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WO2025085664A1 - Conjugués, compositions et méthodes de traitement de la grippe - Google Patents

Conjugués, compositions et méthodes de traitement de la grippe Download PDF

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Publication number
WO2025085664A1
WO2025085664A1 PCT/US2024/051818 US2024051818W WO2025085664A1 WO 2025085664 A1 WO2025085664 A1 WO 2025085664A1 US 2024051818 W US2024051818 W US 2024051818W WO 2025085664 A1 WO2025085664 A1 WO 2025085664A1
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compound
alkyl
influenza
ligand
mmol
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Mary Lynn Niedrauer
Jeffery Jay Howard NIELSEN
Imrul SHAHRIAR
Ananda Kumar KANDULURU
Philip Stewart Low
Madduri SRINIVASARAO
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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
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds

Definitions

  • the present disclosure includes conjugates comprising multiple haptens linked to a targeting ligand for a target protein on a virus or a virus-infected cell, as well as compositions, such as pharmaceutical compositions, comprising such conjugates and processes for making such conjugates. Additionally, the present disclosure further includes methods of treating viral infections.
  • One such viral infection is influenza.
  • Influenza is one of the most life-threatening disseminated diseases resulting in about 3 to 5 million yearly cases of severe illness and about 250,000 to 500,000 yearly deaths. In addition to causing high morbidity and mortality, influenza imposes a substantial social economic burden arising from the productivity lost and medical prevention and treatment.
  • M2 ion channel inhibitors include amantadine and rimantadine.
  • the mechanism of action of these drugs results from blocking the acid-activated viral M2 ion channel, and as a consequence inhibiting the release of viral ribonucleoprotein from virion to host cytosol.
  • H1N1 and H3N2 viruses currently circulating in humans are resistant to these inhibitors.
  • Centers for Disease Control and Prevention advises against their use due to the rapid emergence of drug resistance.
  • neuraminidase inhibitors include oseltamivir and zanamivir and act as competitive inhibitors competing with sialic acid to bind to the active site of neuraminidase. While these inhibitors are effective against both influenza A and influenza B viruses, they have two major limitations. First, only small benefits have been observed for neuraminidase inhibitors in terms of symptom severity alleviation and sickness duration reduction (0.6-0.7 day out of 7 days). Second, this class of antivirals also suffer from the drug resistant problem. An increase in the number of oseltamivir- resistant strains has been noted since 2007 to 2008 season. In light of the limitations of the current anti-influenza chemotherapies, there is an urgent need to develop new anti- influenza drugs with novel mechanisms of action. SUMMARY
  • T is a radical of a ligand for a target protein of an Influenza virus or a virus-infected cell
  • L 1 , L 2 , and L 3 are each, an independently selected linker
  • a 1 and A 2 are each, a radical of an independently selected hapten.
  • compositions comprising one or more compounds of Formula I: and pharmaceutically acceptable salts or solvates thereof, and optionally including one or more diluents, carriers, or excipients, or combinations thereof; wherein:
  • T is a radical of a ligand for a target protein of an Influenza virus or a virus-infected cell
  • L 1 , L 2 , and L 3 are each, an independently selected linker
  • a 1 and A 2 are each, a radical of an independently selected hapten.
  • compositions for use in treating Influenza and Influenza infection comprising one or more compounds of Formula I: and pharmaceutically acceptable salts or solvates thereof, and optionally including one or more diluents, carriers, or excipients, or combinations thereof; wherein:
  • T is a radical of a ligand for a target protein of an Influenza virus or a virus-infected cell
  • L 1 , L 2 , and L 3 are each, an independently selected linker
  • a 1 and A 2 are each, a radical of an independently selected hapten.
  • unit doses for use in treating Influenza and Influenza infection comprising a therapeutically effective amount of one or more compounds of Formula I: and pharmaceutically acceptable salts or solvates thereof, and optionally including one or more diluents, carriers, or excipients, or combinations thereof; wherein:
  • T is a radical of a ligand for a target protein of an Influenza virus or a virus-infected cell
  • T is a radical of a ligand for a target protein of an Influenza virus or a virus-infected cell
  • L 1 , L 2 , and L 3 are each, an independently selected linker
  • L 1 , L 2 , and L 3 are each, an independently selected linker
  • a 1 and A 2 are each, a radical of an independently selected hapten.
  • T is a radical of a neuraminidase inhibitor.
  • T is a radical of a hemagglutinin inhibitor.
  • the haptens recruit endogenous antibodies present in the host animal.
  • compounds, compositions, and unit doses described herein are used in methods for treating Influenza and Influenza infection in a host animal, where the compounds, compositions, and unit doses are administered to the host animal, and after administration A 1 and A 2 , or any epitope of the foregoing are each bound by one or more antibodies.
  • compounds, compositions, and unit doses described herein are used in methods for treating Influenza and Influenza infection in a host animal, where the compounds, compositions, and unit doses are administered to the host animal, and after administration A 1 and A 2 , or any epitope of the foregoing are each bound by a different antibody.
  • each of L 1 , L 2 , and L 3 is independently selected, and each may comprise a single divalent atom, or a chain of atoms.
  • L 2 may be a bond attaching L 1 to A 1 .
  • each of L 3 may be a bond attaching L 1 to A 2 .
  • the compounds, compositions, unit doses, methods, and uses described herein are useful in treating Influenza and Influenza infection. It is to be understood that such treatment includes prophylactic and preventative treatment of host animals, including host animals at risk of Influenza or Influenza infection, or host animals exposed to Influenza or Influenza infection, including when symptoms of Influenza or Influenza infection have not been observed.
  • the compounds, compositions, unit doses, methods, and uses described herein are useful in preventing the onset of Influenza or Influenza infection, and/or decreasing the severity of later arising symptoms associated with Influenza and Influenza infection.
  • FIG. 1 shows example compounds of the invention.
  • FIG. 2A shows plots of effectiveness of Compound 24 and other compounds with various routes of administration.
  • FIG. 2B shows plots of effectiveness of Compound 24 and other compounds with various routes of administration.
  • FIG. 3 is a synthetic scheme for compound 11, which is used in the synthesis of compound 24.
  • FIG. 4 is a synthetic scheme for compound 24.
  • FIG. 5 is a plot of fluorescence intensity as a function of zanamivir-rhodamine. It shows that zanamivir-rhodamine binds to neuraminidase with a binding affinity of 8.253 nM.
  • FIG. 6 are plots of fluorescence intensity as a function of the log[concentration] (nM) for compound 24 and zanamivir.
  • FIG. 6 shows the competitive binding of compound 24 (left panel) and zanamivir (right panel) to neuraminidase-expressing HEK cells. It shows retained affinity for neuraminidase after incorporation into the conjugate compound 24.
  • FIG. 7 is from the study in Method Example 2 is a plot of TCIDso/mL (Tissue Culture Infectious Dose) as a function of dose (mg/kg).
  • FIG. 7 shows the viral titer measured by hemagglutination 24 h after treatment with Tamiflu (oseltamivir phosphate), vehicle (phosphate- buffered saline; “PBS”), or increasing concentrations of compound 24 (the four measurements shown with a “mg/kg” value reported) .
  • Tamiflu oseltamivir phosphate
  • vehicle phosphate- buffered saline
  • increasing concentrations of compound 24 the four measurements shown with a “mg/kg” value reported
  • FIG. 8 shows the cytokine response to various treatments, including compound 24, in human PBMC (Method Example 6).
  • FIG. 9 shows a plot of normalized fluorescence (RFU) as a function of the log of the concentration of compound 24.
  • FIG. 9 shows neuraminidase inhibition activity of compound 24 against multiple influenza strains.
  • FIG. 10 shows a plot of normalized fluorescence (RFU) as a function of the log of the concentration of Compound 41.
  • FIG. 10 shows neuraminidase inhibition activity of compound 41 against multiple influenza strains (Method Example 7).
  • FIG. 12 from Method Example 5 and shows graphs of cytokine and chemokine levels measured in the lungs of mice from treatment (Compound 24 or Tamiflu) or control groups.
  • FIG. 13 from Method Example 5 and graphs of cytokine and chemokine levels measured in the serum of mice from treatment (Compound 24 or Tamiflu) or control groups.
  • Compound 24 demonstrates a statistically significant improvement over both vehicle control and oseltamivir phosphate positive control.
  • FIG. 15B shows the body weight % (B) following administration of compound 24, oseltamivir phosphate, or vehicle treated mice infected with Influenza A/Hong Kong/2369/2009 (HlNlpdmO9, oseltamivir phosphate (Tamiflu) Resistant).
  • FIG. 15C shows the viral titers present in the lungs of mice treated with compound 24, oseltamivir phosphate, or vehicle 24 h after drug administration and 48 h after infection with 10xLD50 of Influenza A strain - A/Hong Kong/2369/2009 (HlNlpdmO9, oseltamivir phosphate (Tamiflu) Resistant).
  • FIG. 16 shows the prevention of influenza transmission in co-housed guinea pigs by Compound 24.
  • FIG. 17A the change in efficacy of compound 24 compared to oseltamivir phosphate after 5 cycles of in vivo mutation as measured by neuraminidase inhibition.
  • FIG. 17B shows the change in virulence of influenza virus A/PR8/34 (H1N1) after 5 cycles of in vivo mutation using compound 24 compared to oseltamivir phosphate as measured by LDSO measured in vivo.
  • FIG. 18A shows IVIS imaging of Group 1 (mock) and Group 2 (infected and vehicle treated mice).
  • FIG. 18B shows IVIS imaging of Group 3 (infected and oseltamivir phosphate treated mice) and Group 4 (infected and compound 24 treated mice) from Method Example 12.
  • FIG. 19A shows antibody-dependent cellular cytotoxicity (ADCC) induced killing of virus-infected HEK293 cells by Compound 24.
  • FIG. 19B shows antibody-dependent cellular phagocytosis (ADCP) induced killing of virus-infected HEK293 cells by Compound 24.
  • FIG. 19C shows complement dependent cytotoxicity (CDC) induced killing of virus- infected HEK293 cells by Compound 24.
  • FIG. 20 shows the neuraminidase inhibition activity of Compound 24 intranasally (“IN”) against seasonal and drug-resistant Influenza A and B strains.
  • FIG. 21 shows the in vivo efficacy of Compound 24 against A/Dlinois/37/2018 (H1N1, Baloxavir Resistant) - Survival over Time.
  • T is a radical of a ligand for a target protein of an Influenza virus or a virus-infected cell
  • L 1 , L 2 , and L 3 are each, an independently selected linker
  • a 1 and A 2 are each, a radical of an independently selected hapten.
  • hapten is selected from a rhamnose, a nitrophenyl, a nitrophenol, a nitroaniline, a dinitrophenyl (DNP), a dinitrophenol, a dinitroaniline, a trinitrophenyl (TNP), a trinitrophenol, a trinitroaniline, chloronitrophenyl, a chloronitrophenol, a chloronitroaniline, an iodonitrophenyl, an iodonitrophenol, an iodonitroaniline, a nitrotyrosine, an hydroxynitrotyrosine, an aminonitrotyrosine, 4-hydroxy-3 -nitrophenyl acetic acid, an a- galactosyl moiety, a sulfated Gal, a phosphorylcholine, a bacterial antigen, a viral antigen, A 1 and A 2 are each an independently selected radical of a rhamnose, a-galacto
  • a 1 and/or A 2 is a radical of a rhamnose, including L-rhamnose.
  • L 1 , L 2 , and L 3 is a single divalent atom selected from N, O, P, and S, where N and P are optionally substituted.
  • L 1 , L 2 , and L 3 includes or also includes ethoxy, ethylamino, ethylene glycol, aza-ethylene glycol, (PEG)n, or aza-(PEG)n, or a combination thereof, where n is in the range from 2 to about 36.
  • L 1 , L 2 , and L 3 includes or also includes (PEG)n, or aza-(PEG)n, or a combination thereof, where n is 2-36.
  • each of L 1 , L 2 , and L 3 includes or also includes ethoxy, ethylamino, ethylene glycol, aza-ethylene glycol, (PEG)n, or aza-(PEG)n, or a combination thereof, where n is 2-36.
  • L 1 , L 2 , and L 3 includes or also includes O-alkyl-O, N-alkyl-N, C(O)-alkyl-C(O), or NC(O)-alkyl-C(O)N, or a combination thereof.
  • L 1 , L 2 , and L 3 includes or also includes O-alkyl-C(O), N-alkyl-C(O), O-alkyl-N-alkyl-C(O), N-alkyl-O-alkyl-C(O), or C(O)alkyl-C(O), or a combination thereof.
  • C(O)N-diyl O-alkyl-O-alkyl-C(O)N-diyl, orN-alkyl-O-alkyl-O-alkyl-C(O)N-diyl.
  • L 1 , L 2 , and L 3 includes or also includes one or more hydrophilic amino acids selected from Arg, Asn, Asp, Cys, Glu, Gin, His, Lys, Met, Om, Ser, or Thr, including the naturally occurring L-enantiomers of each of the foregoing.
  • L 1 , L 2 , and L 3 includes or also includes one or more hydrophilic amino acids selected from P-NH 2 -Ala, Arg, Asn, Asp, Cys, Glu, Gin, His, Lys, Met, Om, Ser, or Thr, including the naturally occurring L-enantiomers of each of the foregoing.
  • L 1 , L 2 , and L 3 includes or also includes one or more amino acids selected from glycine, serine, proline, ornithine, and lysine.
  • L 1 , L 2 , and L 3 includes or also includes an ornithine or lysine, including L-omithine and L-lysine.
  • L 1 , L 2 , and L 3 includes or also includes a (Pro)s-Lys, including (L-Pro)3-(L-Lys).
  • L 1 , L 2 , and L 3 includes or also includes a (Pro)e-Lys, including (L-Pro) 6 -(L-Lys).
  • L 1 , L 2 , and L 3 includes or also includes glycine and serine, including L-serine.
  • L 1 , L 2 , and L 3 includes or also includes (Gly-Ser)n, including [Gly-(L-Ser)]n, where n is 1-3.
  • L 1 , L 2 , and L 3 includes or also includes (Gly-Ser)2-Lys, including [Gly-(L-Ser)]2-(L-Lys).
  • L 1 , L 2 , and L 3 includes or also includes (Gly-Ser)3-Lys, including [Gly-(L-Ser)]3-(L-Lys).
  • L 1 , L 2 , and L 3 includes or also includes a C(O), C(O)O, C(O)NH, OC(O)NH, or NHC(O)NH group.
  • L 1 , L 2 , and L 3 includes or also includes l,2,3-triazol-l,4-diyl, l,2,3-triazol-l,4-diyl, or a combination thereof.
  • L 1 , L 2 , and L 3 includes or also includes a maleimid-diyl or thiomaleimid-N,S-diyl.
  • L 1 includes a region that is capable of forming an a-helical conformation.
  • L 2 and/or I? includes or also includes (PEG)n or aza-(PEG)n, where n is in the range from about 3 to about 36, from about 4 to about 36, from about 5 to about 36, from about 6 to about 36, or from about 7 to about 36.
  • L 2 and/or L 3 includes or also includes one or more divalent cycloalkyl, including adamantyl, heterocyclyl, including maleimidyl, aryl, heteroaryl including triazolyl, , stilbene, oligoproline, or oligopiperidine groups.
  • T-L 1 The compound of any preceding clause wherein the extended conformation of T-L 1 is at least 5 A, 6 A, 7 A, 8 A, 9 A, 10 A, 11 A, or 12 A in length.
  • L 1 includes or also includes (PEG)n or aza-(PEG)n, where n is in the range from about 3 to about 36, from about 4 to about 36, from about 5 to about 36, or from about 6 to about 36.
  • a compound of the formula or a pharmaceutically acceptable salt or solvate thereof is a compound of the formula or a pharmaceutically acceptable salt or solvate thereof.
  • the conjugate compounds described herein may be formed from a ligand that is capable of targeting a protein of an Influenza or virus-infected cell. In many embodiments, said is protein on the surface of an Influenza or virus-infected cell, or is otherwise accessible from the surface of an Influenza or virus-infected cell.
  • the conjugates described herein may also formed from two or more haptens, each comprising an immune system responsive epitope or antigen.
  • the conjugates described herein may also formed from a polyvalent linker that covalently attaches the ligand to the two or more haptens. Described herein are numerous selections for each of the ligand, the haptens, and the linker.
  • Each of the ligands and haptens described herein may be proteinaceous or small molecules.
  • Each of the linkers described herein may be proteinaceous or small molecules, or combinations thereof. It is to be understood that any ligand, any haptens, and any linker described herein may be combined to form the conjugates.
  • the linkers themselves are formed from various building blocks, including single atoms, functional groups, and chemical fragments. It is to be understood that every combination of building blocks is described herein for forming the linkers included in the conjugates.
  • the conjugates have a dual mechanism of action, where the conjugate inhibits the virus, and also labels or decorates virus-infected cells for intervention by the immune system of the host animal, including humans.
  • the conjugates elicit host animal immune response against the virus or virus-infected cell by recruiting antibodies in the host animal.
  • the antibodies are endogenous.
  • the anti-hapten antibodies bind to the hapten and activate the innate immune system against the target virus and virus-infected cells.
  • the haptens may be the same or different. In the conjugates where the haptens are the same, the immune system of the host animal may be multiplied. In the conjugates where the haptens are different, the immune system of the host animal may respond in multiple ways.
  • the conjugates described herein provide a therapeutic delivery system for selectively or specifically delivering haptens to target virus and virus-infected cells. In certain embodiments, the haptens are selected to activate the innate immune system of the subject to recruit immune cells and/or otherwise leverage the subject’s own immune system against the virus.
  • the targeting ligand can selectively or specifically recognize a target protein or receptor, such as an envelope protein of a virus, which can be highly or exclusively expressed on the virus, the surface of an infected cell, or accessible from the surface of an infected cell.
  • the conjugate compound of Formula I can have fragments L ⁇ -A 1 and L 3 -A 2 bound to the same atom on L 1 .
  • the same atom is not a carbon atom.
  • the same atom is a nitrogen atom.
  • the conjugate of Formula I can have fragments I ⁇ -A 1 and L 3 -A 2 bound to a different atom on L 1 .
  • the compounds, compositions, unit doses, and methods described herein are usefill in treating Influenza A, Influenza B, and Avian Influenza.
  • Illustrative strains of Influenza that are treatable using the compounds, compositions, unit doses, and methods described herein include, but are not limited to, H1N1, HlNlpdmO9, H3N2, and avian strains H5N1 and H7N9, and resistant strains, such as HlNlpdmO9, oseltamivir-resistant, and HlNlpdmO9, baloxavir- resistant.
  • the method can elicit an immune response leading to clearance of an antibody (Ab)-coated virus or an Ab-coated-virally infected cell via Ab- dependent cellular phagocytosis (ADCP), Ab-dependent cellular cytotoxicity (ADCC), and/or complement-dependent cytotoxicity (CDC) which works in conjunction with the inhibition of viral budding by neuraminidase inhibition leading to viral eradication.
  • the method for activating an immune response can further comprise administering to the subject autologous antibodies, allogeneic IgG antibodies, or human IVIG. In these and other embodiments, the subject may be further treated with anti-hapten antibodies.
  • T is a radical of a neuraminidase inhibitor.
  • Illustrative neuraminidase inhibitors include, but are not limited to, sialic acid and analogs and derivatives thereof, zanamivir, peramivir, laninamivir, oseltamivir, 2,3-dehydro-2-deoxy-n-acetylneuraminic acid, and analogs and derivatives of the foregoing, such as compounds disclosed in .US 6340702, WO 1991/016320, WO 1996/026933, WO 1999/033781, and the like.
  • the ligand is zanamivir or an analog or derivative thereof.
  • a radical may be formed on any atom of the foregoing to form the radical T.
  • T is a radical of the formula having any specific stereochemical configuration, or a having a mixture of two or more stereochemical configurations.
  • T is a radical of the formula
  • the ligand is peramivir or an analog or derivative thereof, including compounds described in Chand et al. J Med Chem 44(25):4379-92 (2001) Bai et al. Viruses 13(624): 1-13 (2021), and the like.
  • T is a radical of the formula
  • the ligand is an amino analog of peramivir or an analog or derivative thereof.
  • T is a radical of the formula
  • the ligand is laninamivir or an analog or derivative thereof.
  • the ligand is a compound of the formula or an analog or derivative thereof.
  • the ligand is oseltamivir
  • the ligand is of the formula wherein R is alkyl, H, or salt; R 1 is NHz, NHCH 2 CH 2 OH, azido, guanidino, NHallyl, N(aHyl)z, or a heterocyclyl group; and R 2 is NHz or acetamido.
  • the ligand is of the formula wherein R is Me, H, or salt; Ri is NHz or, guanidino; R 2 is NH 2 or acetamido; and R4 is alkyl (C- 4 to C-14), and ether, acyl, and carbamoyl derivatives thereof.
  • the ligand is of the formula where R is Me or Et; R 2 is H or OH; Ri is alkyl (primary and secondary, C-3 to C-10); and R3 is NH 2 or optionally substituted guanidino.
  • the ligand is of the formula wherein R is H, Me, Et or a salt; Ri is NH2, azido, or guanidino; R 2 is C(O)CH 3 , C(O)CF 3 , or SOaMe; and R3 is alkyl (primary or secondary, C-1 to C-5), or CH 2 OMe.
  • T is a radical of a hemagglutinin inhibitor.
  • Illustrative hemagglutinin inhibitors include, but are not limited to, flufirvitide 3, umifenovir, arbidol, tert-butyl hydroquinone, and the like.
  • the ligand is of the formula and analogs and derivatives thereof, including compounds described in Kitamura et al. PNAS 121(22): 1-9 (2024), Bai et al. Viruses 13(624): 1-13 (2021), and the like.
  • a radical may be formed on any atom of any of the foregoing ligands to form the radical T.
  • T is a radical of flufirvitide 3 of the formula VEDTKIDLWSYNAELL (SEQ ID NO: 1).
  • radicals may be formed at the N-terminus, the C-terminus, or on an interior atom on the peptide chain to form the radical T. It is also to be understood that other radicals may be formed on a side chain of an amino acid to form the radical T.
  • each hapten described herein can each be bound by an antibody. After administration to a subject, each hapten (A 1 and A 2 ) can be bound by an antibody. In certain embodiments, the two haptens, A 1 and A 2 can each be bound by a different antibody after administration to a subject. It is to be understood that any hapten, including peptide sequences, that elicit an immunological response in the host animal may be used to form the conjugates described herein. It is to be further understood an immunological response in the host animal may be elicited or supplemented via vaccination as a co-therapy to the methods and uses described herein.
  • a 1 and A 2 are each an independently selected radical of an antigen where for each antigen there is an endogenous antibody present in the host animal.
  • Illustrative haptens include, but are not limited to, a rhamnose, including L-rhamnose, a nitrophenyl, a nitrophenol, a nitroaniline, a dinitrophenyl (DNP), a dinitrophenol, a dinitroaniline, a trinitrophenyl (TNP), a trinitrophenol, a trinitroaniline, chloronitrophenyl, a chloronitrophenol, a chloronitroaniline, an iodonitrophenyl, an iodonitrophenol, an iodonitroaniline, a nitrotyrosine, an hydroxynitrotyrosine, an aminonitrotyrosine, 4-hydroxy-3 -nitrophenyl acetic acid, an a- galactosyl moiety, a sulfated Gal
  • the hapten is selected from a dinitrophenyl (DNP), a dinitrophenol, and a dinitroaniline.
  • a 1 and/or A 2 is a radical of a dinitrophenyl (DNP), a dinitrophenol, or a dinitroaniline, including radicals of the formulae where L 2 or L 3 is attached at (*).
  • the hapten is selected from a rhamnose, including L-rhamnose.
  • a 1 and/or A 2 is a radical of a rhamnose, including L-rhamnose, and including a radicals of the formulae where L 2 or L 3 is attached at (*).
  • a 1 is a DNP radical and A 2 is a rhamnose radical, or vice versa.
  • a 1 is a DNP radical and A 2 is an L-rhamnose radical, or vice versa.
  • neither A 1 nor A 2 comprises a fluorescein, such as FITC, and the like.
  • linker generally refers to a chain of atoms that connects two or more functional parts of a molecule to form a conjugate.
  • the chain of atoms is selected from C, N, O, S, Si, and P, or C, N, O, S, and P, or C, N, O, and S.
  • the chain of atoms covalently connects different functional capabilities of the conjugate.
  • the linker may have a wide variety of lengths, such as in the range from about 2 to about 100 atoms in the contiguous backbone.
  • the atoms used in forming the linker may be combined in all chemically relevant ways, such as chains of carbon atoms forming alkylene, alkenylene, and alkynylene groups, and the like; chains of carbon and oxygen atoms forming ethers, polyoxyalkylene groups, or when combined with carbonyl groups forming esters and carbonates, and the like; chains of carbon and nitrogen atoms forming amines, imines, polyamines, hydrazines, hydrazones, or when combined with carbonyl groups forming amides, ureas, semicarbazides, carbazides, and the like; chains of carbon, nitrogen, and oxygen atoms forming alkoxyamines, alkoxylamines, or when combined with carbonyl groups forming urethanes, amino acids, acyloxylamines, hydroxamic acids, and the like; and many others.
  • the atoms forming the chain in each of the foregoing illustrative embodiments may be either saturated or unsaturated, thus forming single, double, or triple bonds, such that for example, alkanes, alkenes, alkynes, imines, and the like may be radicals that are included in the linker.
  • the atoms forming the linker may also be cyclized upon each other or be part of cyclic structure to form divalent cyclic structures that form the linker, including cycloalkanes, cyclic ethers, cyclic amines, and other heterocycles, arylenes, heteroarylenes, and the like in the linker.
  • the linker length may be defined by any pathway through the one or more cyclic structures.
  • the linker length is defined by the shortest pathway through the each one of the cyclic structures.
  • the linkers may be optionally substituted at any one or more of the open valences along the chain of atoms, such as optional substituents on any of the carbon, nitrogen, silicon, or phosphorus atoms.
  • the linker may connect the two or more functional parts of a molecule to form a conjugate at any open valence, and it is not necessary that any of the two or more functional parts of a molecule forming the conjugate are attached at any apparent end of the linker.
  • L 1 , L 2 , and L 3 each, independently comprises a chain of atoms from 3 atoms to about 60 atoms in length. In many embodiments, L 1 , L 2 , and L 3 each, independently comprises a chain of atoms from about 4 A to about 72 A in length.
  • the chain of atoms are part of the backbone of the conjugate of Formula I.
  • the term “backbone” of the linker L refers to the shortest chain of contiguous atoms forming a covalently bonded connection between T and L 2 , between T and L 3 , between A 1 and L 1 , or between A 2 and L 1 .
  • L 1 , L 2 , or L 3 each, independently, comprise a chain of atoms at least 3 atoms in length, at least 7 atoms in length, at least 10 atoms in length, at least 14 atoms in length, or at least 20 atoms in length.
  • L 1 , L 2 , or L 3 each, independently, have a chain of between 3 and 7 atoms in length, between 7 and 10 atoms in length, between 10 and 14 atoms in length, between 14 and 20 atoms in length, between 20 and 30 atoms in length, between 30 and 40 atoms in length, between 40 and 50 atoms in length, or between 50 and 60 atoms in length.
  • L 1 , L 2 , or L 3 each, independently, comprise a chain of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 atoms in length.
  • the term “between” is inclusive of the endpoints meaning that between 3 and 5 atoms in a chain length includes 3 atoms, 4 atoms, and 5 atoms.
  • L 1 , L 2 , or L 3 can each, independently, comprise one or more amino acid or peptide residues.
  • amino acid refers generally to beta, gamma, and longer amino acids, and including cyclic groups, that have both an amino group and an acid group from each of which a radical can be formed.
  • Illustrative acyclic amino acids include the formula: -N(R>(CR'R") q -C(O)- where R is hydrogen, alkyl, acyl, or a suitable nitrogen protecting group, R' and R" are hydrogen or a substituent, each of which is independently selected in each occurrence, and q is an integer such as 1, 2, 3, 4, or 5.
  • R' and/or R" independently correspond to, but are not limited to, hydrogen or the side chains present on naturally occurring amino acids, such as methyl, benzyl, hydroxymethyl, thiomethyl, carboxyl, carboxylmethyl, guanidinopropyl, and the like, and derivatives and protected derivatives thereof.
  • the above described formula includes all stereoisomeric variations.
  • the amino acid may be selected from asparagine, aspartic acid, cysteine, glutamic acid, lysine, glutamine, arginine, serine, ornithine, threonine, and the like.
  • L 1 , L 2 , or L 3 can each, independently, comprise one or more alkyl groups.
  • L 1 , L 2 , or L 3 can each, independently, comprise one or more or sugar moieties, glycan residues, or peptidoglycan residues.
  • bonds connecting atoms in the chain can be either saturated or unsaturated, such that for example, alkanes, alkenes, alkynes, cycloalkanes, arylenes, imides, and the like can be divalent radicals that are included in L.
  • the atoms of L in the chain can be substituted or unsubstituted.
  • the atoms forming the linker may also be cyclized to form saturated or unsaturated divalent cyclic radicals in the linker, such as radicals of the formulae: wherein each X 1 is independently CH 2 , NR’, or O wherein R’ is alkyl or hydrogen and each X 2 is independently S, O, N, NH, CR” wherein R” is alkyl or hydrogen.
  • radicals include:
  • L 1 , L 2 , or I? comprises suitable substituents that change the hydrophobicity or hydrophilicity of the linker.
  • Illustrative hydrophobic groups include alkyl, cycloalkyl, aryl, and arylalkyl, each of which is optionally substituted.
  • L 1 , L 2 , or I? can each, independently, comprise alkylene-amino-alkylenecarbonyl, alkylene-thio-(carbonylalkylsuccinimid-3-yl) moieties, including the following formulae: wherein x and y are each independently 1, 2, 3, 4, or 5, where the asterisk identifies points of attachment either to other linker fragments of to T or A 1 or A 2 .
  • linkers include 1- alkylsuccinimid-3-yl, carbonyl, thionocarbonyl, alkyl, cycloalkyl, alkylcycloalkyl, alkylcarbonyl, cycloalkylcarbonyl, carbonylalkylcarbonyl, 1 -alkyl succinimid-3-yl, 1- (carbonylalkyl)succinimid-3-yl, alkylsulfoxyl, sulfonylalkyl, alkylsulfoxylalkyl, alkylsulfonylalkyl, carbonyltetrahydro-2H-pyranyl, carbonyltetrahydrofuranyl, 1- (carbonyltetrahydro-2H-pyranyl)succinimid-3-yl, and 1-
  • each group can be substituted or unsubstituted.
  • one or more of the aforementioned groups can be used in combination (or more than once) (e.g., -alkyl-C(O)-alkyl) and may further comprise an additional nitrogen (e.g., alkyl-C(O)-NH-, -NH-alkyl-C(O)- or -NH-alkyl-), oxygen (e.g., - alkyl-O-alkyl-) or sulfur (e.g., -alkyl-S-alkyl-).
  • nitrogen e.g., alkyl-C(O)-NH-, -NH-alkyl-C(O)- or -NH-alkyl-
  • oxygen e.g., - alkyl-O-alkyl-
  • sulfur e.g., -alkyl-S-alkyl-
  • Examples include alkylcarbonyl, cycloalkylcarbonyl, carbonylalkylcarbonyl, l-(carbonylalkyl)succinimid-3-yl, and succinimid-3-ylthiol, wherein each group can be substituted or unsubstituted.
  • L 1 , L 2 , or I? can be formed via click chemistry or be click chemistry-derived.
  • L 1 , L 2 , or L 3 can be derived from copper-catalyzed azidealkyne cycloaddition (CuAAC), strain promoted azide-alkyne cycloaddition (SPAAC), inverse electron demand Diels-Alder reaction (lEDDA), and Staudinger ligation (SL).
  • CuAAC copper-catalyzed azidealkyne cycloaddition
  • SPAAC strain promoted azide-alkyne cycloaddition
  • lEDDA inverse electron demand Diels-Alder reaction
  • SL Staudinger ligation
  • T can be a moiety of the formula T-N3.
  • T-N3 can then be reacted with an alkyne as shown in the following Scheme: where the wavy line connected to T and to A 1 / A 2 represents a linker between T and AVA 2 and
  • L 1 , L 2 , or L 3 can include where x is an integer from 0 to 50 and y is an integer from 0 to 50 [0100] In other embodiments, L 1 , L 2 , or L 3 can include wherein each of R 2 and R 3 is independently H or C 1-6 alkyl; and z is an integer from 1 to 8.
  • L 1 , L 2 , or L 3 can include an amide, ester, urea, carbonate, carbamate, amino acid, amine, ether, alkyl, alkene, alkyne, heteroalkyl cycloalkyl, aryl, heterocycloalkyl, heteroaryl, carbohydrate, glycan, peptidoglycan, polypeptide, or any combination thereof.
  • L 1 , L 2 , or L 3 can include a glycosylated amino acid.
  • L 1 , L 2 , or L 3 can include one or more monosaccharide, disaccharide, polysaccharide, glycan, or peptidoglycan.
  • L 1 , L 2 , and L 3 do not comprise a glycan.
  • L 1 , L 2 , and L 3 do not comprise a sugar.
  • L 1 , L 2 , or L 3 can include a rigid functionality such as an oligoproline or oligopiperidine.
  • an oligoproline or oligopiperidine has about two up to and including about fifty, about two to about forty, about two to about thirty, about two to about twenty, about two to about fifteen, about two to about ten, or about two to about six repeating units (e.g., prolines or piperidines).
  • L 1 , L 2 , or L 3 can comprise (-CH 2 CH 2 -O-)n, where n is an integer between and including 1 and 36 (e.g., 1 to 2, 2 to 6, 3 to 8, 6 to 12, and 4 to 10) a peptide, an alkylamido group (e.g., C(O)N(H)C 2 -C 18 alkyl- or C 2 -C 18 alkyl-C(O)N(H)-), an alkylamidoalkyl group (e.g., a C 2 -C 18 alkyl-C(O)N(C 2 -C 18 alkyl) 2 or a C 2 -C 18 alkyl-C(O)N(H)-C 2 -C 18 alkyl group, such as a -CH 2 CH 2 C(O)N(CH 2 CH 2 ) 2 or a -CH 2 CH 2 C(O)N(H)(CH 2 CH 2 )- group
  • L 1 , L 2 , or L 3 can comprise wherein m is an integer from 0 to 20, such as from 1 to 20, 0 to 15, 1 to 10, 2 to 10, 2 to 8 or 3 to 9.
  • m can be 0 or 1.
  • m can be 5 or 6.
  • m can be 7 or 8.
  • L 1 , L 2 , or L 3 can comprise wherein p and q are each, independently, an integer from 0 to 20, such as from 1 to 20, 0 to 15, 1 to 10, 2 to 10, 2 to 8 or 3 to 9.
  • p can be 2 or 3.
  • q can be 2 or 3.
  • L 1 , L 2 , or L 3 can comprise wherein d is an integer from 0 to 20, such as from 1 to 20, 0 to 15, 1 to 10, 2 to 10, 2 to 8 or 3 to 9.
  • d can be 1, 2 or 3.
  • L 1 , L 2 , and L 3 taken together can comprise
  • m, p, d, and q are each, independently, an integer from 0 to 20, such as from 1 to 20, 0 to 15, 1 to 10, 2 to 10, 2 to 8 or 3 to 9.
  • m can be 0 or 1.
  • m can be 5 or 6.
  • m can be 7 or 8.
  • p can be 2 or 3.
  • q can be 2 or 3.
  • d can be 1, 2 or 3.
  • L 1 , L 2 , and L 3 taken together can comprise [0108] In another embodiment, the following compounds are described
  • the target protein can be an envelope protein of an influenza or an influenza envelope protein on the surface of a virus-infected cell.
  • the target protein can be influenza neuraminidase or influenza hemagglutinin.
  • the compounds described herein may contain one or more chiral centers, or may otherwise be capable of existing as multiple stereoisomers. It is to be understood that in one embodiment, the invention described herein is not limited to any particular stereochemical requirement, and that the compounds, and compositions, methods, uses, and medicaments that include them may be optically pure, or may be any of a variety of stereoisomeric mixtures, including racemic and other mixtures of enantiomers, other mixtures of diastereomers, and the like. It is also to be understood that such mixtures of stereoisomers may include a single stereochemical configuration at one or more chiral centers, while including mixtures of stereochemical configuration at one or more other chiral centers.
  • composition generally refers to any product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combinations of the specified ingredients in the specified amounts. It is appreciated that certain functional groups, such as the hydroxy, amino, and like groups form complexes and/or coordination compounds with water and/or various solvents, in the various physical forms of the compounds. It is to be understood that the compositions described herein may be prepared from isolated compounds described herein or from salts, solutions, hydrates, solvates, and other forms of the compounds described herein. It is also to be understood that the compositions may be prepared from various amorphous, non-amorphous, partially crystalline, crystalline, and/or other solid forms of the compounds described herein.
  • compositions can be prepared by combining one or more conjugates with one or more pharmaceutically acceptable excipients, carriers, or diluents, or any combination thereof, and, optionally, one or more additional pharmaceutically active agents
  • n is an integer from 0 to 8
  • the individual and selectable values of 0, 1, 2, 3, 4, 5, 6, 7, and 8 such as n is 0, or n is 1, or n is 2, etc.
  • the recitation that n is an integer from 0 to 8 also describes each and every subrange, each of which may for the basis of a further embodiment, such as n is an integer from 1 to 8, from 1 to 7, from 1 to 6, from 2 to 8, from 2 to 7, from 1 to 3, from 2 to 4, etc.
  • the recitation of a numerical value necessarily reflects the relative precision of the numerical value.
  • the recitation of a number with a specified precision based on significant figures necessarily includes a range of values that would match that number after appropriate rounding.
  • the recitation of the number 1 with a single significant figure is understood to properly refer to a range of values from 0.5 to 1.4.
  • the recitation of the number 1.0 with two significant figures is understood to properly refer to a range of values from 0.95 to 1.04.
  • the relative precision of the numerical value can be further indicated by modifying with the term “about” to indicate that the modified number has lower precision.
  • the term “about” when used with numerical values or limits generally means that the number is approximate and that, as recited, it is understood to include a range of values.
  • a real number that is recited with a single significant figure would by definition include a so-called rounding range; the number about 5 would at the very least include the range 4.5-S.4, as each of those values rounds to 5.
  • the same is to be understood for real numbers expressed with additional significant figures, where the corresponding rounding range applies to the last significant figure. Integers are to be understood to at least include the values ⁇ 1 for single-digit numbers, ⁇ 10 for two-digit numbers, etc.
  • the term “about” is also interpreted to contemplate a range based on a percentage of the recited number, such as about 5 construed to include 5 ⁇ 10% or 5 ⁇ 20%. Notwithstanding the foregoing, it is understood that the range of values, unless otherwise indicated, should not be interpreted to include a negative range for a positively recited number, and vice-versa. In addition, depending up on the context, the recited number, unless otherwise indicated, should not be interpreted to include a value of zero when used in conjunction with an added component.
  • radical refers to a ligand for a target protein or hapten, respectively, as described herein, where one or more atoms or groups, such as a hydrogen atom, or an alkyl group on a heteroatom, and the like, is removed to provide a radical for covalent linking or conjugation to the polyvalent linkers L 1 , L 2 , and L 3 .
  • one or more groups such as a hydrogen atom, or an alkyl group on a heteroatom, and the like
  • Illustrative analogs include, but are not limited to, those compounds that share functional and in some cases structural similarity to those compounds described hereinlt is to be understood that such radicals can also be formed on acid, ester, or amide groups, such as carboxy, phosphoryl, and sulfuryl acids, by removing the OH, ester, or amide group. It is also to be understood that such radicals can be formed by removing other fragments, such as halo, alkoxy, amino, heterocyclyl, or heteroaryl groups.
  • alkyl group is a saturated, partially saturated, or unsaturated straight chain or branched non-cyclic hydrocarbon having from 1 to 10 carbon atoms (C 1 -C 10 alkyl), from 1 to 8 carbons (C 1 -C 8 alkyl), from 1 to 6 (C 1 -C 6 alkyl), 1 to 4 (C 1 -C 4 alkyl), 1 to 3 (C 1 -C 3 alkyl), or 2 to 6 (C 2 -C 6 alkyl) carbon atoms.
  • the alkyl group has monovalency. Examples of alkyl groups with monovalency include -CH 3 , -CH 2 CH 3 , and the like.
  • Monovalent alkyls may be found on substitutions in the chain of linker, L, for example.
  • the alkyl group has bivalency, such as when found in the chain of the linker, L. Examples of alkyl groups with bivalency include, but are not limited to, -CH 2 -, -CH 2 CH 2 -, and the like.
  • the alkyl group is a saturated alkyl group.
  • an alkyl group is an unsaturated alkyl group, also termed an alkenyl group or an alkynyl group.
  • heteroalkyl by itself or in combination with another term means, unless otherwise stated, a stable straight or branched chain, or combination(s) thereof, consisting of at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quartemized.
  • the heteroatom(s) O, N, P, S, and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule.
  • Up to two heteroatoms may be consecutive, such as, for example, — CH 2 — NH— OCH3.
  • aryl includes monocyclic and polycyclic aromatic carbocyclic groups, each of which may be optionally substituted.
  • Illustrative aromatic carbocyclic groups described herein include, but are not limited to, phenyl, naphthyl, and the like.
  • heteroaryl includes aromatic heterocyclic groups, each of which may be optionally substituted.
  • Illustrative aromatic heterocyclic groups include, but are not limited to, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, tetrazinyl, quinolinyl, quinazolinyl, quinoxalinyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, benzimidazolyl, benzoxazolyl, benzthiazolyl, benzisoxazolyl, benzisothiazolyl, and the like.
  • optionally substituted includes the replacement of hydrogen atoms with other functional groups on the radical that is optionally substituted.
  • Such other functional groups illustratively include, but are not limited to, amino, hydroxyl, halo, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic acids and derivatives thereof, carboxylic acids and derivatives thereof, and the like.
  • any of amino, hydroxyl, thiol, alkyl, haloalkyl, heteroalkyl, aryl, aiylalkyl, arylheteroalkyl, heteroaiyl, heteroarylalkyl, heteroarylheteroalkyl, and/or sulfonic acid is optionally substituted.
  • the compounds described herein can be used for both human clinical medicine and veterinary applications.
  • the host animal treated with the compounds described herein can be human or, in the case of veterinary applications, can be a laboratory, agricultural, domestic, or wild animal.
  • the present invention can be applied to host animals including, but not limited to, humans, laboratory animals such rodents (e.g., mice, rats, hamsters, etc.), rabbits, monkeys, chimpanzees, domestic animals such as dogs, cats, and rabbits, agricultural animals such as cows, horses, pigs, sheep, goats, and wild animals in captivity such as bears, pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas, dolphins, and whales.
  • rodents e.g., mice, rats, hamsters, etc.
  • rabbits, monkeys, chimpanzees domestic animals
  • domestic animals such as dogs, cats
  • rabbits agricultural animals
  • therapeutically effective amount refers to that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.
  • the therapeutically effective amount is that which may treat or alleviate the disease or symptoms of the disease at a reasonable benefit/risk ratio applicable to any medical treatment.
  • the total daily usage of the compounds and compositions described herein may be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically-effective dose level for any particular patient will depend upon a variety of factors, including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, gender and diet of the patient: the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidentally with the specific compound employed; and like factors well known to the researcher, veterinarian, medical doctor or other clinician of ordinary skill.
  • the conjugates and compositions may be delivered by suitable methods of delivery including, for example but not limited to, intranasally, orally, and intravenously.
  • the method can further comprise administering autologous antibodies or allogeneic Immunoglobulin G (IgG) antibodies.
  • IgG Immunoglobulin G
  • the monotherapy may include co-administration of one or more carriers, vehicles, diluents, adjuvants, excipients, and the like, and combinations thereof, and/or include co-administration of one or more additional active pharmaceutical ingredients
  • those latter additional active pharmaceutical ingredients are to be understood to be for treating diseases and/or symptoms distinct from treating the underlying conditions described herein, such as the treatment of the viral infection itself.
  • Illustrative additional active pharmaceutical ingredients may include, for example, active ingredients for treating pain, inflammation, cough, congestion, and the like.
  • EXAMPLE 1 (3S,4R)-3-acetamido-2-((14S,40S,41R)-14-(4-(3-(2-((2,4- dinitrophenyl)amino)ethoxy)propanamido)butyl)-41,42-dihydroxy-13,1638-trioxo-l- (((2R3R,4R3R,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)- 3,6,9,19,22,25,28,31,34,39-decaoxa-12,15,37-triazadotetracontan-40-yl)-4-guanidino-3,4- dihydro-2H-pyran-6-carboxylic acid (Compound 24) (FIGS. 3-4)
  • Compound 7 a-L-rhamnose monohydrate (6, Sigma- Aldrich, 1.0 g, 5.49 mmol) was dissolved in 9.2 mL of anhydrous pyridine. The solution was stirred in an ice bath and purged with nitrogen gas prior to the dropwise addition of acetic anhydride (4.15 mL, 43.92 mmol, 8.0 eq.) for 15 min, maintaining the internal temperature below 10 °C. The reaction was slowly warmed to ambient temperature over 2 h. After 20 h, the reaction mixture was poured into EtOAc and extracted twice with 1.0 M HC1.
  • Compound 24 is prepared by deprotection of compound 22. It is understood that the deprotection of compound 22 may be accomplished by initial removal of the Boc and ketal protecting groups using TFA in DCM, followed by deprotection of the methyl ester and acetyl ester protecting groups using LiOH H2O in MeOH. Alternatively, the deprotection of compound 22 may be accomplished by initial removal of the methyl ester and acetyl ester protecting groups using NaOMe in MeOH, followed by deprotection of the Boc and ketal protecting groups using TFA in DCM, as follows.
  • Compound 6 was prepared using the same procedure to make compound 3 in this Example. Compound 6 was made from compound 5 (0.11 g, 0.37 mmol) and compound 4 (0.35 g, 0.37 mmol) in DMF (10.0 mL), and HATU (0.15 g, 0.40 mmol) and ethylbi s(propan-2- yl)amine (0.34 mL, 1.85 mmol). After reaction completion, the reaction mixture was concentrated and the remainder was purified by silica gel column chromatography eluting in 10% MeOH/DCM to afford compound 6 as a yellowish liquid. Yield: 0.20 g, 44.03 %; LCMS (ESI) m/z 1225.30 [M+1] + .
  • Compound 11 was prepared using the same procedure to make compound 3 in this Example. Compound 11 was made from compound 10 (0.11 g, 0.14 mmol) and compound 7 (0.17 g, 0.15 mmol) in DMF (5.0 mL), and HATU (0.08 g, O.lSmmol) and ethylbis(propan-2-yl)amine (GLR, 0.13 mL, 0.70 mmol). After reaction completion, the reaction mixture was concentrated, and the remainder was purified by silica gel column chromatography eluting in 10% MeOH/DCM to afford compound 11 as a yellowish liquid. Yield: 0.12 g, 45.42 %; LCMS (ESI) m/z 1896.00 [M+1] + .
  • Compound 41 Compound 7 (0.079 g, 0.048 mmol) was dissolved in MeOH (1.2 mL) and treated dropwise with 0.5 M sodium methoxide in MeOH (0.5 mL, 3.5 eq.) and stirred Ih. After reaction completion, the reaction mixture was neutralized by adding Dowex® 50WX8 (H+) resin, filtered, washed with MeOH and concentrated. The remainder was purified by prep- UPLC on a C 18 column 5-95% gradient B over 45 min, flow 15 mL/min (A: 20 mM NH*OAc, pH 7 buffer and B: ACN; UV @ 360 nm and 240 nm) to give Compound 41 (3.3 mg, 50% yield).
  • Compound 3 was prepared using the same procedure to make compound 10 in Example 2. Compound 3 was made from compound 1 (0.10 g, 0.13 mmol) in dry THF (5 ml), DIPEA (0.12 ml, 0.66 mmol), and compound 2 (Havtech, 82.2 mg, 0.13 mmol) in diy THF (2 ml). After reaction completion, the mixture was concentrated and the remainder was purified by column chromatography eluting in 10% MeOH/DCM to afford compound 3 as off white solid. Yield: 0.08 g, 48.88 %; LCMS (ESI) m/z 1230.15 [M+1] + .
  • Compound 5 was prepared using the same procedure to make compound 3 in Example 2. Compound 5 was made from compound 3 (0.10 g, 0.08 mmol) and compound 4 (78.2 mg, 0.12 mmol) in DMF (3.0 mL), and HATU (0.034 g, 0.09 mmol) and ethylbis(propan- 2-yl)amine (GLR, 0.08 mL, 0.41 mmol). After reaction completion, the reaction mixture was concentrated, and the remainder was purified by silica gel column chromatography eluting in 10% MeOH/DCM to afford compound 5 as a yellowish liquid. Yield: 0.09 g, 53.05 %; LCMS (ESI) M/2 1043.85 [M+1] + .
  • Compound 43 was prepared using the same procedure to make compound 42 in Example 2. Compound 43 was made from compound 5 (0.09 g, 0.07 mmol) in THF (3 mL), MeOH (1 mL) and 1.0 M aqueous sodium hydroxide. After reaction completion, the mixture was concentrated and dried, and the remainder was purified by prep HPLC purification using water/ACN in 0.1% TFA to afford Compound 43 as a yellowish solid. Yield: 0.016 g, 21.74 %; LCMS (ESI) m/z 1706.80 [M+l] + .
  • Compound (1) was prepared as described in Carbohydrate Research, 342 (2007) 1636- 1650.
  • Compound 44 was prepared using the same procedure to make compound 42 from Example 2. Compound 44 was made from compound 5 (0.09 g, 0.05 mmol) in THF (3 mL) MeOH (1 mL), and 1.0 M aqueous NaOH. After reaction completion, the mixture was concentrated and dried, and the remainder was purified by prep HPLC purification using water/ACN in 0.1% TFA to afford Compound 44 as a yellowish solid. Yield: 0.024 g, 34.44 %; LCMS (ESI) m/z 1304.9 [M+l] + .
  • Compound 3 was prepared using the same procedure to make compound 3 in Example 2.
  • Compound 3 was made from compound 1 (0.24 g, 0.42 mmol, Habotech) and compound 2 (0.40 g, 0.42 mmol) in DMF (8.0 mL) with HATU (0.17 g, 0.46 mmol) and ethylbis(propan-2-yl)amine (0.22 mL, 1.25 mmol). After reaction completion, the mixture was concentrated, and the remainder was purified by silica gel column chromatography eluting in 5% MeOHZDCM to afford compound 3 as a yellowish liquid. Yield: 0.35 g, 58.6 %; LCMS (ESI) mZz 1449.20 [M+18] + .
  • Compound 6 was prepared using the same procedure to make compound 3 from Example 2. Compound 6 was made from compound 4 (0.20 g, 0.16 mmol) and compound 5 (0.09 g, 0.16 mmol) in DMF (5.0 mL) with HATU (0.07 g, 0.18mmol) and ethylbis(propan-2- yl)amine (0.09 mL, 0.48 mmol). After reaction completion, the mixture was concentrated and the remainder was purified by silica gel column chromatography eluting in 10% MeOHZDCM to afford compound 6 as a yellowish liquid. Yield: 0.11 g, 38.61 %; LCMS (ESI) M/Z 1741.48 [M+l] + .
  • Compound 9 was prepared using the same procedure to make compound 3 in Example 2.
  • Compound 3 was made from compound 7 (0.08 g, 0.05 mmol) and compound 8 (0.04 g, 0.05 mmol) in DMF (4.0 mL) with HATU (0.02 g, 0.06 mmol) and ethylbis(propan-2- yl)amine (0.03 mL, 0.15 mmol).
  • HATU 0.2 g, 0.06 mmol
  • ethylbis(propan-2- yl)amine (0.03 mL, 0.15 mmol).
  • the mixture was concentrated and the remainder was purified by silica gel column chromatography eluting in 10% MeOHZDCM to afford compound 9 as a yellowish liquid. Yield: 0.09 g, 74.86 %; LCMS (ESI) mZz 1137.60 [M/2+1]+
  • Compound 45 was prepared using the same procedure to make compound 42 in Example 2. Compound 45 was made from compound 9 (0.09 g, 0.04 mmol) in THF (3 mL) and MeOH (1 mL). After reaction completion, the mixture was concentrated and dried, and the remainder was purified by prep HPLC purification using waterZACN in 0.1% TFA to afford Compound 45 as a yellowish color solid. Yield: 20 mg, 26.69 %; HRMS (ESI) m/z 1892.91 [M+l] + .
  • EXAMPLE 7 (2R,3R,4S)-3-acetamido-2-((14R,17R,23R,29R ⁇ 8R 1 59R)-14-(4-(3-(2- ((2,4-dinitrophenyl)amino)ethoxy)propanamido)butyl)-59,60-dihydroxy-17,23,29- tris(hydroxymethyl)-13,16,19,22,25,28 , 31,34,56-nonaoxo-l-(((2R3R,4R,5R,6S)-3,4,5- trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-3,6,9,37,40,43,46,49,52,57-decaoxa- 12, 15, 18,21, 24,27, 30,33, 55-nonaazahexacontan-58-yl)-4-guanidino-3,4-dihydro-2H-pyran- 6-carbox
  • Compound 8 To the reaction vessel containing swollen 2CTC resin loaded compound 6 was added a solution of compound 7 (2.0 eq.) in dry THF followed by DIPEA (GLR, 3 eq.) dropwise. The reaction vessel was shaken for 24 h.
  • Compound 10 was prepared using the same procedure to make compound 3 in Example 2. Compound 10 was made from compound 8 (0.25 g, 0.10 mmol) and compound 9 (0.11 g, 0.11 mmol) in DMF (6.0 mL) with HATU (Chempure, 0.043 g, 0.33 mmol) and ethylbis(propan-2-yl)amine (GLR, 0.08 mL, 0.11 mmol). After reaction completion, the mixture was concentrated and the remainder was purified by silica gel column chromatography eluting in 5% MeOH/DCM to afford compound 10 as a yellowish liquid. Yield: 0.16 g, 63.65 %; LCMS m/z 1211.85 [M72+l] + .
  • Compound 46 was prepared using the same procedure to make compound 42 in Example 2. Compound 46 was made from compound 10 (0.12 g, 0.05 mmol) in THF (3 mL) and MeOH (1 mL). After reaction completion, the mixture was concentrated and dried, and the remainder was purified by prep HPLC purification using water/ACN in 0.1% TFA to afford Compound 46 as a yellowish solid. Yield: 0.019 g, 20.47 %; HRMS (ESI) m/z 1874.82 [M+l] + .
  • EXAMPLE 8 Ethyl (3R,4R,5S)-4-acetamido-5-(3-(25-(18-((2,4- dinitrophenyl)amino)-13-oxo-3,6,9,16-tetraoxa-12-azaoctadecyl)-13,26-dioxo-l- (((2R,3 R,4R,5 R,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)- 3,6,9,16,19,22,29,32-octaoxa-12,25-diazatetratriacontan-34-yl)ureido)-3-(pentan-3- yloxy)cyclohex-l-ene-l-carboxylate (Compound 47)
  • Compound 3 was prepared as described in Eur. J. Med. Chem., 2020, 200, 112423 using commercially available Oseltamivir (1, BLD) and Amino-Peg2-t-butyl ester (2, BLD).
  • Compound 6 was prepared using the same procedure to make compound 3 in Example 2. Compound 6 was made from compound 4 (0.15 g, 0.29 mmol) and compound 5 (0.36 g, 0.32 mmol) in DMF (8.0 mL) with HATU (0.17 g, 0.32 mmol) and ethylbis(propan-2- yl)amine (GLR, 0.27 mL, 1.45 mmol). After reaction completion, the mixture was concentrated and the remainder was purified by silica gel column chromatography eluting in 10% MeOH/DCM to afford compound 6 as a yellowish liquid. Yield: 0.095 g, 22.05 %; LCMS (ESI) m/z 1622.65 [M+l] + .
  • METHOD EXAMPLE 1 Binding affinity to HEK expressing neuraminidase.
  • the Ki of Compound 24 when competed against zanamivir-rhodamine was determined using a binding and competition assay. First, the Ka of zanamivir-rhodamine binding to neuraminidase is determined.
  • the Ki of compound 24 and zanamivir can be determined.
  • Cells are plated as described above.
  • One set of serial dilutions is made for compound 24 and a separate set is made for zanamivir.
  • Each dilution is mixed with an equal volume of 80 nM zanamivir-rhodamine.
  • the compound mixture is added to the cells (50 pL/well) and incubated on a shaker at room temperature for 1 h. Cells are washed and resuspended as described above. Fluorescence is measured using an Attune NxT flow cytometer (fisher).
  • the Ki can be calculated using Graph Pad Prism, One site - Fit Ki.
  • FIG. 5 shows that zanamivir-rhodamine binds to neuraminidase with a binding affinity of 8.253 nM. It is well established in the literature that zanamivir binds to neuraminidase, and excess zanamivir blocking fluorescence shows that zanamivir-rhodamine is binding to neuraminidase. This same rhodamine conjugate is used to quantify inhibitor constants for compound 24 and zanamivir (FIG. 6). Zanamivir has a Ki of 0.496 nM. The Ki of compound 24 is in the low nanomolar range at 7.152 nM.
  • mice lungs are homogenized in 5 pL of cold phosphate buffered saline per milligram of tissue. 100 uL of lysate is saved for RNA isolation, and the remaining lysate is used for hemagglutination. All samples are stored at -80 °C.
  • MDCK-London Mesh-Darby canine kidney cells are plated in a 96-well plate and incubated till confluent. On the day of the assay, lung lysate is serially diluted 3-fold in DMEMZF12 (1:1) supplemented with 0.3% bovine serum albumin. MDCK-London cells are washed and 50 uL of diluted lysate is added to each well, so that each dilution has 4 replicates.
  • Cells are incubated for 1 h at 37 °C, 5% CO2, and then 200 uL of DMEM/F12 (1:1) supplemented with 0.3% bovine serum albumin and 1 pg/ml of trypsin are added to each well. Cells are incubated 48 h at 37 °C and 5% CO2. Each well is tested for hemagglutination by incubating 50 uL of tissue culture supernatant with 50 uL of 0.5% turkey red blood cells in phosphate buffered saline in a v-bottom plate. After 30 min at room temperature, the plate is tilted 45-degrees and wells are counted as negative or positive.
  • Negative wells have a red dot, which runs when the plate is tilted, resembling the negative control. Positive wells will appear hazy and will not have a red dot at the bottom of the well.
  • TCID50 is calculated for each lung sample using the Reed-Muench method, and values are plotted using Graph pad prism.
  • FIG. 7 shows that much of the live, infectious virus is cleared after treatment with compound 24. This is supported by the high survival rates and quick weight recovery of mice treated with compound 24 as seen in Method Example 5 below and the corresponding figures.
  • METHOD EXAMPLE 3 Neuraminidase inhibition assay: To evaluate the neuraminidase inhibition activity of compound 24, a standard neuraminidase inhibition assay was performed using the NA-FluorTM Influenza Neuraminidase Assay Kit (Catalog no. 4457091, InvitrogenTM). The assay was performed according to the manufacturer’s protocol.
  • a standard curve was generated first using the 4-methylumbelliferone sodium salt (4-MU(SS)) to determine the linear range of substrate turnover detection on the Synergy Neo2 HTS MultiMode Microplate Reader (Biotek).
  • a Relative Fluorescence Unit (RFU) value was identified within the linear range of fluorescence detection on the instrument.
  • the dilution factor to be used in the assay was decided that best corresponds to the chosen Relative Fluorescence Unit (RFU) value from the 4-methylumbelliferone sodium salt (4-MU(SS)) standard curve. Then the virus stock solutions were titrated by a neuraminidase activity assay.
  • serial dilutions of the virus stock solutions were prepared in a black, 96-well, flat bottom plate. Then 50 pL of 200uM of the NA-FluorTM substrate (MUNANA, 4- (methylumbelliferyl)-N-acetylneuraminic acid) was added to the designated wells. The plate was incubated at 37°C for 60 min, protected from light. The plate was placed on a shaker inside the incubator. Then the reaction was terminated by adding 100 pL of NA-fluorTM stop solution (0.2 M sodium carbonate) to each well. Then the fluorescence intensities were measured using an excitation wavelength of 350 nm and emission wavelength of 440 ran. The Relative
  • Fluorescence Unit (RFU) values were plotted against the respective virus dilutions and for each vims strain, the dilution factor that yields the RFU chosen from the 4-methylumbelliferone sodium salt (4-MU(SS)) standard curve was selected to use for neuraminidase activity normalization in the neuraminidase inhibition assay. Finally, for the neuraminidase inhibition assay, serial tenfold dilutions of the test compounds were prepared in NA-FluorTM assay buffer (66.6 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer, 8 mM CaCh, pH 6.5).
  • NA-FluorTM assay buffer (66.6 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer, 8 mM CaCh, pH 6.5).
  • 25 pL of the 4X compound 24 dilution series was added to all rows of a 96-well plate followed by addition of 25 pL of IX assay buffer to the no vims control wells.
  • 25 pL of diluted vims samples were added to the designated wells and mixed with the compound 24 dilutions.
  • the plate was incubated for 30 min at 37°C with shaking.
  • 50 pL of the diluted 200 uM NA- FluorTM substrate was added to each well and incubated for 60 min at 37°C protected from light. The reaction was terminated by adding 100 pL of NA-FluorTM stop solution to all wells.
  • the plate was read for measuring fluorescence intensities using an excitation wavelength of 350 nm and emission wavelength of 440 nm.
  • the data was plotted using GraphPad Prism 10.0.0 software to generate the sigmoid dose-response curve.
  • the graph was analyzed using a built-in nonlinear regression curve-fitting program to determine the half-maximal inhibitory concentration ( IC 50 ).
  • IC 50 half-maximal inhibitory concentration
  • mice were weighed and monitored daily for 14 days post-infection and counted as dead when they lost 25% of their body weight or were diagnosed as moribund.
  • mice treated with 1.5 pmol/kg single intranasal dose of Compound 24 resulted in 100% survival
  • 1.5 pmol/kg single intravenous dose of Compound 24 resulted in 100% survival
  • the cohort treated with single oral dose resulted in 80% survival.
  • Mice treated with Tamiflu with 5 mg/kg dosage, dosed twice daily for five days resulted in 60% survival
  • mice treated with Xofluza with 10 mg/kg single oral dose resulted in 80% survival.
  • mice treated with 1.5 pmol/kg single intranasal dose of Compound 24 resulted in 100% survival
  • 1.5 pmol/kg single intravenous dose of Compound 24 resulted in 80% survival
  • the cohort treated with single oral dose resulted in 60% survival.
  • Mice treated with Tamiflu with 5 mg/kg dosage, dosed twice daily for five days resulted in 20% survival and mice treated with Xofluza with 10 mg/kg single oral dose resulted in 60% survival.
  • METHOD EXAMPLE 5 Dose range finding study - Efficacy/Toxicity. In the study of Method Example 2, survival, and cytokine response were also measured. With respect to weight gain, mice were weighed and monitored daily for 14 days post-infection and counted as dead when they lost 25% of their body weight or were diagnosed as moribund.
  • mice treated with 2.2 mg/kg, 0.72 mg/kg and 0.24 mg/kg single intranasal dose of compound 24 resulted in 100% survival and the cohort treated with 0.081 mg/kg single intranasal dose resulted in 60% survival.
  • mice treated with Tamiflu with 5 mg/kg dosage, dosed twice daily for five days also resulted in 60% survival. It indicates the superior efficacy of compound 24 over Tamiflu and demonstrates that 0.24 mg/kg dosage can be considered as the minimal effective dose of compound 24 to achieve a 100% survival in mice. Mice in all cohorts had a gradual loss in body weight but the ones that survived could recover the weight loss induced by viral infection. This data shows that compound 24 is 100% effective and even at 9- fold lower dose, a single dose of compound 24 is comparable to Tamiflu dosed 10 times in a period of five days.
  • FIGS. 12 (lungs) and 13 (serum) show graphs of specific cytokine expression levels in the lungs and serum respectively, measured for each group at 24 h after drug administration.
  • a dose-dependent decrease in IFN-y was observed in the lungs.
  • the level of TNF- a and 12 other inflammatory cytokines not reported here but included in BioLegend’s LEGENDplex kit was unaltered in both lungs and serum.
  • the data shown were not statistically significant.
  • the drop in IFN- y in lungs is indicative of a reduction in systemic inflammation due to faster clearance of the infection upon treatment with compound 24.
  • the IFN- y showed an increase, but with large error bars which are not statistically significant.
  • METHOD EXAMPLE 6 A cytokine release assay was performed on Compound 24 (results in FIG. 8) to assess the risk of causing a cytokine storm in humans.
  • human PBMCs pooled from at least 4 donors were washed and plated at 5x105 cells/well. 1000 nM of Compound 24 was added to the PBMCs with and without 10 mg/mL IVIG to reach a final volume of 200 uL. 1000 nM of a TLR7 agonist was used as a positive control, and media was used as a negative control.
  • the PBMCs were incubated with compound at 37 C, 5% CO2, and samples of supernatant were collected at 2, 6, and 24 h. Supernatant was centrifuged at 450xg for 10 min to remove cells, and stored at -80 C.
  • neuraminidase inhibition activity of Compound 41 was evaluated using the NA-FluorTM Influenza Neuraminidase Assay Kit (Catalog no. 4457091, InvitrogenTM). The assay was performed according to the manufacturer’s protocol. In brief, a standard curve was generated first using the 4-methylumbelliferone sodium salt (4-MU(SS)) to determine the linear range of substrate turnover detection on the Synergy Neo2 HTS Multi-Mode Microplate Reader (Biotek). A Relative Fluorescence Unit (RFU) value was identified within the linear range of fluorescence detection on the instrument.
  • 4-MU(SS) 4-methylumbelliferone sodium salt
  • the dilution factor to be used in the assay was decided that best corresponds to the chosen Relative Fluorescence Unit (RFU) value from the 4- methylumbelliferone sodium salt (4-MU(SS)) standard curve. Then the virus stock solutions were titrated by a neuraminidase activity assay. For this assay, serial dilutions of the virus stock solutions were prepared in a black, 96-well, flat bottom plate. Then 50uL of 200uM of the NA- FluorTM substrate (MUNANA, 4-(methylumbelliferyl)-N-acetylneuraminic acid) was added to the designated wells. The plate was incubated at 37°C for 60 min, protected from light.
  • REU Relative Fluorescence Unit
  • the plate was placed on a shaker inside the incubator. Then the reaction was terminated by adding lOOuL of NA-fluorTM stop solution (0.2 M sodium carbonate) to each well. Then the fluorescence intensities were measured using an excitation wavelength of 350 nm and emission wavelength of 440 nm.
  • the Relative Fluorescence Unit (RFU) values were plotted against the respective virus dilutions and for each virus strain, the dilution factor that yields the RFU chosen from the 4- methylumbelliferone sodium salt (4-MU(SS)) standard curve was selected to use for neuraminidase activity normalization in the neuraminidase inhibition assay.
  • FIG. 10 demonstrates the sigmoidal dose-response curves for each viral strain and the table therein denotes their respective half-maximal inhibitory concentration (IC 50 ) values related to Compound 41.
  • IC 50 half-maximal inhibitory concentration
  • METHOD EXAMPLE 8 Efficacy of Compound 24 IN against Multiple Influenza Strains (FIG. 14A and FIG. 14B). Efficacy of an intranasal formulation of Compound 24 (0.6% n-dodecyl ⁇ -D-maltoside, “DDM” and 2% Avicel 591 in PBS), referred to as “Compound 24 IN”) was evaluated in 3 seasonal Influenza strains: A/H3N2/Wisconsin/15/2009, A/Califomia/07/2009(H1N1) pdm09, a pandemic strain of H1N1 that represents a large portion of the seasonal flu burden every year, and B/Brisbane/60/2008). In FIG. 14A and FIG.
  • mice were infected with 10x LD50 of 1 of 3 seasonal Influenza strains and given either 2.2 mg/kg Compound 24 IN, oseltamivir phosphate orally, or a vehicle control at 48 h post-infection intranasally. Sub-cohorts were sacrificed 24 h post-infection to detect viral titers in the lungs. The rest of the group was observed for the following 14 days as part of a survival study with body weight recorded daily. [0220] As shown in FIG.
  • Compound 24 IN against A/H3N2/Wisconsin/15/2009 showed 100% survival in contrast to the group that received oseltamivir phosphate (60% survival) and the vehicle control (0% survival).
  • a Mantel-Cox Log-rank test shows the compound 24 survival curve to be significant with a p-value of 0.0013 for Influenza A/Wisconsin/15/2009 (H3N2).
  • Compound 24 IN against A/Califomia/07/2009(H1N1) pdm09 showed 100% survival in contrast to 40% of the oseltamivir phosphate group and 0% in the vehicle control group.
  • FIG. 14A shows the corresponding viral lung titers.
  • METHOD EXAMPLE 9 Efficacy against Influenza A/Hong Kong/2369/2009 (H1N1pdmO9, Tamiflu Resistant) (FIG. ISA and FIG. 15B). The efficacy of Compound 24 IN was tested in a mouse model against a representative Influenza A strain - A/Hong
  • H1N1 a pandemic strain of H1N1 that represents a large portion of the seasonal flu burden every year that is also resistant to Tamiflu.
  • All mice were first infected with lOx LD?o of the A/Hong Kong/2369/2009 (HlNlpdmO9, Oseltamivir [Tamiflu] Resistant) strain of influenza virus.
  • IVIG 6 g/kg was administered to the Compound 24 IN and Vehicle groups via intraperitoneal injection (IP) to simulate human levels of anti-DNP and anti-rhamnose antibody titers.
  • Treatment began at 24 hpi (1 dpi). Mice receiving either 2.2 mg/kg of Compound 24 IN or a vehicle control were first anesthetized with isoflurane. Each mouse then received 0.25 mL/kg of vehicle intranasally or 0.25 mL/kg of Compound 24 IN to each nostril, then released back into their cage. Mice receiving Tamiflu (oseltamivir phosphate) were administered 5 mg/kg via oral gavage, then released back to their cage.
  • mice from each group were selected, euthanized, and the lungs were collected and snap frozen in liquid nitrogen. Lungs were homogenized and a TCID50 assay was performed in triplicate on MDCK cells looking for cytopathic effects and the virus titer were calculated using the Reed-Muench method.
  • mice were observed and weighed daily during the two-week study period, at which point they were sacrificed. Mice that lost 25% of their initial weight at any timepoint during the study were euthanized according to IACUC protocol.
  • Compound 24 IN was shown to provide protection against Influenza A strain - A/Hong Kong/2369/2009 (HlNlpdmO9, Oseltamivir Resistant) by the complete elimination of detectable virus titer and the survival of all mice out to study completion (Day 14).
  • the oseltamivir and vehicle-treated mice were found to contain comparable high viral titers in collected lung samples 24 h post-drug administration (-2x106 TCID50) as shown in FIG. 15C, and all mice in these groups (but not in the Compound 24 IN group) were sacrificed by Day 7 due to a body weight loss of >25%, as shown in FIG. 15B.
  • METHOD EXAMPLE 10 Compound 24 Prevention of Transmission of H1N1 (FIG. 16).
  • Compound 24 IN was evaluated for its ability to prevent transmission of influenza. It was found that a single dose of Compound 24 IN outperformed vehicle control.
  • guinea pigs were infected with human H1N1 influenza and received 4g/kg IVIG intraperitoneally. Then the guinea pigs were treated 24 h post infection with vehicle, or 4 mg/kg intranasally of Compound 24 IN. Twenty-four h after treatment, infected guinea pigs were cohoused with naive guinea pigs for 14 days. Nasal swabs were taken every other day, and viral RNA was measured using PCR. At the end of the study, anti-neuraminidase and antihemagglutinin antibody titers were measured to assess influenza exposure.
  • Mitigating viral shedding through reduced transmission is believed equally important to symptom management in public health interventions. While symptomatic relief can improve patient well-being, it may inadvertently lead to a resumption of normal activities during the infectious phase. These data suggest that a single dose treatment with compound 24 reduces secondary infection rates of human H1N1 by 90%.
  • mice Three groups of five mice each were used for each passage cycle. All groups received 10 LD50 influenza virus A/PR8/34 (H1N1). Group 1 was administered a single dose of 81 pg/kg of Compound 24 intranasally 48 h post-infection. Group 2 was administered vehicle (20mg/ml Avicel, 6mg/ml DDM, lx PBS, pH 7.4) once intranasally, 48h post infection. Group 3 was administered 5 mg/kg of oseltamivir phosphate twice daily orally for 2 days starting 48h post infection.
  • H1N1 LD50 influenza virus A/PR8/34
  • FIG. ISA shows the images of Groups 1 and 2 and FIG. 18B of Groups 3 and 4.
  • METHOD EXAMPLE 13 Pharmacological Mechanism of Action of Compound 24.
  • the mechanism of action of Compound 24 is evaluated by the ability to recruit sufficient naturally occurring anti-hapten antibodies to mediate antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement dependent cytotoxicity (CDC) induced killing of virus-infected cells.
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • CDC complement dependent cytotoxicity
  • Human embryonic kidney cells HEK293 are modified to express influenza neuraminidase (HEK293-NA), mimicking influenza-infected cells.
  • Compound 24 is evaluated for the ability to activate specific immune system components (FcyRHIa, FcyRIIa) in effector cells and the complement system.
  • HEK293-NA cells If activated, these components are expected to trigger ADCC, ADCP, and CDC, ultimately killing the HEK293-NA cells.
  • a control group using unmodified HEK293 cells (HEK293-WT) is used for comparison.
  • the HEK293-N1 -neuraminidase cell cultures are also supplemented with or without human anti- DNP antibody and incubated with serial dilutions of Compound 24.
  • Compound 24 is effective in mediating ADCC (FIG. 19A), and ADCP (FIG. 19B) of the neuraminidase-transduced HEK293 cells, displaying the anticipated bell-shaped dependence on Compound 24 concentration as detected through changes in luminescence because of activation of the human FcgRIIIa expressing effector cells using a Promega ADCC kit.
  • Cell death can be blocked by either deletion of IVIG or the absence of neuraminidase on the target cell surface, suggesting that ADCC -mediated and ADCP- mediated killing rely on two key elements: 1) the presence of anti-hapten antibodies and 2) cell surface expression of influenza neuraminidase.
  • the dependence upon cell surface expression of influenza neuraminidase is an important safety attribute, suggesting that uninfected cells should not be damaged by Compound 24.
  • Neuraminidase inhibition activity is measured based on the IC 50 in relative fluorescence unit (RFU) of the enzymatic product resulting from neuraminidase activity of the virus particles.
  • REU relative fluorescence unit
  • Compound 24 is found to inhibit neuraminidase activity of the three seasonal influenza A strains with IC 50 ranging from 12.7-16.4 nM. Compound 24 also inhibits neuraminidase activity of the seasonal influenza B strain (Victoria Lineage) with an IC 50 of 34.5 nM. Compound 24 is found to effectively inhibit the neuraminidase activity of oseltamivir-resistant and baloxavir-resistant flu strains with an IC 50 of 17.9 and 27.2 nM, respectively).
  • METHOD EXAMPLE 15 Neuraminidase Inhibition Activity against Influenza A/PR/8/1934 (H1N1).
  • a 10-fold serial dilution of test compounds were prepared in IX NA- FluorTM Assay Buffer.
  • the 4x dilution series and diluted virus samples were added into the corresponding wells in a black, 96-well, clear flat bottom plate.
  • the plate was incubated on a plate shaker for 30 min at 37°C.
  • 200 ⁇ M NA-FluorTM Substrate working solution was added and incubated on a plate shaker for an additional 1 hr at 37°C to detect the viral neuraminidase activity.
  • the reaction was terminated by a stop solution (60% NA-FluorTM Stop Solution/40% ethanol).
  • the fluorescence signal was detected with an excitation wavelength of 350 nm and an emission wavelength of 440 nm by BioTek Synergy Neo2 HTS Multi-Mode Microplate Reader. Data were analyzed using GraphPad Prism 10 to determine the half-maximal inhibitory concentration (IC50) of the test compound.
  • Activity in this assay demonstrates that the tested conjugates retained the direct antiviral activity of the ligand after being conjugated to the linker and two haptens.
  • the IC50 values for the tested compounds are shown in the following table.
  • METHOD EXAMPLE 16 Viral Cytopathic Effect Inhibition Activity of Compound 24. In the presence of viral infections, host cells undergo changes at the cellular level in response to infection. This phenomenon is known as the cytopathic effect (CPE). The effectiveness of Compound 24 in preventing CPE caused by an influenza virus infection and its associated cytotoxicity was determined through a viral CPE-inhibition assay. Virus stock titers were measured by performing a TCID 50 to determine the viral inoculation required for the CPE inhibition assay.
  • CPE cytopathic effect
  • Compound 24 effectively prevented CPE induced by H1N1 and the oseltamivir-resistant strain (EC 50 ⁇ 0.9 ⁇ M). Compound 24 also inhibited CPE of HINlpdm09, H3N2, and influenza B with EC 50 ⁇ 8 ⁇ M. It is important to note that these experiments only consider the activity of the zanamivir moiety, and Compound 24 is expected to have greater than 1,000-fold activity when the immune system can be recruited in vivo.
  • METHOD EXAMPLE 17 Efficacy against Avian Influenza. The efficacy of Compound 24 against the A/Duck/MN/1525/81 (H5N1), A/HK/61/2016 (H7N9), and
  • A/Vietnam/1203/2004 (H5N1) strains of avian influenza was evaluated in vitro. Cells were cultured and then exposed to varying dilutions of Compound 24 and an Avian strain. Cytopathic effects were observed microscopically, measured with a red dye and EC 50 (concentration of 50% CPE inhibition) values were calculated, as shown in the following table.
  • METHOD EXAMPLE 18 Efficacy against A/Illinois/37/2018 (H1N1, Baloxavir Resistant). Baloxavir treatment has very high mutation emergence rate and presents a clinical issue for baloxavir use. Mice were infected with lOLDso of mouse adapted AZIllinois/37/2018 (HlNlpdmO9, baloxavir resistant) in a pilot efficacy study against baloxavir mutants. As shown in FIG. 21, mice that were treated with Compound 24 had 100% survival. The vehicle control and baloxavir treatment groups had similar survival rates suggesting no efficacy from baloxavir. Compound 24 may be useful in treatments against baloxovir resistant strains of influenza.
  • METHOD EXAMPLE 19 Compliment Dependent Cytotoxicity (CDC) Assay. Neuraminidase expressing HEK293 cells (HEK293-NA) were plated at 10,000 cells per well in a 96-well poly-D lysine coated, white-walled plate. The cells were allowed to incubate overnight at 37°C, aiming for 50% confluency on the day of the assay. Serial dilutions of compound were prepared in DMEM, adjusted to be 4x higher than the target concentration. The cells were washed with 200 pL of PBS, followed by the addition of 25 pL of DMEM per well.
  • METHOD EXAMPLE 20 Summary of Nonclinical Pharmacokinetics and Metabolism.
  • the pharmacokinetics (PK) of Compound 24 were characterized in BALB/c mice across four routes of administration: intravenous (IV), subcutaneous (SC), oral, and IN. While IV and SC have the highest level of drug absorbed, IN was chosen as a preferred route of administration (ROA) for ease of administration in human patients.
  • ROA preferred route of administration
  • PK studies were also conducted in mice to determine the minimum pharmacologically active dose (PAD).
  • Compound 24 After IN administration, Compound 24 demonstrates rapid absorption across species with a time to maximum plasma concentration (Tmax) ranging from approximately 0.42 to 1.10 hours in preclinical PK studies.
  • the bioavailability (F) of Compound 24 is dose- and formulationdependent and was found to be highest at clinically relevant dose levels in rodents. At similar doses and across formulations, mean F was moderate to high in mouse (21% - -100%), low to moderate in rat (-20% - -50%), and low (4—20%) in dogs. In each instance, the higher F was a concentration that was £% the maximum feasible dose (MFD) and the lower range F is the MFD.
  • MFD maximum feasible dose
  • the mean ti/2 estimate was -1 hr after IV and IN administration at doses up to -20 mg/kg.
  • Compound 24 was found to have low cellular permeability and is not a substrate of human P-glycoprotein (P-gp).
  • Compound 24 showed low to moderate plasma protein binding. The percentage of unbound Compound 24 ranged from 74.0% to 89.4% across mice, rats, dogs, monkeys, and humans with human plasma showing the highest binding. Partitioning work indicated that Compound 24 had blood to plasma ratios less than or equal to 0.72, suggesting limited partitioning into red blood cells. Compound 24 was metabolically stable across various species in vitro. Profiling pooled rat plasma detected an apparent minor circulating metabolite (0.71%) resulting from amide hydrolysis (Ml 160), with intact Compound 24 appearing to be the predominant circulating entity.
  • Compound 24 did not directly (reversibly) inhibit the major human cytochrome P450s (CYPs) 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, or 3A4/5. In addition, coincubation with Compound 24 did not result in time dependent inhibition (TDI) of these same CYPs. Thus, Compound 24 is not expected to perpetrate a CYP-mediated drug-drug interaction.
  • CYPs major human cytochrome P450s
  • TDI time dependent inhibition
  • Compound 24 was not mutagenic in a bacterial reverse mutation (Ames) test or an in vitro micronucleus assay in vitro. Compound 24 absorbs light between 290 and 700 nm, with an absorption maximum at 360 nm.
  • Non-GLP dose tolerability studies at the MFD were conducted in rats and dogs to support dose selection for 14-Day GLP toxicology studies.
  • the MFD was 100 mg/kg by the IN route, the intended route of clinical administration.
  • a dose of 100 mg/kg was well tolerated in the single dose tolerability phase.
  • the 100 mg/kg dose was selected for further evaluation across a 3-day tolerability study with daily dosing.
  • rats were administered vehicle or doses up to 100 mg/kg/day by the IN route.
  • NOAEL was 100 mg/kg/day in male and female rats dosed daily by the IN route for 14 days.
  • NOAEL was 20 mg/kg/day in male and female dogs dosed daily by the combined IN and SC routes for 14 days.
  • Compound 24 is a small molecule neuraminidase inhibitor covalently conjugated to dinitrophenyl (DNP) and ct-L-rhamnose with PEG-based linkers.
  • DNP dinitrophenyl
  • Compound 24 is manufactured in a four-step process from key starting materials and has been manufactured in batches up to 100g.
  • the structure, including the absolute stereochemistry of 11 chiral centers is confirmed through analysis by infrared spectroscopy (IR), Ultraviolet- Visible Absorbance Spectroscopy, High-resolution Mass Spectrometry (HRMS), Elemental Analysis, and 1-D/2-D Nuclear Magnetic Resonance (NMR) spectroscopy.
  • Compound 24 is packaged in HDPE plastic bottles protected from light, and stored at -20°C ⁇ 5°C for up to 12 months.

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Abstract

L'invention concerne des conjugués comprenant un ligand de ciblage viral, par exemple à la grippe, un lieur et de multiples haptènes. L'invention concerne également des compositions pharmaceutiques comprenant les conjugués et des méthodes de traitement d'infections virales ou de prévention de telles infections à l'aide des conjugués.
PCT/US2024/051818 2023-10-17 2024-10-17 Conjugués, compositions et méthodes de traitement de la grippe Pending WO2025085664A1 (fr)

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US63/590,999 2023-10-17
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004094613A2 (fr) * 2003-04-22 2004-11-04 Ibc Pharmaceuticals Complexe proteinique polyvalent
US20130280204A1 (en) * 2007-08-27 2013-10-24 Massachusetts Institute Of Technology Polymer-Attached Inhibitors of Influenza Virus
US20210393786A1 (en) * 2018-07-26 2021-12-23 Purdue Research Foundation Small molecule ligand-targeted drug conjugates for anti-influenza chemotherapy and immunotherapy

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004094613A2 (fr) * 2003-04-22 2004-11-04 Ibc Pharmaceuticals Complexe proteinique polyvalent
US20130280204A1 (en) * 2007-08-27 2013-10-24 Massachusetts Institute Of Technology Polymer-Attached Inhibitors of Influenza Virus
US20210393786A1 (en) * 2018-07-26 2021-12-23 Purdue Research Foundation Small molecule ligand-targeted drug conjugates for anti-influenza chemotherapy and immunotherapy

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