Solid formulations and methods for treating influenza infection

Abstract

Solid pharmaceutical compositions comprising a dual hapten conjugate for treating influenza infections, processes for making such compositions, and methods for use for treating influenza infections, are disclosed herein.

Description

Docket No. 1165.071126WOO 1SOLID FORMULATIONS AND METHODS FOR TREATING INFLUENZA INFECTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority from Appl. No. 63 / 789,859, filed April 16, 2025, which is incorporated by reference as if fully set forth hereinTECHNICAL FIELD

[0002] The invention described herein pertains to solid formulations of pharmaceutically active ingredients and methods for making such formations, and for using such formulations to treat influenza infections.BACKGROUND

[0003] 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.

[0004] Because influenza virus constantly changes via antigen shift and drift, vaccines often become ineffective against mutating strains. Though vaccines against common influenza antigens have proven successful in limiting the severity and spread of the virus during years when the most aggressive viral strains are correctly predicted, due to the rapid evolution of the vims they have been only 19-60% effective over the past decade, and annual formulations of the vaccine often fail to match the most virulent strains, resulting in many vaccinated patients still contracting an infection. Further complicating this issue is that generally the highest risk populations have the highest numbers of people who either are ineligible for the vaccine or are unable to develop immune responses to the vaccine.

[0005] Chemotherapeutic applications presently available are also limited. Approved anti¬ influenza drugs are either M2 ion channel inhibitors or neuraminidase inhibitors

[0006] 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, inhibit the release of viral ribonucleoprotein from virion to host cytosol. However, both H1N1 and H3N2 viruses currently circulating in human populations are resistant to these inhibitors. Thus, Centers for Disease Control and Prevention (CDC) has advised against their use, due to the potential for rapid emergence of drug resistance.Docket No. 1165.071126WOO 1

[0007] The commonly used 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.

[0008] While prophylactic treatments, such as vaccines, play an important role in managing influenza’s medical burden, both current vaccines and antiviral agents should be supplemented with new antiviral therapies to address future pandemic influenza years. A potent and broadspectrum anti-influenza therapy is needed In view of the foregoing, it is an object of the present disclosure to provide such a therapy including the use of solid intranasal pharmaceutical compositions. This and other objects and advantages, as well as inventive features, will be apparent from the detailed description provided herein.SUMMARY

[0009] Solid pharmaceutical compositions comprising (i) conjugates of the formula:zT-LAl\^2and pharmaceutically acceptable salts thereof, wherein T is a targeting ligand for a target protein on the surface of a vims or a virus-infected cell; L is a linker; and Al and A2 are each, independently, a hapten; and (ii) non-ionic surfactants are described herein. In some embodiments, the compounds are of Formula I:or a pharmaceutically acceptable salt or solave thereof wherein T is a radical of a ligand for a target protein of an Influenza virus or a virus-infected cell;L1, L2, and L3are each, an independently selected linker; andA1and A2are each, a radical of an independently selected hapten. In an illustrative embodiment,7Docket No. 1165.071126WOO 1each of L', L2, and L3is independently selected, and each may comprise a single divalent atom, or a chain of atoms. In another illustrative embodiment, L2may be a bond attaching L1to A1. In another illustrative embodiment, each of L3may be a bond attaching L1to A2.

[0010] Solid pharmaceutical compositions for use in treating an influenza infection are described herein, where the solid pharmaceutical composition comprises (i) a therapeutically effective amount of a conjugate of the formula:ZT-LAlA2and pharmaceutically acceptable salts thereof, wherein T is a targeting ligand for a target protein on the surface of a virus or a virus-infected cell; L is a linker; and Al and A2 are each, independently, a hapten; and (ii) a non-ionic surfactant.

[0011] Solid unit doses and unit dosage forms in single or divided form are described herein, where the unit doses and unit dosage forms comprise (i) a therapeutically effective amount of a conjugate of the formula:and pharmaceutically acceptable salts thereof, wherein T is a targeting ligand for a target protein on the surface of a virus or a virus-infected cell; L is a linker; and Al and A2 are each, independently, a hapten; and (ii) a non-ionic surfactant.

[0012] Methods for treating an influenza infection in a host animal are described herein, where the methods comprise administering to the host animal a solid pharmaceutical composition comprising (i) a therapeutically effective amount of a conjugate of the formula:zT-LAlA2^and pharmaceutically acceptable salts thereof, wherein T is a targeting ligand for a target protein on the surface of a virus or a virus-infected cell; L is a linker; and Al and A2 are each, independently, a hapten; and (ii) a non-ionic surfactant.

[0013] Uses of solids of pharmaceutical compositions in the manufacture of medicaments for treating an influenza infection are described herein, where the solid pharmaceutical compositions comprise (i) a therapeutically effective amount of a conjugate of the formula:Docket No. 1165.071126WOO 1and pharmaceutically acceptable salts thereof wherein T is a targeting ligand for a target protein on the surface of a virus or a virus-infected cell, L is a linker; and Al and A2 are each, independently, a hapten and (ii) a non-ionic surfactant.

[0014] In a first illustrative embodiment, T is a radical of a neuraminidase inhibitor,

[0015] In a second illustrative embodiment, T is a radical of a hemagglutinin inhibitor.

[0016] In another illustrative embodiment, the haptens recruit endogenous antibodies present in the host animal.

[0017] In another aspect, the solid pharmaceutical compositions, unit doses, and unit dosage forms described herein include a conjugate of the formula:and diastereomers thereof.

[0018] In several aspects of the disclosure, amorphous Compound 24 is provided

[0019] In other aspects of the disclosure, spray-dried formulations comprising a compound of Compound 24 are provided.

[0020] In these and other aspects of the disclosure, spray-dried formulations comprising a compound of Compound 24; HPMC, and DDM are provided.

[0021] In still further aspects of the disclosure pharmaceutical compositions comprising spray- dried formulations of the disclosure are provided.

[0022] In these and other aspects, feed solutions for spray drying comprising solutions of Compound 24 and one or more spray-drying excipients are provided.

[0023] In various aspects of the disclosure, processes for making feed solutions and spray-dried formulations comprising Compound 24 and one or more spray-drying excipients are provided.

[0024] Other aspects of the disclosure are directed to methods of treating viral diseases, such as influenza, with spray-dried formulations of the disclosure.

[0025] Methods for treating an influenza infection in a host animal, the method comprising theDocket No. 1165.071126WOO 1step of administering to the host animal a solid pharmaceutical composition comprising (i) a therapeutically effective amount of a compound of 24 and (ii) a spray-drying excipient are provided.

[0026] Uses of a solid pharmaceutical composition in the manufacture of a medicament for treating an influenza infection, the solid phar aceutical composition comprising (i) a therapeutically effective amount of a compound of 24 and (ii) a spray-drying excipient are provided.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 shows plots of effectiveness of Compound 24 and other compounds with various routes of administration.

[0028] FIG. 2 shows plots of effectiveness of Compound 24 and other compounds with various routes of administration.

[0029] FIG. 3 from Method Example 5 shows a graph of days after infection vs. survival (%) and days after infection vs. body weight (%) for mice (n = 5 / group) infected with 10 LDso of influenza A H3N2 / Wi scon sin / 15 / 2009, intraperitoneal administration of human IgGQVIg (GAMUNEX®- C) at 24 hours post-infection (hpi), and administration of Compound 24 at 48 hpi.

[0030] FIG. 4 is from the study in Method Example 2 is a plot of TClD50 / mL (Tissue Culture Infectious Dose) as a function of dose (mg / kg). FIG. 4 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). Mice treated with Compound 24 have a 100-fold reduction in infectious virus, as compared to the vehicle, and a greater than 10-fold reduction in infectious virus as compared to the standard of care.

[0031] FIG. 5 is a synthetic scheme for Compound 11, which is used in the synthesis of Compound 24.

[0032] FIG. 6 is a synthetic scheme for Compound 24.

[0033] FIG. 7 shows example compounds of the disclosure.

[0034] FIG. 8 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.

[0035] FIG. 9 are plots of fluorescence intensity as a function of the log[concentration] (nM) for Compound 24 and zanamivir. FIG. 9 shows the competitive binding of Compound 24 (left panel)Docket No. 1165.071126WOO 1and zanamivir (right panel) to neuraminidase-expressing HEK cells. It shows retained affinity for neuraminidase after incorporation into the conjugate Compound 24

[0036] FIG. 10 shows the cytokine response to various treatments, including Compound 24, in human PBMC (Method Example 6).

[0037] FIG. 11 shows a plot of normalized fluorescence (RFU) as a function of the log of the concentration of Compound 24. FIG. 11 shows neuraminidase inhibition activity of Compound 24 against multiple influenza strains

[0038] FIG. 12 shows a plot of normalized fluorescence (RFU) as a function of the log of the concentration of Compound 41. FIG. 12 shows neuraminidase inhibition activity of Compound 41 against multiple influenza strains (Method Example 7).

[0039] FIG. 13 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.

[0040] FIG. 14 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.

[0041] FIG. 14A shows the in vivo efficacy of Compound 24 against seasonal flu strains A / California / 07 / 2009(H1N1) pdra09, B / Brisbane / 60 / 2008, and A / H3N2 / Wi scon sin / 15 / 2009 in mice infected with lOx LDso titers compared to oseltamivir phosphate and vehicle control (N=5 / cohort).

[0042] FIG. 14B shows the viral titer measured by hemagglutination 24 h after treatment with vehicle (PBS), oseltamivir phosphate, or Compound 24 in vivo against seasonal flu strains A / California / 07 / 2009(H1N1) pdm09, A / H3N2AVisconsin / l 5 / 2009, and B / Brisbane / 60 / 2008 in mice infected with lOx LDso titers (N=5 / cohort). Compound 24 demonstrates a statistically significant improvement over both vehicle control and oseltamivir phosphate positive control.

[0043] FIG 15A shows the in vivo efficacy of Compound 24 against A / Hong Kong / 2369 / 2009 (HlNlpdmO9), an Tamiflu resistant Influenza A, in mice infected with lOx LDso titers compared to oseltamivir phosphate and vehicle control (N=5 / cohort).

[0044] FIG. 15B show's 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).

[0045] 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 withDocket No. 1165.071126WOO 110xLD50 of Influenza A strain - A / Hong Kong / 2369 / 2009 (HlNlpdmO9, oseltamivir phosphate (Tamiflu) Resistant).

[0046] FIG. 16 shows the prevention of influenza transmission in co-housed guinea pigs by Compound 24.

[0047] 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.

[0048] FIG. 17B shows the change in virulence of influenza virus AZPR8 / 34 (H1N1) after 5 cycles of in vivo mutation using Compound 24 compared to oseltamivir phosphate as measured by LD50 measured in vivo.

[0049] FIG. 18A shows IVIS imaging of Group 1 (mock) and Group 2 (infected and vehicle treated mice).

[0050] 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..

[0051] FIG. 19A shows antibody-dependent cellular cytotoxicity (ADCC) induced killing of virus-infected HEK293 cells by Compound 24.

[0052] FIG. 19B shows antibody-dependent cellular phagocytosis (ADCP) induced killing of virus-infected HEK293 cells by Compound 24.

[0053] FIG. 19C shows complement dependent cytotoxicity (CDC) induced killing of virus-infected HEK293 cells by Compound 24.

[0054] FIG. 20 shows the neuraminidase inhibition activity of Compound 24 intranasally (“IN”) against seasonal and drug-resistant Influenza A and B strains

[0055] FIG. 21 shows the in vivo efficacy of Compound 24 against A / Illinois / 37 / 2018 (H1N1, Baloxavir Resistant) - Survival over Time.

[0056] FIG. 22 shows an x-ray powder diffraction (XRPD) pattern of solid Compound 24.

[0057] FIG. 23 shows two differential scanning calorimetry (DSC) thermograms of solid Compound 24 - (a) is a first heat flow cycle and (b) is a second heat flow cycle

[0058] FIG. 24 shows a Particle Size Distribution of solid Compound 24.

[0059] FIG. 25 shows an XRPD pattern of a spray-dried formulation that is 85% Compound 24; 13.5% HPMC. and 1.5% DDM by weight (top) overlaid with an XRPD pattern of Compound 24 (bottom).

[0060] FIG. 26 shows a loss on drying measurement indicating a loss of about 1.7% by weight of a spray-dried formulation that is 85% Compound 24; 13.5% ITPMC, and 1.5% DDM by weight.Docket No. 1165.071126WOO 1

[0061] FIG. 27 shows a particle size distribution of a spray-dried formulation that is 85% Compound 24; 13.5% HPMC, and 1.5% DDM by weight.

[0062] FIG. 28 shows a DSC thermogram of a first heat cycle on a sprav-dried formulation that is 85% Compound 24; 13.5% HPMC, and 1.5% DDM by weight (bottom) overlaid with a Compound 24 DSC thermogram (top).

[0063] FIG. 29 shows a DSC thermogram of a second heat cycle on a spray-dried formulation that is 85% Compound 24; 13.5% HPMC, and 1.5% DDM by weight, (bottom) overlaid with a Compound 24 DSC thermogram (top).

[0064] FIG. 30 shows particle size distribution of three spray-dried formulations (6A, 6B, and 6C) of Compound 24.

[0065] FIG. 31 shows an XRPD pattern of 6A, 6B, and 6C.

[0066] FIG. 32 shows a DSC thermogram for a first heating cycle of 6A, 6B, and 6C.

[0067] FIG. 33 shows a DSC thermogram for a second heating cycle of 6A, 6B, and 6C

[0068] FIG. 34 shows a AINT-NGI nasal deposition profile for 6A, 6B, and 6C.

[0069] FIG. 35 shows an XRPD pattern of 6A, 6B, and 6C between 2-8°C.

[0070] FIG. 36 shows an XRPD pattern of 6A, 6B, and 6C at 25°C and 60% Relative Humidity (RH).

[0071] FIG. 37 shows an XRPD pattern of 6A, 6B, and 6C at 40°C and 75% RH.

[0072] FIG. 38 shows a DSC thermogram plot of a first heat cycle of 6A during a stability study at time = 0, 4 weeks at 2-8°C; 4 weeks at 25°C / 60%RH; and 4 weeks at 40°C / 75%RH.

[0073] FIG. 39 shows a DSC thermogram plot of a second heat cycle of 6A during a stability study at time = 0; 4 weeks at 2-8°C; 4 weeks at 25cC / 60%RH; and 4 weeks at 40°C / 75%RH.

[0074] FIG. 40 shows a DSC thermogram plot of a first heat cycle of 6B during a stability study at time = 0; 4 weeks at 2-8°C; 4 weeks at 25°C / 60%RH; and 4 weeks at 40cC / 75%RH.

[0075] FIG. 41 shows a DSC thermogram plot of a second heat cycle of 6B during a stability¬ study at time = 0; 4 weeks at 2-8°C; 4 weeks at 25°C760%RH; and 4 weeks at 40°C / 75%RH.

[0076] FIG. 42 shows a DSC thermogram plot of a first heat cycle of 6C during a stability study at time = 0; 4 weeks at 2-8°C; 4 weeks at 25°C / 60%RH; and 4 weeks at 40°C / 75%RH.

[0077] FIG. 43 shows a DSC thermogram plot of a second heat cycle of 6C during a stabilitystudy at time::::0; 4 weeks at 2-8°C; 4 weeks at 25°C / 60%RH; and 4 weeks at 40°C / 75%RH.

[0078] FIG. 44 shows an AINI-NGI Nasal Deposition profile of 6A from a stability study at time = 0; 4 weeks at 2-8°C; 4 weeks at 25°C / 60%RH; and 4 weeks at 40°C / 75%RH.Docket No. 1165.071126WOO 1

[0079] FIG. 45 shows an AINI-NGI Nasal Deposition profile of 6B from a stability study at time = 0; 4 weeks at 2-8°C; 4 weeks at 25°C / 60%RH; and 4 weeks at 40°C / 75%RH.

[0080] FIG. 46 shows an AINI-NGI Nasal Deposition profile of 6C from a stability study at time = 0; 4 weeks at 2-8°C; 4 weeks at 25°C / 60%RH; and 4 weeks at 40°C / 75%RH.

[0081] FIG. 47 shows the mean time vs plasma concentration profile of Compound 24 following intra-nasal administration of spray-dried formulations comprising Compound 24 to male Beagle dogs.DETAILED DESCRIPTION

[0082] Pharmaceutical formulations for intranasal delivery may be formulated as solution-based formulations or dry powder formulations. While liquid nasal delivery formulations are prevalent for intranasal delivery, they often suffer from short nasal residence time and may be unstable in solution. Dry powder formulations typically have longer residence time, but are often challenging to manufacture,

[0083] Compounds of the disclosure, may be formulated as dry powders as described herein. In many embodiments, dry powder formulations of Compound 24 were successfully prepared with suitable powder characteristics for intranasal delivery. As a dry powder, they can be prepared by spray drying a solution comprising Compound 24 and one or more spray-drying excipients to prepare a dry powder, such as a spray-dried formulation, of Compound 24.

[0084] In many embodiments, the dry powders have a water content of less than 5% by weight. In these and other embodiments, the water content by weight is between about 0.5% and about 3% by weight, including between about 1% and 2% by weight including between about 1.4% and about 2.4% by weight. In many such embodiments, the dry / powders are spray-dried formulations.

[0085] Several illustrative embodiments of the present disclosure are described by the following enumerated embodiments:1. A solid pharmaceutical composition comprising (i) a conjugate of the formula:and pharmaceutically acceptable salts thereof wherein T is a targeting ligand for a target protein on the surface of a virus or a virus-infected cell: L is a linker; and Al and A2 are each, independently, a hapten; and (ii) a non-ionic surfactant.2 A solid pharmaceutical composition for use in treating an influenza infection, theDocket No. 1165.071126WOO 1composition comprising (i) a therapeutically effective amount of a conjugate of the formula:and pharmaceutically acceptable salts thereof wherein T is a targeting ligand for a target protein on the surface of a virus or a virus-infected cell; L is a linker; and Al and A2 are each, independently, a hapten, and (ii) a non-ionic surfactant.3. A solid unit dose or unit dosage form in single or divided form, the unit dose or unit dosage comprising (i) a therapeutically effective amount of a conjugate of the formula:zT-LAlA2and pharmaceutically acceptable salts thereof wherein T is a targeting ligand for a target protein on the surface of a vims or a vims-infected cell; L is a linker; and Al and K2 are each, independently, a hapten and (ii) a non-ionic surfactant.4. A method for treating an influenza infection in a host animal, the method comprising the step of administering to the host animal a solid pharmaceutical composition comprising (i) a therapeutically effective amount of a conjugate of the formula:and pharmaceutically acceptable salts thereof wherein T is a targeting ligand for a target protein on the surface of a vims or a vims-infected cell; L is a linker; and Al and K2 are each, independently, a hapten; and (ii) a non-ionic surfactant5. Use of a solid pharmaceutical composition in the manufacture of a medicament for treating an influenza infection, the solid pharmaceutical composition comprising (i) a therapeutically effective amount of a conjugate of the formula:and pharmaceutically acceptable salts thereof wherein T is a targeting ligand for a target protein on the surface of a vims or a vims-infected cell; L is a linker; and Al and K are each, independently, a hapten and (ii) a non-ionic surfactant.6. The solid phar aceutical composition, unit dose, unit dosage form, method, or use of any one of the preceding clauses comprising (i) a conjugate of the formula:Docket No. 1165.071126WOO 1L3-A1T— L1L2-A2and pharmaceutically acceptable salts thereof, wherein T is a targeting ligand for a target protein on the surface of a virus or a virus-infected cell; LI-L3 are each, independently, a linker; and Al and A2 are each, independently, a hapten; and (ii) a non-ionic surfactant. 7. The solid pharmaceutical composition, unit dose, unit dosage form, method, or use of any one of the preceding clauses wherein T is a radical of the formula:8 The solid pharmaceutical composition, unit dose, unit dosage form, method, or use of any one of the preceding clauses wherein T is a radical of the formula:9. The solid pharmaceutical composition, unit dose, unit dosage form, method, or use of any one of the preceding clauses where L or L1, L2, and L3, comprise a group of the formula:10. The solid pharmaceutical composition, unit dose, unit dosage form, method, or use of any one of the preceding clauses comprising (i) a conjugate of the formula:Docket No. 1165.071126WOO 1and diastereomers thereof, and (ii) one or more pharmaceutically acceptable excipients.11. The solid pharmaceutical composition, unit dose, unit dosage form, method, or use of any one of the preceding clauses, further comprising a spray-drying excipient.12. The solid pharmaceutical composition, unit dose, unit dosage form, method, or use of any one of the preceding clauses, further comprising a spray-drving excipient.13. The solid pharmaceutical composition, unit dose, unit dosage form, method, or use of clause 12, wherein at least one spray-drying excipient is a cyclodextrin.14. The solid pharmaceutical composition, unit dose, unit dosage form, method, or use of clause 12 or clause 13, wherein at least one spray-drying excipient is hydroxy-propyl-beta- cyclodextrin.15. The solid pharmaceutical composition, unit dose, unit dosage form, method, or use of clause 12, wherein at least one spray-drying excipient is a cellulose-based compound.16. The solid pharmaceutical composition, unit dose, unit dosage form, method, or use of clause 15, wherein the cellulose-based compound is hydroxypropyl methylcellulose, also known as HPMC17. The solid pharm ceutical composition, unit dose, unit dosage form, method, or use of clause 12, wherein at least one spray-drying excipient is a sugar.18. The solid pharmaceutical composition, unit dose, unit dosage form, method, or use of clause 17, wherein the sugar is mannitol.19. The solid pharmaceutical composition, unit dose, unit dosage form, method, or use of clause 12, wherein at least one spray-drying excipient is an ionic surfactant.20. The solid pharmaceutical composition, unit dose, unit dosage form, method, or use of clause 19, wherein the ionic surfactant is n-dodecyl-P-D-maltoside, also known as DDM 21. The solid pharmaceutical composition, unit dose, or unit dosage form of any of the preceding clauses wherein the solid pharmaceutical composition, unit dose, or unit dosage form is a spray-dried dispersion.

[0086] In many embodiments of the disclosure, a solid pharmaceutical composition is providedDocket No. 1165.071126WOO 1comprising Compound 24 and a spray-drying excipient. In many embodiments, a spray-drying excipient is DDM. In many such embodiments, a second spray-drying excipient is present such as a saccharide or a polymer. An example of such a saccharide is mannitol. An example of such a saccharide may be an oligosaccharide such as a cyclic oligosaccharide. Cyclodextrins are examples of such cyclic oligosaccharides. An exemplary cyclodextrin is hydroxypropyl-P-cyclodextrin. An exemplary' polymer is cellulosic polymer such as hydroxypropyl methyl cellulose, otherwise known as HPMC.

[0087] In many embodiments, the spray dried formulations comprising Compound 24, DDM, and HPMC are configured to have a dose of between about 30 mg and 300 mg of Compound 24. Other ranges include 40 mg to 250 mg and 50 mg to 200 mg and ail values in between 30 mg and 300 mg such as 50 mg. Doses are given intranasally and may be given as single or multiple doses in one or both nostrils.

[0088] In another embodiment. A1is a rhamnose fragment.

[0089] In another embodiment, A2is a dinitrophenyl (DNP) fragment.

[0090] In many embodiments, the non-ionic surfactant is n-dodecyl P-D-maltoside (DDM).

[0091] In many embodiments, the solid formulations described herein include one or more excipients, diluents, or combinations of two or more thereof. In another embodiment, the formulations described herein include one or more carriers.

[0092] In many embodiments, unit doses of the solid pharmaceutical compositions containing one or more of the compounds are also described herein. The unit doses include a therapeutically effective amount of the one or more compounds for treating a host animal with an influenza infection. The unit doses are in single or divided form, and may correspond to a daily dosage amount, or adjusted to a periodic amount that is shorter, including for multiple daily doses, or longer, including weekly or monthly doses It is to be understood that the solid pharmaceutical compositions may include other components and / or ingredients, including, but not limited to, other therapeutically active compounds, and / or one or more carriers, vehicles, diluents, adjuvants, excipients, and the like, and combinations thereof.

[0093] In many embodiments, methods for treating host animals with an influenza infection are also described herein, where the methods include administering one or more of the solid pharmaceutical compositions described herein to a host animal with. In another embodiment, uses of the solid pharmaceutical compositions in the manufacture of a medicament for treating host animals with an influenza infection are also described herein. In another embodiment, theDocket No. 1165.071126WOO 1methods, uses, and medicaments include a therapeutically effective amount of the one or more solid pharmaceutical compositions described herein for treating a host animal with an influenza infection.

[0094] It is to be understood herein that the solid pharmaceutical compositions, unit doses, and methods described herein may be used alone or in combination with other compounds useful for treating an influenza infection, including those compounds that may be therapeutically effective by the same or different modes of action. In addition, it is to be understood herein that the solid pharmaceutical compositions comprising the compounds described herein may be used in combination with other compound s and / or formulations thereof that are administered to treat other symptoms or complications of an influenza infection.

[0095] Conjugates described herein include a small molecule neuraminidase inhibitor covalently conjugated to haptens, such as DNP and rhamnose. Conjugates described herein utilize the neuraminidase inhibitor to both suppress viral budding from the host cells, and to bind to the surface of viral particles and virus infected cells, decorating them with haptens. The viral-bound haptens recruit naturally occurring antibodies, such as anti-DNP and anti-rhamnose antibodies, resulting in opsonization of the virus / virus-infected cell and immune-mediated clearance. The mechanism of action of compounds described herein differs from other neuraminidase inhibitors which are susceptible to resistance development via mutations affecting enzymatic activity. The dual mechanism of action of the compound’s neuraminidase-targeted anti-influenza therapy holds promise for significantly reducing the time and severity of influenza-associated symptoms in clinical settings. Conjugates described herein employ that dual -mechanism immunotherapy that targets the viral surface for immune recognition, potentially hindering the emergence of resistance because mutations are less likely to impede this binding compared with enzyme inhibition alone.

[0096] In another embodiment, the solid pharmaceutical compositions described are configured for intranasal (IN) administration, optionally using a device loaded with the composition.

[0097] In another embodiment, the therapeutically effective amount is in the range from about 1 mg to about 1,000 mg for treating a human. In another embodiment, the therapeutically effective amount is in the range from about 10 mg to about 900 mg, from about 20 mg to about 800 mg, from about 20 mg to about 700 mg, from about 20 mg to about 600 mg, from about 20 mg to about 500 mg, or from about 20 mg to about 400 rag.

[0098] In another embodiment, the therapeutically effective amount is in the range from about 100 mg to about 1,000 mg for treating a human. In another embodiment, the therapeuticallyDocket No. 1165.071126WOO 1effective amount is in the range from about 100 mg to about 900 mg, from about 100 mg to about 800 mg, from about 100 mg to about 700 mg, from about 100 mg to about 600 mg, from about 100 mg to about 500 mg, or from about 100 mg to about 400 mg.[00991 another embodiment, the therapeutically effective amount is in the range from about 200 mg to about 1,000 mg for treating a human. In another embodiment, the therapeutically effective amount is in the range from about 200 mg to about 900 mg, from about 200 mg to about 800 mg, from about 200 mg to about 700 mg, from about 200 mg to about 600 mg, from about 200 mg to about 500 mg, or from about 200 mg to about 400 mg.

[0100] In another embodiment, the therapeutically effective amount is in the range from about 300 mg to about 1,000 mg for treating a human. In another embodiment, the therapeutically effective amount is in the range from about 300 mg to about 900 mg, from about 300 mg to about 800 mg, from about 300 mg to about 700 mg, from about 300 mg to about 600 mg, from about 300 mg to about 500 mg, or from about 300 mg to about 400 mg.

[0101] In another embodiment, solid pharmaceutical compositions are described herein that include the active compound in the range from about 0.1% to about 95% by weight. In another embodiment, the compositions include the compound in the range from about 60% to about 95%, or from about 70% to about 92% by weight.

[0102] In another embodiment, solid pharmaceutical compositions are described herein that include the active compound in the range from about 70% to about 80% by weight. In another embodiment, the compositions include the compound in the range from about 70% to about 75%, from about 71 % to about 74%, or from about 72% to about 73% by weight.

[0103] In another embodiment, solid pharmaceutical compositions described herein are administered using a device, such as an Aptar Unidose nasal device.

[0104] In another embodiment, methods are described herein where the compositions are administered for a total treatment duration of 1 day, and dosed either q.d. or b.i.d.

[0105] In another embodiment, the virus is Influenza In another embodiment, the vims is an H1N1 virus, an H3N2 vims, an H3N3 virus, or an oseltamivir-resistant virus.

[0106] In other embodiments, solid pharmaceutical compositions comprising a conjugate of any of the embodiments recited herein, and optionally comprising one or more carriers, diluents, excipients, and the like, and combinations thereof are provided.

[0107] Solid pharmaceutical compositions comprising a conjugate of any of the embodiments recited herein, and optionally comprising one or more carriers, diluents, excipients, and the like,Docket No. 1165.071126WOO 1and combinations thereof for use in treating a host animal having an Influenza infection are also provided.

[0108] Unit doses comprising a solid pharmaceutical composition thereof and optionally comprising one or more carriers, diluents, excipients, and the like, and combinations thereof, where the unit dose is in single or divided form, and includes a therapeutically effective amount of the compound for treating a host animal having an Influenza infection are also provided.

[0109] Further provided, are methods for treating a host animal having an influenza infection, the method comprising administering a therapeutically effective amount of a solid pharmaceutical composition thereof and optionally comprising one or more carriers, diluents, excipients, and the like, and combinations thereof, or a unit dose comprising any of the foregoing, in single or divided form, to the host animal.

[0110] Further embodiments include uses of solid pharmaceutical compositions thereof and optionally comprising one or more carriers, diluents, excipients, and the like, and combinations thereof, in the manufacture of a medicament, including a unit dose, in single or divided form, for treating a host animal having an influenza infection.

[0111] Additional embodiments include kits comprising solid pharmaceutical compositions, or unit dose, of any one of the preceding embodiments; an optional carrier; an optional container for preparing a composition for administration comprising the conjugate or composition or unit dose and the optional carrier; and instructions for administering the conjugate or composition or unit dose or composition for administration to a host animal having an influenza infection.

[0112] The solid pharmaceutical compositions and methods of the disclosure can be used for both human clinical medicine and veterinary applications. Thus, the host animal harboring the population of pathogenic cells and 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. In the embodiments presented herein, the solid pharmaceutical compositions are typically dry¬ powders. In many embodiments, said dry powders are spray-dried compositions comprising a compound of the disclosure and one or more spray-drying excipients In many embodiments, the compound of the disclosure is Compound 24 and tire one or more spray-drying excipients include DDM and HPMC.

[0113] Unless otherwise indicated, the recitation of a numerical value necessarily reflects the relative precision of the numerical value. For example, the recitation of a number with a specified precision based on significant figures necessarily includes a range of values that would match thatDocket No. 1165.071126WOO 1number after appropriate rounding. For example, the recitation of the number I with a single significant figure is understood to properly refer to a range of values from 0.5 to 1.4. Similarly, 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.

[0114] 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. For example, 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 4.5-5.4, 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. Depending upon the context and the variable recited, 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. For DSC, “about” means on the order of ±1 °C on a well maintained instrument.

[0115] The terms “dry' powder formulation” or “dry powder” as used herein mean a solid pharmaceutical composition comprising a compound of the disclosure and one or more pharmaceutically acceptable excipients. The formulation is typically amorphous and may be made, for example, by spray drying or by lyophilization. When made by spray drying, the pharmaceutically acceptable excipients are termed herein as “spray-drying excipients” and the dry powder is referred to herein as a “spray-dried composition” or “spray-dried formulation” or “spray- dried dispersion”. The dry powder formulations of the disclosure are prepared typically for use in intranasal drug delivery. The term dry powder does not require that the powder be 100% without moisture. Indeed, dry powders of the disclosure prepared herein show' loss on drying experiments wherein the amount of moisture int he dry' powder has been measured to be between about 1.46% and about 1.7% on batches not put on stability7. Even on stability, the dry powders may take up some moisture such as to about 2.4% or higher. Even higher moisture contents may occur and theDocket No. 1165.071126WOO 1powder may still be classified as a dry powder.

[0116] The term “spray-drying excipient” means an excipient in a solid pharmaceutical composition of the disclosure which is suitable for use in a spray-dryer in combination with the compound of the disclosure for spray drying so as to make a solid pharmaceutical composition by spray drying.

[0117] The terms “spray-dried dispersion” or “spray-dried composition” or “spray-dried formulation” all mean a solid pharmaceutical composition made by spray-drying a combination of a compound of this disclosure and one or more spray-drying excipients. Typically, the compound of the disclosure and the one or more spray-drying excipients are dissolved or dispersed, as in the case with HPMC, in a solution, often referred to as a feed solution. The feed solution is then fed to a spray dryer for spray drying resulting in said solid pharmaceutical composition. The feed solution solvent is typically deionized water.

[0118] The term “therapeutically effective amount,” as used herein, 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. In one aspect, 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. However, it is to be understood that 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.

[0119] The terms “nasal administration” and / or “intranasal administration” refer to a route of administration where the compound or composition described herein in insufflated through the nose or into the nasal cavity. Nasal administration and / or intranasal administration may use the compound or composition in a variety of physical forms, including, but not limited to dry / powdersDocket No. 1165.071126WOO 1such as spray-dried compositions of the disclosure, suspensions, gels, solutions, and the like. Nasal administration and / or intranasal administration may also use devices loaded with the compound or composition.

[0120] The compound and optionally one or more other therapeutic agents can be administered as a free compound or as a salt. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof.

[0121] The conjugates hereof can be “deuterated,” meaning one or more hydrogen atoms can be replaced with deuterium. As deuterium and hydrogen have nearly the same physical properties, deuterium substitution is the smallest structural change that can be made. Deuteration is well known to those of ordinary skill in the art.

[0122] The conjugates, in some embodiments, can contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that are defined, in terms of absolute stereochemistry, as (R) or (S). In certain embodiments, the conjugate is of R-configuration. In certain embodiments, the conjugate is of S-configuration. Unless stated otherwise, it is intended that all stereoisomeric forms of the conjugates are contemplated. When the conjugates contain alkene double bonds, and unless specified otherwise, it is intended that both E and Z geometric isomers (e.g., cis or trans) and / or optical isomers are included. In certain embodiments, for example, D and A of a conjugate are arranged in a relative cis orientation. In certain embodiments, D and A of a conjugate are arranged in a relative trans orientation. Likewise, all possible isomers, as well as their racemic and optically pure forms, and all tautomeric forms are also intended to be included. The term “geometric isomer” refers to E or Z geometric isomers (e.g., cis or trans) of an alkene doubl e bond. The term “positional isomer” refers to structural isomers around a central ring, such as ortho-, meta-, and para- isomers around a benzene ring.

[0123] Further, in each of the foregoing and following embodiments, it is to be understood that the formulae include and represent not only all pharmaceutically acceptable salts of the conjugates, but also include hydrates and / or solvates of the conjugate formulae or salts thereof. Indeed, hydrates and solvates of the conjugates are also contemplated. The term “solvate” means a conjugate, or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent i termolecular forces. Where the solvent is water, the solvate is a hydrate.

[0124] The following examples further illustrate specific embodiments of the present disclosure;Docket No. 1165.071126WOO 1however, the following illustrative examples should not be interpreted in any way to limit the scope of the claimed invention. Unless otherwise indicated, all starting compounds, reagents, and solvents described in the following examples are obtainable from commercial suppliers.

[0125] The solid pharmaceutical compositions and methods of use and / or treatment described herein may be used with a number of different compounds such as those set forth in the following enumerated clauses. The clauses describe various features and aspects of the compounds used in the pharmaceutical compositions and corresponding methods of the invention. The following enumerated clauses are understood to illustrate many compounds within embodiments of the solid pharmaceutical compositions of the disclosure comprising a compound of the disclosure and one or more spray-drying excipients, and are not to be construed as limiting the scope of the invention in any way.1. A compound of the formulaL2-A1T-UV-A2or a pharmaceutically acceptable salts or solvates thereof, whereinT is a radical of a ligand for a target protein of an Influenza virus or a virus-infected cell;L1, L2, and L3are each, an independently selected linker; andA1and A2are each, a radical of an independently selected hapten.2. The compound of clause 1 wdierein the target protein is a neuraminidase.3. The compound of clause 1 wherein the ligand is a neuraminidase inhibitor,4. The compound of clause 1 wherein the ligand is selected from sialic acid and analogs thereof.5. The compound of clause 1 wherein the ligand is selected from zanamivir, peramivir, laninamivir, oseltamivir, and 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, and analogs and derivatives of the foregoing.6. The compound of clause 1 wherein the ligand is zanamivir, or analog or derivative thereof.7. The compound of clause 1 wherein the ligand is peramivir, or an analog or derivative thereof.8. The compound of clause 1 wherein the ligand is a compound of the formulaDocket No. 1165.071126WOO 1or an analog or derivative thereof.9. The compound of clause 1 wherein the ligand is laninamivir, or an analog or derivative thereof.10. The compound of clause 1 wherein the ligand is of the formulaor an analog or derivative thereof.11. The compound of clause 1 wherein the ligand is oseltamivir, or an analog or derivative thereof.12. The compound of clause 1 wherein the target protein is a hemagglutinin.13. The compound of clause 1 wherein the ligand is a hemagglutinin inhibitor.14. The compound of clause 1 wherein the ligand is aand analogs and derivatives thereof.15. The compound of clause 1 wherein the ligand is umifenovir, arbidol, tert-butyl hydroquinone, flufirvitide 3 (VEDTKIDLWSYNAELL (SEQ ID NO: 1)), cyclo(Ac-YWHKNKYVLTYSC)LFAAG-CONH2 (SEQ ID NO: 2), cyclo(Ac-YRWVWTSFFSEPYFVVC)G-CONH2(SEQ ID NO: 3), cyclo(Ac-Docket No. 1165.071126WOO 1YLKIYWSKIHGLVSEWC)G- CONH2(SEQ ID NO: 4), or cyclo(Ac- YVLFRWDHGTLATHWVC)G-CONH2(SEQ ID NO: 5).16. The compound of clause 1 wherein the ligand is not a folate.17. The compound of any preceding clause wherein A1and A2are the same hapten.18. The compound of preceding clause wherein A1and A2are different haptens.19. The compound of any preceding clause wherein the hapten is selected from a rhamnose, a nitrophenyl, a nitrophenol, a nitroaniline, a dinitrophenyl (DNP), a di nitroph enol, 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,A1and A2are each an independently selected radical of a rhamnose, a-galactosyl moiety, dinitrobenzene, dinitroaniline, trinitrobenzene, and dinitroaniline.20. The compound of any preceding clause wherein A1and / or A2is a radical of a dinitrophenyl (DNP), a dinitrophenol, or a dinitroaniline.21. The compound of any preceding clause wherein A1and / or A2is a radical of a rhamnose, including L-rhamnose.22. The compound of any preceding clause wherein one of A1and A2is a radical of L-rhamnose; and the other of A1and A2is a radical of a dinitroaniline.23. The compound of any preceding clause wherein the hapten has an epitope that has an endogenous Ab, B or T cell.24. The compound of any preceding clause wherein the hapten is not FITC.25. The compound of any preceding clause wherein L2and L3are attached to the same atom of L!.26. The compound of any preceding clause wherein L2and L3are bound to the same atom on L1.27. The compound of any preceding clause wherein L2and L3are bound to the same non-carbon atom on L1.28. The compound of any preceding clause wherein L2and L3are bound to the same nitrogen atom on L1.29. The compound of any preceding clause wherein L2and L3are attached to different atoms of L1.30. The compound of any preceding clause wherein one or more of L1, L2, and L3is a singleDocket No. 1165.071126WOO 1divalent atom selected from N, O, P, and S, where N and P are optionally substituted.31. The compound of any preceding clause wherein one or more of L1, L2, and L3is a chain of atoms, where the length of each chain is independently selected and in the range of about 2 to about 60.32. The compound of any preceding clause wherein one or more of L1, L2, and L3includes 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 36.33. The compound of any preceding clause wherein one or more of L1, L2, and L3includes or also includes (PEG)n, or aza-(PEG)n, or a combination thereof where n is 2-36.34. The compound of any preceding clause wherein each of L1, L2, and L3includes or also includes ethoxy, ethylamino, ethylene glycol, aza-ethylene glycol, (PEG)n, or aza-(PEG)n, or a combination thereof, where n is 2-36.35. The compound of any preceding clause wherein one or more of L1, L2, and L3includes 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.36. The compound of any preceding clause wherein one or more of L1, L2, and L3includes 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.37. The compound of any preceding clause wherein L1includes or also includes O-alkyl-C(O)N-diyl, O-alkyl-O-alkyl-C(O)N-diyl, or N-alkyl-O-alkyl-O-alkyl-C(O)N-diyl.38. The compound of any preceding clause wherein one or more of L1, L2, and L3includes or also includes one or more amino acids.39. The compound of any preceding clause wherein one or more of L1, L2, and L3includes or also includes one or more hydrophilic amino acids selected from Arg, Asn, Asp, Cys, Glu, Gin, His, Lys, Met, Orn, Ser, or Thr, including the naturally occurring L-enantiomers of each of the foregoing.40. The compound of any preceding clause wherein one or more of L1, L2, and L3includes or also includes one or more hydrophilic amino acids selected from p-NH2-Ala, Arg, Asn, Asp, Cys, Glu, Gin, His, Lys, Met, Orn, Ser, or Thr, including the naturally occurring L-enantiomers of each of the foregoing.41. The compound of any preceding clause wherein one or more of L1, L2, and L3includes or also includes one or more amino acids selected from glycine, serine, proline, ornithine, and lysine.Docket No. 1165.071126WOO 142. The compound of any preceding clause wherein one or more of L’, L2, and L3includes or also includes an ornithine or lysine, including L-ornithine and L-lysine.43. The compound of any preceding clause wherein one or more of L1, L2, and L3includes or also includes a lysine, including L-lysine.44. The compound of any preceding clause wherein one or more of L1, L2, and L3includes or also includes a proline, including L -proline.45. The compound of any preceding clause wherein one or more of L1, L2, and L3includes or also includes a (Pro)n, including (L-Pro)n, where n is 1-6.46. The compound of any preceding clause wherein one or more of L1, L2, and LJincludes or also includes a (Pro)3, including (L-Pro)3.47. The compound of any preceding clause wherein one or more of L1, L2, and L3includes or also includes a (Pro)3-Lys, including (L-Pro)3-(L-Lys).48. The compound of any preceding clause wherein one or more of L1, L2, and L3includes or also includes a (Pro)6, including (L-Pro)6.49. The compound of any preceding clause wherein one or more of L1, L2, and L3includes or also includes a (Pro)6-Lys, including (L-Pro)6-(L-Lys)50. The compound of any preceding clause wherein one or more of L1, L2, and L3includes or also includes glycine and serine, including L-serine.51. The compound of any preceding clause wherein one or more of L1, L2, and L3includes or also includes (Gly-Ser)n, including [Gly-(L-Ser)]n, where n is 1-3.52. The compound of any preceding clause wherein one or more of L1, L2, and L3includes or also includes (Gly-Ser), including [Gly-(L-Ser)]2.53. The compound of any preceding clause wherein one or more of L1, L2, and L3includes or also includes (Gly-Ser)2-Lys, including [Gly-(L-Ser)]2-(L-Lys).54. The compound of any preceding clause wherein one or more of L1, L2, and L3includes or also includes (Gly-Ser), including [Gly-(L-Ser)]3.55. The compound of any preceding clause wherein one or more of L1, L2, and L3includes or also includes (Gly-Ser)3-Lys, including [Gly-(L-Ser)]3-(L-Lys).56. The compound of any preceding clause wherein one or more of L1, L2, and L3includes or also includes a C(O), C(O)O, C(O)NH, OC(O)NH, or NHC(O)NH group.57. The compound of any preceding clause wherein one or more of L1, L2, and L3includes or also includes 1,2,3-triazol-1,4-diyl, 1,2,3-triazol-1,4-diyl, or a combination thereof.- 4 ■■Docket No. 1165.071126WOO 158. The compound of any preceding clause wherein one or more of L’, L2, and L3includes or also includes a maleimid-diyl or thiomaleimid-N, S-diyl.59. The compound of any preceding clause wherein L2and / or L3are hydrophilic.60. The compound of any preceding clause wherein L1includes a region that is capable of forming an a-helical conformation.61. The compound of any preceding clause wherein the extended conformation of L2and / or L3is at least 8 A, 9 A, 10 A, 11 A, 12 A, 13 A, 14 A, 15 A, 20 A, 25 A, 30 A, 35 A, or 40 A, in length.62. The compound of any preceding clause wherein L and / or L3includes or also includes (PE(j)nor aza-(PEG)«, 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 36.63. The compound of any preceding clause wherein L2and / or L3includes or also includes one or more divalent cycloalkyl, including adamantyl, heterocyclyl, including maleimidyl, aryl, heteroaryl including triazolyl,, stilbene, oligoproline, or oligopiperidine groups,.64. The compound of any preceding clause wherein the extended conformation of T-Llis at least 5 A, 6 A, 7 A, 8 A, 9 A, 10 A, 11 A, or 12 A in length.65. The compound of any preceding clause wherein L1includes or also includes (PEG)nor 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 36.66. The compound of any preceding clause wherein L1does not include L-lysine.67. The compound of any preceding clause wherein L1does not include DBCO68. The compound of any preceding clause wherein the conjugate is notwhere L1, L2, and L3 are each, independently linkers; orDocket No. 1165.071126WOO 169. A compound of any formula shown in FIG. 7, or a pharmaceutically acceptable salt or solvate thereof.70. A compound of the formulaor a pharmaceutically acceptable salt or solvate thereof.71. A compound of the formulaor a pharmaceutically acceptable salt or solvate thereof.72. A compound of the formulaDocket No. 1165.071126WOO 1or a pharmaceutically acceptable salt or solvate thereof.73. A compound of the formulaor a pharmaceutically acceptable salt or solvate thereof.

[0126] 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. In addition, 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

[0127] In some embodiments, 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.Illustratively, the antibodies are endogenous.

[0128] Without being bound by theory, it is believed that once recruited, 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 conjugatesDocket No. 1165.071126WOO 1described 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 vinis. 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 vims, the surface of an infected cell, or accessible from the surface of an infected cell.

[0129] In other embodiments, the conjugate compound of Formula I can have fragments L2-A!and L / '-A2bound to the same atom on L1. In some embodiments, the same atom is not a carbon atom. In some embodiments, the same atom is a nitrogen atom. Alternatively, the conjugate of Formula I can have fragments L2-A!and L3-A2bound to a different atom on L1.

[0130] The compounds, compositions, unit doses, and methods described herein are useful 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.

[0131] Without being bound by theory, it is believed 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.

[0132] In many illustrative embodiments, 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.

[0133] In many embodiments, the ligand is zanamivirDocket No. 1165.071126WOO 1or an analog or derivative thereof.

[0134] It is to be understood that a radical may be formed on any atom of the foregoing to form the radical T.

[0135] In many embodiments, T is a radical of the formulahaving any specific stereochemical configuration, or having a mixture of two or more stereochemical configurations.

[0136] In many embodiments, T is a radical of the formula

[0137] In many embodiments, the ligand is peramiviror 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.

[0138] In many embodiments, T is a radical of the formulaDocket No. 1165.071126WOO 1

[0139] In many embodiments, the ligand is an amino analog of peramiviror an analog or derivative thereof.

[0140] In many embodiments, T is a radical of the formula

[0141] In many embodiments, the ligand is laninamiviror an analog or derivative thereof.

[0142] In many embodiments, the ligand is a compound of the formulaor an analog or derivative thereofDocket No. 1165.071126WOO 1

[0143] In many embodiments, the ligand is oseltamivir

[0144] In many embodiments, the ligand is of the formulawherein R is alkyl, H, or salt, R₁ is NH₂, NHCH₂CH₂OH, azido, guanidino, NHallyl, N(allyl)₂, or a heterocyclyl group; and R₂ is NH₂ or acetamido.

[0145] In many embodiments, the ligand is of the formulawherein R is Me, H, or salt; R₁ is NH₂ or, guanidino; R₂ is NH₂ or acetamido; and R₄ is alkyl (C-4 to C-14), and ether, acyl, and carbamoyl derivatives thereof.

[0146] In many embodiments, the ligand is of the formulawhere R is Me or Et; R₂ is H or OH; R₁ is alkyl (primary and secondary, C-3 to C-10); and R₃ is NH₂ or optionally substituted guanidino.

[0147] In many embodiments, the ligand is of the formulawherein R is H, Me, Et or a salt; R₁ is NH₂, azido, or guanidino; R₂ is C(O)CH₃, C(O)CF₃, or SO₂Me; and R is alkyl (primary or secondary, C-1 to C-5), or CH₂OMe.

[0148] In another illustrative embodiment, 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.Docket No. 1165.071126WOO 1

[0149] In many embodiments, the ligand is of the formulaand 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.

[0150] It is to be understood that a radical may be formed on any atom of any of the foregoing ligands to form the radical I.

[0151] In many embodiments, T is a radical of flufirvitide 3 of the formula VEDTKIDLWSYNAELL (SEQ ID NO: 1).20. The compound of clause 1 wherein the ligand is cyclo(Ac-YWHKNKYVLTYSC)LFAAG-CONH₂ (SEQ ID NO: 2), cyclo(Ac-YRWVWTSFFSEPYFWC)G-CONH2(SEQ ID NO: 3), cyclo(Ac-YLKIYWSKIHGLVSEWC)G- CONH₂ (SEQ ID NO: 4), or cyclo(Ac- YVLFRWDHGTLATHWVC)G-CONH2(SEQ ID NO: 5), including compounds described in Pascha et al. ACS Chem Biol 17:2425-36 (2022).

[0152] It is to be understood that 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.

[0153] The haptens described herein can each be bound by an antibody. After administration to a subject, each hapten (A1and A2) can be bound by an antibody. In certain embodiments, the two haptens, A1and A2can 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.

[0154] In another embodiment, A1and A2are 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, aDocket No. 1165.071126WOO 1nitrophenol, a nitroaniline, a dinitrophenyl (DNP), a di nitrophenol, 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, compounds of the formulaea phosphorylcholine, a bacterial antigen, a viral antigen, and the like, and any combination of two or more of the foregoing.

[0155] In many embodiments, the hapten is selected from a dinitrophenyl (DNP), a dinitrophenol, and a dinitroaniline. In many embodiments, A1and / or A2is a radical of a dinitrophenyl (DNP), a di nitrophenol, or a dinitroaniline, including radicals of the formulaewhere L2or L3is attached at (*).

[0156] In many embodiments, the hapten is selected from a rhamnose, including L-rhamnose. In many embodiments, A1and / or A2is a radical of a rhamnose, including L-rhamnose, and including a radicals of the formulaewhere L2or L3is attached at (*).Docket No. 1165.071126WOO 1

[0157] In many embodiments, A1is a DNP radical and A2is a rhamnose radical, or vice versa. In many embodiments, A1is a DNP radical and A2is an L-rhamnose radical, or vice versa.

[0158] In another embodiment, neither A1nor A2comprises a fluorescein, such as FITC, and the like.

[0159] As used herein, the term “linker” generally refers to a chain of atoms that connects two or more functional parts of a molecule to form a conjugate. Illustratively, 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, polyoxy alkylene 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. In addition, it is to be understood that 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. In addition, it is to be understood that 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. In this latter arrangement, it is to be understood that the linker length may be defined by any pathway through the one or more cyclic structures. Illustratively, the linker length is defined by the shortest pathway through the each one of the cyclic structures. It is to be understood that 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. It is also to be understood that 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 linkerDocket No. 1165.071126WOO 1

[0160] In many embodiments, L1, L2, and L3each, independently comprises a chain of atoms from 3 atoms to about 60 atoms in length. In many embodiments, L1, L2, and L3each, independently comprises a chain of atoms from about 4 Å to about 72 Å in length. The chain of atoms are part of the backbone of the conjugate of Formula I. As used herein, the term "‘backbone” of the linker L refers to the shortest chain of contiguous atoms forming a covalently bonded connection between T and L2, between T and L3, between A1and L1, or between A2and L1.

[0161] In many embodiments, L1, L2, or L3each, 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. In many embodiments, L1, L2, or L3each, 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. In many embodiments, L1, L2, or L3each, 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. As used herein, the term “betw’een” is inclusive of the endpoints meaning that between 3 and 5 atoms in a chain length includes 3 atoms, 4 atoms, and 5 atoms.

[0162] It is to be understood that while both L2and L3are covalently attached to L1, both L2and L may be attached to the same atom of L1, or L2and L3may each be attached to different atoms of L1. In another embodiment, L2and L3are attached to the same atom on L1where that atom is not a carbon atom.

[0163] In many embodiments, L1, L2, or L3can each, independently, comprise one or more amino acid or peptide residues.

[0164] As used herein, the term “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. Illustratively, R' and / or R" independently correspond to, but are not limited to,Docket No. 1165.071126WOO 1hydrogen 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. For example, the amino acid may be selected from asparagine, aspartic acid, cysteine, glutamic acid, lysine, glutamine, arginine, serine, ornithine, threonine, and the like.

[0165] For example, L1, L2, or L3can each, independently, comprise one or more lysine residues, each of which is independently optionally substituted. For example, L1can comprise a lysine residue of the formula:wherein A3or A2is attached to the lysine residue via a linker. In another embodiment, the lysine residue is L-Lys. In one example, L1comprises at least one lysine residue, such as L-Lys.

[0166] In many embodiments, L1, L2, or L3can each, independently, comprise a polyethylene glycoln(PEGn) moiety, wherein n = 1-36. In many embodiments, a linker comprises one or more PEG moieties where all carbon and oxygen atoms of the one or more PEG moieties are part of the backbone of the linker.

[0167] In many embodiments, L1, L2, or L3can each, independently, comprise one or more alkyl groups.

[0168] In many embodiments, L1, L2, or L3can each, independently, comprise one or more or sugar moieties, glycan residues, or peptidoglycan residues

[0169] It is to be understood that the 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. In each of the foregoing, the atoms of L in the chain can be substituted or unsubstituted.

[0170] In addition, it is to be understood that 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:Docket No. 1165.071126WOO 1wherein each X1is independently CHz, NR’, or O wherein R’ is alkyl or hydrogen and each X2is independently S, O, N, NH, CR” wherein R” is alkyl or hydrogen. Examples of such radicals include:

[0171] In some embodiments, L1, L2, or L3comprises 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.

[0172] L1, L2, or L3can 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 or to T or A1or A2.

[0173] Additional examples of groups which may be present in the linkers include 1-alkylsuccinimid-3-yl, carbonyl, thionocarbonyl, alkyl, cycloalkyl, alkylcycloalkyl, alkylcarbonyl, cycloalkylcarbonyl, carbonylalkyl carbonyl, l-alkylsuccinimid-3-yl, 1-(carbonylalkyl)succinimid-3-yl, alkyl sulfoxyl, sulfonylalkyl, alkylsulfoxylalkyl,al kylsulfonylal kyl, carbonyltetrahydro-2H-pyranyl, carbony I tetrahydrofuranyl, 1 -(carbonyltetrahydro-2H-pyranyl)succinimid-3-yl, and 1 - (carbonyltetrahydrofuranyr)succinimid-3-yl, wherein each group can be substituted or unsubstituted. In some embodiments, one or more of the aforementioned groups can be used inDocket No. 1165.071126WOO 1combination (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-). Examples include alkylcarbonyl, cycloalkylcarbonyl, carbonylalkylcarbonyl, 1-(carbonylalkyl)succinimid-3-yl, and succinimid-3-ylthiol, wherein each group can be substituted or unsubstituted.

[0174] In some embodiments, L1, L2, or L3can be formed via click chemistry or be click chemistry-derived. For example, L1, L2, or L3can be derived from copper-catalyzed azidealkyne cycloaddition (CuAAC), strain promoted azide-alkyne cycloaddition (SPAAC), inverse electron demand Diels- Alder reaction (IEDDA), and Staudinger ligation (SL). For example, 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’ / A2represents a linker between T and A' / A2and the groups to which they are attached.(0175 ] In other embodiments, L’, L2, or L3can includewhere x is an integer from 0 to 50 and y is an integer from 0 to 50

[0176] In other embodiments, L1, L2, or L3can includewherein each of R2and R3is independently H or Ci-6alkyl; and z is an integer from 1 to 8.Docket No. 1165.071126WOO 1

[0177] In other embodiments, L1, L2, or L3can include an amide, ester, urea, carbonate, carbamate, amino acid, amine, ether, alkyl, alkene, alkyne, heteroalkyl cycloalkyl, and, heterocycloalkyl, heteroaryl, carbohydrate, glycan, peptidoglycan, polypeptide, or any combination thereof. In some embodiments, L1, L2, or L3can include a glycosylated amino acid. In some embodiments, L1, L2, or L,3can include one or more monosaccharide, di saccharide, polysaccharide, glycan, or peptidoglycan. In some embodiments, L1, L2, and L3do not comprise a glycan. In some embodiments, L1, L2, and L3do not comprise a sugar.

[0178] In some embodiments, L1, L2, or L can include a rigid functionality such as an oligoproline or oligopiperidine.

[0179] In some embodiments, 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).

[0180] In some embodiments, L1, L2, or I.,3can comprise (“CIl2CIl2-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)C2-Ci8 alkyl- or C2-C18 alkyl-C(O)N(H)-), an alkylamidoalkyl group (e.g., a C2-C18 alkyl-C(O)N(C2-Ci8 alkyl)2 or a C2-C18 alkyl-C(O)N(H)-C2-Ci8 alkyl group, such as a -CH₂CH₂C(O)N(CH₂CH₂)₂ or a -CH₂CH₂C(O)N(H)(CH₂CH₂)- group), or a combination of two or more of the foregoing.

[0181] In some embodiments, L1, L2, or L3can comprisewherein 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. For example, m can be 0 or 1. Or m can be 5 or 6. Or m can be 7 or 8. In some embodiments, L1, L2, or L3can comprisewherein 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, For example, p can be 2 or 3. For example, q can be 2 or 3, In some embodiments, L1, L2, or L3can compriseDocket No. 1165.071126WOO 1wherein 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. For example, d can be 1, 2 or 3.

[0182] In some embodiments, L1, L2, and L3taken together can comprisesuch as wherein 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. For example, m can be 0 or 1. Or m can be 5 or 6. Or m can be 7 or 8. For example, p can be 2 or 3, For example, q can be 2 or 3. For example, d can be 1, 2 or 3.

[0183] In some embodiments, L1, L2, and L3taken together can compriseDocket No. 1165.071126WOO 1

[0184] In another embodiment, the following compounds are describedDocket No. 1165.071126WOO 1

[0185] In many embodiments, the target protein can be an envelope protein of an influenza or an influenza envelope protein on the surface of a virus-infected cell. In many embodiments, the target protein can be influenza neuraminidase or influenza hemagglutinin.

[0186] The disclosure is further directed to solid pharmaceutical formulations of a Compound 24. Multiple formulations comprising of Compound 24 alongside the excipients HPMC and DDM were spray dried at scales ranging from 0.5 g up to 4.5 g. The resulting powders were all fully amorphous and within the particle size range suitable for nasal deliver}'. The Compound 24 in the powder was unchanged, with respect to content and impurity profile, by the spray drying process. The spray dried powders, when filled into Aptar Unidose devices, were also shown to successfully actuate to produce emitted fractions >90%.

[0187] On stability, three different formulations filled in Aptar Unidose devices were evaluated over a 4-week period. The formulations differed with respect to their Compound 24 loading (ranging from 72.5% w / w up to 90% w / w) and the percentage of DDM (ranging from 2.0% up to 6.1% w / w). The data generated showed that when devices were stored at the accelerated conditions of 40°C / 75%RH, the two formulations with the lower Compound 24 loading of 72.5% w / w were less prone to physical changes such as powder aggregation and chemical degradation. When stored at refrigerated (2-8°C) and 25°C / 60%RH, the formulations were more chemically and physically stable. The particle size distribution by laser diffraction was unchanged at the T=4 week testing timepoint, while the powders also remained fully amorphous. Using the AINI-NGI set up, the majority of the formulation deposited in the highly vascularized Turbinate region of the nasal cavity. This was largely unchanged when tested at T=4 weeks for all the devices / formulations stored at 2-8°C and 25°C / 60%RH.

[0188] All three formulations set down on stability were also evaluated in an animal pharmacokinetic study. Here, 6B showed an improvement in Compound 24 bioavailability when compared against batch 6A, prepared with the same Compound 24 loading and 2.0% w / w DDM.

[0189] The disclosure further includes the following embodiments set forth as clauses

[0190] Clause 1. Amorphous Compound 24.Docket No. 1165.071126WOO 1

[0191] Clause 2. The amorphous Compound 24 of clause 1, having an x-ray powder diffraction pattern substantially the same as that of FIG. 22

[0192] C Clause 3. The amorphous Compound 24 of clauses 1 or 2, having a glass transition temperature at one or more of about 41°C, about 43°C, and about 85°C.

[0193] Clause 4. The amorphous Compound 24 of clause 3, having substantially the same DSC thermogram as that of FIG. 23(a) or FIG. 23(b).

[0194] C Clause 5. The amorphous Compound 24 of any one of clauses 1-4, having a particle size distribution with an X?o of between about 35 and 45 microns or about 20 to 40 microns.

[0195] Clause 6. The amorphous Compound 24 of any one of clauses 1-5, where between 5% and 20% of the particles have a size of less than 10 microns.

[0196] Clause 7. The amorphous Compound 24 of any one of clauses 1-6, having a solubility of between about 90 and 100 mg / g in deionized water

[0197] Clause 8. A dry powder comprising Compound 24 and one or more pharmaceutically acceptable excipients.

[0198] Clause 9. A dry' powder comprising Compound 24 of clause 8, and one or more pharmaceutically acceptable excipients.

[0199] Clause 10. A spray-dried formulation comprising Compound 24.

[0200] Clause 11. The spray-dried formulation of clause 10 further comprising one or more spray-drying excipients.

[0201] Clause 12. The spray-dried formulation of clause 11, wherein at least one spray-drying excipient is a cyclodextrin.

[0202] Clause 13. The spray-dried formulation of clause 12, wherein the cyclodextrin is hydroxy -propyl-beta-cyclodextrin.

[0203] Clause 14. The spray-dried formulation of clause 11, wherein at least one spray-drying excipient is a cellulose-based compound.

[0204] Clause 15. The spray-dried formulation of clause 14, wherein the spray-drying excipient is HPMC.

[0205] Clause 16. The spray-dried formulation of clause 11, wherein the spray-drying excipient is a sugar.

[0206] Clause 17. The spray-dried formulation of clause 16, wherein the sugar is mannitol.

[0207] Clause 18. The spray-dried formulation of clause 11, wherein at least one spray drying excipient is DDM.Docket No. 1165.071126WOO 1

[0208] Clause 19. The spray-dried formulation of clause 11, comprising two spray-drying excipients.

[0209] Clause 20. The spray-dried formulation of clause 19, wherein one spray-drying excipient is HPMC.

[0210] Clause 21. The spray-dried formulation of clause 20, wherein the second spray-drying excipient is DDM.

[0211] C Clause 22. The spray-dried formulation of any one of clauses 11 to 20 further comprising DDM

[0212] Clause 23. A spray-dried formulation comprising Compound 24, HPMC, and DDM.

[0213] Clause 24. The spray-dried formulation of clause 23, wherein Compound 24 is present in an amount of between about 60% and about 90% by weight.

[0214] Clause 25. The spray-dried formulation of clauses 23 or 24, wherein the amount of Compound 24 by weight is between about 60% and about 80% by weight.

[0215] Clause 26. The spray -dried formulation of any one of clauses 23-25, wherein the amount of Compound 24 by weight is between about 65% and about 75% by weight.

[0216] Clause 27, The spray-dried formulation of any one of clauses 23-26, wherein the amount of Compound 24 by weight is between about 70% and about 73% by weight.

[0217] Clause 28. The spray-dried formulation of any one of clauses 23-26, wherein the amount of Compound 24 by weight is about 72.5% by weight.

[0218] Clause 29. The spray-dried formulation of any one of clauses 23-28, wherein the amount of HPMC by weight is between about 2% and about 30% by weight.

[0219] Clause 30. The spray-dried formulation of any one of clauses 23-29, wherein the amount of HPMC by weight is between about 8% and about 30% by weight.

[0220] Clause 31. The spray-dried formulation of any one of clauses 23-30, wherein the amount of HPMC by weight is between about 18% and about 30% by weight.

[0221] Clause 32. The spray-dried formulation of any one of clauses 23-31, wherein the amount of HPMC by weight is between about 20% and about 27% by weight.

[0222] Clause 33. The spray-dried formulation of any one of clauses 23-32, wherein the amount of HPMC by weight is between about 21% and about 26% by weight.

[0223] Clause 34, The spray-dried formulation of any one of clauses 23-33, wherein the amount of HPMC by weight is between about 21% and about 22% by weight.

[0224] Clause 35. The spray-dried formulation of any one of clauses 23-34, wherein the amountDocket No. 1165.071126WOO 1of HPMC by weight is about 21.4% by weight.

[0225] Clause 36. The spray-dried formulation of any one of clauses 23-34, wherein the amount of HPMC by weight is about 25.0% by weight.

[0226] Clause 37. The spray-dried formulation of any one of clauses 23-34, wherein the amount of HPMC by weight is between about 6% and about 10% by weight.

[0227] Clause 38. The spray-dried formulation of any one of clauses 23-37, wherein the amount of DDM by weight is between about 1% and about 10% by weight.

[0228] Clause 39. The spray-dried formulation of any one of clauses 23-38, wherein the amount of DDM by weight is between about 1% and about 3% by weight.

[0229] Clause 40. The spray-dried formulation of any one of clauses 23-39, wherein the amount of DDM by weight is about 2% by weight.

[0230] Clause 41. The spray-dried formulation of any one of clauses 23-38, wherein the amount of DDM by weight is between about 4% and about 8% by weight

[0231] Clause 42. The spray-dried formulation of any one of clauses 23-38 or 41, wherein the amount of DDM by weight is between about 5% and about 7% by weight.

[0232] Clause 43. The spray-dried formulation of any one of clauses 23-38 or 41-42, wherein the amount of DDM by weight is about 6.1% by weight.

[0233] Clause 44. A spray-dried formulation comprising Compound 24, HPMC, and DDM wherein the Compound 24 is amorphous.

[0234] Clause 45. The spray-dried formulation of clause 23 or 44 in amorphous form.

[0235] Clause 46. The spray-dried formulation of clauses 44 or 45, wherein the amount of Compound 24 is between about 60% and about 85% by weight, the amount of HPMC is between about 10% and about 30% by weight, and the amount of DDM is between about 2% and about 10% by weight.

[0236] Clause 47. The spray-dried formulation of clause 46, wherein the amount of Compound 24 is between about 68% and about 76% by weight, the amount of HPMC is between about 17% and about 25% by weight, and the amount of DDM is between about 4% and about 8% by weight.

[0237] Clause 48. The spray-dried formulation of clause 47, wherein the amount of Compound 24 is between about 71% and about 74% by weight, the amount of HPMC is between about 20% and about 23% by weight, and the amount of DDM is between about 5.5% and 6.5% by weight.

[0238] Clause 49. The spray-dried formulation of clause 47, wherein the amount of Compound 24 is between about 72% and about 73% by weight, the amount of HPMC is between about 21%Docket No. 1165.071126WOO 1and about 22% by weight, and the amount of DDM is between about 5.5% and 6.5% by weight.

[0239] Clause 50. The spray-dried formulation of clause 47, wherein the amount of Compound 24 is about 72.5% by weight, the amount of HPMC is about 21.4% by weight, and the amount of DDM is about 6.1% by weight.

[0240] Clause 51. The spray-dried formulations of any one of clauses 44 to 50, having an x-ray powder diffraction pattern substantially the same as that of the bottom x-ray powder diffraction pattern of FIG. 25 or any x-ray powder diffraction pattern in FIG, 31.

[0241] Clause 52. The spray-dried formulations of any one of clauses 44 to 1, having a DSC thermogram substantially the same as that of any one of the DSC thermograms of FIG. 33.

[0242] Clause 53. The spray-dried formulations of any one of clauses 44-52, having a water content of less than 3% by weight.

[0243] Clause 54. The spray-dried formulations of any one of clauses 44-53, having a water content of between about 1% and about 2% by weight.

[0244] Clause 55. The spray-dried formulations of any one of clauses 44-54, having a glass transition temperature of between about 46.9°C and about 49.0°C.

[0245] Clause 56. The spray-dried formulations of any one of clauses 44-55, having a glass transition temperature of about 47.4°C.

[0246] Clause 57. A solution comprising Compound 24 and one or more spray-drying excipients.

[0247] Clause 58. The solution of clause 57, wherein one spray-drying excipient is DDM.

[0248] Clause 59. The solution of clauses 57 or 58 further comprising spray-drying excipient HPMC.

[0249] Clause 60. The solution of any one of clauses 57-59 further comprising water.

[0250] Clause 61. The solution of clause 60, wherein the water is deionized water.

[0251] Clause 62. A process of preparing a spray-dried formulation comprising Compound 24 and one or more spray-drying excipients comprising spray-drying a solution of any one of clauses 53-61 to form a spray-dried composition.

[0252] Clause 63. Spray-dried formulations comprising Compound 24 prepared by the process of clause 62.

[0253] Clause 64, A method of treating an influenza infection comprising the step of administering to a patient in need a spray-dried formulation of any one of clauses 10 to 56 to the patient in need thereofDocket No. 1165.071126WOO 1

[0254] Clause 65. Use of a dry powder formulation in the manufacture of a medicament for treating an influenza infection, the composition comprising (i) a therapeutically effective amount of Compound 24 and (ii) one or more spray-drying excipients.

[0255] Clause 66. A pharmaceutical composition comprising the spray-dried formulations of any one of clauses 10 to 56.

[0256] Clause 67. The spray-dried formulation of any one of clauses 10 to 56, having a water content of less than 5% by weight.

[0257] Clause 68. The spray-dried formulation of any one of clauses 10 to 56, having a water content of between about 0.5% and about 3% by weight.

[0258] Clause 69. The spray-dried formulation of any one of clauses 10 to 56, having a water content of between about 1% and about 2% by weight.

[0259] Clause 70. The spray-dried formulation of any one of clauses 10 to 56, having a water content of between about 1.4% and about 2.4% by weight.EXAMPLES

[0260] The following examples serve to further illustrate the invention described herein. The examples are illustrative of the many embodiments of the invention and are not to be constmed as limiting the scope of the invention in any way.LIST OF ABBREVIATIONSDocket No. 1165.071126WOO 1Docket No. 1165.071126WOO 1

[0261] General. Unless otherwise indicated, all starting compounds and reagents are commercially available. Unless otherwise indicated, reactions are performed under an ambient atmosphere, and at ambient temperature and pressure, and reaction progress is monitored by TLC, LCMS, or both. Unless otherwise indicated, solutions are concentrated and compounds are isolated by rotary evaporation under reduced pressure, under high vacuum, or both.COMPOUND EXAMPLES

[0262] EXAMPLE 1. Compound 3 (FIG. 5): To a solution of Compounds 1 (0.5 g, 2.47 mmol) and 2 (0.33 g, 2.47 mmol) (both commercially available) dissolved in ethyl alcohol (25 mL) was added trietliylamine (1.38 mL, 9.87 mmol). The reaction mixture was heated to 55 °C for 16 hours and reaction progress was monitored by LC-MS. After completion of reaction was confirmed by disappearance of one of the starting materials, i.e., dinitrobenzene, the reaction mixture was cooled and concentrated under reduced pressure. The crude mixture was purified by flash column chromatography on a Teledyne CombiFlash RE Lumen (silica-gel, 12 g column, 0-20% methanol in dichloromethane) to yield Compound 3 as a yellow solid, yield, 90%). LC-MS [M+H]+= 300.24. Additional details are described in Chemistry - An Asian Journal (2012), 7, (2), 272-276.

[0263] EXAMPLE 2. Compound 5. To a solution of 3-(2-((2,4~ dinitrophenyl)amino)ethoxy)propanoic acid (3, 0.1 g, 0.33 mmol) in dimethyl sulfoxide (2 mL) was added l-[bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate, (0.11 g, 0.28 mmol, 0.85 equiv.) followed by N, N’-diisopropylethyl amineDocket No. 1165.071126WOO 1(0.29 mL, 1.67 mmol, 5.0 equiv.) under argon atmosphere and stirred for 10 min at room temperature. Compound 4 (0.11 g, 0.27 mmol, 0.8 equiv.) was added to the reaction mixture and stirred for 2-3 hours at room temperature. Progress of the reaction was monitored by LC-MS. After completion of the reaction was confirmed by LC-MS, the reaction mixture quenched by adding water and extracting with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate and concentrated under vacuum. The resulting crude was purified by silica-gel (4 g) column chromatography on Teledyne CombiFlash Rf+ Lumen (0-10% methanol in dichloromethane) and fractions were analyzed by LC-MS. The solvent evaporated from combined pure fractions using a rota-evaporator under reduced vacuum and Compound 5 was isolated in 70% yield. LC-MS [M+H]+= 650.67

[0264] EXAMPLE 3. Compound 7 (FIG. 6): Compound 6 (1.0 g, 5.49 mmol) (commercially available) was dissolved in 9.2 mL of anhydrous pyridine. The solution was stirred in an ice bath and purged with nitrogen prior to the dropwise addition of acetic anhydride (4.15 mL, 43 92 mmol, 8,0 equiv.) for 15 minutes by maintaining the internal temperature below 10 °C. The reaction allowed to warm to room temperature slowly in 2 hours and progress of the reaction was monitored via TLC (Hexane / EtOAc, 65:35) and LC-MS, indicated complete consumption of a-L-rhamnose monohydrate after 20 hours of reaction under an inert atmosphere. The reaction mixture was poured into ethyl acetate and extracted twice with 1.0 M HC1. The combined organic layers were washed with saturated sodium carbonate solution, water and brine and dried over anhydrous sodium sulfate, and concentrated under vacuum. The resulting crude product, 7 (yield, 98%) was used in the next step. LC-MS [M+H]+= 333.32 and / or LC-MS [M+H2O] = 350.32.

[0265] EXAMPLE 4. Compound 9: To a solution of Compound 7 (0.50 g, 1.50 mmol) in dichloromethane (DCM, 7,5 mL) was added H2N-PEG4-OH (8, 0.35 g, 1.81 mmol, 1.2 equiv.) (commercially available) under an inert atmosphere. The reaction flask was placed in an ice bath, and boron trifluoride di ethyl etherate (0.56 mL, 4.51 mmol, 3.0 equiv.) was added dropwise over 30 min at 4 °C. The reaction mixture stirred at ice bath temperature for 2 hours before the reaction was allowed to warm to room temperature. The progress of the reaction was monitored by TLC (Hexane / EtOAc, 30:70, Rf = 0.30) and LC-MS indicated complete consumption of 1,2,3,4-tetra-O-acetyl-a-L -rhamnose after 16 h of reaction. The reaction mixture was poured into ice water and extracted with DCM (3x10 mL). The combined organic layers were washed twice with saturated sodium bicarbonate solution, water, brine and dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was purified by silica-gel (12 g) columnDocket No. 1165.071126WOO 1chromatography on Teledyne CombiFlash Rf+ Lumen (0-10% methanol in dichloromethane) and Compound 9 was isolated, yield, 90%. LC-MS [M+H]+= 466.51. Additional details are described in Biomacromolecules (2020), 21, 793-802.

[0266] EXAMPLE 5. Compound 10 (FIG. 5): To a solution of Compound 5 (0.06 g, 0.09 mmol) and 9 (0.04 g, 0.09 mmol, 1.0 equiv.) in dimethyl sulfoxide (1.5 mL) was added benzotri azole- 1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (0.05 g, 0.10 mmol, 1.1 equiv.) followed by N, N-diisopropyl ethyl amine (DIPEA, 0.081 mL, 0.46 mmol, 5.0 equiv.) under argon atmosphere at room temperature. Progress of the reaction monitored by LC / MS After completion of the reaction confirmed by LC-MS, the reaction mixture quenched by adding water and extracting with ethyl acetate. The combined organic layers rvashed with brine, dried over anhydrous sodium sulfate and concentrated under vacuum The resulting crude was purified by silica gel (4 g) column chromatography on Teledyne CombiFlash Rf+ Lumen (0-10% MeOH in dichloromethane) and Compound 10 was isolated in 93% yield. LC-MS [M+H]+= 1098.15

[0267] EXAMPLE 6. Compound 11: To Compound 10 (0.01 g, 0.01 mmol) in dry' dichloromethane (0.2 ml) was added diethylamine (DEA) (lOOpL) under argon at room temperature. The solution stirred for 1 hour at rt. until the reaction was complete as demonstrated by LC-MS. The DEA was removed by rotary evaporation under reduced pressure and the crude product precipitated in diethyl ether to give as a yellow solid (Compound 11) with a quantitative yield and was used for next step without further purification. LC-MS [M + H]+= 875.91.

[0268] EXAMPLE 7. Compound 13: To Compound 12 (5g, 11.0 mmol) (commercially available) in tetrahydrofuran (THF, 40mL) was added triphenylphosphine (3.67g, 14mmol, 1 27 equiv.), and the resulting solution was stirred at r.t. for 12 hours. Subsequently, water (lOmL) was added, and the solution was stirred at r.t for another 2-4 hours. The reaction mixture was diluted and extracted with EtOAc (2x30 mL), followed by DCM (3x25 mL) The combined organic layers were washed with saturated aq. NaCl separately, dried over anhydrous sodium sulfate, and concentrated under vacuum. The resulting crude product was purified by silica-gel (24 g) flash column chromatography on a Teledyne CombiFlash Rf+ Lumen (0-100% EtOAc in hexanes) to give Compound 13 as a yellow powder. Compound 13 was isolated, 2.89g, yield, 61 %. LC-MS [M+H]+= 431.42. The starting material, Compound 12, was purchased from commercial sources.

[0269] EXAMPLE 8. Compound 15: To a solution of Compound 13 (2.74 g, 6.37 mmol) and N, N’-bis(tertbutoxycarbonyl)-lH-pyrazole-l-carboxamidine (14, CAS number, 152120-54-2, Sigma-Aldrich, 2.57 g, 8.28 mmol, 1.30 equiv.) dissolved in THF (20 mL) was added triethylamineDocket No. 1165.071126WOO 1(1.5 mL). The reaction mixture was stirred overnight at r.t. and progress of the reaction was monitored by LC / MS. After completion of the reaction, quenched with water, diluted and extracted with EtOAc (3x20 mL). The combined organics were washed with saturated brine solution, followed by dried over anhydrous sodium sulfate (anhy. NajSCh) and evaporated under vacuum. The resulting crude product purified by flash column chromatography on a Teledyne Combi Flash Rf+ Lumen (silica gel, 24 g column, 0-100% EtOAc in hexanes) to give Compound 15 as a white solid (4.14 g, 97 %). LC-MS [M+H]1- 673.70

[0270] EXAMPLE 9. Compound 17: 0.5 M sodium methoxide in methanol (2.9mL, 0.5M, 1.414mmol) was added to a stirred solution of Compound 15 (4.225g, 6.281mmol) in anhydrous methanol (70mL). The reaction mixture was then stirred for Ihour. Dowex 50XW8 (H+) resin added to the reaction mixture to lower the pH ~3 and filtered, washed with methanol and concentrated under reduced pressure to lead compound, 16, and was used in the next step without further purification. LC-MS [M+H]4= 547.58

[0271] To Compound 1 in anhydrous acetone (70mL) was added 2,2-dimethoxypropane (7.7mL, 6.54g, 62.81mmol, 10 equiv.), followed by p-toluenesulfonic acid (120mg, 0.628mmol, 0.1 equiv.), and the resulting mixture was stirred overnight at room temperature The reaction mixture was then concentrated and purified by flash column chromatography on a Teledyne CombiFlash Rf+ Lumen (silica gel, 12 g column, 0-100% EtOAc in hexanes) to give Compound 17 as a white solid, product isolated 2.4g and yield, 65 %. LC-MS [M+H]4= 587.64.

[0272] EXAMPLE 10. Compound 19: To a solution of Compounds 17 (1.46 g, 2.49 mmol) and 18 (3.51 g, 17.43 mmol) (commercially available) in pyridine (30 mL) was added DMAP (2 13 g, 17.43 mmol). The reaction mixture was stirred overnight at r.t. and progress of the reaction followed by LC / MS. After completion of the reaction confirmed by LC / MS by disappearance of Compound 17, the reaction mixture is diluted and extracted with EtOAc (3x 25 mL). The combined organic layer was then washed with water, brine and dried over anhydrous NazSCh then concentrated and purified by flash column chromatography on a Teledyne CombiFlash Rf+ Lumen (silica gel, 24 g column, 0-100% EtOAc in hexanes) to give Compound 19 as a white solid (1.62 g, 87%). LC-MS [M+H]4= 752.45.

[0273] EXAMPLE 11. Compound 21: To Compound 19 (0.168 g, 0.223 mmol), in tetrahydrofuran (THF, 1,1 mL) was added 20 (0.079 g, 0.223 mmol, 1.0 equiv.) (commercially available) followed by N, N’-diisopropylethyl amine (DIPEA, 0.1 mL, 0.56 mmol, 2.5 equiv.) at r.t. under argon and stirred for 1 hours. Progress of the reaction was monitored by TI C andDocket No. 1165.071126WOO 1LC7MS. After completion of the reaction was confirmed by LC / MS by the complete disappearance of Compound 19, the reaction was diluted and extracted with EtOAc (3x 25 mL). The combined organic layers were then washed with water, brine and dried over anhydrous NasSOr then concentrated under vacuum. The crude product was purified by flash column chromatography on a Teledyne CombiFlash Rf+ Lumen (silica gel, 12 g column, 0-10% MeOH in DCM) to give Compound 21 as a white solid (0.205 g, 90 %). LC-MS [M+H]+= 967.06.

[0274] EXAMPLE 12. Compound 22. To a solution of Compound 21 (0,025 g, 0,026 mmol) in dichloromethane:dimethyl sulfoxide (10:1, 0.3 mL) was added l-[bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate, (HATU, 0.01g, 0.026 mmol, 1.0 equiv.) followed by N, N’ -di isopropylethyl amine (0.012 uL, 0.065 mmol, 2.5 equiv.) under argon atmosphere and stirred for 3-5 min at room temperature. Compound 11 (0.023 g, 0.026 mmol, 1.0 equiv.) in dichloromethane (0.2 mL) was added to the reaction mixture and stirred for 2-3 hours at room temperature. Progress of the reaction was monitored by LC-MS After completion of the reaction was confirmed by LC-MS, the solvent evaporated, and the resulting crude was purified by silica-gel (4 g) column chromatography on Teledyne CombiFlash Rf+ Lumen (0-10% methanol in di chloromethane) and fractions were analyzed by LC-MS. The solvent evaporated from combined pure fractions using rotaevaporator under reduced pressure and product (22) was isolated in 61% yield. LC-MS [M+H]!= 1822.94.

[0275] EXAMPLE 13, Compound 24: 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 22 (38 mg, 0.021 mmol) was dissolved in methanol (0.5 mL) and treated drop wise with 0.5 M sodium methoxide in methanol (190 pL). The reaction mixture stirred at r.t. for 1 hour at which time LC- MS analysis revealed that hydrolysis of methyl ester as well as -OAc groups were completed. The reaction mixture was neutralized by adding Dowex® 50WX8 (H^) resin, filtered, and concentrated under reduced pressure. The crude product (Compound 23) was used directly for the next step without further purification. LC-MS analysis revealed with multiple products, i.e., [M+H]+= 1683.89, 1642 (loss of acetonide) and 1542 (loss of -boc).Docket No. 1165.071126WOO 1

[0276] To the crude compound, Compound 23, in dichloromethane (DCM, 0.8 mL) and trifluoroacetic acid (TFA, 0.8 mL) was added at ice-cold temperature. The reaction mixture was stirred for 15 min at r.t. and LC-MS indicated the reaction was complete. TFA was removed by rotary evaporation under reduced pressure and washed with cold diethyl ether. The crude product purified by prep-HPLC (UPLC) on a C18 column (5-95% B over 60 rain, flow 7 mL / rain; B: acetonitrile; A: 20 mM NFLOAc, pH 7 buffer, UV @ 360 nm and 220 nm) to afford Compound 24 and yield, 42 %. Purity: 95% by LC-MS [M + H]’ - 1443.83.

[0277] EXAMPLE 14 (2R,3R,4S)-3-acetamido-2-((38R,39R)-25-(18-((2,4-dinitrophenyl)amino)-13-oxo-3, 6,9, 16-tetraoxa-12-azaoctadecyl)-39,40-dihydroxy-13, 26,36-trioxo-l-(((2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-3,6,9,16,19,22,29,32,37-nonaoxa-12,25,35-triazatetracontan-38-yI)-4-guanidino-3,4-dihydro-2H-pyran-6-carboxylic acid (Compound 42).Docket No. 1165.071126WOO 1

[0278] Compound 3. To a stirred solution of Compound 1 (Angene, 1.0 g, 1.91 mmol) and Compound 2 (1.07 g, 2.03 mmol) in DMF (15.0 mL) HATU (0.034 g, 0.09 mmol) was added at 0 °C. After 10 min ethylbis(propan-2-yl)amine (GLR, 1.77 mL, 9.57 mmol) was added dropwise to the reaction mixture at 0 °C. Cooling was discontinued and the mixture was stirred for 12 h. After reaction completion, the reaction mixture was concentrated, and the remainder was purified by silica gel column chromatography eluting in 5% MeOH / DCM to afford Compound 3 as a yellowish liquid. Yield: 1.20 g, 64.65 %; LCMS (ELSD) m / z 970.60 [M+l ].

[0279] Compound 4. To a solution of Compound 3 (1.0 g, 1.03 mmol) in MeOH (50.0 mL), 10% Palladium on carbon (Johnson Matthey, 1.00 g) was added and stirred under an hydrogen atmosphere for 6 h. After reaction completion, the reaction mixture was filtered through a celite bed, washed with MeOH and the filtrate was evaporated to dryness. The remainder was triturated with diethyl ether to afford tert-butyl Compound 4 as a pale yellow viscous liquid. Yield: (0.80 g, 82.2 %); LCMS (ELSD) m / z. 944.65 [M+l]+.

[0280] Compound 6.. 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 ethylbis(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+l]1.

[0281] Compound 7. To a stirred solution of Compound 6 (0.31 g, 0.25 mmol), in anhydrous DCM (5.0 mL), TFA (3.0 ml.,) was added at 0 °C. Cooling was discontinued and the reaction mixture was stirred for 2 h. After reaction completion, the reaction mixture was evaporated to Compound 7 as a light yellow liquid which was used without further purification. Yield: 0.22 g; LCMS (ESI) m / z 1125.10 [M+l]1.

[0282] Compound 8 was prepared as described in Carbohydrate Research, 342 (2007) 1636-1650).

[0283] Compound 10. To a flask containing Compound 8 (0.21 g, 0.28 mmol) in dry THF (5 ml) was added DIPEA (GLR, 0.26 ml, 1.44 mmol) dropwise at 25-30°C under a N? atmosphere. After 5 mins, Compound 9 (Angene, 56 mg, 031 mmol) in dry THF (2 ml) was added to the reaction mixture and the resulting suspension was stirred for another 24 h at 25-30°C. Progress of the reaction was monitored by TI C and LCMS. After reaction completion, the reaction mixture wasDocket No. 1165.071126WOO 1concentrated and the remainder was purified by column chromatography eluting in 10% MeOHDCM to afford Compound 10 as off white solid Yield: 0.16 g, 70.05 %; LCMS (ESI) m / z 790.10 [M+l]+.

[0284] Compound 11. 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, 0.15mmol) 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+l ]+.

[0285] Compound 42 To a stirred solution of Compound 5 (0.11 g, 0.06 mmol) in THF (3 mL) and MeOH (1 mL) was added a 1.0 M solution of sodium hydroxide (0.1 mL) at 25-30 °C over 10 mins. An homogeneous solution was observed and stirred for 4 h at 25-30 °C. Completion of the reaction was confirmed by TLC and I, CMS and the reaction mixture pH was slowly adjusted to pH 7, by using Dowex 50XW8 (H ) resin. The reaction mixture was filtered through celite-bed and evaporated. To the stirred solution of the remainder in anhydrous DCM (3.0 mL), TF (1.0 mL) was added at 0 °C and the reaction mixture was stirred at 15 °C for 2 h. After reaction completion, the solvent was evaporated and the remainder was purified by prep HPLC using water / ACN in 0.1% TFA to afford Compound 42 as a yellowish solid. Yield: 0.015 g, 37.07 %; LCMS (ESI) m / z 1516.35 [M+l]+. ’HNMR (400 MHz, DMSO-de): 8.86 (d, J = 2.8 Hz, 1H), 8.82 (brs, 1H), 8.25 (dd, J = 92 & 2.4 Hz, 1H), 7.93 (m, 2H), 7.86 (d,.1 - 8.80 Hz, 1 H), 7.35 (brs, 1H), 7.27 (d, J - 960 Hz, 1H), 7.18 (brs, 3H), 5.27 (s, 1H), 5.13 - 4.84 (m, 5H), 4.72 (brs, 1H), 4.56 (s, 1H), 4.17 (d, J = 10.40 Hz, 1H), 3.93 - 3.85 (m, 2H), 3.67 - 3.35 (ra, 60H), 3 21 - 3.14 (m, 6H), 3.02 (d, J === 5.20 Hz, 1H),2.59 (t, J = 8.40 Hz, 1H), 2.54 - 2.51 (m, 16H), 2.36 - 2.29 (m, 4H), 1.77 (s, 3H), 1.11 (d, J = 6.0 Hz, 3H).

[0286] EXAMPLE 15. (2R,3R,4S)-3-acetamido-2-((14S,45R,46R)-14-(4-(3-(2-((2,4-dinitrophenyl)amino)ethoxy) propanamido)butyl)-46,47~dihydroxy-13,43-dioxo-l- (((2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-3,6,9,18,21,24,27,30,33,36,39,44-dodecaoxa-12,15,42-triazaheptatetracontan-45-yI)-4-guanidino-3,4-dihydro-2H-pyran-6-carboxylic acid (Compound 41)Docket No. 1165.071126WOO 1

[0287] Compound 1 was prepared as described in WO 2023 / 205669 A2)

[0288] Compound 3: To Compound 1 (0.0758 g, 0.087 mmol) and N-Boc-PEG8-bromide, (2, Broad Phann, 0.05 g, 0.087 mmol, 1.0 eq.) in DMF (0.87 ml,) were added DIPEA (0,038 pL, 0.216 mmol, 2.5 eq.) followed by potassium iodide (0.014 g, 0.087 mmol, 1.0 eq.) under argon atmosphere and stirred for 1-2 h. The reaction temperature was raised to 50°C using oil bath and stirred for 16 h. After reaction completion, the reaction mixture was quenched with water and extracted with EtOAc (3x 5 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4 and concentrated. The remainder was purified by column chromatography on a Teledyne CombiFlash Rf+ Lumen (silica gel, 4 g column, 5-10% MeOH in DCM) to give 34 as a yellow solid (0.083 g, 65 %). LCMS [M+H]+ == 1371.52

[0289] Compound 4: To Compound 3 (0.083 g, 0.061 mmol) in DCM (2.0 mL) was added TFA (0.6 mL) at ice-cold temperature Cooling was removed and stirring continued for 1 h. After reaction completion, TFA was evaporated, and the remainder was washed with cold diethyl ether (3x5 mL) and hexane (2x3 ml,) and dried under vacuum to give Compound 4 (quantitative yield), which was used without further purification. LCMS [M+H]+ = 1271.41.

[0290] Compound 6: To Compound 5 (0.046 g, 0.061 mmol) in DCM (0.6 mL) was added Compound 4 (0.077 g, 0.061 mmol, 1.0 eq.), followed by TEA (0.025 pL, 0.182 mmol, 3.0 eq.) under argon atmosphere and stirred for 2-6 h. After reaction completion, the reaction mixture diluted with water and extracted with EtOAc (3x 10 mL), The combined organic layers were washed with brine, dried over anhydrous Na2SO4, and concentrated. The remainder was purified by flash column chromatography on a Teledyne CombiFlash Rf+ Lumen (silica gel, 12 g column, 5-Docket No. 1165.071126WOO 110% MeOH in DCM) to give 6 as a yellow amorphous solid (0.09 g, 79 %). LCMS [M+H]+ = 1884.14.

[0291] Compound 7: To Compound 6 (0.09 g, 0.048 mmol) dissolved in DCM (2.1 mL), TFA (0.6 mL) was added at ice-cold temperature. Cooling was removed and stirring continued for Ih. After reaction completion, TFA was evaporated and the remainder was washed with cold diethyl ether (3x5 mL) and hexane (2x3 mL) to give 7 (0.079 g, quantitative yield), which was used without further purification. LCMS [M+H]+::::1643.74.

[0292] 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 Cl 8 column 5-95% gradient B over 45 min, flow 15 mL / min (A: 20 mM NHiOAc, pH 7 buffer and B: ACN; UV @ 360 nm and 240 nm) to give Compound 41 (3.3 mg, 50% yield). Purity 89% @360 nm and 84% @ 240 nm. LCMS [M + H]+ - 1503.62.

[0293] EXAMPLE 16. (2R,3R,4S)-3-acetamido-2-((14R,58R,59R)-14-(4-(3-(2-((2,4-dinitrophenyl)amino)ethoxy)propanamido)butyl)-59,60-dihydroxy-13,1,56-trioxo-l-(((2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)- 3,6, 9, 19, 22,25, 28, 31,34, 37, 40,43,46,49,52, 57-hexadecaoxa-12, 15, 55-triazahexacontan-58-yl)-4-guanidino-3,4-dihydro-2H-pyran-6-carboxylic acid (Compound 43)Docket No. 1165.071126WOO 1

[0294] Compound 1 was prepared as described in Carbohydrate Research, 342 (2007) 1636-1650.

[0295] Compound 3, Compound 3 was prepared using the same procedure to make Compound 10 in Example 14. 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 dry THF (2 ml). After reaction completion, the mixture was concentrated and the remainder was purified by¬ column chromatography eluting in 10% MeOHZDCM to afford Compound 3 as off white solid. Yield: 0.08 g, 48.88 %; LCMS (ESI) m / z 1230.15 [M+l]!.

[0296] Compound 5. Compound 5 was prepared using the same procedure to make Compound 3 in Example 14. Compound 5 was made from Compound 3 (0.10 g, 0.08 mmol) and compound 4 (782 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% MeOHZDCM to afford Compound 5 as a yellowish liquid. Yield: 0.09 g, 53.05 %; LCMS (ESI) M / 2 1043.85 ] X! i ].

[0297] Compound 43 was prepared using the same procedure to make Compound 42 in Example 14. Compound 43 was made from Compound 5 (009 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% TFAto afford Compound 43 as a yellowish solid. Yield: 0.016 g, 21.74 %; LCMS (ESI) m / z 1706.80 [M+l]+. ’H NMR (400 MHz, DMSO-de): 8 8.86 (d, J = 2.72 Hz, 1H), 8.82 (brs, IH), 8.26 - 8.23 (m, 2H), 7.99 (d, J = 8 12 Hz, IH), 7.94 (t, J =:5.48 Hz, IH), 7.88 (d, J = 9.12 Hz, IH), 7.79 (t, J = 5.24 Hz, IH), 7.40 (d, J = 7.0 Hz, 1H), 7.27 (d, J = 9.72 Hz, IH), 7.18 - 7.04 (tn, 21:1), 5.34 (brs, IH), 4.86 - 4.79 (m, 5H), 455 (s, IH), 4.22 - 4.17 (m, 3H), 3.94 - 3.83 (m, 2H), 3 66 - 3.60 (m, 6H), 3.59 - 3.56 (m, 4H), 3.53 - 3.40 ( m, 56H), 3 39 -3.32 (m, 8H), 3 23 - 3.15 (m, 4H), 3.04 - 3.01 (m, 2H), 2.97 - 2.95 (m, 214), 2.91 - 2.76 (m, IH), 2.61 - 2.55 (m, 5H), 2.37 (q, J = 6.52 Hz, 2H), 2.33 - 2.99 (m, 2H), 1.90 (s, 1H), 1.77 - 1.76 (m, 3H), 1.58 - 1.56 (m, IH), 1.49 -1.43 (m, IH), 1.35 - 1.29 (m, 2H), 1.28 - 1.19 (m, 3H), 1.16 - 1.11 (m, 5H), 0.99 (t, J = 7.16 Hz, 8H), 0.92 - 0.90 (m, IH).

[0298] EXAMPLE 17. (2R,3R,4S)-3-acetamido-2-((14R,31R,32R)-14-(4-(3-(2-((2,4-dinitrophenyl)amino)etlioxy)propanamido)butyl)-32y33-dihydroxy-13,16,29-trioxo-l-(((2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-3,6,9,30-Docket No. 1165.071126WOO 1tetraoxa-12,15,28-triazatritriacontan-31-yl)-4-guaiiidino-3,4-dihydro-2H-pyran-6-carboxylic acid (Compound 44)

[0299] Compound 1 was prepared as described in Carbohydrate Research, 342 (2007) 1636-1650.

[0300] Compound 3. Compound 3 was prepared using the same procedure to make Compound 10 in Example 14. Compound 10 was made from Compound 1 (0.20 g, 0.27 mmol) in dry THF (5 ml), DIPEA (0.25 ml, 1.33 mmol), and 12-aminododecanoic acid (2, 53.7 mg, 0.27 mmol) in dry 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: 0.09 g, 40.86 %; LCMS (ESI) m / z 828.20 [M+l]+.

[0301] Compound 5. Compound 5 was prepared using the same procedure as described in Example 14 to make Compound 1. Compound 5 was made from Compound 3 (0.09 g, 0.11 mmol) and Compound 4 (0.10 mL, 0.12 mmol) in DMF (3.0 m ) with HATU (0.045 g, 0.12 mmol) and ethylbis(propan-2-yl)amine (GLR, 0.09 L, 0.54 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: 0.08 g, 43.68 %; LCMS (ESI) m / z 1684.30 | M I ].

[0302] Compound 44. Compound 44 was prepared using the same procedure to make Compound 42 from Example 14. 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]+.]H NMR (400 MHz, DMSO-de): 5 13.15 (brs, 1H), 8.86 (d, JDocket No. 1165.071126WOO 1= 2.8 Hz, 1 H), 8.82 (brs, 1H), 8.25 (dd, J = 9.28, 2.32 Hz, I H), 7.96 (d, J = 9.0 Hz, 1H), 7.87 (t, J = 1.6 Hz, 1H), 7.78 (q, J = 8.36 Hz, 2H), 7.53 (d, J = 8.72 Hz, 1H), 7.26 (d, J = 9.68 Hz, 2H), 7.16 - 7.08 (m, 4H), 5.67 (s, 1H), 4.91 (d, J = 1.8 Hz, 1H), 5.15 (d, J = 1.2 Hz, 1H), 4.85 (dd, J = 3.4, 1.0 Hz, 1H), 4.71 (brs, 2H), 4.56 - 4.55 (m, 2H), 4.52 - 4.12 (m, 6H), 4.01 (q, J = 2.3 Hz, 2H), 3.85 (brs, 2H), 3.66- 3.31 (m, 26H), 3.19 - 3.17 (m, 4H), 2.90 - 2.82 (m, 5H), 2.29 (t, J - 6.36 Hz, 2H), 2.09 - 2.08 (m, 2H), 1.78 (s, 3H), 1.45 - 1.21 (m, 24H), 1.12 (d, J = 6.24 Hz, 3H).

[0303] EXAMPLE 18. (2R R,4S)-3-acetamido-2-((2R,3R)-15-((S)-2-((S)-2-((S)-2-(((R)-23-(4-(3-(2-((2,4-dinitrophenyl)amino)ethoxy)propanamido)butyl)-21,24-dioxo-36- (((2R,3R-4R,5R,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-3,6,9,12,15,18,28,31,34-nonaoxa-22,25-diazahexatriacontyl)carbamoyl)pyrrolidine-l-carbonyl)pyrroIidine-l-carbonyl)pyrroIidin-l-yl)-l,2-dihydroxy-5,15-dioxo-4,9,12-trioxa-6-azapentadecan-3-yl)-4-guanidino-3,4-dihydro-2H-pyran-6-carboxylic acid (Compound 45)

[0304] Compound 3. Compound 3 was prepared using the same procedure to make Compound 3 in Example 14. 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 ethylbi s(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%Docket No. 1165.071126WOO 1MeOHZDCM to afford Compound 3 as a yellowish liquid. Yield: 0.35 g, 58.6 %, LCMS (ESI) m / z 1449.20 [M+18]+

[0305] Compound 4. To a stirred solution of Compound 3 (0.30 g, 0.21 mmol), in anhydrous dimethyl formamide (3.0 mL), piperidine (0.6 mL) was added and the reaction mixture was stirred for 30 min. After reaction completion, the solvent was evaporated and the remainder was triturated with diethyl ether to afford Compound 4 as a light yellow liquid which was used without further purification. Yield: 0.16 g, 63.13%; LCMS (ESI) m / z 1210.20 [M + 1]+.

[0306] Compound 6. Compound 6 was prepared using the same procedure to make Compound 3 from Example 14. 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, 0.18 mmol) and ethyl bi s(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% MeOH / DCM to afford Compound 6 as a yellowish liquid. Yield: 0.11 g, 38.61 %; LCMS (ESI) M / Z 1741.48 [M+1]+

[0307] Compound 7. To a stirred solution of Compound 6 (0.11 g, 0.17 mmol) in anhydrous dimethyl formamide (2.0 mL) piperidine (0.4 mL) was added and the reaction mixture was stirred for 30 min After reaction completion, the solvent was evaporated and the remainder was triturated with diethyl ether to afford Compound 7 as a light yellow liquid which was used without further purification. Yield: 0.085 g, 88.71%; LCMS (ESI) m / z 1500.90 [M+1]+

[0308] Compound 9. Compound 9 was prepared using the same procedure to make Compound 3 in Example 14. 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). 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 9 as a yellowish liquid. Yield: 0.09 g, 74.86 %; LCMS (ESI) m / z 1137.60 [M / 2+1]+

[0309] Compound 45 was prepared using the same procedure to make Compound 42 in Example 14. 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 water / ACN in 0.1% TF to afford Compound 45 as a yellowish color solid. Yield: 20 mg, 26.69 %; HRMS (ESI) m / z 1892.91 [M+l]+.!H NMR (400 MHz, DMSO-ds): 8.86 (d, J - 2.80 Hz, 1H), 8.83 (brs, 1H), 8.26 (dd, J === 9.2 & 24 Hz, 1H), 8.00 - 7.90 (m, 3H), 7.83 - 7.78 (m, 2H), 7.53 (brs, 1H), 7.27 (d, J = 9.6 Hz, 2H), 7.22 - 7.20 (m, 1H), 5.66 (brs, 1H), 5.09 (brs, 1H), 484 (dd, J - 10 8 & 2.0 Hz, 1H), 4.76 - 4.73 (m, 2H), 4.61 - 4.51 (m, 4H),Docket No. 1165.071126WOO 14.36 -4.28 (m, 1H), 4.26 - 4.19 (m, 3H), 3.99 (qt, J = 8.8 Hz, 1H), 3.81 (brs, 1H), 3.65 - 3.58 (m, 10H), 3.53 - 3.24 (m, 64H), 2.49 - 2.33 (m, 4H), 2.25 - 2.1.83 (m, 7H), 1.80 (s, 3H), 1.62 - 1.155 (m, 1H), 1.45 - 1.30 (m, 3H), 1.25 - 1.18 (m, 1H), 1.11 (d, J = 6.4 Hz, 3H).

[0310] EXAMPLE 19. (2R,3R,4S)-3-acetamido-2-((14R,17R,23R,29R,58R,59R)-14-(4-(3-(2-((2, 4-dinitrophenyl)amino)ethoxy)propanamido)butyl)-59,60-dihydroxy-l 7,23,29-tris(hydroxymethyI)-13,16,19,22,25,28,31,34,56-nonaoxo-l-(((2R,3R,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-carboxylic acid (Compound 46)Docket No. 1165.071126WOO 1

[0311] Compound 8a. Solid phase peptide synthesis (SPS) of Compound 8 was performed manually in a peptide reaction vessel using 2-CTC resin under standard peptide coupling and deprotection protocol using Fmoc and tBu protection groups.

[0312] General Procedure for Amide coupling: To the reaction vessel containing swollen 2CTC resin is added activated amino acid solution, prepared by weighing out an appropriate amount of Fmoc-Gly-OH (Chemscene, 2.0 eq) or Fmoc-Ser(OtBu)-OH (BLD pharma, 2.0 eq.) or 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22-heptaoxa-4-azapentacosan-25-oic acid (Habotech; 2.0 eq) in DMF, and HATU (2.5 eq.) and DIPEA (3.0 eq.). The reaction vessel is shaken for 2 h.

[0313] General Procedure for Fmoc De-protection: Reaction vessel containing resins in 20% piperidine in DMF is shaken at for 0.5 h to de-protect Fmoc groups.

[0314] 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.

[0315] Compound 8a (Resin Cleavage): 2CTC resin loaded with Compound 8 was suspended in 20% 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) in DCM in a peptide reaction vessel. The reaction vessel was shaken for 30 min. The resin was filtered and washed three times with 10 ml portions of 20% HFIP in DCM. To the combined filtrates was added chilled diethyl ether to give 8a as a white solid. Yield: 250 mg. LC-MS (ESI) m / z 1567.45 [M+1]+

[0316] Compound 10. Compound 10 was prepared using the same procedure to make Compound 3 in Example 14. 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). Aft er 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 [M / 2+l]+.

[0317] Compound 46. Compound 46 was prepared using the same procedure to make Compound 42 in Example 14. 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+1]+rH NMR (400 MHz, DMSO-d6): 8.86 (d, J = 2.80 Hz, IH), 8.83 (brs, 1H), 8.27 - 8.24 (m, 3H), 8.14 (brs, IH), 8.02 - 7.92 (m, 4H), 7.84 - 7.79 (m, 2H), 7.51 (brs, IH), 7.27 (d, J = 10.6 Hz, 2H), 7.07 -Docket No. 1165.071126WOO 17.01 (m, 3H), 5.60 (brs, 1H), 5.16 - 5.06 ( m, 3H), 4.83 (d, J = 10.6 Hz, 1H), 4.74 - 4.73 (m, 2H), 4.56 (s, 2H), 4.48 (brs, 1H), 4.33 - 4.20 (m, 5H), 4.15 - 4.14 (m, 1H), 3.96 (qt, J = 9.2 Hz, 1H), 3.84 - 3.72 (m, 7H), 3.66 - 3.55 (m, 15H), 3.54 - 3.45 (m, 30 H), 3.44 - 3.36 (m, 7H), 3.23 - 3.16 (m, 4H), 3.05 - 2.95 (m, 4H), 2.38 (t, J = 8.4 Hz, 1H), 2.30 (t, J = 8.4 Hz, 1H), 1.78 (s, 3H), 1.70 - 1.60 (m, 1H), 1.55 - 1.45 (m, 1H), 1.38 - 1.23 (m, 4H), 1.12 (d, J = 6.4 Hz, 3H).Docket No. 1165.071126WOO 1

[0319] EXAMPLE 20. 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,3R,4R,5R,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)

[0320] Compound 3. 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).

[0321] Compound 4. To a stirred solution of Compound 3 (0.10 g, 0.17 mmol) in anhydrous DCM (3.0 mL), TFA (1.0 mL) was added at 0 °C. Cooling was discontinued and the reaction mixture was stirred for 12 h. After reaction completion, the solvent was evaporated to Compound 4 as a light yellow liquid which was used for the next reaction. Yield: 0.08 g; LCMS (ESI) m / z 516.20 [M+l]+.

[0322] Compound 6. Compound 6 was prepared using the same procedure to make Compound 3 in Example 14. 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 ethylbi s(propan-2-Docket No. 1165.071126WOO 1yl)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]+.

[0323] Compound 47. To a stirred solution of Compound 6 (0.10 g, 0.061 mmol) in ethanol (5.0 mL) was added sodium ethoxide (0.41 mg, 0.002 mmol) at 0 °C. Cooling was discontinued, and the reaction mixture was stirred for 1 h. After reaction completion, the reaction mixture was treated with acidic (Dowex 50, H+) to pH~7 and the suspension was filtered through a sintered funnel with MeOH wash. The filtrate was concentrated and the remainder was purified by prep HPLC to afford Compound 47 as a yellowish color solid. Yield: 25 mg, 27.11 %; LCMS (ESI) m / z 1496.80 [M+1]+.1H NMR (400 MHz, DMSO-d6): 8.86 (d, J = 2.80 Hz, 1H), 8.83 (brs, 1H), 8.25 (dd, J = 9.2 & 2.4 Hz, 1H), 7.90 (m, 2H), 7.80 (d, J - 8.8 Hz, 1H), 7.27 (d, J = 9.6 Hz, 1H), 6.10 (brs, 1H), 5.77 (d, J = 7.2 Hz, 1H), 4.55 (d, J = 1.6 Hz, 1H), 4.16 - 4.10 (m, 2H), 4.08 - 4.03 (m, 1H), 3.76 - 3.55 (m, 22H), 3.53 - 3.33 (m, 51H), 3.20 - 3.13 (m, 8H), 2.61 - 2.57 (m, 4H), 2.34 - 2.29 (m, 5H), 1.78 (s, 3H), 1.47 - 1.32 (m, 4H), 1.21 (d, J = 7.2 Hz, 3H), 1.11 (d, J = 6.0 Hz, 3H), 0.83 (t, J = 7.2 Hz, 3H), 0.76 (t, J = 7.2 Hz, 3H).METHOD EXAMPLES

[0324] METHOD EXAMPLE 1: Binding affinity to HEK expressing neuraminidase. The Ki of Compound 24 when competed against zanamivir-rhodamine (Nature Communications 11, Article number: 5597 (2020)) was determined using a binding and competition assay. First, the Kd of zanamivir-rhodamine binding to neuraminidase is determined Human Embryonic Kidney cells (HEK293) transfected with influenza viral neuraminidase are suspended in staining buffer (2% fetal bovine serum in phosphate-buffered saline) at a density of 2 million cells / mL, and then plated in triplicate (50 pL / well) in a u-bottom plate Next, two sets of serial dilutions of zanamivir- rhodamine are made in staining buffer. In one set, lOOx concentration of zanamivir is included to determine non-specific binding. The diluted compounds are added to the cells (50 ul / well) and incubated on a shaker at room temperature for 1 h. Cells are washed twice with 200 uL of phosphate-buffered saline and resuspended in 150 uL of staining buffer. Fluorescence is measured using an Attune NxT flow cytometer (Fisher), and Kd is calculated using Graph Pad Prism, One site - total and non-specific binding.

[0325] Next 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 madeDocket No. 1165.071126WOO 1for 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). Using the Ka calculated above and a final concentration of 20 nM zanamivir-rhodamine, the Ki can be calculated using Graph Pad Prism, One site - Fit Ki.

[0326] FIG. 8 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. 9). Zanamivir has a Ki of 0.496 nM. The Ki of Compound 24 is in the low nanomolar range at 7.152 nM.

[0327] METHOD EXAMPLE 2: Dose range finding study - Viral Titers. A group of 6-8 weeks old female B ALB / c mice (n = 5 / group) were infected with 10 LD50 of Influenza virus H3N2 / Wisconsin / 15 / 2009 on day 0 of the experiment. Mice were given an intraperitoneal injection of 6 g / kg human IVIg at 24 hpi to achieve humanized titer of anti-DNP and anti-Rhamnose antibodies at the time of test article administration. Mice were treated with test articles at 48 hpi.Compound 24 was administered as a single intranasal dose of 2.2 mg / kg, 0.72 mg / kg, 0.24 mg / kg or, 0.081 mg / kg in the designated cohorts. Mice in the two control groups received Tamiflu® (oseltamivir phosphate) as a positive control ( 5 mg / kg b.i.d. for 5 days) and Vehicle (PBS) as placebo. Viral titers in mouse lungs were measured in 2 ways: live, infectious virus is measured using a hemagglutination assay, and total viral RNA is measured using reverse transcription polymerase chain reaction (rt-PCR). However, the rt-PCR method cannot distinguish between live and dead viruses so is not valuable in determining efficacy.

[0328] First the mouse lungs are homogenized in 5 mL of cold phosphate buffered saline per milligram of tissue. 100 pL of lysate is saved for RNA isolation, and the remaining lysate is used for hemagglutination. All samples are stored at -80 °C.

[0329] For the hemagglutination assay, MDCK-London (Madin-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 DMEM / F12 (1:1) supplemented with 0.3% bovine serum albumin. MDCK- London cells are washed and 50 pL of diluted lysate is added to each well, so that each dilution has 4 replicates. Cells are incubated for 1 hour at 37 °C, 5% CO2, and then 200 pL of DMEM / F12Docket No. 1165.071126WOO 1(1:1) supplemented with 0.3% bovine serum albumin and 1 pg / ml of trypsin are added to each well. Cells are incubated 48 hours at 37 °C and 5% CO2. Each well is tested for hemagglutination by incubating 50 pL of tissue culture supernatant with 50 pL 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.

[0330] FIG. 4 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 and the corresponding figures.

[0331] 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-Fluor™ Influenza Neuraminidase Assay Kit (Catalog no. 4457091, Invitrogen1M). 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. For each viral strain, 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 wrere 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 50 pL of 200uM: of the NA-Fluor™ 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-fluor™ 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. Finally, for the neuraminidase inhibitionDocket No. 1165.071126WOO 1assay, serial tenfold dilutions of the test compounds were prepared in NA-Fluor™ assay buffer (66.6 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer, 8 mM CaCh, pH 6.5) Then 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 virus control wells. 25 pL of diluted virus 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. Then 50 pL of the diluted 200 pM NA-Fluor™ 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-Fluor™ 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. Then the graph was analyzed using a built-in nonlinear regression curve-fitting program to determine the half-maximal inhibitory concentration (ICso). Lower IC50 values indicate higher potency of the compound.

[0332] FIG. 11 demonstrates the sigmoidal dose-response curves for each viral strain and the table therein denotes their respective half-maximal inhibitory concentration (IC50) values. These data illustrate that the zanamivir targeting ligand moiety in Compound 24 is not only able to engage the target enzyme but also inhibit its function and act as a direct acting antiviral across many strains. Neuraminidase inhibition suppresses the budding of virus particles from infected cells and limits the spread of the infection to neighboring healthy cells.

[0333] Table 5 shows the half-maximal inhibitory concentration (IC50) values for Compound 24 against multiple influenza strains.Table 5. ICso values for Compound 24 against seasonal and multiple influenza strains

[0334] METHOD EXAMPLE 4. A group of 6-8 weeks old female BALB / c mice (n = 5 / group) were infected with 10 LD50 of influenza virus A / Hl N 1 / PR8 / 1934 on day 0 of the experiment. Mice were given an intraperitoneal injection of 6 g / kg human IVIg 24 hours before drug administration to achieve humanized titer of anti-DNP and anti-Rhamnose antibodies at the time of test articleDocket No. 1165.071126WOO 1administration. Mice were treated with test articles at 48 hpi (FIG. 1) or, 96 hpi (FIG. 2).Compound 24 (no DDM or Avicel® RC 591was administered as a single intranasal dose of 1.5 pmol / kg, or single intravenous dose of 1.5 pmol / kg, or single oral dose of 13.5 mol / kg in the designated cohorts. Mice in the three control groups received Tamiflu® (oseltamivir phosphate), or Xofluza® (baloxavir marboxil) or, PBS as placebo.

[0335] For evaluation of drug efficacy, mice were weighed and monitored daily for 14 days postinfection and counted as dead when they lost 25% of their body weight or were diagnosed as moribund.

[0336] Results of this experiment are depicted body weight plots, which graph days after infection vs. survival (%) and days after infection vs. body weight (%) for mice (n = 5 / group).

[0337] At the early-stage treatment (48hpi) 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 and 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 and mice treated with Xofluza® with 10 mg / kg single oral dose resulted in 80% survival. On the other hand, At the late-stage treatment (96 hpi) 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 and 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.

[0338] It indicates the superior efficacy of Compound 24 over Tamiflu® and Xofluza® at both early and late stages of infection and demonstrates that all the three routes of administration are effective with intranasal being the best route of administration at 1.5 (imol / kg dose.

[0339] 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.

[0340] Results of this experiment are depicted in FIG. 3. FIG. 3 is a graph of days after infection vs. survival (%) and days after infection vs. body weight (%) for mice (n = 5 / group) infected with 10 LD50 of influenza A H3N2 / Wisconsin / 15 / 2009, intraperitoneal administration of human IgG (IVIg (GAMUNEX®-C) at 24 hpi, and administration of conjugate at 48 hpi.Docket No. 1165.071126WOO 1

[0341] Mice treated with 2.2 mg / kg, 0.72 mg / kg and 0.24 mg / kg single intranasal dose of Compound 24 (no DDM or Avicel® RC 591) 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. These data show 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.

[0342] The cytokines from each of the treatment and control groups were measured in lung tissue samples (n=3 / group) via BioLegend’s LEGENDplex™ bead-based immunoassay using standard protocol provided by the manufacturer (BioLegend, San Diego, CA).

[0343] 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 Compound 24, administered intranasally, did not induce a cytokine storm in the lungs or serum. 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. With respect to IL-6, the data shown were not statistically significant. By comparison, 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. In serum, the IFN- y showed an increase, but with large error bars which are not statistically significant. These data indicate that Compound 24 does not induce a cytokine storm.

[0344] METHOD EXAMPLE 6. A cytokine release assay was performed on Compound 24 (results in FIG. 10) to assess the risk of causing a cytokine storm in humans. For this assay, 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.

[0345] Cytokines in the supernatant were measured using a Human Anti-Virus Response PanelDocket No. 1165.071126WOO 1(biolegend). This kit measures 13 different proteins: IL-lp, IL-6, IL-8, IL-10, IL-12p70, IFN-a2, IFN-p, IFN-λ1, IFN-λ2 / 3, IFN-y, TNF-a, IP-10, GM-CSF, using fluorescently coded beads and fluorescently labeled antibodies. Fluorescence was measured using an Attune NxT flow cytometer (Fisher), and the concentration of each protein is quantified using LEGENDplex™ software (biolegend). Finally, data was graphed with GraphPad prism

[0346] In all the cytokines tested, Compound 24 had similar levels to the negative control, indicating no inherent, immunogenicity from the molecule by itself. Because Compound 24 could potentially bind 2 antibodies, IVIG was included to determine the risk of forming an aggregate, which would trigger an immune response. IVIG was at a physiologically relevant concentration of 10 mg / mL, which caused a higher background in some of the proteins tested. Considering this, Compound 24 again had similar levels to the negative control, indicating no immunogenicity from the molecule when bound to antibodies. This suggests that Compound 24 would not recruit immune cells in circulation and is unlikely to induce cytokine release if not bound to neuraminidase expressing cells or virus.

[0347] METHOD EXAMPLE 7. To evaluate the neuraminidase inhibition activity of Compound 41, 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. 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. For each viral strain, 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 50 iiL 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 nm. The Relative Fluorescence Unit (RFU) values were plotted against the respective virus dilutions and forDocket No. 1165.071126WOO 1each 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. 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 CaC12, pH 6.5). Then 25 pL of the 4X Compound 41 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 virus control wells. 25 pL of diluted virus samples were added to the designated wells and mixed with the Compound 41 dilutions. The plate was incubated for 30 min at 37°C with shaking. Then 50 pL of the diluted 200uM 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 440nm The data was plotted using GraphPad Prism 10.0.0 software to generate the sigmoid dose-response curve. Then the graph was analyzed using a built-in nonlinear regression curve-fitting program to determine the half-maximal inhibitory concentration (IC50). Lower IC50 values indicate higher potency of the compound.

[0348] FIG. 12 demonstrates the sigmoidal dose-response curves for each viral strain and the table therein denotes their respective half-maximal inhibitory concentration (ICso) values related to Compound 41. This data illustrates that the zanamivir targeting ligand moiety in Compound 41 is not only able to engage the target enzyme but also inhibit its function and act as a direct-acting antiviral agent across many strains. Neuraminidase inhibition suppresses the budding of virus particles from infected cells and limits the spread of the infection to neighboring healthy cells. METHOD EXAMPLES WITH NON-SOLID PHARMACEUTICAL FORMULATIONS

[0349] METHOD EXAMPLE 8. Efficacy of Compound 24 intranasal (IN) in non-solid formulation against Multiple Influenza Strains (FIG. 14A and FIG, 14B). Efficacy of an intranasal formulation of Compound 24 (0.6% n-dodecyl P-D-maltoside, “DDM” and 2% Avicel 591 in PBS), this non-solid formulation referred to as “Compound 24 IN”) was evaluated in 3 seasonal Influenza strains: A / H3N2 / Wisconsin / 15 / 2009, A / California / 07 / 2009(H1N1) pdm09, a pandemic strain of H INI that represents a large portion of the seasonal flu burden every year, and B / Brisbane / 60 / 2008), In FIG. 14A and FIG. 14B, Compound 24 IN was compared with oseltamivir phosphate (oral) and vehicle control (0.6% DDM and 2% Avicel 591 in PBS and intranasal). In each study, mice were infected with lOx LD50 of 1 of 3 seasonal Influenza strainsDocket No. 1165.071126WOO 1and 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.

[0350] As shown in FIG. 14A, 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 / California / 07 / 2009(H1N1) pdm09 showed 100% survival in contrast to 40% of the oseltamivir phosphate group and 0% in the vehicle control group. A Mantel-Cox log-rank test shows the Compound 24 survival curve to be significant with a p-value of 0.0006 for A / California / 07 / 2009 (HlNlpdmO9) strain Compound 24 against influenzaB showed 100% survival in contrast to 0% in the vehicle control group. In the oseltamivir phosphate-treated group, 40% were euthanized before the study ended as they lost more than 25% of their initial weight during the two-week study period. A Mantel-Cox log-rank test shows the Compound 24 survival curve to be significant with a p-value of 0.0065 for the B / Brisbane / 60 / 2008 Strain.

[0351] In addition, Compound 24 IN appeared to provide a better quality of life for infected mice as no large changes in body weight were detected. Weight loss was observed in all other treatment groups. FIG. 14.4 shows the corresponding viral lung titers.

[0352] METHOD EXAMPLE 9. Efficacy against Influenza A / Hong Kong / 2369 / 2009 (HlNlpdmO9, 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 Kong / 2369 / 2009 (H1NlpdmO9, Oseltamivir [Tamiflu] Resistant), 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 LDJO of the A / Hong Kong / 2369 / 2009 (HlNlpdmO9, Oseltamivir [Tamiflu] Resistant) strain of influenza virus.?\t infection 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.

[0353] Treatment began at 24 hpi (I 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 oralDocket No. 1165.071126WOO 1gavage, then released back to their cage.

[0354] At 24 h after treatment, three mice from each group were selected, euthanized, and the lungs were collected and snap frozen in liquid nitrogen. Lungs were homogenized and a TC1D50 assay was performed in triplicate on MDCK cells looking for cytopathic effects and the virus titer were calculated using the Reed-Muench method.

[0355] The remaining 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.

[0356] As shown in FIG. 15A, 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. A Mantel-Cox log-rank shows the Compound 24 survival curve to be significant with a p-value of 0.0009 for the Influenza A strain - A / Hong Kong / 2369 / 2009 (HlNlpdmO9, Oseltamivir Resistant).

[0357] 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. For this study, 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 anti-hemagglutinin antibody titers were measured to assess influenza exposure.

[0358] The results are shown in FIG. 16. In the group treated with vehicle, all naive co-housed animals became infected by day 4, and infection was confirmed by increased antibody titers. In the Compound 24 IN treatment group only 1 of the 10 naive co-housed guinea pigs became infected, and not until day 8, which was confirmed by seropositivity of anti -hem agglutinin and anti¬ neuraminidase. The data suggest that not only did Compound 24 IN protect 90% of the guinea pigs in this prolonged exposure transmission model, the transmission was also delayed in the oneDocket No. 1165.071126WOO 1instance of transmission relative to vehicle transmission.

[0359] 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%.

[0360] METHOD EXAMPLE 11. Compound 24 Mutation Emergence (FIG. 17A and FIG.17B). Compound 24 IN was evaluated for its ability to produce drug-resistant mutants of influenza A virus (IAV) after serial passaging the virus in the presence of Compound 24. Traditionally, drug resistance emergence studies are performed in vitro to streamline experimental design and quickly identify mutations. However, due to the immunological dependent nature of Compound 24, the mutation emergence study described here was performed in vivo to more rigorously test whether Compound 24 produces drug-resistant IAV in conjunction with a functioning immune system. Furthermore, due to the potency of Compound 24 in eliminating nearly all traces of IAV, a sub- optimal dose was necessarily used to maintain measurable viral load and encourage mutation during passaging. It was found that an 81 pg / kg dose of Compound 24 could reduce, but not eliminate, IAV infection in mice and was therefore chosen as the sub-optimal dose.

[0361] Three groups of five mice each were used for each passage cycle. All groups received 10 LD50 influenza virus A / PR.8 / 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 Twenty-four h post-infection 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 antirhamnose antibody titers. Body weight and survival monitoring was recorded each day, and each group was sacrificed 4 days after drug administration for viral titer determination by TCID50 and RNA extraction from lung tissue. After titer determination to maintain consistent 10 LD50 infections between passages, the process was repeated for each consecutive cycle.

[0362] It was found that Compound 24 IN does not produce viable drug-resistant mutants after 5 passages and was shown to maintain efficacy with a modest ~1.5-fold decrease in IC50 from ~12 nM to ~20 nM as seen in neuraminidase inhibition assays (NIA), as shown in FIG. 17B. In contrast, oseltamivir phosphate treatment during IAV passaging resulted in a >1,500-fold decrease in NAIDocket No. 1165.071126WOO 1efficacy, from ~2 nM (Hong, B. T., et al. Oseltamivir hydroxamate and acyl sulfonamide derivatives as influenza neuraminidase inhibitors Bioorganic & medicinal chemistry 2014, 22(23), 6647 6654) to -3,000 nM. These results suggest that Compound 24 does not encourage the emergence of drug-resistant mutations in vivo, even at sub-optimal doses like oseltamivir does. Moreover, Compound 24 was shown to maintain NIA efficacy during the duration of the study, while the efficacy of the industry standard oseltamivir was significantly impaired. Additionally when an LD50 study was run on the mutants after 5 cycles it was found the LD50 of the Tamiflu treated mice showed a 4,000-fold increase in virulence compared to a 4-fold increase in Compound 24 treated, mice, as shown in FIG. 17A. Altogether, this study suggests that extended use of Compound 24 would not encourage the emergence of drug-resistant IAV mutants.

[0363] METHOD EXAMPLE 12. Compound 24 IN Avian Influenza efficacy (FIG. 18A and FIG. 18B). Three groups of mice (n=4 / cohort) were infected with a labeled version of the avian flu currently spreading in Texas - rH5Nl Nluc A / Texas / 37 / 2024 with 10E2 PFU / mouse. A fourth group was mock infected with viral infection media containing no virus as a negative control. Mice were treated with 2mg / kg of Compound 24 IN intranasally, or 5 mg / kg BID for 5 days of Tamiflu orally or Compound 24 vehicle intranasally Mice were serially imaged with an IVIS imager to track the luminescence of the virus to quantify the viral load. At Day 6, Compound 24 IN treated mice had almost no virus left in their body after a single dose while Tamiflu had much higher amounts. The mice were sacrificed and the viral load was quantified in each via TCID50 assay. The viral load in the lungs of the Tamiflu treated mice was found to be significantly higher than in the Compound 24 IN treated mice, suggesting that even at very low doses, Compound 24 is effective at clearing avian influenza infections, including high pathogenic avian influenza infections. FIG. 18A shows the images of Groups 1 and 2 and FIG. 18B of Groups 3 and 4.

[0364] 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. 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 (FcyRIIIa, FcyRIIa) in effector cells and the complement system. If activated, these components are expected to trigger ADCC, ADCP, and CDC, ultimately killing the HEK293-NA cells. A control group usingDocket No. 1165.071126WOO 1unmodified 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.

[0365] As shown in the figures of FIG. 19, 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 FcgRlIIa expressing effector cells using a Promega ADCC kit. Cell death can be blocked by either deletion of I VIG 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.

[0366] As shown in FIG. 19C, Compound 24 is also found to promote CDC-mediated killing of neuraminidase expressing target cells. Cell death is blocked by introducing a 100-fold excess of free zanamivir, suggesting that Compound 24-mediated activation of CDC-mediated killing should also be limited to cells / virions that express viral neuraminidase.

[0367] The data in the figures of FIG. 19 validate the immune activity of Compound 24 and demonstrate that Compound 24 can recruit naturally occurring anti-hapten antibodies to activate ADCC-mediated (Panel A), ADCP-mediated (Panel B), and CDC-mediated (Panel C) killing of influenza-infected cells. Compound 24-mediated killing is dependent on the presence of both antihapten antibodies and viral neuraminidase on the target cell surface, which is a desirable safety feature.

[0368] METHOD EXAMPLE 14. Neuraminidase Inhibition Activity of Compound 24 IN Against Seasonal and Drug-resistant Influenza A and B Strains (FIG. 20).

[0369] The neuraminidase inhibition activity of Compound 24 IN is evaluated against 4 seasonal influenza strains (A / PR / 8 / 1934 (H1N1), A / California / 07 / 2009 (HlNlpdmO9), A / Wisconsin / 15 / 2009 (H3N2), and B / Brisbane / 60 / 2008 (Victoria Lineage)) and 2 drug-resistant influenza virus strains (A / Hong Kong / 2369 / 2009 (HlNlpdmO9, oseltamivir-resistant), and A / Illinois / 37 / 2018 (HlNlpdmO9, baloxavir-resistant). Neuraminidase inhibition activity is measured based on the ICso in relative fluorescence unit (RFU) of the enzymatic product resulting from neuraminidase activity of the virus particles.Docket No. 1165.071126WOO 1

[0370] The ICso against all six strains are shown in FIG. 20. Compound 24 is found to inhibit neuraminidase activity of the three seasonal influenza A strains with IC50 ranging from 127-16.4 nM. Compound 24 also inhibits neuraminidase activity of the seasonal influenza B strain (Victoria Lineage) with an IC50 of 34.5 nM. Compound 24 is found to effectively inhibit the neuraminidase activity of oseltamivir-resistant and baloxavir-resistant flu strains with an IC50 of 17.9 and 27.2 nM, respectively).

[0371] 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 pM 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.

[0372] 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 TCID50 to determine the viral inoculation required for the CPE inhibition assay.

[0373] Cell viability from the viral CPE inhibition assay was quantified with neutral red staining toDocket No. 1165.071126WOO 1calculate the EC 50 of Compound 24 against 4 wild-type (A / PR / 8 / 1934 (H1N1), A / Wisconsin / 629- D00015 / 2009 (HlNlpdm09), A / Hong Kong / H090-756-Vl(0) / 2009 (H3N2), and B / Brisbane / 60 / 2008 (Victoria Lineage)) and 1 drug-resistant influenza strains (A / Hong Kong / 2369 / 2009 (HlNlpdmO9, oseltamivir-resistant).

[0374] The summary of Viral CPE Inhibition of Compound 24 Against Wild-Type and Drug-resistant Strains is shown in the following table.

[0375] Compound 24 effectively prevented CPE induced by H1N1 and the oseltamivir-resistant strain (EC50 < 0.9 pM). Compound 24 also inhibited CPE of HlNlpdmO9, H3N2, and influenza B with EC50 < 8 uM. 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.

[0376] 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 ECso (concentration of 50% CPE inhibition) values were calculated, as shown in the following table.Docket No. 1165.071126WOO 1It was found that Compound 24 inhibits H5NI and H7N9-induced cytopathic effect and production of nascent virions in vitro with single digit micromolar potency. Additionally, the results confirm the direct-acting antiviral property of Compound 24 against the avian influenza strains. The direct antiviral action of Compound 24 was also validated in vivo, where very low doses of Compound 24 were 100% protective against the Texas 2024 isolated H5N1.

[0377] 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 A / Illinois / 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.

[0378] 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. 25 pL of the serially diluted compounds were added to each well, and the plates were then incubated at 37°C for 30 minutes. Rabbit anti-DNP (Invitrogen, cat: A6430) or rabbit anti alpha-gal(Enzo m86) was diluted to 40 pg / mL and rabbit complement (Sigma Aldrich, cat: S7764) was diluted to 20% 50 pL of this mixture, was added to the cells for a final concentration of 20 pg / mL rabbit anti-DNP and 10% rabbit complement. The cells were incubated at 37°C for 4 hours. After the incubation period, the cells were washed twice with 200 pL PBS. Subsequently, 200 pL of Cell Titer-Gio reagent (Promega, cat: G7570) in PBS was added to each well. Luminescence was measured using a plate reader equipped with glow-type luminescence read capabilities. The percent cell death was calculated and plotted, giving a bell-shaped curve. In the lower concentration range, the compound stimulates the complement proteins to kill cells. In the higher concentration range, the compound blocks complement activity by saturating the receptors on to the target protein and the antibodies. Activity in this assay demonstrates the effective ability of the full conjugate to bind to the target viral protein and recruit the immune system with the other domain of the molecule. EC50 values for both the low concentration range and the high concentration ranges for the tested compounds in the followingDocket No. 1165.071126WOO 1table.

[0379] 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, PK studies were also conducted in mice to determine the minimum pharmacologically active dose (PAD).

[0380] 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 £10% the maximum feasible dose (MFD) and the lower range F is the MFD The mean elimination half-life (t1 / 2) ranged from 0.603 to 6.81 hours (0.603 to 4.2 hours excluding a single outlier) with escalating IN doses in rats. These data suggested that absorption had become rate-limiting in the in vivo elimination of Compound 24 (i.e., flip-flop kinetics). In dogs, the mean t1 / 2 estimate was ~1 hr after IV and IN administration at doses up to -20 mg / kg. The Day 14 / 1 ratios for Cmax and AUC0-24 were close to 1 in rats and dogs, indicating there is no accumulation following once daily administration. Compound 24 was found to have low cellular permeability and is not a substrate of human P -glycoprotein (P-gp).

[0381] 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 partitioningDocket No. 1165.071126WOO 1into 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.

[0382] 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.

[0383] METHOD EXAMPLE 21. Summary of Nonclinical Safety Pharmacology and Toxicology. Safety pharmacology studies were conducted to identify any unintended effects on organ systems acutely critical for life including the central nervous system (CNS), cardiovascular, and respiratory systems. Compound 24 had no effects on these organ systems. Additionally, there were no changes in electrocardiogram (ECG) parameters in dogs administered Compound 24 for 14 days in a GLP toxicology study.

[0384] 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.

[0385] Non-GLP dose tolerability studies at the MFD were conducted in rats and dogs to support dose selection for 14-Day GLP toxicology studies. In rats, 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. There were no effects of treatment on standard in-life and post-life assessments which supported the selection of 100 mg / kg as the high dose for a 1 -day GLP study. In the 14-day study, rats were administered vehicle or doses up to 100 mg / kg / day by the IN route. There was no morbidity or mortality, no effect of treatment on any in-life assessments including clinical observations, body weight, food consumption, ophthalmology, CNS (Functional observational battery' (FOB)), or clinical pathology parameters (hematology, coagulation, chemistry, urinalysis) and no effect on post-life assessments including necropsy, organ weights and microscopic examination of a standard list of tissues. Accordingly, the NOAEL was 100 mg / kg / day in male and female rats dosed daily by the IN route for 14 days.

[0386] In dogs, the MFD by the IN route was 10 mg / kg. Consequently, to evaluate the potential systemic toxicity of Compound 24, exposures were increased by supplementing the IN dose with aDocket No. 1165.071126WOO 110 mg / kg SC dose administered simultaneously. combined dose of 20 mg / kg was well tolerated in the single dose tolerability phase. The combined 20 mg / kg dose was selected for further evaluation across a 3-day tolerability study wdth daily dosing. There were no effects of treatment on standard in-life and post-life assessments which supported the selection of 20 mg / kg as the high dose for a 14-day GLP study. In the 14-day study, dogs were administered vehicle or doses up to 20 mg / kg / day. There was no morbidity or mortality, no effect of treatment on any in-life assessments including clinical observations, body weight, food consumption, ophthalmology, ECGs, or clinical pathology parameters (hematology, coagulation, chemistry, urinalysis) and no effect on post-life assessments including necropsy, organ weights and microscopic examination of a standard list of tissues. Accordingly, the NOAEL was 20 mg / kg / day in male and female dogs dosed daily by the combined IN and SC routes for 14 days.

[0387] METHOD EXAMPLE 22. Summary of Chemistry, Manufacturing, and Controls.Compound 24 is a small molecule neuraminidase inhibitor covalently conjugated to dinitrophenyl (DNP) and a-L-rhamnose with PEG-based linkers. 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.

[0388] METHOD EXAMPLE 23. Single-dose pharmacokinetics of compound 24 after intravenous (IV), subcutaneous (SC), and intranasal (IN) administration in mice. Female BALB / c mice were randomly assigned and dosed with Compound 24 according to the following groups:1. IV at 2.22 mg / kg in PB S2. IN at 2.2 mg / kg in PBS3. IN at 0.24 mg / kg in 20 mg / mL Avicel® RC 591, 6 mg / mL DDM in IX PBS4. IN at 0.24 mg / kg in PBS5. IN at 2.2 mg / kg in 20 mg / mL Avicel® RC 591, 6 mg / mL DDM in IX PBS6. SC 2.2 mg / kg in PBS

[0389] Sparse sampling was employed (n=3 / timepoint / dose group). The PK findings are summarized in Table 1. Following IV administration, the clearance (CL) was low relative to theDocket No. 1165.071126WOO 1hepatic blood flow (Qh) in a mouse (e.g, - 18%, Table 1). The mean volume of distribution at steady state (Vss) was higher than plasma volume but lower than total body water for a mouse, suggesting limited distribution into tissues. The corresponding tl / 2 was short (<1 hr).

[0390] After IN administration in PBS, Compound 24 was readily bioavailable (F=47.8%) compared with IV. The absorption was rapid with an observed Tmax of approximately 30 minutes. When dosed IN with a formulation including Avicel® (thickener) and DDM (permeability agent), the exposure when measured by Cmax and AUC is comparable to SC administration, suggesting that Compound 24 is readily absorbed by either ROA. This improved IN formulation doubled the F when compared with PBS IN administration (47.8% vs. 110%).

[0391] Finally, the 0.24 mg / kg IN dose was shown to have good exposure. The AUCinf increase between 0.24 and 2.2 mg / kg in the Avicel / DDM formulation was trending greater than dose proportional but given the sparse sampling, the significance of this trend was unknown.Table 1: PK Comparison of IN and SC Compound 24 in Mice Using Different Formulations

[0392] METHOD EXAMPLE 24. Pharmacokinetics after single IV and IN administration in Sprague Dawley rats. The PK of Compound 24 w7as determined following single IV and IN administration of Compound 24 to female SD rats (n=3 per dose group, serial sampling) to identify the impact of different Compound 24 formulations on the PK exposure profile of Compound 24 when dosed IN versus IV. Broad classes of excipients commonly used in nasal formulations were examined, including thickeners, mucoadhesives, solubility agents, and permeability agents.

[0393] Following a single IV dose of I mg / kg Compound 24 to female SD rats, the elimination tl / 2 of Compound 24 was short (<1 hr). The Vss was higher than rat serum volume but lower than total body water at 0.202 L / kg, indicating that Compound 24 was not extensively distributed in rats (10). Compound 24 was cleared slowly relative to rat Qh (10) with a mean CL of9.43 mL / min / kg. (Table 2)Docket No. 1165.071126WOO 1

[0394] Through the IN route, exposure (Cmax, AUCinf) increased with increasing dose, however, the increases were less than dose proportional. All tl / 2 by IN were longer than found by IV, suggesting absorption becomes rate limiting in the elimination of Compound 24 (e.g., flip-flop kinetics). Finally, systemic exposure achieved was equivalent with and without Avicel as a thickener (Table 2), suggesting that DDM alone is sufficient to obtain the exposure levels desired. Table 2: Mean (± SD) Pharmacokinetic Parameters of Compound 24 in Female SD Rats following Single IV and IN Doses of 1, 10 and 100 mg / kg Compound 24aA single aberrant time point or an unexpectedly high value at 24 hours in a single rat results in a half-life of 13.81 hours for one animal, causing the group average half-life to be reported as 6.81 h. Exclusion of this single data point as an outlier results in a calculation of 4.2 hours, which is closer to the expected half-life.

[0395] METHOD EXAMPLE 25 Pharmacokinetics after single IV and IN administration in dog. The PK parameters of Compound 24 were determined following single IV and IN administration of Compound 24 to male beagle dogs (n=3 per dose group). Serial sampling was employed. The actual doses of 2.06 mg / kg IV and 2.46 or 24.6 mg / kg IN were administered using solutions / suspensions. Groups were tested to compare IN vs IV, to test the necessity of a thickener in the nasal formulation, to explore the optimal concentration of DDM, and to examine PK profile after administration with a nasal device compared with a pipette.

[0396] Following a single IV dose of 2.06 mg / kg Compound 24 to male beagle dogs, the t1 / 2 of Compound 24 was short with a mean value of 0.802 hr. The mean Vss was higher than dog serum volume but was lower than total body water at 0.193 L / kg (10), indicating that Compound 24 was not extensively distributed in dogs. Compound 24 was cleared slowly relative to dog Qh with aDocket No. 1165.071126WOO 1mean CL of 3.43 mL / min / kg. (9)

[0397] Following single IN doses of 2.46 and 24.6 mg / kg Compound 24 to male beagle dogs, absorption of Compound 24 was rapid with a tmax of 0.500 hr for both dose levels, t1 / 2 is similar across dose levels and routes of administration, suggesting absorption was not rate limiting in the elimination of Compound 24 in the dog. Exposure (Cmax, AUCinf) increased with increasing dose; but not dose proportionally. Mean F was low (< 10%) after IN doses of 2.46 and 24.6 mg / kg. (Table 3)

[0398] When Compound 24 was administered through the IN route using either a Uni spray Single-use nasal device or a pipette, no significant difference was seen in PK parameters suggesting that spray pattern is not a significant effector of absorption.

[0399] To determine the effects of varying the DDM concentration, a group of dogs received a single Compound 24 dose of 10 mg / kg with either 1.5 mg / mL or 6 rng / mL DDM in PBS. The higher DDM concentration performed better than the lower concentration (Table 4)Table 3: Mean (± SD) Pharmacokinetic parameters of Compound 24 in beagle dogs following single IV and IN doses of 1, 2.46 and 24.6 mg / kg Compound 24Table 4: Mean (± SD) Pharmacokinetic parameters of Compound 24 in female beagle dogs following single IN doses of 2 and 10 mg / kg Compound 24 using the Unispray Device or via pipette

[0400] METHOD EXAMPLE 26. Randomized, double-blind, placebo-controlled, single-doseDocket No. 1165.071126WOO 1study to assess the safety, tolerability, pharmacokinetics (PK) of intranasal Compound 24 in normal healthy volunteers. Illustratively, healthy volunteers are between 18 and 65 years old at screening, extremes included and have a body weight of at least 50.0 kg at screening, with a body mass index (BMI) within the range of 18.5 to 32.0 kg / m2, extremes included, at screening.

[0401] Safety and tolerability shall be assessed via adverse events (AEs), laboratory safety assessments, 12-lead electrocardiogram (ECG) findings, vital signs, physical examination findings, and intranasal administration site examination findings.

[0402] Single-dose PK profile of Compound 24 intranasal administration in healthy participants shall be assessed by plasma PK parameters of Compound 24 following single intranasal dose administration including, but not limited to, area under the plasma concentration-time curve extrapolated to infinity (AUCoo), area under the plasma concentration-time curve calculated between time of administration of the (first) spray and the last quantifiable concentration (AUClast), maximum plasma concentration (Cmax), time to reach the maximum plasma concentration (tmax), apparent first order terminal elimination rate constant (kz), apparent terminal elimination half-life (t 1 / 2), apparent systemic clearance (CL / F), and apparent volume of di stri buti on (V z / F).

[0403] In each cohort, healthy participants will be randomized to receive a single dose of Compound 24 or placebo intranasally, for example at an active to placebo ratio of 3:1. Illustrative single doses of Compound 24 for the SAD shall be:Cohort 1: 30 mg Compound 24 (100 pL of 150 mg / mL formulation / nostril) or placebo (up to 100 uL DDM-PBS vehicle / nostril)Cohort 2: 100 mg Compound 24 (200 pL of 250 mg / mL formulation / nostril) or placebo(up to 200 pL DDM-PBS vehicle / nostril)Cohort 3: 300 mg Compound 24 (600 µL of 250 mg / mL formulation / nostril) or placebo(up to 600 µL DDM-PBS vehicle / nostril)

[0404] Optionally, a sentinel approach shall be included where only a portion of Cohort 1 is dosed, and the remainder of the participants are dosed at a later date, for example 24 hours later, and following an assessment of safety and tolerability in the sentinel participants. The remainder of participants in Cohort 1 shall be dosed if there is a favorable assessment of safety and tolerability in the sentinel participants based on study criteria. The sentinel participants shall be illustratively randomized at an active to placebo ratio of 1: 1 and the remaining participants at a higher active to placebo ratio, for example 5:1.Docket No. 1165.071126WOO 1

[0405] Screening visits will be performed between Day -28 to Day -2. Eligible participants shall visit the clinical site in the morning of Day -1 for assessment of clinical laboratory tests and urinalysis and shall be admitted to the clinical site in the evening of Day -1, and shall be discharged at the discretion of the investigator after performing the last assessment on Day 2. Follow-up visits and / or phone calls shall be optionally performed on Day 7 (±2) and Day 28 (±2), respectively.

[0406] An analysis shall be performed once all participants have completed follow-up or have discontinued earlier to develop topline data on the safety, tolerability, PK, and PD of Compound 24.

[0407] Pharmacokinetics assessment shall include a pre-dose blood sample taken for baseline, and periodic, for example at 15- and / or 30-minute intervals, and taken post-dose on day 1 Samples shall be analyzed for Compound 24.

[0408] Pharmacodynamics assessment shall include a pre-dose blood sample taken for baseline, and periodic, for example at 12 hours and 24 hours, samples taken post-dose on day 1, and at follow-up. Samples shall be analyzed for one or more cytokines, including but not limited to TNFa, IFNy, IL-6, IL-8, and IL-10.

[0409] Additional optional analyses may be performed at screening (day -28 to day -2), day -1, pre-dose, post-dose on day 1, and at follow-up, including blood samples for hematology, biochemistry, coagulation; urinalysis; vital signs; ECG; physical examination; and intranasal administration site examination.

[0410] METHOD EXAMPLE 27. Randomized, double-blind, placebo-controlled, single-dose challenge study to assess the safety, tolerability, PK, pharmacodynamics (PD), and efficacy of intranasal Compound 24 in influenza challenge participants. Illustratively, influenza challenge participants are between 18 and 55 years old at screening, extremes included and shall have a body weight of at least 50.0 kg at screening, with a BMI within the range of 18.5 to 30.0 kg / m2, extremes included, at screening. Eligible participants must not have pre-existing antibodies to influenza virus H3N2 subtype, strain A / Belgium / 4217 / 2015, at a level, as determined by a microneutralization titer, of >14 at screening. The A / Belgium (H3N2) challenge strain is a live, wild-type influenza A virus initially isolated from a nasopharyngeal swab from a pediatric patient in Antwerp, 2015.

[0411] Safety and tolerability shall be assessed via Adverse events (AEs), laboratory safety assessments, 12-lead electrocardiogram (ECG) findings, vital signs, physical examination findings, intranasal administration site examination findings, peak expiratory flow (PEF), and peripheral oxygen saturation (SpO2%).Docket No. 1165.071126WOO 1

[0412] Efficacy of single doses of Compound 24 on viral AUC compared to placebo in healthy participants exposed to influenza H3N2 with positive predose values shall be assessed by Viral AUC, calculated on 50% tissue culture infective dose / mL (TCID50) results from nasopharyngeal swab sample among participants with positive pre-dose values to be determined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR)

[0413] Single-dose PK profiles of Compound 24 intranasal administration in healthy participants exposed to influenza H3N2 shall be determined by plasma PK parameters of Compound 24 following single intranasal dose administration including, but not limited to, AUCoo, AUClast, Cmax, tmax, AZ, tl / 2, CL / F, and Vz / F.

[0414] Efficacy of single doses of Compound 24 shall be assessed by viral shedding compared to placebo in healthy participants exposed to influenza H3N2, as measured by viral load parameters, including viral load over time, attack rate, time to onset of viral shedding, time to resolution of viral shedding, peak viral load, and time to peak viral load.

[0415] Efficacy of single doses of Compound 24 on measurements of disease severity compared to placebo in healthy participants exposed to influenza H3N2 shall be based on (1) the inFLUenza Participant Reported Outcome (FLU-PRO®) questionnaire domain and total scores, including scores over time, AUC, time to onset of symptoms, time to symptom resolution, peak symptom score, time to peak symptom score; (2) Proportion of participants with at least one FLU-PRO® score of 2 or higher over time.; (3) Proportion of participants with a mean FLU-PRO® score of 1 or higher over time; and (4) Proportion of participants with fever. FLU-PRO® is a validated patient-reported questionnaire that assesses symptoms associated with influenza that assesses symptoms across multiple body systems over the disease course.

[0416] Safety, tolerability, PK, PD, and efficacy of single doses of Compound 24 following an influenza challenge in up to 2 dose levels and in up to 3 cohorts shall be evaluated.

[0417] In each cohort, healthy participants shall be randomized to receive Compound 24 or placebo intranasally, for example at an active to placebo ratio of 2: 1.

[0418] After randomization, participants shall be inoculated with 1.0x106 TCID50 / mL Influenza Type A Virus (A / Belgium / 4217 / 2015 H3N2) on Day 1 and approximately 24 hours after inoculation, participants receive a single dose of either Compound 24 or placebo. Illustrative single doses of Compound 24 for the SAD shall beCohort 1: H3N2 challenge + 30 mg Compound 24 or placeboCohort 2: H3N2 challenge r 300 mg Compound 24 or placeboDocket No. 1165.071126WOO 1

[0419] A microneutralization titer (MNT) test shall be performed from Day -42 to Day -3 and other screening tests from Day -28 to Day -3. If the MNT test result is obtained before performing the other screening tests and is positive, no other screening tests will be performed and the participant shall not be included in the study. Between Day -28 and Day -3, the MNT test and the other screening tests shall be optionally performed on the same day.

[0420] Eligible participants shall be admitted to the clinical site in the morning of Day -2 and are discharged at the discretion of the investigator after performing the last assessment on Day 11. A follow-up visit shall be performed on Day 28 (±2).

[0421] Participants shall be monitored for disease progression. Participants assess any challenge virus or influenza-related signs and symptoms using the FLU-PRO® and the FLU-PRO© Additional Daily Diary. As part of the quantitative validation analyses of the FLU-PRO©, participants complete additional diary items using the FLU-PRO® Additional Daily Diary Items. Optional additional questions are related to Patient Global Rating of Flu Severity; Patient Global Assessment of Interference with Daily Acti vities; Patient Global Assessment of Physical Health; Return to ‘Usual’ Health; and Return to ‘Usual’ Activities. The FLU-PRO® questionnaire shall be performed twice daily, once in the morning and once in the evening, approximately 12 hours apart. The FLU-PRO® Additional Daily Diary shall be completed once daily in the morning. On Day 11, participants only report in the morning prior to discharge.

[0422] Virus levels shall be measured throughout the study by nasopharyngeal swab samples by qRT-PCR. Positive qRT-PCR tests are optionally confirmed by TCID50.

[0423] Pharmacokinetics assessment shall include periodic, for example at. day 2 and day 3, samples taken post-dose. Samples shall be analyzed for Compound 24.

[0424] Pharmacodynamics assessment shall include periodic, for example at each day -1 to day 11, samples taken post-dose on day 1, and at follow-up. Samples shall be analyzed for one or more cytokines, including but not limited to TNFa, ZFNy, IL-6, IL-8, and IL-10. Additional periodic blood samples shall also be taken for humoral immunogenicity (MNT), and to determine antihapten titer.

[0425] Nasopharyngeal swab for viral sequencing shall be taken periodically, for example at day 3 and day 7.

[0426] Additional optional analyses shall be performed at screening (day -28 to day -2), day -1, pre-dose, post-dose on day 1, and at follow-up, including blood samples for hematology, biochemistry, coagulation; urinalysis; vital signs; pulse oximetry, ECG; physical examination;Docket No. 1165.071126WOO 1intranasal administration site examination, spirometry, and peak expiratory flow measurements (PEF).SOLID PHARMACEUT TCAL COMPOSITION EXAMPLES

[0427] Compound 24 was initially stored at -20°C, and also at 2-8°C. When required for use, it was allowed to equilibrate to ambient and handled in a low % RH environment (<25% RH) unless otherwise stated.

[0428] SOLID PHARMACEUTIC L COMPOSITION EXAMPLE I. Feed Solution Preparation for Spray-Dried Formulations

[0429] Feed solutions were prepared using the below method of combination:1. Required mass of deionized water was weighed into a Duran.2. Excipients were added and stirred to fully dissolve, or disperse in the case of HPMC, to produce a clear, colorless, slightly viscous solution.3. Compound 24 was weighed and added to the excipient solution (or dispersal), and stirred to fully dissolve and produce a clear, yellow-colored solution,

[0430] SOLID PHARMACEUTICAL COMPOSITION EXAMPLE 2. Spray-Drying Feed Solutions

[0431] All feed solutions above in SOLID PHARMACEUTICAL COMPOSITION EXAMPLE 1 were spray dried using the ProCepT 4M8-Trix spray dryer, fitted with a 25 kHz ultrasonic nozzle.

[0432] The below7spray drying parameters in the following Table were used for all formulations and also 5°C as shown.*S7?ray drying parameters

[0433] SOLID PHARMACEUTICAL COM POSITION EXAMPLE 3. Particle Size Distribution by Laser Diffraction.Docket No. 1165.071126WOO 1

[0434] Particle size analysis on the spray-dried formulations was performed in duplicate, using a Sympatec HELOS particle size analyzer with ASPIROS disperser. For these spray dried formulations, an R3 lens was used (0.5 175.0 pm range) with a 1 bar dispersal pressure.Approximately 30 mg of material was used for each measurement, and samples were prepared in glass Aspiros tubes under reduced humidity (RH<25%).

[0435] SOLID PHARMACEUTICAL COMPOSITION EXAMPLE 4. Differential Scanning Calorimetry.

[0436] ~5 mg of sample was weighed into a T Zero Aluminum pan and sealed. Samples were analyzed using the heat-cool-heat method below:1. Equilibrate at -40.00 °C2. Isothermal for 5.00 min3. Ramp 10.00 °C to 150.00 °C4. Isothermal for 5 00 min5. Ramp 10.00 °C to -40.00 °C6. Isothermal for 5.00 min7. Ramp 10.00 °C to 150.00 °C

[0437] SOLID PHARMACEUTICAL COMPOSITION EXAMPLE 5. Loss on Drying.

[0438] Residual moisture content was determined by measuring the loss on drying. Analysis was performed using a TA Instruments TGA550. Approximately 20 mg of sample was analyzed in single measurement using the following method:1. Ramp 1.00 °C / min to 60.00 °C2. Isothermal for 120.00 min

[0439] The residual moisture was calculated as a percentage of the weight lost on drying.

[0440] SOLID PHARMACEUTICAL COMPOSITION EXAMPLE 6 X-Ray Powder Diffraction.

[0441] X-Ray powder diffraction was performed using a Rigaku MiniFlex Samples were prepared under low' humidity (RH<25%) then transferred immediately before analysis: sample was placed onto the center of a small sample holder and firmly compressed using a glass plate. Any excess powder was removed from the holder, ensuring the sample in the center was not disturbed. Samples were analyzed using the below parameters:0 / 29 Range: 3-90°Step Width: 0.01°Docket No. 1165.071126WOO 1Step Duration: 5° / min

[0442] SOLID PHARMACEUTICAL COMPOSITION EXAMPLE 7. HPLC Analysis

[0443] Chromatographic conditions for both assay / related substances and AINI-NGI methods are summarized in the Table below.HPLC Chromatographic Conditions for Assay / RS

[0444] SOLID PHARMACEUTICAL COMPOSITION EXAMPLE 8. Device Testing with Aptar Unidose Nasal Devices.

[0445] The Aptar containers were hand-filled with spray dried formulation under low humidity (RH<25%), and the device assembled.

[0446] A 100 mL volumetric flask was used for collection of emitted powder. The device was inserted downwards into the neck of the flask. The emitted dose was calculated gravimetrically by weighing the volumetric flask pre- and post- actuation. AINI stands for Alberta Idealized Nasal Inlet device.

[0447] The AINI is an anatomical model of the nasal airways, developed at the University ofDocket No. 1165.071126WOO 1Alberta and commercialized as a test device by Copley Instruments (Nottingham, UK). It was developed from a series of CT scans of 10 human nasal geometries and creating a “best fit” to represent a patient nasal airway geometric model. The AINI breaks down into four basic sections and enables the practitioner to assess drug deposition behavior in each region, The AINI has a four- region resolution:the vestibule (nostril),the turbinates,the olfactory7region,the nasopharynx.

[0448] To evaluate lung deposition the AINI can be attached to a Next Generation Impactor (NGI) so that the proportion of the delivered dose that ends up in the lungs (ie on the different stages of the NGI) can also be determi ned.

[0449] Spray dried nasal formulations of different sizes can be filled into a nasal delivery' device and delivered into a simulated nasal / lung system. Following delivery by the nasal device, the two impactors representing the nose (AINI) and the lung (NGI) can be opened, and drug deposition determined.

[0450] The AINI was assembled and coated with a solution containing 12 g of Brij and 20 g of glycerol in 80 m of ethanol. The assembled AINI was rotated in a series of rotations to ensure full coating of the internal surfaces, then left to drain and dry overnight. The NGI collection cups were also coated with the Brij and glycerol in ethanol coating solution, drained, and left to dry overnight.

[0451] A pre-separator was used in between the NGI and AINI to collect any large, non-inhalable particles that may deposit because of particle bounce through the AINI. A flow rate of 7.5 L / min was set through the system with an actuation time of 15 seconds. The device was angled at 45° to the AINI and clamped to hold in a fixed position. The nozzle of the device was inserted -1 cm into the nostril. The device was actuated simultaneously with the airflow7. Gravimetric emitted dose w7as determined by weighing the device pre- and post-actuation. Deposition in each region of the AINI, as well as the pre-separator and NGI was determined by HPLC.

[0452] SOLID PHARMACEUTICAL COMPOSITION EXAMPLE 9. Initial Compound 24 Solids Analysis.

[0453] Compound 24 was prepared in accordance with the disclosure. FIG. 22 show's an XRPD pattern of Compound 24 indicating it is amorphous. In FIG. 23, a first (a) and second (b) heat¬ flow are shown in the DSC thermogram. A second-heat flow is often used to measure a glassDocket No. 1165.071126WOO 1transition temperature. Here, the glass transition temperature, Tg, is about 42.3°C, further indicating the amorphous nature of Compound 24. FIG.24 shows the particle size distribution of Compound 24. It has abroad unimodal distribution profile with an Xso value of 40.9 microns and a volume mean diameter of 44.3 microns. 15.7% of the particles were less than 10 microns.

[0454] SOLID PHARMACEUTICAL COMPOSITION EXAMPLE 10 Solubility of Compound 24

[0455] Compound 24 was prepared in accordance with the disclosure.

[0456] The solubility of the Compound 24 was initially assessed in deionized (DI) water.Ethanol was also screened as part of this solubility assessment.

[0457] The solubility assessment was performed by weighing -10 mg of Compound 24 into a vial and adding DI water or ethanol in 100 pL additions. The solution was vortex mixed between additions to wet and dissolve any solids.

[0458] Solubility was assessed visually, and the total mass and volume of solvent added were recorded once a visibly clear solution was produced. As a feed solution solids content of >5% w / w was being targeted, addition of solvent was stopped after a total of 1 mL. Results are summarized in the Table below.Solubility of Compound 24 in Deionized Water & Ethanol

[0459] SOLID PHARMACEUTICAL COMPOSITION EXAMPLE 11. Spray-Dried Compound 24 with HPMC and DDM

[0460] A feed solution of 85% Compound 24, 13.5% HPMC and 1.5% DDM (w / w / w) as per the Table below.Feed solution composition of initial batchDocket No. 1165.071126WOO 1

[0461] The feed solution was observed as a clear, yellow-colored solution. Once prepared, it was immediately spray dried, in accordance with SOLID PHARMACEUTICAL COMPOSITION EXAMPLE 2.

[0462] The spray-dried formulation had the general characteristics in the table below and was in the form of a dry powder.Spray drying yield and product appearance

[0463] The feed solution was spray-dried successfully with a processing yield of about 66%. There was minimal cyclone deposition, and the collected powder was observed as bright yellow with slightly cohesive properties.

[0464] FIG. 25 shows an XRPD pattern of the spray-dried formulation (top) overlaid with an XRPD pattern of Compound 24 (bottom). FIG. 26 shows a loss on drying measurement generated by thermal gravimetric analysis which indicates a weight loss of 1.69% indicating relatively low residual moisture in the spray-dried powder. The table below, and FIG. 27 illustrate the particle size distribution of the spray-dried formulation.Particle size distribution

[0465] For nasal drag delivery, an X50 of approximately 20-40 pm is typically targeted, to ensure that particles deposit in the nasal cavity. In addition, less than 10% of particles with a diameter below 10 pm is also targeted to ensure that delivery of the spray dried powder is controlled with minimal lung deposition. Here a suitable particle size for nasal delivery was achieved, with an X50 of 27.2 pm and only 2.4% of particles below 10 pm.

[0466] FIG. 28 is a first heat cycle DSC thermogram for the spray-dried formulation (bottom) with an overlay of the corresponding DSC thermogram of Compound 24 (top). FIG. 29 is a second heat cycle DSC thermogram for the spray-dried formulation (bottom) with an overlay of theDocket No. 1165.071126WOO 1corresponding DSC thermogram of Compound 24 (top). A single Tg in the second heat cycle (about 46.5°C) indicates an amorphous material and no melting event was observed.

[0467] Assay and related substances analysis was performed using the method outlined in SOLID PHARMACEUTICAL COMPOSITION EXAMPLE 7. Duplicate samples were prepared for repeatability of results, and the data are reported below.Assay of Compound 24 in initial spray dried formulation

[0468] The average assay value of 103.9%, indicate full retention of Compound 24 after spray¬ drying. Assay data also showed good repeatability between replicate sample preparations. No impurity peaks were observed greater than the limit of quantification, which showed no impurities were generated during the spray drying process.

[0469] The spray dried powder was filled into an Aptar Unidose to evaluate the powder’s device filling capability and performance with respect to emitted dose. It was observed that 40mg filled approximately 50% of the container, indicating the dose could be increased beyond 40mg. No issues relating to electrostatic charges were observed.

[0470] To determine gravimetric emitted dose using the filled container (with 40.7 mg of spray dried powder), an Aptar Uni dose device was made up and actuated into a 100 mL volumetric flask. A 99% gravimetric emitted dose (40.3 mg) was calculated by weighing the volumetric flask pre-and post-actuation. A small amount of yellow powder was noted on the device nozzle, likely due to powder returning / blowing back off the volumetric flask.

[0471] SOLID PHARMACEUTICAL COMPOSITION EXAMPLE 12. Spray-Dried Compositions of Compound 24

[0472] Feed Solutions of Compound 24 and HPMC, DDM and Mannitol in deionized water were prepared to the following specifications.Feed solution compositions of various batchesDocket No. 1165.071126WOO 1

[0473] The spray drying yields are shown in the table below.Spray drying yields and product appearance

[0474] Spray drying yields for the 6 batches ranged from 50% up to 75%. The highest yield batch (prepared with 72.5% Compound 24) also had particularly good powder flow and handling properties.

[0475] Particle-size distribution for these batches are in the table below.Particle-size distributionDocket No. 1165.071126WOO 1

[0476] The particle size for all formulations were suitable for nasal delivery with an X50 between 24-31 um and less than 10% of particles with a diameter below 10 pm. The batch prepared with 72.5% Compound 24 loading and the highest percentage HPMC, had the largest overall particle size and lowest percentage of particles <10 pm. The favorable powder properties noted for this batch may be in part at least due to this larger particle size distribution.

[0477] The fill weight and gravimetric fractions are shown below.Fill weight & Gravimetric Emitted Fractions

[0478] The 72.5% Compound 24 formulation had the highest emitted fraction.

[0479] Assay and related substances analysis were performed in duplicate or three batches.AssayDocket No. 1165.071126WOO 1

[0480] SOLID PHARMACEUTICAL COMPOSITION EXAMPLE 13. Stability Study - Sample Preparation and Testing

[0481] The following feed solutions in deionized water were prepared to the specifications indicated.Feed solution compositions

[0482] The spray drying yields are shown in the table belowSpray drying yields and product appearance

[0483] For all batches, the spray dried powder was noted as bright yellow in color with relatively low cohesiveness. Spray drying yields were broadly comparable for the three batches and ranged from 65% up to 69%. Losses of product during the spray drying process were mainly due to deposition on the lower drying chamber glassware, the bottom of the S-bend tubing and on theDocket No. 1165.071126WOO 1temperature probe at the bottom of the drying chamber. A summary of the average particle size distribution data for the three spray dried formulations at T=0 is shown in the table below.Particle size distribution

[0484] FIG. 30 shows the particle distribution profile of the three batches below. The table below shows the loss on drying data.Loss on drying

[0485] FIG. 31 shows XRPD patterns at T=0 for each of the three batches. A halo XRPD diffractogram was observed for all three spray dried powders indicating them to be amorphous. FIG. 32 is a first heating cycle DSC thermogram for the three batches and FIG. 33 a second heating cycle DSC thermogram indicating a Tg between about 47°C to about 49°C.

[0486] Assay and related substances analysis was performed on duplicate sample preparations for all three batches. The data for T=0 is reported in the table below.Assay of Compound 24Docket No. 1165.071126WOO 1

[0487] Assay values ranged from 101-102%, indicating full retention of Compound 24 after spray drying. Assay data showed good repeatability between replicate sample preparations and no impurity peaks were observed greater than the LoQ (>0.05) which were not present in the unprocessed Compound 24 standard solution.

[0488] Nasal deposition profiling was performed on each formulation in duplicate. Aptar Unidose devices were filled under low humidity (RH<25%) using the following masses, equivalent to 50mg of the Compound 24.« 6A - 50 mg Dose Fill Target & Range: 68.97 mg ±5% (65.52 - 72.42)• 6B - 50 mg Dose Fill Target & Range: 68.97 mg ±5% (65.52 - 72.42)* 6C - 50 mg Dose Fill Target & Range: 55.56 mg ±5% (52.78 - 58.34)

[0489] Alberta Idealized Nasal Inlet-Next Generation Impactor (AINI-NGI) evaluations were performed using the method detailed in SOLID PHARMACEUTICAL EXAMPLE 8. AINI-NGI deposition profiles for the spray dried formulations at T=0 is shown in FIG. 34.

[0490] FIG. 34 shows the deposition profiles for 6A, 6B, and 6C. The average mass balance recoveries were >90% for all three batches. For all three formulations, the highest percentage powder deposition was seen in the turbinates, with 37.7-39.5 % recovery in this region. 0% recovery was observed in the NGI stages 1 & 2, confirming that all powder remained within the AINI and pre-separator on actuation. From rinsing the device post-actuation, only a small quantity was retained, equivalent to approximately 0.86-1.63% of the total dose.

[0491] SOLID PHARMACEUTICAL COMPOSITION EXAMPLE 14. Stability StudyDocket No. 1165.071126WOO 1Conditions and Testing

[0492] For stability, Aptar Unidose devices were filled under low humidity (RH<25%) using the following masses, equivalent to 50mg of Compound 24.• 6A - 50 nig Dose Fill Target & Range: 68.97 mg ±5% (65.52 - 72.42)* 6B - 50 mg Dose Fill Target & Range: 68.97 mg ±5% (65.52 - 72.42)• 6C - 50 mg Dose Fili Target & Range: 55.56 mg ±5% (52.78 - 58.34)

[0493] Filled devices were individually sealed in Ml 2 foil laminate pouches with a 1 g desiccant sachet. The table below details the total number of devices filled for each storage condition.Total Aptar Unidose Devices Filled

[0494] At T=2 weeks, only devices stored at 40°C / 75%RH were tested. At T=4 weeks devices at all storage conditions were tested.

[0495] A summary of the average particle size distribution data for all batches at T=0, T=2 weeks and T=4 weeks is shown below in the following tables.Particle size distributionDocket No. 1165.071126WOO 1Particle size distributionParticle size distribution

[0496] For 6 A, following storage after 2 and 4 weeks, particle size for the bulk powder for all three conditions were comparable to T O. All results gave an acceptable size for nasal delivery, with X50 values remaining between 29-31 pm and <4.0% of particles <10 pm. For 6B, following storage after 2 and 4 weeks, particle size for the bulk powder for all three conditions were comparable to T=0. All results gave an acceptable size for nasal delivery, with X50 values remaining between 28-30 pm and <3.0% of particles <10 pm. For 6C, only one replicate was conducted at the 4 week 45°C / 75% RH time point due to difficulties of reaching a high concentration which may have been due to aggregation. Despite the issues relating to analysis of powders held at 40°C / 75%RH, particle sizes for all three conditions were comparable to T=0. All results gave an acceptable size for nasal delivery with X50 values remaining between 27-28 pm andDocket No. 1165.071126WOO 1<3.0% of particles <10 pm.

[0497] The table below summarize the data generated by TGAto assess loss on drying / residual moisture content for the three spray dried powders at T=0, T=2 weeks and T=4 weeks.Loss on drying

[0498] Overlay diffractograms of all three batches comparing T=0 vs. T=4 weeks at the three different storage conditions are shown below in FIG. 35, FIG, 36, and FIG. 37. No structural changes were observed at T=4 weeks, where halo XRPD diffractograms were observed for all three spray dried powders at all three storage conditions.

[0499] By DSC, all spray dried powders after 4 weeks at all three different storage conditions showed the absence of any sharp endothermic (melt) peaks in the thermograms. Each formulationDocket No. 1165.071126WOO 1had glass transition (Tg) events in both the 1st and 2nd heating cycles. The mid- point temperatures for all three formulations are summarized in the table below.DSC Tg Temperatures

[0500] The Tg values in the first heating cycle ranged from 23.0°C up to 46.5°C, with the DSC thermogram profile may be influenced by the loss of moisture and endothermic events associated with the excipient components. By comparison, the second heat cycle saw a single Tg ranging from 46.9°C up to 50.2°C Batch 6C showed evidence of powder aggregation following 4 weeks at 40°C / 75%RH. FIG.38 and FIG. 39 are the DSC thermograms for 6A. FIG. 40 and FIG. 41 are the DSC thermograms for 6B. FIG. 42 and FIG. 43 are the DSC thermograms for 6C.Docket No. 1165.071126WOO 1

[0501] Assay and related substances analysis was performed on duplicate sample preparations for all three batches. The data for T=2 weeks and T=4 weeks are reported alongside T=0 in the table below.Assay of Compound 24Docket No. 1165.071126WOO 1Docket No. 1165.071126WOO 1

[0502] Average assay values ranged from 89.3-101%. Impurity peaks increased on storage (when compared to T=0) and had higher levels for the devices held at 40°C / 75%RH and for batch 6C, formulated with the highest Compound 24 loading of 90% w / w.

[0503] On review of data at T:::4 weeks, it was noted that the Compound 24 peak’s retention time had shifted from -22-24 minutes down to -17.7 minutes. Some partly resolved impurity peaks had also merged into single peaks. The assay and related substances data for T::6 weeks at 25°C is reported alongside T=0 and T=4 weeks at 25°C / 60%RH in the table below for 6B.Docket No. 1165.071126WOO 1Assay of Compound 24

[0504] The data generated at T=6 weeks showed that the total percentage impurities had not changed from the data generated at T::=4 weeks. Nasal deposition profiling was performed on each formulation in duplicate at T=4 weeks at each storage condition. AINI-NGI deposition profiles for each spray dried formulations at T:::4 weeks (and T-0) is in FIG. 44 for 6A, in FIG. 45 for 6B, and in FIG. 46 for 6C.

[0505] For 6A, the average mass balance recoveries were >90% for all three conditions and >98% for devices held at 2-8°C & 25°C / 60%RH. For all three storage conditions, the highest percentage powder deposition was seen in the turbinates, with 41-49 % recovery' in this region.

[0506] Less than 0.05% recovery wras observed in the NGI stages 1 & 2 at all storage conditions, confirming that the powder remained within the AINI and pre-separator on actuation. From rinsing the device post-actuation, it was found that only a small quantity was retained, at around 0.83- 1.25% of the total dose. Overall, there was no notable change to the deposition profile between T=0 and devices reassessed after 4 weeks.

[0507] For 6B, average mass balance recoveries 'ere >90% for all three conditions and >98% forDocket No. 1165.071126WOO 1devices held at 2-8°C & 25°C / 60%RH. For all three storage conditions, the highest percentage powder deposition was seen in the turbinates, with 41-49 % recovery in this region. <0.05% recovery was observed in the NGI stages 1 & 2 at all storage conditions, confirming that the powder remained within the AINI and pre-separator on actuation. From rinsing the device post¬ actuation, it was found that a minimal quantity was retained, at around 034-2.24% of the total dose.

[0508] As shown for batch 6A. overall, there was no notable change to the deposition profile between T=0 and devices reassessed after 4 weeks for batch 6B. For 6C, formulated with 90% Compound 24 loading, devices held at 2-8°C and 25°C / 60% RH conditions had >97% mass balance. For devices held at 40°C / 75% RH however, the powder did not successfully evacuate from the device. On disassembly of the device, it was observed that the powder had formed large agglomerates.

[0509] Of the average 47% total powder emitted from the devices for the 40cC / 75%RH samples, 94% of the total dose was recovered. For all storage conditions, the highest percentage powder deposition was again seen in the turbinates, with 41-43 % recovery in this region for 2-8°C and 25°C / 60% RH. For the device held at 40°C / 75% RH, 50% of the total dose was recovered in the turbinates. For all devices, <0.05% was recovered on NGI stages 1 & 2, confirming that powder remained within the AINI & pre-separator on actuation. For the devices held at 2-8°C and 25°C / 60% RH, only a small amount of powdered material was retained post actuation (0.46 & 0.93% of the total dose). Overall, there was no notable change to the deposition profile between T O and devices reassessed after 4 weeks.

[0510] SOLID PHARMACEUTICAL COMPOSITION EXAMPLE 15. Beagle Dog Study

[0511] To test the PK profile of a dry powder formulation of Compound 24, several variations were administered to male dogs. Blood was collected at several timepoints over 24 hours and analyzed for Compound 24 using LC-MS / MS. It was found that the PK profile of dry powder was generally superior to any liquid formulation tested in previous nonclinical studies. By increasing the concentration of DDM to 6.1% from 2%, the bioavailability reached 36%.Docket No. 1165.071126WOO 1

[0512] Amounts of Compound 24, hydroxy propyl methyl cellulose and N-dodecyl beta-d-maltoside were prepared as feed solution in water for injection. The Compound 24 and excipients were dissolved at 10% with solid in solution. The feed solution was then spray dried using a ProCepT 4M8-Trix fitted with an ultra-sonic nozzle. Once the powder was produced, they were then loaded into an Aptar Unidose Powder Nasal Device.

[0513] Male Beagle dogs were sedated using a combination of ketamine and xylazine (5 mg / kg and 1.5 mg / kg / IM respectively) before intranasal administration. Dogs were restrained manually by a hold in the sitting position with the head tilted slightly up at a 20-30° angle followed by insertion of the device past the nasal vestibule, approximately 0.5 to 1 cm deep. The device was positioned parallel to the ground and pointing directly towards the back of the head. The position was held for 30 to 45 seconds between each nostril.

[0514] The anesthesia was maintained for 20-30 minutes post dosing to avoid sneezing. Animals were monitored for sneezing and vomiting. Dosing devices were weighed before and after dosing to calculate the exact dose delivered. Animals were also dosed in 3 cohorts in the order of: Cohort-1: Animal # 1, 4, 7, 10; Cohort-2: Animal #2, 5, 8, 11 and Cohort-3: Animal #3, 6, 9, 12. A total active dose of 50mg was used.

[0515] Blood was sampled and collected at 5, 15, and 30 minutes as well as 1, 2, 4, 6, 8, & 24 hours for a total of 9 time points. Briefly, the cephalic vein was punctured and approximately 1 mb of blood was collected into K2EDTA tubes. Tubes were then inverted -8 times to ensure adequate mixing with the anticoagulant. Blood samples were then maintained on wet ice until centrifugation. Blood samples were centrifuged at 5000 rpm for 10 minutes at 4°C within 15 minutes of collection. Plasma was then harvested and transferred into pre-labeled micro-centrifuge tubes. Samples were then stored at -70 ± 10°C until bioanalysis, at which point samples were shipped from the in-life facility to Jubilant on dry ice. All plasma samples were analyzed for Compound 24 through a fit-or-purpose LC-MS / MS method in the Jubilant Biosys Drug Metabolism and Pharmacokinetics (JBL DMPK) laboratory located in Bangalore. Compound 24 was extracted from plasma by using a pre-optimized extraction procedure and quantified along with calibration standards and quality control samples by LC-MS / MS.

[0516] For the preparation of CC / QC samples: 2.5 pL of working stock solution was spiked intoDocket No. 1165.071126WOO 147.5 pL of blank plasma to obtain the desired concentration. An aliquot of 25 pL was taken and processed alongside samples. Samples were thawed to room temperature and 25 uL of sample was then aliquoted into a pre-labeled plate. 400 pL of Methanol (100%) containing IS (50 ng / mL) was added, vortexed for 5 minutes, and centrifuged at 4000 rpm for 10 minutes at 4°C. Aliquots of the supernatant were separated and injected into the LC-MS / MS for analysis. Parameters for LC- MSZMS are described in table 6.

[0517] Pharmacokinetic parameters were determined by using standard non-compartmental model with Phoenix software ver 8.1 with the individual and mean pharmacokinetic (PK) parameters reported. Graphs were plotted with Microsoft Excel. Dogs were dosed with various dry powder Compound 24 formulations and blood was drawn at specified timepoints over the next 24 hours after administration to evaluate the pharmacokinetics of each formulation. Results are found in the table below and visualized in FIG, 47.Mean Pharmacokinetic Summary of Compound 24 Following intra-nasal RoAin Male BeagleDogs

[0518] The formulation for groups 1 and 2 were meant to test the loading capacity of the nasal device and whether doubling the loading volume may impact the drug administration negatively asDocket No. 1165.071126WOO 1well as dosing in just one nostril. The device was very consistent in the amount of compound delivered as the mean dose in the two device 25 mg dose was 49.7±05 mg while the single device 50 mg dose resulted in 49.6±0.6 mg delivered. Furthermore, the PK profile of the two formulations were similar in nearly all parameters tested, whether it be in Tmax or in T1 / 2. There was an initially higher Cmax observed in group 2, resulting in better bioavailability overall when compared against group 1.

[0519] To test the possible effect of increasing the -dodecyl [3-D-maltoside (DDM) concentration on the PK of Compound 24 in dogs, formulation 3 (which contains 3x the amount of DDM used in all other groups) was tested and compared against group 1. While the time to maximum was the same between all groups at 30 minutes, all other parameters seemed to be improved. There was a higher amount of compound absorbed as seen in the Cmax as well as an improved T12, leading to a higher AUC and bioavailability overall compared to the other groups. It was found that the concentration of HPMC in the formulation does not impact the PK profile of Compound 24. In previous preclinical PK studies, a liquid formulation was used to determine the PK profile of Compound 24. When comparing the results from previous solution studies and this work, the bioavailability of Compound 24 in the dry powder formulation is significantly higher. With the liquid formulation, the bioavailability was measured to be below 10% while the dry powder had bioavailability above 20% and even 30% with some formulations. Overall, the PK of Compound 24 in male dogs with a dry powder formulation is superior to liquid formulation, which was characterized as ha ving worse bioa vailability (20-36% vs <10%). While the loading of the device or the concentration of HPMC had litle impact on the PK properties of Compound 24, adjustment of the DDM concentration led to the greatest impact on bioavailability and may very well represent the final formulation that will be used in clinical studies for Compound 24.

[0520] The following publications, and each of the additional publications cited herein are incorporated herein by reference:Docket No. 1165.071126WOO 1(1) Kim, Y.; Hong, K.; Kim, H.; Nam, J. Influenza Vaccines: Past, Present, and Future. Rev. Med. Virol. 2022, 32 (1), e2243. https: / / doi [dot] org / 10.1002 / rmv.2243.(2) Gubareva, L. V.; Fry, A. M. Baloxavir and Treatment-Emergent Resistance: Public Health Insights and Next Steps. J. Infect. Dis. 2020, 221 (3), 337-339. https: / / doi[dot]org / 10. ] 093 / infdis / j iz245.(1) McKimm-Breschkin, J. L. Influenza Neuraminidase Inhibitors: Antiviral Action and Mechanisms of Resistance. Influenza Other Respir. Viruses 2013, 7 (si), 25-36 https: / / doi[dot] org / 10.1111 / irv.12047.(2) Moscona, A. Neuraminidase Inhibitors for Influenza. N. Engl. J. Med. 2005, 353 (13), 1363-1373. https: / / doi[dot] org / 10.1056 / NEJMra050740.

Claims

Docket No. 116.071126 WOO 1 WHAT IS CLAIMED IS:

1. Amorphous Compound 24.

2. The amorphous Compound 24 of claim I, having an x-ray powder diffraction pattern substantially the same as that of FIG. 22.

3. The amorphous Compound 24 of claims 1 or 1, having a glass transition temperature at one or more of about 41 °C. about 43°C. and about 85CC.

4. The amorphous Compound 24 of claim 3, having substantially the same DSC thermogram as that of FIG. 23(a) or FIG. 23(b).

5. The amorphous Compound 24 of any one of claims 1 -4. having a particle size distribution with an X50 of between about 35 and 45 microns or about 20 to 40 microns.

6. The amorphous Compound 24 of any one of claims 1-5, where betw een 5% and 20% of the particles have a size of less than 10 microns.

7. The amorphous Compound 24 of any one of claims 1-6, having a solubility of between about 90 and 100 mg / g in deionized water.

8. A dry powder comprising Compound 24 and one or more pharmaceutically acceptable excipients9. A dry powder comprising Compound 24 of claim 8, and one or more pharmaceutically acceptable excipients,10. A spray-dried formulation comprising Compound 24.

11. The spray-dried formulation of claim 10 further comprising one or more spray-dry ing excipients.

12. The spray -dried formulation of claim 11, wherein at least one spray -dry ing excipient is a cyclodextrin13. The sprav-dried formulation of claim 12, wherein the cvclodextrin is hydroxy-propyl-beta- cyclodextrin.

14. The spray-dried formulation of claim 11, wherein at least one spray -drying excipient is a cellulose-based compound.

15. The spray-dried formulation of claim 14, wherein the spray-drying excipient is HPMC.Docket No. 116.071126 WOO 1 16. The spray-dried formulation of claim 11, wherein the spray-drying excipient is a sugar.

17. The spray-dried formulation of claim 16, wherein the sugar is mannitol.

18. The spray-dried formulation of claim 11, wherein at least one spray drying excipient is DDM.

19. The spray-dried formulation of claim 11. comprising twzo spray-drying excipients.

20. The spray-dried formulation of claim 19, wherein one spray -diying excipient is HPMC.

21. The spray-dried formulation of claim 20, wherein the second spray -drying excipient is - dodecyl P-D-maltoside (DDM).

22. The spray-dried formulation of any one of claims 11 to 20 further comprising DDM.

23. A spray-dried formulation comprising Compound 24, HPMC, and DDM.

24. The spray-dried formulation of claim 23, wherein Compound 24 is present in an amount of between about 60% and about 90% by weight.

25. The spray-dried formulation of claims 23 or 24, wherein the amount of Compound 24 by weight is between about 60% and about 80% by weight.

26. The spray-dried formulation of any one of claims 23-25, wherein the amount of Compound 24 by weight is betw een about 65% and about 75% by weight.

27. The spray-dried formulation of any one of claims 23-26, wherein the amount of Compound 24 by weight is between about 70% and about 73% by weight.

28. The spray -dried formulation of any one of claims 23-26, wherein the amount of Compound 24 by w eight is about 72.5% by weight.

29. The spray-dried formulation of any one of claims 23-28. w herein the amount of HPMC by weight is betw een about 2% and about 30% by weight,30. The spray-dried formulation of any one of claims 23-29, w herein the amount of HPMC by weight is between about 8% and about 30% by weight.

31. The spray -dried formulation of any one of claims 23-30, wherein the amount of HPMC by weight is between about 18% and about 30% by weight.

32. ’The spray-dried formulation of aiw one of claims 23-31, wherein the amount of HPMC by’ weight is between about 20% and about 27% by » eightDocket No. 116.071126 WOO 1 33. The spray -dried formulation of any one of claims 23-32, wherein the amount of HPMC by weight is between about 21% and about 26% by weigh;34. The spray-dried formulation of any one of claims 23-33, wherein the amount of HPMC by weight is between about 21% and about 22% by weight.

35. The spray-dried formulation of any one of claims 23-34, w herein the amount of HPMC by weight is about 21.4% by weight.

36. The spray-dried formulation of any one of claims 23-34, wherein the amount of HPMC by weight is about 25.0% by weight.

37. The spray-dried formulation of any one of claims 23-34. wherein the amount of HPMC by weight is between about 6% and about 10% by weight.

38. The spray -dried formulation of any one of claims 23-37, wherein the amount of DDM by weight is between about 1% and about 10% by weight.

39. The spray-dried formulation of any one of claims 23-38. wherein the amount of DDM by weight is between about 1% and about 3% by w eight.

40. The spray-dried formulation of any one of claims 23-39, wherein the amount of DDM by eight is about 2% by weight.

41. The spray-dried formulation of any one of claims 23-38, wherein the amount of DDM by weight is betw een about 4% and about 8% by weight.

42. The spray -dried formulation of any one of claims 23-38 or 41, wherein the amount of DDM by weight is between about 5% and about 7% by weight.

43. The spray-dried formulation of any one of claims 23-38 or 41 -42, w herein the amount of DDM by weight is about 6.1% by weight.

44. A spray -dried formulation comprising Compound 24, HPMC, and DDM wherein the Compound 24 is amorphous.

45. The spray-dried formulation of claim 23 or 44 in amorphous form.

46. The spray-dried formulation of claims 44 or 45, wherein the amount of Compound 24 is betw een about 60% and about 85% by weight, the amount of HPMC is between about 10% and about 30% by weight, and the amount of DDM is bet een about 2% and about 10% by weight.Docket No. 1165.071126 WOO 1 47. The spray -dried formulation of claim 46, wherein the amount of Compound 24 is between about 68% and about 76% by weight, the amount of HPMC is between about 17% and about 25% by weight, and the amount of DDM is between about 4% and about 8% by¬ weight.

48. The spray-dried formulation of claim 47. wherein the amount of Compound 24 is betw een about 71% and about 74% by weight, die amount of HPMC is between about 20% and about 23% by weight, and the amount of DDM is between about 5.5% and 6,5% by weight.

49. ’The spray-dried formulation of claim 47, wherein the amount of Compound 24 is between about 72% and about 73% by weight, the amount of HPMC is between about 21% and about 22% by weight, and the amount of DDM is between about 5.5% and 6.5% by weight.

50. The spray -dried formulation of claim 47, wherein the amount of Compound 24 is about 72.5% by weight, the amount of HPMC is about 21.4% by weight, and the amount of DDM is about 6.1% by weight.

51. The spray -dried formulations of any one of claims 44 to 50, having an x-ray powder diffraction pattern substantially the same as that of the bottom x-ray powder diffraction pattern of FIG. 25 or any x-ray powder diffraction pattern in FIG. 31.

52. The spray-dried formulations of any one of claims 44 to 51, having a DSC thermogram substantially the same as that of any one of the DSC thermograms of FIG. 33.

53. The spray -dried formulations of any one of claims 44-52. having a water content of less than 3% by weight.

54. The spray-dried formulations of any one of claims 44-53, having a water content of betw een about 1% and about 2% by weight.

55. The spray-dried formulations of any one of claims 44-54. having a glass transition temperature of between about 46.9°C and about 49.0°C.

56. The spray-dried formulations of any one of claims 44-55, having a glass transition temperature of about 47.4°C.

57. A solution comprising Compound 24 and one or more spray -drying excipients.

58. The solution of claim 57. wherein one spra -drying excipient is DDM.

59. The solution of claims 57 or 58 further comprising spray -drying excipient HPMC.Docket No. 1165.071126WO01 60. The solution of any one of claims 57-59 further comprising water.

61. The solution of claim 60. wherein the water is deionized water.

62. A process of preparing a spray-dried formulation comprising Compound 24 and one or more spray -drying excipients comprising spray -drying a solution of any one of claims 53-61 to form a spray -dried composition.

63. Spray -dried formulations comprising Compound 24 prepared by the process of claim 62.

64. A method of treating an influenza infection comprising the step of administering to a patient in need a spray -dried formulation of any one of claims 10 to 56 to the patient in need thereof.

65. Use of a dry' pow der formulation in the manufacture of a medicament for treating an influenza infection, the composition comprising (i) a therapeutically effective amount of Compound 24 and (ii) one or more spray-diving excipients.

66. A pharmaceutical composition comprising the spray -dried formulations of any one of claims 10 to 56.

67. The spray-dried formulation of any one of claims 10 to, having a water content of less than 5% by weight.

68. The spray-dried formulation of any one of claims 10 to 56. having a water content of between about 0.5% and about 3% by weight.

69. The spra -dried formulation of any one of claims 10 to 56, having a water content of between about 1% and about 2% by weiglit.

70. The spray-dried formulation of any one of claims 10 to 56, having a water content of between about 1.4% and about 2.4% by weight.

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