[go: up one dir, main page]

WO2021211738A1 - Methods and compositions for antiviral treatment - Google Patents

Methods and compositions for antiviral treatment Download PDF

Info

Publication number
WO2021211738A1
WO2021211738A1 PCT/US2021/027324 US2021027324W WO2021211738A1 WO 2021211738 A1 WO2021211738 A1 WO 2021211738A1 US 2021027324 W US2021027324 W US 2021027324W WO 2021211738 A1 WO2021211738 A1 WO 2021211738A1
Authority
WO
WIPO (PCT)
Prior art keywords
cov
clofazimine
sars
cells
infective
Prior art date
Application number
PCT/US2021/027324
Other languages
French (fr)
Inventor
Sumit Chanda
Arnab Chatterjee
Original Assignee
Sanford Burnham Prebys Medical Discovery Institute
The Scripps Research Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sanford Burnham Prebys Medical Discovery Institute, The Scripps Research Institute filed Critical Sanford Burnham Prebys Medical Discovery Institute
Publication of WO2021211738A1 publication Critical patent/WO2021211738A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A61K9/0075—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a dry powder inhaler [DPI], e.g. comprising micronized drug mixed with lactose carrier particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/07—Retinol compounds, e.g. vitamin A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/498—Pyrazines or piperazines ortho- and peri-condensed with carbocyclic ring systems, e.g. quinoxaline, phenazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
    • A61K31/685—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols one of the hydroxy compounds having nitrogen atoms, e.g. phosphatidylserine, lecithin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/006—Oral mucosa, e.g. mucoadhesive forms, sublingual droplets; Buccal patches or films; Buccal sprays
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A61K9/0078—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a nebulizer such as a jet nebulizer, ultrasonic nebulizer, e.g. in the form of aqueous drug solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/12—Aerosols; Foams
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4841—Filling excipients; Inactive ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals

Definitions

  • Coronavirus infections result in substantial morbidity and death worldwide. While vaccination can protect against certain viral infections, vaccines are not always fully effective. There are many pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2), for which there is no effective vaccine. Therefore, prevention of infection and transmission of disease are of paramount importance; especially in certain young, elderly, or immunocompromised populations.
  • SARS- CoV-2 severe acute respiratory syndrome coronavirus 2
  • anti-infective compositions comprising a pharmaceutically acceptable carrier or excipient and an effective amount of a compound selected from the group consisting of amopyroquine, nelfmavir mesylate hydrate, GR 127935 hydrochloride hydrate, LG-1550, tretinoin, tamibarotene, acitretin, tazarotene, RBAD, AL 3152, ZK-93426, zaleplon GR, pagoclone, MDL 28170, 8-(3-Chlorostyryl)caffeine, Apilimod (APY0201), Clofazimine, Z LVG CHN2, AQ-13, Haggingchin A, astemizole, , SL-11128, ELOPIPRAZOLE, SDZ-62-434, analog of MLN-3897, SB-616234-A, YH-1238, VBY-825, ONO 5334, and
  • the pathogen is a coronavirus.
  • the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • disclosed here are anti -infective compositions comprising a pharmaceutically acceptable carrier or excipient and an effective amount of a compound selected from the group consisting of amopyroquine, nelfmavir mesylate hydrate, GR 127935 hydrochloride hydrate, LG-1550, tretinoin, tamibarotene, acitretin, tazarotene, RBAD, AL 3152, ZK-93426, zaleplon GR, pagoclone, MDL 28170, 8-(3- Chlorostyryl)caffeine, Apilimod (APY0201), Clofazimine, Z LVG CHN2, AQ-13, Haggingchin A, astemizole, , SL-11128, ELOPIPRAZ
  • the anti-viral agent comprises remdesivir (GS-5734). In some embodiments, the anti-viral gent comprises favipiravir (T-705) In some embodiments, disclosed herein are anti -infective compositions comprising a pharmaceutically acceptable carrier or excipient and an effective amount of a compound selected from the group consisting of nelfmavir mesylate hydrate, MDL28170 and GR 127935 hydrochloride hydrate, wherein the anti-infective composition reduces the risk of absorption, infectivity, or transmission of a pathogen.
  • anti -infective compositions comprising a pharmaceutically acceptable carrier or excipient and an effective amount of clofazimine, wherein the anti-infective compositions reduce the risk of absorption, infectivity, or transmission of a pathogen.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • a therapeutically effective amount of an anti-infective composition comprising a pharmaceutically acceptable carrier or excipient and an effective amount of a compound selected from the group consisting of amopyroquine, nelfmavir mesylate hydrate, GR 127935 hydrochloride hydrate, LG-1550, tretinoin, tamibarotene, acitretin, tazarotene, RBAD, AL 3152, ZK-93426, zaleplon GR, pagoclone, MDL 28170, 8-(3-Chlorostyryl)caffeine, Apilimod (APY0201), Clofazimine, Z LVG CHN2, AQ-13, Haggingchin A, astemizole, , SL-11128, ELOPIPRAZOLE, SDZ
  • administering to the patient comprises administration via a route selected from the group consisting of inhalation, oral, parenteral, intranasal, buccal, topical or transdermal administration routes. In some embodiments, the route is inhalation.
  • anti-infective compositions comprising an effective amount of a compound listed in Table 1 and a pharmaceutically acceptable carrier or excipient.
  • the anti-infective composition further comprises an additional anti-infective agent.
  • the additional anti-infective active agent selected from the group consisting of entry-inhibiting drugs, uncoating inhibiting drugs, reverse transcriptase inhibiting drugs, antisense drugs, ribozyme drugs, protease inhibitors, assembly inhibiting drugs, and release inhibiting drugs.
  • the composition treats a respiratory syncytial virus (RSV), metapneumovirus (MPV), rhinovirus, influenza virus, parainfluenza virus, coronavirus, norovirus, rotavirus, hepatitis A virus, adenovirus, astrovirus, S. aureus , methicillin-resistant S. aureus (MRSA), vancomycin-resistant enterococci (VRE), Enterococcus spp ., Enter obacter spp ., C. difficile , Campylobacter, E. faecali, E.faecium , or Salmonella.
  • RSV respiratory syncytial virus
  • MPV metapneumovirus
  • rhinovirus influenza virus
  • parainfluenza virus coronavirus
  • norovirus norovirus
  • rotavirus hepatitis A virus
  • adenovirus astrovirus
  • S. aureus methicillin-resistant S. aureus
  • VRE vancomycin
  • anti-infective compositions comprising an effective amount of a compound listed in Table 1 and a pharmaceutically acceptable carrier or excipient, wherein the anti -infective composition reduces the risk of absorption, infectivity, or transmission of a pathogen.
  • the composition treats at least one strain of a coronavirus.
  • the composition treats severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
  • inhalation devices comprising an effective amount of a compound selected from the group consisting of amopyroquine, nelfmavir mesylate hydrate, GR 127935 hydrochloride hydrate, LG-1550, tretinoin, tamibarotene, acitretin, tazarotene, RBAD, AL 3152, ZK-93426, zaleplon GR, pagoclone, MDL 28170, 8-(3-Chlorostyryl)caffeine, Apilimod (APY0201), Clofazimine, Z LVG CHN2, AQ-13, Haggingchin A, astemizole, , SL-11128, ELOPIPRAZOLE, SDZ-62-434, analog of MLN-3897, SB-616234-A, YH-1238, VBY-825, ONO 5334, and AMG-2674; and a pharmaceutically acceptable propellant.
  • FIG. 1 Panels A-C depict an exemplary embodiment of the screening schematic disclosed herein.
  • Panel A depicts the distribution of the approximately 13,000 compounds in the ReFRAME collection across different stages of clinical development and a schematic of the screening process.
  • Panel B depicts results from the LOPAC®1280 library primary screen.
  • Panel C depicts results from the ReFRAME collection primary screen.
  • FIG. 2 Panels A-B depict Gene Set Enrichment Analysis (GSEA) of primary screening data according to the average Z’ factor.
  • GSEA enrichment plots of six target clusters that are enriched are represented, including retinoic acid receptor agonists, benzodiazepine receptor inhibitors, aldose reductase agonists, potassium channel agonists, cholesterol inhibitors, and antimalarials (P -value ⁇ 0.05, FDR q-value ⁇ 0.25) (panel A) and expression of ACE2, TMPRSS2, and select target genes (panel B). Expression was analyzed using single-cell RNA profiling data from human airway samples of healthy donors. Clustered heat maps in panel B show the fraction of gene-expressing cells separated by sampling location (left-hand side) or cell type (right-hand side).
  • FIG. 3 Panels A-C results from the methods and compositions disclosed herein.
  • Panel A depicts a heat map of the indicated 17 compounds in dose-response, on a scale from 0 to 1, on the average of three independent experiments. Compounds are associated in clusters, based on their classification category. Concentrations are rounded. The symbol # indicates compounds evaluated at a concentration of 0.85 mM instead of 1 mM.
  • Panel C depicts representative immunofluorescence images corresponding to one of the three dose-responses in Panel B. For each condition, the corresponding entire well is shown (4x objective)
  • FIG. 4 depicts Z-scores for ReFRAME collection primary screen.
  • the left graph represents the Z-score of ATP levels after normalization to the median of each plate for all positive (APY0201) and negative (DMSO) controls as well as for non-infected cells, across all the screening plates.
  • the correlation plot in the middle panel indicates the Z-score of each compound in the two replicates.
  • the distribution of each compound according to the average of the Z-score of each replicate (right panel) is also represented.
  • Each dot indicates the Z-score of each drug in each replicate of the screen (black dots). Values corresponding to DMSO (orange dots), APY0201 (cyan dots) and non-infected cells (purple dots) are also represented.
  • FIG. 5 depicts gene set enrichment analysis (GSEA) of primary screening data according to the average Z' factor.
  • GSEA enrichment plots of additional seven target clusters that are enriched were represented including estrogen receptor antagonist, GABA-A receptor modulator, ANGIOTENSIN II 1 antagonist, beta adrenoceptor antagonist, 5-hydroxytryptamine 3 receptor antagonist, serine protease inhibitor, and phosphodiesterase inhibitor (P -value ⁇ 0.05, FDR q-value ⁇ 0.25).
  • FIG. 6 depicts a bar plot of enriched terms across the enriched genes targeted by the compounds.
  • the x-axis corresponds to -logl0(p value) while the y-axis indicates the enriched terms.
  • the analysis was performed using Metascape.
  • FIG. 7 depicts a heatmap of treatment of Vero E6 cells.
  • 24 h post-infection cells were fixed and an immunofluorescence was performed, followed by imaging. For each condition, the total amount of cells stained with DAPI was calculated. Data are normalized to the average of DMSO-treated wells.
  • the heatmap represents the normalized cell number of the indicated 17 compounds in dose-response, on a scale from 0 to 1, on the average of three independent experiments. Compounds are associated in clusters, based on their classification category. Concentrations are rounded. The # symbol indicates compounds were evaluated at a concentration of 0.85 mM instead of 1 mM.
  • FIG. 8 depicts dose-response curves obtained with the compounds identified herein.
  • 24 h post-infection cells were fixed and an immunofluorescence was performed.
  • the percentage of infection was calculated as the ratio between the number of infected cells stained for CoV NP and the total amount of cells stained with DAPI.
  • FIG. 9 Panels A-E depicts that clofazimine inhibited a broad spectrum of human- pathogenic coronavirus (CoV) replication in human cellular models.
  • CoV coronavirus
  • FIG. 10 Panels A-F depicts that clofazimine interferes with multiple steps of the virus life cycle.
  • FIG. 11 Panels A-D depicts transcriptional analysis of clofazimine treatment.
  • FIG. 12 Panels A-I depicts that prophylactic and therapeutic treatment with clofazimine reduces SARS-CoV-2 disease.
  • FIG. 13 Panels A-I depicts that clofazimine exhibits antiviral synergy with remdesivir in vitro and in vivo.
  • FIG. 14 depicts cytotoxicity measurements of clofazimine in matching cells for antiviral evaluation.
  • FIG. 15 Panels A-D depicts exploration of possible effects of clofazimine on virus entry and replication.
  • FIG.16 Panels A-C depicts transcriptional analysis of clofazimine treatment on Caco-
  • FIG. 17 Panels A-E depicts transcriptional analysis of hamster lung tissues with clofazimine administration.
  • FIG. 18 depicts the histological score indicating lung pathological severity in each group.
  • FIG. 19 depicts clofazimine exhibits antiviral synergy with remdesivir for every combination of drug doses in vitro.
  • FIG. 20 depicts that clofazimine exhibits antiviral synergy with remdesivir in hamster lungs.
  • FIG. 21 depicts that clofazimine inhibited SARS-CoV-2 infection in human stem cell derived pneumocyte-like cells.
  • FIG. 22 depicts that clofazimine exhibits antiviral activity in the adenovirus hACE2 mouse model of SARS-CoV-2 infection.
  • compositions effective at treating coronavirus are effective at treating SARS coronavirus 2 (SARS-CoV-2), a novel coronavirus identified in December 2019 as the causative agent of a severe pneumonia-like coronavirus disease (COVID-19) outbreak in Wuhan in the Hubei province of China (X. Yang et ah, Clinical course and outcomes of critically ill patients with SARS-CoV-2 pneumonia in Wuhan, China: a single-centered, retrospective, observational study. Lancet Respir Med, (2020)).
  • SARS coronavirus 2 SARS coronavirus 2
  • COVID-19 severe pneumonia-like coronavirus disease
  • the anti -infective compositions disclosed herein comprise a pharmaceutically acceptable carrier or excipient and a compound identified via a high-throughput cell-based screen for inhibitors of SARS-CoV-2 replication, profiling a library of known drugs encompassing approximately 13,000 clinical-stage or FDA-approved small molecules.
  • the anti -effective compositions disclosed herein comprise a pharmaceutically acceptable carrier or excipient and a compound selected from the group consisting of PIKfyve kinase inhibitor Apilimod, cysteine protease inhibitors (including MDL- 28170, Z LVG CHN2, VBY-825, and ONO 5334), and compound SL-11128.
  • SARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA betacoronavirus, related to the viruses that caused severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) in 2002-2004 and 2012, 2013, respectively.
  • SARS severe acute respiratory syndrome
  • MERS Middle East respiratory syndrome
  • the World Health Organization declared the rapidly spreading disease a pandemic on March 11th, 2020, and the disease has resulted in more than 1.98 million confirmed cases and more than 126,500 deaths have been reported worldwide in 213 countries.
  • the WHO estimated the global case fatality rate (CFR) at 3.4% of those infected, though the number of actual infections is likely much higher than the number of reported cases.
  • Typical COVID-19 symptoms include fever, cough, headache, anorexia, myalgia, and, in the most severe cases, viral-induce pneumonia accompanied by prolonged and systemic cytokine release.
  • the levels of IL-6 have been reported to highly correlate with respiratory failure, and inhibitors are currently being pursued in clinical studies for the amelioration of virus-induced inflammatory responses.
  • GS-5734 adenosine analogue
  • Gilead Sciences to treat Ebola virus
  • Remdesivir an adenosine analogue is a viral RNA polymerase inhibitor that causes premature termination of transcription when incorporated into nascent viral RNA
  • T. K. Warren et al. Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys. Nature 531, 381-385 (2016)).
  • the drug has demonstrated in vitro and in vivo activity in animal models against both MERS and SARS, as well as potent antiviral activity in Vero E6 against a clinical isolate of SARS-CoV-2 (M. Wang et al., Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019- nCoV) in vitro Cell Res 30, 269-271 (2020)). Pending results of several randomized (n > 308) clinical trials are expected to provide definitive insight into the efficacy of remdesivir as a therapeutic solution for the treatment of COVID-19.
  • the ReFRAME library as a comprehensive drug repurposing library and its application to the treatment of cryptosporidiosis. Proc Natl Acad Sci EISA 115, 10750-10755 (2016); Y. J. Kim et al., The ReFRAME library as a comprehensive drug repurposing library to identify mammarenavirus inhibitors. Antiviral Res 169, 104558 (2019)).
  • the ReFRAME library is unique in that nearly 50% of the library was derived from custom synthesis, as commercially available sources of these clinical molecules were not available (Corsello, S. M. et al. The Drug Repurposing Hub: a next-generation drug library and information resource.
  • anti-infective compositions comprising a compound selected from the group consisting of aldose reductase inhibitors, retinoic acid receptor antagonists, benzodiazepine receptor agonists, regulators of cholesterol homeostasis, and antimalarial compounds.
  • anti -infective compositions comprising a compound identified from the ReFRAME library, including PIKfyve kinase inhibitor Apilimod, cysteine protease inhibitors (including MDL-28170, Z LVG CHN2, VBY-825, and ONO 5334), calcium channel blockers (including AMG-2674 and Haggingchin A), proton pump inhibitors (including YH-1238), G-protein receptor antagonists (including MLN-3897 and SDZ-62-434) and anti-viral agents that inhibit viral replication at concentrations were identified, and are expected to be achievable in patients.
  • PIKfyve kinase inhibitor Apilimod cysteine protease inhibitors
  • cysteine protease inhibitors including MDL-28170, Z LVG CHN2, VBY-825, and ONO 5334
  • calcium channel blockers including AMG-2674 and Haggingchin A
  • proton pump inhibitors including YH-1238
  • G-protein receptor antagonists including MLN
  • Combination therapy using two or more clinically approved or evaluated molecules that act synergistically, may offer a powerful approach for treating SARS- CoV-2 infection, similar to the method of treatment for HIV-1 infection.
  • Disclosed herein are methods and compositions comprising the results of a comprehensive repositioning analysis of nearly 13,000 known drugs from the ReFRAME collection. Some of the top candidate compounds identified include those described herein, used as single agents, or in combination therapy with other compounds identified herein or other antiviral compounds.
  • Vero E6 cells derived from African Green Monkeys
  • cell-based assays can be biased towards capturing inhibitors of viral entry, based on these parameters, this assay can elucidate inhibitors of each step of the viral life cycle.
  • one potential limitation of Vero cells is that, due to species differences, pro-drugs that require the host cell machinery for processing into their active form, such as some nucleoside inhibitors, may not harbor the same potency as in human cells. Consistently, it is found that remdesivir inhibits SARS-CoV-2 replication 30-fold more potently in human cells in comparison to Vero E6 cells (data not shown).
  • Clofazimine is a lipophilic riminophenazine antibiotic, with described antimycobacterial and anti-inflammatory activity used for the treatment of leprosy. Main adverse effects include changes in skin pigmentation, nausea and vomiting. Its antibacterial activity is described to be related to its ability to bind to the bacterial DNA. Interestingly, this compound was also identified as a potent antiparasitic drug active against Cryptosporidium, during a repurposing screen of the ReFRAME library (Love, M. S. et al.
  • a high-throughput phenotypic screen identifies clofazimine as a potential treatment for cryptosporidiosis.
  • Acitretin is an approved orally bioavailable retinoid used for the treatment of psoriasis.
  • retinoic acid agonists were highly enriched in the GSEA analysis (as shown in Fig.
  • Antimalarial drugs have been reported to effectively block several viral infections (D’ Allesandro S et al., The Use of Antimalarial Drugs against Viral Infection, Microorganisms 8(1): 85 (2020)), including SARS-CoV-2 (Wang M et al., Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro, Cell Res 30(3):269-271 (2020)).
  • SARS-CoV-2 Wang M et al., Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro, Cell Res 30(3):269-271 (2020)
  • Confirmation of compounds with membership enriched target classes underscore the importance of these molecular circuits in the regulation of SARS-CoV-2 replication and suggest that additional molecules that target over represented mechanisms should be evaluated for antiviral activities.
  • Z-LVG-CHN2 might target SARS-CoV-23CL pro to restrict its replication (Zhang, L. et al. Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved a- ketoamide inhibitors. Science, eabb3405, doi: 10.1126/science. abb3405 (2020)). Structure based docking of 3CL pro against Z-LVG-CHN2 can also be investigated. Moreover, Z-LVG-CHN2 potentially inactivates the host cysteine proteases that are required for SARS-CoV-2 infection to block viral infection.
  • MDL 28170 Another preclinical molecule that exhibits potent antiviral activity, MDL 28170, is a potent cell permeable calpain I and II inhibitor. Interestingly, MDL 28170 was previously found to impair infection by SARS-COV-1 and EBOV. Additionally, astemizole a registered anti -histamine HI receptor antagonist that also reported to have anti-malarial properties, inhibited replication at an EC50 concentration of 900 nM. However, due to fatal arrhythmias when given in high doses or in combination with certain other common drugs, astemizole has been withdrawn in many countries. Therefore, thorough safety studies are required to determine if there exists a sufficient therapeutic index for the treatment of acute SARS-CoV-2 infection.
  • MLN-3897 (AVE-9897) was determined to inhibit SARS-CoV-2 replication an estimated EC50 concentrations of 170 nm (as shown in Fig. 2), and the Cmax of the compound has been reported at 9.0 nM (10 mg QD). Therefore, additional in vivo studies will be required to determine if sufficient systemic concentrations can be reached to promote antiviral activities.
  • This compound is an orally active chemokine CCR1 antagonist and was evaluated in phase-2 clinical studies for the treatment of rheumatoid arthritis (RA) and multiple sclerosis (MS).
  • MLN3897 at a dose of 10 mg one daily was well tolerated. The mechanism by which CCR1 antagonism inhibits SARS-CoV-2 infection requires further investigation.
  • Raf/MEK/ERK signal pathways are employed by SARS-CoV-1 to support its replication via multiple well -documented mechanisms, and thus this signaling axis may also represent a critical therapeutic target for host-directed SARS-CoV-2 antivirals.
  • Human cysteinyl cathepsins including cathepsin B, cathepsin L, and cathepsin K, are required for the proteolytic processing of virally encoded proteins during infection.
  • SARS-CoV-2 requires endosomal acidification and acid-dependent endosomal proteases such as cathepsins for infection.
  • cathepsins For infection, blocking the activity of these proteases in the endosomal/lysosomal compartment can efficiently inhibit virus entry and uncoating at an early stage of viral replication.
  • Inhibition of cathepsin L activity has been previously shown to efficiently suppress SARS-CoV-1 infection (G. Simmons et ah, Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry. Proceedings of the National Academy of Sciences of the United States of America 102, 11876- 11881 (2005)).
  • ONO 5334 (a cathepsin K inhibitor) and VBY-825 (a reversible cathepsin protease inhibitor) were identified in the screen to inhibit SARS-CoV-2 infection in a dose-dependent manner, however it is not currently understood if the observed antiviral activities are due to inhibition of proteolysis of viral or host cellular proteins.
  • ONO 5334 was in phase II clinical trials for the treatment of osteoporosis as a potent inhibitor of Cathepsin K with Ki value of 0.1 nM.
  • ONO-5334 on bone mineral density and biochemical markers of bone turnover in postmenopausal osteoporosis: 2-year results from the OCEAN study. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 29, 458-466, doi: 10.1002/jbmr.2047 (2014).
  • ONO 5334 harbored an antiviral EC50 of -500 nM, which is in range of a previously reported 85% activity observed at 100 nM in an osteoclast- mediated bone resorption assay.
  • the Cmax of this compound is 1.6 uM (300 mg QD), and treatment with ONO-5334 was well tolerated up to daily doses of 300 mg and for up to 12 months without any clinically relevant safety concerns.
  • VBY-825 which is in preclinical development, is another cathepsin inhibitor harboring potential antiviral activities against SARS- CoV-2 with IC50 of -300 nM, and it shows high potency against cathepsins B, L, S and V in vitro.
  • VBY-825 is another cathepsin inhibitor discovered in the screening disclosed herein. VBY-825 shows high potency against cathepsins B, L, S and V in both mouse and human cell lines.
  • apilimod a specific PIKfyve kinase inhibitor, was found to inhibit SARS-CoV-2 replication at an EC50 concentrations of 23 nM (as shown in Fig. 3). More importantly, apilimod is found to be well tolerated in humans showing a desirable safety profile at doses of ⁇ 125 mg BID, and the Cmax of this compound is 0.265 +/- 0.183 mM (70 mg QD). These data indicate that therapeutic dosing of apilimod in patients can achieve concentrations that are likely to promote antiviral activity.
  • Apilimod was evaluated in phase II clinical trials for the treatment of active Crohn's disease and rheumatoid arthritis (RA), and an additional phase II trials for the oral treatment of common variable immunodeficiency (CVID) but did not show efficacy for either indication.
  • RA Crohn's disease and rheumatoid arthritis
  • CVID common variable immunodeficiency
  • 2019, orphan drug designation was granted to apilimod in the U.S. for the treatment of follicular lymphoma.
  • apilimod efficiently inhibit Ebola virus (EBOV), Lassa virus (LASV), and Marburg virus (MARV) virus infection in human cell lines, underscoring its potential broad-spectrum antiviral activity.
  • EBOV Ebola virus
  • LASV Lassa virus
  • MARV Marburg virus
  • PIKfyve predominately resides in early endosomes and plays an essential role in maintenance of endomembrane homeostasis, apilimod likely blocks viral low pH-dependent entry through inhibition of the lipid kinase activity of PIKfyve.
  • Aldose reductase is a monomeric NADPH-dependent cytosolic enzyme, involved in various physiological processes via regulation of polyol pathway of glucose metabolism, oxidative stress signaling, as well as lipid aldehyde mediated cell signaling (K. V. Ramana, ALDOSE REDUCTASE: New Insights for an Old Enzyme. Biomol Concepts 2, 103-114 (2011)). Inhibition of aldose reductase could prevent inflammatory complications as well as oxidative stress-induced cell death. More importantly, in hepatitis C virus (HCV)-infected patients, aldose reductase activity is significantly enhanced, indicating that HCV potentially manipulates the host metabolism to promote its infection and transmission (N. Semmo, T.
  • HCV hepatitis C virus
  • the endosomal system functions as an intracellular sorting network for homeostatic regulation. It is also hijacked by many viruses, including SARS-CoV-2, to enter target cells. Thus, manipulation of the cellular endosomal machinery with small molecules can slow down virus replication and allow the immune system to develop defenses.
  • small molecules selectively regulating endosomal recycling or trafficking such as chloroquine derivatives and PIKfyve inhibitors were significantly enriched in primary screening (Qiu, Z. et al. Endosomal proteolysis by cathepsins is necessary for murine coronavirus mouse hepatitis virus type 2 spike-mediated entry.
  • PIKfyve a lipid and protein kinase that predominately resides in the early endosomes, plays an essential role in maintenance of endomembrane homeostasis.
  • Apilimod a 1,3,5-triazine derivative was identified as an antagonist of PIKfyve by specifically inhibiting its lipid kinase activity. It has been shown that apilimod efficiently inhibits Ebola vims (EBOV), Lassa vims (LASV), and Marburg vims (MARV) infection in primary human macrophage cells with an IC50 of 10 nM.
  • anti-infective compositions comprising a pharmaceutically acceptable carrier or excipient and an effective amount of a PIKfyve inhibitor. In some embodiments, disclosed herein are anti-infective compositions comprising a pharmaceutically acceptable carrier or excipient and an effective amount of apilimod. In some embodiments, disclosed herein are anti -infective compositions comprising a pharmaceutically acceptable carrier or excipient and an effective amount of YM201636.
  • MDL 28170 is a potent cell permeable calpain I and II inhibitor that inhibits calpain with Ki values of lOnM and cathepsin B with Ki values of 25 nM.
  • Treatment with MDL 28170 resulted in severely impaired infection of SARS-CoV-1 and EBOV in Vero E6 cells (Schneider, M. et al. Severe acute respiratory syndrome coronavims replication is severely impaired by MG132 due to proteasome-independent inhibition of M-calpain. J Virol 86, 10112-10122, doi: 10.1128/jvi.01001-12 (2012); Hoffmann, M. et al.
  • MDL 28170 is an anti-SARS-CoV-2 dmg suggests that calpain is involved in the SARS-CoV-2 life cycle.
  • multiple molecules that regulate the ion channel activity such as YH-1238, H rotatingchin A, and AMG-2674 displayed potential antiviral activity against SARS-CoV-2 in a dose-response manner.
  • H frustratingchin A is a calcium channel blocker which could reduce intracellular Ca 2+ level upon treatment. Impairing calcium channel with treatment of H frustratingchin A inhibited SARS-CoV-2 infection, indicating the beneficial role of increased intracellular Ca 2+ level for viral infection. In addition, since cellular or viral factors function in a Ca 2+ -dependent manner, intracellular Ca 2+ might regulate the activity of cellular dependence factors or viral proteins of SARS-CoV-2 to facilitate viral infection. It has been reported that SARS-CoV 3 A and E protein forms an ion channel and modulates vims release (Verdi a-Baguena, C. et al. Coronavims E protein forms ion channels with functionally and structurally-involved membrane lipids.
  • Retinoic acid (RA) signaling plays an important role in controlling the cellular development, homeostasis, and metabolism, particularly in the lungs (Hind, M. & Maden, M. Retinoic acid induces alveolar regeneration in the adult mouse lung. European Respiratory Journal 23, 20-27, doi: 10.1183/09031936.03.00119103 (2004)). It binds to retinoic acid receptor (RAR), a nuclear transcriptional regulator to modulate the expression of specific genes involved in glucose and lipid metabolism.
  • RAR retinoic acid receptor
  • a number of studies have shown that retinoic acid receptor (RAR) agonists exhibit broad antiviral activities against a wide-range of viruses including hepatitis B virus (HBV) (B.
  • Translocator protein is a mitochondrial outer membrane protein previously known as the peripheral benzodiazepine receptor.
  • TSPO has been reported to trigger the degradation of the HIV-1 envelope glycoprotein by interfering with the glycoprotein folding process (T. Zhou, D. A. Frabutt, K. W. Moremen, Y. H. Zheng, ERManI (Endoplasmic Reticulum Class I alpha-Mannosidase) Is Required for HIV-1 Envelope Glycoprotein Degradation via Endoplasmic Reticulum -associated Protein Degradation Pathway. J Biol Chem 290, 22184-22192 (2015)).
  • TSPO agonists namely benzodiazepine receptor agonists
  • GSEA analysis shows a role in inhibiting viral protein biosynthesis through degradation of the proteins by TSPO.
  • benzodiazepine receptor agonists may have potential for clinical use as antivirals against SARS-CoV-2.
  • the compounds disclosed herein will be useful against SARS-CoV-2 in relevant human cells ex vivo , animal models in vivo, as well as directly in clinical trials for the set of FDA approved drugs, either as mono-therapeutics or in combination with a potent antiviral such as remdesivir.
  • a pathogenic infection in an individual or reducing transmission of a pathogen to other individuals comprising administering an anti-infective composition, wherein the anti-infective composition comprises an effective amount of a compound listed in Table 1 and a pharmaceutically acceptable carrier or excipient, and wherein the anti-infective composition reduces the risk of absorption, transmission, or function of a pathogen in the individual or transmission of a pathogen to another individual.
  • the anti-infective composition comprises an effective amount of a compound selected from the group consisting of nelfmavir mesylate hydrate, MDL 28170, GR 127935 hydrochloride hydrate, 8-(3- Chlorostyryl)caffeine, Apilimod, Clofazimine, Z LVG CHN2, AQ-13, H frustratingchin A, astemizole, SL-11128, ELOPIPRAZOLE, SDZ-62-434, analog of MLN-3897, SB-616234-A, YH-1238, VBY-825, ONO 5334, and AMG-2674.
  • the anti-infective composition comprises an effective amount of a compound selected from the group consisting of apilimod, VBY-825, ONO 5334, MLN-3897, SDZ-62-434, YH-1238, SB-616234-A, elopiprazole, and astemizole.
  • the EC50 range (mM) is 0.01 - 0.03 for apilimod, -0.3 for VBY-825, 0.3-1 for ONO 5334, 0.3-1 for MLN-3897, -1 for SDZ-62-434, 1- 2.5 for YH-1238, 1-2.5 for SB-616234-A, 1-2.5 for elopiprazole, and 1.5-2.5 for astemizole Table 1.
  • the terms “effective amount” or “therapeutically effective amount,” as used herein, generally refer to a sufficient amount of an agent or a compound (e.g ., the anti-infective composition described herein) which will relieve, to some extent, or reduce the likelihood of the occurrence of one or more of the symptoms of the disease or condition being treated. The result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.
  • the terms “effective amount” or “therapeutically effective amount” typically include, for example, a prophylactically effective amount.
  • a “prophylactically effective amount” is the amount of the anti-infective composition described herein that is required to reduce the risk of absorption, transmission, or function of a pathogen in an individual or transmission of a pathogen to another individual.
  • a pathogenic infection in an individual or reducing transmission of a pathogen to other individuals comprising administering an anti-infective composition, wherein the anti-infective composition comprises a compound listed in Table 1 and a pharmaceutically acceptable carrier or excipient, wherein the anti -infective composition reduces the risk of absorption, transmission, or function of a pathogen in the individual or transmission of a pathogen to another individual.
  • the pathogen includes, but is not limited to, respiratory syncytial virus (RSV), metapneumovirus (MPV), rhinovirus, influenza virus, parainfluenza virus, coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), norovirus, rotavirus, metapneumovirus (MPV), hepatitis A virus, adenovirus, astrovirus, S. aureus , methicillin-resistant S. aureus (MRSA), vancomycin-resistant enterococci (VRE), Enterococcus spp., Enterobacter spp., C. difficile , Campylobacter, E. faecali, E.faecium , or Salmonella.
  • Coronavirus is transmitted through direct contact with infected individuals, by contact with coronavirus-contaminated objects, and through inhalation of virus-laden, aerosolized respiratory droplets. Vaccination is currently the most effective method to prevent viral infection, but certain coronavirus strains do not have effective vaccines.
  • a pathogenic infection in an individual or reducing transmission of a pathogen to other individuals comprising administering an anti-infective composition to an individual, wherein the anti-infective composition comprises a compound listed in Table 1 and a pharmaceutically acceptable carrier or excipient, wherein the anti-infective composition reduces the risk of absorption, transmission, or function of a pathogen in the individual or transmission of a pathogen to another individual.
  • the anti-infective composition further comprises an additional anti-infective agent.
  • the additional anti-infective agent is an anti-viral agent selected from the group consisting of entry-inhibiting drugs (including enfuvirtide), uncoating inhibiting drugs (including amantadine, rimantadine, and pleconaril), reverse transcriptase inhibiting drugs (including acyclovir, zidovudine, and lamivudine), antisense drugs (including fomivirsen), ribozyme drugs, protease inhibitors, assembly inhibiting drugs (including rifampicin), and release inhibiting drugs.
  • entry-inhibiting drugs including enfuvirtide
  • uncoating inhibiting drugs including amantadine, rimantadine, and pleconaril
  • reverse transcriptase inhibiting drugs including acyclovir, zidovudine, and lamivudine
  • antisense drugs including fomivirsen
  • the additional anti-infective agent is an anti-viral agent.
  • the anti-viral agent is selected from the group consisting of Abacavir, Acyclovir (Aciclovir), Adefovir, Amantadine, Ampligen, Amprenavir (Agenerase), Arbidol, Atazanavir, Atripla, Balavir, Baloxavir marboxil (Xofluza), Biktarvy, Boceprevir (Victrelis), Cidofovir, Cobicistat (Tybost), Combivir, Daclatasvir (Daklinza), Darunavir, Delavirdine, Descovy, Didanosine, Docosanol, Dolutegravir, Doravirine (Pifeltro), Ecoliever, Edoxudine, Efavirenz, Elvitegravir, Emtricitabine, Enfuvirtide, Entecavir, Etravirine (Intelence
  • compositions disclosed herein are formulated in any suitable manner for administration. Any suitable technique, carrier, and/or excipient is contemplated for use with the compositions disclosed herein.
  • suitable technique, carrier, and/or excipient is contemplated for use with the compositions disclosed herein.
  • Non-limiting examples of cosmetic, dermatological, or pharmaceutically acceptable carriers and excipients suitable for formulation can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington ’s Pharmaceutical Sciences , Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms , Marcel Decker, New York, N.Y., 1980; Pharmaceutical Dosage Forms and Drug Delivery Systems , Eighth Ed.
  • compositions described herein can be administrable to a subject in a variety of ways by multiple administration routes, including but not limited to, inhalation, oral, parenteral (e.g. , intravenous, subcutaneous, intramuscular, intramedullary injections, intrathecal, direct intraventricular, intraperitoneal, intralymphatic, intranasal injections), intranasal, buccal, topical or transdermal administration routes.
  • parenteral e.g. , intravenous, subcutaneous, intramuscular, intramedullary injections, intrathecal, direct intraventricular, intraperitoneal, intralymphatic, intranasal injections
  • intranasal buccal
  • topical or transdermal administration routes e.g., topical or transdermal administration routes.
  • compositions described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations.
  • the compositions described herein can be formulated for use as an aerosol, a mist or a powder.
  • the compositions described herein are delivered in the form of an aerosol spray from a pressurized container or dispenser that contains a suitable propellant, for example a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant for example a gas such as carbon dioxide, or a nebulizer.
  • the compositions may take the form of tablets, lozenges, or gels formulated in a conventional manner.
  • the compositions described herein can be prepared as transdermal dosage forms.
  • the compositions described herein can be formulated into a pharmaceutical composition suitable for intramuscular, subcutaneous, or intravenous injection.
  • compositions described herein can be administered topically and can be formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams or ointments.
  • compositions described herein can be formulated in rectal compositions such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly suppositories, or retention enemas.
  • the pharmaceutically acceptable carriers or excipients disclosed herein include, but are not limited to one or more: pH modifying agent (e.g ., buffering agents), stabilizing agents, thickening agents, colorant agents, preservative agents, emulsifying agents, solubilizing agents, antioxidant agents, or any combination thereof.
  • pH modifying agent e.g ., buffering agents
  • stabilizing agents e.g., thickening agents, colorant agents, preservative agents, emulsifying agents, solubilizing agents, antioxidant agents, or any combination thereof.
  • the compositions disclosed herein comprise one or more preservatives.
  • the preservative when utilized, is in an amount sufficient to extend the shelf-life or storage stability, or both, of the topical formulations disclosed herein.
  • exemplary preservatives include, but are not limited to: tetrasodium ethylene-diamine tetraacetic acid (EDTA), methyl, ethyl, butyl, and propyl parabens, benzophenone-4, methylchloroisothiazolinone, methylisothiazolinone, sodium benzoate, paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenylethylalcohol, dehydroacetic acid, sorbic acid, benzalkonium chloride (BKC), benzethonium chloride, phenol, phenylmercuric nitrate, and thimerosal.
  • EDTA tetrasodium ethylene-diamine
  • SARS-CoV-2 HKU-OOla strain was isolated from the nasopharyngeal aspirate specimen of a laboratory-confirmed COVID-19 patient in Hong Kong (K. K. To et ah,
  • SARS-CoV-2 USA-WA1/2020 strain isolated from an oropharyngeal swab from a patient with a respiratory illness who developed clinical disease (COVID-19) in January 2020 in Washington, USA, was obtained from BEI Resources (NR-52281).
  • the virus was propagated in Vero E6 (ATCC® CRL-1586TM) cells transfected with exogenous ACE2 and TMPRSS2 and kept at -80 °C in aliquots.
  • Plaque forming unit (PFU) and TCID50 (Median Tissue Culture Infectious Dose) assays were performed to titrate the cultured virus.
  • Vero E6 and Huh-7 cells were maintained in Dulbecco’s modified eagle medium (DMEM, Gibco) supplemented with 10 % heat-inactivated fetal bovine serum (FBS, Gibco), 50 U/mL penicillin, 50 pg/mL streptomycin, 1 mM sodium pyruvate (Gibco), 10 mM HEPES (Gibco), and IX MEM non- essential amino acids solution (Gibco).
  • Huh7 cells were transfected with PLVX-ACE2 and PLX304-TMPRSS2 prior to infection.
  • the LOP AC® library is a collection of 1,280 pharmacologically active compounds, covering all the major target classes, including kinases, GPCRs, neurotransmission and gene regulation (Sigma).
  • the ReFRAME (Repurposing, Focused Rescue, and Accelerated Medchem) library contains approximately 13,000 high-value molecules assembled by combining three databases (Clarivate Integrity, GVK Excelra GoStar and Citeline Pharmaprojects) for fast-track drug discovery. As shown in Fig. 1, Panel A, this library contains US Food and Drug Administration (FDA)-approved/registered drugs (-39%), investigational new drugs (-58%), and preclinical compounds (-3%).
  • FDA US Food and Drug Administration
  • Cytopathic effect was indirectly quantified as the presence of ATP in live cells by using the CellTiter-Glo (Promega) luminescent cell viability assay at 72 h post-infection. Data were normalized to the median of each plate.
  • the Z-score was calculated based on the log base 2 fold change (Log2FC) with the average and standard deviation of each plate. The screen was performed in duplicate by running the assay in parallel for the LOPAC®1280 library or as two independent experiments for the ReFRAME collection. Twenty-eight compounds from the LOPAC®1280 were selected according to the cutoff of >5*Stdev Log2FC and included in a dose-response confirmation assay.
  • the selected hits were further validated by immunofluorescence in an 8-point dose response experiment to determine EC so and CC50 through a cell-based high-content imaging assay, labeling the viral nucleoproteins within infected cells.
  • Three thousand Vero E6 cells were added into 384-well plates pre-spotted with compounds, in a volume of 40 m ⁇ . The final concentration of compound ranged from 1.1 nM to 2.5 mM.
  • Sixteen hours post-seeding, 10 m ⁇ of SARS-CoV-2 USA-WA1/2020 were added to each well, at an MOI of 0.75.
  • cells were fixed with 5% paraformaldehyde for 4 hours and permeabilized with 0.5% Triton X-100 for 5 minutes. After blocking with 3% bovine serum albumin (BSA) for 30 mins, the cells were incubated for 1 hour at room temperature with rabbit-anti-SARS-CoV-1 nucleoprotein serum, which exhibits strong cross-reactivity with SARS-CoV-2. After two washes with phosphate-buffered saline (PBS), the cells were incubated with Alexa Fluor 488- conjugated goat-anti -rabbit IgG (Thermo Fisher Scientific, USA) for 1 hour at room temperature.
  • BSA bovine serum albumin
  • Proc Natl Acad Sci U S A 117, 7001-7003 (2020)) and viral replication can be assessed through indirect measurement of viral-induced cytopathic effects (CPE) (W. B. Park et ah, Virus Isolation from the First Patient with SARS-CoV-2 in Korea. J Korean Med Sci 35, e84 (2020)).
  • CPE viral-induced cytopathic effects
  • a clinical isolate of the SARS-CoV-2 virus SARS-CoV-2 HKU-OOla was utilized for assay development and screening.
  • the assay parameters, including cell seeding density, multiplicity of infection (MOI), and timepoints were optimized in Vero E6 cells by measuring virus-induced CPE using CellTiter-glo in a 384-well format.
  • Repositioning Analysis of the ReFRAME Drug Repurposing Library [0079] Due to favorable results of the LOP AC HTS screen, the same experimental design was used to screen the comprehensive ReFRAME drug repurposing collection.
  • This library is an inclusive collection of nearly 13,000 chemical compounds, which have been either FDA approved, entered clinical trials or undergone significant pre-clinical characterization (J. Janes et al., The ReFRAME library as a comprehensive drug repurposing library and its application to the treatment of cryptosporidiosis. Proc Natl Acad Sci U S A 115, 10750-10755 (2016)).
  • GSEA Gene Set Enrichment Analysis
  • enriched targets and biological processes include allosteric modulators of the benzodiazepine receptor, cytosolic NADPH- dependent oxidoreductase aldose reductase, cholesterol homeostasis, serine proteases, and as anticipated, antimalarials, including chloroquine derivatives such as Amopyroquine and AQ-13 (as shown in Fig. 2, Panel A and Fig. 5).
  • the hits enriched in the GSEA analysis are provided in Table 3, and the hits validated in Vero single point include LG-1550, Tretinoin, Tamibarotene, acitretin, tazarotene, RBAD, AL 3152, ZK-93426, zaleplon GR, pagoclone, AQ-13, and H frustratingchin A.
  • the hits confirmed in Vero DR include LG-1550, RBAD, AL 3152, ZK-93426, AQ-13 and Haggingchin A, and both AQ-13 and H movingchin A were in the final list.
  • Table 3 Hits enriched in GSEA analysis.
  • SARS-CoV-2 primarily infects the epithelial cells in the respiratory tract.
  • the expression of drug target genes enriched in the compound screen was compared across cell types within the respiratory tract (Cascella, M., Rajnik, M. Cuomo, A., Duleboh, S.C., & Di Napoli, R., Features, Evaluation and Treatment Coronavirus (COVID-19), StatPearls [Internet] (2020)).
  • ACE2 entry receptor for SARS-CoV-2
  • TMRPSS22 priming protease TMRPSS22
  • This validation step in addition to assessing false positives, represents a filtering of compounds that function to block CPE, only inhibit at the screening compound concentration (5 mM), require longer timepoints to impact replication (24 hpi vs 72 hpi), or, although unlikely, specifically inhibit the SARS-CoV-2 HKU-OOla clinical isolate. 27% of the compounds (89 hits) were found to reduce viral replication by at least 40% at 2.5 pm (data not shown) when averaging data from at least two replicates. These include compounds that were found to belong to enriched target classes (as shown in Fig.
  • Panel A including retinoic acid receptor agonists (including LG-1550, Tretinoin, Tamibarotene, acitretin, tazarotene, RBAD), the aldose reductase inhibitor AL 3152, benzodiazepine receptor agonists (including ZK-93426, zaleplon GR, and pagoclone) and antimalarial drugs (including AQ-13 and Hguidingchin A), as well as the FDA- approved anti mycobacterial clofazimine
  • Example 3 Clofazimine inhibition of human pathogenic coronaviruses in human cellular models.
  • Clofazimine whose structure is demonstrated on the left side of FIG. 9a, was introduced into a number of human cellular models to measure its efficacy against coronavirus pathogens.
  • Clofazimine brand name LAMPRENE®
  • EC50 half maximal effective concentration
  • Cmax peak serum concentration
  • Clofazimine inhibited MERS-CoV replication in a dose-dependent manner. Clofazimine was introduced to VeroE6 cells infected with MERS-CoV. As shown in the graph in FIG. 9a, clofazimine demonstrated an EC50 of 1.48+0.17, as measured by plaque reduction when plotted using a logistic non-linear regression model (GraphPad).
  • Clofazimine’ s effectiveness against MERS-CoV-nucleocapsid protein (NP) in Huh7 cells was compared against dimethyl sulfoxide (“DMSO”) as a control and measured by fluorescent imaging and cell quantification.
  • MERS-CoV-infected Huh7 cells (0.01 multiplicity of infection (“MOI”)) were treated for 24 hours with either DMSO or 5 mM of clofazimine. After treatment, cells were viewed using fluorescent imaging. The MERS-CoV-NP antigen was stained green and the Huh7 cell nuclei were stained blue.
  • FIG. 9B (Upper panel): immunofluorescence staining of MERS-CoV-NP antigen (green), and Huh 7 cell nucleus (blue). Scale bar: 20pm.
  • FIG. 9B (Lower panel): MERS-CoV-NP positive cells quantitated by flow cytometry. The experiments were performed twice with representative images and quantifications shown.
  • CMs Human primary cardiomyocytes (CMs) infected with SARS-CoV-2 (0.1 MOI) and human primary human small airway epithelial cells (HSAEpC) infected with MERS-CoV (1 MOI) were treated with 10 pm of remdesivir, or 10 pm, 5 pm or 2.5 pm of clofazimine, or DMSO. Cell lysates were collected at 24 hours post-infection and viral load was determined using RT-qPCR assays.
  • FIG. 9c shows the effect of 5 pm, 2.5 pm and 1.25 pm doses of clofazimine inhibition of SARS-CoV-2 replication in human primary cardiomyocytes and MERS-CoV in human primary human small airway epithelial cells when compared to DMSO.
  • clofazimine or remdesivir were introduced to VeroE6 cells infected with SARS-CoV-1 (0.01 multiplicity of infection (MOI) at 48 hours post-infection (hpi)), HELF cells infected with hCoV-229E (0.001 MOI at 72 hours hpi), and BSC1 cells infected with hCoV-OC43 (0.001 MOI at 72 hours hpi).
  • MOI multiplicity of infection
  • Viral loads in the cell culture supernatants were quantified by RT-qPCR assays and a one-way ANOVA was used to compare the clofazimine and remdesivir groups with the 0 pM group.
  • the left graph in FIG. 9e shows the results of the analysis when , 5 pM, 2.5 pM, 1.25 pM, and 0.625 pM of clofazimine, 10 pM of remdesivir, and 0 pM were introduced to SARS- CoV-1 infected VeroE6 cells.
  • the middle graph in FIG. 9e shows the results of the analysis when 5 mM, 2.5 mM, 1.25 mM, and 0.625 mM of clofazimine, 10 mM of remdesivir, and 0 mM, were introduced to the hCoV-229E infected HELF cells.
  • the right graph in FIG. 9e shows the results of the introduction of 5 mM, 2.5 mM, 1.25 mM, and 0.625 mM of clofazimine, 10 mM of remdesivir, and 0 mM, were introduced to the hCoV-OC43 BSC1 cells. ***p ⁇ 0.001, **p ⁇ 0.01 and *p ⁇ 0.05.
  • Clofazimine’ s effect on virus life cycle was studied using a time-of-addition assay. VeroE6 cells were inoculated with SARS-CoV-2 for 1 hour and then the inoculum was removed. Cells were treated with clofazimine at a concentration of 5 mM at the following time points: 16 hours before to 10 hours after inoculation, 16 hours before to 0 hours before inoculation, 0 hours to 1 hour after inoculation, 0 hours to 2 hours after inoculation, 0 hours to 10 hours after inoculation, 1 hour after to 10 hours after inoculation, 2 hours after to 10 hours after inoculation, and 5 hours after to 10 hours after inoculation.
  • the graph on the left in FIG. 10a shows the time frame over which the cells were treated with clofazimine, which is represented by the shaded bar.
  • VSV Vesicular stomatitis virus
  • Clofazimine’ s inhibitory effects on pseudotype entry into cells. Clofazimine’ s effect was compared to cells treated with DMSO and MDL28170, a positive control entry inhibitor. VeroE6 cells were pre-treated with 2.5 mM of clofazimine, DMSO, or DML28170 for two hours and then infected with SARS-CoV-1 S or SARS-CoV-2 S or MERS-CoV S pseudotyped particles harboring firefly luciferase for two hours. Luciferase signals were quantified at 24 hours post inoculation.
  • Clofazimine’ s effect on SARS-CoV-2 spike-mediated cell-to-cell fusion was measured by calculation of GFP area of confocal images. Vero cells were co-transfected with 1 pg of SARS- CoV-2 spike plasmid and 0.4 pg of EGFP plasmid for 8 hours, at which time the cells were treated with 0.625 pg, 1.25 pg, 2.5 pg, 5 pg, or 10 pg of clofazimine or DMSO (0.1%). After 48 hours, confocal images were acquired. FIG. 10c (Left panel) shows the confocal images acquired at 48 hours post-transfection. Scale bar: 400 pm.
  • Example 5 Transcriptional analysis of clofazimine treatment.
  • Clofazimine’ s effect on the viral load of SARS-CoV-2 in Caco-2 cells was studied using RT-qPCR analysis.
  • Caco-2 cells were infected with 0.1 MOI of SARS-CoV-2 and then treated with 10 pM, 5 pM, 2.5 pM, 1.25 pM, or 0.625 pM of clofazimine, 10 pM remdesivir, or 0.1% DMSO (control group, labelled as “0” mM).
  • Cell culture supernatants were collected at 48 hours post-infection and subjected to viral load determination by RT-qPCR analysis. Results of the experiment are shown in FIG 11a. *p ⁇ 0.05 and ** p ⁇ 0.01 as compared to the DMSO group by one-way ANOVA.
  • PC A Principal component analysis
  • FIG. 11c shows information related to transcription factors regulated by clofazimine.
  • FIG. 11c (Right panel) shows a heatmap of 197 transcription factors regulated by clofazimine treatment without infection, while FIG. 11c (Left panel) displays known interactions among the transcription factors contained within transcription factors shown in the right panel of FIG. 11c.
  • FIG. lid shows the top enriched pathways of significantly up-regulated genes (false discovery rate ⁇ 0.05 and fold change >2) of mock-infected cells receiving clofazimine treatment at 6 hour post treatment as compared to SARS-CoV-2 infected cells receiving clofazimine at 6 hours post-infection. Pathway analysis was performed by Metascape. *denotes innate immunity related pathways.
  • Example 6 Prophylactic and therapeutic treatment with clofazimine reduces SARS-CoV-2 Disease.
  • the experimental design consisted of prophylactic treatment with clofazimine or post-exposure administration of clofazimine.
  • Prophylactic treatment consisted of oral administration of 25mg/kg/day of clofazimine given at 3, 2, and 1 days pre-infection, followed by virus challenge of 10 L 5 plaque forming unit (PFU) per hamster through the intranasal route at 0 days.
  • Therapeutic post-exposure administration of clofazimine was performed at 1, 2, and 3 days post-infection and consisted of applying the same drug dosage and virus inoculum as applied to the prophylactic treatment group.
  • Lung tissue, nasal wash, fecal, and serum samples were collected at 4- and 14-days post-infection.
  • Remdesivir (15mg/kg, indicated by the blue symbols) was administered as a control in the therapeutic regimen through intraperitoneal injection.
  • Statistical analysis of the prophylaxis groups was conducted using Student’s t-test and statistical analysis for the therapeutic groups was conducted using one-way ANOVA, the groups were compared with the vehicle group (indicated by the black symbols). For statistical analysis, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, when compared with vehicle.
  • FIG. 12i displays representative images of H&E-stained lung tissue section from hamsters treated with vehicle, clofazimine, remdesivir, or mock-infection. Numbered circled areas are shown in magnified images on the right-hand portion of each panel, illustrating the severity of: (1) bronchiolar and/or peribronchiolar cell death; (2) alveoli destruction and/or alveolar infiltration; (3) blood vessel and perivascular infiltration. Black arrows indicate sites of infiltration. These representative images were selected from a pool of over 15 images captured in three randomly selected hamsters per group. Scale bar: 200 pm.
  • Table 6 shows multiple pharmacokinetic parameters of VBY-825 after a single dose was administered to hamsters.
  • Table 7 shows multiple pharmacokinetic parameters of aplilimod after a single dose was administered to hamsters.
  • Table 8 shows multiple pharmacokinetic parameters of remdesivir (GS-5734) after a single dose was administered to hamsters.
  • Table 9 shows multiple pharmacokinetic parameters of remdesivir metabolite (GS- 441524) after a single dose of remdesivir was administered to hamsters.
  • Clofazimine exhibits antiviral synergy with remdesivir in vitro and in vivo.
  • VeroE6 cells were pre-treated for 16 hours with increasing concentrations of the indicated compound and then infected with SARS-CoV-2 at a multiplicity of infection (“MOI”) of 0.01. Thirty hours after infection, cells were fixed and analyzed by immunofluorescence imaging. For each concentration, the percentage of infection was calculated as the ratio of the number of infected cells stained for SARS-CoV-2 nucleocapsid protein (NP) to the number of cells stained with DAPI.
  • MOI multiplicity of infection
  • FIG. 13a shows a topographic two-dimensional map of synergy scores determined in SynergyFinder. The color gradient indicates synergy score (red: highest synergy score; blue: highest antagonism score).
  • X-axis remdesivir up to 10 mM
  • y-axis clofazimine up to 10 pM.
  • FIG. 13b shows a graph of a dose response analysis of remdesivir alone (black) and in combination with 0.15625 pM (blue) or 0.625 pM (red) clofazimine. The observed compounds’ activities are represented by solid lines, while the predicted additive combinatorial activities are indicated by dashed lines. The dotted black line denotes 90% inhibition of infection. Data are normalized to mean values for DMSO-treated wells and represent mean ⁇ SEM of 2 independent experiments.
  • FIG. 13c The experimental design used in studying in vivo combination therapy administered to hamsters is shown in FIG. 13d. After virus challenge of 10 L 5 plaque forming unit (PFU) per hamster at day 0, oral administration of clofazimine (15 mg/kg) and/or intraperitoneal injection of remdesivir (1.5 mg/kg) was performed at 1, 2, and 3 days post-infection (dpi).
  • PFU plaque forming unit
  • Vims in the hamster lung tissue harvested at 4 dpi was titrated by plaque assay and shown in FIG. 13f. One-way ANOVA when compared with the vehicle control group, *p ⁇ 0.05, **p ⁇ 0.01 and n.s. (non-significant) .
  • the graph in FIG. 13g shows the results of a virus titer in the nasal wash determined by plaque assays.
  • FIG. 13h shows representative images of infected cells by immunofluorescence staining in nasal turbinate at 4 dpi.
  • SARS-CoV-2 N protein (NP) was detected by specific antibody (green) and cell nuclei were stained by DAPI (blue). Scale bar: 200 pm.
  • Example 8 Cytotoxicity measurement of clofazimine in matching cells for antiviral evaluation.
  • the cell viability was determined using CellTiter-Glo and in the absence of virus infection.
  • the drug-incubation time in the cytotoxicity assay was consistent with that in the antiviral assay, e.g., 24 hours post-treatment for Huh7 cells, primary human small airway epithelial cells (HSAEpC) and human embryonic stem cells-derived cardiomyocytes (CM); 48 hours post-treatment for VeroE6 and Caco-2 cells; and at 72 hours post-treatment for BSC1 and human embryonic lung fibroblasts (HELF), respectively.
  • Example 9 Exploration of possible effects of clofazimine on virus entry and replication.
  • Clofazimine did not display an effect on ACE2 and DPP4 expression.
  • Caco-2 cells were treated with clofazimine for 16 hours prior to collection for western blotting analysis.
  • the expression of ACE2 and DPP4 was determined using anti-ACE2 antibody and anti-DPP4 antibody, respectively. The results of the analysis are shown in FIG. 15a.
  • Clofazimine showed no inhibition on the binding between either ACE2 or heparin with SARS-CoV-2 Spike protein, which are two critical cellular components for viral attachment and infection.
  • ELISA-based detection was used to determine binding of recombinant SARS-CoV-2 Spike protein to the immobilized Heparin-BSA or ACE2.
  • FIG. 15b displays the results of the experiment. Clofazimine was titrated at the indicated concentrations. Dashed line represents binding without inhibitor (i.e. OmM).
  • Clofazimine shows a marginal effect against Mpro and PLPro protease activity.
  • Purified SARS-CoV-2 Mpro (Nsp5) and SARS-CoV-2 PLpro (Nsp3) enzymes were incubated with varying concentrations of clofazimine.
  • the activity of purified SARS-CoV-2 Mpro and SARS- CoV-2 PLpro enzymes was measured using the substrate Dabcyl-KTSAVLQSGFRKM- E(Edans)-NH2 and Arg-Leu-Arg-Gly-Gly-AMC (RLRGG-AMC), respectively.
  • Clofazimine did not display an inhibition on the polymerase activity of the nsp7/nsp8/nspl2 RdRp complex.
  • the scaffold used in this experiment’s in vitro transcription inhibition assay is listed in the upper panel of FIG. 15d.
  • the inhibitory effect of clofazimine or SL-11128 on the RdRp core complex was analyzed by a primer elongation assay.
  • SL-11128 showed some inhibitory effect as time progressed (0, 15s, lmin, 5min and 20min), while the inhibitory effect of clofazimine was minor, ranging from 5-40 mM. All reactions were performed at 30 °C.
  • Example 10 Transcriptional analysis of clofazimine treatment on Caco-2 cells.
  • MOI multiplicity of infection
  • FIG. 16b shows a heat map of the genes enriched in MAPK signaling, TNF signaling, Interleukins (ILs) signaling, or cytokine-cytokine receptor interaction. These genes were up- regulated (fold change >2, FDR ⁇ 0.05) by either 6 hours clofazimine (without infection) or 6 hpi clofazimine (with infection) compared to mock-infection.
  • FIG. 16c shows the network of enriched terms represented as pie charts. Pies are color- coded based on the identities of the gene lists. “6 h. clofazimine vs mock” (red color) represents the genes that were up-regulated by clofazimine treatment without infection at 6 hours as compared to mock. “6 hpi. Clofazimine vs mock” (blue color) represents the up-regulated genes by clofazimine treatment at 6 hours post-infection as compared with mock.
  • Example 11 Transcriptional analysis of hamster lung tissues with clofazimine administration.
  • Fig. 17b presents Gene Ontology Biological Process (GO-BP) analysis results showing up-regulated genes comparing prophylactic clofazimine administration with its corresponding vehicle controls.
  • RNA expression Reads Per Kilobase of transcript per Million mapped reads, “RPKM”
  • FIG. 17c RNA expression (Reads Per Kilobase of transcript per Million mapped reads, “RPKM”) of the 13 genes enriched in the “Leukocyte differentiation” category of GO-BP analysis is shown in FIG. 17c. These genes were up-regulated (fold change > 1.5, p value ⁇ 0.01) by prophylactic clofazimine group when compared to vehicle controls.
  • MHCII molecules are labeled with an asterisk (*).
  • Transcription factors up-regulated by clofazimine on both Caco-2 cells and hamster lung tissues are labeled with an up arrow ( ).
  • FIG. 17d shows a heat map of immune response related genes in uninfected and infected hamster lungs administered prophylactic clofazimine or vehicle controls.
  • Fig. 17e presents Gene Ontology Biological Process (GO BP) analysis results showing up-regulated genes comparing clofazimine and vehicle-treated hamster lungs without virus infection.
  • GO BP Gene Ontology Biological Process
  • Table 10 provides the list of up-regulated genes in hamster lung tissues that were observed when clofazimine was administered prophylactically. All genes demonstrated a p-value of ⁇ 0.05 when compared to vehicle control at 4 days post-infection.
  • Table 10 List of up-regulated genes in hamster lung tissues when comparing prophylactic administration of clofazimine versus vehicle.
  • Example 12 Lung pathological severity using histological scores.
  • Example 13 In vitro antiviral synergy of clofazimine and remdesivir.
  • Clofazimine exhibited in vitro antiviral synergy with remdesivir for every combination of drug dose, as displayed in FIG. 19. Remdesivir at the indicated doses was combined with clofazimine at indicated doses or a negative control (DMSO), and antiviral dose-response relationships were determined in VeroE6 cells by immunofluorescence imaging. VeroE6 cells were pre-treated for 16 hours with increasing concentrations of the indicated compound and then infected with SARS-CoV-2 at a multiplicity of infection (“MOI”) of 0.01. Thirty hours after infection, the infected cells were analyzed by immunofluorescence imaging.
  • MOI multiplicity of infection
  • the percentage of infection was calculated as the ratio of the number of infected cells stained for SARS-CoV-2 NP protein to the number of cells stained with DAPI.
  • EC50 for compound alone black solid line with circles
  • predicted dashed line
  • observed solid line with squares
  • Example 14 Antiviral synergy of clofazimine and remdesivir in hamster lungs.
  • FIG. 20 shows viral N protein expression (green) in diffuse alveolar areas (shown by the thick white arrow) and in the focal bronchiolar epithelial cells (thin white arrow) of the vehicle-treated hamster lungs, whereas standard (“std”) and low dosing (“low”) remdesivir (“Rem”) groups as well as the clofazimine group (“Clo”) exhibited reduced N expression.
  • the representative images of FIG. 20 were selected from a pool of over 15 images captured in three randomly selected hamsters per group. Scale bar: 200 pm.
  • Example 15 Clofazimine inhibition of SARS-CoV-2 infection in human stem cell derived pneumocyte-like cells.
  • Clofazimine demonstrated inhibition of SARS-CoV-2 infection in human stem cell derived pneumocyte-like cells.
  • Cells were induced by alveolar differentiation of human embryonic stem cells. Briefly, cells were collected at 70-80% confluency, and 2 million cells per 10 cm 2 were plated on Vitronectin-coated tissue culture plates in mTeSR. The next day, definitive endoderm differentiation was induced following the protocol previously described (Jacob etal, Differentiation of human pluripotent stem cells into functional lung alveolar epithelial cells , Cell Stem Cell 21(4):472-488 (2017)) for four days.
  • Cells were split and further differentiated following an adapted alveolar differentiation protocol in IMDM medium supplemented with 10% FBS, 2 mM L-glutamine, 0.5 pM all-trans-retinoic acid, 10 ng/ml FGF- 10, 10 ng/ml EGF, 100 ng/ml Wnt3a, 10 ng/ml KGF and 5 ng/ml BMP -4.
  • IMDM medium supplemented with 10% FBS, 2 mM L-glutamine, 0.5 pM all-trans-retinoic acid, 10 ng/ml FGF- 10, 10 ng/ml EGF, 100 ng/ml Wnt3a, 10 ng/ml KGF and 5 ng/ml BMP -4.
  • clofazimine (10 uM
  • remdesivir 10 uM
  • DMSO SARS-CoV2 infection (40000 plaque forming unit) for 48 hours at 37°C.
  • the cells were dissociated using cell dissociation buffer, and fixed in 4% methanol-free formaldehyde for FACS analysis using anti-mouse SARS-CoV-2 - nucleocapside protein antibody.
  • the duplicate set of cells were treated with the same drug concentrations, but the cells were left uninfected. After 48 hours of incubation at 37°C, the cells were analyzed for viability using MTT assay kit. The results of the experiment are shown in FIG. 21. Black indicates the percent infection, whereas red indicates the percent viability.
  • Example 16 Clofazimine effect on antiviral activity in adenovirus hACE2 mouse model of SARS-COV-2 infection.
  • Clofazimine exhibited antiviral activity in an adenovirus hACE2 mouse model of SARS-COV-2 infection.
  • BALB/c mice were infected intranasally with 2.5c10 L 8 plaque forming unit (PFU) of an adenovirus carrying the gene for hACE2.
  • PFU plaque forming unit
  • Doses of clofazimine (50 mg/kg) were administered orally (p.o.), while remdesivir (50 mg/kg) was administered subcutaneously (s.c.). Each intervention was administered once per day for 3 days.
  • mice were put under anesthesia with a mixture of ketamine/xylazine, and infected with 1 c 10 L 4 PFU of SARS-CoV-2 in 50 m ⁇ of PBS.
  • animals were humanely euthanized.
  • Whole left lungs were harvested and homogenized in PBS with silica glass beads and then frozen at -80°C for viral titration via TCID50.
  • Infectious titers were quantified by limiting dilution titration using Vero E6 cells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Dispersion Chemistry (AREA)
  • Otolaryngology (AREA)
  • Pulmonology (AREA)
  • Dermatology (AREA)
  • Nutrition Science (AREA)
  • Physiology (AREA)
  • Virology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Described herein are compositions and methods useful for reducing the likelihood of a pathogenic infection, or for reducing transmission of a pathogen to others, through use of an anti-infective composition that comprises a compound listed in Table 1. The anti-infective compositions disclosed herein are suitable for administration to a subject.

Description

METHODS AND COMPOSITIONS FOR ANTIVIRAL TREATMENT
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/010,630, filed on April 15, 2020. Priority is claimed pursuant to 35 U.S.C. § 119. The above noted patent application is incorporated by reference as if set forth fully herein.
STATEMENT AS TO FEDERALLY SPONSPORED RESEARCH [0002] This invention was made with government support under Grant No. W81XWH-20-1- 0270, awarded by Department of Defense and Grant No. U19 All 35972, awarded by National Institutes of Health. The government has certain rights in the invention.
BACKGROUND
[0003] Coronavirus infections result in substantial morbidity and death worldwide. While vaccination can protect against certain viral infections, vaccines are not always fully effective. There are many pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2), for which there is no effective vaccine. Therefore, prevention of infection and transmission of disease are of paramount importance; especially in certain young, elderly, or immunocompromised populations.
SUMMARY
[0004] Disclosed herein, in some embodiments, are anti-infective compositions comprising a pharmaceutically acceptable carrier or excipient and an effective amount of a compound selected from the group consisting of amopyroquine, nelfmavir mesylate hydrate, GR 127935 hydrochloride hydrate, LG-1550, tretinoin, tamibarotene, acitretin, tazarotene, RBAD, AL 3152, ZK-93426, zaleplon GR, pagoclone, MDL 28170, 8-(3-Chlorostyryl)caffeine, Apilimod (APY0201), Clofazimine, Z LVG CHN2, AQ-13, Hanfangchin A, astemizole, , SL-11128, ELOPIPRAZOLE, SDZ-62-434, analog of MLN-3897, SB-616234-A, YH-1238, VBY-825, ONO 5334, and AMG-2674, wherein the anti-infective composition reduces the risk of absorption, infectivity, or transmission of a pathogen. In some embodiments, the pathogen is a coronavirus. In some embodiments, the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In some embodiments, disclosed here are anti -infective compositions comprising a pharmaceutically acceptable carrier or excipient and an effective amount of a compound selected from the group consisting of amopyroquine, nelfmavir mesylate hydrate, GR 127935 hydrochloride hydrate, LG-1550, tretinoin, tamibarotene, acitretin, tazarotene, RBAD, AL 3152, ZK-93426, zaleplon GR, pagoclone, MDL 28170, 8-(3- Chlorostyryl)caffeine, Apilimod (APY0201), Clofazimine, Z LVG CHN2, AQ-13, Hanfangchin A, astemizole, , SL-11128, ELOPIPRAZOLE, SDZ-62-434, analog ofMLN-3897, SB-616234- A, YH-1238, VBY-825, ONO 5334, and AMG-2674, and further comprising an additional anti- infective agent. In some embodiments, the additional anti-infective agent is an anti-viral agent.
In some embodiments, the anti-viral agent comprises remdesivir (GS-5734). In some embodiments, the anti-viral gent comprises favipiravir (T-705) In some embodiments, disclosed herein are anti -infective compositions comprising a pharmaceutically acceptable carrier or excipient and an effective amount of a compound selected from the group consisting of nelfmavir mesylate hydrate, MDL28170 and GR 127935 hydrochloride hydrate, wherein the anti-infective composition reduces the risk of absorption, infectivity, or transmission of a pathogen. In some embodiments, disclosed herein are anti -infective compositions comprising a pharmaceutically acceptable carrier or excipient and an effective amount of clofazimine, wherein the anti-infective compositions reduce the risk of absorption, infectivity, or transmission of a pathogen.
[0005] Disclosed herein, in some embodiments, are methods for treating a patient having severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) comprising: administering to the patient a therapeutically effective amount of an anti-infective composition comprising a pharmaceutically acceptable carrier or excipient and an effective amount of a compound selected from the group consisting of amopyroquine, nelfmavir mesylate hydrate, GR 127935 hydrochloride hydrate, LG-1550, tretinoin, tamibarotene, acitretin, tazarotene, RBAD, AL 3152, ZK-93426, zaleplon GR, pagoclone, MDL 28170, 8-(3-Chlorostyryl)caffeine, Apilimod (APY0201), Clofazimine, Z LVG CHN2, AQ-13, Hanfangchin A, astemizole, , SL-11128, ELOPIPRAZOLE, SDZ-62-434, analog ofMLN-3897, SB-616234-A, YH-1238, VBY-825,
ONO 5334, and AMG-2674, wherein the anti-infective composition is effective against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In some embodiments, disclosed herein are methods for treating a patient having severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) comprising: administering to the patient a therapeutically effective amount of an anti-infective composition comprising a pharmaceutically acceptable carrier or excipient and an effective amount of clofazimine, wherein the anti-infective composition is effective against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In some embodiments, administering to the patient comprises administration via a route selected from the group consisting of inhalation, oral, parenteral, intranasal, buccal, topical or transdermal administration routes. In some embodiments, the route is inhalation.
[0006] Disclosed herein, in some embodiments, are anti-infective compositions comprising an effective amount of a compound listed in Table 1 and a pharmaceutically acceptable carrier or excipient. In some embodiments, the anti-infective composition further comprises an additional anti-infective agent. In some embodiments, the additional anti-infective active agent selected from the group consisting of entry-inhibiting drugs, uncoating inhibiting drugs, reverse transcriptase inhibiting drugs, antisense drugs, ribozyme drugs, protease inhibitors, assembly inhibiting drugs, and release inhibiting drugs. In some embodiments, the composition treats a respiratory syncytial virus (RSV), metapneumovirus (MPV), rhinovirus, influenza virus, parainfluenza virus, coronavirus, norovirus, rotavirus, hepatitis A virus, adenovirus, astrovirus, S. aureus , methicillin-resistant S. aureus (MRSA), vancomycin-resistant enterococci (VRE), Enterococcus spp ., Enter obacter spp ., C. difficile , Campylobacter, E. faecali, E.faecium , or Salmonella.
[0007] Disclosed herein, in some embodiments, are anti-infective compositions comprising an effective amount of a compound listed in Table 1 and a pharmaceutically acceptable carrier or excipient, wherein the anti -infective composition reduces the risk of absorption, infectivity, or transmission of a pathogen. In some embodiments, the composition treats at least one strain of a coronavirus. In some embodiments, the composition treats severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In some embodiments, disclosed herein are inhalation devices comprising an effective amount of a compound selected from the group consisting of amopyroquine, nelfmavir mesylate hydrate, GR 127935 hydrochloride hydrate, LG-1550, tretinoin, tamibarotene, acitretin, tazarotene, RBAD, AL 3152, ZK-93426, zaleplon GR, pagoclone, MDL 28170, 8-(3-Chlorostyryl)caffeine, Apilimod (APY0201), Clofazimine, Z LVG CHN2, AQ-13, Hanfangchin A, astemizole, , SL-11128, ELOPIPRAZOLE, SDZ-62-434, analog of MLN-3897, SB-616234-A, YH-1238, VBY-825, ONO 5334, and AMG-2674; and a pharmaceutically acceptable propellant. In some embodiments, the compound is clofazimine. In some embodiments, the device further comprises remdesivir.
INCORPORATION BY REFERENCE
[0008] All publications, patents, and patent applications mentioned in this specification are incorporated by reference herein to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS [0009] The patent application contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
[0010] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
[0011] FIG. 1, Panels A-C depict an exemplary embodiment of the screening schematic disclosed herein. Panel A depicts the distribution of the approximately 13,000 compounds in the ReFRAME collection across different stages of clinical development and a schematic of the screening process. Panel B depicts results from the LOPAC®1280 library primary screen.
Panel C depicts results from the ReFRAME collection primary screen.
[0012] FIG. 2, Panels A-B depict Gene Set Enrichment Analysis (GSEA) of primary screening data according to the average Z’ factor. GSEA enrichment plots of six target clusters that are enriched are represented, including retinoic acid receptor agonists, benzodiazepine receptor inhibitors, aldose reductase agonists, potassium channel agonists, cholesterol inhibitors, and antimalarials (P -value < 0.05, FDR q-value < 0.25) (panel A) and expression of ACE2, TMPRSS2, and select target genes (panel B). Expression was analyzed using single-cell RNA profiling data from human airway samples of healthy donors. Clustered heat maps in panel B show the fraction of gene-expressing cells separated by sampling location (left-hand side) or cell type (right-hand side).
[0013] FIG. 3, Panels A-C results from the methods and compositions disclosed herein.
Panel A depicts a heat map of the indicated 17 compounds in dose-response, on a scale from 0 to 1, on the average of three independent experiments. Compounds are associated in clusters, based on their classification category. Concentrations are rounded. The symbol # indicates compounds evaluated at a concentration of 0.85 mM instead of 1 mM. Panel B depicts dose-response curves for both infectivity (black) and cell number (red). Data are normalized to the average of DMSO- treated wells and represent mean ± SEM for n=3. Panel C depicts representative immunofluorescence images corresponding to one of the three dose-responses in Panel B. For each condition, the corresponding entire well is shown (4x objective)
[0014] FIG. 4 depicts Z-scores for ReFRAME collection primary screen. The left graph represents the Z-score of ATP levels after normalization to the median of each plate for all positive (APY0201) and negative (DMSO) controls as well as for non-infected cells, across all the screening plates. The correlation plot in the middle panel indicates the Z-score of each compound in the two replicates. The distribution of each compound according to the average of the Z-score of each replicate (right panel) is also represented. Each dot indicates the Z-score of each drug in each replicate of the screen (black dots). Values corresponding to DMSO (orange dots), APY0201 (cyan dots) and non-infected cells (purple dots) are also represented. R squared indicates the correlation coefficient for the replicates. [0015] FIG. 5 depicts gene set enrichment analysis (GSEA) of primary screening data according to the average Z' factor. GSEA enrichment plots of additional seven target clusters that are enriched were represented including estrogen receptor antagonist, GABA-A receptor modulator, ANGIOTENSIN II 1 antagonist, beta adrenoceptor antagonist, 5-hydroxytryptamine 3 receptor antagonist, serine protease inhibitor, and phosphodiesterase inhibitor (P -value < 0.05, FDR q-value < 0.25).
[0016] FIG. 6 depicts a bar plot of enriched terms across the enriched genes targeted by the compounds. The x-axis corresponds to -logl0(p value) while the y-axis indicates the enriched terms. The analysis was performed using Metascape.
[0017] FIG. 7 depicts a heatmap of treatment of Vero E6 cells. Vero E6 cells were pre-treated for 16 h with increasing concentrations of the indicated compound and then infected with SARS- CoV-2 at an MOI = 0.75 always in the presence of the compound. 24 h post-infection, cells were fixed and an immunofluorescence was performed, followed by imaging. For each condition, the total amount of cells stained with DAPI was calculated. Data are normalized to the average of DMSO-treated wells. The heatmap represents the normalized cell number of the indicated 17 compounds in dose-response, on a scale from 0 to 1, on the average of three independent experiments. Compounds are associated in clusters, based on their classification category. Concentrations are rounded. The # symbol indicates compounds were evaluated at a concentration of 0.85 mM instead of 1 mM.
[0018] FIG. 8 depicts dose-response curves obtained with the compounds identified herein. Vero E6 cells were pre-treated for 16 h with increasing concentrations of the indicated compound and then infected with SARS-CoV-2 at an MOI = 0.75 always in the presence of the compound. 24 h post-infection, cells were fixed and an immunofluorescence was performed. For each condition, the percentage of infection was calculated as the ratio between the number of infected cells stained for CoV NP and the total amount of cells stained with DAPI. Dose-response curves for both infectivity (black) and cell number (red) are shown. Data are normalized to the average of DMSO-treated wells and represent mean ± SEM for n=3.
[0019] FIG. 9, Panels A-E depicts that clofazimine inhibited a broad spectrum of human- pathogenic coronavirus (CoV) replication in human cellular models.
[0020] FIG. 10, Panels A-F depicts that clofazimine interferes with multiple steps of the virus life cycle.
[0021] FIG. 11, Panels A-D depicts transcriptional analysis of clofazimine treatment.
[0022] FIG. 12, Panels A-I depicts that prophylactic and therapeutic treatment with clofazimine reduces SARS-CoV-2 disease. [0023] FIG. 13, Panels A-I depicts that clofazimine exhibits antiviral synergy with remdesivir in vitro and in vivo.
[0024] FIG. 14 depicts cytotoxicity measurements of clofazimine in matching cells for antiviral evaluation.
[0025] FIG. 15, Panels A-D depicts exploration of possible effects of clofazimine on virus entry and replication.
[0026] FIG.16, Panels A-C depicts transcriptional analysis of clofazimine treatment on Caco-
2 cells.
[0027] FIG. 17, Panels A-E depicts transcriptional analysis of hamster lung tissues with clofazimine administration.
[0028] FIG. 18 depicts the histological score indicating lung pathological severity in each group.
[0029] FIG. 19 depicts clofazimine exhibits antiviral synergy with remdesivir for every combination of drug doses in vitro.
[0030] FIG. 20 depicts that clofazimine exhibits antiviral synergy with remdesivir in hamster lungs.
[0031] FIG. 21 depicts that clofazimine inhibited SARS-CoV-2 infection in human stem cell derived pneumocyte-like cells.
[0032] FIG. 22 depicts that clofazimine exhibits antiviral activity in the adenovirus hACE2 mouse model of SARS-CoV-2 infection.
DETAILED DESCRIPTION
[0033] Disclosed herein are compositions effective at treating coronavirus. In some embodiments, the compositions disclosed herein are effective at treating SARS coronavirus 2 (SARS-CoV-2), a novel coronavirus identified in December 2019 as the causative agent of a severe pneumonia-like coronavirus disease (COVID-19) outbreak in Wuhan in the Hubei province of China (X. Yang et ah, Clinical course and outcomes of critically ill patients with SARS-CoV-2 pneumonia in Wuhan, China: a single-centered, retrospective, observational study. Lancet Respir Med, (2020)). In some embodiments, the anti -infective compositions disclosed herein comprise a pharmaceutically acceptable carrier or excipient and a compound identified via a high-throughput cell-based screen for inhibitors of SARS-CoV-2 replication, profiling a library of known drugs encompassing approximately 13,000 clinical-stage or FDA-approved small molecules. In some embodiments, the anti -effective compositions disclosed herein comprise a pharmaceutically acceptable carrier or excipient and a compound selected from the group consisting of PIKfyve kinase inhibitor Apilimod, cysteine protease inhibitors (including MDL- 28170, Z LVG CHN2, VBY-825, and ONO 5334), and compound SL-11128. As most of these molecules have advanced into the clinic, the known pharmacological and human safety profiles of these molecules will accelerate preclinical and clinical evaluation for COVID-19 treatment. [0034] SARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA betacoronavirus, related to the viruses that caused severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) in 2002-2004 and 2012, 2013, respectively. The emergence of novel SARS-CoV-2 in 2019 has triggered an ongoing global pandemic of severe pneumonia-like disease designated as coronavirus disease 2019 (COVID-19). The World Health Organization (WHO) declared the rapidly spreading disease a pandemic on March 11th, 2020, and the disease has resulted in more than 1.98 million confirmed cases and more than 126,500 deaths have been reported worldwide in 213 countries. The WHO estimated the global case fatality rate (CFR) at 3.4% of those infected, though the number of actual infections is likely much higher than the number of reported cases. Typical COVID-19 symptoms include fever, cough, headache, anorexia, myalgia, and, in the most severe cases, viral-induce pneumonia accompanied by prolonged and systemic cytokine release. Notably, the levels of IL-6 have been reported to highly correlate with respiratory failure, and inhibitors are currently being pursued in clinical studies for the amelioration of virus-induced inflammatory responses. Patients with pre-existing chronic conditions such as hypertension, diabetes, and asthma, as well as those 65 years or older are at a higher risk of severe disease outcome (Center For Disease Control, https://www.cdc.gov/coronavirus/2019-ncov/need-extra-precautions/people-at-higher-risk.html (2020)). The underlying basis for these differential outcomes is yet unknown. Together, the still accelerating rate of community transmission and severity of the symptoms have placed an unprecedented burden on the medical supply chain and health care system in Italy, Spain, and the United States (E. Livingston, K. Bucher, Coronavirus Disease 2019 (COVID-19) in Italy. JAMA, (2020); A. Remuzzi, G. Remuzzi, COVID-19 and Italy: what next? Lancet, (2020)), with similar scenarios playing out or anticipated in other countries. While the FDA has recently granted the antimalarial drug hydroxychloroquine sulfate (also known as hydroxychloroquine) emergency use authorization (EUA) for a COVID-19 treatment, at present, there is no vaccine or approved antiviral therapeutic agent available (https://www.fda.gov/media/136538/download). Thus, there is an urgent and critical need to identify novel medical countermeasures both for prophylactic and treatment use. Since, the production of a vaccine for SARS-CoV-2 could to take 12-18 months, and de novo development of therapies usually requires 10-17 years, repositioning clinically evaluated drugs represents one of the most practicable approaches for the rapid development and deployment of treatments for emerging infectious diseases such as SARS-CoV- 2 [0035] Toward this end, many investigational clinical trials using repurposed drugs, including multiple antiviral and antimalarial medicines, have already been launched. Early results of a multicenter trial in China suggested that the antimalarial drug, chloroquine, may limit exacerbation of pneumonia and shorten viral replication and course of disease (J. Gao, Z. Tian,
X. Yang, Breakthrough: Chloroquine phosphate has shown apparent efficacy in treatment of COVID-19 associated pneumonia in clinical studies. Biosci Trends 14, 72-73 (2020)). A French study that used hydroxychloroquine (M. F. Marmor et al., Recommendations on Screening for Chloroquine and Hydroxychloroquine Retinopathy (2016 Revision) Ophthalmology 123, 1386- 1394 (2016)), together with azithromycin reported a significant reduction in viral load in COVID-19 patients when used in combination (P. GAUTRET et al., Hydroxychloroquine and Azithromycin as a treatment of COVID-19: preliminary results of an open-label non-randomized clinical trial. medRxiv, 2020.2003.2016.20037135 (2020)). However, a sufficiently powered case case-control study has not yet been reported, and thus it is unclear if there are therapeutic benefits of chloroquine administration to COVID-19 patients.
[0036] The repurposing of several approved antiviral therapies have all been the focus of clinical investigations, including HIV-1 protease inhibitors lopinavir/ritonavir (Kaletra, Aluvia by Abb Vie) (clinicaltrials.gov) hepatitis C virus protease inhibitor danoprevir (Ganovo, Ascletis Pharma) (H. Chen et al., First Clinical Study Using HCV Protease Inhibitor Danoprevir to Treat Naive and Experienced COVID-19 Patients. medRxiv, 2020.2003.2022.20034041 (2020)) and the influenza antiviral favipiravir (T-705, Avigan) (clinicaltrials.gov).
[0037] Most notably, ten clinical trials at more than 50 global sites are underway to investigate remdesivir (GS-5734), an investigational antiviral originally developed by Gilead Sciences to treat Ebola virus (clinicaltrials.gov). Remdesivir, an adenosine analogue is a viral RNA polymerase inhibitor that causes premature termination of transcription when incorporated into nascent viral RNA (T. K. Warren et al., Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys. Nature 531, 381-385 (2016)). The drug has demonstrated in vitro and in vivo activity in animal models against both MERS and SARS, as well as potent antiviral activity in Vero E6 against a clinical isolate of SARS-CoV-2 (M. Wang et al., Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019- nCoV) in vitro Cell Res 30, 269-271 (2020)). Pending results of several randomized (n > 308) clinical trials are expected to provide definitive insight into the efficacy of remdesivir as a therapeutic solution for the treatment of COVID-19. However, a well-powered randomized controlled trial has yet to demonstrate definitive evidence of antiviral efficacy for remdesivir or any other potential therapeutic [0038] While these repurposing strategies provide potentially rapid trajectories toward an approved treatment, an unbiased large-scale evaluation of known drugs and clinical candidates can identify therapeutic options that can be rapidly evaluated for the treatment of COVID-19 disease. Disclosed herein are methods and compositions utilizing a high-throughput repositioning screen using the ReFRAME (Repurposing, Focused, Rescue, and Accelerated Medchem) drug collection, a comprehensive open-access library of -13,000 clinical-staged or approved small molecules, to identify existing drugs that harbor antiviral activity against SARS- CoV-2 in a cell -based assay (J. Janes et al., The ReFRAME library as a comprehensive drug repurposing library and its application to the treatment of cryptosporidiosis. Proc Natl Acad Sci EISA 115, 10750-10755 (2018); Y. J. Kim et al., The ReFRAME library as a comprehensive drug repurposing library to identify mammarenavirus inhibitors. Antiviral Res 169, 104558 (2019)). The ReFRAME library is unique in that nearly 50% of the library was derived from custom synthesis, as commercially available sources of these clinical molecules were not available (Corsello, S. M. et al. The Drug Repurposing Hub: a next-generation drug library and information resource. Nat Med 23, 405-408, doi:10.1038/nm.4306 (2017)). Each of the molecules in this collection has been previously optimized for efficacy, safety, and bioavailability. Therefore, this enables leveraging of the considerable investment in research and development to compress the timeline required for drug discovery (Li, Y. Y. & Jones, S. J. Drug repositioning for personalized medicine. Genome Med 4, 27, doi: 10.1186/gm326 (2012)). In some embodiments, disclosed herein are anti-infective compositions comprising a compound selected from the group consisting of aldose reductase inhibitors, retinoic acid receptor antagonists, benzodiazepine receptor agonists, regulators of cholesterol homeostasis, and antimalarial compounds. As disclosed herein, validation studies further confirmed at least 17 known drugs to inhibit viral replication and dose response studies have characterized 6 known drugs that exhibit a range of effective concentrations (EC50) that are consistent with clinical efficacy. In some embodiments, disclosed herein are anti -infective compositions comprising a compound identified from the ReFRAME library, including PIKfyve kinase inhibitor Apilimod, cysteine protease inhibitors (including MDL-28170, Z LVG CHN2, VBY-825, and ONO 5334), calcium channel blockers (including AMG-2674 and Hanfangchin A), proton pump inhibitors (including YH-1238), G-protein receptor antagonists (including MLN-3897 and SDZ-62-434) and anti-viral agents that inhibit viral replication at concentrations were identified, and are expected to be achievable in patients.
[0039] Since the beginning of January 2020, an extraordinary number of investigational programs and clinical trials has been initiated in a concerted effort to identify therapeutics against the rapidly growing COVID-19 pandemic. Clinical trials using repurposed investigational or approved drugs such as remdesivir, favipiravir, lopinavir/ritonavir, hydroxychloroquine and others have been under investigation for treating COVID-19 patients (Q. Zhang, Y. Wang, C.
Qi, L. Shen, J. Li, Clinical trial analysis of 2019-nCoV therapy registered in China. J Med Virol, (2020); J. Gao, Z. Tian, X. Yang, Breakthrough: Chloroquine phosphate has shown apparent efficacy in treatment of COVID-19 associated pneumonia in clinical studies. Biosci Trends 14, 72-73 (2020); M. F. Marmor et al., Recommendations on Screening for Chloroquine and Hydroxychloroquine Retinopathy (2016 Revision). Ophthalmology 123, 1386-1394 (2016); P. GAUTRET et al., Hydroxychloroquine and Azithromycin as a treatment of COVID-19: preliminary results of an open -label non-randomized clinical trial. medRxiv, 2020.2003.2016.20037135 (2020); Z. Wang, X. Chen, Y. Lu, F. Chen, W. Zhang, Clinical characteristics and therapeutic procedure for four cases with 2019 novel coronavirus pneumonia receiving combined Chinese and Western medicine treatment. Biosci Trends 14, 64-68 (2020);
B. Cao et al., A Trial of Lopinavir-Ritonavir in Adults Hospitalized with Severe Covid-19. N Engl J Med, (2020); S. A. Kujawski et al., First 12 patients with coronavirus disease 2019 (COVID-19) in the United States. medRxiv, 2020.2003.2009.20032896 (2020); H. Chen et al., First Clinical Study Using HCV Protease Inhibitor Danoprevir to Treat Naive and Experienced COVID-19 Patients. medRxiv, 2020.2003.2022.20034041 (2020); H. Bian et al., Meplazumab treats COVID-19 pneumonia: an open-labelled, concurrent controlled add-on clinical trial. medRxiv, 2020.2003.2021.20040691 (2020)). However, most of the reported studies have been conducted in small cohorts and thus should be viewed as preliminary, with larger case-control clinical evaluations still pending. The elucidation of additional candidate therapies would greatly enhance the probability of identifying safe and efficacious treatment options and would enable the development of combinatorial regimens (“cocktails”), as in the case of HIV- 1 and Hepatitis C virus (HCV) current therapies. Combination therapy, using two or more clinically approved or evaluated molecules that act synergistically, may offer a powerful approach for treating SARS- CoV-2 infection, similar to the method of treatment for HIV-1 infection. Disclosed herein are methods and compositions comprising the results of a comprehensive repositioning analysis of nearly 13,000 known drugs from the ReFRAME collection. Some of the top candidate compounds identified include those described herein, used as single agents, or in combination therapy with other compounds identified herein or other antiviral compounds.
[0040] Large-scale surveys of existing drugs that may harbor antiviral activities can significantly facilitate such repositioning efforts. A recently reported SARS-CoV-2-human protein-protein interaction (PPI) analysis identified 332 viral-host interactions, 66 of which are potential druggable human host factors targeted by 69 known drugs that have FDA approval, however activities on SARS-CoV-2 replication were not reported so far (Nelson, E. A. et al. The phosphatidylinositol-3-phosphate 5-kinase inhibitor apilimod blocks filoviral entry and infection. PLoS Negl Trop Dis 11, e0005540, doi: 10.1371/journal. pntd.0005540 (2017)). An additional study reported testing a focused panel of 48 FDA approved drugs, previously shown to have antiviral activity against both SARS-CoV and MERS-CoV, also demonstrated that several known drugs harbor potent antiviral activities against SARS-CoV-2 (Qiu, S. et al. Ebola virus requires phosphatidylinositol (3,5) bisphosphate production for efficient viral entry. Virology 513, 17-28, doi: 10.1016/j.virol.2017.09.028 (2018)).
[0041] Disclosed herein is a high-throughput analysis of approximately 13,000 known drugs tested for activity against SARS-CoV-2 replication. The assay, conducted in Vero E6 cells derived from African Green Monkeys, was designed to capture multicycle replication, as the input of virus was low (MOI=0.01) and the endpoint measurement was captured 72 hours post infection. Although cell-based assays can be biased towards capturing inhibitors of viral entry, based on these parameters, this assay can elucidate inhibitors of each step of the viral life cycle. Of note, one potential limitation of Vero cells is that, due to species differences, pro-drugs that require the host cell machinery for processing into their active form, such as some nucleoside inhibitors, may not harbor the same potency as in human cells. Consistently, it is found that remdesivir inhibits SARS-CoV-2 replication 30-fold more potently in human cells in comparison to Vero E6 cells (data not shown).
[0042] The window of the viral-induced CPE in the assay was small (-2-2.5 fold), but robust and reproducible (as shown in Fig. 1, Panel B). Both the optimization using the LOP AC 1280© library and the first ReFRAME collection screen displayed acceptable Z’ factors (0.4 and 0.51, respectively). The replicate ReFRAME screen, for unknown reasons, possessed a degraded assay window (1.5-fold) and corresponding Z’ factor (0.19). However, the correlation between the two ReFRAME replications was high (R5=0.68), therefore, all datasets were leveraged to select molecules for further validation. In addition to 28 molecules from the LOP AC library, approximately 100 compounds were selected based on their activity in replicate 1, and 75 additional ones were selected from replicate 2. Next, 75 additional compounds were chosen based on the average activity ranking of the two ReFRAME replicates, and an additional 50 compounds that fell within enriched target/MOA sets based on GSEA analysis were also the focus of further study.
[0043] These selected compounds were validated using an endpoint that directly measures viral replication, in contrast to the indirect measurement of replication assessed by CPE. This was enabled through the development of a high-throughput immunofluorescence assay which monitors infection levels through assessment of SARS-CoV-2 N protein expression in individual cells. Importantly, this validation step enables the separation of molecules that function to block CPE (i.e. cell death), instead of direct effects on replication. This assay was found to be most robust at a 24-hour timepoint using an MOI of 0.75, thus, the antiviral activities of compounds were not interrogated under the original 72-hour timepoint conditions. Both the earlier timepoint and higher MOI likely biased the validation screen towards the confirmation of early stage inhibitors. Consistent with this hypothesis, it was found that several molecules with potent EC50s were only able to inhibit replication to approximately 50-60% at multiple high concentrations, including MLN-3897, YH-1238 and SL-11128 (as shown in Fig. 3, Panel A and Fig. 8). While this may represent the maximal ability of these molecules to suppress viral replication, alternatively, analysis of these molecules utilizing lower MOIs at later timepoint may reveal greater inhibition of infection. In addition, validation assays were conducted employing drug concentrations that were half of what was utilized in the screen (2.5uM vs. 5uM) and using a second isolate of SARS-CoV-2. The introduction of these stringencies during the validation step, as well as false positive activities from the HTS assay, likely account for -30% confirmation rate observed at this step of the analysis.
[0044] Among the validated compounds, the two marketed compounds clofazimine and acitretin were identified. Clofazimine is a lipophilic riminophenazine antibiotic, with described antimycobacterial and anti-inflammatory activity used for the treatment of leprosy. Main adverse effects include changes in skin pigmentation, nausea and vomiting. Its antibacterial activity is described to be related to its ability to bind to the bacterial DNA. Interestingly, this compound was also identified as a potent antiparasitic drug active against Cryptosporidium, during a repurposing screen of the ReFRAME library (Love, M. S. et al. A high-throughput phenotypic screen identifies clofazimine as a potential treatment for cryptosporidiosis. PLoS Negl Trop Dis 11, e0005373, doi: 10.1371/journal. pntd.0005373 (2017)). Further studies are required to understand the mechanism by which this molecule blocks the replication of this positive-strand RNA virus. Determination of the dose-response relationship for Clofazimine will enable the determination if the antiviral efficacy can be achieved at therapeutic doses. Acitretin is an approved orally bioavailable retinoid used for the treatment of psoriasis. Critically, retinoic acid agonists were highly enriched in the GSEA analysis (as shown in Fig. 2, Panel A) and five other compounds belonging to this class (LG-155, tretinoin, tamibarotene, tazarotene and RBAD) also confirmed secondary assay. Tretinoin and tamibarotene are both registered in Japan. It is currently unclear how activation of transcriptional program governed by retinoic acid receptors may impinge upon SARS-CoV-2 replication.
[0045] Six additional compounds confirmed in validation studies modulate targets that were enriched in the high-throughput screen, including aldose reductase inhibitor AL 3151, the benzodiazepine receptor agonists ZK-93426, zaleplon GR and pagoclone, and the two antimalarial drugs AQ-13 and Hanfangchin A. Antimalarial drugs have been reported to effectively block several viral infections (D’ Allesandro S et al., The Use of Antimalarial Drugs against Viral Infection, Microorganisms 8(1): 85 (2020)), including SARS-CoV-2 (Wang M et al., Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro, Cell Res 30(3):269-271 (2020)). Confirmation of compounds with membership enriched target classes underscore the importance of these molecular circuits in the regulation of SARS-CoV-2 replication and suggest that additional molecules that target over represented mechanisms should be evaluated for antiviral activities.
[0046] Among the 17 compounds validated to show dose-response relationship, 11 compounds harbored EC50 antiviral activities >800 nM, suggesting that additional preclinical studies will likely be required to determine if administration of these compounds can achieve sufficient systemic exposure to enable antiviral activity (as shown in Fig. 8). Six molecules were found to inhibit viral replication at EC50 concentration <500 nM. These include Z LVG CHN2, a preclinical tripeptide derivative that displays a broad-spectrum bactericidal activity. Previously, it was shown to suppress HSV replication potentially through inhibiting the enzymatic activity of HSV-encoded cysteine protease. Since SARS-CoV-2 encodes the cysteine proteinase known as 3CLpro, Z-LVG-CHN2 might target SARS-CoV-23CLpro to restrict its replication (Zhang, L. et al. Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved a- ketoamide inhibitors. Science, eabb3405, doi: 10.1126/science. abb3405 (2020)). Structure based docking of 3CLpro against Z-LVG-CHN2 can also be investigated. Moreover, Z-LVG-CHN2 potentially inactivates the host cysteine proteases that are required for SARS-CoV-2 infection to block viral infection. Another preclinical molecule that exhibits potent antiviral activity, MDL 28170, is a potent cell permeable calpain I and II inhibitor. Interestingly, MDL 28170 was previously found to impair infection by SARS-COV-1 and EBOV. Additionally, astemizole a registered anti -histamine HI receptor antagonist that also reported to have anti-malarial properties, inhibited replication at an EC50 concentration of 900 nM. However, due to fatal arrhythmias when given in high doses or in combination with certain other common drugs, astemizole has been withdrawn in many countries. Therefore, thorough safety studies are required to determine if there exists a sufficient therapeutic index for the treatment of acute SARS-CoV-2 infection.
[0047] MLN-3897 (AVE-9897) was determined to inhibit SARS-CoV-2 replication an estimated EC50 concentrations of 170 nm (as shown in Fig. 2), and the Cmax of the compound has been reported at 9.0 nM (10 mg QD). Therefore, additional in vivo studies will be required to determine if sufficient systemic concentrations can be reached to promote antiviral activities. This compound is an orally active chemokine CCR1 antagonist and was evaluated in phase-2 clinical studies for the treatment of rheumatoid arthritis (RA) and multiple sclerosis (MS). MLN3897 at a dose of 10 mg one daily was well tolerated. The mechanism by which CCR1 antagonism inhibits SARS-CoV-2 infection requires further investigation. However, it has been reported that CCR1 inhibition with MLN3897 potentially blocks ERK phosphorylation, leading to suppression of the mitogen-activated protein kinase (Raf/MEK/ERK) signal transduction pathway. Interestingly, Raf/MEK/ERK signal pathways are employed by SARS-CoV-1 to support its replication via multiple well -documented mechanisms, and thus this signaling axis may also represent a critical therapeutic target for host-directed SARS-CoV-2 antivirals.
[0048] Human cysteinyl cathepsins, including cathepsin B, cathepsin L, and cathepsin K, are required for the proteolytic processing of virally encoded proteins during infection. Human cysteinyl cathepsins, including cathepsin B, cathepsin L, and cathepsin K, are hijacked by multiple viruses, including SARS-CoV-1, influenza virus, and EBOV, to promote the proteolytic processing of virally encoded proteins (C. T. Pager, R. E. Dutch, Cathepsin L is involved in proteolytic processing of the Hendra virus fusion protein. Journal of virology 79, 12714-12720 (2005); Y. Mori et ah, Processing of Capsid Protein by Cathepsin L Plays a Crucial Role in Replication of Japanese Encephalitis Virus in Neural and Macrophage Cells. Journal of Virology 81, 8477-8487 (2007); M. Brecher et ah, Cathepsin cleavage potentiates the Ebola virus glycoprotein to undergo a subsequent fusion-relevant conformational change. J Virol 86, 364-372 (2012)). Notably, Cathepsin L is required for activation of membrane fusion, mediated by the SARS-CoV Spike (S) protein. Similar to SARS-CoV-1, SARS-CoV-2 requires endosomal acidification and acid-dependent endosomal proteases such as cathepsins for infection. Thus, blocking the activity of these proteases in the endosomal/lysosomal compartment can efficiently inhibit virus entry and uncoating at an early stage of viral replication. Inhibition of cathepsin L activity has been previously shown to efficiently suppress SARS-CoV-1 infection (G. Simmons et ah, Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry. Proceedings of the National Academy of Sciences of the United States of America 102, 11876- 11881 (2005)).
[0049] As disclosed herein, ONO 5334 (a cathepsin K inhibitor) and VBY-825 (a reversible cathepsin protease inhibitor) were identified in the screen to inhibit SARS-CoV-2 infection in a dose-dependent manner, however it is not currently understood if the observed antiviral activities are due to inhibition of proteolysis of viral or host cellular proteins. ONO 5334 was in phase II clinical trials for the treatment of osteoporosis as a potent inhibitor of Cathepsin K with Ki value of 0.1 nM. It also possesses potent inhibitory activity for other cathepsins such as Cathepsin B (Ki: 32 nM), Cathepsin L, (Ki: 17 nM), Cathepsin S, (Ki: 0.83 nM) (Eastell, R. et al. Safety and efficacy of the cathepsin K inhibitor ONO-5334 in postmenopausal osteoporosis: the OCEAN study. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 26, 1303-1312, doi:10.1002/jbmr.341 (2011); Eastell, R. et al. Effect of ONO-5334 on bone mineral density and biochemical markers of bone turnover in postmenopausal osteoporosis: 2-year results from the OCEAN study. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 29, 458-466, doi: 10.1002/jbmr.2047 (2014). ONO 5334 harbored an antiviral EC50 of -500 nM, which is in range of a previously reported 85% activity observed at 100 nM in an osteoclast- mediated bone resorption assay. Importantly, the Cmax of this compound is 1.6 uM (300 mg QD), and treatment with ONO-5334 was well tolerated up to daily doses of 300 mg and for up to 12 months without any clinically relevant safety concerns. 0N05334 reached phase II clinical trials for the treatment of osteoporosis in postmenopausal women, but development was discontinued to an unfavorable competitive landscape. VBY-825, which is in preclinical development, is another cathepsin inhibitor harboring potential antiviral activities against SARS- CoV-2 with IC50 of -300 nM, and it shows high potency against cathepsins B, L, S and V in vitro. VBY-825 is another cathepsin inhibitor discovered in the screening disclosed herein. VBY-825 shows high potency against cathepsins B, L, S and V in both mouse and human cell lines. In a preclinical study, subcutaneous injection of VBY-825 at a dose of 10 mg/kg/day could significantly decrease tumor burden and tumor number in a model of pancreatic islet cell carcinogenesis (Elie, B. T. et al. Identification and pre-clinical testing of a reversible cathepsin protease inhibitor reveals anti-tumor efficacy in a pancreatic cancer model. Biochimie 92, 1618- 1624, doi:10.1016/j. biochi.2010.04.023 (2010)). The identification of VBY-825 as an effective molecule against SARS-CoV-2 encourages the therapeutic application of cathepsin inhibitors in the treatment of COVID-19.
[0050] Overall, the identification of VBY-825 and ONO 5334 as effective antiviral molecules against SAR-COV-2 supports the repositioning of these, and potentially additional protease inhibitors, for the treatment of COVID-19 disease.
[0051] Apilimod, a specific PIKfyve kinase inhibitor, was found to inhibit SARS-CoV-2 replication at an EC50 concentrations of 23 nM (as shown in Fig. 3). More importantly, apilimod is found to be well tolerated in humans showing a desirable safety profile at doses of <125 mg BID, and the Cmax of this compound is 0.265 +/- 0.183 mM (70 mg QD). These data indicate that therapeutic dosing of apilimod in patients can achieve concentrations that are likely to promote antiviral activity. Apilimod was evaluated in phase II clinical trials for the treatment of active Crohn's disease and rheumatoid arthritis (RA), and an additional phase II trials for the oral treatment of common variable immunodeficiency (CVID) but did not show efficacy for either indication. In 2019, orphan drug designation was granted to apilimod in the U.S. for the treatment of follicular lymphoma. Notably, it has been reported that apilimod efficiently inhibit Ebola virus (EBOV), Lassa virus (LASV), and Marburg virus (MARV) virus infection in human cell lines, underscoring its potential broad-spectrum antiviral activity. The underlying mechanism for the inhibition of SARS-CoV-2 infection by apilimod is currently not known. However, since PIKfyve predominately resides in early endosomes and plays an essential role in maintenance of endomembrane homeostasis, apilimod likely blocks viral low pH-dependent entry through inhibition of the lipid kinase activity of PIKfyve.
[0052] Aldose reductase (AR) is a monomeric NADPH-dependent cytosolic enzyme, involved in various physiological processes via regulation of polyol pathway of glucose metabolism, oxidative stress signaling, as well as lipid aldehyde mediated cell signaling (K. V. Ramana, ALDOSE REDUCTASE: New Insights for an Old Enzyme. Biomol Concepts 2, 103-114 (2011)). Inhibition of aldose reductase could prevent inflammatory complications as well as oxidative stress-induced cell death. More importantly, in hepatitis C virus (HCV)-infected patients, aldose reductase activity is significantly enhanced, indicating that HCV potentially manipulates the host metabolism to promote its infection and transmission (N. Semmo, T.
Weber, J. R. Idle, D. Bey oglu, Metabolomics reveals that aldose reductase activity due to AKR1B10 is upregulated in hepatitis C virus infection. Journal of viral hepatitis 22, 617-624 (2015)). As disclosed herein, a cluster of aldose reductase inhibitors with potent antiviral activity against SARS-CoV-2 in Vero E6 cells were identified in primary screening, two of which (AL- 3152 and tolrestat) were further confirmed in the dose-response analysis using an immunofluorescence assay. These results implicate targeting aldose reductase as a considerable therapeutic strategy for the prevention or treatment of SARS-CoV-2 infection.
[0053] The endosomal system functions as an intracellular sorting network for homeostatic regulation. It is also hijacked by many viruses, including SARS-CoV-2, to enter target cells. Thus, manipulation of the cellular endosomal machinery with small molecules can slow down virus replication and allow the immune system to develop defenses. As disclosed herein, small molecules selectively regulating endosomal recycling or trafficking, such as chloroquine derivatives and PIKfyve inhibitors were significantly enriched in primary screening (Qiu, Z. et al. Endosomal proteolysis by cathepsins is necessary for murine coronavirus mouse hepatitis virus type 2 spike-mediated entry. Journal of virology 80, 5768-5776, doi:10.1128/JVI.00442-06 (2006); Jethwa, N. et al. Endomembrane PtdIns(3,4,5)<em>P</em><sub>3</sub> activates the PI3K-Akt pathway. Journal of Cell Science 128, 3456-3465, doi: 10.1242/jcs.172775 (2015); de Lartigue, J. et al. PIKfyve regulation of endosome-linked pathways. Traffic (Copenhagen, Denmark) 10, 883-893, doi:10.1111/j.l600-0854.2009.00915.x (2009)). PIKfyve, a lipid and protein kinase that predominately resides in the early endosomes, plays an essential role in maintenance of endomembrane homeostasis. Apilimod, a 1,3,5-triazine derivative was identified as an antagonist of PIKfyve by specifically inhibiting its lipid kinase activity. It has been shown that apilimod efficiently inhibits Ebola vims (EBOV), Lassa vims (LASV), and Marburg vims (MARV) infection in primary human macrophage cells with an IC50 of 10 nM. Disclosed herein is the novel finding that apilimod dramatically decreases pathogenic human coronavims infection, including SARS-CoV-1, SARS-CoV-2 and MERS-CoV in both Vero E6 cells and Huh-7 cells. These results unravel the critical role of PIKfyve in regulating coronavims endocytosis and offer another target for therapeutic intervention.
[0054] In some embodiments, disclosed herein are anti-infective compositions comprising a pharmaceutically acceptable carrier or excipient and an effective amount of a PIKfyve inhibitor. In some embodiments, disclosed herein are anti-infective compositions comprising a pharmaceutically acceptable carrier or excipient and an effective amount of apilimod. In some embodiments, disclosed herein are anti -infective compositions comprising a pharmaceutically acceptable carrier or excipient and an effective amount of YM201636.
[0055] MDL 28170 is a potent cell permeable calpain I and II inhibitor that inhibits calpain with Ki values of lOnM and cathepsin B with Ki values of 25 nM. Treatment with MDL 28170 resulted in severely impaired infection of SARS-CoV-1 and EBOV in Vero E6 cells (Schneider, M. et al. Severe acute respiratory syndrome coronavims replication is severely impaired by MG132 due to proteasome-independent inhibition of M-calpain. J Virol 86, 10112-10122, doi: 10.1128/jvi.01001-12 (2012); Hoffmann, M. et al. Analysis of Resistance of Ebola Vims Glycoprotein-Driven Entry Against MDL28170, An Inhibitor of Cysteine Cathepsins. Pathogens 8, 192, doi:10.3390/pathogens8040192 (2019)). The discovery disclosed herein that MDL 28170 is an anti-SARS-CoV-2 dmg suggests that calpain is involved in the SARS-CoV-2 life cycle. [0056] Of the validated anti-SARS-CoV-2 dmgs, multiple molecules that regulate the ion channel activity such as YH-1238, Hanfangchin A, and AMG-2674 displayed potential antiviral activity against SARS-CoV-2 in a dose-response manner. Hanfangchin A is a calcium channel blocker which could reduce intracellular Ca2+ level upon treatment. Impairing calcium channel with treatment of Hanfangchin A inhibited SARS-CoV-2 infection, indicating the beneficial role of increased intracellular Ca2+ level for viral infection. In addition, since cellular or viral factors function in a Ca2+-dependent manner, intracellular Ca2+ might regulate the activity of cellular dependence factors or viral proteins of SARS-CoV-2 to facilitate viral infection. It has been reported that SARS-CoV 3 A and E protein forms an ion channel and modulates vims release (Verdi a-Baguena, C. et al. Coronavims E protein forms ion channels with functionally and structurally-involved membrane lipids. Virology 432, 485-494, doi:10.1016/j.virol.2012.07.005 (2012); Lu, W. et al. Severe acute respiratory syndrome-associated coronavims 3a protein forms an ion channel and modulates virus release. Proceedings of the National Academy of Sciences 103, 12540-12545, doi: 10.1073/pnas.0605402103 (2006)). Given the sequence similarity between SARS-CoV-1 and SARS-CoV-2 (Zhou, P. et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270-273, doi:10.1038/s41586-020-2012-7 (2020)), this compound may inhibit viral replication by targeting the 3 A or E protein through inhibiting ion channel activity.
[0057] Retinoic acid (RA) signaling plays an important role in controlling the cellular development, homeostasis, and metabolism, particularly in the lungs (Hind, M. & Maden, M. Retinoic acid induces alveolar regeneration in the adult mouse lung. European Respiratory Journal 23, 20-27, doi: 10.1183/09031936.03.00119103 (2004)). It binds to retinoic acid receptor (RAR), a nuclear transcriptional regulator to modulate the expression of specific genes involved in glucose and lipid metabolism. A number of studies have shown that retinoic acid receptor (RAR) agonists exhibit broad antiviral activities against a wide-range of viruses including hepatitis B virus (HBV) (B. Li et al., Identification of Retinoic Acid Receptor Agonists as Potent Hepatitis B Virus Inhibitors via a Drug Repurposing Screen. Antimicrobial agents and chemotherapy 62, e00465-00418 (2018)), human immunodeficiency virus type 1 (HIV-1) (T. M. Hanley, H. L. B. Kiefer, A. C. Schnitzler, J. E. Marcello, G. A. Viglianti, Retinoid-Dependent Restriction of Human Immunodeficiency Virus Type 1 Replication in Monocytes/Macrophages. Journal of Virology 78, 2819-2830 (2004)), and hepatitis C virus (HCV) (B. R. Bang et al., Regulation of Hepatitis C Virus Infection by Cellular Retinoic Acid Binding Proteins through the Modulation of Lipid Droplet Abundance. J Virol 93, (2019); Y. Murakami et al., Retinoids and rexinoids inhibit hepatitis C virus independently of retinoid receptor signaling. Microbes and infection 16, 114-122 (2014)). As disclosed herein, multiple compounds belonging to the family of retinoic acid receptor (RAR) agonists are able to effectively inhibit SARS-CoV-2 replication in a dose-dependent manner, indicating that RA signaling is involved in the SARS-CoV-2 life cycle.
[0058] Translocator protein (TSPO) is a mitochondrial outer membrane protein previously known as the peripheral benzodiazepine receptor. TSPO has been reported to trigger the degradation of the HIV-1 envelope glycoprotein by interfering with the glycoprotein folding process (T. Zhou, D. A. Frabutt, K. W. Moremen, Y. H. Zheng, ERManI (Endoplasmic Reticulum Class I alpha-Mannosidase) Is Required for HIV-1 Envelope Glycoprotein Degradation via Endoplasmic Reticulum -associated Protein Degradation Pathway. J Biol Chem 290, 22184-22192 (2015)). TSPO agonists, namely benzodiazepine receptor agonists, show a high enrichment score in GSEA analysis, as shown in Fig. 2, panel B, indicating a role in inhibiting viral protein biosynthesis through degradation of the proteins by TSPO. Since high levels of TSPO gene expression are found in the lung (Fig. 2, panel B), benzodiazepine receptor agonists may have potential for clinical use as antivirals against SARS-CoV-2. The compounds disclosed herein will be useful against SARS-CoV-2 in relevant human cells ex vivo , animal models in vivo, as well as directly in clinical trials for the set of FDA approved drugs, either as mono-therapeutics or in combination with a potent antiviral such as remdesivir.
[0059] Disclosed herein, in some embodiments, are methods useful for reducing the likelihood of a pathogenic infection in an individual or reducing transmission of a pathogen to other individuals comprising administering an anti-infective composition, wherein the anti-infective composition comprises an effective amount of a compound listed in Table 1 and a pharmaceutically acceptable carrier or excipient, and wherein the anti-infective composition reduces the risk of absorption, transmission, or function of a pathogen in the individual or transmission of a pathogen to another individual. In some embodiments, the anti-infective composition comprises an effective amount of a compound selected from the group consisting of nelfmavir mesylate hydrate, MDL 28170, GR 127935 hydrochloride hydrate, 8-(3- Chlorostyryl)caffeine, Apilimod, Clofazimine, Z LVG CHN2, AQ-13, Hanfangchin A, astemizole, SL-11128, ELOPIPRAZOLE, SDZ-62-434, analog of MLN-3897, SB-616234-A, YH-1238, VBY-825, ONO 5334, and AMG-2674. In some embodiments, the anti-infective composition comprises an effective amount of a compound selected from the group consisting of apilimod, VBY-825, ONO 5334, MLN-3897, SDZ-62-434, YH-1238, SB-616234-A, elopiprazole, and astemizole. In some embodiments, the EC50 range (mM) is 0.01 - 0.03 for apilimod, -0.3 for VBY-825, 0.3-1 for ONO 5334, 0.3-1 for MLN-3897, -1 for SDZ-62-434, 1- 2.5 for YH-1238, 1-2.5 for SB-616234-A, 1-2.5 for elopiprazole, and 1.5-2.5 for astemizole Table 1. Compounds
Figure imgf000020_0001
Certain Terminology
[0060] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not intended to be limited solely to the recited items. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
[0061] The terms “effective amount” or “therapeutically effective amount,” as used herein, generally refer to a sufficient amount of an agent or a compound ( e.g ., the anti-infective composition described herein) which will relieve, to some extent, or reduce the likelihood of the occurrence of one or more of the symptoms of the disease or condition being treated. The result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. The terms “effective amount” or “therapeutically effective amount” typically include, for example, a prophylactically effective amount. For example, a “prophylactically effective amount” is the amount of the anti-infective composition described herein that is required to reduce the risk of absorption, transmission, or function of a pathogen in an individual or transmission of a pathogen to another individual.
[0062] The terms “about” or “approximately,” as used herein, generally mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part upon how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, and more preferably within 2-fold of a value.
Compositions
[0063] Disclosed herein, in some embodiments, are methods useful for reducing the likelihood of a pathogenic infection in an individual or reducing transmission of a pathogen to other individuals comprising administering an anti-infective composition, wherein the anti-infective composition comprises a compound listed in Table 1 and a pharmaceutically acceptable carrier or excipient, wherein the anti -infective composition reduces the risk of absorption, transmission, or function of a pathogen in the individual or transmission of a pathogen to another individual. [0064] In some embodiments, the pathogen includes, but is not limited to, respiratory syncytial virus (RSV), metapneumovirus (MPV), rhinovirus, influenza virus, parainfluenza virus, coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), norovirus, rotavirus, metapneumovirus (MPV), hepatitis A virus, adenovirus, astrovirus, S. aureus , methicillin-resistant S. aureus (MRSA), vancomycin-resistant enterococci (VRE), Enterococcus spp., Enterobacter spp., C. difficile , Campylobacter, E. faecali, E.faecium , or Salmonella.
[0065] Each year, worldwide epidemics result in substantial morbidity and death, with the young and elderly representing the majority of this mortality. Coronavirus is transmitted through direct contact with infected individuals, by contact with coronavirus-contaminated objects, and through inhalation of virus-laden, aerosolized respiratory droplets. Vaccination is currently the most effective method to prevent viral infection, but certain coronavirus strains do not have effective vaccines.
Combination Therapies
[0066] Disclosed herein, in some embodiments, are methods useful for reducing the likelihood of a pathogenic infection in an individual or reducing transmission of a pathogen to other individuals comprising administering an anti-infective composition to an individual, wherein the anti-infective composition comprises a compound listed in Table 1 and a pharmaceutically acceptable carrier or excipient, wherein the anti-infective composition reduces the risk of absorption, transmission, or function of a pathogen in the individual or transmission of a pathogen to another individual.
[0067] In some embodiments, the anti-infective composition further comprises an additional anti-infective agent. In some embodiments, the additional anti-infective agent is an anti-viral agent selected from the group consisting of entry-inhibiting drugs (including enfuvirtide), uncoating inhibiting drugs (including amantadine, rimantadine, and pleconaril), reverse transcriptase inhibiting drugs (including acyclovir, zidovudine, and lamivudine), antisense drugs (including fomivirsen), ribozyme drugs, protease inhibitors, assembly inhibiting drugs (including rifampicin), and release inhibiting drugs. In some embodiments, the additional anti-infective agent is an anti-viral agent. In some embodiments, the anti-viral agent is selected from the group consisting of Abacavir, Acyclovir (Aciclovir), Adefovir, Amantadine, Ampligen, Amprenavir (Agenerase), Arbidol, Atazanavir, Atripla, Balavir, Baloxavir marboxil (Xofluza), Biktarvy, Boceprevir (Victrelis), Cidofovir, Cobicistat (Tybost), Combivir, Daclatasvir (Daklinza), Darunavir, Delavirdine, Descovy, Didanosine, Docosanol, Dolutegravir, Doravirine (Pifeltro), Ecoliever, Edoxudine, Efavirenz, Elvitegravir, Emtricitabine, Enfuvirtide, Entecavir, Etravirine (Intelence), Famciclovir, Fomivirsen, Fosamprenavir, Foscamet, Fosfonet, Fusion inhibitor, Ganciclovir (Cytovene), Ibacitabine, Ibalizumab (Trogarzo), Idoxuridine, Imiquimod, Imunovir, Indinavir, Inosine, Integrase inhibitor, Interferon type I, Interferon type II, Interferon type III, Interferon, Lamivudine, Letermovir (Prevymis), Lopinavir, Loviride, Maraviroc, Methisazone, Moroxydine, Nelfmavir, Nevirapine, Nexavir, Nitazoxanide, Norvir, Nucleoside analogues, Oseltamivir (Tamiflu), Peginterferon alfa-2a, Peginterferon alfa-2b, Penciclovir, Peramivir (Rapivab), Pleconaril, Podophyllotoxin, Protease inhibitor (pharmacology), Pyramidine, Raltegravir, Remdesivir, Reverse transcriptase inhibitor, Ribavirin, Rilpivirine (Edurant), Rimantadine, Ritonavir, Saquinavir, Simeprevir (Olysio), Sofosbuvir, Stavudine, Synergistic enhancer (antiretroviral), Telaprevir, Telbivudine (Tyzeka), Tenofovir alafenamide, Tenofovir disoproxil, Tenofovir, Tipranavir, Trifluridine, Trizivir, Tromantadine, Truvada, Valaciclovir (Valtrex), Valganciclovir, Vicriviroc, Vidarabine, Viramidine, Zalcitabine, Zanamivir (Relenza), and Zidovudine.
Formulations Carriers and Excipients
[0068] The compositions disclosed herein are formulated in any suitable manner for administration. Any suitable technique, carrier, and/or excipient is contemplated for use with the compositions disclosed herein. Non-limiting examples of cosmetic, dermatological, or pharmaceutically acceptable carriers and excipients suitable for formulation can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington ’s Pharmaceutical Sciences , Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms , Marcel Decker, New York, N.Y., 1980; Pharmaceutical Dosage Forms and Drug Delivery Systems , Eighth Ed. (Lippincott Williams & Wilkins 2004); and Muller, R.H., etal. , Advanced Drug Delivery Reviews 59 (2007) 522-530, each of which is incorporated by reference in its entirety. The compositions described herein can be administrable to a subject in a variety of ways by multiple administration routes, including but not limited to, inhalation, oral, parenteral ( e.g. , intravenous, subcutaneous, intramuscular, intramedullary injections, intrathecal, direct intraventricular, intraperitoneal, intralymphatic, intranasal injections), intranasal, buccal, topical or transdermal administration routes. The compositions described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations. For administration by inhalation, the compositions described herein can be formulated for use as an aerosol, a mist or a powder. In some embodiments, the compositions described herein are delivered in the form of an aerosol spray from a pressurized container or dispenser that contains a suitable propellant, for example a gas such as carbon dioxide, or a nebulizer. For buccal or sublingual administration, the compositions may take the form of tablets, lozenges, or gels formulated in a conventional manner. In some embodiments, the compositions described herein can be prepared as transdermal dosage forms. In some embodiments, the compositions described herein can be formulated into a pharmaceutical composition suitable for intramuscular, subcutaneous, or intravenous injection. In some embodiments, the compositions described herein can be administered topically and can be formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams or ointments. In some embodiments, the compositions described herein can be formulated in rectal compositions such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly suppositories, or retention enemas.
[0069] In some embodiments, the pharmaceutically acceptable carriers or excipients disclosed herein include, but are not limited to one or more: pH modifying agent ( e.g ., buffering agents), stabilizing agents, thickening agents, colorant agents, preservative agents, emulsifying agents, solubilizing agents, antioxidant agents, or any combination thereof. Other suitable compounds contemplated herein and within the knowledge of a practitioner skilled in the relevant art are found in the Handbook of Pharmaceutical Excipients , 4th Ed. (2003), the entire content of which is incorporated by reference herein.
[0070] In some embodiments, the compositions disclosed herein comprise one or more preservatives. The preservative, when utilized, is in an amount sufficient to extend the shelf-life or storage stability, or both, of the topical formulations disclosed herein. Exemplary preservatives include, but are not limited to: tetrasodium ethylene-diamine tetraacetic acid (EDTA), methyl, ethyl, butyl, and propyl parabens, benzophenone-4, methylchloroisothiazolinone, methylisothiazolinone, sodium benzoate, paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenylethylalcohol, dehydroacetic acid, sorbic acid, benzalkonium chloride (BKC), benzethonium chloride, phenol, phenylmercuric nitrate, and thimerosal.
EXAMPLES
[0071] The following examples are illustrative and non-limiting to the scope of the compositions, methods, and formulations described herein.
Example 1 Cells and Viruses
[0072] SARS-CoV-2 HKU-OOla strain was isolated from the nasopharyngeal aspirate specimen of a laboratory-confirmed COVID-19 patient in Hong Kong (K. K. To et ah,
Consistent detection of 2019 novel coronavirus in saliva. Clin Infect Dis, (2020)). SARS-CoV-2 USA-WA1/2020 strain, isolated from an oropharyngeal swab from a patient with a respiratory illness who developed clinical disease (COVID-19) in January 2020 in Washington, USA, was obtained from BEI Resources (NR-52281). The virus was propagated in Vero E6 (ATCC® CRL-1586™) cells transfected with exogenous ACE2 and TMPRSS2 and kept at -80 °C in aliquots. Plaque forming unit (PFU) and TCID50 (Median Tissue Culture Infectious Dose) assays were performed to titrate the cultured virus. Vero E6 and Huh-7 cells (Apath LLC, Brooklyn) were maintained in Dulbecco’s modified eagle medium (DMEM, Gibco) supplemented with 10 % heat-inactivated fetal bovine serum (FBS, Gibco), 50 U/mL penicillin, 50 pg/mL streptomycin, 1 mM sodium pyruvate (Gibco), 10 mM HEPES (Gibco), and IX MEM non- essential amino acids solution (Gibco). Huh7 cells were transfected with PLVX-ACE2 and PLX304-TMPRSS2 prior to infection. All experiments involving live SARS-CoV-2 followed the approved standard operating procedures of the Biosafety Level 3 facility at the University of Hong Kong (S. Yuan et ak, SREBP-dependent lipidomic reprogramming as a broad-spectrum antiviral target. Nat Commun 10, 120 (2019)) and Sanford Burnham Prebys Medical Discovery Institute.
Chemical Libraries
[0073] The LOP AC® library is a collection of 1,280 pharmacologically active compounds, covering all the major target classes, including kinases, GPCRs, neurotransmission and gene regulation (Sigma). The ReFRAME (Repurposing, Focused Rescue, and Accelerated Medchem) library contains approximately 13,000 high-value molecules assembled by combining three databases (Clarivate Integrity, GVK Excelra GoStar and Citeline Pharmaprojects) for fast-track drug discovery. As shown in Fig. 1, Panel A, this library contains US Food and Drug Administration (FDA)-approved/registered drugs (-39%), investigational new drugs (-58%), and preclinical compounds (-3%).
Drug Screening
[0074] Compounds from the LOPAC®1280 and ReFRAME library were transferred into F- BOTTOM, pCLEAR®, BLACK 384-well plates (Greiner) using an Echo 550 Liquid Handler (Labcyte). All compounds were diluted in culture media to a final concentration of 5 mM during screening. Briefly, Vero E6 cells were seeded in 384-well plates, on top of pre-spotted compounds, at a density of 3,000 cells per well in 40 mΐ using a microFlo™ select dispenser (BioTek Instruments). Sixteen hours post-seeding, the cells were infected by adding 10 mΐ of SARS-CoV-2 per well at an MOI of 0.01. Cytopathic effect (CPE) was indirectly quantified as the presence of ATP in live cells by using the CellTiter-Glo (Promega) luminescent cell viability assay at 72 h post-infection. Data were normalized to the median of each plate. For the ReFRAME library, the Z-score was calculated based on the log base 2 fold change (Log2FC) with the average and standard deviation of each plate. The screen was performed in duplicate by running the assay in parallel for the LOPAC®1280 library or as two independent experiments for the ReFRAME collection. Twenty-eight compounds from the LOPAC®1280 were selected according to the cutoff of >5*Stdev Log2FC and included in a dose-response confirmation assay. Compounds from the ReFRAME collection were ranked according to their Z-score. The top 100 hits from each replicate were selected (25 overlapping). Seventy -five additional hits were selected according to their ranking based on the average Z-score. The last 48 hits were selected according to drug target and pathway enrichment analysis. The 298 selected hits were included in a dose-response confirmation assay.
Dose Response Curves, IC50 Calculations, and Orthogonal Validation [0075] The selected hits were further validated by immunofluorescence in an 8-point dose response experiment to determine EC so and CC50 through a cell-based high-content imaging assay, labeling the viral nucleoproteins within infected cells. Three thousand Vero E6 cells were added into 384-well plates pre-spotted with compounds, in a volume of 40 mΐ. The final concentration of compound ranged from 1.1 nM to 2.5 mM. Sixteen hours post-seeding, 10 mΐ of SARS-CoV-2 USA-WA1/2020 were added to each well, at an MOI of 0.75. Twenty-four hours post-infection, cells were fixed with 5% paraformaldehyde for 4 hours and permeabilized with 0.5% Triton X-100 for 5 minutes. After blocking with 3% bovine serum albumin (BSA) for 30 mins, the cells were incubated for 1 hour at room temperature with rabbit-anti-SARS-CoV-1 nucleoprotein serum, which exhibits strong cross-reactivity with SARS-CoV-2. After two washes with phosphate-buffered saline (PBS), the cells were incubated with Alexa Fluor 488- conjugated goat-anti -rabbit IgG (Thermo Fisher Scientific, USA) for 1 hour at room temperature. After two additional washes, PBS supplemented with 0.1 pg/ml antifade-4 6-diamidino-2- phenylindole (DAPI) (BioLegend, USA) was added to the cells at least 30 minutes before imaging. Images were acquired using the Celigo Image Cytometer (Nexcelom). The assay results and data analysis enabled the determination of infectivity and viability/cytotoxicity.
Based on all infectivity and cytotoxicity values, a 4-parameter logistic non-linear regression model was used to calculate IC50 and CC50 concentration values.
Expression analysis
[0076] Gene expression analysis was conducted using single-cell RNA profiling data of samples from four macro-anatomical locations of human airway epithelium in healthy living volunteers, as shown in Fig. 2, Panel B. For each gene, the fraction of cells with non-zero expression values was calculated in nasal, tracheal, intermediate, and distal samples from multiple donors. Values for each sampling location were averaged across donors. To analyze gene expression levels in different cell types, the fractions of cells with non-zero expression values were determined in all cells of a given cell type across samples. Cell types with a total of less than 250 cells detected were excluded from analysis. Clustered heat maps were generated in R using the pheatmap and viridis packages.
Example 2
Optimization of a High-throughput Screen for Inhibitors of SARS-CoV-2 Replication [0077] An efficient way to identify antiviral candidates against an emergent virus, such as SARS-CoV-2, that can be rapidly evaluated in clinical trials, is to repurpose clinically assessed drugs. Given the urgent need for therapeutics to treat SARS-CoV-2 infection, a high-throughput assay was developed to screen a comprehensive repurposing library. Vero E6 cells, kidney epithelial cells derived from an African green monkey, have been shown to be highly permissive to SARS-CoV-2 infection (S. Matsuyama et ah, Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells. Proc Natl Acad Sci U S A 117, 7001-7003 (2020)) and viral replication can be assessed through indirect measurement of viral-induced cytopathic effects (CPE) (W. B. Park et ah, Virus Isolation from the First Patient with SARS-CoV-2 in Korea. J Korean Med Sci 35, e84 (2020)). A clinical isolate of the SARS-CoV-2 virus (SARS-CoV-2 HKU-OOla) was utilized for assay development and screening. The assay parameters, including cell seeding density, multiplicity of infection (MOI), and timepoints were optimized in Vero E6 cells by measuring virus-induced CPE using CellTiter-glo in a 384-well format. Maximal assay windows and reproducibility were found at conditions of 3,000 cells/well, infection at a MOI of 0.01, and CPE measurement at 72 hours post-infection (Fig. 1, Panel A). Importantly, these conditions were established to enable viral multicycle replication, and thus elucidate compounds that inhibit both early and late stages of viral replication.
[0078] In an effort to assess robustness and reproducibility of the optimized assay in a high- throughput screening (HTS) configuration, the assay was initially evaluated utilizing a collection of 1,280 known bioactive molecules (Library of Pharmacologically Active Compounds; LOPAC®1280; (Sigma)). Upon initiation of the screening effort, no compound with activity against SARS-CoV-2 in Vero E6 cells had been reported. Based on studies that indicate that inhibition of the PIKfyve kinase inhibits low pH-dependent entry of viruses such as Ebola (Nelson EA, et ah, The phosphatidylinositol-3-phosphate 5-kinase inhibitor apilimod blocks filoviral entry and infection, PLoS Negl Trop Dis 1 l(4):e0005540 (2017)), the activity of the PIKfyve kinase inhibitor APY0201 was evaluated against SARS-CoV-2. As shown in Fig. 1, Panel B and Panel C, compared to vehicle (DMSO), cells dosed with 1 mM APY0201 harbored a 2.5X increase in cell viability, reflecting reduced cytopathic effect (CPE) after viral challenge, which was comparable to non-infected control. These data confirmed the antiviral activity of APY0201 against SARS-CoV-2, and enabled establishment of a reliable assay window based on the activity of a positive control. Vero E6 cells were seeded in 384-well plates with pre-spotted compounds from the LOP AC® library at a 5mM (final) concentration. After 16 hours, cells were infected with SARS-CoV-2 (MOI = 0.01) in the presence of compound, and at seventy -two hours post-infection, CPE induced by the virus quantified cell viability. Duplicates of each plate were run in parallel and the value corresponding to each well was normalized to the median of each plate and used to calculate the log base 2 of the fold change (Log2FC). Based on the activity of APY0201, the average Z' factor for the 5 plates in duplicate was 0.4, and the correlation coefficient for the duplicates (R2) was 0.81 (as shown in Fig. 4). Using the selection criteria of 5 times the standard deviation of the Log2FC calculated over all DMSO treated samples (5· StdDev[Log2FCuMSo] = 0.35), 28 compounds were selected for further study including the HIV protease inhibitor nelfmavir mesylate hydrate, the calpain and cathepsin B inhibitor MDL28170 and the antagonist of the serotonin receptors 5-HT1B and 5-HT1D GR 127935 hydrochloride hydrate, which have been shown to efficiently block either SARS-CoV-1 or 2 infection in vitro and in vivo (as shown in Fig. 1, Panel B).
Repositioning Analysis of the ReFRAME Drug Repurposing Library [0079] Due to favorable results of the LOP AC HTS screen, the same experimental design was used to screen the comprehensive ReFRAME drug repurposing collection. This library is an inclusive collection of nearly 13,000 chemical compounds, which have been either FDA approved, entered clinical trials or undergone significant pre-clinical characterization (J. Janes et al., The ReFRAME library as a comprehensive drug repurposing library and its application to the treatment of cryptosporidiosis. Proc Natl Acad Sci U S A 115, 10750-10755 (2018)).
Specifically, 12,966 compounds were arrayed in 384-well plates at a final concentration of 5mM. As with the previous assay, 3000 Vero E6 cells were seeded into each well pre-spotted with compound, infected 16 hours later with SARS-CoV-2 (MOI=0.01) and at seventy-two hours post-infection, CPE was determined. Analysis of the average Z' factor calculated on the activity of APY0201 was determined to be 0.51, reflecting favorable screening conditions (Fig. 1, Panels B-C, left-hand side). The screen was then repeated as an independent replicate. Although the window of the second assay was significantly smaller compared to the first replicate (Z’=0.19), the correlation coefficient (R2) for the two replicates was 0.68 (Fig. 1, Panels B-C, middle). Therefore data were normalized to the median of each plate and used to calculate the Log2FC. Z-scores were then calculated per plate, based on the Log2FC values. The distribution of the compounds based on the average of their Log2FC calculated within the replicates is shown in Fig. 1, Panel C. Z-scores were then calculated per plate, based on the Log2FC values (as shown in Fig. 4). The best performing compounds are listed in Table 2 below, showing each ID, Name, Concentration, Fold Change (FC) infection, Fold Change (FC) Cell nb, and DR Set (L = LOP AC® library; RF = ReFRAME library).
Table 2: Best Performing Hits
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
[0080] To elucidate targets, pathways, indications, and mechanisms of actions (MO A), enriched in compounds harboring antiviral activities, compounds in the collection were classified based on their reported target annotation. Gene Set Enrichment Analysis (GSEA) was used to assess the distribution of molecules with similar targets, functional category, or MOA across the screen (A. Subramanian et al., Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. ProcNatl Acad Sci EISA 102, 15545-15550 (2005); V. K. Mootha et al., PGC-1 alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet 34, 267-273 (2003)). Based on a nominal P-value cutoff of 0.05 and FDR q-value < 0.25, 13 target sets were enriched in a ranked hit list (as shown in Fig. 5). As shown in Fig. 2, Panel A, these enriched targets and biological processes include allosteric modulators of the benzodiazepine receptor, cytosolic NADPH- dependent oxidoreductase aldose reductase, cholesterol homeostasis, serine proteases, and as anticipated, antimalarials, including chloroquine derivatives such as Amopyroquine and AQ-13 (as shown in Fig. 2, Panel A and Fig. 5). The hits enriched in the GSEA analysis are provided in Table 3, and the hits validated in Vero single point include LG-1550, Tretinoin, Tamibarotene, acitretin, tazarotene, RBAD, AL 3152, ZK-93426, zaleplon GR, pagoclone, AQ-13, and Hanfangchin A. The hits confirmed in Vero DR include LG-1550, RBAD, AL 3152, ZK-93426, AQ-13 and Hanfangchin A, and both AQ-13 and Hanfangchin A were in the final list.
Table 3: Hits enriched in GSEA analysis.
Figure imgf000032_0002
Figure imgf000033_0001
Figure imgf000034_0001
[0081] SARS-CoV-2 primarily infects the epithelial cells in the respiratory tract. To elucidate the expression pattern of the genes that have been annotated to be targeted by putative antiviral compounds, the expression of drug target genes enriched in the compound screen was compared across cell types within the respiratory tract (Cascella, M., Rajnik, M. Cuomo, A., Duleboh, S.C., & Di Napoli, R., Features, Evaluation and Treatment Coronavirus (COVID-19), StatPearls [Internet] (2020)). Although both the entry receptor for SARS-CoV-2, ACE2, and the priming protease TMRPSS22, were found to be expressed with specific anatomical sampling locations in the respiratory tract (as shown in Fig. 2, Panel B, left-hand side heatmap), ACE2 expression was found to be restricted to epithelial cell types including multiciliated, nasal, deuterosomal, secretory, and basal cells (as shown in Fig. 2, Panel B, right-hand side heatmap). Notably, as shown in Fig. 2, Panel B, a majority of the mapped targets of active compounds also harbored expression in relevant respiratory epithelial cells, suggesting these may be physiologically relevant drug targets (as shown in Fig. 2, Panel B). Further pathway analyses (Zhou Y et al., Metascape provides a biologist-oriented resource for the analysis of systems-level datasets, Nat Commun 10(1): 1523 (2019)) of these enriched MOAs and targets revealed enrichment in genes involved in nuclear hormone receptor pathways, GPCR ligand binding and signaling, and calcium signaling (as shown in Fig. 6) suggesting a role for these molecular circuits in cellular control of the SARS-CoV-2 life cycle. The enriched genes with their description are provided in
Table 4.
Table 4: Enriched Genes
Figure imgf000035_0001
Figure imgf000036_0001
Orthogonal Validation of Selected Anti-SARS-CoV-2 Compounds
[0082] To select candidates for validation studies, compounds were ranked according to their Z-score in the primary screen (as shown in Fig. 4), and the top 100 hits from each replicate were prioritized, including 25 overlapping hits. In addition, seventy-five hits were then selected according to their ranking calculated based on the average Z-score and an additional forty-eight hits were finally selected according to drug target and pathway enrichment analysis (as described above), for a total of 298 hits.
[0083] The activity of hits at half the original screen concentration (2.5 mM) was initially assessed using an orthogonal assay readout. Specifically, three thousand Vero E6 cells were pre incubated with each compound dilution for 16 h, followed by infection with SARS-CoV-2 USA- WA1/2020 (MOI = 0.75) in the presence of compound. Twenty-four hours post-infection, cells were fixed and immunostained for the CoV nucleoprotein (NP). Cellular nuclei were stained with DAPI, prior to automated imaging and analysis. The percentage of infection for each well was calculated as the ratio of infected cells stained with NP antibody, over the total number of cells. Each of these values was normalized to the average of the DMSO control wells in each plate. This validation step, in addition to assessing false positives, represents a filtering of compounds that function to block CPE, only inhibit at the screening compound concentration (5 mM), require longer timepoints to impact replication (24 hpi vs 72 hpi), or, although unlikely, specifically inhibit the SARS-CoV-2 HKU-OOla clinical isolate. 27% of the compounds (89 hits) were found to reduce viral replication by at least 40% at 2.5 pm (data not shown) when averaging data from at least two replicates. These include compounds that were found to belong to enriched target classes (as shown in Fig. 2, Panel A), including retinoic acid receptor agonists (including LG-1550, Tretinoin, Tamibarotene, acitretin, tazarotene, RBAD), the aldose reductase inhibitor AL 3152, benzodiazepine receptor agonists (including ZK-93426, zaleplon GR, and pagoclone) and antimalarial drugs (including AQ-13 and Hanfangchin A), as well as the FDA- approved anti mycobacterial clofazimine
Dose Response Analysis
[0084] Although specific for each compound, therapeutic dose ranges are typically expected to track to cellular EC50s well below 1 mM concentrations. Therefore, a dose response analysis was conducted to determine the relationship between compound concentration and antiviral activity. Compound concentrations tested ranged from 1.1 nM to 2.5 mM in the immunofluorescence assay described previously. Total cell counts were used to assess compound cytotoxicity (as shown in Fig. 7). Treatment with seventeen compounds resulted in discemable dose-dependent antiviral activities, most of which could be segregated based on broad functional, structural, or target-based classes (as shown in Fig. 3, Panel A). While twelve of these known drugs have EC50s > 800 nM or values that could not be extrapolated (see Fig. 3, Panel A and Fig. 8), six compounds harbored EC50s less than 500 nM, indicating effective antiviral potency could likely be achieved during therapeutic dosing of a COVID-19 patient (see Fig. 3, Panels B-C). To enable prioritization of known drugs for preclinical and clinical evaluation for the treatment of SARS-CoV-2, a summary of the publicly disclosed and relevant preclinical and clinical properties of these molecules is provided in Table 5.
Table 5: Identified compounds
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Example 3. Clofazimine inhibition of human pathogenic coronaviruses in human cellular models.
[0085] Clofazimine, whose structure is demonstrated on the left side of FIG. 9a, was introduced into a number of human cellular models to measure its efficacy against coronavirus pathogens. Clofazimine (brand name LAMPRENE®) has a half maximal effective concentration (EC50) of 310 nM against SARS-CoV-2 and a peak serum concentration (Cmax) of >780 nM when administered orally to patients.
[0086] Clofazimine inhibited MERS-CoV replication in a dose-dependent manner. Clofazimine was introduced to VeroE6 cells infected with MERS-CoV. As shown in the graph in FIG. 9a, clofazimine demonstrated an EC50 of 1.48+0.17, as measured by plaque reduction when plotted using a logistic non-linear regression model (GraphPad).
[0087] Clofazimine’ s effectiveness against MERS-CoV-nucleocapsid protein (NP) in Huh7 cells was compared against dimethyl sulfoxide (“DMSO”) as a control and measured by fluorescent imaging and cell quantification. MERS-CoV-infected Huh7 cells (0.01 multiplicity of infection (“MOI”)) were treated for 24 hours with either DMSO or 5 mM of clofazimine. After treatment, cells were viewed using fluorescent imaging. The MERS-CoV-NP antigen was stained green and the Huh7 cell nuclei were stained blue. FIG. 9B (Upper panel): immunofluorescence staining of MERS-CoV-NP antigen (green), and Huh 7 cell nucleus (blue). Scale bar: 20pm. FIG. 9B (Lower panel): MERS-CoV-NP positive cells quantitated by flow cytometry. The experiments were performed twice with representative images and quantifications shown.
[0088] Human primary cardiomyocytes (CMs) infected with SARS-CoV-2 (0.1 MOI) and human primary human small airway epithelial cells (HSAEpC) infected with MERS-CoV (1 MOI) were treated with 10 pm of remdesivir, or 10 pm, 5 pm or 2.5 pm of clofazimine, or DMSO. Cell lysates were collected at 24 hours post-infection and viral load was determined using RT-qPCR assays. FIG. 9c shows the effect of 5 pm, 2.5 pm and 1.25 pm doses of clofazimine inhibition of SARS-CoV-2 replication in human primary cardiomyocytes and MERS-CoV in human primary human small airway epithelial cells when compared to DMSO. The data represents ± standard deviation (SD) for n=3 biological replicates. Data was analyzed by a one-way ANOVA when compared with DMSO group (0 pm). **p<0.01. .
[0089] Viral titers were measured after clofazimine or remdesivir or DMSO were introduced to ex vivo human lung tissues infected with certain pathogens. Measures were obtained by infecting ex vivo human lung tissues with an inoculum of 5x 105 plaque forming unit (PFU) of either SARS-CoV-2 or MERS-Co-V-2 for two hours. The inoculum was then removed and clofazimine (10 pM) or remdesivir (10 pM) or DMSO (0.1%) was introduced to the lung tissues, which were subsequently incubated for 24 hours. After incubation, supernatants were collected for quantification of viral titers by plaque assay. Data analysis was completed with the use of a student’s t-test. FIG 9d displays the results of the analysis. Error bars present in these graphs represent a SD for n=5 biological replicates. *p<0.05 and ***p<0.001.
[0090] To measure antiviral activity, clofazimine or remdesivir were introduced to VeroE6 cells infected with SARS-CoV-1 (0.01 multiplicity of infection (MOI) at 48 hours post-infection (hpi)), HELF cells infected with hCoV-229E (0.001 MOI at 72 hours hpi), and BSC1 cells infected with hCoV-OC43 (0.001 MOI at 72 hours hpi). Clofazimine and remdesivir were compared against a control comprising cells without clofazimine or remdesivir (0 pM). Viral loads in the cell culture supernatants were quantified by RT-qPCR assays and a one-way ANOVA was used to compare the clofazimine and remdesivir groups with the 0 pM group. The data in graphs represent mean data ± SD for n=3 biological replicates. ***p<0.001, **p<0.01 and *p<0.05.
[0091] The left graph in FIG. 9e, shows the results of the analysis when , 5 pM, 2.5 pM, 1.25 pM, and 0.625 pM of clofazimine, 10 pM of remdesivir, and 0 pM were introduced to SARS- CoV-1 infected VeroE6 cells. [0092] The middle graph in FIG. 9e, shows the results of the analysis when 5 mM, 2.5 mM, 1.25 mM, and 0.625 mM of clofazimine, 10 mM of remdesivir, and 0 mM, were introduced to the hCoV-229E infected HELF cells.
[0093] The right graph in FIG. 9e shows the results of the introduction of 5 mM, 2.5 mM, 1.25 mM, and 0.625 mM of clofazimine, 10 mM of remdesivir, and 0 mM, were introduced to the hCoV-OC43 BSC1 cells. ***p<0.001, **p<0.01 and *p<0.05.
Example 4. Clofazimine interferes with multiple steps of the virus life cycle.
[0094] Clofazimine’ s effect on virus life cycle was studied using a time-of-addition assay. VeroE6 cells were inoculated with SARS-CoV-2 for 1 hour and then the inoculum was removed. Cells were treated with clofazimine at a concentration of 5 mM at the following time points: 16 hours before to 10 hours after inoculation, 16 hours before to 0 hours before inoculation, 0 hours to 1 hour after inoculation, 0 hours to 2 hours after inoculation, 0 hours to 10 hours after inoculation, 1 hour after to 10 hours after inoculation, 2 hours after to 10 hours after inoculation, and 5 hours after to 10 hours after inoculation. The graph on the left in FIG. 10a shows the time frame over which the cells were treated with clofazimine, which is represented by the shaded bar. Cells that received clofazimine were compared against cells that received DMSO (“No treatment”). Infection was quantified with the use of immunostaining for CoV nucleocapsid protein at 10 hours post-infection. Data was normalized to the average of DMSO-treated wells for each corresponding time point and are presented as mean ± SD for n=6 independent experiments. Statistical analysis was performed using a two-way ANOVA followed by Tukey post-hoc test. The graph on the right in FIG. 10a shows the results of the experiment. **p <0.01 when compared to cells that received no treatment.
[0095] Vesicular stomatitis virus (VSV)-based pseudotyped viral particles were used to study clofazimine’ s inhibitory effects on pseudotype entry into cells. Clofazimine’ s effect was compared to cells treated with DMSO and MDL28170, a positive control entry inhibitor. VeroE6 cells were pre-treated with 2.5 mM of clofazimine, DMSO, or DML28170 for two hours and then infected with SARS-CoV-1 S or SARS-CoV-2 S or MERS-CoV S pseudotyped particles harboring firefly luciferase for two hours. Luciferase signals were quantified at 24 hours post inoculation. Statistical analysis was performed using a one-way ANOVA followed by Dunnet post-test. The results of the experiment are shown in FIG. 10b, where the error bar represents structural equation modeling (SEM) for n=6 independent experiments. As compared to DMSO, *p< 0.05, **p<0.01, and n.s. (non-significant) p>0.05.
[0096] Clofazimine’ s effect on SARS-CoV-2 spike-mediated cell-to-cell fusion was measured by calculation of GFP area of confocal images. Vero cells were co-transfected with 1 pg of SARS- CoV-2 spike plasmid and 0.4 pg of EGFP plasmid for 8 hours, at which time the cells were treated with 0.625 pg, 1.25 pg, 2.5 pg, 5 pg, or 10 pg of clofazimine or DMSO (0.1%). After 48 hours, confocal images were acquired. FIG. 10c (Left panel) shows the confocal images acquired at 48 hours post-transfection. Scale bar: 400 pm. Quantification of the inhibitory effect of clofazimine was based on the GFP positive area (using ImageJ software), results of which are shown in the right panel of FIG. 10c. Error bars represent SEM for n=5 randomly selected images. ****p<0.0001 and ***p<0.001 as compared to OpM (DMSO) group by one-way ANOVA.
[0097] The effect of clofazimine on in vitro transcribed viral RNA was studied using viral replication assay. The timeline in FIG lOd shows the process used to conduct the experiment. As shown VeroE6 cells were electroporated with in vitro transcribed viral RNA. Two hours post- seeding, the cells were treated with remdesivir or clofazimine at the dose indicated in FIG. lOd. Negative- stranded RNA was then quantified at 12 hours post-electroporation. 24 hours post electroporation, GFP was detected with use of a fluorescence-activated cell sorter. The results of the GFP mRNA detection at 24 hours is shown in the graph in FIG lOd. n.s. indicates a p-value of >0.05 as compared to the OpM group by one-way ANOVA.
[0098] The results of negative-stranded RNA detection by qRT-PCR at 12 hours post electroporation are shown in FIG. lOe. Error bars in the graphs represent SEM for n=3 independent experiments. Cells that received varying doses of clofazimine and remdesivir were compared to cells that received GFP mRNA, a negative control whose translation was not affected by remdesivir or clofazimine, by one-way ANOVA. *p<0.05, **p<0.01, and n.s. p>0.05 as compared to the OpM group by one-way ANOVA.
[0099] Varying concentrations of clofazimine’ s effect on the titration of DNA-unwinding activity and RNA-unwinding activity of SARS-CoV-2 helicase was studied using a fluorescence resonance energy transfer (FRET)-based assay. Results of the study are shown in FIG lOf, where the black curve represents ranitidine bismuth citrate, a positive control inhibitor, using DNA- based substrate. The red line represents double stranded RNA (dsRNA) and the blue line represents double stranded DNA (dsDNA). Data for clofazimine represents the mean ± SD for n=3 biological replicates. The mean and SD values of the control inhibitor are not shown in the graph.
Example 5. Transcriptional analysis of clofazimine treatment.
[0100] Clofazimine’ s effect on the viral load of SARS-CoV-2 in Caco-2 cells was studied using RT-qPCR analysis. Caco-2 cells were infected with 0.1 MOI of SARS-CoV-2 and then treated with 10 pM, 5 pM, 2.5 pM, 1.25 pM, or 0.625 pM of clofazimine, 10 pM remdesivir, or 0.1% DMSO (control group, labelled as “0” mM). Cell culture supernatants were collected at 48 hours post-infection and subjected to viral load determination by RT-qPCR analysis. Results of the experiment are shown in FIG 11a. *p<0.05 and ** p<0.01 as compared to the DMSO group by one-way ANOVA.
[0101] Principal component analysis (PC A) of RNA-sequence datasets was conducted on Caco-2 cells that were introduced to clofazimine or DMSO (control). As shown in the timeline in the left panel of FIG lib, the process of conducting the experiment involved uninfected Caco-2 cells and Caco-2 cells infected with SARS-CoV-2 (MOI=4). One hour after Caco-2 cells were infected, either clofazimine or DMSO were introduced to both infected and uninfected cells. At three hours and six hours post-introduction of either clofazimine or DMSO, samples were acquired from the cells, and PC A was conducted of the RNA-sequence dataset after Reads Per Kilobase of transcript per Million reads mapped (RPKM) normalization on each gene expression level was conducted. Results of the analysis are shown in graph in FIG lib. Within the graph, each dot represents one sample, and the percentage labelled on the x or y-axis represents the proportion of variance explained with each principal component (PC).
[0102] FIG. 11c shows information related to transcription factors regulated by clofazimine. FIG. 11c (Right panel) shows a heatmap of 197 transcription factors regulated by clofazimine treatment without infection, while FIG. 11c (Left panel) displays known interactions among the transcription factors contained within transcription factors shown in the right panel of FIG. 11c. [0103] FIG. lid shows the top enriched pathways of significantly up-regulated genes (false discovery rate <0.05 and fold change >2) of mock-infected cells receiving clofazimine treatment at 6 hour post treatment as compared to SARS-CoV-2 infected cells receiving clofazimine at 6 hours post-infection. Pathway analysis was performed by Metascape. *denotes innate immunity related pathways.
Example 6. Prophylactic and therapeutic treatment with clofazimine reduces SARS-CoV-2 Disease.
[0104] The experimental design, as shown in FIG. 12a, consisted of prophylactic treatment with clofazimine or post-exposure administration of clofazimine. Prophylactic treatment consisted of oral administration of 25mg/kg/day of clofazimine given at 3, 2, and 1 days pre-infection, followed by virus challenge of 10L5 plaque forming unit (PFU) per hamster through the intranasal route at 0 days. Therapeutic post-exposure administration of clofazimine was performed at 1, 2, and 3 days post-infection and consisted of applying the same drug dosage and virus inoculum as applied to the prophylactic treatment group. Lung tissue, nasal wash, fecal, and serum samples were collected at 4- and 14-days post-infection. Remdesivir (15mg/kg, indicated by the blue symbols) was administered as a control in the therapeutic regimen through intraperitoneal injection.
[0105] Daily body weights of the hamsters (n=3) were monitored throughout the experiment. Body weight measurements are shown in FIG. 12b for vehicle (black symbols), remdesivir (blue symbols), and clofazimine (red symbols). Asterisks indicate statistically significant differences (p<0.05) as determined by two-way ANOVA and Tukey’s multiple comparison test.
[0106] Viral yield in the hamster lung tissue after prophylactic (n=5) or therapeutic treatment (n=l 1 or 13) were harvested at 4 days post-infection and titrated by plaque assay and RT-qPCR assay, results of which are shown in FIG. 12c and FIG. 12d, respectively. Statistical analysis of the prophylaxis groups was conducted using Student’s t-test and statistical analysis for the therapeutic groups was conducted using one-way ANOVA, the groups were compared with the vehicle group (indicated by the black symbols). For statistical analysis, *p<0.05, **p<0.01, ***p<0.001, when compared with vehicle.
[0107] Hamster nasal washes were collected on 4 days post-infection while the hamsters were under anesthesia. The nasal washes were subjected to live virus titration by plaque assays (n=5). The results of the experiments are shown in FIG. 12e. For statistical analysis, *p<0.05,
**p<0.01, ***p<0.001 when compared with vehicle.
[0108] Hamster feces was freshly collected at 4 days post-infection and subjected to SARS-CoV- 2 viral copy detection by RT-qPCR assays (n=5). FIG. 12f shows the results of this experiment. [0109] A value of 10-100 was assigned for any data point below the detection line (denoted as the dotted line). For statistical analysis, *p<0.05, **p<0.01, ***p<0.001 when compared with vehicle.
[0110] The disease severity marker IL-6 levels in hamster serum was quantified by ELISA at 4 days post-infection. The results of these measurements are shown in FIG. 12g. For statistical analysis, *p<0.05, **p<0.01, ***p<0.001 when compared with vehicle.
[0111] Hamsters exhibited normal humoral immune response after SARS-CoV-2 infection and clofazimine treatment. Enzyme immunoassay (EIA) for immunoglobulin G against SARS-CoV-2 nucleoprotein was performed in hamster sera of the groups indicated in FIG. 12h, was collected at 14 days post-infection. The sera were serially diluted (from 1:100 to 1: 204800) before adding to the nucleoprotein-coated ELISA plate (n=3). The results of the experiment are shown in FIG. 12h.
[0112] FIG. 12i displays representative images of H&E-stained lung tissue section from hamsters treated with vehicle, clofazimine, remdesivir, or mock-infection. Numbered circled areas are shown in magnified images on the right-hand portion of each panel, illustrating the severity of: (1) bronchiolar and/or peribronchiolar cell death; (2) alveoli destruction and/or alveolar infiltration; (3) blood vessel and perivascular infiltration. Black arrows indicate sites of infiltration. These representative images were selected from a pool of over 15 images captured in three randomly selected hamsters per group. Scale bar: 200 pm.
[0113] Table 6 shows multiple pharmacokinetic parameters of VBY-825 after a single dose was administered to hamsters.
Table 6. PK parameters of VBY-825 after single dosing to hamsters
Figure imgf000045_0002
[0114] Table 7 shows multiple pharmacokinetic parameters of aplilimod after a single dose was administered to hamsters.
Table 7. PK parameters of Apilimod after single dosing to hamsters
Figure imgf000045_0001
[0115] Table 8 shows multiple pharmacokinetic parameters of remdesivir (GS-5734) after a single dose was administered to hamsters.
Table 8. PK parameters of Remdesivir (GS-5734) after single dosing to hamsters
Figure imgf000045_0003
[0116] Table 9 shows multiple pharmacokinetic parameters of remdesivir metabolite (GS- 441524) after a single dose of remdesivir was administered to hamsters.
Table 9. PK parameters of Remdesivir metabolite (GS-441524) after single dosing of Remdesivir to hamsters
Figure imgf000045_0004
Example 7. Clofazimine exhibits antiviral synergy with remdesivir in vitro and in vivo. [0117] VeroE6 cells were pre-treated for 16 hours with increasing concentrations of the indicated compound and then infected with SARS-CoV-2 at a multiplicity of infection (“MOI”) of 0.01. Thirty hours after infection, cells were fixed and analyzed by immunofluorescence imaging. For each concentration, the percentage of infection was calculated as the ratio of the number of infected cells stained for SARS-CoV-2 nucleocapsid protein (NP) to the number of cells stained with DAPI.
[0118] FIG. 13a shows a topographic two-dimensional map of synergy scores determined in SynergyFinder. The color gradient indicates synergy score (red: highest synergy score; blue: highest antagonism score). X-axis: remdesivir up to 10 mM, y-axis: clofazimine up to 10 pM. [0119] FIG. 13b shows a graph of a dose response analysis of remdesivir alone (black) and in combination with 0.15625 pM (blue) or 0.625 pM (red) clofazimine. The observed compounds’ activities are represented by solid lines, while the predicted additive combinatorial activities are indicated by dashed lines. The dotted black line denotes 90% inhibition of infection. Data are normalized to mean values for DMSO-treated wells and represent mean ± SEM of 2 independent experiments.
[0120] Cell numbers were counted in each combination of varying doses of remdesivir and 0.15625 pM (black) or 1.25 pM (red) clofazimine, and for treatment with varying doses of remdesivir alone. Results of the experiment, shown as mean ±SD, are displayed in FIG. 13c. [0121] The experimental design used in studying in vivo combination therapy administered to hamsters is shown in FIG. 13d. After virus challenge of 10L5 plaque forming unit (PFU) per hamster at day 0, oral administration of clofazimine (15 mg/kg) and/or intraperitoneal injection of remdesivir (1.5 mg/kg) was performed at 1, 2, and 3 days post-infection (dpi). Lung tissue, nasal turbinate and nasal wash samples were collected at 4 dpi. Remdesivir alone (dosed at 15 mg/kg) was included as a control through intraperitoneal injection. Vehicle control was also administered, in which hamsters received oral administration of com oil and intraperitoneal injection of 2% DMSO in 12% sulfobutylether-P-cyclodextrin (SBE).
[0122] Daily body weights of the hamsters (n=5) were monitored throughout the experiment, results of which are shown in FIG. 13e. Asterisks indicate statistically significant differences as determined by two-way ANOVA and Tukey’s multiple comparison test; *p< 0.05 and **p<0.01. [0123] Vims in the hamster lung tissue harvested at 4 dpi was titrated by plaque assay and shown in FIG. 13f. One-way ANOVA when compared with the vehicle control group, *p< 0.05, **p<0.01 and n.s. (non-significant) . [0124] The graph in FIG. 13g shows the results of a virus titer in the nasal wash determined by plaque assays. Statistical analysis was performed using a one-way ANOVA when compared with the combinational group that received 1.5 mg/kg of remdesivir and 15 mg/kg of clofazimine. *p< 0.05, **p<0.01 and n.s. indicates non-significant.
[0125] FIG. 13h shows representative images of infected cells by immunofluorescence staining in nasal turbinate at 4 dpi. SARS-CoV-2 N protein (NP) was detected by specific antibody (green) and cell nuclei were stained by DAPI (blue). Scale bar: 200 pm.
[0126] The graph in FIG. 13i shows nucleocapsid protein positive cells per 50 c field per hamster’s nasal turbinate section (n=5). **p<0.01 when compared with the vehicle-treated group by one-way ANOVA.
Example 8. Cytotoxicity measurement of clofazimine in matching cells for antiviral evaluation.
[0127] The cell viability was determined using CellTiter-Glo and in the absence of virus infection. The drug-incubation time in the cytotoxicity assay was consistent with that in the antiviral assay, e.g., 24 hours post-treatment for Huh7 cells, primary human small airway epithelial cells (HSAEpC) and human embryonic stem cells-derived cardiomyocytes (CM); 48 hours post-treatment for VeroE6 and Caco-2 cells; and at 72 hours post-treatment for BSC1 and human embryonic lung fibroblasts (HELF), respectively. The results of the experiment are shown in FIG. 14. Data represents mean ± SD for n=3 biological replicates.
Example 9. Exploration of possible effects of clofazimine on virus entry and replication. [0128] Clofazimine did not display an effect on ACE2 and DPP4 expression. Caco-2 cells were treated with clofazimine for 16 hours prior to collection for western blotting analysis. The expression of ACE2 and DPP4 was determined using anti-ACE2 antibody and anti-DPP4 antibody, respectively. The results of the analysis are shown in FIG. 15a.
[0129] Clofazimine showed no inhibition on the binding between either ACE2 or heparin with SARS-CoV-2 Spike protein, which are two critical cellular components for viral attachment and infection. ELISA-based detection was used to determine binding of recombinant SARS-CoV-2 Spike protein to the immobilized Heparin-BSA or ACE2. FIG. 15b displays the results of the experiment. Clofazimine was titrated at the indicated concentrations. Dashed line represents binding without inhibitor (i.e. OmM).
[0130] Clofazimine shows a marginal effect against Mpro and PLPro protease activity. Purified SARS-CoV-2 Mpro (Nsp5) and SARS-CoV-2 PLpro (Nsp3) enzymes were incubated with varying concentrations of clofazimine. The activity of purified SARS-CoV-2 Mpro and SARS- CoV-2 PLpro enzymes was measured using the substrate Dabcyl-KTSAVLQSGFRKM- E(Edans)-NH2 and Arg-Leu-Arg-Gly-Gly-AMC (RLRGG-AMC), respectively. Enzyme activity in the absence (zero percent inhibition control) and presence of clofazimine was used to calculate the percent inhibition at each concentration. Results of the experiment are shown in FIG. 15c. Data are presented as mean ± s.d (n=3).
[0131] Clofazimine did not display an inhibition on the polymerase activity of the nsp7/nsp8/nspl2 RdRp complex. The scaffold used in this experiment’s in vitro transcription inhibition assay is listed in the upper panel of FIG. 15d. The inhibitory effect of clofazimine or SL-11128 on the RdRp core complex was analyzed by a primer elongation assay. SL-11128 showed some inhibitory effect as time progressed (0, 15s, lmin, 5min and 20min), while the inhibitory effect of clofazimine was minor, ranging from 5-40 mM. All reactions were performed at 30 °C.
Example 10. Transcriptional analysis of clofazimine treatment on Caco-2 cells.
[0132] FIG. 16a shows patterns of transcription levels across all samples. The genes that were significantly and differentially expressed (fold change >2 or <0.5, FDR<0.05) between 6 hours post-infection (hpi) and mock are shown. Conditions included 3 hpi and 6 hpi of Caco-2 cells post infection, multiplicity of infection (“MOI”) =4, with or without clofazimine treatment.
Genes were clustered by K-means method.
[0133] FIG. 16b shows a heat map of the genes enriched in MAPK signaling, TNF signaling, Interleukins (ILs) signaling, or cytokine-cytokine receptor interaction. These genes were up- regulated (fold change >2, FDR<0.05) by either 6 hours clofazimine (without infection) or 6 hpi clofazimine (with infection) compared to mock-infection.
[0134] FIG. 16c shows the network of enriched terms represented as pie charts. Pies are color- coded based on the identities of the gene lists. “6 h. clofazimine vs mock” (red color) represents the genes that were up-regulated by clofazimine treatment without infection at 6 hours as compared to mock. “6 hpi. Clofazimine vs mock” (blue color) represents the up-regulated genes by clofazimine treatment at 6 hours post-infection as compared with mock.
Example 11. Transcriptional analysis of hamster lung tissues with clofazimine administration.
[0135] Transcriptional analysis of hamster lung tissues with clofazimine was studied using the experimental design shown in FIG. 17a. The design shows the different time points when drug was introduced, when virus was introduced, and when tissue samples were obtained.
[0136] Fig. 17b presents Gene Ontology Biological Process (GO-BP) analysis results showing up-regulated genes comparing prophylactic clofazimine administration with its corresponding vehicle controls. [0137] RNA expression (Reads Per Kilobase of transcript per Million mapped reads, “RPKM”) of the 13 genes enriched in the “Leukocyte differentiation” category of GO-BP analysis is shown in FIG. 17c. These genes were up-regulated (fold change > 1.5, p value<0.01) by prophylactic clofazimine group when compared to vehicle controls. MHCII molecules are labeled with an asterisk (*). Transcription factors up-regulated by clofazimine on both Caco-2 cells and hamster lung tissues are labeled with an up arrow ( ).
[0138] FIG. 17d shows a heat map of immune response related genes in uninfected and infected hamster lungs administered prophylactic clofazimine or vehicle controls.
[0139] Fig. 17e presents Gene Ontology Biological Process (GO BP) analysis results showing up-regulated genes comparing clofazimine and vehicle-treated hamster lungs without virus infection.
[0140] Table 10 provides the list of up-regulated genes in hamster lung tissues that were observed when clofazimine was administered prophylactically. All genes demonstrated a p-value of <0.05 when compared to vehicle control at 4 days post-infection.
Table 10: List of up-regulated genes in hamster lung tissues when comparing prophylactic administration of clofazimine versus vehicle.
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Example 12. Lung pathological severity using histological scores.
[0141] To differentiate lung pathology, semi-quantitative histology scores were given to each lung tissue by grading the severity of damages in bronchioles, alveoli and blood vessel and accumulating the total scores (see Yuan et ah, Metallodrug ranitidine bismuth citrate suppresses SARS-CoV-2 replication and relieves virus-associated pneumonia in Syrian hamsters, Nat Microbiol 5(11): 1439-1448 (2020). Results of the scores are shown in FIG. 18. The scores in the chart indicate the accumulation of the following scores: Bronchioles: 0=normal structure; 1= mild peribronchiolar infiltration; 2= peribronchiolar infiltration plus epithelial cell death; 3=score 2 plus intra-bronchi olar wall infiltration and epithelium desquamation. Alveoli: 0=normal structure; l=alveolar wall thicken and congestion; 2=focal alveolar space infiltration or exudation; 3=diffuse alveolar space infiltration or exudation or hemorrhage. Blood vessel: 0=normal structure; l=mild perivascular edema or infiltration; 2=vessel wall infiltration; 3=severe endothelium infiltration. Data shown are means ± SD of three randomly selected slides of each group shown. Statistical analysis was completed with use of an unpaired two-tailed Student’ s t-test between the two prophylactic groups and a one-way ANOVA for the three therapeutic groups. **p < 0.01 and ***p<0.001 when compared with the vehicle control group. Histological scores of the mock infection were set at zero.
Example 13. In vitro antiviral synergy of clofazimine and remdesivir.
[0142] Clofazimine exhibited in vitro antiviral synergy with remdesivir for every combination of drug dose, as displayed in FIG. 19. Remdesivir at the indicated doses was combined with clofazimine at indicated doses or a negative control (DMSO), and antiviral dose-response relationships were determined in VeroE6 cells by immunofluorescence imaging. VeroE6 cells were pre-treated for 16 hours with increasing concentrations of the indicated compound and then infected with SARS-CoV-2 at a multiplicity of infection (“MOI”) of 0.01. Thirty hours after infection, the infected cells were analyzed by immunofluorescence imaging. For each condition, the percentage of infection was calculated as the ratio of the number of infected cells stained for SARS-CoV-2 NP protein to the number of cells stained with DAPI. EC50 for compound alone (black solid line with circles), and predicted (dashed line) and observed (solid line with squares) EC50 for the combined treatment are presented. Data are normalized to mean values for DMSO- treated wells and represent mean ± SEM of n = 2 independent experiments.
Example 14. Antiviral synergy of clofazimine and remdesivir in hamster lungs.
[0143] Clofazimine exhibited antiviral synergy with remdesivir in hamster lungs. FIG. 20 shows viral N protein expression (green) in diffuse alveolar areas (shown by the thick white arrow) and in the focal bronchiolar epithelial cells (thin white arrow) of the vehicle-treated hamster lungs, whereas standard (“std”) and low dosing (“low”) remdesivir (“Rem”) groups as well as the clofazimine group (“Clo”) exhibited reduced N expression. Combinatorial therapy restricted virus replication within the entry gate of lung infection, i.e. bronchiolar epithelial cells (shown by the thin white arrow). The representative images of FIG. 20 were selected from a pool of over 15 images captured in three randomly selected hamsters per group. Scale bar: 200 pm.
Example 15. Clofazimine inhibition of SARS-CoV-2 infection in human stem cell derived pneumocyte-like cells.
[0144] Clofazimine demonstrated inhibition of SARS-CoV-2 infection in human stem cell derived pneumocyte-like cells. Cells were induced by alveolar differentiation of human embryonic stem cells. Briefly, cells were collected at 70-80% confluency, and 2 million cells per 10 cm2 were plated on Vitronectin-coated tissue culture plates in mTeSR. The next day, definitive endoderm differentiation was induced following the protocol previously described (Jacob etal, Differentiation of human pluripotent stem cells into functional lung alveolar epithelial cells , Cell Stem Cell 21(4):472-488 (2017)) for four days. Cells were split and further differentiated following an adapted alveolar differentiation protocol in IMDM medium supplemented with 10% FBS, 2 mM L-glutamine, 0.5 pM all-trans-retinoic acid, 10 ng/ml FGF- 10, 10 ng/ml EGF, 100 ng/ml Wnt3a, 10 ng/ml KGF and 5 ng/ml BMP -4. Nine days after induction of differentiation, cells were treated with clofazimine (10 uM), remdesivir (10 uM), or DMSO for 1 hour at 37°C followed by SARS-CoV2 infection (40000 plaque forming unit) for 48 hours at 37°C. Two days post infection, the cells were dissociated using cell dissociation buffer, and fixed in 4% methanol-free formaldehyde for FACS analysis using anti-mouse SARS-CoV-2 - nucleocapside protein antibody. The duplicate set of cells were treated with the same drug concentrations, but the cells were left uninfected. After 48 hours of incubation at 37°C, the cells were analyzed for viability using MTT assay kit. The results of the experiment are shown in FIG. 21. Black indicates the percent infection, whereas red indicates the percent viability.
Example 16. Clofazimine effect on antiviral activity in adenovirus hACE2 mouse model of SARS-COV-2 infection.
Clofazimine exhibited antiviral activity in an adenovirus hACE2 mouse model of SARS-COV-2 infection. Five days prior to infection with SARS-CoV-2, BALB/c mice were infected intranasally with 2.5c10L8 plaque forming unit (PFU) of an adenovirus carrying the gene for hACE2. Doses of clofazimine (50 mg/kg) were administered orally (p.o.), while remdesivir (50 mg/kg) was administered subcutaneously (s.c.). Each intervention was administered once per day for 3 days. 2 hours after the last dosing, the mice were put under anesthesia with a mixture of ketamine/xylazine, and infected with 1 c 10L4 PFU of SARS-CoV-2 in 50 mΐ of PBS. Three days post-infection, animals were humanely euthanized. Whole left lungs were harvested and homogenized in PBS with silica glass beads and then frozen at -80°C for viral titration via TCID50. Infectious titers were quantified by limiting dilution titration using Vero E6 cells.
Results of the experiment are shown in FIG. 22.
[0145] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.
It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:
1. An anti -infective composition comprising a pharmaceutically acceptable carrier or excipient and an effective amount of a compound selected from the group consisting of amopyroquine, nelfmavir mesylate hydrate, GR 127935 hydrochloride hydrate, LG-1550, tretinoin, tamibarotene, acitretin, tazarotene, RBAD, AL 3152, ZK-93426, zaleplon GR, pagoclone, MDL 28170, 8-(3-Chlorostyryl)caffeine, Apilimod (APY0201), Clofazimine, Z LVG CHN2, AQ-13, Hanfangchin A, astemizole, , SL-11128, ELOPIPRAZOLE, SDZ-62-434, analog of MLN-3897, SB-616234-A, YH-1238, VBY-825, ONO 5334, and AMG-2674, wherein the anti-infective composition reduces the risk of absorption, infectivity, or transmission of a pathogen.
2. The anti-infective composition of claim 1, wherein the pathogen is a coronavirus.
3. The anti-infective composition of claim 2, wherein the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
4. The anti-infective composition of claim 1, wherein the anti-infective composition further comprises an additional anti -infective agent.
5. The anti-infective composition of claim 4, wherein the additional anti -infective agent comprises an anti -viral agent.
6. The anti-infective composition of claim 5, wherein the anti-viral agent is selected from the group consisting of entry-inhibiting drugs, uncoating inhibiting drugs, reverse transcriptase inhibiting drugs, antisense drugs, ribozyme drugs, protease inhibitors, assembly inhibiting drugs, and release inhibiting drugs.
7. The anti-infective composition of claim 5, wherein the anti-viral agent comprises remdesivir (GS-5734).
8. The anti-infective composition of claim 5, wherein the anti-viral agent comprises favipiravir (T-705).
9. The anti-infective composition of claim 1, wherein the compound is selected from the group consisting of apilimod (APY0201), nelfmavir mesylate hydrate, MDL28170 and GR 127935 hydrochloride hydrate.
10. A method for treating a patient having severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) comprising: administering to the patient a therapeutically effective amount of an anti-infective composition comprising a pharmaceutically acceptable carrier or excipient and an effective amount of a compound selected from the group consisting of amopyroquine, nelfmavir mesylate hydrate, GR 127935 hydrochloride hydrate, LG-1550, tretinoin, tamibarotene, acitretin, tazarotene, RBAD, AL 3152, ZK-93426, zaleplon GR, pagoclone, MDL 28170, 8-(3- Chlorostyryl)caffeine, Apilimod (APY0201), Clofazimine, Z LVG CHN2, AQ-13, Hanfangchin A, astemizole, , SL-11128, ELOPIPRAZOLE, SDZ-62-434, analog ofMLN-3897, SB-616234- A, YH-1238, VBY-825, ONO 5334, and AMG-2674, wherein the anti-infective composition is effective against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
11. The method of claim 10, wherein the anti -infective composition further comprises an additional anti -infective agent.
12. The method of claim 11, wherein the additional anti -infective agent comprises an anti viral agent.
13. The method of claim 12, wherein the anti-viral agent comprises remdesivir (GS-5734).
14. The method of claim 12, wherein the anti-viral agent comprises favipiravir (T-705).
15. The anti-infective composition of claim 1, wherein the compound is clofazimine.
16. The method of claim 10, wherein the compound is clofazimine.
17. The method of claim 10, wherein administering to the patient comprises administration via a route selected from the group consisting of inhalation, oral, parenteral, intranasal, buccal, topical or transdermal administration routes.
18. The method of claim 17, wherein the route in inhalation.
19. An inhalation device comprising:
(a) an effective amount of a compound selected from the group consisting of amopyroquine, nelfmavir mesylate hydrate, GR 127935 hydrochloride hydrate, LG-1550, tretinoin, tamibarotene, acitretin, tazarotene, RBAD, AL 3152, ZK- 93426, zaleplon GR, pagoclone, MDL 28170, 8-(3-Chlorostyryl)caffeine, Apilimod (APY0201), Clofazimine, Z LVG CHN2, AQ-13, Hanfangchin A, astemizole, , SL-11128, ELOPIPRAZOLE, SDZ-62-434, analog ofMLN-3897, SB-616234-A, YH-1238, VBY-825, ONO 5334, and AMG-2674; and
(b) a pharmaceutically acceptable propellant.
20. The device of claim 19, wherein the compound is clofazimine.
21. The device of claim 19, further comprising remdesivir.
PCT/US2021/027324 2020-04-15 2021-04-14 Methods and compositions for antiviral treatment WO2021211738A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202063010630P 2020-04-15 2020-04-15
US63/010,630 2020-04-15

Publications (1)

Publication Number Publication Date
WO2021211738A1 true WO2021211738A1 (en) 2021-10-21

Family

ID=78083990

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2021/027324 WO2021211738A1 (en) 2020-04-15 2021-04-14 Methods and compositions for antiviral treatment

Country Status (1)

Country Link
WO (1) WO2021211738A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023118896A1 (en) 2021-12-23 2023-06-29 Subintro Limited Novel antiviral compositions comprising oleic acid
WO2023132842A1 (en) * 2022-01-10 2023-07-13 AiPharma Global Holdings LLC Drug combination to treat microbial infections including coronavirus

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060223847A1 (en) * 2003-07-15 2006-10-05 Arigen, Inc. Anti-coronavirus drug
US20160143907A1 (en) * 2014-11-21 2016-05-26 KamTek, Inc. Methods and Compositions for Treating Clostridium difficile Associated Disease
WO2019079339A1 (en) * 2017-10-18 2019-04-25 Avalon Flaviviral Therapeutics (Hk) Limited Compositions and methods for broad-spectrum antiviral therapy
US20200069588A1 (en) * 2017-05-04 2020-03-05 Nanologica Ab Process for manufacturing porous silica particles loaded with at least one bioactive compound adapted for lung, nasal, sublingual, and/or pharyngeal delivery
US10980756B1 (en) * 2020-03-16 2021-04-20 First Wave Bio, Inc. Methods of treatment

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060223847A1 (en) * 2003-07-15 2006-10-05 Arigen, Inc. Anti-coronavirus drug
US20160143907A1 (en) * 2014-11-21 2016-05-26 KamTek, Inc. Methods and Compositions for Treating Clostridium difficile Associated Disease
US20200069588A1 (en) * 2017-05-04 2020-03-05 Nanologica Ab Process for manufacturing porous silica particles loaded with at least one bioactive compound adapted for lung, nasal, sublingual, and/or pharyngeal delivery
WO2019079339A1 (en) * 2017-10-18 2019-04-25 Avalon Flaviviral Therapeutics (Hk) Limited Compositions and methods for broad-spectrum antiviral therapy
US10980756B1 (en) * 2020-03-16 2021-04-20 First Wave Bio, Inc. Methods of treatment

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
XU JIABAO; ZHAO SHIZHE; TENG TIESHAN; ABDALLA ABUALGASIM ELGAILI; ZHU WAN; XIE LONGXIANG; WANG YUNLONG; GUO XIANGQIAN: "Systematic Comparison of Two Animal-to-Human Transmitted Human Coronaviruses: SARS-CoV-2and SARS-CoV", VIRUSES, vol. 12, no. 2, 244, 22 February 2020 (2020-02-22), pages 1 - 17, XP055853415, DOI: 10.3390/v12020244 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023118896A1 (en) 2021-12-23 2023-06-29 Subintro Limited Novel antiviral compositions comprising oleic acid
WO2023132842A1 (en) * 2022-01-10 2023-07-13 AiPharma Global Holdings LLC Drug combination to treat microbial infections including coronavirus

Similar Documents

Publication Publication Date Title
Kaddoura et al. COVID-19 therapeutic options under investigation
Hu et al. The in vitro antiviral activity of lactoferrin against common human coronaviruses and SARS-CoV-2 is mediated by targeting the heparan sulfate co-receptor
US10434116B2 (en) Methods of treating coronavirus infection
Ashour et al. A systematic review of the global intervention for SARS-CoV-2 combating: from drugs repurposing to molnupiravir approval
Ahidjo et al. Current perspective of antiviral strategies against COVID-19
US20230346820A1 (en) Methods and compositions for the treatment of sars-cov-2
Singh et al. SARS-CoV-2 therapeutics: how far do we stand from a remedy?
US12150971B2 (en) Method and composition for preventing and treating Covid-19 and long Covid
Chen et al. Sertraline is an effective SARS-CoV-2 entry inhibitor targeting the spike protein
WO2021211738A1 (en) Methods and compositions for antiviral treatment
US20230158103A1 (en) Pld for use in combination in the treatment of coronavirus
WO2021164689A1 (en) Application of nelfinavir in preparation of drug for preventing and treating novel coronavirus pneumonia
EP4164749B1 (en) Compound and method for the treatment of coronaviruses
US12083135B2 (en) Use of a nitrogen-containing bisphosphonate in combination with a glucocorticoid in preventing or treating viral pneumonia
Belouzard et al. Large scale screening discovers clofoctol as an inhibitor of SARS-CoV-2 replication that reduces COVID-19-like pathology
Goc et al. Simultaneous inhibition of SARS-CoV-2 infectivity by a specific combination of plant-derived compounds
CN116194114A (en) Cathepsin inhibitors for preventing or treating viral infections
US20240165096A1 (en) Anti Viral Therapy
US11986478B2 (en) Targeting papain-like protease for broad-spectrum coronaviruses inhibition
Zhou et al. Conessine inhibits enveloped viruses replication through up-regulating cholesterol level
US20230002774A1 (en) Methods and compostions for inhibiting coronaviral replication
Wen et al. Homo-harringtonine (HHT) is highly effective against SARS-CoV-2-A potential first-line defense in future coronavirus epidemics
Yuan et al. Salinomycin inhibits porcine epidemic diarrhea virus infection by targeting Wnt/β-catenin pathway
WO2022238886A1 (en) Cyclobenzaprine for use in the treatment of coronavirus
CN117957009A (en) Methods and compositions for preventing and treating severe and severe specific infectious pneumonia (COVID-19) and chronic severe and specific infectious pneumonia (LONG COVID)

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21788469

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21788469

Country of ref document: EP

Kind code of ref document: A1