin [GMR (90% CI): 0.46 (0.40-0.52) and 0.64 (0.54-0.75), respectively]. GMR (90% CI) of intracellular tenofovir-DP AUC0-24 for tenofovir alafenamide/emtricitabine + rifampicin versus tenofovir disoproxil fumarate was 4.21 (2.98-5.95). Rifampicin did not affect emtricitabine pharmacokinetics. CYP3A4*22 rs35599367 was associated with higher plasma tenofovir alafenamide AUC0-24 at day 56. CONCLUSIONS: Following tenofovir alafenamide/emtricitabine administration with rifampicin, intracellular tenofovir-DP concentrations were still 4.21-fold higher than those achieved by tenofovir disoproxil fumarate, supporting further study during HIV/TB co-infection.

Pharmacokinetics, Pharmacodynamics, and Pharmacogenetics of Efavirenz 400 mg Once Daily During Pregnancy and Post-Partum.

Background: A clinical trial showed that efavirenz 400 mg once daily (EFV400) is as effective as the standard adult dose. World Health Organization recommends EFV400 as an alternative first-line agent, but data are lacking in the third trimester of pregnancy (TT). We investigated the pharmacokinetics, efficacy, and CYP2B6 pharmacogenetics in HIV-infected women (WLWH) on EFV400 during TT and post-partum (PP). Methods: An open-label 2-center study (United Kingdom, Uganda) was conducted in WLWH receiving antiretroviral regimens containing efavirenz 600 mg, who had their efavirenz dose reduced to EFV400. Weekly therapeutic drug monitoring (TDM), steady-state pharmacokinetic profiles (TT and PP), safety, virological efficacy, and CYP2B6 polymorphisms at positions 516 (C > T) and 938 (T > C) were evaluated. Results: Twenty-five WLWH of African origin were enrolled. All had viral loads <50 copies/mL at baseline, which were maintained throughout the study. No infant was HIV infected. No WLWH were withdrawn due to low EFV400 TDM results. Geometric mean ratios (TT/PP; 90% confidence interval) for EFV400 maximum observed plasma concentration, area under the curve, and plasma concentration measured 24 hours after the observed dose were 0.97 (.85-1.10), 0.87 (.76-.99), and 0.77 (.65-.91), respectively. Five of 25 WLWH were slow metabolizers. Conclusions: Although EFV400 pharmacokinetic parameters were slightly lower for TT compared with PP values, efavirenz concentrations exceeded cutoff levels established by the study and those measured in antiretroviral-naive patients receiving EFV400 in ENCORE1. All subjects maintained a viral load <50 copies/mL, suggesting that EFV400 can be used in pregnant WLWH.

Pregnancy affects nevirapine pharmacokinetics: evidence from a CYP2B6 genotype-guided observational study.

OBJECTIVES: Previous studies on nevirapine pharmacokinetics during pregnancy reported contradictory findings. METHODS: The magnitude of pregnancy-induced changes in nevirapine pharmacokinetics was investigated in a genotype-guided study preceded by a pharmacogenetic association study of six genes involved in its disposition. RESULTS: CYP2B6 516 G>T and 983 T>C were associated independently with plasma nevirapine concentrations in pregnant (n=110) and postpartum (n=122) women and were used for stratification. NR1I3 540C>T and P450 oxidoreductase 1508C>T were associated with lower and higher plasma concentrations in pregnant and postpartum women, respectively. In the intensive pharmacokinetic phase, apparent clearance (CL/F) was higher in pregnant (n=31) than postpartum (n=28) women (P=0.022) and AUC0-12, Cmax and Cmin were significantly lower. When stratified on the basis of composite CYP2B6 516 G>T and 983 T>C genotypes, CL/F was similar between pregnant (n=6) and postpartum (n=9) women with no variant alleles, but Cmin was below target (3400 ng/ml) in most patients in both groups. In women with one variant allele, clearance was 40.6% higher (P=0.0009) and Cmin was below target in 58% (11/19) of pregnant and 0% (0/10) of postpartum women. Similarly, clearance was 51.7% higher (P=0.008) in pregnant compared with postpartum women with two variant alleles. Cmin was below target in 50% (3/6) of pregnant and 0% (0/10) of postpartum women. CONCLUSION: Nevirapine exposure is significantly reduced during pregnancy. The pharmacodynamic consequences in patients at risk of suboptimal exposure and potential dose optimization strategies warrant further investigation.

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.

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.

Polymer-prodrug conjugates as candidates for degradable, long-acting implants, releasing the water-soluble nucleoside reverse-transcriptase inhibitor emtricitabine.

Circulating, soluble polymer-drug conjugates have been utilised for many years to aid the delivery of sensitive, poorly-soluble or cytotoxic drugs, prolong circulation times or minimise side effects. Long-acting therapeutics are increasing in their healthcare importance, with intramuscular and subcutaneous administration of liquid formulations being most common. Degradable implants also offer opportunities and the use of polymer-prodrug conjugates as implant materials has not been widely reported in this context. Here, the potential for polymer-prodrug conjugates of the water soluble nucleoside reverse transciption inhibitor emtricitabine (FTC) is studied. A novel diol monomer scaffold, allowing variation of prodrug substitution, has been used to form polyesters and polycarbonates by step-growth polymerisation. Materials have been screened for physical properties that enable implant formation, studied for drug release to provide mechanistic insights, and tunable prolonged release of FTC has been demonstrated over a period of at least two weeks under relevant physiological conditions.

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.

Multivalent bicyclic peptides are an effective antiviral modality that can potently inhibit SARS-CoV-2.

COVID-19 has stimulated the rapid development of new antibody and small molecule therapeutics to inhibit SARS-CoV-2 infection. Here we describe a third antiviral modality that combines the drug-like advantages of both. Bicycles are entropically constrained peptides stabilized by a central chemical scaffold into a bi-cyclic structure. Rapid screening of diverse bacteriophage libraries against SARS-CoV-2 Spike yielded unique Bicycle binders across the entire protein. Exploiting Bicycles' inherent chemical combinability, we converted early micromolar hits into nanomolar viral inhibitors through simple multimerization. We also show how combining Bicycles against different epitopes into a single biparatopic agent allows Spike from diverse variants of concern (VoC) to be targeted (Alpha, Beta, Delta and Omicron). Finally, we demonstrate in both male hACE2-transgenic mice and Syrian golden hamsters that both multimerized and biparatopic Bicycles reduce viraemia and prevent host inflammation. These results introduce Bicycles as a potential antiviral modality to tackle new and rapidly evolving viruses.