WO2022072504A1 - Methods and compositions for the treatment of viral diseases - Google Patents

Methods and compositions for the treatment of viral diseases Download PDF

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Publication number
WO2022072504A1
WO2022072504A1 PCT/US2021/052664 US2021052664W WO2022072504A1 WO 2022072504 A1 WO2022072504 A1 WO 2022072504A1 US 2021052664 W US2021052664 W US 2021052664W WO 2022072504 A1 WO2022072504 A1 WO 2022072504A1
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Prior art keywords
peptidyl
virus
protease inhibitor
phe
mammalian protease
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PCT/US2021/052664
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French (fr)
Inventor
Sina Bavari
Simon NEWMAN
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Healion Bio, Inc.
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Publication of WO2022072504A1 publication Critical patent/WO2022072504A1/en
Priority to US18/055,596 priority Critical patent/US20230142126A1/en

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    • 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
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/32—One oxygen, sulfur or nitrogen atom
    • C07D239/42—One nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/14—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D295/155—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with the ring nitrogen atoms and the carbon atoms with three bonds to hetero atoms separated by carbocyclic rings or by carbon chains interrupted by carbocyclic rings

Definitions

  • SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2
  • COVID-19 severe acute respiratory disease 2019-19.
  • SARS-CoV-2 Repurposing previously identified drugs for therapeutic indications represents a potential path towards identifying promising candidate drugs to counteract current viral pathogens and possible emerging viruses.
  • SUMMARY OF THE DISCLOSURE The disclosure provides antiviral compositions and methods.
  • the present disclosure provides a method of inhibiting the growth of a virus for example, a coronavirus.
  • the methods may involve contacting the virus with a mammalian protease inhibitor.
  • the method may further include measuring the growth of the virus.
  • the growth of the virus may be measured by methods known in the art.
  • the present disclosure provides a method of reducing the percentage of virus infected cells in a population of cells. Such methods may include, contacting the virus infected cells with a mammalian protease inhibitor.
  • the method further involves measuring the percentage of virus infected cells. Also provided herein are methods of reducing coronavirus infection in a subject.
  • Such methods may include, contacting a subject in need with a mammalian protease inhibitor.
  • the efficacy of the mammalian protease inhibitor in reducing the coronavirus infection may be measured after providing the subject with mammalian protease inhibitor.
  • the mammalian protease inhibitor has the structure of Formula (I):
  • R1 and R2 are independently H or
  • R1 and R2 together with the carbon atom to which they are attached form a C3-C8 cycloalkyl ring; and R3 is an optionally substituted heterocyclic group comprising at least one nitrogen; and n is between 1 and 3.
  • the mammalian protease inhibitor may have the structure of
  • R4 is H, C1-C7 lower alkyl, C1-C7 lower alkoxy, C5-C10 aryl, or C3-C8 cycloalkyl.
  • the mammalian protease inhibitor may have the structure of [0010] In one aspect, the mammalian protease inhibitor may have the structure of [0011] The mammalian protease inhibitor may be a cathepsin inhibitor.
  • Non-limiting examples of cathepsin inhibitors include The method of claim 4, wherein the cysteine cathepsin inhibitor is Balicatib, E-64, E-64a, E-64b, E-64c, E- 64d, CA-074, CA-074 Me, CA-030, CA-028, peptidyl aldehyde derivatives leupeptin, antipain, chymostatin, Ac-LVK- CHO, Z-Phe-Tyr-CHO, a epoxisuccinate Z-Phe-Tyr(OtBu)-COCHO.H2O, 1- Naphthalenesulfonyl-Ile-Trp-CHO, Z-Phe-Leu-COCHO.H2O; peptidyl semicarbazone derivatives, peptidyl methylketone derivatives, peptidyl trifluoromethylketone, Biotin-Phe- Ala-fluoromethyl ketone, Z-Leu-Leu-Leu fluoro
  • the virus may be a virus in the family, Coronaviridae, or a virus in the sub-family Orthocoronavirinae, or a virus in the order Nidovirales
  • the methods of the disclosure may be used to inhibit the growth of any coronavirus.
  • the methods of the disclosure may be used to inhibit the growth of a coronavirus, such as a SARS-CoV-2 virus, SARS-CoV-1 virus, MERS-CoV virus, 229E virus, NL63 virus, OC43 virus, HKU1 virus, or variants thereof.
  • the coronavirus may be SARS-CoV-2 virus.
  • the concentration of the mammalian protease used may be from about 1 x 10 -12 M to about 1x10 -5 M, for example, from about 0.1 ⁇ M to about 50 ⁇ M.
  • the effective concentration (EC50) of the mammalian protease inhibitor against a coronavirus may be from about 0.25 ⁇ M to about 30 ⁇ M, for example, from about 0.5 ⁇ M to about 30 ⁇ M.
  • the effective concentration of the mammalian protease inhibitor against an enterovirus may be from about 15 ⁇ M to about 30 ⁇ M.
  • the EC50 of the mammalian protease inhibitor may be 0.1 ⁇ M, 0.3 pM, 1 ⁇ M, 3 ⁇ M, 10 ⁇ M or 30 ⁇ M.
  • the EC90 of the mammalian protease inhibitor may be from about I ⁇ M to lOO ⁇ M. As a non-limiting example, the EC90 may be I ⁇ M to 100 ⁇ M.
  • Contacting the coronavirus with the mammalian protease inhibitors may inhibit the growth of the coronavirus by from about 50% to about 100%.
  • the mammalian protease inhibitor may be associated with a selectivity index of at least 300.
  • Figure 1 shows the percentage of virus infected cells treated with varying concentrations of Beautycatib (HB-121).
  • Figure 2 shows the percentage of virus infected cells treated with varying concentrations of ONO-5334.
  • Figure 3 shows the percentage of virus infected cells treated with varying concentrations of Odanacatib (MK-0822).
  • Figure 4 is a Prior Art table showing the inhibition (IC50) by odanacatib and belacatib of different cathepsins relative to different diseases. The data was presented at the 8 th RCS-SCI Symposium on Proteinase Inhibitor Design, on April 16, 2013, hosted by the Royal Society of Chemistry.
  • Figure 5 is a graph depicting an effect of adding a cathepsin inhibitor to a weak antiviral compound.
  • the cathepsin inhibitor was able to enhance the antiviral activity of the compound by more than a log.
  • the large arrow depicts the shift in the Effective Concentration (EC50) of the antiviral.
  • the vertical dotted line depicts cytotoxicity of the drug with or without a potentiator.
  • Viruses comprise a large group of pathogens that are responsible for causing severe infectious diseases.
  • Therapeutic agents targeting viruses may be broadly classified into (i) therapeutic agents that target the viruses themselves or (ii) therapeutic agents that target host cell factors.
  • Virus-targeting therapeutic agents can function directly or indirectly to inhibit the biological functions of viral proteins, such as enzymatic activities, or to block viral replication machinery.
  • Host-targeting therapeutic agents target the host proteins that play a role in the viral life cycle, regulating the function of the immune system or other cellular processes in host cells.
  • the present disclosure provides host -targeting therapeutic agents for the treatment of viral di seases.
  • the present disclosure provides virus- targeting therapeutic agents and the related compositions. Also provided herein are methods of inhibiting the growth of a virus, and methods of reducing the percentage of virus infected cells in a population of cells. II. COMPOSITIONS
  • the compositions of the disclosure may be or may include protease inhibitors.
  • protease inhibitors are small molecule that block or reduce the activity of a protease.
  • proteases may be essential for virus replication. Many human pathogenic viruses use human enzymes to activate the viral proteins and successfully overtake the infected cell processes. For example, human cathepsins assist in the cleavage of viral proteins that are essential for the virus life cycle. These proteases may include, but are not limited to cysteine proteases, serine proteases, aspartic proteases.
  • the compositions of the disclosure may be or may include a “cathepsin inhibitor” which, as used herein may refer to an agent which is capable of reducing, suppressing or inhibiting the activity of the class of endosomal proteases called cathepsins.
  • the cathepsins may require acidic pH for enzyme activity.
  • the cathepsins may be enzymatically active at neutral pH. There are approximately a dozen members of this family, which are distinguished by their structure, catalytic mechanism, and which proteins they cleave. Cathepsins have a vital role in mammalian cellular turnover, e.g. bone resorption. They degrade polypeptides and are distinguished by their substrate specificities.
  • C1 cysteine cathepsins are endopeptidases (L, S, K, V, F), while cathepsin X is a carboxypeptidase and cathepsins B, C and H have both endopeptidase- and exopeptidase activities.
  • the substrate-binding region of cysteine cathepsins is defined as an arrangement of binding subsites (SeSO) for peptide substrate amino acids (PePO) on both sides (N- and C-) of the scissile bond, encompassing the stretch of seven sites from S4 to S30 of papain.
  • S2 and S10 sites are the major determinants of specificity, SI is important for the affinity and efficient catalysis of substrates.
  • the positioning of the P3 residue in site S3 is, as in subsite S20, mediated only by side chain contacts over a relative wide area.
  • Cathepsins K, L, S and V have somewhat overlapping specificities, making it difficult to discriminate between them in vivo.
  • Cathepsin K attacks sites having aliphatic amino acids (Leu, lie, Val), unlike cathepsins L and V (which both rather accept hydrophobic residues with preference for Phe), and also accommodates Pro in the S2 subsite.
  • Cathepsin K is unusual among cysteine cathepsins in that it can cleave substrates with Pro in the P2 position, although it has been reported that congopain, a cysteine protease from Trypanosoma congolense, with an amino acid sequence (65% of homology) and biochemi cal properties similar to cathepsin K, also does so. Another feature of cathepsin K is its preference for Gly at the P3 position.
  • Cathepsin K is a protease, which is defined by its high specificity for kinins, that are involved in bone resorption, as discussed in U.S. Patent 6,642,239, which is hereby incorporated by reference in its entirety.
  • the enzyme's ability to catabolize elastin, collagen, and gelatin allow it to break down bone and cartilage. This catabolic activity is also partially responsible for the loss of lung elasticity and recoil in emphysema.
  • Cathepsin K inhibitors, such as odanacatib show great potential in the treatment of osteoporosis.
  • Cathepsin K is also expressed in a significant fraction of human breast cancers, where it could contribute to tumor invasiveness.
  • Mutations in this gene are the cause of pycnodysostosis, an autosomal recessive disease characterized by osteosclerosis and short stature. Cathepsin K expression is stimulated by inflammatory cytokines that are released after tissue injury.
  • Proteases may be grouped according to the key catalytic group in the active site.
  • the active site of the protease may include a serine (Ser), a threonine (Thr), a cysteine (Cys), an aspartate (Asp), a glutamate (Glu), or a zinc in the case of metal loproteases.
  • the proteases may be a serine protease, a threonine protease, a cysteine protease, an aspartate protease, a glutamate protease, or a zinc protease.
  • the protease may be a mammalian protease and the inhibitor may be a mammalian protease inhibitor.
  • the mammalian protease may be a cathepsin protease and the inhibitor may be a cathepsin protease inhibitor.
  • the cy steine protease inhibitor is a cathepsin inhibitor such as a cathepsin-B inhibitor, a cathepsin-L inhibitor, a cathepsin-S inhibitor, a cathepsin-F inhibitor, a cathepsin-X inhibitor, a cathepsin-K, inhibitor, a cathepsin- V inhibitor, a cathepsin-W inhibitor, a cathepsin-C inhibitor, a cathepsin-0 inhibitor, and a cathepsin- H inhibitor.
  • the cathepsin inhibitor is a cathepsin-K. inhibitor.
  • the SARS-Cov-2 virus has 3-way redundancy for infection and viral entry.
  • Cleavage of S1-S2 protein can be accomplished by serine proteases such as TMPRSS2, or the enzyme furin, or a cathepsin, such as, but not limited to cathepsin L.
  • the cathepsin inhibitor is epoxi succinate and derivative thereof; E-64; E-64a; E-64b; E-64c; E-64d; CA-074; CA-074 Me, CA-030; CA-028; peptidyl aldehyde derivatives leupeptin, antipain, chymostatin, Ac-LVK-CHO, Z-Phe-Tyr-CHO, Z- Phe- Tyr(OtBu)-COCHOH2O, 1 -Naphthal enesulfonyl-He-Trp- CHO, Z-Phe-Leu- COCHO.H2O; peptidyl semicarbazone derivatives; peptidyl methylketone derivatives; peptidyl trifluoromethylketone derivatives Biotin- Phe-Ala-fluoromethyl ketone, Z-Leu-Leu- Leu-fluoromethy!
  • ketone Z-Phe-Phe-fluoromethyl ketone, N-Methoxysuccinyl-Phe-HOMO- Phe-fluoromethyl ketone, Z-Leu-Leu-Tyr-fluorom ethyl ketone, Leupeptin trifluoroacetate, ketone; peptidylchloromethases and derivatives thereof; peptidylhydroxymates and derivatives thereof; peptidylhydroxylamines and derivatives thereof; peptidyl acyloxymethanes and derivatives thereof; peptidylacyl oxy methyl ketones and derivatives thereof; peptidyl aziridines and derivatives thereof; peptidyl aryl vinyl sulfones and derivatives thereof; peptidyl arylvinylsulfonates and derivatives thereof; gallinamide analogs and derivates thereof; peptidyl aldehydes and derivatives thereof; azepinone-based inhibitors and derivatives thereof; nitrile-
  • the cathepsin inhibitor may be Beautycatib (AAE581).
  • proteases are essential for virus replication. Many human pathogenic viruses use human enzymes to activate the viral proteins and successfully overtake the infected cell processes. For example, human cathepsins assist in the cleavage of viral proteins that are essential for the virus life cycle. These proteases may include but are not limited to cysteine proteases and proteinases, serine proteases, aspartic proteases.
  • serine protease inhibitors like Camostat, Odalasvir, Femostat, or any other protease inhibitors currently in use for HIV such as atazanavir (Reyataz), darunavir (Prezista), fosamprenavir (Lexiva), indinavir (Crixivan), lopinavir/ritonavir (Kaletra), nelfinavir (Viracept), ritonavir (Norvir), saquinavir (Invirase), tipranavir (Aptivus), atazanavir/cobicistat (Evotaz), darunavir/cobicistat (Prezcobix); or other protease inhibitors use for other viruses such as HCV for example asunaprevir, boceprevir, grazoprevir, glecaprevir, paritaprevir, simeprevir, telaprevir, and in HBV treatment.
  • HCV protease inhibitors use for other viruses such
  • a cathepsin inhibitor may be an agent whose main pharmacological effect is to inhibit the activity of the class of endosomal cysteine peptidases that may be enzymatically active in acidic pH or neutral pH.
  • human cysteine proteases include but are not limited to cathepsins, which include but are not limited to cathepsin B, cathepsin L, cathepsin S, cathepsin-F, cathepsin-X, cathepsin K, cathepsin V, cathepsin W, cathepsin C, cathepsin O, and cathepsin H.
  • Cathepsin inhibitors useful in nonhuman animals are often categorized differently but are known to those of skill in the art.
  • the inhibitors include cathepsin inhibitors which are known to correspond with human cathepsin inhibitors.
  • Inhibitors of these cathepsins are useful according to methods of the disclosure. Many cathepsin inhibitors have been described in the literature and are well known and are commercially available.
  • Cathepsin inhibitors have a variable level of specificity. There is a reasonable comparison between in-situ peptide assays versus in-vitro cell-based assay. Most of the literature discusses the specificity of cathepsin inhibitors based on the selectivity of cleavage in a peptide assay. The same literature assumes the same level of specificity in an in-cell culture and in-vivo applications. However, the cathepsin inhibitor specificity is different even in in-vitro cell cultures compared to in-situ peptide assays.
  • a cathepsin K inhibitor may be active against cathepsin-K, cathepsin-L, cathepsin- B in a patient, as shown in Figure 4.
  • the non-specific activity may be dose dependent.
  • the non-specific activity may also be affected by other factors such as pH of the endosomes and lysosomes in the cells.
  • Specific cathepsin inhibitors may become broadly acting in cells.
  • compositions of the present disclosure may include dipeptide dinitriles.
  • the mammalian protease inhibitor may be a dipeptide nitrile.
  • any of the compounds described in International Patent Publication WO2001 058886 (the contents of which are herein incorporated by reference in its entirety).
  • the compound has a structure of Formula (I) (I), wherein, R 1 and R2 are independently H or Cl-
  • R1 and R2 together with the carbon atom to which they are attached form a C3-C8 cycloalkyl ring; and R3 is an optionally substituted heterocyclic group comprising at least one nitrogen; n is between 1 and 3.
  • the compound of the present disclosure has a structure of: wherein the protease inhibitor has the structure of Formula (II): (II), wherein X is CH or N; and
  • R4 is H, C1-C7 lower alkyl, C1-C7 lower alkoxy, C5-C10 aiyl, or C3-C8 cycloalkyl.
  • the compound has a structure of
  • the compound has a structure of
  • the compound has a structure of
  • compositions of the disclosure may include a compound of Formula (I) of the International Patent Publication W02001058886 and provided below as Formula (III),
  • compositions of the disclosure may be or may include
  • the compounds have a structure of Formula (IV) (IV), or a pharmaceutically acceptable salt thereof , wherein R1 and R2 are independently H, C1-C3 alkyl, C3-C6 cycloalkyl, or form a C3-C6 cycloalkyl group with the carbon to which they are attached, wherein the Cl-
  • C3 alkyl or the C3-C6 cycloalkyl is optionally substituted;
  • A is a bond, C1-C3 alkyl, C6 aryl, or C6 heteroaryl, wherein the C1-C3 alkyl, C6 aryl, or C6 heteroaryl is optionally substituted;
  • B is a bond, C1-C3 alkyl, an amide, an amine, wherein the C1-C3 alkyl, amide or amine is optionally substituted; and C is a C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, C6 aryl, or C6 heteroaryl, wherein the C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, C6 aryl, or C6 heteroaryl is optionally substituted.
  • A is a C6 heteroaryl comprising one or two nitrogens. In some embodiments, A is optionally substituted
  • B is optionally substituted -CH2-NH-CH2- or optionally substituted -CO-NH-CH2-.
  • C is a substituted piperazine group wherein the substituent may be a C1-C3 alkyl.
  • C is a phenyl group wherein the substituent may be a C1-C3 alkyl, halogen, or -SO2-CH3.
  • compositions of the disclosure may be or may include N-[l - (Cyanomethyl carbamoyl) cyclohexyl] 4-[4-(l-propy 1) piperalin 1 y 1] benzamide, or a pharmaceutically acceptable salt.
  • Non-limiting examples of the compounds useful in the present disclosure include, N ⁇ [ 1 -(Cyanomethyl-carbamoyl)-cyclohexyl] -4-(piperazin- 1 - yl)-benzamide; N-[l-(Cyanomethyl-carbamoyl)-cyclohexyl]-4-(4-methyl-piperazin-l-yl)- benzarnide; N-[r-(Cyanomethyl-carbamoyl)-cyclohexyl]-4-(4-ethyl-piperazin-l-yl)- benzamide; N-[l-(Cyanomethyl-carbamoyl)-cyclohexyl]-4-(4-isopropyl-piperazin-l-yl)- benzamide; N-[l-(Cyanomethyl-carbamoyl)-cyclohexyl]-4-(4 ⁇ benzyI-piperazin-l
  • the compounds of the present disclosure may comprise at least one warhead moiety.
  • the warhead moiety can be any covalent binding modality that is capable of forming a covalent bond with a biological target.
  • the warhead moiety may comprise one or more chemical groups, one or more of which is capable of forming a covalent bond with a biological target.
  • the warhead moiety comprises nitrile (-CN).
  • the present disclosure provides methods wherein a composition provides an increase in bioavailability of a drug, such as an anti-viral, when combined with a protease inhibitor, for example a cathepsin inhibitor, as measured by AUC (Area Under the Curve) of at least 25% relative to dosing of the drug alone.
  • AUC Area Under the Curve
  • the present disclosure also provides methods wherein the composition provides an increase in bioavailability of the drug combination as measured by AUC of at least 50% relative to dosing of the drug alone.
  • the present disclosure further provides methods wherein said composition provides an increase in bioavailability of the drug in combination as measured by AUC of at least 100% relative to dosing of the drug alone.
  • Certain small molecules have unexpected anti-viral activity, as they were developed and examined for non-viral disease states. These small molecules act as an antiviral whether as a monotherapy or in combination with other anti-viral compounds. The small molecules may demonstrate anti-viral activity across all virus classes, in which anti-viral activity has been proven. The efficacy of these small molecules is especially high against Coronaviruses (i.e. SARS-CoV-2.)
  • These small molecules may be effective as a pre-exposure prophylaxis during a viral outbreak, for healthcare workers, first responders, at-risk workers, or travelers. These small molecules may be effective as a post-exposure prophylaxis as well.
  • the post-exposure treatment can be used to prevent a fatal or severe infection and in a “track, trace, and treat” program implementation. Even with a viable vaccine, this form of treatment is necessary.
  • These small molecules may treat mild and severe viral infections. As a treatment for infections, these small molecules may treat the viral load and prevent or reduce hospital stays and reduce the burden on the healthcare system. This treatment may be effective when dealing with multiple non-viral complications including organ failure, ARDS, pneumonia, or other side effects.
  • These small molecules may be effective in a wide array of patient populations include those over 65, patients with comorbidities, and immunosuppressed patients.
  • the disclosure provides methods for the identification of a compound that produces synergistic activity with a drug of choice.
  • the disclosure provides methods for the identification of a compound that reduces the effective dosage of a drug of choice. Any technique well-known to the skilled artisan can be used to screen for a compound that would reduce the effective dose of a drug.
  • a cell is contacted with a test protease inhibitor in combination with a drug of choice, for example an antiviral drug.
  • a control without the test protease inhibitor is provided.
  • the cell can be contacted with a test protease inhibitor before, concurrently with, or subsequent to the administration of the drug.
  • a cell was incubated with multiple concentrations of a drug and test protease inhibitor, for at least 1 minute to at least 10 minutes during the experiment.
  • the effect of the combination on the viral replication was measured at various times during the assay.
  • a time course of viral growth in the culture was determined. If the viral growth is inhibited or reduced in the presence of the test compound at reduced drug concentrations wherein the effect is more than an additive effect, the test protease inhibitor is identified as being effective in producing a synergistic activity.
  • the disclosure provides a composition that increases the bioavailability of the drug when in combination with a protease inhibitor, for example a cathepsin inhibitor, as measured by Cmax of at least 50% relative to dosing of the drug alone, which is shown in Figure 5.
  • the disclosure also provides said composition that increases the bioavailability of the drug when in combination as measured by Cmax of at least 100% relative to dosing of the drug alone.
  • the disclosure further provides said composition which provides an increase in bioavailability of the drug when in combination as measured by Cmax of at least 200% relative to dosing of drug alone.
  • Systemic drug concentrations are measured using standard biochemical drug measurement techniques (Simmons et al., Anal Lett. 39: 2009-2021 (1997)).
  • the combinations of the disclosures may be tested for in vitro activity against a disease or microorganism and sensitivity, and for cytotoxicity in laboratory adapted cell lines or cultured cells such as peripheral blood mononuclear cells (PBMC), human fibroblast cells, hepatic, renal, epithelium cells, according to standard assays developed for testing compounds.
  • PBMC peripheral blood mononuclear cells
  • Combination assays may be performed at varying concentrations of the compounds of the combinations to determine EC 50 by serial dilutions.
  • HEp-2 (CCL-23), PC-3 (CCL-1435), HeLa (CCL-2), U2OS (HTB-96), Vero (CCL-81), HFF-1 (SCRC-1041), and HepG2 (HB-8065) cell lines can be purchased from the American Type Culture Collection.
  • HEp-2 cells can be cultured in Eagle’s Minimum Essential Media (MEM) with GlutaMAX supplemented with 10% fetal bovine serum (FBS) and 100 U ml ⁇ l penicillin and streptomycin.
  • PC-3 cells can be cultured in Kaighn’s F12 media supplemented with 10% FBS and 100 U ml-1 penicillin and streptomycin.
  • HeLa, U2OS, and Vero cells can be cultured in MEM: supplemented with 10% FBS, 1% L- glutamine, 10 mM HEPES, 1% non-essential amino acids, and 1% penicillin/ streptomycin.
  • HFF-1 cells can be cultured in MEM supplemented with 10% FBS and 0.5 mM sodium pyruvate.
  • HepG2 cells can be cultured in Dulbecco’s Modified Eagle Medium (DMEM) with GlutaMAX supplemented with 10% FBS, 100 U ml-1 penicillin and streptomycin, and 0.1 mM non-essential amino acids.
  • DMEM Modified Eagle Medium
  • the MT-4 cell line can be obtained from the NIH AIDS Research and Reference Reagent Program and cultured in R PM 1-1640 medium supplemented with 10% FBS, 100 U ml-1 penicillin and streptomycin, and 2 mM L -glutamine.
  • the Huh-7 cell line can be obtained from C. M. Rice (Rockefeller University) and cultured in DMEM supplemented with 10% FBS, 100 U ml-1 penicillin and streptomycin, and non-essential amino acids.
  • Primary human hepatocytes or other primary cell can be purchased from Invitrogen and cultured in William’s Medium E medium containing cell maintenance supplement.
  • Donor profiles will be limited to 18- to 65-year-old nonsmokers with limited alcohol consumption. Upon delivery, the cells will be allowed to recover for 24 h in complete medium with supplement provided by the vendor at 37°C.
  • Human PBMCs will be isolated from human buffy coats obtained from healthy volunteers (Stanford Medical School Blood Center, Palo Alto, California) and maintained in RPMI-1640 with GlutaMAX supplemented with 10% FBS, 100 U ml-1 penicillin and streptomycin.
  • PBMCs will be isolated from whole blood by Ficoll-Hypaque density gradient centrifugation. Briefly, blood will be overlaid on 15 ml Ficoll-Paque (GE Healthcare Bio-Sciences AB), and centrifuged at 500g for 20 min. The top layer containing platelets and plasma wall be removed, and the middle layer containing PBMCs will be transferred to a fresh tube, diluted with Tris buffered saline up to 50 ml, and centrifuged at 500g for 5 min.
  • Ficoll-Paque GE Healthcare Bio-Sciences AB
  • the supernatant will be removed and the cell pellet will be resuspended in 5 ml red blood cell lysis buffer (155 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA, pH 7.5).
  • red blood cell lysis buffer 155 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA, pH 7.5.
  • freshly isolated quiescent PBMCs will be seeded into a T-150 (150 cm2) tissue culture flask containing fresh medium supplemented with 10 U ml-1 of recombinant human interleukin-2 (IL-2) and 1 pg ml-1 phytohaemagglutinin-P at a density of 2 x 106 cells ml-1 and incubated for 72 h at 37°C.
  • IL-2 human interleukin-2
  • Human macrophage cultures will be isolated from PBMCs that will be purified by Ficoll gradient centrifugation from 50 ml of blood from healthy human volunteers.
  • PBMCs will be cultured for 7 to 8 days in in RPMI cell culture media supplemented with 10% FBS, 5 to 50 ng ml-1 granulocyte-macrophage colony-stimulating factor and 50 ⁇ M p-mercaptoethanol to induce macrophage differentiation.
  • the cryopreserved human primary' renal proximal tubule epithelial cells will be obtained from LifeLine Cell Technology and isolated from the tissue of human kidney.
  • HMVEC-TERT Immortalized human microvascular endothelial cells
  • the intracellular metabolism of nucleoside may be assessed in different cell types (HMVEC and HeLa cell lines, and primary' human and rhesus PBMCs, monocytes and monocyte-derived macrophages) following 2-h pulse or 72-h continuous incubations with 10-1,000 pM of nucleobase or nucleoside.
  • intracellular metabolism during a 72-h incubation with 10-1,000 ⁇ M of Nuc will be completed in human monocyte-derived macrophages.
  • monocyte-derived macrophages isolated from rhesus monkeys or humans will be incubated for 2 h in compound-containing media followed by removal, Uimethylhexylamine (DMH) in water for analysis by liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS).
  • DMH Uimethylhexylamine
  • LC-MS/MS may be performed using low-flow ion-pairing chromatography, similar to methods described previously (Durand-Gasselin L, et al.
  • Nucleotide analogue prodrug tenofovir disoproxil enhances lymphoid cell loading following oral administration in monkeys. Mol. Pharm. 2009; 6: 1145 -1 151).
  • Analytes may be separated using a 50 x 2 mm x 2.5 pm Luna Cl 8(2) HST column (Phenomenex) connected to a LC-20ADXR (Shimadzu) ternary pump system and HTS PAL autosampler (LEAP Technologies).
  • a multi-stage linear gradient from 10% to 50% acetonitrile in a mobile phase containing 3 mM ammonium formate (pH 5.0) with 10 mM dimethylhexylamine over 8 min at a flow rate of 150 ⁇ l min-1 may be used to separate analytes.
  • Detection may be performed on an API 4000 (Applied Biosystems) MS/MS operating in positive ion and multiple reaction monitoring modes.
  • Intracellular metabolites alanine metabolite, Nuc, nucleoside monophosphate, nucleoside diphosphate, and nucleoside triphosphate may be quantified using 7-point standard curves ranging from 0.274 to 200 pmol (approximately 0.5 to 400 pM) prepared in cell extract from untreated cells. Levels of adenosine nucleotides may be also quantified to assure dephosphorylation had not taken place during sample collection and preparation. In order to calculate intracellular concentration of metabolites, the total number of cells per sample may be counted using a Countess automated cell counter (Invitrogen).
  • Ebola Antiviral testing can be conducted in a biosafety level 4 containment (BSL-4), for example at the Centers for Disease Control and Prevention.
  • BSL-4 biosafety level 4 containment
  • EBOV antiviral assays may be conducted in primary/ HMVEC-TERT and in Huh-7 cells. Huh-7 cells will not be authenticated and will not be tested for mycoplasma.
  • Ten concentrations of compound may be diluted in fourfold serial dilution increments in media, and 100 pl per well of each dilution may be transferred in duplicate (Huh-7) or quadruplicate (HMVEC-TERT) onto 96-well assay plates containing cell monolayers.
  • the plates may be transferred to BSL-4 containment, and the appropriate dilution of virus stock may be added to test plates containing cells and serially diluted compounds. Each plate will include four wells of infected untreated cells and four wells of uninfected cells that serve as 0% and 100% virus inhibition controls, respectively.
  • assay plates may be incubated for 3 days (Huh-7) or 5 days (HMVEC-TERT) in a tissue culture incubator. Virus replication may be measured by direct fluorescence using a Biotek HTSynergy plate reader.
  • Huh-7 cells may be infected with wild-type EBOV for 1 h at 0.1 plaque-forming units (PFU) per cell.
  • the virus inoculum may be removed and replaced with 100 pl per well of media containing the appropriate dilution of compound. At 3 days post-infection, supernatants may be collected, and the amount of virus may be quantified by endpoint dilution assay.
  • the endpoint dilution assay may be conducted by preparing serial dilutions of the assay media and adding these dilutions to fresh Vero cell monolayers in 96-well plates to determine the tissue culture infectious dose that caused 50% cytopathic effects (TCID50).
  • total RNA may be extracted using the MagM AX-96 Total RNA Isolation Kit and quantified using a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay with primers and probes specific for the EBOV nucleoprotein gene.
  • qRT-PCR quantitative reverse transcription polymerase chain reaction
  • antiviral assays may be conducted in BSL-4.
  • HeLa or HFF- 1 cells may be seeded at 2,000 cells per well in 384-weH plates.
  • Ten serial dilutions of compound in triplicate may be added directly to the cell cultures using the HP D300 digital dispenser (Hewlett Packard) in twofold dilution increments starting at 10 ⁇ M at 2 h before infection.
  • the DMSO concentration in each weB may be normalized to 1% using an TIP D300 digital dispenser.
  • the assay plates may be transferred to the BSL-4 suite and infected with EBOV Kikwit at a multiplicity of infection of 0.5 PFU per cell for HeLa cells and with EBOV Makona at a multiplicity of infection of 5 PFU per cell for HFF-1 ceils.
  • the assay plates may be incubated in a tissue culture incubator for 48 h. Infection may be terminated by fixing the samples in 10% formalin solution for an additional 48 h before immune-staining, as described.
  • antiviral assays in EBOV human macrophages may be conducted in BSL-4.
  • Primary human macrophage cells may be seeded in a 96-well plate at 40,000 cells per well. Eight to ten serial dilutions of compound in triplicate may be added directly to the cell cultures using an HP D300 digital dispenser in threefold dilution increments 2 h before infection. The concentration of DMSO may be normalized to 1% in all wells.
  • the plates may be transferred into the BSL-4 suite, and the cells may be infected with 1 PFU per cell of EBOV in 100 pl of media and incubated for 1 h. The inoculum may be removed, and the media may be replaced with fresh media containing diluted compounds.
  • virus replication may be quantified by immuno-staining.
  • compounds may be threefold serially diluted in source plates from which 100 ml of diluted compound may be transferred to a 384-web cell culture plate using an Echo acoustic transfer apparatus.
  • HEp-2 cells may be added at a density of 5 x 105 cells per ml, then infected by adding RSV A2 at a titer of 1 x 104.5 tissue culture infectious doses (TCID50) per ml.
  • TCID50 tissue culture infectious doses
  • 20 pl of the virus and cells mixture may be added to the 384-well cell culture plates using a pFlow liquid dispenser and cultured for 4 days at 37°C. After incubation, the cells may be allowed to equilibrate to 25°C for 30 min.
  • the RSV-induced cytopathic effect may be determined by adding 20 pl of CellTiter-Glo Viability Reagent. After a 10-min incubation at 25°C, cell viability may be determined by measuring luminescence using an Envision plate reader.
  • antiviral assays may be conducted in 384-or 96-well plates in B SL-4 using a high-content imaging system to quantify virus antigen production as a measure of virus infection.
  • a ‘no virus’ control and a ‘ 1% DMSO’ control may be included to determine the 0% and 100% virus infection, respectively.
  • the primary and secondary antibodies and dyes used for nuclear and cytoplasmic staining are listed.
  • the primary antibody specific for a particular viral protein may be diluted 1,000-fold in blocking buffer (1 x PBS with 3% BSA) and added to each well of the assay plate.
  • the assay plates may be incubated for 60 min at room temperature.
  • the primary' antibody may be removed, and the cells may be washed three times with 1 x PBS.
  • the secondary' detection antibody may be an anti-mouse (or rabbit) IgG conjugated with Dylight488 (Thermo Fisher Scientific, catalogue number 405310).
  • the secondary antibody may be diluted 1, 000-fold in blocking buffer and may be added to each well in the assay plate. Assay plates may be incubated for 60 min at room temperature. Nuclei may be stained using Draq5 (Biostatus) or 33342 Hoechst (ThermoFisher Scientific) for Vero and HFF-1 cell lines. Both dyes may be diluted in 1 x PBS.
  • the cytoplasm of HFF-1 (EBOV assay) and Vero E6 (MERS assay) cells may be counter-stained with CellMask Deep Red (Thermo Fisher Scientific).
  • Cell images may be acquired using a Perkin Elmer Opera confocal plate reader (Perkin Elmer) using a x 10 air objective to collect five images per well.
  • Virus-specific antigen may be quantified by measuring fluorescence emission at a 488 nm wavelength and the stained nuclei may be quantified by measuring fluorescence emission at a 640 nm wavelength.
  • Acquired images may be analyzed using Harmony and Acapella PE software.
  • the Draq5 signal may be used to generate a nuclei mask to define each nucleus in the image for quantification of cell number.
  • the CellMask Deep Red dye may be used to demarcate the Vero and HFF-1 cell borders for cell-number quantitation.
  • the viral-antigen signal may be compartmentalized within the cell mask. Cells that exhibited antigen signal higher than the selected threshold may be counted as positive for viral infection.
  • the ratio of virus-positive cells to total number of analyzed cells may be used to determine the percentage of infection for each well on the assay plates. The effect of compounds on the viral infection may be assessed as percentage of inhibition of infection in comparison to control wells.
  • the resultant cell number and percentage of infection may be normalized for each assay plate.
  • Analysis of dose-response curve may be performed using GeneData Screener or similar software applying Levenberg -Marquardt algorithm for curve-fitting strategy. The curve-fitting process, including individual data point exclusion, will be pre-specified by default software settings. R2 value quantified goodness of fit and fitting strategy may be considered acceptable at R2 > 0.8.
  • the drug combinations of the disclosure can be combined with other therapeutic agents.
  • Other therapeutic agents can include additional cathepsin inhibitors or protease inhibitors.
  • Drug combinations of the disclosure can include, but are not limited to, for example, Drug combinations according to the disclosure can be, but are not limited to, for example: Relacatib (GSK-462795, SB-462795), in combination with T-705 (Avigan) for use in the treatment of arenavirus infections, Relacatib (GSK -462795, SB- 462795), in combination with T-705 (Avigan) for use in the treatment of SARS-CoV-2 infections, MIV-711, in combination with T-705 (Favipiravir, Avigan) for use in treatment of SARS-CoV-2 and other coronaviruses such as MERS and SARS-CoV; AM-3701, MIV-701, MIV-710, MIV-711 , NC-2300, ORG-219517 or Relacatib (GSK-462795,
  • DMARDs Disease-modifying anti-rheumatic drugs
  • compositions may include gold preparations.
  • the term gold preparations may include auranofin.
  • compositions may include penicillamine, which may include D-penicillamine.
  • compositions may include aminosalicylic acid preparations, which may include sulfasalazine, mesalazine, olsalazine, balsalazide.
  • compositions may include antimalarials, which may include chloroquine.
  • compositions may include pyrimidine synthesis inhibitors, which may include leflunomide.
  • compositions may include prograf.
  • compositions may include protein drugs.
  • protein drugs may include TNF inhibitors such as etanercept, infliximab, adalimumab, certolizumab pegol, golimumab, PASSTNF-alpha, soluble TNF-alpha receptor, TNF-alpha binding protein, anti -TNF-alpha antibody.
  • protein drugs may include interleukin- 1 inhibitors, such as anakinra (interleukin- 1 receptor antagonist), soluble interleukin-1 receptor and the like; interleukin-6 inhibitors such as tocilizumab (antiinterleukin-6 receptor antibody), anti-interleukin-6 antibody.
  • protein drugs may include interleukin- 10 drugs such as interleukin- 10.
  • protein drugs may include interleukin-12/23 inhibitors such as ustekinumab, briakinumab (anti-interleukin- 12/23 antibody).
  • protein drugs may include B cell activation inhibitors such as rituximab, belimumab and the like; co-stimulatory molecules-related protein preparations such as abatacept and the like; complement mediated inhibitors both synthetic and biologic.
  • compositions may include non-protein drugs such as MAPK inhibitors such as BMS-582949.
  • non-protein drugs may include gene modulators; inhibitors of molecule involved in signal transduction, such as NF-kappa, NF- kappaB, IKK- 1, IK K -2, AP-1.
  • non-protein drugs may include cytokine and chemokine production inhibitors, receptor binding inhibitors such as iguratimod, tetomilast.
  • non-protein drugs may include TNF-alpha converting enzyme inhibitors; interleukin-1 beta converting enzyme inhibitors such as VX-765.
  • non-protein drugs may include interleukin-6 antagonists such as HMPL-004.
  • non-protein drugs may include interleukin-8 inhibitors such as IL-8 antagonist, CXCR1 & CXCR2 antagonist, reparixin.
  • non-protein drugs may include Chemokine antagonists such as CCR9 antagonist (CCX-282, CCX-025), MCP-1 antagonist.
  • nonprotein drugs may include interleukin-2 receptor antagonists such as denileukin, diftitox.
  • non-protein drugs may include therapeutic vaccines such as TNF-alpha vaccine.
  • non-protein drugs may include gene therapy drugs such as drugs promoting the expression of a gene having an anti-inflammatory action such as interleukin-4, interleukin-10, soluble interleukin-1 receptor, soluble TNF-alpha receptor.
  • non-protein drugs may include antisense compounds such as ISIS-104838. Integrin inhibitor
  • compositions may include integrin inhibitors such as natalizumab, vedolizumab, AJM300, TRK-170, E-600.
  • compositions may include immunomodulators such as cyclophosphamide, MX-68, atipriniod dihydrochloride, BMS- 188667, CKD-461, rimexolone, cyclosporine, tacrolimus, gusperimus, azathiopurine, antilymphocyte serum, freeze-dried sulfonated normal immunoglobulin, erythropoietin, colony stimulating factor, interleukin, interferon, intravenous immunoglobulin, anti-thymocyte globulin, RSLV-132.
  • immunomodulators such as cyclophosphamide, MX-68, atipriniod dihydrochloride, BMS- 188667, CKD-461, rimexolone, cyclosporine, tacrolimus, gusperimus, azathiopurine, antilymphocyte serum, freeze-dried sulfonated normal immunoglobulin, erythropo
  • compositions may include proteasome inhibitors such as bortezomib.
  • compositions may include JAK inhibitors such as tofacitinib.
  • compositions may include steroids.
  • steroid may include dexamethasone, hexestrol, methimazole, betamethasone, triamcinolone, triamcinolone acetonide, fluocinonide, fluocinolone acetonide, predonisolone, methylpredonisolone, cortisone acetate, hydrocortisone, fluoromethoIone, beclomethasone dipropionate, estriol.
  • compositions may include angiotensin converting enzyme inhibitors.
  • angiotensin converting enzyme inhibitors may include enalapril, captopril, ramipril, lisinopril, cilazapril, perindopril.
  • Angiotensin II receptor antagonists may include enalapril, captopril, ramipril, lisinopril, cilazapril, perindopril.
  • compositions may include angiotensin II receptor antagonists.
  • angiotensin II receptor antagonists may include candesartan, candesartan cilexetil (TCV-116), valsartan, irbesartan, olmesartan, eprosartan.
  • compositions may include a diuretic.
  • a diuretic may include hydrochlorothiazide, spironolactone, furosemide, indapamide, bendrofluazide, cyclopenthiazide .
  • compositions may include a cardiotonic substance.
  • a cardiotonic substance may include digoxin, dobutamine.
  • compositions may include a beta receptor antagonist.
  • a beta receptor antagonist may include carvedilol, metoprolol, atenolol.
  • compositions may include a Ca sensitizer.
  • a CA sensitizer may include MCC-135.
  • compositions may include Ca channel antagonists.
  • a Ca channel antagonist may include nifedipine, diltiazem, verapamil.
  • Anti-platelet drug, anti coagulator
  • compositions may include an anti-platelet substance or anti coagulator.
  • an anti-platelet, substance or anti coagulator may include heparin, aspirin, warfarin.
  • compositions may include an anti-platelet substance or anticoagulator.
  • an anti-platelet substance or anti coagulator may include atorvastati n, simvastatin .
  • compositions may include other substances which improve functionality of the compound.
  • other substances may include T cell inhibitors, inosine monophosphate dehydrogenase (IMPDH) inhibitor mycophenolate mofetil.
  • other substances may include adhesion molecule inhibitor such as ISIS-2302, selectin inhibitor, ELAM-1, VCAM-1, ICAM-1.
  • other substances may include thalidomide, a combination of cathepsin inhibitor or a single cathepsin inhibitor, matrix metalloprotease (MMPs) inhibitor such as V-85546.
  • MMPs matrix metalloprotease
  • other substances may include glucose-6-phosphate dehydrogenase inhibitor, Dihydroorotate dehydrogenase (DHODH) inhibitor, phosphodiesterase IV (PDE IV) inhibitor such as roflumilast, CG-1088.
  • other substances may include a phospholipase A2 inhibitor, iNOS inhibitor such as VAS-203.
  • other substances may include microtubule stimulating compound such as paclitaxel.
  • other substances may include microtubule inhibitor such as reumacon.
  • other substances may include MHC class II antagonist, prostacyclin agonist such as iloprost.
  • other substances may include CD4 antagonist such as zanolimumab.
  • other substances may include CD23 antagonist, LTB4 receptor antagonist such as DW-1305.
  • other substances may include 5 -lipoxygenase inhibitor such as zileuton.
  • other substances may include cholinesterase inhibitor such as galanthamine.
  • other substances may include a tyrosine kinase inhibitor such as Tyk2 inhibitor (WO 2010/142752).
  • other substances may include cathepsin B inhibitor.
  • other substances may include adenosine deaminase inhibitor such as pentostatin.
  • other substances may include osteogenesis stimulator, dipeptidylpeptidase inhibitor, collagen agonist, capsaicin cream, hyaluronic acid derivative synvisc (hylan G-F 20), orthovis.
  • other substances may include glucosamine sulfate, amiprilose.
  • other substances may include CD-20 inhibitors such as rituximab, ibritumomab, tositumomab, ofatumumab.
  • other substances may include BAFF inhibitors such as belimumab, tabalumab, atacicept, A-623.
  • other substances may include CD52 inhibitors such as alemtuzuma
  • other substances may include.
  • compositions may include other substances which improve functionality of the compound.
  • other substances may include antiviral substances such as idoxuridine, acyclovir, vidarabine, gancyclovir.
  • other substances may include anti-HIV agents such as zidovudine, didanosine, zalcitabine, indinavir sulfate ethanolate, ritonavir.
  • 0.001-fold to about 0.01 fold about 0.05-fold to about 0.1-fold, about 0.1-fold to about 0.5-fold, about 0.5-fold to about 1-fold, about 1-fold to about 2-fold, about 3-fold to about.
  • Effective dose is that that achieves 50% of the effect which is also termed Inhibitory' Concentration 50% (IC50) or Effective Concentration (EC50) in assays in vitro, wherein the EC50 is decreased by about 5% or more, or by about 5%-l 0%, about 10%-20%, about 20%-30%, about 30%-40%, about 40%-50%, about 50%-60%, about 60%-70%, about 70%-80%, about 80%-90%, about 90%-100%, about 100%- 150%, about 150%-200%, about 200%-300%, about 300%-400%, about 400%-500%, about 500%-1000%, 1000%-5000%, about 5000%-7000%, about 7000%-10,000% or more.
  • sub-optimal doses refers to doses which do not reach EC50 or IC50.
  • the combination or mixture of the present disclosure can be used together with other drags for the prophylaxis or treatment of various diseases.
  • the combination or mixture of the present disclosure when used as an antiviral therapy, it can be used together with the following drugs:
  • Non-steroidal anti-inflammatory drugs NSAIDs
  • compositions may include classical non-steroidal antiinflammatory drugs (NSAID).
  • NSAID may include, but are not limited to, alcofenac, aceclofenac, sulindac, tolmetin, etodolac, fenoprofen, thiaprofenic acid, meclofenamic acid, meloxicam, tenoxicam, lomoxicam, nabumeton, acetaminophen, phenacetin, ethenzamide, sulpyrine, antipyrine, migrenin, aspirin, mefenamic acid, flufenamic acid, diclofenac sodium, loxoprofen sodium, phenylbutazone, indomethacin, ibuprofen, ketoprofen, naproxen, oxaprozin, flurbiprofen, fenbufen, pranoprofen, floctafen
  • compositions may include cyclooxygenase inhibitors.
  • cyclooxygenase inhibitors may include, but are not limited to, (COX-1 selective inhibitors, COX-2 selective inhibitors, salicylic acid derivatives (e.g., celecoxib, aspirin), etoricoxib, valdecoxib, diclofenac, indomethacin, loxoprofen and the like.
  • compositions may include Nitric oxide-releasing NSAIDs.
  • the inhibitors of the disclosure are useful for treating enveloped viral infection by interfering with the critical role by cathepsins of proteolysis of the GP1 glycoprotein subunit to trigger membrane fusion and cell entry.
  • a new mechanism for activating the enveloped virus fusion machinery has been discovered. It has been demonstrated that papain like cysteine proteases are involved in, and are important components of, the viral entry' process. Inhibition of these proteases is sufficient to inhibit enveloped viral entry into a host cell.
  • cysteine proteases provide an additional important mechanism by which enveloped viruses, such as Ebola or SARS-CoV-2, infect host cells.
  • cathepsins are sufficient for triggering of enveloped viral membrane fusion within the acidic endosomal milieu of target cells.
  • inhibitors of the disclosure are useful for treating enveloped viral infection by interfering with the critical role by cathepsins of proteolysis of the GP1 glycoprotein subunit to trigger membrane fusion and cell entry.
  • the specific data presented herein suggest that GP1 proteolysis is a multistep process.
  • AUC area under the curve
  • Cmax area under the curve
  • AUC is determined by plotting the serum or plasma concentration of a drug along the ordinate (Y-axis) against time along the abscissa (X-axis).
  • Y-axis the ordinate
  • X-axis the abscissa
  • the values for the AUC represent drug concentrations over time in units of mass-time/volume.
  • the amount and form of the drug administered should be the same in both a) the administration of the drug in combination with a protease inhibitor, for example a cathepsin inhibitor, or b) the administration of the drag alone.
  • Insufficient time in the gastrointestinal tract is a common cause of low 7 bioavailability. Ingested drug is exposed to the entire gastrointestinal tract, for no more than one to two days and to the small intestine for only 2 to 4 hours. If the drug does not dissolve readily or cannot penetrate the epithelial membrane (e.g., if it is highly ionized and polar), time at the absorption site may be insufficient. In such cases, bioavailability tends to be highly variable as well as low. Age, sex, activity, genetic phenotype, stress, disease, or previous gastrointestinal surgery' can affect drug bioavailability.
  • Drug products may be considered bioequivalent in extent and rate of absorption if their plasma level curves are essentially super imposable. Drug products that have similar AUCs but differently shaped plasma level curves are equivalent in extent but differ in their absorption rate-time profiles.
  • Absorption occurs by one of three methods, either passive diffusion, active transport or facilitated active transport.
  • Passive diffusion is simply the passage of molecules across the mucosal barrier until the concentration of molecules reaches osmotic balance on both sides of the membrane.
  • active transport the molecule is actively pumped across the mucosa.
  • a carrier generally a protein, is required to convey the molecule across the membrane for absorption.
  • the present disclosure provides methods of use related to the compositions described herein.
  • the methods described herein may include a method of inhibiting the growth of a virus. Such methods may include contacting the virus with the compositions of the disclosure.
  • the compositions of the disclosure may include mammalian protease inhibitors.
  • the methods may involve contacting the virus with a mammalian protease inhibitor.
  • the growth of the virus is inhibited in vivo in a subject.
  • the growth of the virus is inhibited in the presence of a cell or a population of cells infected by the virus.
  • the growth of the virus is inhibited by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and/or 90%. In some embodiments the growth of the virus is inhibited by 5-15%, 10-20%, 15-25%, 20-30%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55-65%, 60-70%, 65-75%, 70-80%, 75-85%, and/or 90-100%.
  • the methods described herein may include a method of reducing the percentage of virus infected cells in a population. Such methods may include contacting the virus infected cells with the compositions of the disclosure.
  • the compositions of the disclosure may include mammalian protease inhibitors.
  • the methods may involve contacting the virus infected cells with a mammalian protease inhibitor.
  • the percentage of the virus infected cells is reduced in vivo in a subject. In some embodiments, the percentage of virus infected cells in a population is reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and/or 90%.
  • the percentage of virus infected cells in a population is reduced by 5-15%, 10- 20%, 15-25%, 20-30%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55-65%, 60- 70%, 65-75%, 70-80%, 75-85%, and/or 90-100%.
  • the amount of the compositions of the disclosure to be utilized may be identified using methods described here.
  • the concentration of the protease inhibitors is determined using an MTS assays and cytotoxicity assays described herein or any other methods known in the art.
  • Assay output may be analyzed to determine EC 50 (50% inhibition of virus replication), EC 90 (90% inhibition of virus replication), EC 95 (95% inhibition of virus replication), CC 50 (50% cytotoxicity), CC95 (95% cytotoxicity).
  • the selectivity index (SI) for each protease inhibitor is determined by dividing the CC 50 by the EC 50 .
  • the methods of the disclosure selectivity index (SI) for each protease inhibitor for each virus infection may be determined.
  • the SI is determined for coronavirus.
  • the SI may be at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900 or more.
  • the methods described herein correspond to a subject or patient or infected by a virus, such as RNA or DNA viruses which are pathogenic to humans and animals.
  • a virus such as RNA or DNA viruses which are pathogenic to humans and animals.
  • the present disclosure provides compounds and compositions may be used to reduce the coronavirus infection levels in a subject.
  • Such methods may include providing the compositions of the disclosure to a subject in need.
  • the levels of coronavirus infection may be measured in the subject by any methods known in the art, such as, but not limited to, measuring coronavirus antigen levels, measuring anticoronavirus antibodies and/or measuring levels of coronavirus nucleic acids in the subject. Treatment with the compositions of the disclosure is expected to reduce the coronavirus infection levels.
  • the subject has an infection by a Type I enveloped virus.
  • the compositions of the disclosure may be provided or administered to a subject with an infection associated with a Type I enveloped virus e.g. a filovirus.
  • the compositions may be used in the treatment of or may be provided to a subject infected with a coronavirus, a filovirus such as an Ebola virus or a Marburg virus.
  • the compositions may be used in the treatment of or may be provided to a subject infected with a Type I enveloped virus such as an orthomyxovirus.
  • compositions may be used in the treatment of or may be provided to a subject infected with a Type I enveloped virus such as a paramyxovirus.
  • Type I enveloped virus is an Arenavirus.
  • the compositions of the disclosure may be used to inhibit a virus and/or reduce the percentage of virus infected cells in a population of cells.
  • the virus may be a coronavirus, an enterovirus, an adenovirus, a dengue virus, human immunodeficiency virus, a parainfluenza virus, a respiratory syncytial virus (RSV), a coxsackie virus, a rhinovirus, Measles, Influenza virus.
  • RSV respiratory syncytial virus
  • the virus may be a virus in the family, Coronaviridae, or a virus in the sub-family Orthocoronavirinae, or a virus in the order Nidovirales, In some embodiments, the methods of the disclosure may be used to inhibit the growth of any coronavirus.
  • the virus may be a coronavirus.
  • the coronavirus may be a SARS-CoV-2 virus, SARS-CoV-1 virus, MERS-CoV virus, 229E virus, NL63 virus, OC43 virus, HKU1 virus, or variants thereof As a non-limiting example, the virus may be a SARS-CoV-2 virus.
  • the virus may be an enterovirus.
  • the subject or patient has, or is at risk of, an infection by a virus.
  • viruses such as RNA or DNA viruses, are pathogenic for humans and animals.
  • the subject has or is at risk of infection by a Type I enveloped virus.
  • the Type I enveloped virus is a filovirus.
  • the filovirus is an Ebola virus or a Marburg virus.
  • the Type I enveloped virus is an orthomyxovirus.
  • the Type I enveloped virus is a paramyxovirus.
  • the Type I enveloped virus is an Arenavirus.
  • the subject has or is at risk of infection by a virus such as, but not limited to, filoviruses, fl avi viruses such as hepatitis-C virus, bunyaviruses, poxvirus, arboroviruses such as Togaviruses, bunyaviruses, orthomyxoviridae, paramyxoviridae, poxviruses, herpesviruses, henipaviruses, hepadnaviruses, rhabdoviruses, bornaviruses, arteri viruses, papillomaviridae, human retroviruses, polyomaviridae, picomaviridae, coronaviruses, and adenoviridae.
  • a virus such as, but not limited to, filoviruses, fl avi viruses such as hepatitis-C virus, bunyaviruses, poxvirus, arboroviruses such as Togaviruses, bunya
  • the disclosure provides for methods of treating infection by a virus of the family Filoviridae, a family of viruses with a single-stranded, unsegmented ( ⁇ ) sense RNA genome.
  • Filoviruses can cause severe hemorrhagic fever in humans and nonhuman primates. So far, only two genuses of this virus family have been identified: Marburg and Ebola. Four species of Ebola virus have been identified: Cote d'Irete (CI), Sudan (S), Zaire (Z), and Reston (R). The Reston subty pe is the only known filovirus that is not known to cause fatal disease in humans; however, it can be fatal in monkeys.
  • the family Orthomyxoviridae may include, without limitation, influenza A virus, influenza B virus, influenza C virus, Thogotovirus, Dhori virus, and infectious salmon anemia virus.
  • Influenza type A viruses may be divided into subtypes based on two proteins on the surface of the virus. These proteins are called hemagglutinin (HA) and neuraminidase (NA). There are 15 different HA subtypes and 9 different NA subtypes. Subtypes of influenza A virus are named according to their HA and NA surface proteins, and many different combinations of HA and NA proteins are possible. For example, an “H7N2 virus” designates an influenza A subtype that has an HA 7 protein and an NA 2 protein.
  • an "115X 1" virus has an HA 5 protein and an NA 1 protein.
  • H1N1, H2N2, and H3N2 are currently in general circulation among people.
  • Other subtypes such as H5 N1 are found commonly in other animal species and in a small number of humans, where it is highly pathogenic.
  • H7N7 and H3N8 viruses cause illness in horses.
  • influenza A virus Humans can be infected with influenza types A, B, and C. However, the only subtypes of influenza A virus that normally infect people are influenza A subtypes H1N1, H2N2, and H3N2 and recently, H5N1.
  • the family Paramyxoviridae may include, without limitation, human parainfluenza virus, human respiratory' syncytial virus (RSV), Sendai virus, Newcastle disease virus, mumps virus, rubeola (measles) virus, Hendra virus, Nipah virus, avian pneumovirus, and canine distemper virus.
  • RSV human respiratory' syncytial virus
  • Sendai virus Sendai virus
  • Newcastle disease virus Newcastle disease virus
  • mumps virus mumps virus
  • rubeola (measles) virus Hendra virus
  • Nipah virus avian pneumovirus
  • canine distemper virus canine distemper virus.
  • the family Rhabdoviridae may include, without limitation, rabies virus, vesicular stomatitis virus (VSV), Mokola virus, Duvenhage virus, European bat virus, salmon infectious hematopoietic necrosis virus, viral hemorrhagic septicaemia virus, spring viremia of carp virus, and snakehead rhabdovirus.
  • the family Bornaviridae may include, without limitation, Borna disease virus.
  • the family Bunyaviridae may include, without limitation, Bunyamwera virus, Hantaan virus, Crimean Congo virus, California encephalitis virus. Rift Valley fever virus, and sandfly fever virus.
  • the family Arenaviridae includes, without limitation.
  • Old World Arenaviruses such as Lassa virus (Lassa fever), Ippy virus, Lymphocytic choriomeningitis virus (LCMV), Mobala virus, and Mopeia virus and New World Arenaviruses, such as Junin virus (Argentine hemorrhagic fever), Sabia (Brazilian hemorrhagic fever), Amapari virus, Flexal virus, Guanarito virus (Venezuela hemorrhagic fever), Machupo virus (Bolivian hemorrhagic fever), Latino virus, Boliveros virus, Parana virus, Pichinde virus, Pirital virus, Tacaribe virus, Tamiami virus, and Whitewater Arroyo virus.
  • Lassa virus Lassa virus
  • Ippy virus Lymphocytic choriomeningitis virus
  • LCMV Lymphocytic choriomeningitis virus
  • Mobala virus Mopeia virus
  • Mopeia virus and New World Arenaviruses such as Junin virus
  • the arboviruses are a large group (more than 400) of enveloped RNA viruses that are transmitted primarily (but not exclusively) by arthropod vectors (mosquitoes, sand-flies, fleas, ticks, lice, etc). More recently, the designated Arborviruses have been split into four virus families, including the togaviruses, flaviviruses, arenaviruses and bunyaviruses.
  • togaviru s refers to members of the family Togaviridae, which includes the genuses Alphavirus (e.g. Venezuela equine encephalitis virus, Sindbis virus, which causes a self-limiting febrile viral disease characterized by sudden onset of fever, rash, arthralgia or arthritis, lassitude, headache and myalgia) and Rubivirus (e.g. Rubella virus, which causes Rubella in vertebrates).
  • Alphavirus e.g. Venezuela equine encephalitis virus, Sindbis virus, which causes a self-limiting febrile viral disease characterized by sudden onset of fever, rash, arthralgia or arthritis, lassitude, headache and myalgia
  • Rubivirus e.g. Rubella virus, which causes Rubella in vertebrates.
  • Flaviviridae is a member of the family of (+)-sense RNA enveloped viruses. Flaviviridae includes flavivirus, Pestivirus, and Hepacivirus. Flavivirus genus including yellow fever virus, dengue fever virus, and Japanese encaphilitis (JE) virus. The Pestivirus genus includes the three serotypes of bovine viral diarrhea, but no known human pathogens. Genus Hepacivirus consists of hepatitis C virus and hepatitis C-like viruses. The Japanese encephalitis antigenic complex includes Alfuy, Japanese encephalitis, Kokobera, Koutango, Kunjin, Murray Valley encephalitis, St.
  • West Nile virus is the most widespread of the flaviviruses, with geographic distribution including Africa and Eurasia.
  • the genus Pestivirus has been divided into bovine viral diarrhea virus (BVDV), classical swine fever virus (CSFV), and border disease virus (BDV).
  • BVDV bovine viral diarrhea virus
  • CSFV classical swine fever virus
  • BDV border disease virus
  • the Hepacivirus genus includes the hepatitis C virus (HCV).
  • .Arenaviridae is a member of the fami ly of ( ⁇ ) sense RNA viruses.
  • the term “Arenavirus” refers to members of the genus Arenavirius, a family of viruses whose members are generally associated with rodent-transmitted disease in humans, including Lymphocytic choriomeningitis virus (LCMV), Lassa virus, Junin virus, which causes Argentine hemorrhagic fever, Machupo virus, which causes Venezuelan hemorrhagic fever, Guanarito virus, which causes Venezuelan hemorrhagic fever, and Sabia, which causes Brazilian hemorrhagic fever.
  • LCMV causes which causes lymphocytic choriomeningitis, a mild disease that is occasionally severe with hemorrhaging.
  • the Phlebovirus Rift valley fever virus produces an acute, flu-like illness and is transmitted by mosquitoes from animal reservoirs (e.g. sheep) to man.
  • Sand fly fever is transmitted to man by Phlebotomous flies (sand-flies) and causes an acute, febrile illness characterized by fever, malaise, eye pain, and headache.
  • Hendra and Nipah virus in the Henipavirus genus of the subfamily Paramyxovirinae are distinguished by fatal disease in both animal and human hosts.
  • Riboviria are all RNA viruses that replicate using RNA-dependent RNA polymerase. Examples of viruses that cause infections in humans include SARS-CoV-1, MERS-CoV, and SARS-CoV-2, 229E, NL63, OC43, KHU1.
  • Herpesviridae is a large family of DNA viruses that cause disease in animals, including humans.
  • Herpesviruses include herpes simplex virus types I and 2, varicella-zoster virus, cytomegalovirus, Esptein-Barr virus, human herpesvirus 6 (variants A and B), human herpesvirus 7, and Kaposi’s sarcoma virus or human herpesvirus 8.
  • Hepadnaviridae is a family of DNA viruses that cause hepatitis in humans and animals. Hepadnaviridae include hepatitis B virus isolated from mammals or birds.
  • Papillomaviridae is a family of non-enveloped DNA viruses with over a hundred species of papillomaviruses including Alpha papillomavirus, Beta papillomavirus. Gamma papillomavirus, Mu papillomavirus and Nupapillomavirus. HPVs are most associated with cutaneous and genital legions, cervical carcinoma and recurrent respiratory papillomatosis, among other diseases.
  • Human retroviruses including human T-cell leukemia virus (HTLV-1, 2, 3 and 4) and adult T-cell leukemia virus (ATLV) Human T-lymphotropic virus cause serious diseases in humans, including adult T-cell leukemia/lymphoma (ATE) and neurological disease (HTLV-associated myelopathy/tropical spastic paraparesis), uveitis, and rheumatic syndromes.
  • HTLV-1, 2, 3 and 4 Human T-lymphotropic virus cause serious diseases in humans, including adult T-cell leukemia/lymphoma (ATE) and neurological disease (HTLV-associated myelopathy/tropical spastic paraparesis), uveitis, and rheumatic syndromes.
  • ATE adult T-cell leukemia/lymphoma
  • neurological disease HTLV-associated myelopathy/tropical spastic paraparesis
  • Poiyomaviridae family of viruses are non-enveloped DNA viruses that cause disease in immunocompromised hosts.
  • Human polyomaviruses BKV and JCV cause hemorrhagic cystitis and leukoencephalopathy.
  • Merkel cell polyomavirus (MCPyV or MCV) shares some traits to piyomaviruses and is thought to be linked to Merkel Cell Carcinoma (MCC), a neuroendocrine cancer.
  • Poxviridae is a large family of DNA viruses including mollusci poxvirus, parapoxvirus (Orf virus, pseudocowpox virus, bovine popular stomatitis virus). Orthopoxvirus (cowpox virus, monkeypox virus, vaccinia virus, variola virus), Yatapoxvirus (tanapoxvirus, yaba monkey tumor poxvirus).
  • Picomaviridae are a family of viruses with single-stranded positive-sense RNA genomes and includes, without limitation, enteroviruses A through L, coxsackieviruses, echoviruses, polioviruses 1-3, and rhinoviruses A and B, hepatoviruses (Hepatitis A virus), cardioviruses (infect rodents, aphthoviruses (food-and-mouth disease virus which infects cloven-hoofed animals and occasionally humans).
  • Adenoviridae is a family of double- stranded DNA viruses and include more than 100 antigenic types with human adenoviruses divided in subgenuses A-F and Serotypes 1-47, e.g. HAdV-B3, -E4, and -B7.
  • HeLa cells may be seeded at 2,000 cells per well in a 384-well plate, and compounds may be added to the assay plates. Assay plates may be transferred to the BSL-4 suite and infected with 1 PFU per cell MARY, which resulted in 50% to 70% of the cells expressing virus antigen in a 48-h period.
  • HeLa cells may be seeded at 2,000 cells per well in a 384-well plate, and compounds may be added to the assay plates.
  • Assay plates may be transferred to the BSL-4 suite and infected with 0.08 PFU SUDV per cell, which resulted in 50% to 70% of the cells expressing virus antigen in a 48-h period.
  • HeLa cells may be seeded at 2,000 cells per well in a 384-well plate, and compounds may be added to the assay plates.
  • Assay plates may be transferred to the BSL-4 suite and infected with 0.1 PFU per cell LASV, which resulted in >60% of the cells expressing virus antigen in a 48-h period.
  • SARS-CoV-1 , and SARS-CoV-2 Vero E6 cells may be seeded at 4,000 cells per weH in a 384-well plate, and compounds may be added to the assay plates in a dose dependent manner. Assay plates may be transferred to the BSL-3/4 suite and infected with 0.5 or other PFU per cell of MERS, SARS-CoV-1 and 2 virus, which resulted in >70% of the cells expressing virus antigen in a 48-h period.
  • U20S cells may be seeded at 3,000 cells per well in a 384-well plate, and compounds may be added to the assay plates. Assay plates may be transferred to the BSL suite and infected with 0.5 PFU per cell of CHIK, which resulted in >80% of the cells expressing virus antigen in a 48-h period.
  • HeLa cells may be seeded at 4,000 cells per well in a 384-well plate, and compounds may be added to the assay plates. Assay plates may be transferred to the BSL-4 suite and infected with 0.1 PFU per cell VEEV, which resulted in >60% of the cells expressing virus antigen in a 20-h period.
  • cytotoxicity assays may be conducted.
  • MI' -4 (2 x 10 3 cells per well) cells may be plated in 384-well plates and incubated with the appropriate medium containing threefold serially diluted compound ranging from 15 nM to 100,000 nM.
  • PC-3 cells (2.5 x 10 3 cells per well), HepG2 cells (4 x 10 3 cells per well), hepatocytes (1 x 10 6 cells per well), quiescent PBMCs (1 x io b cells per well), stimulated PBMCs (2 x 10 5 cells per well), and RPTEC cells (1 x io 3 cells per well) may be plated in 96-well plates and incubated with the appropriate medium containing threefold serially diluted compound ranging from 15 nM to 100,000 nM. Cells may be cultured for 4-5 days at 37 °C. Following the incubation, the cells may be allowed to equilibrate to 25°C, and cell viability may be determined by adding Cell-Titer Gio viability reagent. The mixture may be incubated for 10 min, and the luminescence signal may be quantified using an Envision plate reader. Cell lines may be not authenticated and may be not tested for my coplasma as part of routine use in cytotoxicity assays.
  • RNA synthesis by the RSV polymerase may be reconstituted in vitro using purified RSV L/P complexes and an RNA oligonucleotide template (Dharmacon), representing nucleotides 1-14 of the RSV leader promoter.
  • RNA synthesis reactions may be performed as described previously, except that the reaction mixture contained 250 ⁇ M guanosine triphosphate (GTP), 10 ⁇ M uridine triphosphate (UTP), 10 ⁇ M cytidine triphosphate (CTP), supplemented with 10 pCi [a- 32 P]CTP, and either included 10 ⁇ M adenosine triphosphate (ATP) or no ATP.
  • the polymerase is able to initiate synthesis from the position 3 site of the promoter, but not the position 1 site.
  • the NTP metabolite of GS- 5734 may be serially diluted in DMSO and included in each reaction mixture at concentrations of 10, 30, or 100 ⁇ M as specified.
  • RNA products may be analyzed by electrophoresis on a 25% polyacrylamide gel, containing 7 M urea, in Tris-taurine-EDTA buffer, and radiolabeled RNA products may be detected by autoradiography.
  • RSV A2 polymerase inhibition assay may be performed.
  • Transcription reactions contained 25 pg of crude RSV RNP complexes in 30 ⁇ L of reaction buffer (50 mM Tris-acetate (pH 8.0), 120 mM potassium acetate, 5% glycerol, 4.5 mM MgCl 2, 3 mM DTT, 2 mM EGTA, 50 pg ml" 1 BSA, 2.5 U RNasin, 20 ⁇ M ATP, 100 ⁇ M GTP, 100 ⁇ M UTP, 100 ⁇ M CTP, and 1.5 pCi [ ⁇ - 32 P]ATP (3,000 Ci mmol" 1 ).
  • the radiolabeled nucleotide used in the transcription assay may be selected to match the nucleotide analogue being evaluated for inhibition of RSV RNP transcription.
  • nucleotide analogues inhibited RSV RNP transcription compounds may be added using a six-step serial dilution in fivefold increments. After a 90- min incubation at 30°C, the RNP reactions may be stopped with 350 pl of Qiagen RLT lysis buffer, and the RNA may be purified using a Qiagen RNeasy 96 kit. Purified RNA may be denatured in RNA sample loading buffer at 65°C for 10 min and run on a 1.2% agarose/MOPS gel containing 2 M formaldehyde. The agarose gel may be dried, exposed to a Storm phosphorimaging screen, and developed using a Storm phosphorimager.
  • the compound may be prepared as pharmaceutical compositions. It will be understood that such compositions necessarily comprise one or more active ingredients and, most often, a pharmaceutically acceptable excipient.
  • Relative amounts of the active ingredients may vary', depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 99% (w/w) of the active ingredients.
  • the composition may comprise between 0.1% and 100%, e.g., between .5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredients.
  • the anti-viral and protease inhibitors pharmaceutical compositions described herein may comprise at least one anti-viral and at least one protease inhibitor.
  • the pharmaceutical compositions may contain an antiviral and a cathepsin inhibitor.
  • compositions are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g. non-human mammals. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation.
  • Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, rats, birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.
  • compositions are administered to humans, human patients, or subjects.
  • compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology.
  • pharmaceutical composition refers to compositions comprising at least one active ingredient and optionally one or more pharmaceutically acceptable excipients.
  • Formulations of the anti-viral, protease inhibitors, and pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredients into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.
  • a pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a “unit dose” refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • Relative amounts of the active ingredient (e.g. anti-viral, protease inhibitor), the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may wiry, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 99% (w/w) of the active ingredient.
  • the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, or at least 80% (w/w) active ingredient.
  • the anti-viral and protease inhibitor of the disclosure can be formulated using one or more excipients or diluents to (1 ) increase stability; (2) increase absorption; (3) permit the sustained or delayed release; or (4) alter the biodistribution (e.g., target the compound to specific tissues or cell types).
  • a pharmaceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure.
  • an excipient is approved for use for humans and for veterinary use.
  • an excipient may be approved by United States Food and Drug Administration.
  • an excipient may be of pharmaceutical grade.
  • an excipient may meet the standards of the United States Pharmacopoeia (LISP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
  • Excipients include, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired.
  • Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety).
  • any conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.
  • Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and/or combinations thereof.
  • formulations may comprise at least one inactive ingredient.
  • inactive ingredient refers to one or more agents that do not contribute to the activity of the active ingredient of the pharmaceutical composition included in formulations.
  • all, none or some of the inactive ingredients which may be used in the formulations of the present disclosure may be approved by the US Food and Drug Administration (FDA).
  • FDA US Food and Drug Administration
  • Formulations of the disclosure may also include one or more pharmaceutically acceptable salts.
  • pharmaceutically acceptable salts refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form (e.g., by reacting the free base group with a suitable organic acid).
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • Representative acid addition salts include acetate, acetic acid, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzene sulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2 -naphthalenesulfonate, nicotinate, nitrate,
  • alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary/ ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.
  • the pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • Solvates may be prepared by crystallization, recrystallization, or precipitation from a solution that includes organic solvents, water, or a mixture thereof.
  • solvents examples include ethanol, water (for example, mono-, di-, and tri-hydrates), A ; - methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), N,N ’-dimethylformamide (DMF), N,N ’-dimethylacetamide (DMAC), 1 ,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl- 3,4,5,6-tetrahydro-2-(lH)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, and the like.
  • the solvent When water is the solvent, the solvate is referred to as a “hydrate.”
  • Non-steroidal anti-inflammatory drugs NSAIDs
  • formulations may include classical non-steroidal antiinflammatory' drugs (NS AID).
  • NSAID may include, but are not limited to, alcofenac, aceclofenac, sulindac, tolmetin, etodolac, fenoprofen, thiaprofenic acid, meclofenamic acid, meloxicam, tenoxicam, lornoxicam, nabumeton, acetaminophen, phenacetin, ethenzamide, sulpyrine, antipyrine, migrenin, aspirin, mefenamic acid, flufenamic acid, diclofenac sodium, loxoprofen sodium, phenylbutazone, indomethacin, ibuprofen, ketoprofen, naproxen, oxaprozin, flurbiprofen, fenbufen, pranoprofen, floct
  • alcofenac alcofenac
  • formulations may include cyclooxygenase inhibitors.
  • cyclooxygenase inhibitors may include, but are not limited to, (COX-1 selective inhibitors, COX-2 selective inhibitors, salicylic acid derivatives (e.g., celecoxib, aspirin), etoricoxib, valdecoxib, diclofenac, indomethacin, loxoprofen and the like.
  • formulations may include Nitric oxi de-releasing NSAIDs. Salts
  • a pharmaceutically acceptable salt of the combination or mixture of the disclosure or a citric acid ester thereof is utilized in these compositions, the salt preferably is derived from an inorganic or organic acid or base.
  • suitable salts see, e.g., Berge et al, J. Pharm. Sci. 66: 1-19 (1977) and Remington: The Science and Practice of Pharmacy, 20th Ed., ed. A. Gennaro, Lippincott Williams & Wilkins, 2000.
  • non-limiting examples of suitable acid addition salts include the following: acetate, adipate, alginate, aspartate, benzoate, benzene sulfonate, bisulfate, butyrate, citrate, camphorate, camphor sulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, lucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3 -phenyl -propionate, picrate
  • suitable base addition salts include, without limitation, ammonium salts, alkali metal salts, such as lithium, sodium and potassium salts; alkaline earth metal salts, such as calcium and magnesium salts; other multivalent metal salts, such as zinc salts; salts with organic bases, such as dicyclohexylamine, N-methyl-D-glucamine, t-butylamine, ethylene diamine, ethanolamine, and choline; and salts with amino acids such as arginine, lysine, and so forth.
  • alkali metal salts such as lithium, sodium and potassium salts
  • alkaline earth metal salts such as calcium and magnesium salts
  • other multivalent metal salts such as zinc salts
  • salts with organic bases such as dicyclohexylamine, N-methyl-D-glucamine, t-butylamine, ethylene diamine, ethanolamine, and choline
  • salts with amino acids such as arginine, lysine, and so forth.
  • the pharmaceutical composition comprises the combination, whether as separate compounds or as a mixture, of the present disclosure and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is used herein to refer to a material that is compatible with a recipient subject, preferably a mammal, more preferably a human, and is suitable for delivering an active agent to the target site without terminating the activity of the agent.
  • the toxicity or adverse effects, if any, associated with the carrier preferably are commensurate with a reasonable risk/benefrt ratio for the intended use of the active agent.
  • carrier includes any and all solvents, diluents, and other liquid vehicles, dispersion or suspension aids, surface active agents, pH modifiers, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • Remington The Science and Practice of Pharmacy, 20th Ed., ed. A. Gennaro, Lippincott Williams & Wilkins, 2000 discloses various carriers used in formulating pharmaceutically acceptable compositions and known techniques for the preparation thereof, which is incorporated by reference herein, in its entirety.
  • any conventional carrier medium is incompatible with the compound of the disclosure, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, its use is contemplated to be within the scope of this disclosure.
  • materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, carbonates, magnesium hydroxide and aluminum hydroxide, glycine, sorbic acid, or potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, pyrogen-free water, salts or electrolytes such as protamine sulfate, di sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylenepoly oxypropyl ene-block polymers, wool fat, sugars such as lactose, glucose, sucrose, and mannitol, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethoxyl
  • a two-part or three-part combination may be administered simultaneously or sequentially. When administered sequentially, the combination may be administered in one, two, or three administrations.
  • two-part, or three-part combinations are administered in a single pharmaceutical dosage form. More preferably, a two-part combination is administered as a single oral dosage form and a three-part combination is administered as two identical oral dosage forms.
  • the compounds of the combination may be administered: (1) simultaneously by combination of the compounds in a co-formulation or (2) by alternation, i.e. delivering the compounds serially, sequentially, in parallel or simultaneously in separate pharmaceutical formulations.
  • the delay in administering the second, and optionally a third active ingredient should not be such as to lose the benefit of a synergistic therapeutic effect of the combination of the active ingredients.
  • the combination should be administered to achieve peak plasma concentrations of each of the active ingredients.
  • Effective peak plasma concentrations of the active ingredients of the combination will be in the range of approximately 0.001 ⁇ M to 10 uM.
  • Optimal peak plasma concentrations may be achieved by a formulation and dosing regimen prescribed for a particular patient.
  • active ingredient or the physiologically functional derivatives of either thereof, whether presented simultaneously or sequentially, may be administered individually, in multiples, or in any combination thereof.
  • alternation therapy (2) an effective dosage of each compound is administered serially , where in co-formulation therapy (1), effective dosages of two or more compounds are administered together.
  • the combination may be formulated in a unit dosage formulation comprising a fixed amount of each active pharmaceutical ingredient for a periodic, e.g. daily, dose or subdose of the active ingredients.
  • Pharmaceutical formulations according to the present disclosure comprise a combination according to the disclosure together with one or more pharmaceutically acceptable carriers or excipients and optionally other therapeutic agents.
  • composition of the present disclosure may be administered by any delivery route which results in a therapeutically effective outcome.
  • these include, but are not limited to, enteral (into the intestine), gastroenteric, epidural (into the dura mater), oral (by w'ay of the mouth), transdermal, intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intra-arterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow 7 ), intrathecal (into the spinal canal), intraparenchymal (into brain tissue), intraperitoneal (infusion or injection into the peritoneum), intravesical infusion, intravitreal (through
  • compositions may be administered in a way which allows them to cross the blood-brain barrier, vascular barrier, or other epithelial barrier.
  • the viral particles of the present disclosure may be administered in any suitable form, either as a liquid solution or suspension, as a solid form suitable for liquid solution or suspension in a liquid solution.
  • the viral particles may be formulated with any appropriate and pharmaceutically acceptable excipient.
  • composition of the present disclosure may be delivered to a subject via a single route administration.
  • composition of the present disclosure may be delivered to a subject via a multi-site route of administration.
  • a subject may be administered at 2, 3, 4, 5, or more than 5 sites.
  • a subject may be administered the composition of the present disclosure using a bolus infusion.
  • a subject may be administered the composition of the present disclosure using sustained delivery' over a period of minutes, hours, or days.
  • the infusion rate may be changed depending on the subject, distribution, formulation or another delivery parameter.
  • the composition of the present disclosure may be delivered by oral administration.
  • oral administration include a digestive tract administration and a buccal administration.
  • the composition of the present disclosure may be delivered by intraocular delivery route.
  • intraocular administration include an intravitreal injection.
  • the composition of the present disclosure may be delivered by intranasal delivery route.
  • intranasal delivery include administration of nasal drops or nasal sprays.
  • the composition may be delivered by systemic delivery.
  • the systemic delivery may be by intravascular administration.
  • composition of the present disclosure may be administered to a subject by intraparenchymal administration.
  • composition of the present disclosure may be administered to a subject by intramuscular administration.
  • the composition of the present disclosure is administered to a subject and transduce muscle of a subject.
  • the composition is administered by intramuscular administration.
  • composition of the present disclosure may be administered to a subject by intravenous administration.
  • composition of the present disclosure may be administered to a subject by subcutaneous administration.
  • composition of the present disclosure may be administered to a subject by topical administration.
  • the composition may be delivered by direct injection into the brain.
  • the brain delivery may be by intrastriatal administration.
  • the composition may be delivered by more than one route of administration.
  • composition may be delivered by intrathecal and intracerebroventricular, or by intravenous and i ntraparenchy mal admi ni strati on .
  • the present disclosure additionally provides a method of delivering to a subject, including a mammalian subject, any of the above-described composition comprising administering to the subject said composition, or administering to the subject a formulation comprising said composition, or administering to the subject any of the described compositions, including pharmaceutical compositions.
  • the therapeutically effective amount can be initially determined from preliminary in vitro studies and/or animal models.
  • a therapeutically effective dose can also be determined from human data for inhibitors which have been tested in humans and for compounds which are known to exhibit similar pharmacological activities, such as other related active agents. For instance, many cathepsin inhibitors have been extensively studied.
  • the applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled artisan,
  • the dose of the compositions of the disclosure may be determined based on the concentration of the protease inhibitor required to inhibit the growth of a virus or reduce the percentage of virus infected cells.
  • the concentration of the protease inhibitor may be from about 1 x 10 -12 M to about I xl O'” M, for example, from about 0. 1 ⁇ M to about 50 ⁇ M.
  • the concentration of the protease inhibitor may be 0.01 ⁇ M -0.1 iiM, 0.1 ⁇ M -1 ⁇ M, 1 ⁇ M -10 ⁇ M, 10 ⁇ M -100 ⁇ M.
  • the concentration of the protease inhibitor may be 0. 1 pM, 0.3 ⁇ M, 1 ⁇ M, 3 ⁇ M, 10 ⁇ M or 30 ⁇ M.
  • the mammalian protease inhibitor may have an effective concentration (EC50) of from about 0.25 ⁇ M to about 50 ⁇ M.
  • the effective concentration of the protease inhibitor may be about 0.01 ⁇ M -0.1 ⁇ M, 0.1 ⁇ M -1 ⁇ M, 1 ⁇ M -10 ⁇ M, 10 ⁇ M -100 ⁇ M.
  • the effective concentration (EC50) of the protease inhibitor is from about 15 ⁇ M to about 30 ⁇ M.
  • the effective concentration (EC50) of the protease inhibitor is from about 0.25 ⁇ M to about 0.5 ⁇ M.
  • the mammalian protease inhibitor may have an effective concentration (EC90) of from about 0.25 ⁇ M to about 50 ⁇ M.
  • the effective concentration of the protease inhibitor may be about 0.01 ⁇ M -0.1 ⁇ M, 0.1 ⁇ M -1 ⁇ M, 1 ⁇ M -10 ⁇ M, 10 ⁇ M -100 ⁇ M.
  • the effective concentration (EC90) of the protease inhibitor is from about 1 ⁇ M to about 100 ⁇ M.
  • the effective concentration (EC90) of the protease inhibitor is from about 1 ⁇ M to about 3 ⁇ M.
  • the dose varies depending on the target disease, symptom, subject of administration, administration method and the like, for oral administration as a therapeutic agent, for example, it is generally about 0.01-1000 mg/kg body weight.
  • the dose may be 0.01-0.1 mg/kg, 0.1-1 mg/kg, 1-10 mg/kg, 10-100 mg/kg, 100-1000 mg/kg, 0.05-30 mg/kg body weight, 0.5-10 mg/kg body weight, as one dose of the compound of the present disclosure, which is, for example, administered once to 3 times a day, on a weekly schedule, on a twice-weekly schedule and the like.
  • the dose may be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 mg/kg body weight.
  • the dose may be 400mg oral dosing of Beautycatib (HB-121) which may result in plasma concentrations of Beautycatib (HB-121) above EC 50 for 16-20 hrs.
  • the dosing of 400mg may be provided orally once or twice daily.
  • compositions of the present disclosure or a pharmaceutical composition thereof is administered on a weekly schedule. In some embodiments, the compositions are administered on a weekly schedule.
  • compositions of the present disclosure or a pharmaceutical composition of the present disclosure or a pharmaceutical composition thereof is administered on days 1, 8, and 15 of a 28-day cycle. In some embodiments, the compositions of the present disclosure, are administered on days 1, 8, and 15 of a 28-day cycle.
  • compositions of the present disclosure or a pharmaceutical composition thereof is administered on a twice-weekly schedule.
  • compositions of the present disclosure for example a cathepsin inhibitor, or a pharmaceutical composition thereof is administered on a twice-weekly schedule.
  • compositions of the present disclosure of the present disclosure or a pharmaceutical composition thereof is administered on days 1 , 4, 8, and 11 of a 21 -day cycle.
  • compositions of the present disclosure or a pharmaceutical composition thereof is administered in conjunction with another therapeutic modality.
  • the other therapeutic modality is one that is normally administered to patients with the disease to be treated or prevented.
  • the other therapeutic modality is radiotherapy or plasmapheresis or another therapeutic agent.
  • the other therapeutic modality may be administered in the same dosage form or as a separate dosage form.
  • the other therapeutic agent may be administered prior to, at the same time as, or following administration of the compound of the present disclosure or a pharmaceutical composition thereof.
  • Pharmaceutical formulations containing the active ingredient may be in any form suitable for the intended method of administration. When used for oral use for example, tablets, troches, lozenges, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs may be prepared an in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA, which is incorporated herein by reference in its entirety.
  • compositions intended for oral use may be prepared according to any method known to the art. for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents including antioxidants, sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide a palatable preparation. Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipient which are suitable for manufacture of tablets are acceptable.
  • excipients may be, for example, inert diluents, such as calcium or sodium carbonate, lactose, lactose monohydrate, croscarmellose sodium, povidone, calcium or sodium phosphate; granulating and disintegrating agents, such as maize starch, or alginic acid; binding agents, such as cellulose, microcrystalline cellulose, starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or may be coated by known techniques including microencapsulation to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • inert diluents such as calcium or sodium carbonate, lactose, lactose monohydrate, croscarmellose sodium, povidone, calcium or sodium phosphate
  • granulating and disintegrating agents such as maize starch, or alginic acid
  • binding agents such
  • a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.
  • Formulations for oral use may be also presented as hard gelatin capsules where the active ingredient is mixed with an inert solid diluent, for example pregelatinized starch, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.
  • the present disclosure provides pharmaceutical formulations combining the active ingredients, a mammalian protease inhibitor, or physiologically functional derivatives thereof, in a sufficiently homogenized form, and a method for using this pharmaceutical formulation.
  • An object of the present disclosure is to utilize glidants to reduce the segregation of active ingredients in pharmaceutical compositions during pre-compression material handling.
  • Formulations include those suitable for oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods represent a further feature of the present disclosure and include the step of bringing into association the active ingredients with the carrier, which constitutes one or more accessory ingredients, and maintaining chemical stability.
  • the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
  • Formulations of the present disclosure suitable for oral administration may be presented as discrete units such as capsules, caplets, cachets or tablets each containing a predetermined amount of the active ingredients; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in- water liquid emulsion or a w'ater-in-oil liquid emulsion.
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredients in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. povidone, gelatin, hydroxypropyl methylcellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycollate, cross linked povidone, cross-linked sodium carboxymethyl cellulose) surfaceactive or dispersing agent.
  • Molded tablets may be made by molding a mixture of the powdered compound moistened with an inert liquid diluent in a suitable machine.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredients therein using, for example, cellulose ether derivatives (e.g., hydroxypropyl methylcellulose) or methacrylate derivatives in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach .
  • Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredients in a flavored base, usually sucrose and acacia or tragacanth, pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
  • Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylates. Topical administration may also be by means of a transdermal iontophoretic device.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
  • Formulations suitable for penile administration for prophylactic or therapeutic use may be presented in condoms, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
  • compositions suitable for rectal administration wherein the carrier is a solid are most preferably presented as unit dose suppositories.
  • Suitable carriers include cocoa butter and other materials commonly used in the art.
  • the suppositories may be conveniently formed by admixture of the active combination with the softened or melted carrier(s) followed by chilling and shaping in molds.
  • Formulations suitable for parenteral administration include aqueous and nonaqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents; and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs.
  • the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Exemplary’ unit dosage formulations are those containing a daily dose or daily subdose of the active ingredients, as hereinbefore recited, or an appropriate fraction thereof. It should be understood that in addition to the ingredients particularly mentioned above the formulations of this disclosure may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include such further agents as sweeteners, thickeners and flavoring agents.
  • Aqueous suspensions of the disclosure contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., 1 polyoxyethylene sorbitan monooleate).
  • a suspending agent such as sodium
  • the aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose, sucralose or saccharin.
  • Oil suspensions may be formulated by suspending the active ingredient in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oral suspensions may contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation.
  • compositions may be preserved by the addition of an antioxidant such as ascorbic acid, BHT, etc.
  • Dispersible powders and granules of the disclosure suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending a-gents are exemplified by those disclosed above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.
  • the pharmaceutical compositions of the disclosure may also be in the form of oil in-water emulsions or liposome formulations.
  • the oily phase may be a vegetable oil, such as olive oil or arachis oil, a mineral oil, such as liquid paraffin, or a mixture of these.
  • Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate.
  • the emulsion may also contain sweetening and flavoring agents.
  • Syrups and elixirs may be formulated with sweetening agents, such as glycerol, sorbitol or sucrose.
  • Such formulations may also contain a demulcent, a preservative, a flavoring or a coloring agent.
  • compositions of the disclosure may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension.
  • a sterile injectable preparation such as a sterile injectable aqueous or oleaginous suspension.
  • This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butane diol or prepared as a lyophilized powder.
  • a non-toxic parenterally acceptable diluent or solvent such as a solution in 1,3-butane diol or prepared as a lyophilized powder.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile fixed oils may conventionally be employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid may likewise be used in the preparation of injectables.
  • compositions of the disclosure may be injected parenterally, for example, intravenously, intraperitoneally, intrathecally, intraventricularly, intrasystemically, intracranially, intramuscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary'.
  • suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the an.
  • compositions of the disclosure may also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurized container or a nebulizer with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1,1, 1,2-tetra.fluoroethane (HFC 134a), carbon dioxide or other suitable gas.
  • a suitable propellant e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1,1, 1,2-tetra.fluoroethane (HFC 134a), carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount..
  • the pressurized container or nebulizer may contain a solution or suspension of the composition, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate.
  • a lubricant e.g. sorbitan trioleate.
  • Capsules and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of a pharmaceutical composition of the disclosure and a suitable powder base such as lactose or starch. Aerosol or dry' powder formulations are preferably arranged so that each metered dose or "puff contains from 20 pg to 200 mg of a composition for delivery to the patient.
  • the overall daily dose with an aerosol or nebulizer will be in the range of from 20 pg to 200 mg which may be administered in a single dose or, more usually, in divided doses throughout the day.
  • a time-release formulation intended for oral administration to humans may contain approximately 1 to 1000 mg of active material compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 95% of the total compositions (weight : weight) .
  • the pharmaceutical composition can be prepared to provide easily measurable amounts for administration.
  • an aqueous solution intended for intravenous infusion may contain from about 3 to 500 mg of the active ingredient per milliliter of solution in order that infusion of a suitable volume at a rate of about 30 ml/hr can occur.
  • formulations of the present disclosure suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containi ng a predetermined amount of the acti ve ingredient, as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be administered as a bolus, electuary' or paste.
  • Bioavailability is the degree to which a pharmaceutically active agent becomes available to the target tissue after the agent's introduction into the body. Enhancement of the bioavailability of a pharmaceutically active agent can provide a more efficient and effective treatment for patients because, for a given dose, more of the pharmaceutically active agent will be available at the targeted tissue sites.
  • active ingredients or pharmaceutically active agents.
  • Bioequivalence refers to chemical equivalents that., when administered to the same person in the same dosage regimen, result in equivalent concentrations of drug in blood and tissues.
  • “Chemical equivalence” refers to drug products that contain the same compound in the same amount and that meet current official standards. However, inactive ingredients in drug products may differ.
  • the term "effective amount,” “effective concentration,” or “effective dose” means an amount that is sufficient upon appropriate administration to a patient (a) to cause a detectable decrease in the severity of the disorder or disease state being treated; (b) to ameliorate or alleviate the patient's symptoms of the disease or disorder; or (c) to slow or prevent advancement of, or otherwise stabilize or prolong stabilization of, the disorder or disease state being treated. It should also be understood that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, and diet of the patient, time of administration, rate of excretion, drug combinations, the judgment of the treating physician, and the severity of the particular disease being treated.
  • the “maximum tolerated dose” is the highest possible but still tolerable dose level with respect to a pre-specified clinical limiting toxicity. In general, these limits refer to the average patient population. For instances in which there is a large difference between the MED and MTD, it is stated that the drug has a large therapeutic window. Conversely, if the range is relatively small, or if the MTD is less than the MED, then the pharmaceutical product will have little to no practical value.
  • method refers to manners, means, techniques, processes and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques, processes and procedures either known to, or readily developed from known manners, means, techniques, processes and procedures by practitioners of chemistry and/or pharmacology.
  • MED minimum effective dose
  • physiologically functional derivative means a pharmaceutically active compound with equivalent or near equivalent physiological functionality when administered in combination with another pharmaceutically active compound alone or in combination with another compound.
  • physiologically functional derivative includes any: physiologically acceptable salt, ether, ester, prodrug, solvate, stereoisomer including enantiomer, diastereomer or stereoisomerically enriched or racemic mixture, and any other compound, which upon administration to a recipient, is capable of providing (directly or indirectly) such a compound or an active metabolite or residue thereof.
  • potentiating effect refers to and enhancement of an effect or action of an agent, a drug, or a chemical.
  • a potentiating agent can be a chemical, an agent or a drug that enhances or intensifies an effect or action of another agent, chemical or drug.
  • prodrug refers to any compound that when administered to a biological system generates the drug substance, i.e. active ingredient, as a result of spontaneous chemical reaction(s), enzyme catalyzed chemical reaction(s), and/or metabolic chemical reaction(s).
  • Prodrug moiety means a labile functional group which separates from the active inhibitory compound during metabolism, systemically, inside a cell, by hydrolysis, enzymatic cleavage, or by some other process (Bundgaard, Hans, “Design and Application of Prodrugs” in Textbook of Drug Design and Development (1991), P. Krogsgaard Larsen and H. Bundgaard, Eds. Harwood Academic Publishers, pp. 113-191).
  • Side effects or "toxicity” or “adverse drug reactions” of drugs are side effects which may minor, severe, quite severe, or disabling and reversible or irreversible.
  • a side effect is an adverse effect that is secondary to the one intended; an unintended, consequences of the use of a drug whether in the targeted or untargeted parts of the body.
  • the term “subject” or “patient” is a mammal, and examples thereof include human, dog, cat, bovine, horse, swine, or human.
  • synergy and “synergistic” mean that the effect achieved when the drug and compound are used together is greater than the sum of the effects that results from using the drug and the compound separately, i.e. greater than what would be predicted based on the two active ingredients administered separately.
  • a synergistic effect may be attained when the drug and compound are: (I) co-formulated and administered or delivered simultaneously in a combined formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect may be attained when the drug and compound are administered or delivered sequentially, e.g. in separate tablets, pills or capsules, or by different injections in separate syringes.
  • an effective dosage of each active ingredient is administered sequentially, i.e. serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together.
  • a synergistic antiviral effect denotes an antiviral effect which is greater than the predicted purely additive effects of the individual active ingredients of the combination.
  • treatment means treating a patient having, or at risk of developing or experiencing a recurrence of the relevant disorder being treated, including suppression of progression of the relevant disorder being treated.
  • Therapeutic equivalence refers to drug products that, when administered to the same person in the same dosage regimen, provide essentially the same therapeutic effect or toxicity. Bioequivalent products are expected to be therapeutically equivalent. Sometimes therapeutic equivalence may be achieved despite differences in bioavailability', for example when the therapeutic index is wide (ratio of maximum tolerated dose to the minimum effective dose).
  • Absorption rate is important because even when a drug is absorbed completely, it may be absorbed too slowly to produce a therapeutic blood level quickly enough or so rapidly that toxicity results from high drag concentrations given to achieve the therapeutic level after each dose.
  • Viruses and cells were mixed in the presence of Beautycatib (HB-121) and incubated for the assay duration as specified in Table 1. Each virus was pre-titered such that control wells exhibited 85 to 95% loss of cell viability due to virus replication. An antiviral effect or cytoprotective effect was considered to be observed when therapeutic agent (Balicatib or positive control indicated in Table 1) prevented virus replication.
  • a standardized plate format was used for cytoprotective assays. Each plate contained cell control wells (cells only), virus control wells (cells plus virus), therapeutic agent cytotoxicity wells (cells plus therapeutic agent only), therapeutic agent colorimetric control wells (therapeutic agent only), background control wells (media only), as well as experimental wells (therapeutic agent plus cells plus virus).
  • I' able 2 shows a representative plate format for evaluating test therapeutic agent (Balicatib/ HB-121; labeled “Drug 1” in Table 2) at 6 concentrations (2-fold dilutions) using representative high-test concentrations of 50 ⁇ M and control therapeutic agent (labelled “Drug 2” in Table 2) at 6 concentrations (10-fold dilutions) using representative high-test concentration of 200 IU/mL.
  • Interferon was used as positive controls in the assays were noted as IU/mL (bioactivity defined by International Units/mL was used from the certificate of analysis information provided by the vendor, PBL Assay Science, Piscataway, NJ).
  • IU/mL biological activity defined by International Units/mL was used from the certificate of analysis information provided by the vendor, PBL Assay Science, Piscataway, NJ.
  • fields labeled as “Drug 1” or “Drug 2” indicate Cells + Virus + Drug
  • fields labeled as “Tox T” or “Tox 2” indicate Cells + Datg 1 or Drug 2, respectively (toxicity tested in duplicate)
  • Cells labeled as “Color 1” or “Color 2” Media + Drug 1 or Drug 2, respectively (colorimetric background, no cells).
  • the assay plates were stained with the soluble tetrazolium- based dye MTS (CellTiter®96 Reagent, Promega) to determine cell viability and quantify compound toxicity.
  • MTS is metabolized by the mitochondrial enzymes of metabolically active cells to yield a soluble formazan product, allowing the rapid quantitative analysis of cell viability and compound cytotoxicity.
  • This reagent is a stable, single solution that does not require preparation before use.
  • MTS reagent 10-25 pL of MTS reagent was added per well (10% final concentration based on volume) and the microtiter plates were then incubated for 2-3 hours at 37°C, (except Rhinovirus assay piates, which were incubated at 33°C) 5% CO 2 to assess cell viability.
  • Adhesive plate sealers were used in place of the lids, the sealed plates were inverted several times to mix the soluble formazan product and the plate was read spectrophotometrically at 490/650 nm with a Molecular Devices SpectraMax i3 plate reader.
  • the minimum inhibitory drug concentration that reduced plaque formation by 50% (EC 50 ) and the minimum drug concentration that inhibited cell growth by 50% ( AA 50 ) were calculated.
  • the selectivity index (SI) for each active compound was determined by dividing the CCso by the EC 50 .
  • SARS-CoV-2 strain: BEI USA-WA1/2020
  • RNA viruses must bind, fuse, enter, and use host proteins to be able to productively infect human cells.
  • Many negative strand RNA viruses such as Ebola, Marburg, Nipah, SARS, SARS-CoV-2, and MERS use cathepsin inhibitors to enter and replicate within human cells.
  • Some viruses such as SARS-CoV-2 have their own proteases which allows them to invade host detection or use their proteases to cleave their proteins. However, these proteases alone are not able to sustain cellular infection and the virus must use host protases to be able to infect and replicate within human cells.
  • SARS-CoV-2 has at least two essential proteases and at least one cleavage site in its spike protein for cathepsins. There are several cell proteases/cathepsins that are essential for SARS- CoV-2 productive replication.
  • Browncatib (HB 0121) acts a potent antiviral against SARS-CoV-2 with an effective concentration (EC) which inhibits 50% of SARS-CoV-2 infection in 11M range.
  • EC effective concentration
  • Balcatib (HB 0121) has a (SI) and cell cytotoxicity (CC) profile is below.
  • SI SI of >300 and CC50 of over 100 ⁇ M in Calu-3 human cells.
  • CC cell cytotoxicity
  • Clinical dosing may be further refined based on the exact EC50 and EC90 of Beautycatib against SARS-CoV-2.
  • Browncatib was purchased from MedChemExpress (www.medchemexpress.com). The vial contained 5mg of Balicatib (HB 0121) the compound was over 97% pure and was dissolved in DMSO to obtain a 10mM concentration. The efficacy of Balicatib was determined in two separate studies. In study one 0.1, 1, and lO ⁇ M final concentration was tested and in study two six doses’ concentrations 0.1, 0.3, 1, 3, 10, and 30 ⁇ M of Balicatib (HB 0121) was examined.
  • the experiment was set up as follows (8 Wells total): 1) Control well (no infection and no treatment); 2) Control well treated with drag diluent (DMSO at the same final cone, as 30uM: treatment), 3) Inhibitor at 30uM concentration with infection; 4) Inhibitor at 10uM concentration with infection; 5) Inhibitor at 3uM concentration with infection; 6) Inhibitor at luM concentration with infection; 7) Inhibitor at 0.3uM concentration with infection; 8) Inhibitor at O.1uM concentration with infection. [0260] All reagents and the piate were taken into the BSC hood. The cells were pretreated with 500pl of the preparation for each corresponding labeled well.
  • the plates were placed inside the 37°C75% CO2 incubator and allowed to incubate for 4hrs. After the incubation time, the plate will be taken into the BSC and the contents of each well were removed and stored in a separate tube as backup.
  • the cells were infected with SARS-CoV-2 virus (obtained from ATCC/BEI resources Washington strain) at MOI of 0.1 in 100ul of EMEM containing 10% FBS (original titer of stock virus is 4.5e6/ml; so, 4.44ul of the virus stock will be added to each well to achieve 0.1 MOI).
  • the plates were transferred to 370C Z5% C02 incubator for Jackpot (rock plate gently every 10 minutes for even distribution of virus). The plates were taken back into the BSC and the virus was removed.
  • the discarded virus was bleached based on GMU SOP.
  • Cells were washed two times with PBS.
  • Mixture of 500ul each of the above inhibitor mixed with 500ul of EMEM containing 20% FBS were prepared and added to each of the corresponding wells.
  • the plates were placed in 37°C/5% CO2 incubator and allowed to incubate for 72hrs.
  • the cells were observed daily for CPE, and their viability were measured at the 72 hour time point.
  • the supernatants were collected and spun down at 1200 x g for 5 minutes to remove floating cells, and the remaining supernatants were used for plaque assay analysis.
  • the residual cells, pellets, and other tissue culture reagents were bleached per GMU SOP.
  • the reagents for the plaque assay include one or more cell culture media.
  • Complete EMEM+++++ media was prepared using 1 x bottle 2X EMEM for plaques (500ml), 10% FBS (50mL), 1% Minimum Essential Amino Acids (NEAA) (5ml), 1% Sodium Pyruvate (5ml), 1% L-glutamine (200mM) (5ml), 1% Pen/Strep (5ml).
  • Complete DMEM+++ media was prepared using 1x bottle Dulbecco’s modified eagle medium (DMEM) (500mL), lx 25mL aliquot FBS (5%), 1x L-glutamine (200mM) (5ml), lx Pen/Strep (5ml).
  • Crystal violet solution was prepared using 1% Crystal violet, 20% Ethanol, 79% d H 20, Agarose (0.6%) and 0.6g in 100mL of H2O.
  • test samples were prepared. The day before the assay, 2.5x105 Vero cells/mL were seeded in a 12 well plate and incubated at 37°C/5% CO2 in order to achieve a 90 - 100% confluency the following day. On day 2, samples were prepared. The confluency and health of the Vero cells was checked before starting the assay. Ten-fold dilutions of each test sample in DMEM was performed using deep-well 96 well plates (sample undiluted testing was included as needed). 450 pl DMEM was added to all wells. 50 ⁇ l of each test sample was added to the well in the first row containing DMEM and mixed the content of the well by pipetting up and down multiple times.
  • the pipet tips were changed and 50 pl was transferred from the first dilution wells in the first row to the next row of wells using a multichannel pipette.
  • the samples were mixed, the pipette tips were changed and the process was repeated this step until all desired dilutions are prepared.
  • the agarose was heated on a hot plate or in a microwave until it melted and was cooled down in the water bath to about 56°C.
  • the agarose was added to cold EMEM so that the mixture could reach a temperature below 50°C. It is essential not to add the overlay to the cells when it is hotter than 50°C or the cells will die.
  • HB 0121 was added to cells at various concentrations and after a few hours of incubation the SARS-CoV-2 was added. The infection was stopped at 72 hours and the amount of infection was recorded based on plaque formation.
  • the control sample contained the highest amounts of DMSO which was used to dilute Balicatib and was used as “control”.
  • the plates w z ere checked the plates under microscope again. 0.8-lmL of the agarose overlay and incubated at 37°C/5% CO2 for 2 days. The plates were not shaken or agitated during this period to avoid getting smudged plaques.
  • the cells were fixed with 10% formaldehyde in diH2O ( ⁇ 1mL per well ) using a pasture pipet and left for 1 hour inside the chemical hood. The formaldehyde was then gently removed using a pasture pipet, making sure not to touch the cells with the pipet tip. The formaldehyde was discarded in the appropriate container. The plate was inverted to expel out the overlay onto a sheet of paper towel inside the sink. A few drops of crystal violet were added to each well and allowed to sit for 5 mins. Using a pasture pipet, added enough diH2O to each well to wash out excess crystal violet. Extra washes could be done if needed.
  • plaques in each well were counted, taking the average of technical replicates of the same dilution: The plaques were counted the plaques in each well, taking the average of technical replicates of the same dilution. Pfu/ml was calculated as the average number of plaques divided by the product of dilution (D) and volume of diluted virus added to the plate. After the number of plaques was determined for control and experimental treatments, the following formula was used to obtain % inhibition of the drug. The results are shown in Table 5 and Table 6.
  • Table 5 depicts the data obtained from two experiments in which cells were treated with Bascatib (HB 0121) and infected with SAR.S-CoV-2 virus at MOI (Multiplicity of Infection) of 0.1 .
  • the data clearly shows that Balicatib (HB 0121) inhibited the growth of SARS-CoV-2.
  • Balcatib (HB 0121) inhibited almost 100% of the viral growth at 3, 10, and 30 uM.
  • I ⁇ M the efficacy of Browncatib was reduced but was substantially different than control.
  • the efficacy, as measured by % inhibition of SARS-CoV-2 growth of Balicatib (HB 0121) was about 68% and 57% at 1 pM and 100nM, respectively.
  • plaque assay which is the “gold standard” for analyzing efficacy of antivirals.
  • the plaque assay examines actual infection. Further, the size of the plaque, shape of the plaque, and the quality of the plaques allows one to better understand the quality of antiviral activity of the compounds.
  • the plaque assay is difficult to perform and is within 0.5 log accuracy and reproducibility. Therefore, it is not unusual to see differences between several replicate studies, specially at the low end of the effective concentrations of antivirals. Here, at lower concentration of Browncatib (HB0121), the data was variable between the two studies specifically at the 100-300nM.
  • the above method was used to perform antiviral testing for ONO- 5334 (HB 0119) and Odanacatib (HB 0122) against SARS-CoV-2 infection using human lung cells (Calu-3 cells).
  • ONO-5334 (HB 0119) has EC50 value of less than 1 ⁇ M and EC90 is between 1-10 ⁇ M, as shown in Table 7.
  • Odanacatib (HB 0122) another cysteine cathepsin inhibitor has lower activity against SARS-CoV-2 infection using Calu-3 cells. This antiviral has EC50 of about l ⁇ M and EC90 l-10 ⁇ M.
  • articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.
  • the disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process.
  • the disclosure includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.

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Abstract

The present disclosure relates to antiviral composition and methods. Methods for using the antiviral compositions of the disclosure for inhibiting the growth of viruses and treatment of viral diseases are also described.

Description

METHODS AND COMPOSITIONS FOR THE TREATMENT OF VIRAL DISEASES CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to US Provisional Patent Application No. 63/084,754 entitled “Compositions and Methods for Increasing the Efficacy of a Drug” filed on 29 September 2020 and US Provisional Patent Application No. 63/188,723 entitled “Methods and Compositions for the Treatment of Viral Diseases” filed on 14 May 2021, the contents of each of which are herein incorporated by reference in their entirety. FIELD OF THE DISCLOSURE [0002] The present disclosure provides protease inhibitor based compositions and methods for the treatment of diseases such as diseases caused by virus. BACKGROUND OF THE DISCLOSURE [0003] The emergence of new viruses has exposed the need for innovative strategies to develop antiviral drugs. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emergent coronavirus which causes a severe acute respiratory disease, COVID-19. There is an urgent need for preventive and therapeutic antiviral therapies for SARS-CoV-2 control. [0004] Repurposing previously identified drugs for therapeutic indications represents a potential path towards identifying promising candidate drugs to counteract current viral pathogens and possible emerging viruses. SUMMARY OF THE DISCLOSURE [0005] The disclosure provides antiviral compositions and methods. In some embodiments, the present disclosure provides a method of inhibiting the growth of a virus for example, a coronavirus. In some aspects, the methods may involve contacting the virus with a mammalian protease inhibitor. The method may further include measuring the growth of the virus. The growth of the virus may be measured by methods known in the art. In some embodiments, the present disclosure provides a method of reducing the percentage of virus infected cells in a population of cells. Such methods may include, contacting the virus infected cells with a mammalian protease inhibitor. The method further involves measuring the percentage of virus infected cells. Also provided herein are methods of reducing coronavirus infection in a subject. Such methods may include, contacting a subject in need with a mammalian protease inhibitor. The efficacy of the mammalian protease inhibitor in reducing the coronavirus infection may be measured after providing the subject with mammalian protease inhibitor. [0006] In some embodiments, the mammalian protease inhibitor has the structure of Formula (I):
(I), wherein, R1 and R2 are independently H or
Figure imgf000004_0001
C1-C7 lower alkyl, or R1 and R2 together with the carbon atom to which they are attached form a C3-C8 cycloalkyl ring; and R3 is an optionally substituted heterocyclic group comprising at least one nitrogen; and n is between 1 and 3.
[0007] In some embodiments, the mammalian protease inhibitor may have the structure of
Formula (II):
Figure imgf000004_0002
(II), wherein X is CH or N; and
[0008] R4 is H, C1-C7 lower alkyl, C1-C7 lower alkoxy, C5-C10 aryl, or C3-C8 cycloalkyl.
[0009] In one embodiment, the mammalian protease inhibitor may have the structure of
Figure imgf000004_0003
[0010] In one aspect, the mammalian protease inhibitor may have the structure of
Figure imgf000005_0001
[0011] The mammalian protease inhibitor may be a cathepsin inhibitor. Non-limiting examples of cathepsin inhibitors include The method of claim 4, wherein the cysteine cathepsin inhibitor is Balicatib, E-64, E-64a, E-64b, E-64c, E- 64d, CA-074, CA-074 Me, CA-030, CA-028, peptidyl aldehyde derivatives leupeptin, antipain, chymostatin, Ac-LVK- CHO, Z-Phe-Tyr-CHO, a epoxisuccinate Z-Phe-Tyr(OtBu)-COCHO.H2O, 1- Naphthalenesulfonyl-Ile-Trp-CHO, Z-Phe-Leu-COCHO.H2O; peptidyl semicarbazone derivatives, peptidyl methylketone derivatives, peptidyl trifluoromethylketone, Biotin-Phe- Ala-fluoromethyl ketone, Z-Leu-Leu-Leu fluoromethyl ketone, Z-Phe-Phe-fluoromethyl ketone, N-Methoxysuccinyl-Phe-HOMO-Phe-fluoromethyl ketone, Z-Leu-Leu-Tyr- fluoromethyl ketone, Leupeptin trifluoroacetate, ketone, peptidylchloromethases peptidylhydroxymates, peptidylhydroxylamines, peptidyl acyloxymethanes, peptidylacyloxymethyl ketones, peptidyl aziridines, peptidyl aryl vinylsufones, peptidyl arylvinylsulfonates, gallinamide analogs, peptidyl aldehydes , azepinone-based inhibitors , nitrile-containing inhibitors , thiosemicarbazone , propeptide mimics, thiocarbazate, oxocarbazate, azapaptides , peptidyl halomethylketone derivatives, TLCK; bis(acylamino) ketone, 1,3- Bis(CBZ-Leu-NH)-2-propanone; peptidyl diazomethanes, Z-Phe-Ala-CHN2, Z- Phe-Thr(OBzl)-CHN2, Z-Phe-Tyr (Ot-But)-CHN2, Z-Leu-Leu-Tyr-CHN2; peptidyl acyloxymethyl ketones; peptidyl methylsulfonium salts; peptidyl vinyl sulfones, LHVS; peptidyl nitriles; peptidyl disulfides, 5,5′-dithiobis[2-nitrobenzoic acid], cysteamines, 2,2′- dipyridyl disulfide; N-(4-Biphenylacetyl)-S-methyl cysteine-(D)-Arg-Phe-b phenethylamide; thiol alkylating agents, maleimides, azapeptides, azobenzenes, O-acylhydroxamates, Z-Phe- Gly-NHO-Bz, Z-FG-NHO-BzOME, lysosomotropic agents, chloroquine, ammonium chloride, Cystatins A, Cystatin B, Cystatin C, Cystatin D, Cystatin F, stefins, kininogens, Sialostain L, antimicrobial peptide LL-37, Procathepsin B Fragment 26-50, Procathepsin B Fragment 36-50, Odanacatib (MK-0822), Relacatib (GSK-462795, SB-462795), SLV213 (K777 OR K1777), RO5459072, RWJ-445380, VBY036P1A, AM-3701, MIV-701, MIV- 710, MIV-711, NC-2300, ORG-219517, ONO-5334,MK-0674, GB-111-NH2, L-873724, L- 006235, AZD4996, VBY-036, RWY-445380, AM-3840, Cz-007, VBY-825 (VBY-106; VBY-285;VBY-825), VBY-129, SAR-114137, VBY-891, Petesicatib (RG-7625; RO- 5459072), LY-3000328, MIV-247, CRA-028129, RG-7236, GSK2793660, Al oxi statin (E- 64d, Loxistatin, EST), BI-1181181 (VTP-37948),VBY-376, Aloxistatin (Ab-007; E-64-d), Begacestat (GS 1-953; WAY-210953), AL101 (BMS906024), BMS-986115 (AL- 102), MK- 0752 (L-000891675), EVP-0962 (EVP-0015962), SAR-164653, KGP94, VEL-0230, and/or BLD2660. In one embodiment, the cathepsin inhibitor may be Balicatib, or a pharmaceutically acceptable salt or an ester of Balicatib.
[0012] In some embodiments, the virus may be a virus in the family, Coronaviridae, or a virus in the sub-family Orthocoronavirinae, or a virus in the order Nidovirales, In some embodiments, the methods of the disclosure may be used to inhibit the growth of any coronavirus. As a non-limiting example, the methods of the disclosure may be used to inhibit the growth of a coronavirus, such as a SARS-CoV-2 virus, SARS-CoV-1 virus, MERS-CoV virus, 229E virus, NL63 virus, OC43 virus, HKU1 virus, or variants thereof. As a nonlimiting example, the coronavirus may be SARS-CoV-2 virus.
[0013] According to the methods of the present disclosure the concentration of the mammalian protease used may be from about 1 x 10-12M to about 1x10-5 M, for example, from about 0.1 μM to about 50 μM. In some embodiments, the effective concentration (EC50) of the mammalian protease inhibitor against a coronavirus may be from about 0.25μM to about 30 μM, for example, from about 0.5 μM to about 30 μM. In some embodiments, the effective concentration of the mammalian protease inhibitor against an enterovirus may be from about 15μM to about 30μM. The EC50 of the mammalian protease inhibitor may be 0.1 μM, 0.3 pM, 1 μM, 3 μM, 10 μM or 30 μM. The EC90 of the mammalian protease inhibitor may be from about IμM to lOOμM. As a non-limiting example, the EC90 may be IμM to 100μM. Contacting the coronavirus with the mammalian protease inhibitors may inhibit the growth of the coronavirus by from about 50% to about 100%. The mammalian protease inhibitor may be associated with a selectivity index of at least 300.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The foregoing and other objects, features, and advantages will be apparent from the following description of particular embodiments of the disclosure, as illustrated in the accompanying drawings. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the disclosure. [0015] Figure 1 shows the percentage of virus infected cells treated with varying concentrations of Balicatib (HB-121).
[0016] Figure 2 shows the percentage of virus infected cells treated with varying concentrations of ONO-5334.
[0017] Figure 3 shows the percentage of virus infected cells treated with varying concentrations of Odanacatib (MK-0822).
[0018] Figure 4 is a Prior Art table showing the inhibition (IC50) by odanacatib and belacatib of different cathepsins relative to different diseases. The data was presented at the 8th RCS-SCI Symposium on Proteinase Inhibitor Design, on April 16, 2013, hosted by the Royal Society of Chemistry.
[0019] Figure 5 is a graph depicting an effect of adding a cathepsin inhibitor to a weak antiviral compound. The cathepsin inhibitor was able to enhance the antiviral activity of the compound by more than a log. The large arrow depicts the shift in the Effective Concentration (EC50) of the antiviral. The vertical dotted line depicts cytotoxicity of the drug with or without a potentiator.
DETAILED DESCRIPTION OF THE DISCLOSURE
I. INTRODUCTION
[0020] Viruses comprise a large group of pathogens that are responsible for causing severe infectious diseases. Therapeutic agents targeting viruses may be broadly classified into (i) therapeutic agents that target the viruses themselves or (ii) therapeutic agents that target host cell factors.
[0021] Virus-targeting therapeutic agents can function directly or indirectly to inhibit the biological functions of viral proteins, such as enzymatic activities, or to block viral replication machinery. Host-targeting therapeutic agents target the host proteins that play a role in the viral life cycle, regulating the function of the immune system or other cellular processes in host cells. In some embodiments, the present disclosure provides host -targeting therapeutic agents for the treatment of viral di seases. In some embodiments, the present disclosure provides virus- targeting therapeutic agents and the related compositions. Also provided herein are methods of inhibiting the growth of a virus, and methods of reducing the percentage of virus infected cells in a population of cells. II. COMPOSITIONS
Protease Inhibitors
[0022] In some embodiments, the compositions of the disclosure may be or may include protease inhibitors. Protease inhibitors are small molecule that block or reduce the activity of a protease. In some embodiments, proteases may be essential for virus replication. Many human pathogenic viruses use human enzymes to activate the viral proteins and successfully overtake the infected cell processes. For example, human cathepsins assist in the cleavage of viral proteins that are essential for the virus life cycle. These proteases may include, but are not limited to cysteine proteases, serine proteases, aspartic proteases.
[0023] In some embodiments, the compositions of the disclosure may be or may include a “cathepsin inhibitor” which, as used herein may refer to an agent which is capable of reducing, suppressing or inhibiting the activity of the class of endosomal proteases called cathepsins. In some embodiments, the cathepsins may require acidic pH for enzyme activity. In other aspects, the cathepsins may be enzymatically active at neutral pH. There are approximately a dozen members of this family, which are distinguished by their structure, catalytic mechanism, and which proteins they cleave. Cathepsins have a vital role in mammalian cellular turnover, e.g. bone resorption. They degrade polypeptides and are distinguished by their substrate specificities.
[0024] Most C1 cysteine cathepsins are endopeptidases (L, S, K, V, F), while cathepsin X is a carboxypeptidase and cathepsins B, C and H have both endopeptidase- and exopeptidase activities. The substrate-binding region of cysteine cathepsins is defined as an arrangement of binding subsites (SeSO) for peptide substrate amino acids (PePO) on both sides (N- and C-) of the scissile bond, encompassing the stretch of seven sites from S4 to S30 of papain. Since the crystal structure of numerous substrate analogue inhibitors are available, the definition has been revised and redefined, limiting the binding of substrate residues to subsites S2eS10, in which both main-chain and side-chain atoms are involved. However recent studies have shown the importance of cathepsin K site S3 for determining substrate specificity. Whereas the S2 binding site is a true deep pocket, the other sites provide a binding surface.
Furthermore, while S2 and S10 sites are the major determinants of specificity, SI is important for the affinity and efficient catalysis of substrates. The positioning of the P3 residue in site S3 is, as in subsite S20, mediated only by side chain contacts over a relative wide area. Cathepsins K, L, S and V have somewhat overlapping specificities, making it difficult to discriminate between them in vivo. Cathepsin K attacks sites having aliphatic amino acids (Leu, lie, Val), unlike cathepsins L and V (which both rather accept hydrophobic residues with preference for Phe), and also accommodates Pro in the S2 subsite. Cathepsin K is unusual among cysteine cathepsins in that it can cleave substrates with Pro in the P2 position, although it has been reported that congopain, a cysteine protease from Trypanosoma congolense, with an amino acid sequence (65% of homology) and biochemi cal properties similar to cathepsin K, also does so. Another feature of cathepsin K is its preference for Gly at the P3 position.
[0025] Cathepsin K is a protease, which is defined by its high specificity for kinins, that are involved in bone resorption, as discussed in U.S. Patent 6,642,239, which is hereby incorporated by reference in its entirety. The enzyme's ability to catabolize elastin, collagen, and gelatin allow it to break down bone and cartilage. This catabolic activity is also partially responsible for the loss of lung elasticity and recoil in emphysema. Cathepsin K inhibitors, such as odanacatib, show great potential in the treatment of osteoporosis. Cathepsin K is also expressed in a significant fraction of human breast cancers, where it could contribute to tumor invasiveness. Mutations in this gene are the cause of pycnodysostosis, an autosomal recessive disease characterized by osteosclerosis and short stature. Cathepsin K expression is stimulated by inflammatory cytokines that are released after tissue injury.
[0026] Proteases may be grouped according to the key catalytic group in the active site. For example, the active site of the protease may include a serine (Ser), a threonine (Thr), a cysteine (Cys), an aspartate (Asp), a glutamate (Glu), or a zinc in the case of metal loproteases. Accordingly the proteases may be a serine protease, a threonine protease, a cysteine protease, an aspartate protease, a glutamate protease, or a zinc protease.
Mammalian protease inhibitors
[0027] In one embodiment, the protease may be a mammalian protease and the inhibitor may be a mammalian protease inhibitor. In one aspect, the mammalian protease may be a cathepsin protease and the inhibitor may be a cathepsin protease inhibitor.
[0028] In one embodiment of the disclosure, the cy steine protease inhibitor is a cathepsin inhibitor such as a cathepsin-B inhibitor, a cathepsin-L inhibitor, a cathepsin-S inhibitor, a cathepsin-F inhibitor, a cathepsin-X inhibitor, a cathepsin-K, inhibitor, a cathepsin- V inhibitor, a cathepsin-W inhibitor, a cathepsin-C inhibitor, a cathepsin-0 inhibitor, and a cathepsin- H inhibitor. In yet another aspect, the cathepsin inhibitor is a cathepsin-K. inhibitor. [0029] The SARS-Cov-2 virus has 3-way redundancy for infection and viral entry. Cleavage of S1-S2 protein can be accomplished by serine proteases such as TMPRSS2, or the enzyme furin, or a cathepsin, such as, but not limited to cathepsin L.
[0030] In another aspect, the cathepsin inhibitor is epoxi succinate and derivative thereof; E-64; E-64a; E-64b; E-64c; E-64d; CA-074; CA-074 Me, CA-030; CA-028; peptidyl aldehyde derivatives leupeptin, antipain, chymostatin, Ac-LVK-CHO, Z-Phe-Tyr-CHO, Z- Phe- Tyr(OtBu)-COCHOH2O, 1 -Naphthal enesulfonyl-He-Trp- CHO, Z-Phe-Leu- COCHO.H2O; peptidyl semicarbazone derivatives; peptidyl methylketone derivatives; peptidyl trifluoromethylketone derivatives Biotin- Phe-Ala-fluoromethyl ketone, Z-Leu-Leu- Leu-fluoromethy! ketone, Z-Phe-Phe-fluoromethyl ketone, N-Methoxysuccinyl-Phe-HOMO- Phe-fluoromethyl ketone, Z-Leu-Leu-Tyr-fluorom ethyl ketone, Leupeptin trifluoroacetate, ketone; peptidylchloromethases and derivatives thereof; peptidylhydroxymates and derivatives thereof; peptidylhydroxylamines and derivatives thereof; peptidyl acyloxymethanes and derivatives thereof; peptidylacyl oxy methyl ketones and derivatives thereof; peptidyl aziridines and derivatives thereof; peptidyl aryl vinyl sulfones and derivatives thereof; peptidyl arylvinylsulfonates and derivatives thereof; gallinamide analogs and derivates thereof; peptidyl aldehydes and derivatives thereof; azepinone-based inhibitors and derivatives thereof; nitrile-containing inhibitors and derivates thereof; thiosemicarbazone and derivatives thereof; propeptide mimics and derivatives thereof; thiocarbazate, oxocarbazate, azapaptides and derivatives thereof; peptidyl halomethylketone derivatives, TLCK; bis(acylamino)ketone, 1,3- Bis(CBZ-Leu-NH)-2-propanone; peptidyl diazomethanes, Z-Phe-Ala-CHN2, Z-Phe-Thr(OBzl)-CHN2, Z-Phe-Tyr (Ot- But)-CHN2, Z-Leu-Leu-Tyr- CHN2; peptidyl acyloxymethyl ketones; peptidyl methylsulfonium salts; peptidyl vinyl sulfones, LHVS; peptidyl nitriles; peptidyl disulfides, 5,5'-dithiobis[2- nitrobenzoic acid], cysteamines, 2,2 '-dipyridyl disulfide; non-covalent inhibitors, N-(4- Biphenyl acetyl)- S- methyl cysteine-(D)-Arg-Phe-b-phenethylamide; thiol alkylating agents, maleimides, etc; azapeptides; azobenzenes; O-acylhydroxamates, Z-Phe-Gly-NHO-Bz, Z-FG-NHO-BzOME; ly sosomotropic agents, chloroquine, ammonium chloride; Cy statin A, Cy statin B, Cy statin C, Cystatin D, Cystatin F, stefins, kininogens, Sialostain L, antimicrobial peptide LL- 37, Procathepsin B Fragment 26-50, Procathepsin B Fragment 36-50; Odanacatib (MK-0822), Balicatib (AAE581 ), Relacatib (GSK-462795, SB-462795), SLV213 (K777 or K1777), RO5459072, RWJ-445380, VBY036P1A, AM-3701, MIV- 701 , MIV-710, MIV-711, NC- 2300, ORG-219517, ONO-5334, MK-0674, GB-111-NH2, L-873724, L-006235, AZD4996, VBY-036, RWY-445380, AM-3840, Cz-007, VBY-825 (VBY- 106; VBY-285; VBY-825), VBY-129, SAR-114137, VBY-891, Petesicatib (RG-7625; RO-5459072), LY-3000328, MIV32247, CRA-028129, RG-7236, GSK2793660, Aloxistatin (E-64d, Loxi statin, EST), BI-1181181 (VTP-37948), VBY-376, Aloxistatin (Ab-007; E-64-d), Begacestat (GSI-953; WAY-210953), A.L101 ( BMS906024), BMS-986115 5 (AL-102), MK-0752 (L-000891675), EVP-0962 (EVP- 0015962), SAR-164653, KGP94, VEL-0230, and BLD2660.
[0031] In one embodiment, the cathepsin inhibitor may be Balicatib (AAE581).
[0032] Proteases are essential for virus replication. Many human pathogenic viruses use human enzymes to activate the viral proteins and successfully overtake the infected cell processes. For example, human cathepsins assist in the cleavage of viral proteins that are essential for the virus life cycle. These proteases may include but are not limited to cysteine proteases and proteinases, serine proteases, aspartic proteases. For example serine protease inhibitors like Camostat, Odalasvir, Femostat, or any other protease inhibitors currently in use for HIV such as atazanavir (Reyataz), darunavir (Prezista), fosamprenavir (Lexiva), indinavir (Crixivan), lopinavir/ritonavir (Kaletra), nelfinavir (Viracept), ritonavir (Norvir), saquinavir (Invirase), tipranavir (Aptivus), atazanavir/cobicistat (Evotaz), darunavir/cobicistat (Prezcobix); or other protease inhibitors use for other viruses such as HCV for example asunaprevir, boceprevir, grazoprevir, glecaprevir, paritaprevir, simeprevir, telaprevir, and in HBV treatment.
[0033] In some embodiments, a cathepsin inhibitor may be an agent whose main pharmacological effect is to inhibit the activity of the class of endosomal cysteine peptidases that may be enzymatically active in acidic pH or neutral pH. Examples of human cysteine proteases include but are not limited to cathepsins, which include but are not limited to cathepsin B, cathepsin L, cathepsin S, cathepsin-F, cathepsin-X, cathepsin K, cathepsin V, cathepsin W, cathepsin C, cathepsin O, and cathepsin H. Cathepsin inhibitors useful in nonhuman animals are often categorized differently but are known to those of skill in the art. Thus, the inhibitors include cathepsin inhibitors which are known to correspond with human cathepsin inhibitors. Inhibitors of these cathepsins, are useful according to methods of the disclosure. Many cathepsin inhibitors have been described in the literature and are well known and are commercially available.
[0034] Cathepsin inhibitors have a variable level of specificity. There is a reasonable comparison between in-situ peptide assays versus in-vitro cell-based assay. Most of the literature discusses the specificity of cathepsin inhibitors based on the selectivity of cleavage in a peptide assay. The same literature assumes the same level of specificity in an in-cell culture and in-vivo applications. However, the cathepsin inhibitor specificity is different even in in-vitro cell cultures compared to in-situ peptide assays.
[0035] A cathepsin K inhibitor may be active against cathepsin-K, cathepsin-L, cathepsin- B in a patient, as shown in Figure 4. In some embodiments, the non-specific activity may be dose dependent. The non-specific activity may also be affected by other factors such as pH of the endosomes and lysosomes in the cells. Specific cathepsin inhibitors may become broadly acting in cells.
Dipeptide dinitriles
[0036] In some embodiments, the compositions of the present disclosure may include dipeptide dinitriles. In one aspect, the mammalian protease inhibitor may be a dipeptide nitrile. In some aspects, any of the compounds described in International Patent Publication WO2001 058886 (the contents of which are herein incorporated by reference in its entirety). [0037] In some embodiments, the compound has a structure of Formula (I)
Figure imgf000012_0001
(I), wherein, R 1 and R2 are independently H or Cl-
C7 lower alkyl, or R1 and R2 together with the carbon atom to which they are attached form a C3-C8 cycloalkyl ring; and R3 is an optionally substituted heterocyclic group comprising at least one nitrogen; n is between 1 and 3.
[0038] In some embodiments, the compound of the present disclosure has a structure of: wherein the protease inhibitor has the structure of Formula (II):
Figure imgf000012_0002
(II), wherein X is CH or N; and
R4 is H, C1-C7 lower alkyl, C1-C7 lower alkoxy, C5-C10 aiyl, or C3-C8 cycloalkyl. [0039] In some embodiments, the compound has a structure of
Figure imgf000013_0001
[0040] In some embodiments, the compound has a structure of
Figure imgf000013_0002
[0041] In some embodiments, the compound has a structure of
Figure imgf000013_0003
[0042] As a non-limiting example, the compositions of the disclosure may include a compound of Formula (I) of the International Patent Publication W02001058886 and provided below as Formula (III),
Figure imgf000013_0004
C1-C7 lower alkyl, or R1 and R2 together with the carbon atom to which they are attached form a C3-C8 cycloalkyl ring, and Het is an optionally substituted nitrogen-containing heterocyclic substituent.
[0043] In some embodiments, the compositions of the disclosure may be or may include
Formula (III), pharmaceutically acceptable salts or esters thereof. [0044] In some embodiments, the compounds have a structure of Formula (IV) (IV), or a pharmaceutically acceptable
Figure imgf000014_0001
salt thereof , wherein R1 and R2 are independently H, C1-C3 alkyl, C3-C6 cycloalkyl, or form a C3-C6 cycloalkyl group with the carbon to which they are attached, wherein the Cl-
C3 alkyl or the C3-C6 cycloalkyl is optionally substituted; A is a bond, C1-C3 alkyl, C6 aryl, or C6 heteroaryl, wherein the C1-C3 alkyl, C6 aryl, or C6 heteroaryl is optionally substituted;
B is a bond, C1-C3 alkyl, an amide, an amine, wherein the C1-C3 alkyl, amide or amine is optionally substituted; and C is a C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, C6 aryl, or C6 heteroaryl, wherein the C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, C6 aryl, or C6 heteroaryl is optionally substituted.
[0045] In some embodiments, A is a C6 heteroaryl comprising one or two nitrogens. In some embodiments, A is optionally substituted
Figure imgf000014_0002
[0046] In some embodiments, B is optionally substituted -CH2-NH-CH2- or optionally substituted -CO-NH-CH2-.
[0047] In some embodiments, C is a substituted piperazine group wherein
Figure imgf000014_0003
the substituent may be a C1-C3 alkyl.
[0048] In some embodiments, C is a phenyl group
Figure imgf000014_0004
wherein the substituent may be a C1-C3 alkyl, halogen, or -SO2-CH3.
[0049] In one aspect, the compositions of the disclosure may be or may include N-[l - (Cyanomethyl carbamoyl) cyclohexyl] 4-[4-(l-propy 1) piperalin 1 y 1] benzamide, or a pharmaceutically acceptable salt. Non-limiting examples of the compounds useful in the present disclosure include, N~ [ 1 -(Cyanomethyl-carbamoyl)-cyclohexyl] -4-(piperazin- 1 - yl)-benzamide; N-[l-(Cyanomethyl-carbamoyl)-cyclohexyl]-4-(4-methyl-piperazin-l-yl)- benzarnide; N-[r-(Cyanomethyl-carbamoyl)-cyclohexyl]-4-(4-ethyl-piperazin-l-yl)- benzamide; N-[l-(Cyanomethyl-carbamoyl)-cyclohexyl]-4-(4-isopropyl-piperazin-l-yl)- benzamide; N-[l-(Cyanomethyl-carbamoyl)-cyclohexyl]-4-(4~benzyI-piperazin-l-yl)- benzamide; N-[l-(Cyanomethyl-carbamoyl)-cyclohexyl]-4-[4-(2-methoxy-ethyl)-piperazin- 1- yl]-benzamide; N-[ 1 -(Cyanomethyl-carbamoyl)-cyclohexyl]-4-( 1 -propyl-piperidin-4-yl)- benzamide; N-[l-(Cyanomethyl-carbamoyl)-cyclohexyl]- 4-[l-(2-methoxy-ethyl)-piperidin- 4- yl]-benzamide; N-[l-(Cyanomethyl-carbamoyl)-cyclohexyl]-4-(l-isopropyl-piperidin-4-yl)- benzamide; N-[l-(Cyanomethyl-carbamoyl)-cyclohexyl]-4-(l-cyclopentyl-piperidin-4-yl)- benzamide; N-[l-(Cyanomethyl-carbamoyl)-cyclohexyl]-4-(l-methyl-piperidin-4-yl)- benzamide, or N-[l-(Cyanomethyl-carbamoyl)-cyclohexyl]-4-(piperidin-4-yl)-benzamide; and/or or N-[l-(Cyanomethyl-carbamoyl)-cyclohexyl]-4-[4-(l-propyl)-piperazin-l-yl]- benzamide.
Covalent Warheads
[0050] In some embodiments, the compounds of the present disclosure may comprise at least one warhead moiety. The warhead moiety can be any covalent binding modality that is capable of forming a covalent bond with a biological target. The warhead moiety may comprise one or more chemical groups, one or more of which is capable of forming a covalent bond with a biological target. In one embodiment, the warhead moiety comprises nitrile (-CN).
Combinations
[0051] During the drug development pathway, a vast number of candidate drugs are abandoned because of observed serious adverse reactions. In many cases, these adverse side effects are a result of the large doses required of the intrinsically toxic drug for therapeutic effect when the efficacy of the drug is low. An effective strategy for eliminating the toxicity of these drugs is to lower the required dosage by increasing the drug’s efficacy. In order to prevent or reduce the effects of a pandemic, a need also exi sts to further develop countermeasures against new and virulent strains as a monotherapy or in combination with other anti-virals. Therefore, there is a need for a method for increasing the efficacy of a drag and reducing the required effective dose. In some embodiments, the compositions and methods described herein meet this need.
[0052] The present disclosure provides methods wherein a composition provides an increase in bioavailability of a drug, such as an anti-viral, when combined with a protease inhibitor, for example a cathepsin inhibitor, as measured by AUC (Area Under the Curve) of at least 25% relative to dosing of the drug alone. The present disclosure also provides methods wherein the composition provides an increase in bioavailability of the drug combination as measured by AUC of at least 50% relative to dosing of the drug alone. The present disclosure further provides methods wherein said composition provides an increase in bioavailability of the drug in combination as measured by AUC of at least 100% relative to dosing of the drug alone.
[0053] Certain small molecules have unexpected anti-viral activity, as they were developed and examined for non-viral disease states. These small molecules act as an antiviral whether as a monotherapy or in combination with other anti-viral compounds. The small molecules may demonstrate anti-viral activity across all virus classes, in which anti-viral activity has been proven. The efficacy of these small molecules is especially high against Coronaviruses (i.e. SARS-CoV-2.)
[0054] These small molecules may be effective as a pre-exposure prophylaxis during a viral outbreak, for healthcare workers, first responders, at-risk workers, or travelers. These small molecules may be effective as a post-exposure prophylaxis as well. The post-exposure treatment can be used to prevent a fatal or severe infection and in a “track, trace, and treat” program implementation. Even with a viable vaccine, this form of treatment is necessary. These small molecules may treat mild and severe viral infections. As a treatment for infections, these small molecules may treat the viral load and prevent or reduce hospital stays and reduce the burden on the healthcare system. This treatment may be effective when dealing with multiple non-viral complications including organ failure, ARDS, pneumonia, or other side effects.
[0055] These small molecules may be effective in a wide array of patient populations include those over 65, patients with comorbidities, and immunosuppressed patients.
[0056] The disclosure provides methods for the identification of a compound that produces synergistic activity with a drug of choice. In certain aspects, the disclosure provides methods for the identification of a compound that reduces the effective dosage of a drug of choice. Any technique well-known to the skilled artisan can be used to screen for a compound that would reduce the effective dose of a drug. As an example, a cell is contacted with a test protease inhibitor in combination with a drug of choice, for example an antiviral drug. A control without the test protease inhibitor is provided. The cell can be contacted with a test protease inhibitor before, concurrently with, or subsequent to the administration of the drug. A cell was incubated with multiple concentrations of a drug and test protease inhibitor, for at least 1 minute to at least 10 minutes during the experiment. The effect of the combination on the viral replication was measured at various times during the assay. A time course of viral growth in the culture was determined. If the viral growth is inhibited or reduced in the presence of the test compound at reduced drug concentrations wherein the effect is more than an additive effect, the test protease inhibitor is identified as being effective in producing a synergistic activity.
[0057] The disclosure provides a composition that increases the bioavailability of the drug when in combination with a protease inhibitor, for example a cathepsin inhibitor, as measured by Cmax of at least 50% relative to dosing of the drug alone, which is shown in Figure 5. The disclosure also provides said composition that increases the bioavailability of the drug when in combination as measured by Cmax of at least 100% relative to dosing of the drug alone. The disclosure further provides said composition which provides an increase in bioavailability of the drug when in combination as measured by Cmax of at least 200% relative to dosing of drug alone. Systemic drug concentrations are measured using standard biochemical drug measurement techniques (Simmons et al., Anal Lett. 39: 2009-2021 (1997)).
In vitro assays for testing combinations
[0058] The combinations of the disclosures may be tested for in vitro activity against a disease or microorganism and sensitivity, and for cytotoxicity in laboratory adapted cell lines or cultured cells such as peripheral blood mononuclear cells (PBMC), human fibroblast cells, hepatic, renal, epithelium cells, according to standard assays developed for testing compounds. Combination assays may be performed at varying concentrations of the compounds of the combinations to determine EC 50 by serial dilutions.
[0059] Cells: HEp-2 (CCL-23), PC-3 (CCL-1435), HeLa (CCL-2), U2OS (HTB-96), Vero (CCL-81), HFF-1 (SCRC-1041), and HepG2 (HB-8065) cell lines can be purchased from the American Type Culture Collection. HEp-2 cells can be cultured in Eagle’s Minimum Essential Media (MEM) with GlutaMAX supplemented with 10% fetal bovine serum (FBS) and 100 U ml~l penicillin and streptomycin. PC-3 cells can be cultured in Kaighn’s F12 media supplemented with 10% FBS and 100 U ml-1 penicillin and streptomycin. HeLa, U2OS, and Vero cells can be cultured in MEM: supplemented with 10% FBS, 1% L- glutamine, 10 mM HEPES, 1% non-essential amino acids, and 1% penicillin/ streptomycin. HFF-1 cells can be cultured in MEM supplemented with 10% FBS and 0.5 mM sodium pyruvate. HepG2 cells can be cultured in Dulbecco’s Modified Eagle Medium (DMEM) with GlutaMAX supplemented with 10% FBS, 100 U ml-1 penicillin and streptomycin, and 0.1 mM non-essential amino acids. The MT-4 cell line can be obtained from the NIH AIDS Research and Reference Reagent Program and cultured in R PM 1-1640 medium supplemented with 10% FBS, 100 U ml-1 penicillin and streptomycin, and 2 mM L -glutamine. The Huh-7 cell line can be obtained from C. M. Rice (Rockefeller University) and cultured in DMEM supplemented with 10% FBS, 100 U ml-1 penicillin and streptomycin, and non-essential amino acids.
[0060| Primary human hepatocytes or other primary cell can be purchased from Invitrogen and cultured in William’s Medium E medium containing cell maintenance supplement.
Donor profiles will be limited to 18- to 65-year-old nonsmokers with limited alcohol consumption. Upon delivery, the cells will be allowed to recover for 24 h in complete medium with supplement provided by the vendor at 37°C. Human PBMCs will be isolated from human buffy coats obtained from healthy volunteers (Stanford Medical School Blood Center, Palo Alto, California) and maintained in RPMI-1640 with GlutaMAX supplemented with 10% FBS, 100 U ml-1 penicillin and streptomycin.
[0061] To test viral inhibition in primary' nonhuman primate cells, Rhesus fresh whole blood will be obtained from Valley Biosystems or other suppliers. PBMCs will be isolated from whole blood by Ficoll-Hypaque density gradient centrifugation. Briefly, blood will be overlaid on 15 ml Ficoll-Paque (GE Healthcare Bio-Sciences AB), and centrifuged at 500g for 20 min. The top layer containing platelets and plasma wall be removed, and the middle layer containing PBMCs will be transferred to a fresh tube, diluted with Tris buffered saline up to 50 ml, and centrifuged at 500g for 5 min. The supernatant will be removed and the cell pellet will be resuspended in 5 ml red blood cell lysis buffer (155 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA, pH 7.5). To generate stimulated PBMCs, freshly isolated quiescent PBMCs will be seeded into a T-150 (150 cm2) tissue culture flask containing fresh medium supplemented with 10 U ml-1 of recombinant human interleukin-2 (IL-2) and 1 pg ml-1 phytohaemagglutinin-P at a density of 2 x 106 cells ml-1 and incubated for 72 h at 37°C. Human macrophage cultures will be isolated from PBMCs that will be purified by Ficoll gradient centrifugation from 50 ml of blood from healthy human volunteers. PBMCs will be cultured for 7 to 8 days in in RPMI cell culture media supplemented with 10% FBS, 5 to 50 ng ml-1 granulocyte-macrophage colony-stimulating factor and 50 μM p-mercaptoethanol to induce macrophage differentiation. The cryopreserved human primary' renal proximal tubule epithelial cells will be obtained from LifeLine Cell Technology and isolated from the tissue of human kidney. The cells will be cultured at 90% confluency with RenaLife complete medium in a T-75 flask for 3 to 4 days before seeding into 96-well assay plates. Immortalized human microvascular endothelial cells (HMVEC-TERT) will be obtained from R. Shao at the Pioneer Valley Life Sciences Institute. HMVEC-TERT cells will be cultured in endothelial basal media supplemented with 10% FBS, 5 pg of epithelial growth factor, 0.5 mg hydrocortisone, and gentamycin/amphotericin- B
[0062] In some experiments it may be essential to evaluate the intracellular metabolism (phosphorylation) of nucleobase and nucleoside (Nuc) these studies may be performed as below.
[0063] In some embodiments, the intracellular metabolism of nucleoside may be assessed in different cell types (HMVEC and HeLa cell lines, and primary' human and rhesus PBMCs, monocytes and monocyte-derived macrophages) following 2-h pulse or 72-h continuous incubations with 10-1,000 pM of nucleobase or nucleoside. For comparison, intracellular metabolism during a 72-h incubation with 10-1,000 μM of Nuc will be completed in human monocyte-derived macrophages. For pulse incubations, monocyte-derived macrophages isolated from rhesus monkeys or humans will be incubated for 2 h in compound-containing media followed by removal, Uimethylhexylamine (DMH) in water for analysis by liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS).
[0064] In some embodiments, LC-MS/MS may be performed using low-flow ion-pairing chromatography, similar to methods described previously (Durand-Gasselin L, et al.
Nucleotide analogue prodrug tenofovir disoproxil enhances lymphoid cell loading following oral administration in monkeys. Mol. Pharm. 2009; 6: 1145 -1 151). Analytes may be separated using a 50 x 2 mm x 2.5 pm Luna Cl 8(2) HST column (Phenomenex) connected to a LC-20ADXR (Shimadzu) ternary pump system and HTS PAL autosampler (LEAP Technologies). A multi-stage linear gradient from 10% to 50% acetonitrile in a mobile phase containing 3 mM ammonium formate (pH 5.0) with 10 mM dimethylhexylamine over 8 min at a flow rate of 150 μl min-1 may be used to separate analytes. Detection may be performed on an API 4000 (Applied Biosystems) MS/MS operating in positive ion and multiple reaction monitoring modes. Intracellular metabolites alanine metabolite, Nuc, nucleoside monophosphate, nucleoside diphosphate, and nucleoside triphosphate may be quantified using 7-point standard curves ranging from 0.274 to 200 pmol (approximately 0.5 to 400 pM) prepared in cell extract from untreated cells. Levels of adenosine nucleotides may be also quantified to assure dephosphorylation had not taken place during sample collection and preparation. In order to calculate intracellular concentration of metabolites, the total number of cells per sample may be counted using a Countess automated cell counter (Invitrogen). [0065] In some embodiments, Ebola Antiviral testing can be conducted in a biosafety level 4 containment (BSL-4), for example at the Centers for Disease Control and Prevention. EBOV antiviral assays may be conducted in primary/ HMVEC-TERT and in Huh-7 cells. Huh-7 cells will not be authenticated and will not be tested for mycoplasma. Ten concentrations of compound may be diluted in fourfold serial dilution increments in media, and 100 pl per well of each dilution may be transferred in duplicate (Huh-7) or quadruplicate (HMVEC-TERT) onto 96-well assay plates containing cell monolayers. The plates may be transferred to BSL-4 containment, and the appropriate dilution of virus stock may be added to test plates containing cells and serially diluted compounds. Each plate will include four wells of infected untreated cells and four wells of uninfected cells that serve as 0% and 100% virus inhibition controls, respectively. After the infection, assay plates may be incubated for 3 days (Huh-7) or 5 days (HMVEC-TERT) in a tissue culture incubator. Virus replication may be measured by direct fluorescence using a Biotek HTSynergy plate reader. For virus yield assays, Huh-7 cells may be infected with wild-type EBOV for 1 h at 0.1 plaque-forming units (PFU) per cell. The virus inoculum may be removed and replaced with 100 pl per well of media containing the appropriate dilution of compound. At 3 days post-infection, supernatants may be collected, and the amount of virus may be quantified by endpoint dilution assay. The endpoint dilution assay may be conducted by preparing serial dilutions of the assay media and adding these dilutions to fresh Vero cell monolayers in 96-well plates to determine the tissue culture infectious dose that caused 50% cytopathic effects (TCID50). To measure levels of viral RNA from infected cells, total RNA may be extracted using the MagM AX-96 Total RNA Isolation Kit and quantified using a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay with primers and probes specific for the EBOV nucleoprotein gene.
[0066] In some embodiments, antiviral assays may be conducted in BSL-4. HeLa or HFF- 1 cells may be seeded at 2,000 cells per well in 384-weH plates. Ten serial dilutions of compound in triplicate may be added directly to the cell cultures using the HP D300 digital dispenser (Hewlett Packard) in twofold dilution increments starting at 10 μM at 2 h before infection. The DMSO concentration in each weB may be normalized to 1% using an TIP D300 digital dispenser. The assay plates may be transferred to the BSL-4 suite and infected with EBOV Kikwit at a multiplicity of infection of 0.5 PFU per cell for HeLa cells and with EBOV Makona at a multiplicity of infection of 5 PFU per cell for HFF-1 ceils. The assay plates may be incubated in a tissue culture incubator for 48 h. Infection may be terminated by fixing the samples in 10% formalin solution for an additional 48 h before immune-staining, as described.
[0067] In some embodiments, antiviral assays in EBOV human macrophages may be conducted in BSL-4. Primary human macrophage cells may be seeded in a 96-well plate at 40,000 cells per well. Eight to ten serial dilutions of compound in triplicate may be added directly to the cell cultures using an HP D300 digital dispenser in threefold dilution increments 2 h before infection. The concentration of DMSO may be normalized to 1% in all wells. The plates may be transferred into the BSL-4 suite, and the cells may be infected with 1 PFU per cell of EBOV in 100 pl of media and incubated for 1 h. The inoculum may be removed, and the media may be replaced with fresh media containing diluted compounds. At 48 h post-infection, virus replication may be quantified by immuno-staining.
[0068] For RSV A2 antiviral tests, compounds may be threefold serially diluted in source plates from which 100 ml of diluted compound may be transferred to a 384-web cell culture plate using an Echo acoustic transfer apparatus. HEp-2 cells may be added at a density of 5 x 105 cells per ml, then infected by adding RSV A2 at a titer of 1 x 104.5 tissue culture infectious doses (TCID50) per ml. Immediately following virus addition, 20 pl of the virus and cells mixture may be added to the 384-well cell culture plates using a pFlow liquid dispenser and cultured for 4 days at 37°C. After incubation, the cells may be allowed to equilibrate to 25°C for 30 min. The RSV-induced cytopathic effect may be determined by adding 20 pl of CellTiter-Glo Viability Reagent. After a 10-min incubation at 25°C, cell viability may be determined by measuring luminescence using an Envision plate reader.
[0069] In some embodiments, antiviral assays may be conducted in 384-or 96-well plates in B SL-4 using a high-content imaging system to quantify virus antigen production as a measure of virus infection. A ‘no virus’ control and a ‘ 1% DMSO’ control may be included to determine the 0% and 100% virus infection, respectively. The primary and secondary antibodies and dyes used for nuclear and cytoplasmic staining are listed. The primary antibody specific for a particular viral protein may be diluted 1,000-fold in blocking buffer (1 x PBS with 3% BSA) and added to each well of the assay plate. The assay plates may be incubated for 60 min at room temperature. The primary' antibody may be removed, and the cells may be washed three times with 1 x PBS. The secondary' detection antibody may be an anti-mouse (or rabbit) IgG conjugated with Dylight488 (Thermo Fisher Scientific, catalogue number 405310). The secondary antibody may be diluted 1, 000-fold in blocking buffer and may be added to each well in the assay plate. Assay plates may be incubated for 60 min at room temperature. Nuclei may be stained using Draq5 (Biostatus) or 33342 Hoechst (ThermoFisher Scientific) for Vero and HFF-1 cell lines. Both dyes may be diluted in 1 x PBS. The cytoplasm of HFF-1 (EBOV assay) and Vero E6 (MERS assay) cells may be counter-stained with CellMask Deep Red (Thermo Fisher Scientific). Cell images may be acquired using a Perkin Elmer Opera confocal plate reader (Perkin Elmer) using a x 10 air objective to collect five images per well. Virus-specific antigen may be quantified by measuring fluorescence emission at a 488 nm wavelength and the stained nuclei may be quantified by measuring fluorescence emission at a 640 nm wavelength. Acquired images may be analyzed using Harmony and Acapella PE software. The Draq5 signal may be used to generate a nuclei mask to define each nucleus in the image for quantification of cell number. The CellMask Deep Red dye may be used to demarcate the Vero and HFF-1 cell borders for cell-number quantitation. The viral-antigen signal may be compartmentalized within the cell mask. Cells that exhibited antigen signal higher than the selected threshold may be counted as positive for viral infection. The ratio of virus-positive cells to total number of analyzed cells may be used to determine the percentage of infection for each well on the assay plates. The effect of compounds on the viral infection may be assessed as percentage of inhibition of infection in comparison to control wells. The resultant cell number and percentage of infection may be normalized for each assay plate. Analysis of dose-response curve may be performed using GeneData Screener or similar software applying Levenberg -Marquardt algorithm for curve-fitting strategy. The curve-fitting process, including individual data point exclusion, will be pre-specified by default software settings. R2 value quantified goodness of fit and fitting strategy may be considered acceptable at R2 > 0.8.
Additional Therapeutic Agents
[0070] In some embodiments, the drug combinations of the disclosure can be combined with other therapeutic agents. Other therapeutic agents can include additional cathepsin inhibitors or protease inhibitors. Drug combinations of the disclosure can include, but are not limited to, for example, Drug combinations according to the disclosure can be, but are not limited to, for example: Relacatib (GSK-462795, SB-462795), in combination with T-705 (Avigan) for use in the treatment of arenavirus infections, Relacatib (GSK -462795, SB- 462795), in combination with T-705 (Avigan) for use in the treatment of SARS-CoV-2 infections, MIV-711, in combination with T-705 (Favipiravir, Avigan) for use in treatment of SARS-CoV-2 and other coronaviruses such as MERS and SARS-CoV; AM-3701, MIV-701, MIV-710, MIV-711 , NC-2300, ORG-219517 or Relacatib (GSK-462795, SB-462795), in combination with JNJ-64041575, JNJ-1575, ALS-008176, AL-8176 (Lumicitabine) for use in the treatment of RSV infections; AM-3701, MIV-701, MIV-710, MIV-711, NC-2300, ORG- 219517 or Relacatib (GSK-462795, SB-462795), in combination with JNJ-64041575, JNJ- 1575, ALS-008176, AL-8176 (Lumicitabine) for use in the treatment of SARS-CoV-2 infections; AM-3701, MIV-701 , MIV-710, MIV-711, NC-2300, ORG-219517 or Relacatib (GSK-462795, SB-462795) or Odanacatib (MK-0822) or Balicatib (AAE581), in combination with JNJ-64041575, JNJ-1575, ALS-008176, AL-8176 (Lumicitabine) for use in the treatment of RSV infections; AM-3701, MIV-701, MIV-710, MIV-711, NC-2300, ORG- 219517 or Relacatib (GSK-462795, SB-462795) in combination with T-705 (Avigan, Favipiravir) or BCX 4430 (Galidesivir) for use in the treatment of SARS-CoV-2 infections; AM-3701, MIV-701, MIV-710, MIV-711, NC-2300, ORG-219517 or Relacatib (GSK- 462795, SB-462795), in combination with GS-5734 (Redmdesivir) in the treatment of SARS- CoV-2 infections; Odanacatib (MK-0822) or Balicatib (AAE581), in combination with GS- 5734 (Remdesivir) in the treatment of SARS-CoV-2 infections; Relacatib (GSK-462795, SB- 462795), in combination with T-705 (Avigan, Favipiravir) along with lopinavir/ritonavir (Kaletra) for use in the treatment of filovirus infections; Relacati b (GSK-462795, SB- 462795), in combination with T-705 (Avigan), along with lopinavir/ritonavir (Kaletra) for use in the treatment of coronavirus infections such as SARS-CoV-2 infections; Relacatib (GSK-462795, SB-462795), in combination with T-705 (Avigan), along with lopinavir/ritonavir (Kaletra) for use in the treatment of coronavirus infections such as SARS- CoV-2 and other coronaviruses such as MERS and SARS-CoV; AM-3701, MIV-701, MIV- 710, MIV-711, NC-2300, ORG-219517 or Relacatib (GSK-462795, SB-462795), in combination with JNJ-64041575, JNJ-1575, ALS-008176, AL-8176 (Lumicitabine) for use in the treatment of RSV infections; AM-3701, MIV-701, MIV-710, MIV-711, NC-2300, ORG- 219517 or Relacatib (GSK-462795, SB-462795), in combination with BCX4430 (Galidesivir), T-705 (Avigan, Favipiravir) along with a protease inhibitor such as darunavir (Prezista) in the treatment of coronavirus infections such as SARS-CoV-2 infections; AM- 3701, MIV-701, MIV-710, MIV-711, NC-2300, ORG-219517 or Relacatib (GSK-462795, SB-462795), in combination with GS-5734 (Remdesivir), along with a protease inhibitor such as darunavir (Prezista) in the treatment of coronavirus infections such as SARS-CoV-2 infections; Odanacatib (MK-0822) or Balicatib (AAE581), in combination with GS-5734 (Remdesivir), along with a protease inhibitor such as darunavir (Prezista) in the treatment of coronavirus infections such as SARS-CoV-2 infections.
Disease-modifying anti-rheumatic drugs (DMARDs)
[0071] In some embodiments, compositions may include gold preparations. As used herein, the term gold preparations may include auranofin. In some embodiments, compositions may include penicillamine, which may include D-penicillamine. In some embodiments, compositions may include aminosalicylic acid preparations, which may include sulfasalazine, mesalazine, olsalazine, balsalazide. In some embodiments, compositions may include antimalarials, which may include chloroquine. In some embodiments, compositions may include pyrimidine synthesis inhibitors, which may include leflunomide. In some embodiments, compositions may include prograf.
Anti -cytokine drug
[0072] In some embodiments, compositions may include protein drugs. As used herein, protein drugs may include TNF inhibitors such as etanercept, infliximab, adalimumab, certolizumab pegol, golimumab, PASSTNF-alpha, soluble TNF-alpha receptor, TNF-alpha binding protein, anti -TNF-alpha antibody. As used herein, protein drugs may include interleukin- 1 inhibitors, such as anakinra (interleukin- 1 receptor antagonist), soluble interleukin-1 receptor and the like; interleukin-6 inhibitors such as tocilizumab (antiinterleukin-6 receptor antibody), anti-interleukin-6 antibody. As used herein, protein drugs may include interleukin- 10 drugs such as interleukin- 10. As used herein, protein drugs may include interleukin-12/23 inhibitors such as ustekinumab, briakinumab (anti-interleukin- 12/23 antibody). As used herein, protein drugs may include B cell activation inhibitors such as rituximab, belimumab and the like; co-stimulatory molecules-related protein preparations such as abatacept and the like; complement mediated inhibitors both synthetic and biologic. [0073] In some embodiments, compositions may include non-protein drugs such as MAPK inhibitors such as BMS-582949. As used herein, non-protein drugs may include gene modulators; inhibitors of molecule involved in signal transduction, such as NF-kappa, NF- kappaB, IKK- 1, IK K -2, AP-1. As used herein, non-protein drugs may include cytokine and chemokine production inhibitors, receptor binding inhibitors such as iguratimod, tetomilast. As used herein, non-protein drugs may include TNF-alpha converting enzyme inhibitors; interleukin-1 beta converting enzyme inhibitors such as VX-765. As used herein, non-protein drugs may include interleukin-6 antagonists such as HMPL-004. As used herein, non-protein drugs may include interleukin-8 inhibitors such as IL-8 antagonist, CXCR1 & CXCR2 antagonist, reparixin. As used herein, non-protein drugs may include Chemokine antagonists such as CCR9 antagonist (CCX-282, CCX-025), MCP-1 antagonist. As used herein, nonprotein drugs may include interleukin-2 receptor antagonists such as denileukin, diftitox. As used herein, non-protein drugs may include therapeutic vaccines such as TNF-alpha vaccine. As used herein, non-protein drugs may include gene therapy drugs such as drugs promoting the expression of a gene having an anti-inflammatory action such as interleukin-4, interleukin-10, soluble interleukin-1 receptor, soluble TNF-alpha receptor. As used herein, non-protein drugs may include antisense compounds such as ISIS-104838. Integrin inhibitor
[0074] In some embodiments, compositions may include integrin inhibitors such as natalizumab, vedolizumab, AJM300, TRK-170, E-600.
Immunomodulator (immunosuppressant)
[0075] In some embodiments, compositions may include immunomodulators such as cyclophosphamide, MX-68, atipriniod dihydrochloride, BMS- 188667, CKD-461, rimexolone, cyclosporine, tacrolimus, gusperimus, azathiopurine, antilymphocyte serum, freeze-dried sulfonated normal immunoglobulin, erythropoietin, colony stimulating factor, interleukin, interferon, intravenous immunoglobulin, anti-thymocyte globulin, RSLV-132. Proteasome inhibitor
[0076] In some embodiments, compositions may include proteasome inhibitors such as bortezomib.
JAK inhibitor
[0077] In some embodiments, compositions may include JAK inhibitors such as tofacitinib.
Steroids
[0078] In some embodiments, compositions may include steroids. As used herein, steroid may include dexamethasone, hexestrol, methimazole, betamethasone, triamcinolone, triamcinolone acetonide, fluocinonide, fluocinolone acetonide, predonisolone, methylpredonisolone, cortisone acetate, hydrocortisone, fluoromethoIone, beclomethasone dipropionate, estriol.
Angiotensin converting enzyme inhibitors
[0079] In some embodiments, compositions may include angiotensin converting enzyme inhibitors. As used herein, angiotensin converting enzyme inhibitors may include enalapril, captopril, ramipril, lisinopril, cilazapril, perindopril. Angiotensin II receptor antagonists
[0080] In some embodiments, compositions may include angiotensin II receptor antagonists. As used herein, angiotensin II receptor antagonists may include candesartan, candesartan cilexetil (TCV-116), valsartan, irbesartan, olmesartan, eprosartan.
Diuretic substances
[0081] In some embodiments, compositions may include a diuretic. As used herein, a diuretic may include hydrochlorothiazide, spironolactone, furosemide, indapamide, bendrofluazide, cyclopenthiazide .
Cardiotonic substances
[0082] In some embodiments, compositions may include a cardiotonic substance. As used herein, a cardiotonic substance may include digoxin, dobutamine.
Beta receptor antagonists
[0083] In some embodiments, compositions may include a beta receptor antagonist. As used herein, a beta receptor antagonist may include carvedilol, metoprolol, atenolol.
Ca sensitizers
[0084] In some embodiments, compositions may include a Ca sensitizer. As used herein, a CA sensitizer may include MCC-135.
Ca channel antagonists
[0085] In some embodiments, compositions may include Ca channel antagonists. As used herein, a Ca channel antagonist may include nifedipine, diltiazem, verapamil.
Anti-platelet drug, anti coagulator
[0086] In some embodiments, compositions may include an anti-platelet substance or anti coagulator. As used herein, an anti-platelet, substance or anti coagulator may include heparin, aspirin, warfarin.
HMG-CoA reductase inhibitors
[0087] In some embodiments, compositions may include an anti-platelet substance or anticoagulator. As used herein, an anti-platelet substance or anti coagulator may include atorvastati n, simvastatin .
Other substances
[0088] In some embodiments, compositions may include other substances which improve functionality of the compound. As used herein, other substances may include T cell inhibitors, inosine monophosphate dehydrogenase (IMPDH) inhibitor mycophenolate mofetil. As used herein, other substances may include adhesion molecule inhibitor such as ISIS-2302, selectin inhibitor, ELAM-1, VCAM-1, ICAM-1. As used herein, other substances may include thalidomide, a combination of cathepsin inhibitor or a single cathepsin inhibitor, matrix metalloprotease (MMPs) inhibitor such as V-85546. As used herein, other substances may include glucose-6-phosphate dehydrogenase inhibitor, Dihydroorotate dehydrogenase (DHODH) inhibitor, phosphodiesterase IV (PDE IV) inhibitor such as roflumilast, CG-1088. As used herein, other substances may include a phospholipase A2 inhibitor, iNOS inhibitor such as VAS-203. As used herein, other substances may include microtubule stimulating compound such as paclitaxel. As used herein, other substances may include microtubule inhibitor such as reumacon. As used herein, other substances may include MHC class II antagonist, prostacyclin agonist such as iloprost. As used herein, other substances may include CD4 antagonist such as zanolimumab. As used herein, other substances may include CD23 antagonist, LTB4 receptor antagonist such as DW-1305. As used herein, other substances may include 5 -lipoxygenase inhibitor such as zileuton. As used herein, other substances may include cholinesterase inhibitor such as galanthamine. As used herein, other substances may include a tyrosine kinase inhibitor such as Tyk2 inhibitor (WO 2010/142752). As used herein, other substances may include cathepsin B inhibitor. As used herein, other substances may include adenosine deaminase inhibitor such as pentostatin. As used herein, other substances may include osteogenesis stimulator, dipeptidylpeptidase inhibitor, collagen agonist, capsaicin cream, hyaluronic acid derivative synvisc (hylan G-F 20), orthovis. As used herein, other substances may include glucosamine sulfate, amiprilose. As used herein, other substances may include CD-20 inhibitors such as rituximab, ibritumomab, tositumomab, ofatumumab. As used herein, other substances may include BAFF inhibitors such as belimumab, tabalumab, atacicept, A-623. As used herein, other substances may include CD52 inhibitors such as alemtuzuma As used herein, other substances may include.
[0089] In some embodiments, compositions may include other substances which improve functionality of the compound. As used herein, other substances may include antiviral substances such as idoxuridine, acyclovir, vidarabine, gancyclovir. As used herein, other substances may include anti-HIV agents such as zidovudine, didanosine, zalcitabine, indinavir sulfate ethanolate, ritonavir.
[0090] The simultaneous combination of sub-optimal doses from the drag along with one or more protease inhibitor, for example one or more cathepsin inhibitor, achieves an increase in function or efficacy of the drug, wherein the increase is any increase of about 2% and above, or between about 2%-5%, about 5%-l 0%, about 10%-20%, about 20%-30%, about 30%-40%, about 40%-50%, about 50%-60%, about 60%-70%, about 70%-80%, about 80%- 90%, about 90%-I00%, about 100%-150%, about 150%-200%, about 200%-300%, about 300%-400%, about 400%-500%, about 500%-1000%, 1000%-5000%, about 5000%-7000%, about 7000%-! 0,000% or more, or about 0.001-fold to about 0.01 fold, about 0.05-fold to about 0.1-fold, about 0.1-fold to about 0.5-fold, about 0.5-fold to about 1-fold, about 1-fold to about 2-fold, about 3-fold to about. 5-fold, about 5-fold to about 10-fold, about 10-fold to about 20-fold, about 20-fold to about 40-fold, about 50-fold to about 75-fold, about 80 fold to about. 100-fold, or more, such that the effective dose is decreased for each drag mentioned in this disclosure. Effective dose is that that achieves 50% of the effect which is also termed Inhibitory' Concentration 50% (IC50) or Effective Concentration (EC50) in assays in vitro, wherein the EC50 is decreased by about 5% or more, or by about 5%-l 0%, about 10%-20%, about 20%-30%, about 30%-40%, about 40%-50%, about 50%-60%, about 60%-70%, about 70%-80%, about 80%-90%, about 90%-100%, about 100%- 150%, about 150%-200%, about 200%-300%, about 300%-400%, about 400%-500%, about 500%-1000%, 1000%-5000%, about 5000%-7000%, about 7000%-10,000% or more. In this disclosure, "sub-optimal doses" refers to doses which do not reach EC50 or IC50.
The combination or mixture of the present disclosure can be used together with other drags for the prophylaxis or treatment of various diseases. For example, when the combination or mixture of the present disclosure is used as an antiviral therapy, it can be used together with the following drugs:
Non-steroidal anti-inflammatory drugs (NSAIDs)
[0091] In some embodiments, compositions may include classical non-steroidal antiinflammatory drugs (NSAID). As used herein, the term NSAID may include, but are not limited to, alcofenac, aceclofenac, sulindac, tolmetin, etodolac, fenoprofen, thiaprofenic acid, meclofenamic acid, meloxicam, tenoxicam, lomoxicam, nabumeton, acetaminophen, phenacetin, ethenzamide, sulpyrine, antipyrine, migrenin, aspirin, mefenamic acid, flufenamic acid, diclofenac sodium, loxoprofen sodium, phenylbutazone, indomethacin, ibuprofen, ketoprofen, naproxen, oxaprozin, flurbiprofen, fenbufen, pranoprofen, floctafenine, piroxicam, epirizole, tiaramide hydrochloride, zaltoprofen, gabexate mesylate, ulinastatin, colchicine, probenecid, sulfinpyrazone, benzbromarone, allopurinol, sodium aurothiomalate, hyaluronate sodium, sodium salicylate, morphine hydrochloride, salicylic acid, atropine, scopolamine, morphine, pethidine, levorphanol, oxymorphone or a salt thereof and the like. [0092] In some embodiments, compositions may include cyclooxygenase inhibitors. As used herein the term cyclooxygenase inhibitors may include, but are not limited to, (COX-1 selective inhibitors, COX-2 selective inhibitors, salicylic acid derivatives (e.g., celecoxib, aspirin), etoricoxib, valdecoxib, diclofenac, indomethacin, loxoprofen and the like.
[0093] In some embodiments, compositions may include Nitric oxide-releasing NSAIDs. Mechanism of Action
[0094] Although applicant is not bound by a mechanism, it is believed that, the inhibitors of the disclosure are useful for treating enveloped viral infection by interfering with the critical role by cathepsins of proteolysis of the GP1 glycoprotein subunit to trigger membrane fusion and cell entry. As described in U.S. Patent 8,232,240, which is hereby incorporated by reference in its entirety, a new mechanism for activating the enveloped virus fusion machinery has been discovered. It has been demonstrated that papain like cysteine proteases are involved in, and are important components of, the viral entry' process. Inhibition of these proteases is sufficient to inhibit enveloped viral entry into a host cell. Previously signals involved in these processes in other enveloped viruses have been shown to be due to binding of viruses to a specific receptor and/or exposure of viruses to acidic pH. The discoveries of the disclosure suggest that cysteine proteases provide an additional important mechanism by which enveloped viruses, such as Ebola or SARS-CoV-2, infect host cells. In particular, cathepsins are sufficient for triggering of enveloped viral membrane fusion within the acidic endosomal milieu of target cells.
[0095] Although applicant is not bound by a mechanism, it is believed that the inhibitors of the disclosure are useful for treating enveloped viral infection by interfering with the critical role by cathepsins of proteolysis of the GP1 glycoprotein subunit to trigger membrane fusion and cell entry. The specific data presented herein suggest that GP1 proteolysis is a multistep process.
[0096] Changes in the integrated systemic concentrations over time are indicated by area under the curve (AUC) or Cmax, both parameters well known in the art. AUC is determined by plotting the serum or plasma concentration of a drug along the ordinate (Y-axis) against time along the abscissa (X-axis). Generally, the values for the AUC represent drug concentrations over time in units of mass-time/volume. When efficacy of a drug is being measured, the amount and form of the drug administered should be the same in both a) the administration of the drug in combination with a protease inhibitor, for example a cathepsin inhibitor, or b) the administration of the drag alone. [0097] Clearance of a drug normally occurs from the liver and kidneys and it is assumed that only free and non-protein bound drags are available for clearance. For hepatic clearance, passive diffusion through the lipid core of the hepatocyte membranes, available to lipophilic drugs, is augmented by sinusoidal carrier systems particularly for ionized molecules (anionic and cationic) of molecular weights of approximately 3-400, Likewise, other transporters on the canalicular face transport drugs or their metabolites into bile. This system has two separate processes, hepatic uptake and biliary excretion. With small sized lipophilic drugs that readily traverse membranes hepatic uptake is not a major factor, but with higher molecular weight compounds (above 500) and those containing considerable H-bonding hepatic uptake can become the key clearance process, even if metabolism occurs subsequent to this.
[0098] Low bioavailability may occur when a drug rapidly dissolves and readily crosses the intestinal membranes. This absorption tends to be complete, but absorption of orally administered drags is not always complete. Before reaching the vena cava, a drug must move down the gastrointestinal tract and pass through the gut wal 1 and liver, common sites of drag metabolism. Thus, a drag may be metabolized during first-pass metabolism before it can be measured in the systemic circulation. Many drags have low7 oral bioavailability because of expensive first-pass metabolism.
[0099] Low bioavailability is most common with oral dosage forms of poorly water- soluble, slowly absorbed drugs. More factors can affect bioavailability when absorption is slow or incomplete than when it is rapid and complete. That is, slow or incomplete absorption leads to variable therapeutic responses. Slow absorption in the gastrointestinal tract also leads to increased acute and delayed-phase chemotherapy induced nausea and vomiting.
[0100] Insufficient time in the gastrointestinal tract is a common cause of low7 bioavailability. Ingested drug is exposed to the entire gastrointestinal tract, for no more than one to two days and to the small intestine for only 2 to 4 hours. If the drug does not dissolve readily or cannot penetrate the epithelial membrane (e.g., if it is highly ionized and polar), time at the absorption site may be insufficient. In such cases, bioavailability tends to be highly variable as well as low. Age, sex, activity, genetic phenotype, stress, disease, or previous gastrointestinal surgery' can affect drug bioavailability.
[0101] Reactions that compete with absorption can reduce bioavailability. They include complex formation, hydrolysis by gastric acid or digestive enzymes, conjugation in the gut wall, absorption of other drags and metabolism by luminal micro flora. [0102] Assessment of bioavailability from plasma concentration-time data usually involves determining maximum peak concentration, the time at which maximum peak plasma drug concentration occurs, and the area under the plasma concentration time curve (AUC). The plasma drug concentration increases with the extent of absorption. The peak is reached when the drug elimination rate equals absorption rate. AUC is the most reliable measure of bioavailability. It is directly proportional to the total amount of unchanged drug that reaches the systemic circulation.
[0103] Drug products may be considered bioequivalent in extent and rate of absorption if their plasma level curves are essentially super imposable. Drug products that have similar AUCs but differently shaped plasma level curves are equivalent in extent but differ in their absorption rate-time profiles.
[0104] Absorption occurs by one of three methods, either passive diffusion, active transport or facilitated active transport. Passive diffusion is simply the passage of molecules across the mucosal barrier until the concentration of molecules reaches osmotic balance on both sides of the membrane. In active transport the molecule is actively pumped across the mucosa. In facilitated transport, a carrier generally a protein, is required to convey the molecule across the membrane for absorption.
III. METHODS
[0105] The present disclosure provides methods of use related to the compositions described herein. In some embodiments, the methods described herein may include a method of inhibiting the growth of a virus. Such methods may include contacting the virus with the compositions of the disclosure. As a non-limiting example the compositions of the disclosure may include mammalian protease inhibitors. The methods may involve contacting the virus with a mammalian protease inhibitor. In some embodiments, the growth of the virus is inhibited in vivo in a subject. In some aspects, the growth of the virus is inhibited in the presence of a cell or a population of cells infected by the virus. In some embodiments, the growth of the virus is inhibited by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and/or 90%. In some embodiments the growth of the virus is inhibited by 5-15%, 10-20%, 15-25%, 20-30%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55-65%, 60-70%, 65-75%, 70-80%, 75-85%, and/or 90-100%.
[0106] In some embodiments, the methods described herein may include a method of reducing the percentage of virus infected cells in a population. Such methods may include contacting the virus infected cells with the compositions of the disclosure. As a non-limiting example the compositions of the disclosure may include mammalian protease inhibitors. The methods may involve contacting the virus infected cells with a mammalian protease inhibitor. In some embodiments, the percentage of the virus infected cells is reduced in vivo in a subject. In some embodiments, the percentage of virus infected cells in a population is reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and/or 90%. In some embodiments the percentage of virus infected cells in a population is reduced by 5-15%, 10- 20%, 15-25%, 20-30%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55-65%, 60- 70%, 65-75%, 70-80%, 75-85%, and/or 90-100%.
[0107] The amount of the compositions of the disclosure to be utilized may be identified using methods described here. In some embodiments, the concentration of the protease inhibitors is determined using an MTS assays and cytotoxicity assays described herein or any other methods known in the art. Assay output may be analyzed to determine EC50 (50% inhibition of virus replication), EC90 (90% inhibition of virus replication), EC95 (95% inhibition of virus replication), CC50 (50% cytotoxicity), CC95 (95% cytotoxicity). In some embodiments, the selectivity index (SI) for each protease inhibitor is determined by dividing the CC50 by the EC50.
[0108] In some embodiments, the methods of the disclosure selectivity index (SI) for each protease inhibitor for each virus infection may be determined. In some embodiments, the SI is determined for coronavirus. In one aspect, the SI may be at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900 or more.
[0109] In one embodiment of the disclosure, the methods described herein correspond to a subject or patient or infected by a virus, such as RNA or DNA viruses which are pathogenic to humans and animals.
[0110] In some embodiments, the present disclosure provides compounds and compositions may be used to reduce the coronavirus infection levels in a subject. Such methods may include providing the compositions of the disclosure to a subject in need. The levels of coronavirus infection may be measured in the subject by any methods known in the art, such as, but not limited to, measuring coronavirus antigen levels, measuring anticoronavirus antibodies and/or measuring levels of coronavirus nucleic acids in the subject. Treatment with the compositions of the disclosure is expected to reduce the coronavirus infection levels.
[0111] In another embodiment of the disclosure , the subject has an infection by a Type I enveloped virus. In some embodiments, the compositions of the disclosure may be provided or administered to a subject with an infection associated with a Type I enveloped virus e.g. a filovirus. In still another embodiment, the compositions may be used in the treatment of or may be provided to a subject infected with a coronavirus, a filovirus such as an Ebola virus or a Marburg virus. In yet another embodiment, the compositions may be used in the treatment of or may be provided to a subject infected with a Type I enveloped virus such as an orthomyxovirus. In still another embodiment, the compositions may be used in the treatment of or may be provided to a subject infected with a Type I enveloped virus such as a paramyxovirus. In still another embodiment, the Type I enveloped virus is an Arenavirus. [0112] In some embodiments, the compositions of the disclosure may be used to inhibit a virus and/or reduce the percentage of virus infected cells in a population of cells. In some aspects, the virus may be a coronavirus, an enterovirus, an adenovirus, a dengue virus, human immunodeficiency virus, a parainfluenza virus, a respiratory syncytial virus (RSV), a coxsackie virus, a rhinovirus, Measles, Influenza virus.
[0113] In some embodiments, the virus may be a virus in the family, Coronaviridae, or a virus in the sub-family Orthocoronavirinae, or a virus in the order Nidovirales, In some embodiments, the methods of the disclosure may be used to inhibit the growth of any coronavirus. In one embodiment, the virus may be a coronavirus. In some embodiments, the coronavirus may be a SARS-CoV-2 virus, SARS-CoV-1 virus, MERS-CoV virus, 229E virus, NL63 virus, OC43 virus, HKU1 virus, or variants thereof As a non-limiting example, the virus may be a SARS-CoV-2 virus.
[0114] In one embodiment, the virus may be an enterovirus.
[0115] In an embodiment of the disclosure, the subject or patient has, or is at risk of, an infection by a virus. These viruses, such as RNA or DNA viruses, are pathogenic for humans and animals. In an embodiment of the disclosure, the subject has or is at risk of infection by a Type I enveloped virus. In an embodiment, the Type I enveloped virus is a filovirus. In an embodiment, the filovirus is an Ebola virus or a Marburg virus. In an embodiment, the Type I enveloped virus is an orthomyxovirus. In an embodiment, the Type I enveloped virus is a paramyxovirus. In an embodiment, the Type I enveloped virus is an Arenavirus.
[0116] In an embodiment of the disclosure, the subject has or is at risk of infection by a virus such as, but not limited to, filoviruses, fl avi viruses such as hepatitis-C virus, bunyaviruses, poxvirus, arboroviruses such as Togaviruses, bunyaviruses, orthomyxoviridae, paramyxoviridae, poxviruses, herpesviruses, henipaviruses, hepadnaviruses, rhabdoviruses, bornaviruses, arteri viruses, papillomaviridae, human retroviruses, polyomaviridae, picomaviridae, coronaviruses, and adenoviridae.
[0117] In some embodiments, the disclosure provides for methods of treating infection by a virus of the family Filoviridae, a family of viruses with a single-stranded, unsegmented (~) sense RNA genome. Filoviruses can cause severe hemorrhagic fever in humans and nonhuman primates. So far, only two genuses of this virus family have been identified: Marburg and Ebola. Four species of Ebola virus have been identified: Cote d'Ivoire (CI), Sudan (S), Zaire (Z), and Reston (R). The Reston subty pe is the only known filovirus that is not known to cause fatal disease in humans; however, it can be fatal in monkeys.
[0118] The family Orthomyxoviridae may include, without limitation, influenza A virus, influenza B virus, influenza C virus, Thogotovirus, Dhori virus, and infectious salmon anemia virus. Influenza type A viruses may be divided into subtypes based on two proteins on the surface of the virus. These proteins are called hemagglutinin (HA) and neuraminidase (NA). There are 15 different HA subtypes and 9 different NA subtypes. Subtypes of influenza A virus are named according to their HA and NA surface proteins, and many different combinations of HA and NA proteins are possible. For example, an “H7N2 virus” designates an influenza A subtype that has an HA 7 protein and an NA 2 protein. Similarly an "115X 1" virus has an HA 5 protein and an NA 1 protein. Only some influenza A subtypes (i.e., H1N1, H2N2, and H3N2) are currently in general circulation among people. Other subtypes such as H5 N1 are found commonly in other animal species and in a small number of humans, where it is highly pathogenic. For example, H7N7 and H3N8 viruses cause illness in horses.
Humans can be infected with influenza types A, B, and C. However, the only subtypes of influenza A virus that normally infect people are influenza A subtypes H1N1, H2N2, and H3N2 and recently, H5N1.
[0119] The family Paramyxoviridae may include, without limitation, human parainfluenza virus, human respiratory' syncytial virus (RSV), Sendai virus, Newcastle disease virus, mumps virus, rubeola (measles) virus, Hendra virus, Nipah virus, avian pneumovirus, and canine distemper virus.
[0120] The family Rhabdoviridae may include, without limitation, rabies virus, vesicular stomatitis virus (VSV), Mokola virus, Duvenhage virus, European bat virus, salmon infectious hematopoietic necrosis virus, viral hemorrhagic septicaemia virus, spring viremia of carp virus, and snakehead rhabdovirus. The family Bornaviridae may include, without limitation, Borna disease virus. [0121] The family Bunyaviridae may include, without limitation, Bunyamwera virus, Hantaan virus, Crimean Congo virus, California encephalitis virus. Rift Valley fever virus, and sandfly fever virus. The family Arenaviridae includes, without limitation. Old World Arenaviruses, such as Lassa virus (Lassa fever), Ippy virus, Lymphocytic choriomeningitis virus (LCMV), Mobala virus, and Mopeia virus and New World Arenaviruses, such as Junin virus (Argentine hemorrhagic fever), Sabia (Brazilian hemorrhagic fever), Amapari virus, Flexal virus, Guanarito virus (Venezuela hemorrhagic fever), Machupo virus (Bolivian hemorrhagic fever), Latino virus, Boliveros virus, Parana virus, Pichinde virus, Pirital virus, Tacaribe virus, Tamiami virus, and Whitewater Arroyo virus.
[0122] The arboviruses are a large group (more than 400) of enveloped RNA viruses that are transmitted primarily (but not exclusively) by arthropod vectors (mosquitoes, sand-flies, fleas, ticks, lice, etc). More recently, the designated Arborviruses have been split into four virus families, including the togaviruses, flaviviruses, arenaviruses and bunyaviruses.
[0123] As used herein, the term “togaviru s” refers to members of the family Togaviridae, which includes the genuses Alphavirus (e.g. Venezuela equine encephalitis virus, Sindbis virus, which causes a self-limiting febrile viral disease characterized by sudden onset of fever, rash, arthralgia or arthritis, lassitude, headache and myalgia) and Rubivirus (e.g. Rubella virus, which causes Rubella in vertebrates).
[0124] Flaviviridae is a member of the family of (+)-sense RNA enveloped viruses. Flaviviridae includes flavivirus, Pestivirus, and Hepacivirus. Flavivirus genus including yellow fever virus, dengue fever virus, and Japanese encaphilitis (JE) virus. The Pestivirus genus includes the three serotypes of bovine viral diarrhea, but no known human pathogens. Genus Hepacivirus consists of hepatitis C virus and hepatitis C-like viruses. The Japanese encephalitis antigenic complex includes Alfuy, Japanese encephalitis, Kokobera, Koutango, Kunjin, Murray Valley encephalitis, St. Louis encephalitis, Stratford, Usutu, and West Nile viruses. West Nile virus is the most widespread of the flaviviruses, with geographic distribution including Africa and Eurasia. The genus Pestivirus has been divided into bovine viral diarrhea virus (BVDV), classical swine fever virus (CSFV), and border disease virus (BDV). The Hepacivirus genus includes the hepatitis C virus (HCV).
[0125] .Arenaviridae is a member of the fami ly of (~) sense RNA viruses. As used herein, the term “Arenavirus” refers to members of the genus Arenavirius, a family of viruses whose members are generally associated with rodent-transmitted disease in humans, including Lymphocytic choriomeningitis virus (LCMV), Lassa virus, Junin virus, which causes Argentine hemorrhagic fever, Machupo virus, which causes Bolivian hemorrhagic fever, Guanarito virus, which causes Venezuelan hemorrhagic fever, and Sabia, which causes Brazilian hemorrhagic fever. LCMV causes which causes lymphocytic choriomeningitis, a mild disease that is occasionally severe with hemorrhaging.
[0126] The Phlebovirus Rift valley fever virus produces an acute, flu-like illness and is transmitted by mosquitoes from animal reservoirs (e.g. sheep) to man. Sand fly fever is transmitted to man by Phlebotomous flies (sand-flies) and causes an acute, febrile illness characterized by fever, malaise, eye pain, and headache.
[0127] Hendra and Nipah virus in the Henipavirus genus of the subfamily Paramyxovirinae are distinguished by fatal disease in both animal and human hosts.
[0128] Riboviria are all RNA viruses that replicate using RNA-dependent RNA polymerase. Examples of viruses that cause infections in humans include SARS-CoV-1, MERS-CoV, and SARS-CoV-2, 229E, NL63, OC43, KHU1.
[0129] Herpesviridae is a large family of DNA viruses that cause disease in animals, including humans. Herpesviruses include herpes simplex virus types I and 2, varicella-zoster virus, cytomegalovirus, Esptein-Barr virus, human herpesvirus 6 (variants A and B), human herpesvirus 7, and Kaposi’s sarcoma virus or human herpesvirus 8.
[0130] Hepadnaviridae is a family of DNA viruses that cause hepatitis in humans and animals. Hepadnaviridae include hepatitis B virus isolated from mammals or birds.
[0131] Papillomaviridae is a family of non-enveloped DNA viruses with over a hundred species of papillomaviruses including Alpha papillomavirus, Beta papillomavirus. Gamma papillomavirus, Mu papillomavirus and Nupapillomavirus. HPVs are most associated with cutaneous and genital legions, cervical carcinoma and recurrent respiratory papillomatosis, among other diseases.
[0132] Human retroviruses, including human T-cell leukemia virus (HTLV-1, 2, 3 and 4) and adult T-cell leukemia virus (ATLV) Human T-lymphotropic virus cause serious diseases in humans, including adult T-cell leukemia/lymphoma (ATE) and neurological disease (HTLV-associated myelopathy/tropical spastic paraparesis), uveitis, and rheumatic syndromes.
[0133] Poiyomaviridae family of viruses are non-enveloped DNA viruses that cause disease in immunocompromised hosts. Human polyomaviruses BKV and JCV cause hemorrhagic cystitis and leukoencephalopathy. Merkel cell polyomavirus (MCPyV or MCV) shares some traits to piyomaviruses and is thought to be linked to Merkel Cell Carcinoma (MCC), a neuroendocrine cancer.
[0134] Poxviridae is a large family of DNA viruses including mollusci poxvirus, parapoxvirus (Orf virus, pseudocowpox virus, bovine popular stomatitis virus). Orthopoxvirus (cowpox virus, monkeypox virus, vaccinia virus, variola virus), Yatapoxvirus (tanapoxvirus, yaba monkey tumor poxvirus).
[0135] Picomaviridae are a family of viruses with single-stranded positive-sense RNA genomes and includes, without limitation, enteroviruses A through L, coxsackieviruses, echoviruses, polioviruses 1-3, and rhinoviruses A and B, hepatoviruses (Hepatitis A virus), cardioviruses (infect rodents, aphthoviruses (food-and-mouth disease virus which infects cloven-hoofed animals and occasionally humans).
[0136] Adenoviridae is a family of double- stranded DNA viruses and include more than 100 antigenic types with human adenoviruses divided in subgenuses A-F and Serotypes 1-47, e.g. HAdV-B3, -E4, and -B7.
[0137] To test efficacy of the compositions against Marburg virus, HeLa cells may be seeded at 2,000 cells per well in a 384-well plate, and compounds may be added to the assay plates. Assay plates may be transferred to the BSL-4 suite and infected with 1 PFU per cell MARY, which resulted in 50% to 70% of the cells expressing virus antigen in a 48-h period. [0138] To test efficacy of the compositions against Sudan virus, HeLa cells may be seeded at 2,000 cells per well in a 384-well plate, and compounds may be added to the assay plates.
Assay plates may be transferred to the BSL-4 suite and infected with 0.08 PFU SUDV per cell, which resulted in 50% to 70% of the cells expressing virus antigen in a 48-h period. [0139] To test efficacy against Lassa fever virus, HeLa cells may be seeded at 2,000 cells per well in a 384-well plate, and compounds may be added to the assay plates. Assay plates may be transferred to the BSL-4 suite and infected with 0.1 PFU per cell LASV, which resulted in >60% of the cells expressing virus antigen in a 48-h period.
[0140] To test efficacy against Middle East respiratory' syndrome, SARS-CoV-1 , and SARS-CoV-2 Vero E6 cells may be seeded at 4,000 cells per weH in a 384-well plate, and compounds may be added to the assay plates in a dose dependent manner. Assay plates may be transferred to the BSL-3/4 suite and infected with 0.5 or other PFU per cell of MERS, SARS-CoV-1 and 2 virus, which resulted in >70% of the cells expressing virus antigen in a 48-h period. [0141] To test efficacy against Chikungunya virus, U20S cells may be seeded at 3,000 cells per well in a 384-well plate, and compounds may be added to the assay plates. Assay plates may be transferred to the BSL suite and infected with 0.5 PFU per cell of CHIK, which resulted in >80% of the cells expressing virus antigen in a 48-h period.
[0142] To test efficacy against Venezuelan equine encephalitis virus, HeLa cells may be seeded at 4,000 cells per well in a 384-well plate, and compounds may be added to the assay plates. Assay plates may be transferred to the BSL-4 suite and infected with 0.1 PFU per cell VEEV, which resulted in >60% of the cells expressing virus antigen in a 20-h period.
[0143] In some embodiments, cytotoxicity assays may be conducted. HEp-2
(1.5 x 103 cells per well) and MI' -4 (2 x 103 cells per well) cells may be plated in 384-well plates and incubated with the appropriate medium containing threefold serially diluted compound ranging from 15 nM to 100,000 nM. PC-3 cells (2.5 x 103 cells per well), HepG2 cells (4 x 103 cells per well), hepatocytes (1 x 106 cells per well), quiescent PBMCs (1 x iob cells per well), stimulated PBMCs (2 x 105 cells per well), and RPTEC cells (1 x io3 cells per well) may be plated in 96-well plates and incubated with the appropriate medium containing threefold serially diluted compound ranging from 15 nM to 100,000 nM. Cells may be cultured for 4-5 days at 37 °C. Following the incubation, the cells may be allowed to equilibrate to 25°C, and cell viability may be determined by adding Cell-Titer Gio viability reagent. The mixture may be incubated for 10 min, and the luminescence signal may be quantified using an Envision plate reader. Cell lines may be not authenticated and may be not tested for my coplasma as part of routine use in cytotoxicity assays.
[0144] RNA synthesis by the RSV polymerase may be reconstituted in vitro using purified RSV L/P complexes and an RNA oligonucleotide template (Dharmacon), representing nucleotides 1-14 of the RSV leader promoter. RNA synthesis reactions may be performed as described previously, except that the reaction mixture contained 250 μM guanosine triphosphate (GTP), 10 μM uridine triphosphate (UTP), 10 μM cytidine triphosphate (CTP), supplemented with 10 pCi [a-32P]CTP, and either included 10 μM adenosine triphosphate (ATP) or no ATP. Under these conditions, the polymerase is able to initiate synthesis from the position 3 site of the promoter, but not the position 1 site. The NTP metabolite of GS- 5734 may be serially diluted in DMSO and included in each reaction mixture at concentrations of 10, 30, or 100 μM as specified. RNA products may be analyzed by electrophoresis on a 25% polyacrylamide gel, containing 7 M urea, in Tris-taurine-EDTA buffer, and radiolabeled RNA products may be detected by autoradiography. [0145] In some embodiments, RSV A2 polymerase inhibition assay may be performed. Transcription reactions contained 25 pg of crude RSV RNP complexes in 30 μL of reaction buffer (50 mM Tris-acetate (pH 8.0), 120 mM potassium acetate, 5% glycerol, 4.5 mM MgCl 2, 3 mM DTT, 2 mM EGTA, 50 pg ml"1 BSA, 2.5 U RNasin, 20 μM ATP, 100 μM GTP, 100 μM UTP, 100 μM CTP, and 1.5 pCi [α-32P]ATP (3,000 Ci mmol"1). The radiolabeled nucleotide used in the transcription assay may be selected to match the nucleotide analogue being evaluated for inhibition of RSV RNP transcription.
[0146] To determine whether nucleotide analogues inhibited RSV RNP transcription, compounds may be added using a six-step serial dilution in fivefold increments. After a 90- min incubation at 30°C, the RNP reactions may be stopped with 350 pl of Qiagen RLT lysis buffer, and the RNA may be purified using a Qiagen RNeasy 96 kit. Purified RNA may be denatured in RNA sample loading buffer at 65°C for 10 min and run on a 1.2% agarose/MOPS gel containing 2 M formaldehyde. The agarose gel may be dried, exposed to a Storm phosphorimaging screen, and developed using a Storm phosphorimager.
IV. PHARMACEUTICAL COMPOSITIONS
[0147] According to the present disclosure the compound may be prepared as pharmaceutical compositions. It will be understood that such compositions necessarily comprise one or more active ingredients and, most often, a pharmaceutically acceptable excipient.
[0148] Relative amounts of the active ingredients (e.g. anti-viral, protease inhibitor), a pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary', depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered. For example, the composition may comprise between 0.1% and 99% (w/w) of the active ingredients. By way of example, the composition may comprise between 0.1% and 100%, e.g., between .5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredients.
[0149] In some embodiments, the anti-viral and protease inhibitors pharmaceutical compositions described herein may comprise at least one anti-viral and at least one protease inhibitor. As a non-limiting example, the pharmaceutical compositions may contain an antiviral and a cathepsin inhibitor.
[0150] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g. non-human mammals. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, rats, birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.
[0151] In some embodiments, compositions are administered to humans, human patients, or subjects.
V. FORMULATIONS
[0152] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. As used herein the term “'pharmaceutical composition” refers to compositions comprising at least one active ingredient and optionally one or more pharmaceutically acceptable excipients.
[0153] Formulations of the anti-viral, protease inhibitors, and pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredients into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.
[0154] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
[0155] Relative amounts of the active ingredient (e.g. anti-viral, protease inhibitor), the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may wiry, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered. For example, the composition may comprise between 0.1% and 99% (w/w) of the active ingredient. By way of example, the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, or at least 80% (w/w) active ingredient.
Excipients and Diluents
[0156] The anti-viral and protease inhibitor of the disclosure can be formulated using one or more excipients or diluents to (1 ) increase stability; (2) increase absorption; (3) permit the sustained or delayed release; or (4) alter the biodistribution (e.g., target the compound to specific tissues or cell types).
[0157] In some embodiments, a pharmaceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use for humans and for veterinary use. In some embodiments, an excipient may be approved by United States Food and Drug Administration. In some embodiments, an excipient may be of pharmaceutical grade. In some embodiments, an excipient may meet the standards of the United States Pharmacopoeia (LISP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
[0158] Excipients, as used herein, include, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.
[0159] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and/or combinations thereof.
Inactive Ingredients
[0160] In some embodiments, formulations may comprise at least one inactive ingredient. As used herein, the term “inactive ingredient” refers to one or more agents that do not contribute to the activity of the active ingredient of the pharmaceutical composition included in formulations. In some embodiments, all, none or some of the inactive ingredients which may be used in the formulations of the present disclosure may be approved by the US Food and Drug Administration (FDA).
[0161] Formulations of the disclosure may also include one or more pharmaceutically acceptable salts. As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form (e.g., by reacting the free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, acetic acid, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzene sulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2 -naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenyl propion ate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary/ ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. The pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. [0162] Solvates may be prepared by crystallization, recrystallization, or precipitation from a solution that includes organic solvents, water, or a mixture thereof. Examples of suitable solvents are ethanol, water (for example, mono-, di-, and tri-hydrates), A;- methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), N,N ’-dimethylformamide (DMF), N,N ’-dimethylacetamide (DMAC), 1 ,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl- 3,4,5,6-tetrahydro-2-(lH)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When water is the solvent, the solvate is referred to as a “hydrate.”
Non-steroidal anti-inflammatory drugs (NSAIDs)
[0163] In some embodiments, formulations may include classical non-steroidal antiinflammatory' drugs (NS AID). As used herein, the term NSAID may include, but are not limited to, alcofenac, aceclofenac, sulindac, tolmetin, etodolac, fenoprofen, thiaprofenic acid, meclofenamic acid, meloxicam, tenoxicam, lornoxicam, nabumeton, acetaminophen, phenacetin, ethenzamide, sulpyrine, antipyrine, migrenin, aspirin, mefenamic acid, flufenamic acid, diclofenac sodium, loxoprofen sodium, phenylbutazone, indomethacin, ibuprofen, ketoprofen, naproxen, oxaprozin, flurbiprofen, fenbufen, pranoprofen, floctafenine, piroxicam, epirizole, tiaramide hydrochloride, zaltoprofen, gabexate mesylate, ulinastatin, colchicine, probenecid, sulfinpyrazone, benzbromarone, allopurinol, sodium aurothiomalate, hyaluronate sodium, sodium salicylate, morphine hydrochloride, salicylic acid, atropine, scopolamine, morphine, pethidine, levorphanol, oxymorphone or a salt thereof and the like.
[0164] In some embodiments, formulations may include cyclooxygenase inhibitors. As used herein the term cyclooxygenase inhibitors may include, but are not limited to, (COX-1 selective inhibitors, COX-2 selective inhibitors, salicylic acid derivatives (e.g., celecoxib, aspirin), etoricoxib, valdecoxib, diclofenac, indomethacin, loxoprofen and the like.
[0165] In some embodiments, formulations may include Nitric oxi de-releasing NSAIDs. Salts
[0166] If a pharmaceutically acceptable salt of the combination or mixture of the disclosure or a citric acid ester thereof is utilized in these compositions, the salt preferably is derived from an inorganic or organic acid or base. For reviews of suitable salts, see, e.g., Berge et al, J. Pharm. Sci. 66: 1-19 (1977) and Remington: The Science and Practice of Pharmacy, 20th Ed., ed. A. Gennaro, Lippincott Williams & Wilkins, 2000.
[0167] As used herein, non-limiting examples of suitable acid addition salts include the following: acetate, adipate, alginate, aspartate, benzoate, benzene sulfonate, bisulfate, butyrate, citrate, camphorate, camphor sulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, lucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3 -phenyl -propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate.
[0168] As used herein, suitable base addition salts include, without limitation, ammonium salts, alkali metal salts, such as lithium, sodium and potassium salts; alkaline earth metal salts, such as calcium and magnesium salts; other multivalent metal salts, such as zinc salts; salts with organic bases, such as dicyclohexylamine, N-methyl-D-glucamine, t-butylamine, ethylene diamine, ethanolamine, and choline; and salts with amino acids such as arginine, lysine, and so forth.
[0169] The pharmaceutical composition comprises the combination, whether as separate compounds or as a mixture, of the present disclosure and a pharmaceutically acceptable carrier.
[0170] The term "pharmaceutically acceptable carrier" is used herein to refer to a material that is compatible with a recipient subject, preferably a mammal, more preferably a human, and is suitable for delivering an active agent to the target site without terminating the activity of the agent. The toxicity or adverse effects, if any, associated with the carrier preferably are commensurate with a reasonable risk/benefrt ratio for the intended use of the active agent. [0171] The terms "carrier", "adjuvant", or "vehicle" are used interchangeably herein, and include any and all solvents, diluents, and other liquid vehicles, dispersion or suspension aids, surface active agents, pH modifiers, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington: The Science and Practice of Pharmacy, 20th Ed., ed. A. Gennaro, Lippincott Williams & Wilkins, 2000 discloses various carriers used in formulating pharmaceutically acceptable compositions and known techniques for the preparation thereof, which is incorporated by reference herein, in its entirety. Except insofar as any conventional carrier medium is incompatible with the compound of the disclosure, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, its use is contemplated to be within the scope of this disclosure. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, carbonates, magnesium hydroxide and aluminum hydroxide, glycine, sorbic acid, or potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, pyrogen-free water, salts or electrolytes such as protamine sulfate, di sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylenepoly oxypropyl ene-block polymers, wool fat, sugars such as lactose, glucose, sucrose, and mannitol, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate, powdered tragacanth; malt, gelatin, talc, excipients such as cocoa butter and suppository waxes, oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil, glycols such as propylene glycol and polyethylene glycol, esters such as ethyl oleate and ethyl laurate, agar, alginic acid, isotonic saline, Ringer's solution, alcohols such as ethanol, isopropyl alcohol, hexadecyl alcohol, and glycerol, cyclodextrins such as hydroxypropyl beta- cyclodextrin and sulfobutylether beta-cyclodextrin, lubricants such as sodium lauryl sulfate and magnesium stearate, petroleum hydrocarbons such as mineral oil and petrolatum. Coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.
[0172] While it is possible for the active ingredients of the combination to be administered alone and separately as monotherapies, it is preferable to administer them as a pharmaceutical co-formulation. A two-part or three-part combination may be administered simultaneously or sequentially. When administered sequentially, the combination may be administered in one, two, or three administrations. Preferably, two-part, or three-part combinations are administered in a single pharmaceutical dosage form. More preferably, a two-part combination is administered as a single oral dosage form and a three-part combination is administered as two identical oral dosage forms.
[0173] It will be appreciated that the compounds of the combination may be administered: (1) simultaneously by combination of the compounds in a co-formulation or (2) by alternation, i.e. delivering the compounds serially, sequentially, in parallel or simultaneously in separate pharmaceutical formulations.
[0174] In alternation therapy, the delay in administering the second, and optionally a third active ingredient, should not be such as to lose the benefit of a synergistic therapeutic effect of the combination of the active ingredients. By either method of administration (1) or (2), ideally the combination should be administered to achieve peak plasma concentrations of each of the active ingredients. A one pill once-per-day regimen by administration of a combination co formulation may be feasible for some IV-positive patients. Effective peak plasma concentrations of the active ingredients of the combination will be in the range of approximately 0.001 μM to 10 uM. Optimal peak plasma concentrations may be achieved by a formulation and dosing regimen prescribed for a particular patient. It will also be understood that either active ingredient, or the physiologically functional derivatives of either thereof, whether presented simultaneously or sequentially, may be administered individually, in multiples, or in any combination thereof. In general, during alternation therapy (2), an effective dosage of each compound is administered serially , where in co-formulation therapy (1), effective dosages of two or more compounds are administered together.
[0175] When the individual components of the combination are administered separately they are generally each presented as a pharmaceutical formulation. The references hereinafter to formulations refer unless otherwise stated to formulations containing either the combination or a component compound thereof. It will be understood that the administration of the combination of the disclosure by means of a single patient pack, or patient packs of each formulation, within a package insert diverting the patient to the correct use of the disclosure is a desirable additional feature of this disclosure.
[0176] The combination may be formulated in a unit dosage formulation comprising a fixed amount of each active pharmaceutical ingredient for a periodic, e.g. daily, dose or subdose of the active ingredients. Pharmaceutical formulations according to the present disclosure comprise a combination according to the disclosure together with one or more pharmaceutically acceptable carriers or excipients and optionally other therapeutic agents. VI. DOSING AND ADMINISTRATION
Administration
[0177] The composition of the present disclosure may be administered by any delivery route which results in a therapeutically effective outcome. These include, but are not limited to, enteral (into the intestine), gastroenteric, epidural (into the dura mater), oral (by w'ay of the mouth), transdermal, intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intra-arterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow7), intrathecal (into the spinal canal), intraparenchymal (into brain tissue), intraperitoneal (infusion or injection into the peritoneum), intravesical infusion, intravitreal (through the eye), intracavemous injection (into a pathologic cavity) intracavitary (into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through the intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), transvaginal, insufflation (snorting), sublingual, sublabial, enema, eye drops (onto the conjunctiva), ear drops, auricular (in or by way of the ear), buccal (directed toward the cheek), conjunctival, cutaneous, dental (to a tooth or teeth), electro-osmosis, endocervical, endosinusial, endotracheal, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra-amniotic, intra-articular, intrabiliary, intrabronchial, intrabursal, intracartilaginous (within a cartilage), intracaudal (within the cauda equine), intraci sternal (within the cisterna magna cerebellomedularis), intracorneal (within the cornea), dental intracoronal, intracoronary (within the coronary arteries), intracorporus cavernosum (within the dilatable spaces of the corporus cavernosa of the penis), intradiscal (within a disc), intraductal (within a duct of a gland), intraduodenal (within the duodenum), intradural (within or beneath the dura), intraepidermal (to the epidermis), intraesophageal (to the esophagus), intragastric (within the stomach), intragingival (within the gingivae), intraileal (within the distal portion of the small intestine), intralesional (within or introduced directly to a localized lesion), intraluminal (within a lumen of a tube), intralymphatic (within the lymph), intramedullar}- (within the marrow cavity of a bone), intrameningeal (within the meninges), intramyocardial (within the myocardium), intraocular (within the eye), intraovarian (within the ovary), intrapericardial (within the pericardium), intrapleural (within the pleura), intraprostatic (within the prostate gland), intrapul mon ary (within the lungs or its bronchi), intrasinal (within the nasal or periorbital sinuses), intraspinal (within the vertebral column), intrasynovial (within the synovial cavity of ajoint), intratendinous (within a tendon), intratesticular (within the testicle), intrathecal (within the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (within the thorax), intratubular (within the tubules of an organ), intratumor (within a tumor), intratympanic (within the aurus media), intravascular (within a vessel or vessels), intraventricular (within a ventricle), iontophoresis (by means of electric current where ions of soluble salts migrate into the tissues of the body), irrigation (to bathe or flush open wounds or body cavities), laryngeal (directly upon the larynx), nasogastric (through the nose and into the stomach), occlusive dressing technique (topical route administration which is then covered by a dressing which occludes the area), ophthalmic (to the external eye), oropharyngeal (directly to the mouth and pharynx), parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (within the respiratory tract by inhaling orally or nasally for local or systemic effect), retrobulbar (behind the pons or behind the eyeball), soft tissue, subarachnoid, subconjunctival, submucosal, topical, transplacental (through or across the placenta), transtracheal (through the wall of the trachea), transtympanic (across or through the tympanic cavity), ureteral (to the ureter), urethral (to the urethra), vaginal, caudal block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photopheresis, and spinal.
[0178] In some embodiments, compositions may be administered in a way which allows them to cross the blood-brain barrier, vascular barrier, or other epithelial barrier. The viral particles of the present disclosure may be administered in any suitable form, either as a liquid solution or suspension, as a solid form suitable for liquid solution or suspension in a liquid solution. The viral particles may be formulated with any appropriate and pharmaceutically acceptable excipient.
[0179] In some embodiments, the composition of the present disclosure may be delivered to a subject via a single route administration.
[0180] In some embodiments, the composition of the present disclosure may be delivered to a subject via a multi-site route of administration. A subject may be administered at 2, 3, 4, 5, or more than 5 sites.
[0181] In some embodiments, a subject may be administered the composition of the present disclosure using a bolus infusion.
[0182] In some embodiments, a subject may be administered the composition of the present disclosure using sustained delivery' over a period of minutes, hours, or days. The infusion rate may be changed depending on the subject, distribution, formulation or another delivery parameter.
[0183] In some embodiments, the composition of the present disclosure may be delivered by oral administration. Non-limiting examples of oral administration include a digestive tract administration and a buccal administration.
[0184] In some embodiments, the composition of the present disclosure may be delivered by intraocular delivery route. A non-limiting example of intraocular administration include an intravitreal injection. [0185] In some embodiments, the composition of the present disclosure may be delivered by intranasal delivery route. Non-limiting examples of intranasal delivery include administration of nasal drops or nasal sprays.
[0186] In some embodiments, the composition may be delivered by systemic delivery. As a non-limiting example, the systemic delivery may be by intravascular administration.
[0187] In some embodiments, the composition of the present disclosure may be administered to a subject by intraparenchymal administration.
[0188] In some embodiments, the composition of the present disclosure may be administered to a subject by intramuscular administration.
[0189] In some embodiments, the composition of the present disclosure is administered to a subject and transduce muscle of a subject. As a non-limiting example, the composition is administered by intramuscular administration.
[0190] In some embodiments, the composition of the present disclosure may be administered to a subject by intravenous administration.
[0191] In some embodiments, the composition of the present disclosure may be administered to a subject by subcutaneous administration.
[0192] In some embodiments, the composition of the present disclosure may be administered to a subject by topical administration.
[0193] In some embodiments, the composition may be delivered by direct injection into the brain. As a non-limiting example, the brain delivery may be by intrastriatal administration.
[0194] In some embodiments, the composition may be delivered by more than one route of administration. As non-limiting examples of combination administrations, composition may be delivered by intrathecal and intracerebroventricular, or by intravenous and i ntraparenchy mal admi ni strati on .
Dosing
[0195] The present disclosure additionally provides a method of delivering to a subject, including a mammalian subject, any of the above-described composition comprising administering to the subject said composition, or administering to the subject a formulation comprising said composition, or administering to the subject any of the described compositions, including pharmaceutical compositions.
[0196] For any compound described herein the therapeutically effective amount can be initially determined from preliminary in vitro studies and/or animal models. A therapeutically effective dose can also be determined from human data for inhibitors which have been tested in humans and for compounds which are known to exhibit similar pharmacological activities, such as other related active agents. For instance, many cathepsin inhibitors have been extensively studied. The applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled artisan,
[0197] In some embodiments, the dose of the compositions of the disclosure may be determined based on the concentration of the protease inhibitor required to inhibit the growth of a virus or reduce the percentage of virus infected cells. The concentration of the protease inhibitor may be from about 1 x 10-12 M to about I xl O'” M, for example, from about 0. 1 μM to about 50 μM. In some embodiments, the concentration of the protease inhibitor may be 0.01 μM -0.1 iiM, 0.1 μM -1 μM, 1 μM -10 μM, 10 μM -100 μM. In some embodiments, the concentration of the protease inhibitor may be 0. 1 pM, 0.3 μM, 1 μM, 3 μM, 10 μM or 30 μM.
[0198] The mammalian protease inhibitor may have an effective concentration (EC50) of from about 0.25μM to about 50μM. In some embodiments, the effective concentration of the protease inhibitor may be about 0.01 μM -0.1 μM, 0.1 μM -1 μM, 1 μM -10 μM, 10 μM -100 μM. In some embodiments, the effective concentration (EC50) of the protease inhibitor is from about 15 μM to about 30 μM. In some embodiments, the effective concentration (EC50) of the protease inhibitor is from about 0.25 μM to about 0.5 μM.
[0199] The mammalian protease inhibitor may have an effective concentration (EC90) of from about 0.25μM to about 50μM. In some embodiments, the effective concentration of the protease inhibitor may be about 0.01 μM -0.1 μM, 0.1 μM -1 μM, 1 μM -10 μM, 10 μM -100 μM. In some embodiments, the effective concentration (EC90) of the protease inhibitor is from about 1 μM to about 100 μM. In some embodiments, the effective concentration (EC90) of the protease inhibitor is from about 1 μM to about 3 μM.
[0200] While the dose varies depending on the target disease, symptom, subject of administration, administration method and the like, for oral administration as a therapeutic agent, for example, it is generally about 0.01-1000 mg/kg body weight. The dose may be 0.01-0.1 mg/kg, 0.1-1 mg/kg, 1-10 mg/kg, 10-100 mg/kg, 100-1000 mg/kg, 0.05-30 mg/kg body weight, 0.5-10 mg/kg body weight, as one dose of the compound of the present disclosure, which is, for example, administered once to 3 times a day, on a weekly schedule, on a twice-weekly schedule and the like. The dose may be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 mg/kg body weight.
[0201] In some embodiments, the dose may be 400mg oral dosing of Balicatib (HB-121) which may result in plasma concentrations of Balicatib (HB-121) above EC50 for 16-20 hrs. In some aspects, the dosing of 400mg may be provided orally once or twice daily.
[0202] In some embodiments, the compositions of the present disclosure or a pharmaceutical composition thereof is administered on a weekly schedule. In some embodiments, the compositions are administered on a weekly schedule.
[0203] In some embodiments, the compositions of the present disclosure or a pharmaceutical composition of the present disclosure or a pharmaceutical composition thereof is administered on days 1, 8, and 15 of a 28-day cycle. In some embodiments, the compositions of the present disclosure, are administered on days 1, 8, and 15 of a 28-day cycle.
[0204] In some embodiments, the compositions of the present disclosure or a pharmaceutical composition thereof is administered on a twice-weekly schedule. In some embodiments, the compositions of the present disclosure, for example a cathepsin inhibitor, or a pharmaceutical composition thereof is administered on a twice-weekly schedule.
[0205] In some embodiments, the compositions of the present disclosure of the present disclosure or a pharmaceutical composition thereof is administered on days 1 , 4, 8, and 11 of a 21 -day cycle.
[0206] In some embodiments, the compositions of the present disclosure or a pharmaceutical composition thereof is administered in conjunction with another therapeutic modality.
[0207] In certain such embodiments, the other therapeutic modality is one that is normally administered to patients with the disease to be treated or prevented. In some such embodiments, the other therapeutic modality is radiotherapy or plasmapheresis or another therapeutic agent.
[0208] In the above embodiments, the other therapeutic modality may be administered in the same dosage form or as a separate dosage form. When administered as a separate dosage form, the other therapeutic agent may be administered prior to, at the same time as, or following administration of the compound of the present disclosure or a pharmaceutical composition thereof. [0209] Pharmaceutical formulations containing the active ingredient may be in any form suitable for the intended method of administration. When used for oral use for example, tablets, troches, lozenges, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs may be prepared an in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA, which is incorporated herein by reference in its entirety.
[0210] Compositions intended for oral use may be prepared according to any method known to the art. for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents including antioxidants, sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide a palatable preparation. Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipient which are suitable for manufacture of tablets are acceptable. These excipients may be, for example, inert diluents, such as calcium or sodium carbonate, lactose, lactose monohydrate, croscarmellose sodium, povidone, calcium or sodium phosphate; granulating and disintegrating agents, such as maize starch, or alginic acid; binding agents, such as cellulose, microcrystalline cellulose, starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or may be coated by known techniques including microencapsulation to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed. Formulations for oral use may be also presented as hard gelatin capsules where the active ingredient is mixed with an inert solid diluent, for example pregelatinized starch, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.
[0211] The present disclosure provides pharmaceutical formulations combining the active ingredients, a mammalian protease inhibitor, or physiologically functional derivatives thereof, in a sufficiently homogenized form, and a method for using this pharmaceutical formulation. An object of the present disclosure is to utilize glidants to reduce the segregation of active ingredients in pharmaceutical compositions during pre-compression material handling. Formulations include those suitable for oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods represent a further feature of the present disclosure and include the step of bringing into association the active ingredients with the carrier, which constitutes one or more accessory ingredients, and maintaining chemical stability.
[0212] In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. Formulations of the present disclosure suitable for oral administration may be presented as discrete units such as capsules, caplets, cachets or tablets each containing a predetermined amount of the active ingredients; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in- water liquid emulsion or a w'ater-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredients in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. povidone, gelatin, hydroxypropyl methylcellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycollate, cross linked povidone, cross-linked sodium carboxymethyl cellulose) surfaceactive or dispersing agent. Molded tablets may be made by molding a mixture of the powdered compound moistened with an inert liquid diluent in a suitable machine. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredients therein using, for example, cellulose ether derivatives (e.g., hydroxypropyl methylcellulose) or methacrylate derivatives in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach . Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredients in a flavored base, usually sucrose and acacia or tragacanth, pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
[0213] Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylates. Topical administration may also be by means of a transdermal iontophoretic device. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate. Formulations suitable for penile administration for prophylactic or therapeutic use may be presented in condoms, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate. Pharmaceutical formulations suitable for rectal administration wherein the carrier is a solid are most preferably presented as unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories may be conveniently formed by admixture of the active combination with the softened or melted carrier(s) followed by chilling and shaping in molds. Formulations suitable for parenteral administration include aqueous and nonaqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents; and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
[0214] Exemplary’ unit dosage formulations are those containing a daily dose or daily subdose of the active ingredients, as hereinbefore recited, or an appropriate fraction thereof. It should be understood that in addition to the ingredients particularly mentioned above the formulations of this disclosure may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include such further agents as sweeteners, thickeners and flavoring agents.
Aqueous Suspensions
[0215] Aqueous suspensions of the disclosure contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., 1 polyoxyethylene sorbitan monooleate).
[0216] The aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose, sucralose or saccharin. Oil suspensions may be formulated by suspending the active ingredient in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oral suspensions may contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid, BHT, etc. Dispersible powders and granules of the disclosure suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending a-gents are exemplified by those disclosed above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.
Emulsions
[0217] The pharmaceutical compositions of the disclosure may also be in the form of oil in-water emulsions or liposome formulations. The oily phase may be a vegetable oil, such as olive oil or arachis oil, a mineral oil, such as liquid paraffin, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavoring agents. Syrups and elixirs may be formulated with sweetening agents, such as glycerol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, a flavoring or a coloring agent.
Injectable
[0218] The pharmaceutical compositions of the disclosure may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butane diol or prepared as a lyophilized powder. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils may conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may likewise be used in the preparation of injectables.
[0219] The pharmaceutical compositions of the disclosure may be injected parenterally, for example, intravenously, intraperitoneally, intrathecally, intraventricularly, intrasystemically, intracranially, intramuscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary'. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the an.
Intranasal
[0220] The pharmaceutical compositions of the disclosure may also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurized container or a nebulizer with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1,1, 1,2-tetra.fluoroethane (HFC 134a), carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.. The pressurized container or nebulizer may contain a solution or suspension of the composition, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate.
Oral
[0221] Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a pharmaceutical composition of the disclosure and a suitable powder base such as lactose or starch. Aerosol or dry' powder formulations are preferably arranged so that each metered dose or "puff contains from 20 pg to 200 mg of a composition for delivery to the patient. The overall daily dose with an aerosol or nebulizer will be in the range of from 20 pg to 200 mg which may be administered in a single dose or, more usually, in divided doses throughout the day. The amount of active ingredient that may be combined with the carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a time-release formulation intended for oral administration to humans may contain approximately 1 to 1000 mg of active material compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 95% of the total compositions (weight : weight) .
[0222] The pharmaceutical composition can be prepared to provide easily measurable amounts for administration. For example, an aqueous solution intended for intravenous infusion may contain from about 3 to 500 mg of the active ingredient per milliliter of solution in order that infusion of a suitable volume at a rate of about 30 ml/hr can occur.
[0223] As noted above, formulations of the present disclosure suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containi ng a predetermined amount of the acti ve ingredient, as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be administered as a bolus, electuary' or paste.
VII. DEFINITIONS
[0224] "Bioavailability" is the degree to which a pharmaceutically active agent becomes available to the target tissue after the agent's introduction into the body. Enhancement of the bioavailability of a pharmaceutically active agent can provide a more efficient and effective treatment for patients because, for a given dose, more of the pharmaceutically active agent will be available at the targeted tissue sites. The compounds of the combinations of the disclosure may be referred to as "active ingredients" or pharmaceutically active agents." [0225] "Bioequivalence" refers to chemical equivalents that., when administered to the same person in the same dosage regimen, result in equivalent concentrations of drug in blood and tissues.
[0226] "Chemical equivalence" refers to drug products that contain the same compound in the same amount and that meet current official standards. However, inactive ingredients in drug products may differ.
- 33 - [0227] "Clearance" of drug occurs by perfusion of blood to the organs of extraction. "Extraction" refers to the proportion of drug presented to the organ which is removed irreversibly (excreted) or altered to a different chemical form (metabolism). Clearance (CL) is therefore calculated as the product of the flow of blood through the organ and proportion of the drug extracted by the organ.
[0228] As used herein, the terms "comprising," "including," or grammatical variants thereof, are to be taken as specifying inclusion of the stated features, integers, actions, or components without precluding the addition of one or more additional features, integers, actions, components, or groups thereof.
[0229] As used herein, the term "effective amount," “effective concentration,” or “effective dose” means an amount that is sufficient upon appropriate administration to a patient (a) to cause a detectable decrease in the severity of the disorder or disease state being treated; (b) to ameliorate or alleviate the patient's symptoms of the disease or disorder; or (c) to slow or prevent advancement of, or otherwise stabilize or prolong stabilization of, the disorder or disease state being treated. It should also be understood that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, and diet of the patient, time of administration, rate of excretion, drug combinations, the judgment of the treating physician, and the severity of the particular disease being treated. [0230] As used herein, the “maximum tolerated dose” (MTD) is the highest possible but still tolerable dose level with respect to a pre-specified clinical limiting toxicity. In general, these limits refer to the average patient population. For instances in which there is a large difference between the MED and MTD, it is stated that the drug has a large therapeutic window. Conversely, if the range is relatively small, or if the MTD is less than the MED, then the pharmaceutical product will have little to no practical value.
[0231] The term "method" refers to manners, means, techniques, processes and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques, processes and procedures either known to, or readily developed from known manners, means, techniques, processes and procedures by practitioners of chemistry and/or pharmacology.
[0232] As used herein, the “minimum effective dose” (MED) is defined as the lowest dose level of a pharmaceutical product that provides a clinically significant response in average efficacy, which is also statistically significantly superior to the response provided by the placebo.
[0233] The term "physiologically functional derivative" means a pharmaceutically active compound with equivalent or near equivalent physiological functionality when administered in combination with another pharmaceutically active compound alone or in combination with another compound. As used herein, the term "physiologically functional derivative" includes any: physiologically acceptable salt, ether, ester, prodrug, solvate, stereoisomer including enantiomer, diastereomer or stereoisomerically enriched or racemic mixture, and any other compound, which upon administration to a recipient, is capable of providing (directly or indirectly) such a compound or an active metabolite or residue thereof.
[0234] The term “potentiating” effect as used herein refers to and enhancement of an effect or action of an agent, a drug, or a chemical. A potentiating agent can be a chemical, an agent or a drug that enhances or intensifies an effect or action of another agent, chemical or drug.
[0235] The term "prodrug" as used herein refers to any compound that when administered to a biological system generates the drug substance, i.e. active ingredient, as a result of spontaneous chemical reaction(s), enzyme catalyzed chemical reaction(s), and/or metabolic chemical reaction(s). "Prodrug moiety" means a labile functional group which separates from the active inhibitory compound during metabolism, systemically, inside a cell, by hydrolysis, enzymatic cleavage, or by some other process (Bundgaard, Hans, "Design and Application of Prodrugs" in Textbook of Drug Design and Development (1991), P. Krogsgaard Larsen and H. Bundgaard, Eds. Harwood Academic Publishers, pp. 113-191).
[0236] "Side effects" or "toxicity" or "adverse drug reactions" of drugs are side effects which may minor, severe, quite severe, or disabling and reversible or irreversible. In medicine, a side effect is an adverse effect that is secondary to the one intended; an unintended, consequences of the use of a drug whether in the targeted or untargeted parts of the body.
[0237] As used herein, the term “subject” or “patient” is a mammal, and examples thereof include human, dog, cat, bovine, horse, swine, or human.
[0238] The terms "synergy" and "synergistic" mean that the effect achieved when the drug and compound are used together is greater than the sum of the effects that results from using the drug and the compound separately, i.e. greater than what would be predicted based on the two active ingredients administered separately. A synergistic effect may be attained when the drug and compound are: (I) co-formulated and administered or delivered simultaneously in a combined formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect may be attained when the drug and compound are administered or delivered sequentially, e.g. in separate tablets, pills or capsules, or by different injections in separate syringes. In general, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e. serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together. A synergistic antiviral effect denotes an antiviral effect which is greater than the predicted purely additive effects of the individual active ingredients of the combination.
[0239] As used herein, the term "treatment" means treating a patient having, or at risk of developing or experiencing a recurrence of the relevant disorder being treated, including suppression of progression of the relevant disorder being treated.
[0240] "Therapeutic equivalence" refers to drug products that, when administered to the same person in the same dosage regimen, provide essentially the same therapeutic effect or toxicity. Bioequivalent products are expected to be therapeutically equivalent. Sometimes therapeutic equivalence may be achieved despite differences in bioavailability', for example when the therapeutic index is wide (ratio of maximum tolerated dose to the minimum effective dose).
[0241] "Absorption" rate is important because even when a drug is absorbed completely, it may be absorbed too slowly to produce a therapeutic blood level quickly enough or so rapidly that toxicity results from high drag concentrations given to achieve the therapeutic level after each dose.
[0242] The details of one or more embodiments of the disclosure are set forth in the accompanying description below. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred materials and methods are now described. Other features, objects and advantages of the disclosure will be apparent from the description. In the description, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly- understood by one of ordinary skill in the art to which this disclosure belongs. In the case of conflict, the present description will control. [0243] The present disclosure is further illustrated by the following non-limiting examples.
EXAMPLES
Example 1. Efficacy of Balicatib against viruses
[0244] The efficacy of Balicatib (also referred to herein as HB-121) against several viruses was tested. Many viruses use cathepsins to enter the host cells. Cathepsins e.g. cathepsin B and cathepsin also help activate viral proteins. Accordingly, Balicatib (HB-121) may be effective against. Table 1 provides the list of these viruses tested. In Table 1, CPE indicates cytopathic effect; and IF indicates immunofluorescent assays.
Table 1. Virus Strains
Figure imgf000061_0001
[0245] Viruses and cells were mixed in the presence of Balicatib (HB-121) and incubated for the assay duration as specified in Table 1. Each virus was pre-titered such that control wells exhibited 85 to 95% loss of cell viability due to virus replication. An antiviral effect or cytoprotective effect was considered to be observed when therapeutic agent (Balicatib or positive control indicated in Table 1) prevented virus replication. A standardized plate format was used for cytoprotective assays. Each plate contained cell control wells (cells only), virus control wells (cells plus virus), therapeutic agent cytotoxicity wells (cells plus therapeutic agent only), therapeutic agent colorimetric control wells (therapeutic agent only), background control wells (media only), as well as experimental wells (therapeutic agent plus cells plus virus). Samples were evaluated for antiviral efficacy with triplicate measurements to determine cytotoxicity , if detectable. I' able 2 shows a representative plate format for evaluating test therapeutic agent (Balicatib/ HB-121; labeled “Drug 1” in Table 2) at 6 concentrations (2-fold dilutions) using representative high-test concentrations of 50 μM and control therapeutic agent (labelled “Drug 2” in Table 2) at 6 concentrations (10-fold dilutions) using representative high-test concentration of 200 IU/mL. Interferon (IFN) was used as positive controls in the assays were noted as IU/mL (bioactivity defined by International Units/mL was used from the certificate of analysis information provided by the vendor, PBL Assay Science, Piscataway, NJ). In Table 2, fields labeled as “Drug 1” or “Drug 2” indicate Cells + Virus + Drug; fields labeled as “Tox T” or “Tox 2” indicate Cells + Datg 1 or Drug 2, respectively (toxicity tested in duplicate), Cells labeled as “Color 1” or “Color 2” = Media + Drug 1 or Drug 2, respectively (colorimetric background, no cells).
Table 2. Plate Format for Viral Assays
Figure imgf000062_0001
MTS Staining for Cell Viability
[0246] At assay termination, the assay plates were stained with the soluble tetrazolium- based dye MTS (CellTiter®96 Reagent, Promega) to determine cell viability and quantify compound toxicity. MTS is metabolized by the mitochondrial enzymes of metabolically active cells to yield a soluble formazan product, allowing the rapid quantitative analysis of cell viability and compound cytotoxicity. This reagent is a stable, single solution that does not require preparation before use. At termination of the assay, 10-25 pL of MTS reagent was added per well (10% final concentration based on volume) and the microtiter plates were then incubated for 2-3 hours at 37°C, (except Rhinovirus assay piates, which were incubated at 33°C) 5% CO2 to assess cell viability. Adhesive plate sealers were used in place of the lids, the sealed plates were inverted several times to mix the soluble formazan product and the plate was read spectrophotometrically at 490/650 nm with a Molecular Devices SpectraMax i3 plate reader.
Data Analysis of MTS staining
[0247] Using an in-house computer program, the CPE data analysis was conducted and included the calculation of EC50 (50% inhibition of virus replication), EC95 (95% inhibition of virus replication), CC50 (50% cytotoxicity), CC95 (95% cytotoxicity) and selectivity index values (SI = CC/EC; also referred to as Antiviral Index or Al).
Cytotoxicity Assay
[0248] Cells were seeded at 2,500 to 40,000 cells per well in 96-well plates in growth medium as specified in Table 3. The plates were incubated overnight at 37°C and 5% CO2. The following day, test compound was prepared in assay medium. Growth medium was removed from the plates and replaced with the prepared test compound. Each dilution of compound was tested in triplicate. Each toxicity plate contained the necessary cell control and compound color controls. After the assay readout (duration) as specified in Table 1, cell viability was determined using MTS dye reduction. In Table 3, DMEM indicates Dulbecco’s Modified Eagle’s Medium; FBS indicates Fetal Bovine Serum; and TPCK indicates N-p- Tosyl-L -phenylalanine chloromethyl ketone.
Table 3. Cells and Media used for Antiviral Evaluations
Figure imgf000063_0001
Figure imgf000064_0001
Data Analysis of Cytotoxicity Assay
[0249] The minimum inhibitory drug concentration that reduced plaque formation by 50% (EC50) and the minimum drug concentration that inhibited cell growth by 50% ( AA50) were calculated. The selectivity index (SI) for each active compound was determined by dividing the CCso by the EC50.
Results
[0250] Balicatib (HB-121) was tested against multipie viruses such as Influenza strains listed in Table 1 and Table 3. Balicatib (HB-121) showed antiviral activity against
Enterovirus 71 at about 20μM EC50. The results are shown in Table 4 below:
Table 4.Efficacy of Balicatib against Enterovirus 71
Figure imgf000064_0002
[0251] Balicatib (HB-121) was not effective against the other viruses listed in Table 1 at the concentrations tested.
Example 2. Evaluation assay for SARS-CoV
[0252] Calu-3 cells were seeded in 96 well plate. After 24 h, cells were washed and treated with various concentrations of Balicatib (HB-121 or compound 21 in this assay) or two other cathepsin inhibitors compounds ONO-5334 (compound 19), or Odanacatib (MK- 0822 (compound 22) in a serum-free medium (n::::6). The incubation of cells with PBS served as a negative control (n=6). After 4 h of treatment, cells were washed and replaced with serum-free media for infection with SARS-CoV-2 (strain: BEI USA-WA1/2020) multiplicity of infection (MOI) of 0.01 for 1 h at 37°C. After infection, cells were washed and replaced with 5% FBS containing media. After 48 h post-infection, cells were fixed with 4% buffered paraformaldehyde (Electron Microscopy Sciences) for 15 min at room temperature. The fixed cells were washed with PBS then permeabilized in 0.1% Triton X100 PBS solution for 15 min then blocked in 3% BSA PBS solution. The cells were incubated with anti-S protein Rab (Sino Biological, PA, USA) at 1 : 1000 in the blocking solution overnight at 4°C, followed by incubation with 1 :2000 diluted Alexa Fluor 488 conjugated secondary antibody (Thermo Fisher, MA, USA) for 1 h at room temperature. The ceils were counterstained for nuclei with Hoechst 33342 (Thermo Fisher, MA, USA). The fluorescent images were captured by using an Operetta system. Total and virus-infected cells were counted by imaging and %infection was plotted. The compounds were blinded to the testers in the experiments. [0253] Robust antiviral efficacy of Balicatib (HB-121) with approximate EC50 of 0.25μM to 0.5μM was observed. (See Figure 1). Based on human clinical dosing of Balicatib (HB- 121), 400mg oral dosing of Balicatib (HB-121) will result in plasma concentrations of Balicatib (HB-121) which is above EC50 for 16-20 hrs. This suggests dosing of 400mg orally once or twice daily may be appropriate to observe clinical benefit from Balicatib (HB-121) against SARS-CoV-2. Figure 2 shows that ONO-5334 demonstrated antiviral activity. Figure 3 shows the percentage of virus infected cells treated with varying concentrations of Odanacatib (MK-0822). There was no drug dose dependent antiviral activity observed for Odanacatib.
Example 3. Evaluation assay for SARS-CoV
[0254] Many negative and positive strand RNA viruses must bind, fuse, enter, and use host proteins to be able to productively infect human cells. Many negative strand RNA viruses such as Ebola, Marburg, Nipah, SARS, SARS-CoV-2, and MERS use cathepsin inhibitors to enter and replicate within human cells. Some viruses such as SARS-CoV-2 have their own proteases which allows them to invade host detection or use their proteases to cleave their proteins. However, these proteases alone are not able to sustain cellular infection and the virus must use host protases to be able to infect and replicate within human cells. SARS-CoV-2 has at least two essential proteases and at least one cleavage site in its spike protein for cathepsins. There are several cell proteases/cathepsins that are essential for SARS- CoV-2 productive replication.
[0255] It is hypothesized that proteases such as cysteine cathepsins are essential for viral replication and inhibition of these cathepsins with cysteine cathepsin inhibitor such as Balicatib protects against SARS-CoV-2 infection. An objective is to test clinically advanced cathepsin inhibitors against SARS-CoV-2 infection in relevant human cells and obtaining EC50/90 and determining the best drug-like advanced candidate for repurposing against SARS-CoV-2 infection. This hy pothesis is supported by the data for Balicatib, a cathepsin inhibitor which has been in phase 2 clinical trial for osteoporosis. Balicatib (HB 0121) acts a potent antiviral against SARS-CoV-2 with an effective concentration (EC) which inhibits 50% of SARS-CoV-2 infection in 11M range. Balicatib (HB 0121) has a (SI) and cell cytotoxicity (CC) profile is below. The data shows that Balicatib has a SI of >300 and CC50 of over 100 μM in Calu-3 human cells. Based on these data and Balicatib’ s pharmacokinetics in human after a single dose of 5, 50, or 400mg, the calculated adult human dose is about 50mg-3,600mg daily dosing. Clinical dosing may be further refined based on the exact EC50 and EC90 of Balicatib against SARS-CoV-2.
[0256] Balicatib was purchased from MedChemExpress (www.medchemexpress.com). The vial contained 5mg of Balicatib (HB 0121) the compound was over 97% pure and was dissolved in DMSO to obtain a 10mM concentration. The efficacy of Balicatib was determined in two separate studies. In study one 0.1, 1, and lOμM final concentration was tested and in study two six doses’ concentrations 0.1, 0.3, 1, 3, 10, and 30μM of Balicatib (HB 0121) was examined.
[0257] Below is the protocol that was used to test the efficacy of Balicatib in 6 doses against SARS-CoV-2 using Calu-3 cells. The study number 1, Balicatib (HB 0121) was diluted to 20 μM and then 1 : 10 serial dilutions to achieve 2 μM, 0.2 μM. The rest of the study was similar as outlined below7. Calu-3 Cells were seeded at 2e5cells/well in a 24 w7ell plates in the BSL-2 lab the previous day (cells are at passage 6). Preparation of HB 0121 (Balicatib) inhibitor stock for the experiment: the inhibitor stock vial HB 0121 was prepared at lOmM concentration in DMSO. The inhibitor was tested at concentrations of 30uM, lOuM, 3uM, luM, 0.3uM and O.luM.
[0258] The stock concentrations was prepared according to the following steps: lOmM stock: 10ul + 90ul of direct EMEM media without FBS = 1mM stock; 1mM stock: 150ul + 1.35ml of direct EMEM media without FBS= 100uM stock; 100uM stock: 900ul > 600ul of direct EMEM media without FBS= 60uM stock; 100uM stock: 300ul > 1 ,2ml of direct EMEM media without FBS= 20uM stock; 60uM stock: 150ul + 1.35ml of direct EMEM media without FBS = 6uM stock; 20uM stock: 150ul + 1.35ml of direct EMEM media without FBS= 2uM stock; 6uM stock: 150ul + 1.35ml of direct EMEM^ media without FBS= 0.6uM stock; 2uM stock: 150ul + 1.35ml of direct EMEM media without FBS= 0.2uM stock. [0259] The experiment was set up as follows (8 Wells total): 1) Control well (no infection and no treatment); 2) Control well treated with drag diluent (DMSO at the same final cone, as 30uM: treatment), 3) Inhibitor at 30uM concentration with infection; 4) Inhibitor at 10uM concentration with infection; 5) Inhibitor at 3uM concentration with infection; 6) Inhibitor at luM concentration with infection; 7) Inhibitor at 0.3uM concentration with infection; 8) Inhibitor at O.1uM concentration with infection. [0260] All reagents and the piate were taken into the BSC hood. The cells were pretreated with 500pl of the preparation for each corresponding labeled well. The plates were placed inside the 37°C75% CO2 incubator and allowed to incubate for 4hrs. After the incubation time, the plate will be taken into the BSC and the contents of each well were removed and stored in a separate tube as backup. The cells were infected with SARS-CoV-2 virus (obtained from ATCC/BEI resources Washington strain) at MOI of 0.1 in 100ul of EMEM containing 10% FBS (original titer of stock virus is 4.5e6/ml; so, 4.44ul of the virus stock will be added to each well to achieve 0.1 MOI). The plates were transferred to 370C Z5% C02 incubator for Ihr (rock plate gently every 10 minutes for even distribution of virus). The plates were taken back into the BSC and the virus was removed. The discarded virus was bleached based on GMU SOP. Cells were washed two times with PBS. Mixture of 500ul each of the above inhibitor mixed with 500ul of EMEM containing 20% FBS were prepared and added to each of the corresponding wells. The plates were placed in 37°C/5% CO2 incubator and allowed to incubate for 72hrs. The cells were observed daily for CPE, and their viability were measured at the 72 hour time point. At 72 hours, the supernatants were collected and spun down at 1200 x g for 5 minutes to remove floating cells, and the remaining supernatants were used for plaque assay analysis. The residual cells, pellets, and other tissue culture reagents were bleached per GMU SOP.
Method: Plaque Assay Protocol for SARS-CoV-2
[0261] The reagents for the plaque assay include one or more cell culture media. Complete EMEM+++++ media was prepared using 1 x bottle 2X EMEM for plaques (500ml), 10% FBS (50mL), 1% Minimum Essential Amino Acids (NEAA) (5ml), 1% Sodium Pyruvate (5ml), 1% L-glutamine (200mM) (5ml), 1% Pen/Strep (5ml). Complete DMEM+++ media was prepared using 1x bottle Dulbecco’s modified eagle medium (DMEM) (500mL), lx 25mL aliquot FBS (5%), 1x L-glutamine (200mM) (5ml), lx Pen/Strep (5ml).
[0262] Crystal violet solution was prepared using 1% Crystal violet, 20% Ethanol, 79% d H 20, Agarose (0.6%) and 0.6g in 100mL of H2O.
[0263] On day one, cells were prepared. The day before the assay, 2.5x105 Vero cells/mL were seeded in a 12 well plate and incubated at 37°C/5% CO2 in order to achieve a 90 - 100% confluency the following day. On day 2, samples were prepared. The confluency and health of the Vero cells was checked before starting the assay. Ten-fold dilutions of each test sample in DMEM was performed using deep-well 96 well plates (sample undiluted testing was included as needed). 450 pl DMEM was added to all wells. 50 μl of each test sample was added to the well in the first row containing DMEM and mixed the content of the well by pipetting up and down multiple times. The pipet tips were changed and 50 pl was transferred from the first dilution wells in the first row to the next row of wells using a multichannel pipette. The samples were mixed, the pipette tips were changed and the process was repeated this step until all desired dilutions are prepared.
[0264] On day 3, the infection was carried out. Media was aspirated from the 12 wellplate, and 200 μl of each test sample dilution was added to two separate wells containing the Vero cell monolayer (to prepare technical duplicates). To prevent wells from drying up while all the sample dilutions were added, aspirated-2-4 wells at a time. The plate was incubated for 1 hr at 37°C/5%CO2, rocking it gently every 10-15 mins to prevent drying in the center. The overlay was prepared. A 1:1 mixture of 0.6% agarose in diH2O and EMEM was prepared.
The agarose was heated on a hot plate or in a microwave until it melted and was cooled down in the water bath to about 56°C. The agarose was added to cold EMEM so that the mixture could reach a temperature below 50°C. It is essential not to add the overlay to the cells when it is hotter than 50°C or the cells will die.
[0265] Further, provided 100ul of HB 0121 in DMSO at 10mM concentration was provided. The investigator was blinded to name of the compound to reduce any potential investigator bias on the outcome of the studies. Balicatib (HB 0121) was added to cells at various concentrations and after a few hours of incubation the SARS-CoV-2 was added. The infection was stopped at 72 hours and the amount of infection was recorded based on plaque formation. The control sample contained the highest amounts of DMSO which was used to dilute Balicatib and was used as “control”.
[0266] Once the infection was complete, the plates wzere checked the plates under microscope again. 0.8-lmL of the agarose overlay and incubated at 37°C/5% CO2 for 2 days. The plates were not shaken or agitated during this period to avoid getting smudged plaques.
[0267] The cells were fixed with 10% formaldehyde in diH2O (~1mL per well ) using a pasture pipet and left for 1 hour inside the chemical hood. The formaldehyde was then gently removed using a pasture pipet, making sure not to touch the cells with the pipet tip. The formaldehyde was discarded in the appropriate container. The plate was inverted to expel out the overlay onto a sheet of paper towel inside the sink. A few drops of crystal violet were added to each well and allowed to sit for 5 mins. Using a pasture pipet, added enough diH2O to each well to wash out excess crystal violet. Extra washes could be done if needed. [0268] The plaques in each well were counted, taking the average of technical replicates of the same dilution: The plaques were counted the plaques in each well, taking the average of technical replicates of the same dilution. Pfu/ml was calculated as the average number of plaques divided by the product of dilution (D) and volume of diluted virus added to the plate. After the number of plaques was determined for control and experimental treatments, the following formula was used to obtain % inhibition of the drug. The results are shown in Table 5 and Table 6.
Figure imgf000069_0001
Table 5. Antiviral activity of Baiicatib (HB 0121) against SARS-CoV-2 infection using human lung cells (Calu-3 cells)
Figure imgf000069_0002
[0269] In Table 6, Cell Appearance: No CPEs (no clumping, cell swelling or shrinkage, and no cell detachment). Cells were seeded at 2.0E5 per well of a 24-well plate in a 1 ml volume of media. Cells were at 98.7% viability at seeding.
Table 6. Effect of Balicatib (HB 0121) at 100μM on Calu 3 cell viability and proliferation
Figure imgf000069_0003
[0270] The data clearly shows that Balicatib (HB 0121) is a SARS-CoV-2 antiviral at nM concentrations, as shown in Table 5 and Table 6.
[0271] Table 5 depicts the data obtained from two experiments in which cells were treated with Balicatib (HB 0121) and infected with SAR.S-CoV-2 virus at MOI (Multiplicity of Infection) of 0.1 . The data clearly shows that Balicatib (HB 0121) inhibited the growth of SARS-CoV-2. Balicatib (HB 0121) inhibited almost 100% of the viral growth at 3, 10, and 30 uM. At and below IμM the efficacy of Balicatib was reduced but was substantially different than control. The efficacy, as measured by % inhibition of SARS-CoV-2 growth of Balicatib (HB 0121) was about 68% and 57% at 1 pM and 100nM, respectively. Hence, based on these two studies, it is asserted that the EC50 of Balicatib is in low to high nM range (50- 600nM) and EC90 is in very low uM range (I-3pM). Because in this study Balicatib (HB 0121) was not dosed down enough, below lOOnM, a clear EC50 was not reached. In future studies, Balicatib (HB 0121) may be reduced to lOnM.
[0272] These studies were performed by plaque assay, which is the “gold standard” for analyzing efficacy of antivirals. The plaque assay examines actual infection. Further, the size of the plaque, shape of the plaque, and the quality of the plaques allows one to better understand the quality of antiviral activity of the compounds. However, the plaque assay is difficult to perform and is within 0.5 log accuracy and reproducibility. Therefore, it is not unusual to see differences between several replicate studies, specially at the low end of the effective concentrations of antivirals. Here, at lower concentration of Balicatib (HB0121), the data was variable between the two studies specifically at the 100-300nM. To attempt to decrease variation between studies, Balicatib (HB 0121) will be tested against SARS-CoV-2 and other viruses that use cathepsins for entry and replication by various methods such as QTRPCR and immunostaining or high-content imaging. By using other technologies to test Balicatib against SARS-CoV-2 an opportunity exists to better define Balicatib's EC50 and EC90. Table 5 shows that. Balicatib does not alter cellular viability and it has little to no effect on cell growth. Based on these data Selectivity Index (SI) and Cell Cytotoxicity for Balicatib were calculated to be at >300 and >100μM, respectively.
[0273] The above method (plaque assay) was used to perform antiviral testing for ONO- 5334 (HB 0119) and Odanacatib (HB 0122) against SARS-CoV-2 infection using human lung cells (Calu-3 cells). The data shows that ONO-5334 (HB 0119) has EC50 value of less than 1μM and EC90 is between 1-10 μM, as shown in Table 7. Odanacatib (HB 0122) another cysteine cathepsin inhibitor has lower activity against SARS-CoV-2 infection using Calu-3 cells. This antiviral has EC50 of about lμM and EC90 l-10μM. More studies are being performed to fully define EC50 and EC90 of these compounds by various methods such as QRTPCR and high content imaging. The data clearly show' that both of these compounds are antiviral against SARS-CoV-2 and may be useful for pre or post exposure to the virus in clinical settings.
Table 7. Antiviral activity of ONO-5334 (HB 0119) and Odanacatib (HB 0122) against SARS-CoV-2 infection using human lung cells (Calu-3 cells)
Figure imgf000071_0001
Equivalents and Scope
[0274] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the disclosure described herein. The scope of the present disclosure is not intended to be limited to the above Description, but rather is as set forth in the appended claims.
[0275] In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.
[0276] It is also noted that the term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the term “consisting of’ is thus also encompassed and disclosed.
[0277] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary' skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. [0278] In addition, it is to be understood that any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary' skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the disclosure (e.g., any antibiotic, therapeutic or active ingredient; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.
[0279] It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the disclosure in its broader aspects.
[0280] While the present disclosure has been described at some length and with some particularity with respect to the several described embodiments, it is not intended that, it should be limited to any such particulars or embodiments or any particular embodiment, but it is to be construed with references to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope of the disclosure.

Claims

1. A method of inhibiting the growth of a coronavirus, the method comprising, contacting the coronavirus with a mammalian protease inhibitor; and measuring the growth of the coronavirus, wherein the contacting the coronavirus with the mammalian protease inhibitor, inhibits the growth of the coronavirus.
2. The method of claim 1, wherein the mammalian protease inhibitor has a structure of Formula (I):
Figure imgf000073_0001
wherein,
R1 and R2 are independently H or C1-C7 lower alkyl, or R1 and R2 together with the carbon atom to which they are attached form a C3-C8 cycloalkyl ring;
R3 is an optionally substituted heterocyclic group comprising at least one nitrogen; and n is between 1 and 3.
3. The method of claim 2, wherein the mammalian protease inhibitor has a structure of Formula (II):
Figure imgf000073_0002
wherein X is CH or N; and
R4 is H, C1 -C7 lower alkyl, C1-C7 lower alkoxy, C5-C10 aryl, or C3-C8 cycloalkyl.
4. The method of claim 1, wherein the mammalian protease inhibitor has a structure of:
Figure imgf000074_0001
5. The method of claim 1, wherein the mammalian protease inhibitor has a structure of:
Figure imgf000074_0002
6. The method of claim 1, wherein the mammalian protease inhibitor is a cathepsin inhibitor.
7. The method of claim 6, wherein the cathepsin inhibitor is Balicatib, E-64, E-64a, E- 64b, E-64c, E- 64d, CA-074, CA-074 Me, CA-030, CA-028, peptidyl aldehyde derivatives leupeptin, antipain, chymostatin, Ac-LVK-CHO, Z-Phe-Tyr-CHO, a epoxisuccinate Z-Phe- Tyr(OtBu)-COCHO.H2O, 1-Naphthalenesulfonyl-He-Trp-CHO, Z-Phe-Leu-COCHO.H2O; peptidyl semicarbazone derivatives, peptidyl methylketone derivatives, peptidyl trifluoromethylketone, Biotin-Phe-Ala-fluoromethyl ketone, Z-Leu-Leu-Leu fluoromethyl ketone, Z-Phe-Phe-fluoromethyl ketone, N-Methoxy succinyl -Phe-HOMO-Phe-fluoromethyl ketone, Z-Leu-Leu-Tyr-fluoromethyl ketone, Leupeptin trifluoroacetate, ketone, peptidyl chi oromethases peptidylhydroxymates, peptidylhydroxyl amines, peptidyl acyloxymethanes, peptidyl acyloxymethyl ketones, peptidyl aziridines, peptidyl aryl vinylsufones, peptidyl aryl vinyl sulfonates, gallinamide analogs, peptidyl aldehydes , azepinone-based inhibitors , nitrile-containing inhibitors , thiosemicarbazone , propeptide mimics, thiocarbazate, oxocarbazate, azapaptides , peptidyl halomethylketone derivatives, TLCK; bis(acylamino) ketone, 1,3- Bis(CBZ-Leu-NH)-2-propanone, peptidyl diazomethanes, Z-Phe-Ala-CHN2, Z-Phe-Thr(OBzl)-CHN2, Z-Phe-Tyr (Ot-But)-CHN2, Z- Leu-Leu-Tyr-CHN2; peptidyl acyloxymethyl ketones; peptidyl methylsulfonium salts; peptidyl vinyl sulfones, LHVS; peptidyl nitriles; peptidyl disulfides, 5,5'-dithiobis[2- nitrobenzoic acid], cysteamines, 2,2'-dipyridyl disulfide; N-(4-Biphenylacetyl)-S-methyl cysteine-(D)-Arg-Phe-b phenethyl amide; thiol alkylating agents, maleimides, azapeptides, azobenzenes, O-acylhydroxamates, Z-Phe-Gly-NHO-Bz, Z-FG-NHO-BzOME, lysosomotropic agents, chloroquine, ammonium chloride, Cystatins A, Cystatin B, Cystatin C, Cystatin D, Cystatin F, stefins, kininogens, Sialostain L, antimicrobial peptide LL-37, Procathepsin B Fragment 26*50, Procathepsin B Fragment 36-50, Odanacatib (MK-0822), Relacatib (GSK-462795, SB-462795), SLV213 (K777 OR K1777), RO5459072, RWJ- 445380, VBY036P1 A, AM-3701, MIV-701, MIV-710, MIV-711, NC-2300, ORG-219517, ONO-5334, MK-0674, GB-111-NH2, L-873724, L-006235, AZD4996, VBY-036, RWY- 445380, AM-3840, Cz-007, VBY-825 (VBY-106; VBY-285;VBY-825), VBY-129, SAR- 114137, VBY-891, Petesicatib (RG-7625; RO-5459072), LY-3000328, MIV-247, CRA- 028129, RG-7236, GSK2793660, Aloxistatin (E-64d, Loxi statin, EST), BI-1181181 (VTP- 37948), VBY-376, Aloxistatin (Ab-007; E-64-d), Begacestat (GSI-953; WAY-210953), AL101 (BMS906024), BMS-986115 (AL- 102), MK-0752 (L-000891675), EVP-0962 (EVP- 0015962), SAR-164653, KGP94, VEL-0230, or BLD2660.
8. The method of claim 7, wherein the cathepsin inhibitor is Balicatib.
9. The method of any one of claims 1-8, wherein the coronavirus is contacted with the mammalian protease inhibitor at a concentration of from about 0.1 μM to about 50 μM.
10. The method of claim 9, wherein the concentration of the mammalian protease inhibitor is 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM or 30 μM.
11. The method of claim 9, wherein the mammalian protease inhibitor comprises an effective concentration (EC50) of from about 0.25μM to about 30μM.
12. The method of claim 11, wherein the mammalian protease inhibitor comprises an effective concentration (EC50) of from about 15 μM to about 30 μM.
13, The method of claim 11, wherein the mammalian protease inhibitor comprises an effective concentration (EC50) of from about 0.25μM to about 0.5μM.
14. The method of any one of claims 1-8, wherein the mammalian protease inhibitor comprises an EC90 of from about 1μM to 100μM.
15. The method of claim 14, wherein the EC90 is from about. 1 μM to about. 3μM.
16. The method of claim 1, wherein contacting the coronavirus with the mammalian protease inhibitor inhibits the growth of the coronavirus by from about 50% to about 100%.
17. The method of claim 1, wherein the mammalian protease inhibitor comprises a. selectivity index of at least 300.
18. The method of claim 1, wherein the coronavirus is a SARS-CoV-2 virus, a SARS- CoV-1 virus, a MERS-CoV virus, a 229E virus, a NL63 virus, a OC43 virus, a HKU1 virus, or variants thereof.
19. The method of claim 18, wherein the coronavirus is a. SARS-CoV-2 virus.
20. A method of reducing the percentage of virus infected cells in a population, the method comprising, contacting the virus infected cells with a mammalian protease inhibitor; and measuring the percentage of virus infected cells in the population; wherein the contacting the virus infected cells with the protease inhibitor, inhibits the growth of the virus.
21. The method of claim 20, wherein the mammalian protease inhibitor has a structure of Formula (I):
Figure imgf000077_0001
(I) wherein,
R1 and R2 are independently H or C1-C7 lower alkyl, or R1 and R2 together with the carbon atom to which they are attached form a C3-C8 cycloalkyl ring; and
R3 is an optionally substituted heterocyclic group comprising at least one nitrogen; n is between 1 and 3.
22 The method of claim 21, wherein the mammalian protease inhibitor has a structure of
Formula (II):
Figure imgf000077_0002
(II), wherein X is CH or N; and
R4 is H, C1-C7 lower alkyl, C1-C7 lower alkoxy, C5-C10 aryl, or C3-C8 cycloalkyl.
23. The method of claim 20, wherein the mammalian protease inhibitor has a structure of:
Figure imgf000077_0003
24. The method of claim 20, wherein the mammalian protease inhibitor has a structure of:
Figure imgf000078_0001
25. The method of claim 20, wherein the mammalian protease inhibitor is a cathepsin inhibitor.
26. The method of claim 25, wherein the cathepsin inhibitor is Balicatib, E-64, E-64a, E- 64b, E-64c, E- 64d, CA-074, CA-074 Me, CA-030, CA-028, peptidyl aldehyde derivatives leupeptin, antipain, chymostatin, Ac-LVK-CHO, Z-Phe-Tyr-CHO, a epoxisuccinate Z-Phe- Tyr(OtBu)-COCHO.H2O, 1 -Naphthal enesulfonyl-He-Trp-CHO, Z-Phe-Leu-COCHO.H2O; peptidyl semicarbazone derivatives, peptidyl methylketone derivatives, peptidyl trifluoromethylketone, Biotin-Phe-Ala-fluoromethyl ketone, Z-Leu-Leu-Leu fluoromethyl ketone, Z-Phe-Phe-fluoromethyl ketone, N-Methoxysuccinyl-Phe-HOMO-Phe-fluoromethyl ketone, Z-Leu-Leu-Tyr-fluoromethyl ketone, Leupeptin trifluoroacetate, ketone, peptidyl chloromethases peptidylhydroxymates, peptidylhydroxyl amines, peptidyl acyloxymethanes, peptidylacyloxymethyl ketones, peptidyl aziridines, peptidyl aryl vinylsufones, peptidyl arylvinylsulfonates, gallinamide analogs, peptidyl aldehydes , azepinone-based inhibitors , nitrile-containing inhibitors , thiosemicarbazone , propeptide mimics, thi ocarb azate, oxocarbazate, azapaptides , peptidyl halomethylketone derivatives, TLCK; bis(acylamino) ketone, 1,3- Bis(CBZ-Leu-NH)-2-propanone; peptidyl diazomethanes, Z-Phe-Ala-CHN2, Z-Phe-Thr(OBzl)-CHN2, Z-Phe-Tyr (Ot-But)-CHN2, Z- Leu-Leu-Tyr-CHN2; peptidyl acyloxymethyl ketones; peptidyl methylsulfonium salts; peptidyl vinyl sulfones, LHVS; peptidyl nitriles; peptidyl disulfides, 5,5'-dithiobis[2- nitrobenzoic acid], cysteamines, 2,2'-dipyridyl disulfide; N-(4-Biphenylacetyl)-S-methyl cysteine-(D)-Arg-Phe-b phenethyl amide; thiol alkylating agents, maleimides, azapeptides, azobenzenes, O-acylhydroxamates, Z-Phe-Gly-NHO-Bz, Z-FG-NHO-BzOME, lysosomotropic agents, chloroquine, ammonium chloride, Cystatins A, Cystatin B, Cystatin C, Cystatin D, Cystatin F, stefins, kininogens, Sialostain L, antimicrobial peptide LL-37, Procathepsin B Fragment 26-50, Procathepsin B Fragment 36-50, Odanacatib (MK-0822), Relacatib (GSK-462795, SB-462795), SLV213 (K777 OR K1777), RO5459072, RWJ- 445380, VBY036P1 A, AM-3701, M1V-701, MIV-710, MIV-711, NC-2300, ORG-219517, ONO-5334, MK-0674, GB-111-NH2, L-873724, L-006235, AZD4996, VBY-036, RWY- 445380, AM-3840, Cz-007, VBY-825 (VBY-106; VBY-285;VBY-825), VBY-129, SAR- 114137, VBY-891, Petesicatib (RG-7625; RO-5459072), LY-3000328, MIV-247, CRA- 028129, RG-7236, GSK2793660, Aloxistatin (E-64d, Loxi statin, EST), BM 181181 (VTP- 37948), VBY-376, Aloxistatin (Ab-007; E-64-d), Begacestat (GSI-953; WAY-210953), AL101 (BMS906024), BMS-986115 (AL- 102), MK-0752 (L-000891675), EVP-0962 (EVP- 0015962), SAR-164653, KGP94, VEL-0230, or BLD2660.
27. The method of claim 26, wherein the cathepsin inhibitor is Balicatib.
28. The method of any one of claims 20-27, wherein the coronavirus is contacted with the mammalian protease inhibitor at a concentration of from about 0.1 μM to about 50 μM.
29. The method of claim 28, wherein the concentration of the mammalian protease inhibitor is 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM or 30 μM.
30. The method of claim 28, wherein the mammalian protease inhibitor comprises an effective concentration (EC50) of from about 0.25μM to about 30μM.
31. The method of claim 30, wherein the mammalian protease inhibitor comprises an effective concentration (EC50) of from about 15 μM to about 30 μM.
32. The method of claim 30, wherein the mammalian protease inhibitor comprises an effective concentration (EC50) of from about 0.25μM to about 0.5μM.
33. The method of any one of claims 20-27, wherein the mammalian protease inhibitor comprises an EC90 of from about IμM to lOOμMl
34. The method of claim 33, wherein the EC90 is from about 1 to about 3μMi.
35. The method of claim 20, wherein the contacting the coronavirus with the mammalian protease inhibitor inhibits the growth of the coronavirus by from about 50% to about 100%.
36. The method of claim 20, wherein the mammalian protease inhibitor comprises a selectivity index of at least 300.
37, The method of claim 20, wherein the coronavirus is a SARS-CoV-2 virus, a SARS- CoV-1 virus, a MERS-CoV virus, a 229E virus, a NL63 virus, a OC43 virus, a HKU1 virus, or variants thereof.
38. The method of claim 37, wherein the coronavirus is a SARS-CoV-2 virus.
39. A method of reducing coronavirus infection in a subject, the method comprising: contacting the subject with a mammalian protease inhibitor; and measuring the coronavirus infection in the subject, wherein the contacting the subject with the mammalian protease inhibitor, reduces the coronavirus infection in the subject.
40. The method of claim 39, wherein the mammalian protease inhibitor has a structure of
Formula (I):
Figure imgf000080_0001
(I), wherein,
R1 and R2 are independently H or C1-C7 lower alkyl, or R1 and R2 together with the carbon atom to which they are attached form a C3-C8 cycloalkyl ring; and
R3 is an optionally substituted heterocyclic group comprising at least one nitrogen; n is between 1 and 3.
41. The method of claim 40, wherein the mammalian protease inhibitor has a structure of Formula (II):
Figure imgf000081_0001
(II), wherein X is CH or N; and
R4 is H, C1-C7 lower alkyl, C1-C7 lower alkoxy, C5-C10 aryl, or C3-C8 cycloalkyl.
42. The method of claim 39, wherein the mammalian protease inhibitor has a structure of:
Figure imgf000081_0002
43. The method of claim 39, wherein the mammalian protease inhibitor has a structure of:
Figure imgf000081_0003
44. The method of claim 39, wherein the mammalian protease inhibitor is a cathepsin inhibitor.
45. The method of claim 44, wherein the cathepsin inhibitor is Balicatib, E-64, E-64a, E- 64b, E-64c, E- 64d, CA-074, CA-074 Me, CA-030, CA-028, peptidyl aldehyde derivatives leupeptin, antipain, chymostatin, Ac-LVK-CHO, Z-Phe-Tyr-CHO, a epoxi succinate Z-Phe- Tyr(OtBu)-COCHO.H2O, 1-Naphthalenesulfonyl-He-Trp-CHO, Z-Phe-Leu-COCHO.H:2O; peptidyl semicarbazone derivatives, peptidyl methylketone derivatives, peptidyl trifluoromethylketone, Biotin-Phe-Ala-fluoromethyl ketone, Z-Leu-Leu-Leu fluoromethyl ketone, Z-Phe-Phe-fluoromethyl ketone, N-Methoxysuccinyl-Phe-HOMO-Phe-fluoromethyl ketone, Z-Leu-Leu-Tyr-fluoromethyl ketone, Leupeptin tri fluoroacetate, ketone, peptidylchloromethases peptidylhydroxymates, peptidylhydroxylamines, peptidyl acyloxymethanes, peptidyl acyloxymethyl ketones, peptidyl aziridines, peptidyl aryl vinylsufones, peptidyl arylvinylsulfonates, gallinamide analogs, peptidyl aldehydes , azepinone-based inhibitors , nitrile-containing inhibitors , thiosemicarbazone , propeptide mimics, thiocarbazate, oxocarbazate, azapaptides , peptidyl halomethylketone derivatives, TLCK; bis(acylamino) ketone, 1,3- Bis(CBZ-Leu-NH)-2-propanone; peptidyl diazomethanes, Z-Phe-Ala-CHN2, Z-Phe-Thr(OBzl)-CHN2, Z-Phe-Tyr (Ot-But)-CHN2, Z- Leu-Leu-Tyr-CHN2; peptidyl acyloxymethyl ketones; peptidyl methylsulfonium salts; peptidyl vinyl sulfones, LHVS; peptidyl nitriles; peptidyl disulfides, 5,5'-dithiobis[2- nitrobenzoic acid], cysteamines, 2,2'-dipyridyl disulfide; N-(4-Biphenylacetyl)-S-methyl cysteine-(D)-Arg-Phe-b phenethylamide, thiol alkylating agents, maleimides, azapeptides, azobenzenes, O-acylhydroxamates, Z-Phe-Gly-NHO-Bz, Z-FG-NHO-BzOME, lysosomotropic agents, chloroquine, ammonium chloride, Cystatins A, Cystatin B, Cystatin C, Cystatin D, Cystatin F, stefms, kininogens, Sialostain L, antimicrobial peptide LL-37, Procathepsin B Fragment 26-50, Procathepsin B Fragment 36-50, Odanacatib (MK-0822), Relacatib (GSK-462795, SB-462795), SLV213 (K777 OR KI 777), RO5459072, RWJ- 445380, VBY036P1A, AM-3701, MIV-701, MIV-710, MTV-711, NC-2300, ORG-219517, ONO-5334, MK-0674, GB-111-NH2, L-873724, L-006235, AZD4996, VBY-036, RWY- 445380, AM-3840, Cz-007, VBY-825 (VBY-106; VBY-285;VBY-825), VBY- 129, SAR- 114137, VBY-891, Petesicatib (RG-7625; RO-5459072), LY-3000328, MIV-247, CRA- 028129, RG-7236, GSK2793660, Aloxistatin (E-64d, Loxistatin, EST), BI-1181181 (VTP- 37948), VBY-376, Aloxistatin (Ab-007; E-64-d), Begacestat (GSI-953; WAY-210953), AL101 (BMS906024), BMS-986115 (AL-102), MK-0752 (L-000891675), EVP-0962 (EVP- 0015962), SAR-164653, KGP94, VEL-0230, or BLD2660.
46. The method of claim 45, wherein the cathepsin inhibitor is Balicatib,
47. The method of any one of claims 39-46, wherein the coronavirus is contacted with the mammalian protease inhibitor at a concentration of from about 0.1 μM to about 50 μM.
48. The method of claim 47, wherein the concentration of the mammalian protease inhibitor is 0.1 itM, 0.3 μM, 1 μM, 3 μM, 10 μM or 30 μM.
49. The method of claim 47, wherein the mammalian protease inhibitor comprises an effective concentration (EC50) of from about 0.25p.M to about 30μM.
50. The method of claim 47, wherein the mammalian protease inhibitor comprises an effective concentration (EC50) of from about 15 μM to about 30 μM.
51. The method of claim 49, wherein the mammalian protease inhibitor comprises an effective concentration (EC50) of from about 0.25μM to about 0.5μM.
52. The method of any one of claims 39-46, wherein the mammalian protease inhibitor comprises an EC90 of from about IμM to 100μM.
53. The method of claim 52, wherein the EC90 is from about 1 to about. 3μM.
54. The method of claim 39, wherein the contacting the coronavirus with the mammalian protease inhibitor inhibits the growth of the coronavirus by from about 50% to about 100%.
55. The method of claim 39, wherein the mammalian protease inhibitor comprises a selectivity index of at least 300.
56. The method of claim 39, wherein the coronavirus infection is a SARS-CoV-2 virus infection, a SARS-CoV-1 virus infection, a MERS-CoV virus infection, a 229E virus infection, NL63 virus infection, OC43 virus infection, or HKU1 virus infection.
57. The method of claim 56, wherein the coronavirus infection is a SARS-CoV-2 virus infection.
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