WO2023059809A2 - Méthodes et agents pour la prévention de la prolifération virale - Google Patents

Méthodes et agents pour la prévention de la prolifération virale Download PDF

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WO2023059809A2
WO2023059809A2 PCT/US2022/045913 US2022045913W WO2023059809A2 WO 2023059809 A2 WO2023059809 A2 WO 2023059809A2 US 2022045913 W US2022045913 W US 2022045913W WO 2023059809 A2 WO2023059809 A2 WO 2023059809A2
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acid
poly
nanoparticles
caprolactone
nanoparticle
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WO2023059809A3 (fr
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Mark Grinstaff
Brett TINGLEY
Anton PETCHERSKI
Aaron COLBY
Orian S. SHIRIHAI
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The Regents Of The University Of California
Trustees Of Boston University
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Publication of WO2023059809A2 publication Critical patent/WO2023059809A2/fr
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    • 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
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4706—4-Aminoquinolines; 8-Aminoquinolines, e.g. chloroquine, primaquine
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • 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
    • A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
    • A61K31/635—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide having a heterocyclic ring, e.g. sulfadiazine
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

Definitions

  • Endocytosis is a highly conserved cell process which allows cells to uptake extracellular material and is essential for cell growth, function and repair. This process, and specifically receptor- mediated endocytosis, is a mechanism which many viruses leverage togain entry to the cell cytosol for replication (Boulant et al. (2015) Viruses 7: 2794-2815).
  • ligands present on the surface of viral particles bind to specific host cell receptors which triggers formation of membrane coated endosomes which shuttle the viral particles into the cytosol (Rennick et al. (2021) Nat. Nano. 16: 266-276). These endosomes subsequently fuse with acidic lysosomes to create acidified ‘endolysosomes’.
  • proteases e.g., cathepsin L
  • Coronaviruses which are enveloped viruses with long single-stranded RNAs ( ⁇ 32 kb), are one family of viruses that leverage endocytic pathway to infect cells. Included in this family is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has emerged into the leading global health crisis, termed the coronavirus disease- 19 (COVID-19) pandemic (Zhu et al. (2020) N. Engl. J. Med. 382: 727-733). To date, SARS-CoV-2 has infected > 500 million people and killed > 6 million individuals worldwide, causing a devastating socioeconomic burden (WHO COVID- 19 dashboard).
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the spike (S) protein present on the viral coat of SARS-CoV-2 binds the cell surface receptor angiotensin-converting enzyme 2 (ACE2), resulting in receptor-mediated endocytosis (Hoffmann et al. (2020) Cell 181: 271-280; Walls et al. (2020) Cell 181: 281-292; Yan, et al. (2020) Science 367: 1444-1448; Zhou et al. (2020) Nature 579: 270-273).
  • ACE2 cell surface receptor angiotensin-converting enzyme 2
  • the positivesense genome of CoVs can act as messenger RNA (mRNA) and be directly translated into viral proteins using the host cell's ribosomes, typically encoding for an RNA-dependent RNA polymerase (RdRp) that generates further viral copies.
  • mRNA messenger RNA
  • RdRp RNA-dependent RNA polymerase
  • RdRp inhibitors such as Remdesivir
  • endosomal pH modulators such as Chloroquine/Hydroxychloroquine
  • Coronavirus protease 3CL1 inhibitors such as Lopinavir I Ritonavir
  • S protein inhibitors such as recombinant human ACE2
  • COVID-19 coronavirus disease-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to infect and kill millions of individuals worldwide.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • SARS-CoV-2 infects cells through a highly conserved process known as receptor-mediated endocytosis.
  • endosomal fusion with lysosomes exposes the virus to acidic pH-dependent proteases.
  • proteases e.g., cathepsin L
  • cleave the viral spike protein triggering a fusion event between the SARS-CoV-2 envelope and endolysosomal membrane which releases viral RNA into the cytosol, resulting in infection and proliferation of the virus.
  • Palmitic acid a natural fatty acid
  • delivery of palmitate to the endosome/lysosome will result in enzymatic conjugation of palmitate to V-ATPase proton pumps, thereby inactivating the proton pumps and resulting in an increase in pH (less acidic).
  • proteases reliant on low pH for function will decrease in activity, thereby inhibiting cleavage of the viral spike protein and preventing the virus from releasing its viral RNA into the cytosol.
  • NPs novel, biocompatible, palmitate nanoparticles
  • the disclosure presented herein describes novel, biocompatible, palmitate nanoparticles (NPs) that, like the virus, enter the cell via endocytosis whereupon the NPs release palmitate and perform desired inhibition of viral proliferation.
  • the NPs are fabricated as either solid lipid nanoparticles (SLNs) composed of a solid fatty acid core, or in some embodiments, polymeric poly(glycerol monostearate-co-e- caprolactone) (PGC-C18) NPs which physically encapsulate fatty acids and/or synergistic antiviral agents.
  • SSNs solid lipid nanoparticles
  • PLC-C18 polymeric poly(glycerol monostearate-co-e- caprolactone)
  • the proposed delivery of palmitate via a NP formulation represents a potential paradigm shift in the management of SARS-CoV-2.
  • the generality of targeting a highly conserved pathway as opposed to specific viral genome sequences suggests that this approach may be applied broadly to other viral infections reliant on acidic pH-dependent proteases for infection.
  • these NPs are capable of localized delivery to lung tissue, allowing for greater local concentration of drug in the lungs while mitigating off-target toxicity.
  • this includes but is not limited to other coronaviruses, influenza A and Respiratory Syncytial Virus.
  • a composition for treating or preventing viral infection in a subject in need thereof comprising nanoparticles that comprise palmitic acid and/or palmitate.
  • the viral infection is caused by coronavirus, influenza virus, or respiratory syncytial virus.
  • the coronavirus is human coronavirus 229E, Middle East respiratory syndrome coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2), human coronavirus OC43, human coronavirus NL63, or human coronavirus HKU1.
  • the nanoparticles further comprise a surfactant.
  • the surfactant is dodecyl sulfate (SDS), poly(vinyl alcohol) (PVA), Triton X-100, Tween 80, Sodium taurocholate hydrate (STH), L-a-phosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) or triblock poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (PEG-PPG- PEG).
  • SDS dodecyl sulfate
  • PVA poly(vinyl alcohol)
  • Triton X-100 Triton X-100
  • Tween 80 Triton X-100
  • STH Sodium taurocholate hydrate
  • L-a-phosphatidylcholine L-a-phosphatidylcholine
  • DPPC dipalmitoylphosphatidylcholine
  • the nanoparticles further comprise a fatty acid that is liquid at room temperature.
  • the fatty acid is oleic acid.
  • the nanoparticles have a diameter of about 10 to about 1000 nm. In certain embodiments, the nanoparticles have a diameter of about 50 to about 250 nm.
  • the nanoparticles are solid lipid nanoparticles or polymeric nanoparticles.
  • the polymeric nanoparticles comprise a polymer functionalized by conjugation with at least one fatty acid.
  • the fatty acid-functionalized polymer is polycarbonate, a mixture or copolymer of polycarbonates, a mixture or copolymer of polycarbonates and polycaprolactone, a mixture or copolymer of polycarbonates and poly(lactic acid), or a mixture or copolymer of polycarbonates and poly(glycolic acid).
  • the polymeric nanoparticles comprise a fatty acid functionalized poly( 1,3 -glycerol carbonate-co-e-caprolactone).
  • the fatty acid is a saturated or unsaturated fatty acid of about 4 to about 20 carbons.
  • the fatty acid is lauric acid, myristic acid, palmitic acid, stearic acid and arachidic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, a-linolenic acid, arachidonic acid eicosapentanoic acid or any combination thereof.
  • the polymeric nanoparticles comprise poly (glycerol monostearate-co-e-caprolactone), poly(glycerol monopalmitate-co-e-caprolactone) or poly(glycerol monomyristate-co-e-caprolactone).
  • the nanoparticles further comprise one or more antiviral agents, pharmacological agents, fluorophores, or any combination thereof.
  • the antiviral agent or pharmacological agent is mefloquine, sulfadoxine, nitazoxanide, chloroquine, palmitic acid and/or palmitate or stearic acid and/or stearate.
  • the nanoparticles comprise palmitic acid and sodium dodecyl sulfate. In certain embodiments, the nanoparticles comprise palmitic acid and poly(vinyl alcohol). In certain embodiments, the nanoparticles comprise poly(glycerol monostearate-co-e- caprolactone) and mefloquine, or poly(glycerol monostearate-co-e-caprolactone) and chloroquine.
  • composition comprising nanoparticles is administered via inhalation.
  • a method for treating or preventing viral infection in a subject in need thereof comprising administering to the subject a composition comprising nanoparticles that comprises palmitic acid.
  • the viral infection is caused by coronavirus, influenza virus, or respiratory syncytial virus.
  • the coronavirus is human coronavirus 229E, Middle East respiratory syndrome coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2), human coronavirus OC43, human coronavirus NL63, or human coronavirus HKU1.
  • the nanoparticles further comprise a surfactant.
  • the surfactant is dodecyl sulfate (SDS), poly(vinyl alcohol) (PVA), Triton X-100, Tween 80, Sodium taurocholate hydrate (STH), L-a-phosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) or triblock poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (PEG-PPG- PEG).
  • the nanoparticles further comprise a fatty acid that is liquid at room temperature.
  • the fatty acid is oleic acid.
  • the nanoparticles have a diameter of about 10 to about 1000 nm. In certain embodiments, the nanoparticles have a diameter of about 50 to about 250 nm.
  • the nanoparticles are solid lipid nanoparticles or polymeric nanoparticles.
  • the polymeric nanoparticles comprise a polymer functionalized by conjugation with at least one fatty acid.
  • the fatty acid-functionalized polymer is polycarbonate, a mixture or copolymer of polycarbonates, a mixture or copolymer of polycarbonates and polycaprolactone, a mixture or copolymer of polycarbonates and poly(lactic acid), or a mixture or copolymer of polycarbonates and poly(glycolic acid).
  • the polymeric nanoparticles comprise a fatty acid functionalized poly( 1,3 -glycerol carbonate-co-e-caprolactone).
  • the fatty acid is a saturated or unsaturated fatty acid of about 4 to about 20 carbons.
  • the fatty acid is lauric acid, myristic acid, palmitic acid, stearic acid and arachidic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, a-linolenic acid, arachidonic acid eicosapentanoic acid or any combination thereof.
  • the polymeric nanoparticles comprise poly (glycerol monostearate-co-e-caprolactone), poly(glycerol monopalmitate-co-e-caprolactone) or poly(glycerol monomyristate-co-e-caprolactone).
  • the nanoparticles further comprise one or more antiviral agents, pharmacological agents, fluorophores, or any combination thereof.
  • the antiviral agent or pharmacological agent is mefloquine, sulfadoxine, nitazoxanide, chloroquine, palmitic acid and/or palmitate, or stearic acid and/or stearate.
  • the nanoparticles comprise palmitic acid and/or palmitate and sodium dodecyl sulfate.
  • the nanoparticles comprise palmitic acid and/or palmitate and poly(vinyl alcohol).
  • the nanoparticles comprise poly(glycerol monostearate-co-e- caprolactone) and mefloquine, or poly(glycerol monostearate-co-e-caprolactone) and chloroquine.
  • composition comprising nanoparticles is administered via inhalation.
  • a composition for treating or preventing viral infection in a subject in need thereof comprising nanoparticles that comprise a fatty acid functionalized polymer.
  • the viral infection is caused by coronavirus, influenza virus, or respiratory syncytial virus.
  • the coronavirus is human coronavirus 229E, Middle East respiratory syndrome coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2), human coronavirus OC43, human coronavirus NL63, or human coronavirus HKU1.
  • the nanoparticles have a diameter of about 10 to about 1000 nm. In certain embodiments, the nanoparticles have a diameter of about 50 to about 250 nm.
  • the fatty acid- functionalized polymer is polycarbonate, a mixture or copolymer of polycarbonates, a mixture or copolymer of polycarbonates and polycaprolactone, a mixture or copolymer of polycarbonates and poly (lactic acid), or a mixture or copolymer of polycarbonates and poly (glycolic acid).
  • the polymeric nanoparticles comprise a fatty acid functionalized poly(l,3-glycerol carbonate-co-e-caprolactone).
  • the fatty acid is a saturated or unsaturated fatty acid of about 4 to about 20 carbons.
  • the fatty acid is lauric acid, myristic acid, palmitic acid, stearic acid and arachidic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, a-linolenic acid, arachidonic acid eicosapentanoic acid or any combination thereof.
  • the polymeric nanoparticles comprise poly (glycerol monostearate-co-e-caprolactone), poly(glycerol monopalmitate-co-e-caprolactone) or poly(glycerol monomyristate-co-e-caprolactone).
  • the nanoparticles further comprise one or more antiviral agents, pharmacological agents, fluorophores, or any combination thereof.
  • the antiviral agent or pharmacological agent is mefloquine, sulfadoxine, nitazoxanide, chloroquine, palmitic acid and/or palmitate, or stearic acid and/or stearate.
  • the polymeric nanoparticles comprise poly (glycerol monostearate-co-e-caprolactone) and mefloquine, or poly(glycerol monostearate-co-e- caprolactone) and chloroquine.
  • the nanoparticles further comprise a surfactant such as dodecyl sulfate (SDS), poly(vinyl alcohol) (PVA), Triton X-100, Tween 80, Sodium taurocholate hydrate (STH), L-a-phosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) or triblock poly(ethylene glycol) -block-poly (propylene glycol) -block-poly (ethylene glycol) (PEG-PPG-PEG).
  • SDS dodecyl sulfate
  • PVA poly(vinyl alcohol)
  • Triton X-100 Triton X-100
  • Tween 80 Sodium taurocholate hydrate
  • STH L-a-phosphatidylcholine
  • DPPC dipalmitoylphosphatidylcholine
  • DSPC distearoylphosphatidy
  • composition comprising nanoparticles is administered via inhalation.
  • method for treating or preventing viral infection in a subject in need thereof, comprising administering to the subject a composition comprising nanoparticles that comprises a fatty acid functionalized polymer.
  • the viral infection is caused by coronavirus, influenza virus, or respiratory syncytial virus.
  • the coronavirus is human coronavirus 229E, Middle East respiratory syndrome coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human coronavirus OC43, human coronavirus NL63, or human coronavirus HKU 1.
  • the nanoparticles have a diameter of about 10 to about 1000 nm.
  • the nanoparticles have a diameter of about 50 to about 250 nm.
  • the fatty acid- functionalized polymer is polycarbonate, a mixture or copolymer of polycarbonates, a mixture or copolymer of polycarbonates and polycaprolactone, a mixture or copolymer of polycarbonates and poly (lactic acid), or a mixture or copolymer of polycarbonates and poly (glycolic acid).
  • the polymeric nanoparticles comprise a fatty acid functionalized poly (1,3 -glycerol carbonate-co-e-caprolactone).
  • the fatty acid is a saturated or unsaturated fatty acid of about 4 to about 20 carbons.
  • the fatty acid is lauric acid, myristic acid, palmitic acid, stearic acid and arachidic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, a-linolenic acid, arachidonic acid eicosapentanoic acid or any combination thereof.
  • the polymeric nanoparticles comprise poly (glycerol monostearate-co-e-caprolactone), poly(glycerol monopalmitate-co-e-caprolactone) or poly(glycerol monomyristate-co-e-caprolactone).
  • the nanoparticles further comprise one or more antiviral agents, pharmacological agents, fluorophores, or any combination thereof.
  • the antiviral agent or pharmacological agent is mefloquine, sulfadoxine, nitazoxanide, chloroquine, palmitic acid and/or palmitate, or stearic acid and/or stearate.
  • the nanoparticles comprise poly(glycerol monostearate-co-e-caprolactone) and mefloquine, or poly(glycerol monostearate-co-e-caprolactone) and chloroquine.
  • the nanoparticles further comprise a surfactant such as dodecyl sulfate (SDS), poly(vinyl alcohol) (PVA), Triton X-100, Tween 80, Sodium taurocholate hydrate (STH), L-a-phosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) or triblock poly(ethylene glycol) -block-poly (propylene glycol) -block-poly (ethylene glycol) (PEG-PPG-PEG).
  • SDS dodecyl sulfate
  • PVA poly(vinyl alcohol)
  • Triton X-100 Triton X-100
  • Tween 80 Sodium taurocholate hydrate
  • STH L-a-phosphatidylcholine
  • DPPC dipalmitoylphosphatidylcholine
  • DSPC distearoylphosphatidy
  • composition comprising nanoparticles is administered via inhalation.
  • a nanoparticle that consists of palmitic acid and/or palmitate.
  • the nanoparticle has a diameter of about 10 to about 1000 nm. In certain embodiments, the nanoparticle has a diameter of about 50 to about 250 nm.
  • a nanoparticle that comprises palmitic acid and/or palmitate.
  • the nanoparticle has a diameter of about 10 to about 1000 nm. In certain embodiments, the nanoparticle has a diameter of about 50 to about 250 nm. In certain embodiments, further comprises a surfactant.
  • the surfactant is dodecyl sulfate (SDS), poly(vinyl alcohol) (PVA), Triton X-100, Tween 80, Sodium taurocholate hydrate (STH), L-a-phosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) or triblock poly(ethylene glycol) -block-poly (propylene glycol) -block-poly (ethylene glycol) (PEG-PPG-PEG).
  • SDS dodecyl sulfate
  • PVA poly(vinyl alcohol)
  • Triton X-100 Triton X-100
  • Tween 80 Triton X-100
  • STH Sodium taurocholate hydrate
  • L-a-phosphatidylcholine L-a-phosphatidylcholine
  • DPPC dipalmitoylphosphatidylcholine
  • the nanoparticle further comprises a fatty acid that is liquid at room temperature.
  • the fatty acid is oleic acid.
  • the nanoparticle further comprises one or more antiviral agents, pharmacological agents, or combination thereof.
  • the antiviral or pharmacological agent is mefloquine, sulfadoxine, nitazoxanide, chloroquine, palmitic acid and/or palmitate, or stearic acid and/or stearate.
  • the nanoparticle comprises palmitic acid and/or palmitate and mefloquine, or palmitic acid and/or palmitate and chloroquine.
  • a pharmaceutical composition comprising the foregoing nanoparticles.
  • the pharmaceutical composition is formulated for administration by inhalation.
  • a nanoparticle consisting of a polymer functionalized by conjugation with at least one fatty acid.
  • the nanoparticle has a diameter of about 10 to about 1000 nm.
  • the nanoparticle has a diameter of about 50 to about 250 nm.
  • the fatty acid-functionalized polymer is polycarbonate, a mixture or copolymer of polycarbonates, a mixture or copolymer of polycarbonates and polycaprolactone, a mixture or copolymer of polycarbonates and poly(lactic acid), or a mixture or copolymer of polycarbonates and poly (glycolic acid).
  • the nanoparticles comprise a fatty acid functionalized poly(l,3- glycerol carbonate-co-e-caprolactone).
  • the fatty acid is a saturated or unsaturated fatty acid of about 4 to about 20 carbons.
  • the fatty acid is lauric acid, myristic acid, palmitic acid, stearic acid and arachidic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, a-linolenic acid, arachidonic acid eicosapentanoic acid or any combination thereof.
  • the nanoparticles consist of poly(glycerol monostearate-co-e- caprolactone), poly(glycerol monopalmitate-co-e-caprolactone) or poly(glycerol monomyristate- co-e-caprolactone).
  • the nanoparticle further comprises one or more antiviral agents, pharmacological agents, fluorophores, or any combination thereof.
  • the antiviral agent or pharmacological agent is mefloquine, sulfadoxine, nitazoxanide, chloroquine, palmitic acid and/or palmitate, or stearic acid and/or stearate.
  • the nanoparticles comprise poly(glycerol monostearate-co-e- caprolactone) and mefloquine, or poly(glycerol monostearate-co-e-caprolactone) and chloroquine.
  • the nanoparticle further comprise a surfactant.
  • the surfactant is dodecyl sulfate (SDS), poly(vinyl alcohol) (PVA), Triton X-100, Tween 80, Sodium taurocholate hydrate (STH), L-a-phosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) or triblock poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (PEG-PPG- PEG).
  • a pharmaceutical composition comprising the aforementioned nanoparticles.
  • the pharmaceutical composition is formulated for administration by inhalation.
  • Figure 1 illustrates the use of novel palmitate NPs to decrease the infection rate of Coronaviruses (CoVs) and respiratory viruses that leverage receptor- mediated endocytosis for infection (e.g., Influenza A, Respiratory Syncytial Virus, etc.).
  • Coronaviruses Coronaviruses
  • Pathogenic viral infection depends on acidification of the endosome by V-ATPases. Lowered pH activates proteolysis and releases viral nucleic acids to the cytosol.
  • the novel anti-viral treatment utilizes NPs that enter the endosome through endocytosis. The NPs release palmitate and, in some cases, other small molecule antivirals, which synergistically raise the pH or act via other mechanisms to prevent the release of viral nucleic acids to the cytosol.
  • DLS dynamic light scattering
  • PDI poly dispersity index
  • Figure 4 shows representative scanning electron microscopy (SEM) images of PGC-C18 NPs.
  • HPLC partition coefficient
  • Figure 7 shows quantification of encapsulation efficiency of fatty acids within solid lipid NPs using gas chromatography-mass spectrometry (GC-MS).
  • DPPC dipalmitoylphosphatidylcholine
  • DSPC distearoylphosphatidylcholine
  • the emulsion was placed into SnakeSkin dialysis tubing (MWCO lOKDa) and dialyzed against 25mM pH 9 borate buffer for 7 days with daily buffer changes. Aliquots of the emulsion are taken at each timepoint for HPLC quantification of chloroquine.
  • Figure 10 shows representative population shift in fluorescence in HFL1, Calu-3 and Vero E6 cells after incubation with 75 pg/mL Rho-NPs. Histograms represent fluorescence intensity across all cells present in one well of a 96-well plate, as measured by flow cytometry. Over a 24 hr period, population histograms shift rightward indicating an increase in fluorescence as a result of increased accumulation of fluorescent Rho-NPs.
  • FIG 11 shows Rho-NP (100 pg/mL) uptake into cells and the lysosomes.
  • Colocalization channel represents overlap between Rho-NPs and LysoTrackerTM (magenta).
  • Nuclei staining (cyan), cell membrane (green), and merged image (far right) are also provided. Pearson correlation coefficient (0 ⁇ r ⁇ 1) values were determined and show a strong positive correlation between NPs and lysosomal compartments.
  • Figures 12A-12B illustrate the characterization of NPs size and distribution before and after nebulization.
  • PGC-C18-12.5% Mefloquine NPs are measured via A) DLS (bars with +SD) and PDI(dots with ⁇ SD); and B) SEM undiluted (24 mg/mL) or diluted in lOmM pH 7.4 phosphate buffer before and after nebulization using an Aerogen® Pro nebulizer and controller. Note that size, distribution, and spherical morphology did not change significantly after being nebulized.
  • Figure 13 shows cytotoxicity after 72 hours of NP treatment in Calu 3 and Vero E6 cells for PGC-C18 NPs loaded with free fatty acids, mefloquine, or free fatty acid and mefloquine. Actinomycin D was utilized as apoptosis inducing positive control (500 nM) (Representative of 3 experiments).
  • Figure 14 shows cytotoxicity after 72 hours of NP treatment in Vero E6 cells for SLNs coated in low molecular weight poly(vinyl alcohol) (LMW PVA) and PGC-C18 NPs loaded with free fatty acids or fatty acids and chloroquine.
  • Actinomycin D was utilized as apoptosis inducing positive control (500 nM) and lysosomal inhibitor controls were chloroquine (1 and 10 pM) and bafilomycin Al (100 nM).
  • Figure 15 illustrates PGC-C18 and PGC-C18-12.5% Mefloquine (PGC-C18 MFQ) NPs effect on proteolytic activity in HFL1 cells as measured by DQ Red BSA assay (Invitrogen).
  • Pepstatin A (10 pg/mL) and E64d (10 pg/mL) were used as a protease inhibitor positive control.
  • Bafilomycin Al (100, 200 nM) was used as a lysosomal inhibitor control, and mefloquine (MFQ, 10 or 15 pM) was used to compare to NPs. Cells were treated with NPs for 24 hrs, and controls were incubated for time indicated.
  • mefloquine loaded PGC- C18 MFQ NPs exhibit a dosage dependent reduction in protease activity as indicated by a reduction in mean fluorescence intensity, ns, *, and **** indicate no statistical difference, P ⁇ 0.05, and P ⁇ 0.0001, respectively.
  • Figure 16B shows quantification of lysosomal pH. Data are displayed as box and whiskers graph indicating median, quartiles and interquartile ranges.
  • Figure 17B shows quantification of lysosomal accumulation. Data are displayed as means +SEM.
  • Figures 18B-18D show quantification of cell numbers, GFP-area per image, and syncytia for treatment groups. Statistical significance was determined by two-way ANOVA: a, p ⁇ 0.05 against MHV-Control cell numbers; b, p ⁇ 0.05 against MHV-Control infected cell area; c, p ⁇ 0.05 against MHV-Control syncytia count.
  • Figures 20B-20D show quantification of cell numbers and fraction of infected cells. Statistical significance was determined by two-way ANOVA: a, p ⁇ 0.05 against SARS-CoV-2 Control cell numbers; b, p ⁇ 0.05 against SARS-CoV-2-Control infected cell area.
  • Figure 24 shows cytotoxicity after 72 hours of NP treatment in Vero E6 cells for unloaded PGC-C18 NPs, PGC-C18 NPs loaded with mefloquine, or free molecular mefloquine at escalated dosages. (Representative of 3 experiments)
  • Figure 25A shows in vivo localization of Alexa-Fluor 750 conjugated NP to the thoracic area in C57/BL6J mice.
  • Figure 25B shows quantification of NP levels in mouse lung homogenates normalized to protein content.
  • Figure 25C shows Alexa-Fluor 750 conjugated NP localization in dissected mouse lungs.
  • Endocytosis is a highly conserved cell process which allows cells to uptake extracellular material and is essential for cell growth, function and repair. This process, and specifically receptor-mediated endocytosis, is a mechanism which many viruses leverage to gain entry to the cell cytosol for replication.
  • Coronaviruses are one family of viruses which leverage endocytic pathway to infect cells. Included in this family is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has emerged as the leading global health crisis termed the coronavirus disease-19 (COVID-19) pandemic.
  • the replication cycle of Co Vs involves three specific intracellular compartments: 1) endosomes and lysosomes; 2) endoplasmic reticulum; and, 3) Golgi.
  • the endocytic pathway which is responsible for the intake and digestion of extracellular materials, is a highly conserved mechanism that many viruses leverage to gain entry to the cytosol.
  • the spike protein of SARS-CoV-2 binds the angiotensin-converting enzyme 2 (ACE2) receptor on the surface of the cell resulting in receptor-mediated endocytosis.
  • ACE2 angiotensin-converting enzyme 2
  • CoV particles are engulfed at the cell surface by endosomes. These endosomes subsequently fuse with acidic lysosomes to create acidified ‘endolysosomes’.
  • SARS-CoV-2 relies on the acidic pH and activated proteases of the endocytic pathway for replication.
  • SARS-CoV-2 enters cells through endocytosis and leverages the low pH of the late endosome to activate proteases which facilitate membrane fusion and release of viral nucleic acids/proteins into the cytosol. While there are several pharmacological and molecular tools that may inhibit endosomal acidification, they are toxic to patients on a timescale of hours to days, potentially limiting the achievement of doses sufficient to halt viral replication.
  • palmitic acid a natural fatty acid present in many plants, organisms, and food products
  • palmitic acid is an inhibitor of endosome/lysosome acidification via inhibition of the V-ATPase proton pump.
  • the disclosure presented herein describes novel, biocompatible, solid lipid and polymeric nanoparticles that, like the virus, enter the cell via endocytosis whereupon the NPs release palmitate and/or small molecule antivirals.
  • the intra-endosomal release of palmitate results in enzymatic conjugation of palmitate to V-ATPase proton pumps, which are responsible for regulating endosomal pH.
  • the V-ATPase pumps are rendered inactive, thereby increasing the pH of the endosome and, subsequently, decreasing the proteolytic activity within endosomes.
  • the virus remains locked in its own envelope and does not replicate.
  • solid lipid nanoparticles refer to nanoparticles primarily comprised of a solid lipid such as but not limited to one or more of palmitic acid, stearic acid, or other fatty acids and their derivatives which are in a solid phase at room temperature (20 °C).
  • the lipids disclosed herein may be present in the NP as a free acid and/or deprotonated form, e.g., palmitate and stearate.
  • These particles may be prepared using a surfactant coating for improved stability and may encapsulate one or more fatty acids or their derivatives which are a liquid at room temperature to a lesser degree (e.g., >50% mole/mole of the total lipid is a solid lipid ).
  • Oleic acid is one such example of liquid lipid which acts to break up the local crystalline structure otherwise present in the SLN as a means to accelerate the degradation rate.
  • solid fatty acids and liquid fatty acids useful for the purposes disclosed herein include saturated fatty acids such as but not limited to lauric acid, myristic acid, palmitic acid, stearic acid and arachidic acid, and unsaturated fatty acids such as but not limited to myristoleic acid, palmitoleic acid, sapienic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, a-linolenic acid, arachidonic acid and eicosapentanoic acid. Any of the foregoing may be present in the NP as a free acid and/or deprotonated form.
  • saturated fatty acids such as but not limited to lauric acid, myristic acid, palmitic acid, stearic acid and arachidic acid
  • unsaturated fatty acids such as but not limited to myristoleic acid, palmitoleic acid, sapienic acid, elaidic acid, vaccenic acid,
  • SLNs are spherical in size and can range from about 10 - 1000 nm in diameter, and in some embodiments, from about 50 to about 250 nm in diameter.
  • the lipid encapsulated within the SLN may serve a biological function upon NP degradation, such as but not limited to palmitic acid inactivation of V-ATPase proton pumps.
  • active compounds can be loaded into the NPs which serve a biological function upon NP degradation and release.
  • polymeric nanoparticles refer to nanoparticles comprising functionalized poly (1,3 -glycerol carbonate) such as but not limited to poly(glycerol monostearate- co-e-caprolactone) (PGC-C18), and that may be surfactant coated.
  • Polymeric nanoparticles may be composed of, but are not limited to, functionalized polycarbonate alone, a mixture or copolymer of polycarbonates, a mixture or copolymer of polycarbonates and polycaprolactone, a mixture or copolymer of polycarbonates and poly(lactic acid), a mixture or copolymer of polycarbonates and poly (glycolic acid).
  • the hydroxyl group of the glycerol monomer may be functionalized with saturated or unsaturated fatty acyl chains of various lengths (e.g., 4 - 20 carbons) such as but not limited to saturated fatty acids such as but not limited to lauric acid, myristic acid, palmitic acid, stearic acid and arachidic acid, or unsaturated fatty acids such as but not limited to myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, a-linolenic acid, arachidonic acid and eicosapentanoic acid.
  • saturated fatty acids such as but not limited to lauric acid, myristic acid, palmitic acid, stearic acid and arachidic acid
  • unsaturated fatty acids such as but not limited to myristoleic acid, palmitoleic acid, sapienic acid,
  • Conjugation may comprise any one, or any combination of any fatty acids or derivatives such as but not limited to those described herein.
  • the fatty acyl chain conjugated to the polymer backbone may serve as a biologically active component upon hydrolysis such as but not limited to palmitic acid. Varying the fatty acyl chain length conjugated to the glycerol monomer also serves as a modulator for physical encapsulation of biologically active small molecule compounds such as but not limited to sulfadoxine, nitazoxanide, chloroquine or mefloquine.
  • the hydroxyl group of the glycerol monomer may be conjugated to a fluorophore such as but not limited to rhodamine B.
  • the polymeric nanoparticles may comprise both a fatty acid and fluorophore conjugated thereto.
  • the hydroxyl group of the glycerol monomer may be conjugated to a biologically active small molecule such as but not limited to sulfadoxine, nitazoxanide, chloroquine or mefloquine.
  • a biologically active small molecule such as but not limited to sulfadoxine, nitazoxanide, chloroquine or mefloquine.
  • These polymeric nanoparticles are spherical in size and can range from about 10 - 1000 nm in diameter, and in some embodiments, from about 50 to about 250 nm in diameter.
  • the lipid encapsulated within the NP or conjugated to the glycerol monomer may serve a biological function upon NP degradation, such as but not limited to palmitic acid inactivation of V-ATPase proton pumps.
  • active compounds can be loaded into the NPs which serve a biological function upon NP degradation and release.
  • Such polymeric nanoparticles may further encapsulate a fatty acid such as palmitic acid or stearic acid and/or may encapsulate an antiviral agent such as but not limited to those disclosed herein.
  • Non-limited examples include poly(glycerol monostearate-co-e-caprolactone) with a sodium dodecyl sulfate surfactant and encapsulating palmitic acid and/or palmitate, encapsulating stearic acid and/or stearate, encapsulating mefloquine, encapsulating chloroquine, encapsulating sulfadoxine, encapsulating nitazoxanide, encapsulating palmitic acid and/or palmitate and mefloquine, encapsulating palmitic acid and/or palmitate and chloroquine, encapsulating palmitic acid and/or palmitate and sulfadoxine, encapsulating palmitic acid and/or palmitate and nitazoxanide, or encapsulating stearic acid and/or stearate and mefloquine.
  • any of the foregoing may also be provided as encapsulated with poly(glycerol monolaurate-co-e- caprolactone) or poly (glycerol monopalmitate-co-e-caprolactone) polymeric nanoparticles.
  • solid lipid nanoparticles composed of palmitic acid can be formed via a number of techniques and variations of the techniques generally known in the art, including but not limited to mini-emulsion, high shear homogenization, high pressure homogenization, sonication, solvent displacement, and nanoprecipitation.
  • these NPs may be formed using an array of surfactant coatings, including but not limited to sodium dodecyl sulfate (SDS), poly(vinyl alcohol) (PVA), Triton X-100, Tween 80, Sodium taurocholate hydrate (STH), L-a-phosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) and triblock poly(ethylene glycol)-block-poly(propylene glycol) -block-poly (ethylene glycol) (PEG-PPG-PEG).
  • SDS sodium dodecyl sulfate
  • PVA poly(vinyl alcohol)
  • Triton X-100 Triton X-100
  • Tween 80 Triton X-100
  • STH Sodium taurocholate hydrate
  • L-a-phosphatidylcholine L-a-phosphatidylcholine
  • NPs may be formed using a number of different aqueous buffers including but not limited to deionized (DI) water, phosphate buffer solutions and borate buffer solutions. As it is known in the art, NPs can be formed at an array of temperatures above palmitate's melting temperature (62.9°C) and using various amounts of lipid and various ratios of lipid to surfactant and lipid to aqueous buffer.
  • DI deionized
  • NPs can be formed at an array of temperatures above palmitate's melting temperature (62.9°C) and using various amounts of lipid and various ratios of lipid to surfactant and lipid to aqueous buffer.
  • NPs have been synthesized using modifications of published heat/melt emulsion procedures. Briefly, palmitic (Cl 6 chain) and stearic acid (Cl 8 chain) were used to fabricate NPs. Fatty acids were heated above their melt temperature (85°C) and combined with heated solutions (85°C) of various surfactants followed by sonication to generate an emulsion. Various surfactants have been tested to generate stable surfactant-coated SLNs including PVA, SDS, Triton X-100, Tween 80, DPPC, DSPC and PEG-PPG-PEG.
  • Emulsification parameters can be modulated to optimize nanoparticle sizing as 50-100 nm NPs are known to be readily taken into the lysosome via the endocytic pathway. These parameters include the amplitude (e.g., about 5% - 35%) and duration of sonication (e.g., about 5 min - 30 min) as well as the quenching method and temperature. These NPs are thought to enable localized de-acidification of lysosomal pH, resulting in reduced proteolytic activity and stunting viral replication by preventing membrane fusion with the endosome/lysosomal membrane.
  • polymeric NPs composed of fatty acid functionalized poly(glycerol- co-e-caprolactone) (PGC) described herein and illustrated in the Examples are loaded with free fatty acids and/or synergistic antivirals to further reduce viral proliferation.
  • PGC NPs can be formed by a number of techniques and variations of the techniques generally known in the art, including but not limited to mini-emulsion, high shear homogenization, high pressure homogenization, sonication, solvent displacement, and nanoprecipitation.
  • the benzyl-protected secondary hydroxyl groups on the glycerol monomer were removed using Pd catalyzed hydrogenolysis and functionalized with stearic (Cl 8) acid.
  • various functionalized analogues of poly(glycerol-co-e-caprolactone) can be prepared using any method of reacting an alcohol known in the art. This includes but is not limited to functionalization with alkyl chains of various length (e.g. lauric, C12; myristic, C14; or palmitic acid, C16) to the copolymer backbone.
  • these PGC NPs are loaded with multiple pharmacological agents as well as bioactive molecules, including but not limited to mefloquine, sulfadoxine, nitazoxanide, chloroquine, palmitic acid and/or palmitate and stearic acid and/or stearate.
  • NP loading can be achieved either by physical encapsulation into the NP or via chemical conjugation of the desired agent onto the copolymer backbone or surfactant coating.
  • PGC is functionalized with stearic acid to form PGC-C18.
  • antivirals and or free fatty acids or their deprotonated forms have been physically encapsulated within the NPs.
  • PGC-C18 and/or fatty acids (e.g. palmitate, stearate) and/or antivirals (e.g. mefloquine, nitazoxanide, chloroquine, sulfadoxine) are first solubilized in methylene chloride.
  • This solution is added to a sonochemical reaction vessel along with a solution of sodium dodecyl sulfate (SDS) solubilized in buffer.
  • SDS sodium dodecyl sulfate
  • the size, morphology and stability of both SLNs and PGC -based NPs can be characterized by dynamic light scattering (DLS), scanning electron microscopy (SEM), Zeta Potentializer and tunable resistive pulse sensing (e.g., Izon qNano).
  • DLS dynamic light scattering
  • SEM scanning electron microscopy
  • Zeta Potentializer e.g., Izon qNano
  • Cell viability assays have assessed NP dosedependent toxicity on monkey kidney epithelium (Vero E6) cells. Using non-toxic doses, NP uptake has been evaluated using fluorescently-tagged NPs and flow cytometry. Additionally, cell staining and confocal microscopy has elucidated intracellular localization of NPs once endocytosed.
  • NPs of the present invention are relatively non-cytotoxic to the cells and are rapidly taken up by the cells ( ⁇ 1 hr) via one or a mixture of micropinocytosis, endocytotosis (e.g. clathrin or caveolin-mediated), or phagocytosis. After entry into the cells, the NPs are readily localized to acidic vesicular organelles (e.g., endosomes, lysosomes).
  • NPs The ability of NPs to de-acidify endosomes and lysosomes in cells is quantified by monitoring the cell via confocal microscopy and using LysoSensor Yellow/Blue dye.
  • Bafilomycin a known inhibitor of V-ATPase proton pumping activity, is used as a positive control while studies include untreated negative controls.
  • the efficacy of the NP formulations disclosed herein in reducing viral load the number of infected cells was quantified using a semi- quantitative imaging method. Briefly, immunofluorescence staining of the SARS-N protein was performed at the completion of the study followed by cell nuclei counterstaining with DAPI.
  • PGC-C18 NPs have been nebulized using an Aerogen® Pro nebulizer and controller and collected via centrifugation. DLS and SEM measurements indicated that the NPs retained structure, size and distribution after nebulization. This supports the clinical application of the NPs disclosed herein for localized delivery to the lungs, larynx and nasopharynx to treat the organs at greatest risk for respiratory viral infection.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • the term “about” refers to a deviance of between 0.1-5% from the indicated number or range of numbers. In another embodiment, the term “about” refers to a deviance of between 1-10% from the indicated number or range of numbers. In another embodiment, the term “about” refers to a deviance of up to 20% from the indicated number or range of numbers. In one embodiment, the term “about” refers to a deviance of ⁇ 10% from the indicated number or range of numbers. In another embodiment, the term “about” refers to a deviance of ⁇ 5% from the indicated number or range of numbers.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • the terms “treat”, “treatment”, or “therapy” refer to therapeutic treatment, including prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change associated with a disease or condition.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of the extent of a disease or condition, stabilization of a disease or condition (i.e., where the disease or condition does not worsen), delay or slowing of the progression of a disease or condition, amelioration or palliation of the disease or condition, and remission (whether partial or total) of the disease or condition, whether detectable or undetectable.
  • Those in need of treatment include those already with the disease or condition as well as those prone to having the disease or condition or those in which the disease or condition is to be prevented.
  • composition As used herein, the terms “component,” “composition,” “formulation”, “composition of compounds,” “compound,” “drug,” “pharmacologically active agent,” “active agent,” “therapeutic,” “therapy,” “treatment,” or “medicament,” are used interchangeably herein, as context dictates, to refer to a compound or compounds or composition of matter which, when administered to a subject (human or animal) induces a desired pharmacological and/or physiologic effect by local and/or systemic action.
  • a personalized composition or method refers to a product or use of the product in a regimen tailored or individualized to meet specific needs identified or contemplated in the subject.
  • the terms "subject,” “individual,” and “patient” are used interchangeably herein, and refer to an animal, for example a human, to whom treatment with a composition or formulation in accordance with the present invention is provided.
  • the term “subject” as used herein refers to human and non-human animals.
  • the terms "non-human animals” and “non-human mammals” are used interchangeably herein and include all vertebrates, e.g., mammals, such as non-human primates (e.g. higher primates) or rodent (e.g. mouse or rat).
  • the compositions described herein can be used to treat any suitable mammal, including primates, such as monkeys and humans, and rodents such as rats and mice.
  • the mammal to be treated is human.
  • the human can be any human of any age. In one embodiment, the human is an adult. In another embodiment, the human is a child.
  • the human can be male, female, pregnant, middle- aged, adolescent, or elderly.
  • the subject is canine, feline, bovine, equine, laprine or porcine.
  • compositions suitable for use in the methods disclosed herein include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose.
  • a “therapeutically effective amount” means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease (e.g., viral infection) or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.
  • the therapeutically effective amount or dose can be estimated initially from in vitro assays.
  • a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.
  • toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by individual physician in view of the patient's condition.
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is affected or diminution of the disease state is achieved.
  • the term "therapeutically effective amount” may encompass total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
  • a meaningful patient benefit i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
  • the term refers to that ingredient alone.
  • the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
  • compositions are formulated in a unit dosage form.
  • unit dosage forms refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
  • compositions of the invention may be formulated in a variety of ways, including for example, solid, semi-solid and liquid dosage forms, such as tablets, pills, powders, capsules, gels, liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes and suppositories.
  • the composition can also be in a form suitable for oral, intravenous, intraarterial, intramuscular, subcutaneous, parenteral, transmucosal, transdermal, or topical administration.
  • a single bolus may be administered.
  • several divided doses may be administered over time.
  • a dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for treating mammalian subjects. Each unit may contain a predetermined quantity of active compound calculated to produce a desired therapeutic effect. In some embodiments, the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved.
  • composition of the invention may be administered only once, or it may be administered multiple times.
  • the composition may be, for example, administered three times a day, twice a day, once a day, once every two days, twice a week, weekly, once every two weeks, or monthly.
  • dosage values may vary with the type and severity of the condition to be alleviated.
  • specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
  • administering refers to bringing in contact with a composition of the present invention. Administration can be accomplished to cells or tissue cultures, or to living organisms, for example humans. In one embodiment, the present invention encompasses administering the compositions of the present invention to a human subject. In another embodiment, methods of the present invention comprise the step of contacting one or more cells of a subject with a composition described herein.
  • compositions of the present invention may be administered prophylactically before infection, may be administered after suspected or known virus exposure but prior to the appearance of symptoms of infection, administered during an incubation period of a virus, or any combination thereof.
  • compositions of the present invention are administered to the patient in hospital. In one embodiment, the compositions of the present invention are administered to the patient in the intensive care unit. In one embodiment, the compositions of the present invention are administered when the patient is receiving mechanical ventilation. In one embodiment, the compositions of the present invention are administered when the patient is undergoing extracorporeal membrane oxygenation.
  • any of the therapeutic or prophylactic drugs or compositions described herein may be administered simultaneously. In another embodiment, they may be administered at different time points. In one embodiment, they may be administered within a few minutes of one another. In another embodiment, they may be administered within a few hours of one another. In another embodiment, they may be administered within 1 hour of one another. In another embodiment, they may be administered within 2 hours of one another. In another embodiment, they may be administered within 5 hours of one another. In another embodiment, they may be administered within 12 of one another. In another embodiment, they may be administered within 24 hours of one another.
  • an enzyme or "at least one enzyme” may include a plurality of enzymes, including mixtures thereof.
  • the subject in addition to treating the subject with the composition disclosed herein, the subject can be further treated with a second agent (e.g. an antiviral drug) prior to, after, or concurrently with treatment with the above composition.
  • a second agent e.g. an antiviral drug
  • the antiviral drug can be Remdesivir.
  • the coronavirus infection is caused by human coronavirus 229E (HCoV-229E), Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human coronavirus OC43 (HCoV-OC43), human coronavirus NL63 (HCoV- NL63), or human coronavirus HKU1 (HCoV-HKUl).
  • the coronavirus is SARS-CoV-2.
  • NPs containing palmitic acid were formed via sonication. Sonication is a simple, fast method that circumvents the use of highly toxic solvents to form a mono-disperse nanoparticle emulsion.
  • nanoparticle fabrication begins with separately melting palmitic acid and heating surfactant (sodium dodecyl sulfate, poly(vinyl alcohol), Triton X-100, Tween 80, dipalmitoylphosphatidylcholine, or distearoylphosphatidylcholine) solubilized in DI water, lOrnM pH 7.4 phosphate buffer, 25mM pH 8.0 borate buffer, or IX phosphate buffered saline on a hot plate set to 85°C.
  • surfactant sodium dodecyl sulfate, poly(vinyl alcohol), Triton X-100, Tween 80, dipalmitoylphosphatidylcholine, or distearoylphosphatidylcho
  • a preliminary hot emulsion was formed by mixing both phases under magnetic stirring. This mixture was then sonicated with a 1/8” probe at 35% amplitude in a constant (5min) or pulsatile (15min total, 2s on I Is off) manner to form a nano-emulsion. This solution was then quenched with cold (4°C) DI water, lOmM pH 7.4 phosphate buffer, 25mM pH 8.0 borate buffer or IX phosphate buffered saline to “lock-in” nanoparticle structure. Excess surfactant was removed by dialyzing overnight in 5mMpH 7.4 phosphate buffer, 25mM pH 8.0 borate buffer or IX phosphate buffered saline.
  • Palmitate NPs having different sizes (40 to 1800 nm in diameter) and stability (days to weeks) were varied, e.g., (1) amount of palmitic acid, (2) type of buffer, (3) lipid to buffer solution ratio, (3) type of surfactant, (4) lipid to surfactant ratio and (5) sonication time and method (Table 1).
  • the size, morphology and stability of the Palmitate NPs were characterized by dynamic light scattering (DLS), scanning electron microscopy (SEM) and Zeta Potentializer.
  • Ratios of palmitate:aqueous buffer below 1 :25 formed nanoparticles, and further lowering the amount of palmitate typically led to smaller NPs (from 350nm to 275nm as lipid mass is decreased from 375 mg to 25 mg). Mass of surfactant solubilized in buffer seemed to have minimal effect on particle sizing, but surfactant type appeared to have a significant effect.
  • palmitate NPs were quenched in an excess volume (e.g., 1:1 - 20:1 ratio of cold bufferhot NP emulsion) with cold (4°C) DI water, lOrnM pH 7.4 phosphate buffer, 25mM pH 8.0 borate buffer, or IX PBS.
  • NPs containing stearic acid were formed via sonication.
  • nanoparticle fabrication begins with separately melting stearic acid and heating surfactant (sodium dodecyl sulfate, poly(vinyl alcohol), Triton X-100, Tween 80, dipalmitoylphosphatidylcholine, or distearoylphosphatidylcholine) solubilized in DI water, lOmM pH 7.4 phosphate buffer, 25mM pH 8.0 borate buffer, or IX phosphate buffered saline on a hot plate set to 85°C.
  • a preliminary hot emulsion was formed by mixing both phases under magnetic stirring.
  • This mixture was then sonicated with a 1/8” probe at 35% amplitude in a constant (5min) or pulsatile (15min total, 2s on / Is off) manner to form a nano-emulsion.
  • This solution was then quenched with cold (4°C) DI water, lOmM pH 7.4 phosphate buffer, 25mM pH 8.0 borate buffer or IX phosphate buffered saline to “lock-in” nanoparticle structure. Excess surfactant was removed by dialyzing overnight in 5mMpH 7.4 phosphate buffer, 25mM pH 8.0 borate buffer or IX phosphate buffered saline.
  • stearate NPs having different sizes and stability
  • parameters e.g., (1) amount of palmitic acid, (2) type of buffer, (3) lipid to buffer solution ratio, (3) type of surfactant, (4) lipid to surfactant ratio and (5) sonication time and method.
  • the size, morphology and stability of the stearate NPs were characterized by dynamic light scattering (DLS), scanning electron microscopy (SEM) and ZetaPotentializer. Ratios of stearate:aqueous buffer below 1:25 (mass:mass) formed nanoparticles, and further lowering the amount of stearate led to smaller NPs.
  • DLS dynamic light scattering
  • SEM scanning electron microscopy
  • ZetaPotentializer ZetaPotentializer
  • stearate NPs were quenched in an equivalent volume or 2x volume (e.g., 1:1 or 2:1 ratio of cold buffer:hot NP emulsion) with cold (4°C) DI water, lOmM pH 7.4 phosphate buffer or 25mM pH 8.0 borate buffer.
  • PGC-C18 Poly(glycerol monostearate-co-e-caprolactone) (PGC-C18) was used to form nanoparticles as this hydrophobic polymer was capable of physically encapsulating both fatty acids and synergistic antivirals.
  • NPs were formed via sonication.
  • nanoparticle fabrication begins by solubilizing PGC-C18 (200mg) in methylene chloride (2mL). This solution was placed in a sonication bath for 5min to quickly form a homogenous solution. Next, a solution of sodium dodecyl sulfate (SDS, 80mg) in lOmM pH 7.4 phosphate buffer (8mL) was prepared.
  • SDS sodium dodecyl sulfate
  • PGC-C18 Poly(glycerol monostearate-co-e-caprolactone) (PGC-C18) was used to form nanoparticles as this hydrophobic polymer is capable of physically encapsulating both fatty acids and synergistic antivirals.
  • NPs were formed via sonication.
  • nanoparticle fabrication begins by solubilizing PGC-C18 (200mg) and palmitic acid (22.2mg) in methylene chloride (2mL). This solution was placed in a sonication bath for 5min to quickly form a homogenous solution.
  • PGC-C18 Poly(glycerol monostearate-co-e-caprolactone) (PGC-C18) was used to form nanoparticles as this hydrophobic polymer is capable of physically encapsulating both fatty acids and synergistic antivirals.
  • NPs were formed via sonication.
  • nanoparticle fabrication begins by solubilizing PGC-C18 (200mg), palmitic acid (22.2mg) and nitazoxanide (25mg) in methylene chloride (2mL). This solution was placed in a sonication bath for 5min to quickly form a homogenous solution.
  • PGC-C18 Poly(glycerol monostearate-co-e-caprolactone) was characterized by its ability to form nanoparticles as this hydrophobic polymer is capable of physically encapsulating both fatty acids and synergistic antivirals.
  • NPs were formed via sonication.
  • nanoparticle fabrication begins by solubilizing PGC-C18 (200mg), palmitic acid (22.2mg) and sulfadoxine (25mg) in methylene chloride (2mL). This solution was placed in a sonication bath for 5min to quickly form a homogenous solution.
  • PGC-C18 Poly(glycerol monostearate-co-e-caprolactone) was used to form nanoparticles as this hydrophobic polymer is capable of physically encapsulating both fatty acids and synergistic antivirals.
  • NPs were formed via sonication.
  • nanoparticle fabrication begins by solubilizing PGC-C18 (200mg), palmitic acid (22.2mg) and mefloquine (25mg) in methylene chloride (2mL). This solution was placed in a sonication bath for 5min to quickly form a homogenous solution.
  • PGC-C18 Poly(glycerol monostearate-co-e-caprolactone) was used to form nanoparticles as this hydrophobic polymer is capable of physically encapsulating both fatty acids and synergistic antivirals.
  • NPs were formed via sonication.
  • nanoparticle fabrication begins by solubilizing PGC-C18 (200mg), palmitic acid (22.2mg) and chloroquine (25mg) in methylene chloride (2mL). This solution was placed in a sonication bath for 5min to quickly form a homogenous solution.
  • PGC-C18 Poly(glycerol monostearate-co-e-caprolactone) was used to form nanoparticles as this hydrophobic polymer is capable of physically encapsulating both fatty acids and synergistic antivirals.
  • NPs were formed via sonication.
  • nanoparticle fabrication begins by solubilizing PGC-C18 (200mg) and stearic acid (22.2mg) in methylene chloride (2mL). This solution was placed in a sonication bath for 5min to quickly form a homogenous solution.
  • PGC-C18 Poly(glycerol monostearate-co-e-caprolactone) was characterized by its ability to form nanoparticles as this hydrophobic polymer is capable of physically encapsulating both fatty acids and synergistic antivirals.
  • NPs were formed via sonication.
  • nanoparticle fabrication begins by solubilizing PGC-C18 (200mg) and mefloquine (25mg) in methylene chloride (2mL). This solution was placed in a sonication bath for 5min to quickly form a homogenous solution.
  • PGC-C18 Poly(glycerol monostearate-co-e-caprolactone) was characterized by its ability to form nanoparticles as this hydrophobic polymer is capable of physically encapsulating both fatty acids and synergistic antivirals.
  • NPs were formed via sonication.
  • nanoparticle fabrication begins by solubilizing PGC-C18 (200mg) and chloroquine (25mg) in methylene chloride (2mL). This solution was placed in a sonication bath for 5min to quickly form a homogenous solution.
  • Poly (glycerol monostearate-co-e-caprolactone) (PGC-C18) was characterized by its ability to form nanoparticles as this hydrophobic polymer is capable of physically encapsulating both fatty acids and synergistic antivirals.
  • NPs were formed via sonication.
  • nanoparticle fabrication begins by solubilizing PGC-C18 (200mg) and nitazoxanide (25mg) in methylene chloride (2mL). This solution was placed in a sonication bath for 5min to quickly form a homogenous solution.
  • Poly (glycerol monostearate-co-e-caprolactone) (PGC-C18) was characterized by its ability to form nanoparticles as this hydrophobic polymer is capable of physically encapsulating both fatty acids and synergistic antivirals.
  • NPs were formed via sonication.
  • nanoparticle fabrication begins by solubilizing PGC-C18 (200mg) and sulfadoxine (25mg) in methylene chloride (2mL). This solution was placed in a sonication bath for 5min to quickly form a homogenous solution.
  • Solid lipid nanoparticles (e.g., palmitate, stearate) were formed via a heat/melt emulsion and sonication approach. Nanoparticle fabrication begins with separately melting 50 - 375 mg of fatty acid (palmitate or stearate) and heating a solution of 50 - 400 mg surfactant (sodium dodecyl sulfate, poly(vinyl alcohol), Triton X-100, Tween 80, dipalmitoylphosphatidylcholine or distearoylphosphatidylcholine) solubilized in 2 - 10 mL of DI water, lOmM pH 7.4 phosphate buffer, or IX phosphate-buffered saline (PBS) on a hot plate set to 85°C.
  • surfactant sodium dodecyl sulfate, poly(vinyl alcohol), Triton X-100, Tween 80, dipalmitoylphosphatidylcholine or
  • a preliminary hot emulsion was formed by mixing both phases under magnetic stirring ( ⁇ lk rpm). This mixture was then sonicated (Sonics & Materials Vibra-CellTM) with a 1/8” probe tip at 35% amplitude in a constant (5min) or pulsatile (15min total, 2s on / Is off) manner to form a nano-emulsion. This solution was then quenched with 10 - 30 mL cold (4°C) DI water, lOmM pH 7.4 phosphate buffer or IX PBS to “lock-in” nanoparticle structure.
  • PGC-C18 Poly(glycerol monostearate-co-e-caprolactone) (PGC-C18) nanoparticles were formed using a solvent evaporation technique and sonication.
  • nanoparticle fabrication begins by solubilizing PGC-C18 (200mg), fatty acid (palmitate or stearate, 22.2mg) and/or pharmacologic antivirals (25mg) in methylene chloride (2mL). This solution was placed in a sonication bath for 5min to quickly form a homogenous solution. Next, a solution of sodium dodecyl sulfate (80mg) in lOrnM pH 7.4 phosphate buffer (8mL) was prepared.
  • NPs were diluted 100 or 1000 times in DI water. Aliquots were plated on silicon wafers and allowed to air dry overnight. The wafers were affixed to aluminum stubs with copper tape and sputter coated with 5 nm Au/Pd. These samples were imaged using a Supra 55VP field emission scanning electron microscope (ZEISS) with an accelerating voltage of 3 kV and working distance of 6.0 mm.
  • ZEISS Supra 55VP field emission scanning electron microscope
  • TRPS resistive pulse sensing
  • Antiviral drug loading (e.g., mefloquine, chloroquine, nitazoxanide, sulfadoxine) was measured using high performance liquid chromatography (HPLC). Serial dilution standards were prepared in a mobile phase composed of: 45% - 0.1% triethylamine I phosphate buffer (pH 3.0) and 55% - acetonitrile. Standard samples for each pharmacologic agent were run for 8 min through a Zorbax SB300 - C18 column (150 mm length) and detected through UV absorbance to generate a standard curve.
  • HPLC high performance liquid chromatography
  • NPs were disrupted by adding acetonitrile to a final volume of 90% (v/v). This solution was then re- equilibrated by adding aqueous buffer to match mobile phase composition. This solution was filtered through a 0.22 pm syringe filter (Millipore) to remove large aggregates or dust prior to running samples. Samples were run similar to standards.
  • Fatty acid loading (e.g., palmitate, stearate, oleate) was measured using gas chromatography-mass spectrometry (GC-MS). Fatty acids were first extracted from aqueous suspensions of SLNs using chloroform and centrifugation. Pentadecanoic acid (C15 fatty acid) was then spiked into samples solubilized in chloroform (5 mg/mL) to serve as an internal standard for loading quantification. Fatty acids are then converted to fatty acid methyl esters (FAMEs) by mixing this solution with methanol and hydrochloric acid at 50 oC for 1 hr.
  • FAMEs fatty acid methyl esters
  • FAMEs are volatile enough to be readily separated by gas chromatography to separate fatty acids with chain lengths differing by as little as 1-2 carbons.
  • the FAMEs are then extracted from the chloroform phase using cyclohexane and the cyclohexane solution is then mixed 1:1 with ethyl acetate.
  • Samples were then run on a HP Agilent 6890 GC/MS System with 5973 MSD equipped with Agilent 7683 Series sample injector. Samples were run through a ZB-5MSPLUS (Zebron, # 7HG-G030-11) column.
  • the GC temperature program was as follows: 70 oC hold for one minute, then +30 oC/min ramp from 70 oC to 290 oC, and 290 oC hold for one minute.
  • Helium head pressure constant linear velocity mode 18 cm/s was 0.592 bar.
  • FACS analysis of rhodamine-labelled PGC-C18 treated cells was done with an Attune NxT Flow Cytometer (Invitrogen). HFL1, Calu 3, and Vero E6 were cultured in a 96- well plate at 12,000 cells/well for 1 day, after which the media was exchanged for media containing 75 pg/mL PGC-C18-Rho NPs. The cells were then incubated with treatment for up to 24 hours, after which cells were trypsinized, washed with IX PBS by centrifugation, resuspended in FACS buffer (IX PBS + 2% FBS) and then subjected to flow cytometry. Cell debris was excluded by gating on the forward and side scatter plot. FACS data analysis was performed using FlowJo software.
  • HFL1 cells were seeded at 50,000 cells/well in 12-well plates and grown overnight. Cells were then incubated with 100 pg/mL Rho-NPs (Ex:Em, 544:576 nm) for 1, 2, 4, or 24 hours. Following incubation, cells were washed with IX PBS and 50 nM LysotrackerTM Deep Red (Ex:Em, 647:668 nm) was added according to manufacturer’s protocol for 2 hours.
  • This solution was aspirated and replaced with a solution of 1 pg/mL Hoechst 33342 (Ex:Em, 361:486 nm), 50 nM LysotrackerTM Deep Red, and a 1:200 dilution of 1 mg/mL wheat germ agglutinin Oregon GreenTM 488 (Ex:Em, 496:524 nm) in warm (37°C) live cell imaging solution for 10 min.
  • This solution was aspirated and cells were washed with warm live cell imaging solution to remove unbound wheat germ agglutinin conjugates. After washing, cells were incubated with 1 pg/mL Hoechst 33342 in live cell imaging solution forimaging.
  • Vero E6 cells were seeded at a density of 10,000 cells/well into Greiner CellView 4 compartment dishes. After 24 h cells were stained with 5 mg/mL Lysosensor-Dextran Yellow/Blue dye in EMEM for 3 h following an overnight media chase. The next day cells were incubated for 24 h with 100 pg/mL PGC-NPs (+MFQ) or 10 pM mefloquine. Bafilomycin Al was added at a concentration of 200 nM as alkalization agent 2-4 h before imaging.
  • Imaging was performed using a Zeiss LSM880 equipped with a coherent 2-photon laser at a 2-photon excitation of 720 nm and 2 emission bands at 400-480 nm (blue) and 510-620 nm (yellow) using a 63X oil immersion objective. pH standard curves were generated by permeabilizing Lysosensor-Dextran Yellow/Blue stained cells with 10 pM nigericin, 20 pM monensin in pH clamped buffers ranging from pH 4.5 - 6.0. Lysosomal ROIs and Yellow/Blue staining intensity were determined using CellProfiler.
  • HFL1 cells were cultured in a 96- well plate at 15,000 cells/well for 1 day, after which the media was exchanged for media containing no treatment or PGC-C18 NPs (12.5, 25, 50, 75, 100 pg/mL NPs) or PGC-C18 NPs + 12.5 wt% MFQ (12.5, 25, 50, 75, 100 pg/mL NPs) for 24 h.
  • Control treatments include Bafilomycin Al (100, 200 nM), free MFQ (15 pM), and Pepstatin A (10 pg/mL) + E64d (10 pg/mL) for 4 h, or free MFQ (10, 15 pM) for 24 h.
  • PGC-C18 Mefloquine NPs were first fabricated as described earlier and concentration was established by lyophilizing 1 mL of nanoparticle solution and relating the resulting mass to said volume. NPs were then diluted to respective concentrations using lOmMpH 7.4 phosphate buffer as to prevent clogging of the vibrating meshes in the nebulizer at high concentrations. Diluted NPs were added to the top reservoir of an Aerogen® Pro nebulizer. A conical tube was used to collect nebulized sample, and then centrifuged to condense the vapor. Nebulized NPs were compared to the pre-diluted sample as well as pre-nebulized dilutions.
  • HFL1 cells were cultured in F12K media supplemented with 10% FBS, 50 units/ml penicillin, and 50 pg/ml streptomycin.
  • Vero-E6 and Calu3 cells were cultured in EMEM media supplemented with 10% FBS, 50 units/ml penicillin, and 50 pg/ml streptomycin.
  • cells were seeded at a density of 5,000 cells/well into 96- well plates. After 24 hours cells were treated with the indicated concentration of nanoparticles for 72 hours.
  • SARS-CoV-2 USA-WA1/2020 was obtained from the Biodefense and Emerging Infections (BEI) Resources of the National Institute of Health.
  • SARS-CoV-2 isolate hCoV- 19/USA/MD-HP20874/2021 (Lineage B.1.1.529; Omicron Variant) was used in later assays at MOI of 0.1. All work with SARS-CoV-2 was performed at the UCLA high containment laboratory at biosafety level 3.
  • SARS-CoV-2 was propagated and passaged in Vero E6 cells.
  • HCoV-OC43 was obtained from ATCC (VR-1558) and propagated in HCT-8 cells.
  • MHV-A59-GFP was propagated in 17CL-1 cells.
  • Viral titers were determined by assessing viral cytopathic effect (CPE) by microscopy in cells infected with serial 10-fold dilutions respectively.
  • TCIDso/ml was calculated using the Reed-Muench method.
  • L929 or Vero E6 cells are plated into 96-well Corning imaging plates 24 hrs later, cells undergo a 1 hr prophylactic pre-treatment with unloaded and PGC-C18-MFQ NPs (12.5-100 pg/mL), free MFQ (1.25-20 pM), positive control treatment (10 pM remdesivir), or no treatment. Following pre-treatment, media is replaced with serum free media containing virus (HCoV-OC43 at MOI of 1 or MHV at MOI of 0.1) for 1 hr.
  • Cells are then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, blocked with 2% BSA, 5% NDS, and infection is visualized by immuno-fluorescence staining of SARS- CoV-2 N protein with a rabbit polyclonal primary and a donkey-anti-rabbit AlexaFluor568 conjugated secondary antibody and cell nuclei counterstaining with DAPI. Plates were imaged with an Operetta High-Content imager using a 10X air objective, and images were processed using CellProfiler. Positive cells were determined as expanded nucleus ROIs containing above threshold virus positive staining, syncytia were defined as irregularly close nucleus clusters of >40 pm diameter.
  • Calu-3 cells were also used in a post-infection treatment model. Calu-3 cells were plated into 96-well, clear-bottom imaging plates. 24 hrs later, cells were treated with 100 pL of media containing virus (SARS-CoV-2 at MOI of 0.1). After 1 hr, cells were washed with IX PBS and cells were incubated with 100 pL of media containing IX concentrated unloaded and PGC-C18-MFQ NPs (25-200 pg/mL), free MFQ (2.5-40 pM), positive control treatment (20 pM remdesivir), or no treatment for 48 hrs.
  • SARS-CoV-2 virus
  • IX PBS IX concentrated unloaded and PGC-C18-MFQ NPs (25-200 pg/mL), free MFQ (2.5-40 pM), positive control treatment (20 pM remdesivir), or no treatment for 48 hrs.
  • cells are fixed after the last timepoint with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, blocked with 2% BSA, 5% NDS, and infection is visualized by immuno-fluorescence staining of SARS-CoV-2 N protein with a rabbit polyclonal primary and a donkey-anti-rabbit AlexaFluor568 conjugated secondary antibody and cell nuclei counterstaining with DAPI.
  • mice were euthanized using isoflurane overdose and cervical dislocation and lungs were extracted with or without inflation for IVIS imaging of whole lungs or whole mount imaging of lungs with a Zeiss LSM880 confocal microscope using a lOx objective.
  • Lung homogenates were used to quantify AF75O fluorescence using an NP standard curve on a Tecan Spark 10M plate reader. Protein content of the lungs was determined using a BCA assay.

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Abstract

Dans divers modes de réalisation, de nouvelles nanoparticules lipidiques solides biodégradables (SLN) et nanoparticules de poly(monostéarate de glycérol-co-ε-caprolactone) (PGC-C18) sont utilisées pour administrer de l'acide palmitique et/ou des antiviraux pour retarder la réplication virale. Ces nanoparticules (NP) se concentrent rapidement dans des lysosomes où elles sont dégradées et où elles libèrent des acides gras constitutifs qui augmentent le pH lysosomal, ce qui permet de diminuer la capacité de protéases dépendantes du pH de cliver des protéines virales qui initie la libération du génome viral dans le cytosol cellulaire. Ces NP servent d'agent thérapeutique pour réduire la prolifération de virus dépendant de la voie d'infection endocytaire, par exemple de virus respiratoires tels que des coronavirus, le virus de la grippe A ou le virus respiratoire syncytial.
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