New formulation of antiviral peptide conjugates

Abstract

The application provides a pharmaceutical composition, which comprises an antiviral peptide conjugate, for the treatment or prevention of a condition or disease associated with a coronaviral infection. The coronavirus includes human coronavirus OC43, human coronavirus HKU1, human coronavirus 229E, human coronavirus NL63, Middle East respiratory syndrome-related coronavirus (MERS-CoV), SARS-CoV, SARS-CoV-2, or any variant thereof. The pharmaceutical composition is designed for, inter alia, intranasal administration.

Description

[0001] Docket No. : 4314.3006 WO

[0002] NEW FORMULATION OF ANTIVIRAL PEPTIDE CONJUGATES

[0003] RELATED APPLICATION

[0004] This application claims the benefit of U.S. Provisional Application No. 63 / 640,329, filed on April 30, 2024. The entire teachings of the above application are incorporated herein by reference.

[0005] BACKGROUND OF THE INVENTION

[0006] Coronaviruses constitute a viral family responsible for a range of illnesses, including the common cold, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and CO VID-19. The global impact of the CO VID-19 pandemic, triggered by the SARS-CoV-2 virus, has been profound, affecting millions worldwide. In response, the medical community has been tirelessly working to develop treatments and vaccines to combat this virus.

[0007] A key driver of the global pandemic and the persistent influence of SARS-CoV-2 on our daily lives is its exceptional transmissibility. The virus primarily spreads through respiratory droplets released during activities such as talking, coughing, sneezing, or even breathing. Laden with viral particles, these droplets can be inhaled by individuals in close proximity, typically within a range of about six feet (two meters). Additionally, SARS-CoV-2 can spread through aerosols — smaller respiratory particles capable of lingering in the air for extended periods. In indoor settings with poor ventilation, these aerosols pose an increased risk, potentially reaching individuals further away from the infected person. Furthermore, respiratory viruses, including SARS-CoV-2, can undergo rapid mutation, leading to the emergence of new strains that may evade existing treatments and vaccines. Consequently, there is an urgent need to develop new therapeutic strategies that can adapt to the evolving nature of respiratory viruses, including not only SARS-CoV-2 but also other viruses like RSV and emerging zoonotic viruses such as SARS-CoV, MERS-CoV, and SARS-CoV-2.

[0008] Despite the various therapeutic and preventive approaches targeting SARS-CoV-2, nasal administration has seen limited use globally and has not been widely employed in the U.S. However, when compared to alternative approaches, nasal administration stands out as a non-invasive and easily administered method. It offers rapid absorption, reduces pain and discomfort, and improves overall compliance. Notably, nasal administration holds significant potential for self-administration, presenting exciting new opportunities for the prevention and treatment of coronaviruses. SUMMARY OF THE INVENTION

[0009] The present invention is based on the discovery of a novel formulation for pancoronavirus peptide conjugates, which exhibits significantly enhanced storage stability and additional enhanced pharmaceutical properties for effective delivery, preferably for intranasal administration. Also provided is a method to treat, prevent, or reduce an infection associated with a coronavirus, such as a SARS-Cov-2 variant. The method comprises intranasally administrating the formulation to a subject in need, preferably via an intranasal spray, an inhaler, or a nebulizer. Preferably, the formulation of the invention is in liquid form.

[0010] BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.

[0012] Figure 1. pH effects on stability for DCOY101, DCOY102, and DCOY103 (from left to right) with pH ranging from 2.0 - 8.0 analyzed for A pH (top row), A concentration (middle row), and A% purity (bottom row) at t = 0, 1, 4, & 7 days.

[0013] Figure 2. Purity of DCOY101 (F1-F5), DCOY102 (F7, F9), and DCOY103 (F12) isotonic formulations at t = 0, 3, & 7 days.

[0014] Figure 3. Purity change of DCOY102 in D-mannitol only (blue) and 1 :3 NaCl:D- mannitol isotonic formulations at timepoints t = 0, 3, 7, 14, 21, and 28 days. All data points were normalized to t = 0.

[0015] Figure 4. Purity change of DCOY102 with 0.01% w / v and 0.10% w / v benzalkonium chloride, with 0.01% v / v and 0.10% v / v phenethyl alcohol, and without preservatives at t = 0, 2, & 7 days at room temperature. All samples were normalized using initial purity of control sample of 95.2% (see F7 t = 0 purity from Table 5).

[0016] Figure 5. Purity change of DCOY102 with 0.01% w / v and 0.30% w / v methylparaben, with 0.01% w / v propylparaben, and without preservatives at t = 0, 2 or 4, & 7 days at room temperature. All samples were normalized using initial purity of control sample of 95.2% (see F7 t = 0 purity from Table 5).

[0017] Figure 6. Total impurities (%) of DCOY101 (left), DCOY102 (middle), and DCOY103 (right) stressed with 0. IM and 1 ,0M HC1 at t = 0, 1 or 2, & 7 days at room temperature. Figure 7. Total impurities (%) of DCOY101 (left), DCOY102 (middle), and

[0018] DCOY103 right) stressed with 0. IM and 1 ,0M NaOH at t = 0, 1 or 2, & 7 days at room temperature.

[0019] Figure 8. Total impurities (%) of DCOY101 left), DCOY102 (middle), and DCOY103 (right) stressed with 3.0% H2O2 at t = 0, 1 or 2, & 7 days at room temperature.

[0020] Figure 9. Optical density measurements at 600 nm for formulation Fl -Fl 2 with pH ranging from 2.0 - 8.0 analyzed at t = 0, 1, 4, & 7 days. Formulations at pH 2.0 showed cloudy solutions: F2 (DCOY102, blue), F6 (DCOY103, brown), and F10 (DCOY101, green) while the other formulations remained optically clear.

[0021] Figure 10. Images of DCOY101, DCOY102, and DCOY103 in isotonic solutions at timepoint t = 0.

[0022] DETAILED DESCRIPTION OF THE INVENTION

[0023] Formulation of the Invention

[0024] Coronaviruses, particularly the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for COVID-19, have had a significant impact on human life globally. Other human-infecting coronaviruses encompass a range of strains, including human coronavirus OC43 (HCoV-OC43), human coronavirus HKU1 (HCoV-HKUl), human coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome- related coronavirus (MERS-CoV). The dynamic nature of coronaviruses is evident through their continual evolution, giving rise to new variants that swiftly outpace established therapies and pose ongoing risks to human health. The global pandemic caused by SARS- CoV-2 has led to changes in healthcare systems, increased emphasis on vaccine distribution and preparedness, and ongoing efforts to address mental health challenges and other consequences. However, among the available therapeutic or prophylactic approaches (e.g., intramuscular vaccination), intranasal administration, though being the least intrusive and the easiest to administer, has not been approved for treatment of coronaviral infection in the United States.

[0025] This invention discovers a novel platform formulation for delivery of anti-coronaviral peptide conjugates, preferably via intranasal or pulmonary administration. As used herein, the terms “formulation”, “pharmaceutical composition”, “composition”, and “pharmaceutical formulation” all refer to the formulation of the invention and thus are used interchangeably. In other preferred embodiments, the formulation of the invention is used for oral inhalation for delivery to a subject in need. In yet additional preferred embodiments, the formulation of the invention is used for pulmonary delivery to a subject in need.

[0026] Intranasal administration includes but is not limited to nasal spray, nasal drops, nasal gels, nasal powders, nasal aerosols, nasal pumps, nasal nebulizers, and nasal inhalers. Preferably the intranasal administration of this invention is achieved using an intranasal spray, an inhaler, or a nebulizer. In some embodiments, the formulation can be designed for drug delivery using an inhaler, which results in the absorption of peptide conjugates into the bloodstream via the lung mucous membrane. An inhaler includes all available types of inhalers on the market, such as metered-dose inhalers (MDI), dry powder inhalers (DPI), soft mist inhalers (SMI) and nebulizers (e.g., a jet nebulizer, an ultrasonic wave nebulizer). Where delivery to the pulmonary system, or lungs, is desired, it can be efficacious to aerosolize a low concentration solution of the active agent for an extended period, such as overnight.

[0027] In some preferred embodiments, the formulation of the invention is designed to be compatible with intranasal administration, oral inhalation administration, and pulmonary delivery. In some cases, more than one route of administration are combined for the delivery of the formulation of the invention to a subject in need to achieve enhanced therapeutic effectiveness, such as combining a conventional nebulizer and an oral inhaler, a conventional nebulizer and a nasal spray.

[0028] To achieve a desirable formulation for intranasal administration, a suitable solvent that provides sufficient solubility and storage stability for the ani-coronaviral peptide conjugate must be identified.

[0029] A solvent of the invention is a non-aqueous solvent, including but not limited to ethanol, glycerol, polyethylene glycol, propanediol (propane- 1,3 -diol), butylene glycol (butane- 1,3 -diol), butane-2,3,-diol, butane- 1,2-diol, triacetin, dimethyl sulfoxide, isopropyl alcohol, propylene glycol, propylene glycol derivatives, vegetable oils, and any combination thereof. Preferably, the solvent is ethanol, glycerol, polyethylene glycol, propanediol (propane-1, 3-diol), butylene glycol (butane- 1,3 -diol), butane-2,3,-diol, butane- 1,2-diol, isopropyl alcohol, propylene glycol, propylene glycol derivative, or any combination thereof.

[0030] As a non-limiting example, propylene glycol derivatives include propylene glycol phenyl ether, propylene glycol diacetate, dipropylene glycol butyl ether, tripropylene glycol butyl ether, propylene glycol methyl ether, dipropylene glycol ethyl ether, tripropylene glycol methyl ether, propylene glycol butyl ether, isostearyl isononanoate, diisobutyl adipate, vinyl chloride copolymer, 1,2-cyclohexane dicarboxylic acid diisononyl ester, and mixtures thereof.

[0031] Preferably, the solvent is propylene glycol.

[0032] In its simplest form, the formulation comprises an anti-coronaviral peptide conjugate dissolved in a mixed aqueous and organic solution (preferably water: propylene glycol). The peptide conjugate of the invention is as defined herein. The formulation is used for storing, stabilizing, and / or transporting the anti-coronaviral peptide conjugate. The formulation can be directly used for administration without reconstitution. In other embodiments, the formulation comprises an anti-coronaviral peptide conjugate dissolved in a non-aqueous solvent as described herein; preferably, an anti-coronaviral peptide conjugate dissolved in propylene glycol. The formulation is preferably used for storing, stabilizing, and / or transporting the anti-coronaviral peptide conjugate and will be preferably reconstituted or diluted with an aqueous solvent before administering to a subject in need.

[0033] In some embodiments, the formulation is capable of storing the peptide conjugate for about 36 months, 24 months, 12 months, 6 months, 5 months, 4 months, 3 months, 2 months, or 1 month. In some embodiments, the formulation is capable of storing the peptide conjugate for about 90 days, 60 days, 45 days, 30 days, 20 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day. In some embodiments, the formulation is capable of storing the peptide conjugate for about 72 hours, 56 hours, 48 hours, 36 hours, 24 hours, 12 hours, or 6 hours. During the storage period, the peptide conjugate preferably does not undergo any substantial degradation.

[0034] In some embodiments, the formulation further optionally comprises a preservative. The preservative is preferably benzalkonium chloride, phenethyl alcohol, methylparaben, or propylparaben. In some embodiments, the preserve is phenethyl alcohol. In some embodiments, the preserve is phenethyl alcohol at a concentration of <0.1% v / v. In some embodiments, the preserve is methylparaben. In some embodiments, the preserve is methylparaben at a concentration of <0.3% w / v. In some embodiments, the preserve is propylparaben. In some embodiments, the preserve is propylparaben at a concentration of <0.01% w / v.

[0035] In preferred embodiments, the formulation is used without further reconstitution or dilution. The vehicle solution is a mixed aqueous and organic solution. In additional embodiments, the formulation comprises the peptide conjugate dissolved in an organic solvent, such as propylene glycol, before administering to a subject in need, the formulation is reconstituted or diluted with a second solvent. The second solvent is preferably an aqueous solvent. Aqueous solvents include, for example, water and isotonic saline. Preferably, the aqueous solvent includes, without limitation, water, saline solution (NaCl, 0.9%), diluted saline solution such as half saline solution (NaCl, 0.45%), tris-buffered saline (TBS), phosphate-buffered saline (PBS), or any combination thereof. Preferably, the aqueous solution is saline solution (NaCl, 0.9%), PBS, or a mixture of both. Preferably, the aqueous solution is saline solution (NaCl, 0.9%). Preferably, the aqueous solution is PBS.

[0036] The non-aqueous solvent as described herein and the aqueous solvent have a ratio ranging from 100: 1 to 1 : 100. The ratio of the non-aqueous solvent to the aqueous solvent is about 5: 100, about 10: 100, about 15: 100, about 20: 100, about 25: 100, about 30: 100, about 35: 100, about 40: 100. Or the ratio of the non-aqueous solvent to the aqueous solvent is about 11 : 100, 12: 100, 13: 100. 14: 100, 15:100, 16: 100, 17:100, 18: 100, 19: 100, 20: 100, 21 : 100, 22: 100, 23: 100, 24: 100, 25: 100, 26: 100, 27: 100, 28:100, or 29: 100.

[0037] In some embodiments, the formulation comprises an anti-coronaviral peptide conjugate, a non-aqueous solvent, and an aqueous solvent. The peptide conjugate is dissolved in the non-aqueous solvent and later reconstituted or diluted with the aqueous solvent. In some embodiments, the formulation comprises an anti-coronaviral peptide conjugate, propylene glycol, and an aqueous solvent selected from saline solution (NaCl, 0.9%), PBS, and a mixture of both. In some embodiments, the formulation comprises an anti-coronaviral peptide conjugate, propylene glycol, and PBS.

[0038] The concentration of the peptide conjugate is about Img / mL to about 50mg / mL. In some embodiments, the concentration of the peptide conjugate is about 5mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, or 20 mg / mL. Preferably, the concentration of the peptide conjugate is about 10 mg / mL.

[0039] The formulation further optionally comprises a surfactant. The surfactant is preferably a non-ionic surfactant to stabilize drug formulations, particularly in biopharmaceuticals and injectable medications. The surfactant for example includes Polysorbate-20 (PS-20), Polysorbate-80 (PS-80), Tween Series such as Tween 20 and Tween 80, sorbitan esters such as Span 20 and Span 80, Cremophor EL, Pluronic block copolymers such as Pol oxamer 188 and Pol oxamer 407, Brij Series such as Brij 35 and Brij 58, are polyoxyethylene alkyl ethers used in pharmaceuticals to enhance the stability of emulsions and suspensions. PEG (Polyethylene Glycol) such as PEG derivatives PEG-400 and PEG-600. Preferably, the surfactant is PS-20. The formulation further optionally comprises a mucoadhesive. Exemplary mucoadhesive include synthetic or biological polymers; and lipids. Examples of natural and / or synthetic polymers include: cellulosic derivatives (such as methylcellulose, carboxymethyl cellulose, hydroxyethyl cellulose, and hydroxyethylmethyl cellulose); natural gums (such as guar gum, xanthan gum, locust bean gum, karaya gum, veegum); polyacrylates (such as carbopol and polycarbophil); alginates, polyoxyethylenes, polyethylene glycols (PEG) of all molecular weights (preferably between 1000 and 40,000 Da, of any chemistry, linear or branched); dextrans of all molecular weights (preferably between 1000 and 40,000 Da of any source); block copolymers, such as those prepared by combinations of lactic & glycolic acid (PLA, PGA, PLGA of various viscosities, molecular weights and lactic-to- glycolic acid ratios); polyethylene glycol-polypropylene glycol block copolymers of any number and combination of repeating units (such as Pluronics, Tektronix or Genapol block copolymers); and a combination of the above copolymers with units either physically or chemically linked (for example PEG-PLA or PEG-PLGA copolymers). A composition for nasal transmucosal delivery may contain one or more different mucoadhesives in any combination. Preferably, the mucoadhesive is carboxymethyl cellulose sodium (CMC -Na) and / or hydroxypropyl methylcellulose (HPMC). Preferably, the mucoadhesive is CMC -Na at a concentration of <0.1% w / v in the formulation. Preferably, the mucoadhesive is HPMC at a concentration of <0.1% w / v in the formulation. In some preferred embodiments, a mucoadhesive is selected from CMC -Na, HPMC, polyacrylamide, Carbopol, PVP, or any combination thereof. In preferred embodiments, a mucoadhesive is CMC -Na. In other preferred embodiments, a mucoadhesive is a mixture of HPMC, polyacrylamide, and Carbopol. In yet other preferred embodiments, a mucoadhesive is a mixture of HPMC, polyacrylamide, and PVP.

[0040] The formulation has a pH in the range of 4.0 to 8.0, preferably 5.0-7.0. In some cases, the pH of the formulation is about 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or 6.9. In some embodiments, the pH of the formulation is about 6.0.

[0041] Anti-Coronaviral Peptide Conjugate

[0042] The anti-coronaviral peptide conjugate of the invention is represented by: (Peptide-Linker)n-B-Hydrophobic Moiety, wherein the Linker is independently optional and independently a bivalent linking moiety, each Peptide is independently a short amino acid sequence, a therapeutic peptide, such as a HRC peptide derived from a coronavirus spike protein and / or target protein, B is a multimeric core which covalently links each peptide moiety to the hydrophobic moiety, and n is an integer selected from 1, 2, 3 or more. Hydrophobic moiety has a function of anchoring to the cell membrane and can be for example a membrane integrating lipid.

[0043] As used here, the term “peptide conjugate” specifically refers to the anti-coronaviral peptide conjugate, and in some embodiments, they are used interchangeably.

[0044] In some cases, the peptide conjugate can be designed to serve as a pan-inhibitor, capable of targeting multiple related viruses or proteins across the coronavirus family. For example, the conjugate may incorporate peptides that collectively inhibit a broad spectrum of coronaviruses, including SARS-CoV, MERS-CoV, SARS-CoV-2, and other emerging strains, by engaging conserved regions critical for viral entry or replication — preferably conserved domains within the spike protein, such as the HR1 and HR2 regions.

[0045] In some cases, the peptide conjugate is engineered to function as a selective inhibitor, wherein the conjugated peptides are specifically tailored to block a particular coronavirus with high specificity. Such selective conjugates may enhance antiviral potency, improve pharmacokinetic properties, extend serum half-life, promote multivalent binding to viral targets, or enable targeted delivery to specific tissues or cell types.

[0046] In some cases, the peptide conjugate comprises peptides that act as pan-coronavirus inhibitors, capable of neutralizing SARS-CoV, MERS-CoV, SARS-CoV-2, and related coronaviruses by interfering with conserved regions of the viral spike protein, preferably the HR1 / HR2 domains involved in membrane fusion.

[0047] In some cases, the peptide conjugate comprises selective inhibitors targeting the same virus, such as peptides that are individually selective for SARS-CoV, MERS-CoV, or SARS- CoV-2. The use of highly selective peptides may reduce off-target effects and maximize therapeutic precision.

[0048] In some cases, while the individual peptides of the invention are selective for specific coronaviruses, a peptide conjugate comprising two or more different selective peptides can function collectively as a pan-inhibitor. For example, a conjugate combining a MERS-CoV- selective peptide with a SARS-CoV-selective peptide and a SARS-CoV-2-selective peptide may exhibit broad-spectrum inhibitory activity across the coronavirus family.

[0049] In some cases, the peptide conjugate comprises one or more peptides that are selectively engineered to inhibit SARS-CoV-2, the causative agent of COVID-19. These peptides may target specific structural features of the SARS-CoV-2 spike protein, such as the receptor-binding domain (RBD) or the highly conserved HR1 and HR2 regions within the S2 subunit, to block viral entry and membrane fusion. The peptide conjugate may enhance antiviral efficacy through multivalent binding, improved stability, and extended half-life, offering a targeted therapeutic strategy against SARS-CoV-2 infection.

[0050] Peptide

[0051] Coronaviruses target human cells via the spike protein binding domain (RBD) attaching to the human angiotensin converting enzyme 2 (hACE2) receptor on host cells. The coronavirus spike (S) glycoprotein is a class I viral fusion protein on the outer envelope of the virion that plays a critical role in viral infection by recognizing host cell receptors and mediating fusion of the viral and cellular membranes. Coronavirus entry into host cells is mediated by the transmembrane spike (S) glycoprotein that forms homotrimers protruding from the viral surface. S comprises two functional subunits responsible for binding to the host cell receptor (SI subunit) and fusion of the viral and cellular membranes (S2 subunit).

[0052] SI serves the function of receptor-binding and contains a signal peptide (SP) at the N terminus, an N-terminal domain (NTD), and receptor-binding domain (RBD). S2 functions in membrane fusion to facilitate cell entry, and it contains a fusion peptide (FP) domain, internal fusion peptide (IFP), two heptad-repeat domains (HR1 and HR2), transmembrane domain, and a C-terminal domain.

[0053] After binding, the spike protein is activated by the host cell transmembrane protease / serine subfamily member 2 (TMPRSS2) and consequently the virus undergoes fusion with the endosomal membrane for entry into the cell. The membrane fusion domain of the spike protein in coronaviruses is highly conserved so targeting membrane fusion may result in durable long-lasting therapeutics. The mechanisms of the viral entry have been illustrated in Jackson et al, “Mechanisms of SARS-CoV-2 entry into cells.” Nat Rev Mol Cell Biol 23, 3-20 (2022). https: / / doi.org / 10.1038 / s41580-021-00418-x.

[0054] Each Peptide is independently a short amino acid sequence, including therapeutic peptides for coronavirus or peptide inhibitors inhibiting coronavirus (such as peptide inhibitors inhibiting the fusion process). In some embodiments, each Peptide independently has a length ranging from 5 to 100 amino acid residues, from 6 to 80 amino acid residues, from 8 to 60 amino acid residues, or from 10 to 50 amino acid residues. In preferred embodiments, each Peptide independently has a length of 12-40 amino acid residues. In preferred embodiments, each Peptide independently has a length of 18-39 amino acid residues.

[0055] In some embodiments, each Peptide is independently derived from a coronavirus spike fusion protein and / or target protein, preferably from C-terminus heptad repeat (HRC) region of a spike fusion protein. The HRC region of the coronavirus spike fusion protein is involved in the formation of the six-helix bundle structure that drives viral fusion. HRC peptides mimic the HRC region and can bind to the N-terminal heptad repeat (HRN) region of the fusion protein, blocking the formation of the six-helix bundle and preventing viral fusion and entry into host cells. In some embodiments, a HRC peptide is a wild type peptide derived from the HRC region of a coronavirus spike fusion protein. In other embodiments, an HRC peptide is a mutant or variant peptide derived from the HRC region of a coronavirus spike fusion protein, which can comprise genetic mutations that occur in nature, and / or modifications made in a laboratory setting.

[0056] In some embodiments, each peptide is independently a HRC peptide derived from a coronavirus spike protein and / or a targeting peptide, provided that there is at least one HRC peptide derived from a coronavirus spike protein. While preferred embodiments of the invention utilize native or wild-type peptides, non-natural peptides can be used as well. For example, amino acids found in one or more mutations (e.g., omicron mutations) can be combined with the native sequences of other viruses (e.g., the delta virus). The so-called HRC peptide or region of the coronavirus spike protein is preferred. The HRC peptides inhibit viral fusion, an important early step in the infection process. The wild type HRC peptide is a conserved region of the spike, or S, protein across coronaviruses. The conserved nature of the fusion regions (HRC / HRN) and mechanism of the class I enveloped viruses make it an ideal target to develop a pan-coronavirus inhibitor.

[0057] Preferred wild type HRC peptides comprise the sequence and binding fragments thereof:

[0058] Acn-DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL (SEQ ID NO. 1), wherein n is 0 or 1.

[0059] With regard to the HRC peptide in SEQ ID NO.1, the conventional numbering of the amino acids begins with 1168 at D. 11691, 11721 and 1176V are in the hydrophobic interface pre-fusion and 1177V is exposed. These amino acids stabilize a helix. When the conformation change occurs (e.g., protease clipping to release FP), 1176V is exposed and 1177 V presents in the hydrophobic surface interacting with HRN trimer. 1173N, and 1194N are implicated in N-linked glycosylation conserved in coronavirus. The first 7 amino acids are implicated in HRN binding. The “N-Cap” region spans 1177V and 1189V. The 1189V is a conserved hydrophobe in coronaviruses and stabilizes the HRC hydro-core and is involved in the HRN interaction. The hydrophobic core spans 11791 and 1193L and is helical pre- and post-fusion. The isoleucines, leucines and alanine are important in folding and stability of a coiled coil. The C-Cap region spans 1194N and 1203L. 1197L, 1200L and 1203L are in the hydrophobic interface pre-fusion and 11801 is exposed. These amino acids stabilize the helix. 1203L may be implicated in hydrophobic packing between three polypeptide chains in a trimeric coiled coil. When confirmation change occurs (protease clipping to release FP), 1197L is exposed and 11981 is in the hydrophobic surface interacting with a HRN trimer. 1182E and 1202E form a salt bridge between HRC and HRN. Further, 1177V, 1178N, 1189V and 11981 have been shown to interact with HRN in crystal structures.

[0060] In some embodiments, the peptides are selected from variants of a wild type HRC peptides comprising the sequence and binding fragments thereof:

[0061] Acn-DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL (SEQ ID NO. 1), wherein n is 0 or 1.

[0062] In the context of protein variants, the term “variant” is defined as a peptide which has at least one amino acid deleted, added, or substituted in comparison with a wild type sequence, such as SEQ ID NO. 1 or other native sequence described herein. Variants preferably bind the cognate ligand of the wild type sequence. For example, a peptide wherein 1, 2, 3, 4 or 5 amino acids of SEQ ID NO. 1 are substituted can be used. Such substituted amino acids can preferably be selected from one or more corresponding amino acids identified in a different coronavirus strain via a sequence alignment, such as shown above. For example, one or both underlined isoleucines can be substituted with leucine and / or methionine, as described in the alignment provided above. The underlined alanine can be substituted by valine, leucine or isoleucine. One or both underlined leucines can be independently substituted by isoleucine, tyrosine, alanine or valine. Other conservative or nonconservative substitutions, (lysine and glutamine or aspartic acid and glutamic acid) can be selected as well. In some embodiments, amino acids that are conserved amongst 2, 3, 4, 5 or more coronavirus (e.g., coronavirus isolated from bats or SARS-CoV2 mutants or variants) remain conserved in the non-natural HRC peptide.

[0063] For example, the wild type HRC sequence can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more additional amino acids native to the S protein at the N- and / or C termini. For example, glycine can be added to the N-terminus. Additionally, the wild type HRC peptide fragment can delete 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the N and / or C termini and inhibit infection. Typically, not more than ten total amino acids are deleted in total. For example, the 10 amino acids at the C terminus can be deleted and be expected to retain inhibitory activity.

[0064] In some embodiments, modifications to wild type sequences are desirable. For example, using one or more D amino acids can improve pharmacokinetics and the half-life of the peptide. Thus, in some embodiments, the invention includes peptides characterized by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more D-amino acids. The D-amino acids can preferably be a corresponding L-amino acid of the wild type sequence. In some embodiments, the D-amino acid is an amino acid located at or near (e.g., within 1, 2, or 3 amino acids) a protease degradation site. In some embodiments, the D-amino acid is a hydrophobic amino acid participating in binding with the HRN peptide and preferably at a higher affinity than the corresponding wild type sequence. Alternatively, or additionally, the D-amino acid is a hydrophilic amino acid, such as lysine, aspartic acid, glutamic acid or arginine. Alternatively, or additionally, the D-amino acid can be selected from the 7 amino acids at the N-terminus of SEQ ID NO: 1. Peptides that have been improved by incorporating D-amino acids are described in USSN 63 / 140,387, filed on January 22, 2021, which is incorporated by reference in its entirety.

[0065] However, swapping one or more D-amino acids for the corresponding L-amino acid can change the topology of the peptide and impact function. Therefore, a preferred nonnatural HRC peptide is a Retro-Inversion HRC peptide, or “RI HRC peptide”. Retro- Inversion HRC peptides are preferably characterized by a binding affinity of at least about 50% of the wild-type HRC peptide with its cognate ligand in a standard binding assay and decreased susceptibility to mammalian protease degradation. Retroinversion is defined as reversing a D-peptide sequence of a helical peptide or “flipping” the termini thereby restoring the presentation of the side chains to the binding ligand or target. See Kim et al, Method to generate highly stable D-amino acid analogs of bioactive helical peptides using a mirror image of the entire PDB, PNAS, February 13, 2018, 115 (7) 1505-1510, which is incorporated herein by reference in its entirety. Therefore, a non-natural peptide of the invention can include a peptide having the sequence of SEQ ID NO.: 1 wherein amino acids are D-amino acids, such as the amino acids within a region, flipping the N-terminus for a C terminus. For example, the N termini can be subjected to retroinversion as shown in SEQ ID NO. 2 where each D amino acid is preceded by a “d”: dldGdSdldD NASVVNIQKEIDRLNEVAKNLNESLIDLQEL (SEQ ID NO. 2)

[0066] This example offers a single RI region of 5 amino acids. However, as few as two amino acids can be selected (e.g., the 2 N-terminal amino acids). For example, the RI region can span the hydrophobic core, 1160N to 1176N, or the C-cap region or a portion thereof. Alternatively, the entire peptide can be an RI peptide. Additionally, two, three or more RI regions can be included. For example, both the N-terminus and C-Cap region can be RI regions, retaining the hydrophobic core with L-amino acids.

[0067] For example, in using mirror-image phage display to screen for HRC variants, a first D-peptide can be synthesized from a HRN coronavirus peptide, or first L-peptide. The first L- peptide can be a naturally occurring L-peptide or can be a chimera of a peptide. The methods can further comprise screening for a HRC peptide, or second L-peptide, that specifically binds to the first D-peptide; then, a second D-peptide that is the mirror image of the second L-peptide can be synthesized. In one aspect of the D-peptide screening methods described herein, an N-trimer target can first be synthesized with D-amino acids, creating the mirror image of the natural L-N-trimer target. The D-N-trimer target can be used in standard peptide-based screens such as phage display, ribosome display, and / or CIS display to identify L-peptides that bind to the D-N-trimer. The identified L-peptides can then be synthesized with D-amino acids. By the law of symmetry, the resulting D-peptides bind the natural L-N- trimer and will thus target the N-trimer region of the coronavirus HRN intermediate, thereby inhibiting infection. This screening method is also described in Schumacher, et al., Identification of D-peptide ligands through mirror-image phage display, Science, 1996 Mar 29; 271(5257): 1854-7, which is hereby incorporated in its entirety by this reference.

[0068] The hotspot residues of the HRC peptide can be identified by crystal structure or NMR solution structure of the HRC peptide. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids selected from 1150D, 11511, 11541, 1155N, 1158V, 1159V, 11611, 1164E, 1171V, 1175L, 1176L, 1179L, 11801, 1182L, 1184E and / or 1185 Y, such as one or more amino acids selected from 1159V, 1160N, 1163E, 1171V, 11801, 1184E and / or 1185L of SEQ ID NO. 1 can be designated hotspot residues.

[0069] In some embodiments, the Peptide comprises a cell targeting peptide and at least one HRC peptide. In some embodiments, the targeting peptide is selected from but not limited to a receptor binding domain targeting peptide, an ACE2 targeting peptide. In some embodiments, the targeting peptide is an ACE2 targeting peptide.

[0070] In some embodiments where the peptide conjugate comprises only one Peptide, the peptide is HRC peptide derived from a coronavirus spike protein. In some embodiments where the peptide conjugate comprises 2 or more Peptides, each peptide can be a HRC peptide derived from a coronavirus spike protein. In some embodiments where the peptide conjugate comprises 2 or more Peptides, the Peptide can be selected from a HRC peptide derived from a coronavirus spike protein and / or a targeting peptide, provided that there is at least one HRC peptide derived from a coronavirus spike protein. Preferably, only one of the Peptides is a targeting peptide and the other peptide(s) are a HRC peptide derived from a coronavirus spike protein.

[0071] In some embodiments, the peptide conjugate of the invention is synthesized using click chemistry, and the Peptide comprises modified peptides such as modified HRC peptides or modified ACE2 targeting peptides, for example, proteins comprising a C-terminal or an N- terminal click chemistry handle. Such Peptides can then be covalently conjugated to B comprising a moiety that can react with the click chemistry handle of the Peptides. The term “chemistry handle” is defined as herein.

[0072] Linker

[0073] Each Linker is independently optional. Each Linker is independently a bivalent moiety that covalently binds to a Peptide and to B. Each Linker independently comprises ester, amide, disulfide, thiol, peptide, or polymeric moiety (such as polyethylene glycol (PEG)).

[0074] Each Linker can independently have 1, 2, 3, 4, 5 or more subunits or segments. In some embodiments, each Linker independently comprises a subunit with one or more amino acids. The amino acids may be naturally occurring or synthetic. Thus, the Linker may comprise (Gly)n+i, (GlySerGly)n or (Gly-Pro)n where n is 1 or greater, for example, 1 to 12, 1 to 6 or 1 to 4. GlySerGly is one example of a sequence of amino acids which may form the Linker or part of the Linker.

[0075] In some embodiments, the Linker may comprise a non-amino acid subunit. In some embodiments, examples of the non-amino acid subunit of the linker are -(OC LC Ljm- where m is from 1 to 15, for example 2 to 10, 2 to 6 or 4. Introduction of a (poly)ethyleneglycol group assists solubility in aqueous media. In some embodiments, examples of the non-amino acid portion of the linker are -CH2C(O)- and -CH2C(O)NHCH2CH2(OCH2CH2)4C(O)-.

[0076] For example, it can be advantageous to use a Linker with 3 subunits. A first optional subunit which comprises a flexible peptide, such as -(G)m- or -(GS)mG-, where m is an integer of 1, 2, 3, 4, 5 or more, such as 2. A second subunit can be a residue of a chemical reaction (such as an automated flow), such as a peptide bond, ester, or ether involving the N- terminus, C-terminus or side chain of the Peptide or first subunit. The residue can be non- cleavable, such as that formed with carbodiimide or sulfhydryl maleimide. A third optional subunit can be a hydrophilic spacer, a PEG spacer, such as polyethyleneglycol, polyethyleneamine, polyacetal polymer, poly(l -hydroxymethylethylene hydroxymethyl- formal) (PHF) or a carbohydrate. The hydrophilic spacers can generally be polymeric and comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more monomers. Polyethyleneglycol with 4 monomers (PEG4) is satisfactory. When the Peptide that a linker is connected to is a HRC peptide, the length of the hydrophilic spacer can correspond to the span of the protein gap to facilitate the orientation of the HRC peptide to bind the HRN domain.

[0077] Each Linker can be independently designed to modify the activities of the peptide conjugate, such as flexible linkers to increase flexibility or rigid linkers to maintain a fixed distance. Chen et al. “Fusion Protein Linkers: Property, Design and Functionality”, Adv Drug Deliv Rev. 2013 65(10): 1357-69. Preferably, when the Peptide is a HRC peptide, the Linker is selected from a flexible linker to facilitate the orientation of the HRC peptide to bind the HRN domain.

[0078] Suitable peptide linkers include polypeptides of between about 1 amino acid and about 40 amino acids in length, or between about 3 amino acids and about 25 amino acids in length. Peptide linkers with a degree of flexibility can be used. The use of small amino acids, such as glycine (G or Gly), serine (S or Ser), and alanine (A or Ala), are of use in creating a flexible peptide. A variety of different linkers are commercially available and are considered suitable for use.

[0079] Examples of each Linker include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n, (GGSGGS)n, (GGGS)n, where n is an integer of at least one), glycine-alanine (G-A) polymers, alanine-serine (A-S) polymers. Exemplary Linker can comprise amino acid sequences including, but not limited to, GGSG, GGSGG, GSGSG, GSGGG, GGGSG, GSSSG, and the like.

[0080] B moiety

[0081] B is a multimeric core which provides a framework that covalently links the one or more Peptide-Linker) moieties to the hydrophobic moiety.

[0082] In some embodiments, the peptide conjugate of the invention is synthesized using click chemistry. Click chemistry handles are chemical moieties that provide a reactive group that can partake in a click chemistry reaction. Click chemistry reactions and suitable chemical groups for click chemistry reactions are well known to those of skill in the art, and include, but are not limited to terminal alkynes, azides, strained alkynes, dienes, dieneophiles, alkoxyamines, carbonyls, phosphines, hydrazides, thiols, and alkenes. For example, in some embodiments, an azide and an alkyne are used in a click chemistry reaction. In some embodiments where copper-catalyzed azide-alkyne cycloaddition (CuAAC) is the click-chemistry employed for functionalizing materials as disclosed herein, the “clickchemistry compatible” compounds include a terminal alkyne and / or terminal azide functional group.

[0083] An exemplary click-chemistry reaction is CuAAC, although skilled artisans will appreciate that other click-chemistry compatible reactions that would be appreciated as equivalent to CuAAC may be employed without departing from the scope of the inventive concepts described herein. For instance, in various embodiments click-chemistry compatible reactions may include CuAAC, strain-promoted azide-alkyne cycloaddition (SPAAC), strain- promoted alkyne-nitrone cycloaddition (SPANC), strained alkene reactions such as alkeneazide cycloaddition, etc. Click-chemistry compatible reactions may also be considered to include alkene-tetrazine inverse-demand Diers-Alder reactions, alkene-tetrazole photoclick reactions, Michael additions of thiols, nucleophilic substitution of thiols with amines, and certain Diels-Alder reactions, etc. such as disclosed by Becer, et al. “Click chemistry beyond metal-catalyzed cycloaddition.” Angew. Chem. Int. Ed. 2009, 48: p. 4900-4908, and equivalents thereof as would be understood by a person having ordinary skill in the art upon reading the present disclosures.

[0084] Accordingly, click-chemistry compatible groups, compounds, etc. should be understood to include one or more suitable chemical moieties conveying capability to participate in any combination of the foregoing exemplary click chemistries, in various embodiments.

[0085] In some embodiments, B comprises a moiety that is derived from a compound comprising a thiol group that facilitates the click-chemistry reactions described herein. The compound for example is cysteine. In some embodiments, B comprises one or more cysteine residue, one or more X, and optionally Y, and / or optionally Z, wherein X, Y and Z are defined herein.

[0086] The one or more cysteine residue, one or more X, optional Y, and optional Z can be in any order, wherein the component of B listed first is bound to the Peptide-Linker and the component listed last is bound to the Hydrophobic Moiety. For example, wherein B comprises, in order, one or more cysteine residue, one or more X, and Z, the Peptide-Linker is bound to the one or more cysteine and Z is bound to the Hydrophobic Moiety. In some embodiments, B comprises, in order, one or more cysteine and one or more X.

[0087] In some embodiments, B comprises cysteine and X. In some embodiments, B comprises one or more cysteine, one or more X and Z. In some embodiments, B comprises Z, one or more cysteine, and one or more X.

[0088] In some embodiments, B comprises Y, one or more cysteine, and one or more X. In some embodiments, B comprises Y, one or more cysteine, one or more X, and Z.

[0089] In some embodiments, the one or more cysteine binds the one or more Peptide-Linker moieties to the other components of B. In some embodiments, the one or more cysteine is attached to the one or more X via a thioether bond. In some embodiments, the one or more cysteines have the following structure: , wherein R4 is OH or NH2, the -S- bond is covalently linked with X, the -NH- bond is covalently linked with a Peptide-Linker directly or indirectly via one or more Y.

[0090] In some embodiments, X comprises one or more sulfur aryl linkage, nitrogen aryl linkage or other linkages such as triazoles, amides, sulfur-sp3 carbon bonds, or a hydrophilic linker. In some embodiments, hydrophilic linker is selected from such as polyethyleneglycol (PEG), polyethyleneimine, polyacetal polymer, poly(l -hydroxymethylethylene hydroxymethyl-formal) (PHF) or a carbohydrate. In some embodiments, the hydrophilic linker can be polymeric and comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more monomers. In some embodiments, the hydrophilic linker is polyethyleneglycol (PEG). In some embodiments, the hydrophilic linker is polyethyleneglycol with 4 monomers (PEG4). In one embodiment X comprises one or more sulfur aryl linkage. In one embodiment X comprises one or more nitrogen aryl linkage. In one embodiment, X comprises one or more sulfur-sp3 carbon bonds.

[0091] In some embodiments, X is represented by -RA-XI-RB-, wherein the left of RA is covalently linked to cysteine via a thioether bond and the right of RB is covalently linked to Hydrophobic Moiety directly or indirectly via one or more Z; RA is selected from a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted cycloheteroalkyl group, such as a -(CH2)I-6-, a substituted or unsubstituted phenyl, a substituted or unsubstituted 5- to 8-membered cycloalkyl, a substituted or unsubstituted 5- or 6-membered cycloheteroalkyl; Xi is -C(O)O-, -OC(O)-, - SC(O)-, -C(O)NH-, -NHC(O)-, -(O)CS-, -SONH-, -NHCONH-, -S(O)-, or -S(O)2-; RB is a substituted or unsubstituted alkyl group, a -(CH2O)i-8-, -(CH2CH2O)i-8-, -(OCH2)I-8-, or - (OCH2CH2)I-8-.

[0092] In some embodiments, the one or more X is attached via a thioether bond directly with the hydrophobic moiety. In some embodiments, the one or more X is attached to Z, when present, which is then attached to the hydrophobic moiety.

[0093] In some embodiments, B further comprises Y. In some embodiments, Y, when present, the one or more Peptide-Linker moieties to the one or more cysteine, which then binds to the other components of B.

[0094] In some embodiments, Y comprises one or more amino acids. The amino acids may be naturally occurring or synthetic. Y may comprise 1 or more amino acids, for example, 1 to 12, 1 to 6 or 1 to 4. The one or more amino acids can be added to the linker in stepwise fashion. For example, a first amino acid is added to the cysteine of B and then, prior to the addition of a second amino acid, a Peptide-Linker is attached to the first amino acid. After attachment of the Peptide-Linker to the first amino acid, a subsequent amino acid is attached to the previous amino acid and allows for the attachment of a further Peptide-Linker and so on. In some embodiments, the amino acid of the linker is one or more diamino acids, such as lysine, arginine, ornithine, diaminopimelic acid (DAP). In some embodiments, the amino acid of the linker is one or more lysine.

[0095] In some embodiments, Z, when present, comes between X and the hydrophobic moiety and binds B to the hydrophobic moiety.

[0096] In some embodiments, Z, when present, comprises a moiety having a structure according to formula (I): wherein each of Ri and R2 is independently selected from the group consisting of: (i) absent

[0097] (ii) a structure according to formula (II): wherein represents a bond covalently linked to X;

[0098] W is in each instance independently selected from -C(O)O-, -OC(O)-, -O-, C(O)-, - (CH2)m-, and -NHC(O)-, most preferably W is -C(O)NH-;

[0099] V is in each instance independently selected from -(CH2)m-, -(CH2)m- C(O)-, -C(O)- (CH2)m-, -(CH2)m-C(O)-O-, -0-C(0)-(CH2)m-, -(CH2)m-C(0)-0-, -O-C(O)-(CH2)m-, -C(O)O-, and -OC(O)-; most preferably

[0100] V is -CH2CH2-C(O)NH-; D is in each instance either -O- or -S-;

[0101] A is in each instance independently selected from -C(O)CH2-, -CH2C(O)-, -CH2-, - C(O)-, -CH2C(O)-, and -C(O)CH2-; most preferably A is -CH2-;

[0102] Q is in each instance independently selected from -CH2-, -O-, -CH2O-, and -OCH2-; most preferably Z is -O-; R3 is in each case independently selected from any of said polypeptides, which may be the same or different; m is in each instance independently selected from an integer of between 0 and 5, i.e., 0, 1, 2, 3, 4, or 5; preferably between 0 and 3, preferably m is the same in each instance; n is in each instance independently selected from an integer of between 0 and 40, i.e., 0, 1, 2, 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, or 40; preferably between 3 and 10, preferably n is the same in each instance; o is in each case independently selected from an integer of between 0 and 5, i.e., 0, 1, 2, 3, 4, or 5; preferably 1 or 2, preferably o is the same in each instance; p is in each instance independently selected from an integer of between 0 and 5, i.e., 0, 1, 2, 3, 4, or 5; preferably between 0 and 3, preferably p is the same in each instance; q is in each instance independently selected from an integer of between 0 and 5, i.e., 0, 1, 2, 3, 4, or 5; preferably between 0 and 3; preferably q is the same in each instance and / or preferably q < p;

[0103] M represents the Hydrophobic Moiety; and wherein * marks, where the structures (II-III) are linked to structure (I).

[0104] In some embodiments, Z, when present, comprises a moiety having a structure according to formula (IV): wherein Rs is selected from hydrophilic linker such as polyethyleneglycol (PEG), polyethyleneimine, polyacetal polymer, poly(l -hydroxymethylethylene hydroxymethyl- formal) (PHF) or a carbohydrate; and

[0105] W is in each instance independently selected from direct bond, hydrophilic linker is selected from such as polyethyleneglycol (PEG), polyethyleneimine, polyacetal polymer, poly(l -hydroxymethylethylene hydroxymethyl-formal) (PHF) or a carbohydrate. In some embodiments, the hydrophilic linker of Rs can be polymeric and comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more monomers. In some embodiments, the hydrophilic linker of Rs is polyethyleneglycol (PEG). In some embodiments, the hydrophilic linker of Rs is polyethyleneglycol with 4 monomers (PEG4).

[0106] In some embodiments, the hydrophilic linker of W can be at each instance independently polymeric and comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more monomers. In some embodiments, the hydrophilic linker is polyethyleneglycol (PEG). In some embodiments, the hydrophilic linker of W can be at each instance independently polyethyleneglycol with 4 monomers (PEG4).

[0107] In some embodiments, the peptide conjugates are synthesized using automated flow chemistry, preferably using an automated flow peptide synthesis (AFPS) system. An automated flow peptide synthesis (AFPS), as disclosed in U.S. Patent No. 10,683,325 B2 is a solid phase peptide synthesis system equipped with feedback control and can afford a high degree of control over individual coupling reactions for making peptides and / or minimize side reactions.

[0108] In any embodiments where the peptide-conjugates are synthesized using automated flow chemistry, B is a multivalent moiety that is designed to allow the rapid synthesis of the peptide conjugates via automated flow chemistry and can be tailored to covalently link the Peptide, the Hydrophobic Moiety, and other optional modules to improve anti-viral activities. B preferably comprises at least one diaminoaliphatic acids (such as diamino acids, lysine, arginine, ornithine, diaminopimelic acid (DAP)), optionally spacers, and optionally function groups such as amides, esters, and ethers. In some embodiments, B comprises 1, 2, 3, 4, 5, 6 or more diamino acids each independently selected from lysine, arginine, ornithine, and DAP. In some embodiments, B comprises 1, 2, 3, 4, 5, 6, or more lysines.

[0109] In some embodiments, B comprises one diamino acid selected from lysine, arginine, ornithine, and DAP; preferably one lysine. In some embodiments, B comprises two diamino acids each independently selected from lysine, arginine, ornithine, and DAP; preferably two lysines. In some embodiments, B comprises three diamino acids each independently selected from lysine, arginine, ornithine, and DAP; preferably three lysines. In some embodiments, B comprises four diamino acids each independently selected from lysine, arginine, ornithine, and DAP; preferably four lysines. In some embodiments, B comprises five diamino acids each independently selected from lysine, arginine, ornithine, and DAP; preferably five lysines. In some embodiments, B comprises six diamino acids each independently selected from lysine, arginine, ornithine, and DAP; preferably six lysines. When two or more diamino acids are present in B, the two or more diamino acids can be covalently linked to each other via amide bonds or via amino acid linkers such as GS linkers or polymeric linkers such as PEG linkers. In one preferable embodiment, B comprises two lysines covalently linked to each other via an amide bond. In additional preferable embodiments, B comprises one lysine. In yet additional embodiments, B comprises three lysines, covalently linked to each other via two amide bonds. In another embodiment, B comprises four lysines, covalently linked to each other via three amide bonds.

[0110] In some embodiments, when the diamino acid is lysine, B is represented by the Formula (Bl):

[0111] Formula (Bl), wherein represents covalent bonds linking to the moieties of the compound that comprise one or more fusion peptide inhibitors, a membrane anchoring moiety, optionally one or more spike binding peptide, and optionally one or more targeting peptide; m is an integer that can be 0, 1, 2, 3, 4, or more. Preferably, represents a covalent bond to -CO- group. Preferably, m is 0, 1, or 2.

[0112] In some embodiments, the peptide conjugate is represented by Formula (V):

[0113] Peptide - Linker Hydrophobic Moiety

[0114] Peptide - Linker

[0115] Formula (V), wherein Peptide, Linker, and Hydrophobic Moiety are as defined above, including all and preferable embodiments; m is 0, 1, 2, 3, 4, preferably, m is 0, 1, and 2; and each DA is independently selected from a diaminoaliphatic acid, preferably a diamino acid. In some embodiments, each Linker can be independently absent.

[0116] In some embodiments, each DA is independently selected from lysine, arginine, ornithine, and DAP. Preferably, each DA is lysine.

[0117] In some embodiments, B comprises one or more lysines, one or more spacers, and one or more additional function groups such as amides, esters, or ethers. The term “spacer”, as used herein, refers to a hydrophilic and biocompatible molecule or a chemical group that is inserted between two lysines, a lysine and a fusion peptide inhibitor, a lysine and a membrane anchoring moiety, a lysine and a spike binding peptide, or a lysine and a targeting peptide, to increase the distance between them. The spacer is used to avoid steric hindrance, reduce aggregation, improve solubility and the accessibility of the compound to the target. A common type of spacer used in the compounds is polyethylene glycol (PEG), which is a hydrophilic and biocompatible polymer that can increase the solubility and stability of the compounds in vivo. When a hydrophilic spacer PEG is present in B, a variety of PEG derivatives can be used for synthesizing the compound, such as, without limitation, amine- PEG-carboxyl acid, amine-PEG-maleimide, amine-PEG-biotin, amine-PEG-azido, azido- PEG-carboxyl acid, amine-PEG-NHS ester, maleimide-PEG-NHS ester, and biotin-PEG- NHS ester. An amine-PEG-carboxyl acid is preferably used for synthesizing the compound, preferably via automated flow chemistry, such as H2N-PEG1-40-COOH, H2N-PEG1-40- CH2COOH, or H2N-PEG1-40- CH2CH2COOH. Other hydrophilic spacers can be used, such as polyethyleneamine, polyacetal polymer, poly(l -hydroxymethylethylene hydroxymethyl- formal) (PHF) or a carbohydrate. The length of the hydrophilic spacer can correspond to the span of the protein gap to facilitate the orientation of the HRC peptide to bind the HRN domain. B and the spacer, Linker and the spacer, or Peptide and the spacer can be joined to each other by the residue of a chemical reaction (such as an automated flow chemistry reaction).

[0118] Therefore, in some embodiments, B can be for example represented by the Formula

[0119] (B2):

[0120] Formula (B2), wherein and m are as defined above, including all and preferable embodiments, p is an integer selected from 0 to 40.

[0121] When a spacer PEG is present in B, in some embodiments, the compound can be therefore represented by Formula (VI): Peptide - Linker Peg - Lys - Lys - Peg - Hydrophobic Moiety

[0122] Linker

[0123] Peptide

[0124] Formula (VI), wherein Peptide, Linker, Hydrophobic Moiety are as defined above, including all and preferable embodiments; m is 0, 1, 2, 3, 4, preferably, m is 0, 1, and 2; and each Peg is independently selected from a PEG spacer.

[0125] With reference to Formula (VI), Hydrophobic Moiety is cholesterol and B is Formula

[0126] (B2), resulting in the structure of the peptide conjugate as shown by Formula (VII):

[0127] Peptide — Linker

[0128] Formula (VII), wherein Peptide, Linker, m, and p are as defined above, including all and preferable embodiments.

[0129] Hydrophobic Moiety

[0130] Hydrophobic Moiety plays a role as an anchoring agent to anchor the peptide conjugate to cellular membrane so that the peptide can target the fusion process of the coronavirus and inhibit viral entry.

[0131] Hydrophobic moiety can be a lipid-based moiety including fats, waxes, steroids, cholesterol, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, phospholipids, sphingolipids, glycolipids, cationic or anionic lipids, derivatized lipids. Preferably Hydrophobic Moiety is a membrane integrating lipid including cholesterol, sphingolipid, sphingomyelin, glycolipid, glycerophospholipid (such as phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine), ergosterol, 7-dihydrocholosterol and stigmasterol.

[0132] In some embodiments, the Hydrophobic Moiety can be cholesterol. Cholesterols can include cholesterol, esters of cholesterol including cholesterol hemi-succinate, salts of cholesterol including cholesterol hydrogen sulfate and cholesterol sulfate, ergosterol, esters of ergosterol including ergosterol hemi-succinate, salts of ergosterol including ergosterol hydrogen sulfate and ergosterol sulfate, lanosterol, esters of lanosterol including lanosterol hemi-succinate, salts of lanosterol including lanosterol hydrogen sulfate and lanosterol sulfate. In any embodiments wherein Hydrophobic Moiety is cholesterol, B is preferably linked directly or indirectly to a cholesterol hydroxyl group, such as 3 -OH.

[0133] In some embodiments, the Hydrophobic Moiety can be a phospholipid. Phospholipids that can be used in this application include, without limitation, phosphatidylethanolamine (EPE), and phosphatidic acid (EP A); the soya counterparts, soy phosphatidylcholine (SPC); SPG, SPS, SPI, SPE, and SPA; the hydrogenated egg and soya counterparts (e.g., HEPC, HSPC), other phospholipids made up of ester linkages of fatty acids in the 2 and 3 of glycerol positions containing chains of 12 to 26 carbon atoms and different head groups in the I position of glycerol that include choline, glycerol, inositol, serine, ethanolamine, as well as the corresponding phosphatidic acids. The chains on these fatty acids can be saturated or unsaturated, and the phospholipid may be made up of fatty acids of different chain lengths and different degrees of unsaturation. In particular, the compositions of the formulations can include dipalmitoylphosphatidylcholine (DPPC), a major constituent of naturally-occurring lung surfactant. Other examples include dimyristoylphosphatidycholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) dipalmitoylphosphatideholine (DPPQ) and dipalmitoylphosphatidylglycerol (DPPG) distearoylphosphatidylcholine (DSPQ) and distearoylphosphatidylglycerol (DSPG), dioleylphosphatidyl-ethanolarnine (DOPE) and mixed phospholipids like palmitoylstearoylphosphatidyl-choline (PSPC) and palmitoylstearolphosphatidylglycerol (PSPG), and single acylated phospholipids like monool eoyl -phosphati dyl ethanol amine (MOPE) .

[0134] In some embodiments, the Hydrophobic Moiety can be a sphingolipid, including sphingosine, sphingomyelins, cerebroside, sulfatides, globosides, gangliosides, galactocerebroside, glucocerebroside, GM2 ganglioside, GM1 ganglioside, and glycoplipids including ceramide trihexoside. In some embodiments, the Hydrophobic Moiety can be a tocopherol. The tocopherols can include tocopherols, esters of tocopherols including tocopherol hemi-succinates, salts of tocopherols including tocopherol hydrogen sulfates and tocopherol sulfates.

[0135] In some preferable embodiments, the Hydrophobic Moiety is cholesterol. B can be covalently connected to a convenient position on the Hydrophobic Moiety.

[0136] In some embodiments, connection is via a hydroxy group of the Hydrophobic Moiety. For example, when the Hydrophobic Moiety is cholesterol, B can be connected to the cholesterol by a group — C(O) — or — Ci-4 alkylene C(O) — , such as — CH2C(0) — .

[0137] Nonlimiting examples of the peptide conjugate structure are presented below:

[0138]

[0139] H N g g g p

[0140] Peptide Conjugate 6 (left) and Peptide Conjugate 7 (right), The NH group with ^is bound to cholesterol via a linker.

[0141]

[0142] Peptide Conjugate DCOY103,

[0143] Peptide — Linker

[0144] Peptide Conjugate DCOY104. Peptide-Linker for any one of the peptide conjugates (Peptide Conjugates 1-5 and DOY101-DCOY104) is SEQ ID NO. 5:

[0145] H2N-DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGSGSG (SEQ ID NO. 5).

[0146] In additional embodiments, provided are also peptide conjugates that respectively comprise the B-Hydrophobic Moiety structures as presented above with Peptide-Linker different from SEQ ID NO. 5. In those embodiments, with reference to the core structures of Peptide Conjugates 1-5 and DOY101-DCOY104, each Peptide and each Linker are independently as defined above, including all additional and preferred embodiments.

[0147] Preferably, each Peptide is independently selected from SEQ ID NO. 1 and SEQ ID NO. 2:

[0148] Acn-DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL (SEQ ID NO. 1), wherein n is 0 or 1. dldGdSdldD NASVVNIQKEIDRLNEVAKNLNESLIDLQEL (SEQ ID NO. 2)

[0149] In some embodiments, the Peptide-Linker is independently selected from SEQ ID NO. 3 and SEQ ID NO. 4:

[0150] DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGSGSG (SEQ ID NO. 3) dldGdSdldD NASVVNIQKEIDRLNEVAKNLNESLIDLQELGSGSG (SEQ ID NO. 4).

[0151] Preferably, with reference to the core structures of Peptide Conjugates 1-5 and DOY101-DCOY104, each Peptide-Linker is the same for the same peptide conjugate. Additionally, with reference to the core structures in Table 1, one Peptide-Linker is selected from a receptor binding domain targeting peptide, an ACE2 targeting peptide, and the other (Peptide-Linker)s are the same HRC peptide for the same peptide conjugate.

[0152] Method of Use

[0153] Provided is also a method of treating an infection associated with a coronavirus or variant thereof in a subject in need, wherein the method comprises administering the formulation of the invention to the subject in need. Provided is also a method of preventing or reducing the transmission of a coronavirus or variant thereof (e.g., a SARS-CoV-2 variant) from an infected subject to other otherwise uninfected subjects, even when close contact occurs between the infected and uninfected groups. Consequently, it offers individuals an opportunity to maintain their normal daily activities in the event of a virus infection or contact with an infected person. The term “preventing or reducing transmission” as used herein refers to a variety of situations where, in contact with the infected subject, the uninfected subject remains negative for covid test; the uninfected subject does not show symptoms of infection or only shows minor symptoms; the uninfected subject remains negative for covid test and does not show symptoms of infection or only shows minor symptoms; and the uninfected subject does not show symptoms of infection or only shows minor symptoms not withstanding a positive test result.

[0154] The term “contact” as used herein direct or indirect physical interaction or exposure between an infected subject (the source) and a susceptible subject (the recipient) that can result in the transfer of the virus from the infected subject to the uninfected subject.

[0155] The term "infected" as used herein refers to the incident wherein a subject or organism that has been exposed to the virus and has had the virus enter their body, where it can potentially multiply and cause illness. Infection with, for example, SARS-CoV-2 can lead to a wide range of symptoms, from mild or asymptomatic cases to severe respiratory distress and other complications. The infection can be an infection of the gastrointestinal tract or upper or lower respiratory tract, including the common cold, influenza, respiratory syncytial virus infection, Severe Acute Respiratory Syndrome, Middle East Respiratory Syndrome, COVID-19 or a disease caused by another emerging zoonotic virus, such as a zoonotic coronavirus. In specific aspects, the methods of the invention treat a viral respiratory infection, such as a SARS-CoV-2 (COVID-19) respiratory infection.

[0156] The term “transmission” used as herein refers to the process by which the virus is passed from an infected subject to an otherwise uninfected subject, resulting in the viral infection of the otherwise uninfected subject.

[0157] The SARS-Cov-2 variant can comprise one or more mutations in the viral fusion protein with reference to the wild type. The SARS-Cov-2 variant can comprise at least 1, at least 2, at least 3, at least 4, at least 5 mutations. In some embodiments, the variant comprises at least 10 mutations. In some embodiments, the variant comprises at least 15 mutations. In some embodiments, the variant comprises at least 20 mutations.

[0158] In some embodiments, the SARS-Cov-2 variant comprises at least 5 mutations wherein the at least 5 mutations are independently in the spike protein SI subunit or the S2 subunit or combinations thereof.

[0159] In some embodiments, the at least 5 mutations are independently in N-Terminal domain (NTD), the receptor binding domain (RBD), the fusion peptide (FP) domain, the heptad repeat 1 (HR1) domain, or combinations thereof. In some embodiments, the at least 5 mutations are independently selected from at least 5 mutations from the SAR-Cov-2 Alpha variant; at least 5 mutations from the SAR- Cov-2 Beta variant; at least 5 mutations from the SAR-Cov-2 Delta variant; or at least 5 mutations from the SAR-Cov-2 Omicron variant. In some embodiments, the at least 5 mutations are independently selected from at least 5 mutations from the SAR-Cov-2 Alpha variant. In some embodiments, the at least 5 mutations are independently selected from at least 5 mutations from the SAR-Cov-2 Beta variant. In some embodiments, the at least 5 mutations are independently selected from at least 5 mutations from the SAR-Cov-2 Delta variant. In some embodiments, the at least 5 mutations are independently selected from at least 5 mutations from the SAR-Cov-2 Omicron variant.

[0160] In some embodiments, the SARS-CoV-2 variant comprises at least one variant selected from B.1.1.7 (Alpha), B.1.351 (Beta), P.l (Gamma), B.1.617.2 (Delta), B.1.429 / B.1.427 (Epsilon), B.1.617.1 (Kappa), B.1.525 (Eta), B.1.526 (Iota), P.3 (Theta), P.2 (Zeta), and B.1.1.529 (Omicron).

[0161] In some embodiments, the SARS-CoV-2 variant comprises at least one variant selected from A.1-A.6, B.3-B.7, B.9, B.10, B.13-B.16, B.2, B.l lineage, P.l, P.2, P.3, and R. l.

[0162] In some embodiments, the B.l lineage comprises at least one of (including, but not limited to), B. l, B.1.1, B. l.1.7, B.l.1.7 with E484K, B.l.2, B.1.5-B.1.72, B.1.9, B.l.13, B.1.22, B.l.26, B.l.37, B.1.3-B.1.66, B. l.177, B.1.243, B.1.313, B.1.351, B.1.427, B.1.429, B.1.525, B.l.526, B.l.526.1, B.l.526.2, B.1.617, B.l.617.1, B.1.617.2, B.1.617.3, B.1.619, B.1.620, and B. l.621.

[0163] In some embodiments, the administration is achieved via nasal administration such as nasal spray, nasal drops, nasal gels, nasal powders, nasal aerosols, nasal pumps, nasal nebulizers, nasal inhalers. Preferably nasal administration is achieved using an intranasal spray, an inhaler, or a nebulizer.

[0164] In some embodiments, the formulation is administered in combination with at least one other antiviral active agent or therapy.

[0165] The infected subject, preferably a human, can be an individual diagnosed with the infection and is either symptomatic, pre-symptomatic, or asymptomatic, or at risk for developing infection. For example, the subject can be at risk for developing the viral respiratory infection due to direct or indirect exposure or possible exposure to the virus (such as SARS-CoV-2 or a mutant thereof), such as via exposure to an infected individual or a virus-contaminated fomite. The subject can be a resident of, or a visitor to, a community in which the viral respiratory infection has been identified, for example, the subject can be a family member of an infected individual or the subject can work in a health care setting caring for infected individuals. In some embodiments, the subject at risk for infection is asymptomatic and has tested negative for presence of the virus prior to the commencement of therapy. In specific examples, the subject can be at risk for developing COVID-19 due to exposure to the SARS-CoV-2 virus, for example, from the respiratory droplets or aerosols of an infected individual and / or contact with a contaminated fomite. In yet further aspects, the subject is suffering from COVID-19 including subjects suffering from mild, moderate, or severe COVID-19.

[0166] In some embodiments of the method of the invention, the infected subject suffers from another disease or condition, such as chronic obstructive pulmonary disease (COPD) or ulcerative colitis, which can be exacerbated by an infection.

[0167] The formulation is administered to the infected subject upon the discovery of the infection, before infection as a prophylactic measure, or within 72 hours of the discovery of the infection. The peptide conjugate is preferably administered to the infected subject before the infected subject comes into contact with other uninfected subjects. The peptide conjugate is preferably administered to the infected subject within 48 hours, 36 hours, 24 hours, 12 hours, or 8 hours of the discovery of the infection, or 48 hours, 36 hours, 24 hours, 12 hours, or 8 hours before the infected subject comes into contact with other uninfected subjects. Preferably the administration continues until the infected subject test negative for the coronavirus. Contact may involve sharing a residence, workplace, classroom, car or any enclosed area. The term “enclosed area” as used herein refers to any space that is enclosed or substantially enclosed by physical barriers, such as walls, fences, doors, or other structures. Enclosed areas can vary widely in size and purpose, ranging from small rooms or compartments to large buildings and structures.

[0168] In additional embodiments, the method further comprises an optional step of administering an effective amount of formulation to the other uninfected subject(s) before the uninfected subject(s) come into contact with the infected subject. In some cases, the method further comprises an optional step of administering an effective amount of peptide conjugate to the other uninfected subject(s) 48 hours, 36 hours, 24 hours, 12 hours, 8 hours before the uninfected subject(s) come into contact with the infected subject. In some cases, the administration to the uninfected subject(s) continues throughout the duration of physical contact with the infected subject or until the infected subject tests negative, whichever occurs first. In alternative embodiments, provided is also a method for preventing or reducing the transmission of a coronavirus or variant thereof (preferably a SARS-CoV-2 variant) from an infected subject to other otherwise uninfected subjects when in contact with the infected subject, comprising administrating an effective amount of the formulation to the uninfected subject. The peptide conjugate is preferably administered to the uninfected subject 48 hours, 36 hours, 24 hours, 12 hours, 8 hours before the uninfected subject(s) come into contact with the infected subject. Preferably the administration continues throughout the duration of physical contact with the infected subject or until the infected subject tests negative, whichever occurs first.

[0169] The formulation is preferably administered to the infected subject before the infected subject is symptomatic (e.g., pre-symptomatic), at the onset of symptoms, or within 24 hours of the onset of symptoms. The pharmaceutical composition can be administered at a variety of dosing schedules. For example, the pharmaceutical composition can be administered one or more times and over a course of one or more days. In some embodiments, the pharmaceutical composition is administered one or more times per day for one to 10 days. In some embodiments, the pharmaceutical composition is administered one or more times per day until the subject is asymptomatic and / or testing for the virus is negative.

[0170] The pharmaceutical composition can be administered to the nasal passages using routine methods and devices (see D. Marx et al., IntechOpen, DOI: 10.5772 / 59468. Available from: https: / / www.intechopen.com / books / drug-discovery-and-development-from-molecules- to-medicine / intranasal-drug-administration-an-attractive-delivery-route-for-some-drugs). For example, the pharmaceutical composition can be administered to the nasal passages as drops or as an aerosol spray, for example, using an aerosol bottle or a multi-dose spray pump, which can provide a uniform metered dose. The volume per dose can be varied, but is typically from about 50 to about 150 pl. The desired volume will depend on the desired dose of the active agent and the concentration of the active agent in the composition.

[0171] Where delivery to the pulmonary system, or lungs, is desired it can be efficacious to aerosolize a low concentration solution of the active agent for an extended period, such as overnight.

[0172] In addition to the prevention or reduction of viral transmission from the infected subject to other uninfected subject(s), the method as described above also has a therapeutic effect on the treatment of the infected subject. Additional Routes of Administration

[0173] Preferably, the formulation of this invention is designed for intranasal administration. In additional embodiments, the formulation can be directly used or slightly modified to be compatible with other routes of administration. For example, the composition can be administered systemically or locally. The composition can be administered for oral, intravenous, intramuscular, rectal, cutaneous, subcutaneous, topical, transdermal, sublingual, nasal, inhalation, or vaginal delivery, for example. The compositions may be modified according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy, 22ndedition, 2013, ed. L. V. Allen, Pharmaceutical Press, Philadelphia, and Encyclopedia of Pharmaceutical Technology, 4. sup. th Edition, ed. J. Swarbrick, 2013, CRC Press, New York).

[0174] The formulation can be incorporated for administration orally include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

[0175] Formulations suitable for parenteral administration (e.g., by injection), include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), in which the peptide conjugate is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticulate). Such liquids may additionally contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the peptide conjugate in the liquid is from about 1 ng / ml to about 10 ug / ml, for example from about 10 ng / ml to about 1 ug / ml. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0176] The formulation can comprise a liquid vehicle which is suitable for nasal administration. The vehicle is preferably an aqueous solution. More preferably, the vehicle is an aqueous solution which includes a viscosity enhancing agent and, optionally one or more additional excipients which, for example, improve formulation stability and / or comfort upon administration.

[0177] A variety of viscosity enhancing agents are known in the art. Viscosity enhancing agent is preferably a mucoadhesive as described here. Additional viscosity enhancing agents include hydrophilic polymers, such as polysaccharides, polysaccharide derivatives, proteins and synthetic polymers. Examples include, but are not limited to, acacia, tragacanth, alginic acid, carrageenan, locust bean gum, guar gum, gelatin, hyaluronic acid, polyacrylate, polyacrylate / alkylacrylate copolymers, polyvinyl alcohol, polyvinylpyrrolidone, starch, propylene glycol alginate, maltodextrin, and cellulose ether derivatives, such as methylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, and carboxymethylcellulose. Where possible, salt forms of any of the foregoing are preferred. Preferred viscosity enhancing agents include hyaluronic acid, including sodium hyaluronate; carboxymethylcellulose, including sodium carboxymethylcellulose and calcium carboxymethylcellulose; methylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, and hydroxypropylcellulose.

[0178] The composition optionally includes one or more additional excipients which, for example, increase the ease of administration, the comfort of the subject, or the stability of the composition. Suitable additional excipients include, but are not limited to, tonicity modifiers, such as mannitol, sodium chloride and dextrose; antioxidants, such as butylated hydroxyanisole; buffers, such as sodium bicarbonate, sodium citrate and sodium phosphate; preservatives, such as benzalkonium chloride, ethanol, propylene glycol, benzoyl alcohol, phenethyl alcohol, chlorobutanol or methylparaben; pH adjusters, such as hydrochloric acid, sulfuric acid and sodium hydroxide; surfactants, such as Polysorbate 80, Polysorbate 20, and polyoxyl 400 stearate; chelating agents, such as disodium EDTA; antioxidants; co-solvents, such as ethanol, PEG 400, and propylene glycol; penetration enhancers, such as oleic acid; and humectants, such as glycerin (see S. Thorat, Sch. J. App. Med. Sci. 2016, 4(8D):2976- 2985; D. Marx et al., IntechOpen, DOI: 10.5772 / 59468. Available from: intechopen.com / books / drug-discovery-and-development-from-molecules-to- medicine / intranasal-drug-administration-an-attractive-delivery-route-for-some-drugs).

[0179] In one embodiment, the vehicle consists of sodium hyaluronate, aloe vera, allantoin, sodium chloride, sodium bicarbonate, glycerin, propylene glycol, benzalkonium chloride and USP grade purified water. A suitable vehicle is sold by NEILMED™ under the tradename NASOGEL™. The amount of active agent in the composition can vary, for example, from about 0.5% by weight to about 25% by weight.

[0180] The pH of the formulation is tolerable in the nasal cavity and preferably in the range of about 5.0 to about 8.0. Buffers that can be used in the formulation include, but are not limited to phosphate, TRIS, [tris(hydroxymethyl) methylamino] propanesulfonic acid, 2- (bis(2-hydroxyethyl)amino)acetic acid, and N-[tris(hydroxymethyl)methyl]glycine, and Alkaline Buffer (Seachem).

[0181] A pharmaceutical composition suitable for nasal or pulmonary administration comprising a water soluble solvent selected from the group consisting of propylene glycol, glycerin, polyethylene glycol, and combinations thereof. The composition can further comprise one or more of a polysaccharide gum, a non-ionic surfactant, and a preservative. An exemplary polysaccharide gum is sclerotium gum. Exemplary surfactants are poloxamers, including, but not limited to poloxamer 188. The preservative can, for example, be benzalkonium chloride.

[0182] The formulation can be modified into a dry powder form and delivered by a dry powder inhaler, suspended in a propellant or in an aqueous suspension or solution and delivered via a nebulizer. In some cases, the formulation can be modified into a solid dispersion form.

[0183] For example, a solution or suspension of the active agent and a pulmonary excipient, such as lactose, can be spray dried to form particles having a fine particle fraction sufficient to deliver to the lungs or upper respiratory system. Alternatively, an aqueous solution or suspension can be sonicated, thereby aerosolizing the solution / suspension to a droplet size that can be inhaled, e.g., via a nebulizer.

[0184] Excipients include carbohydrates including monosaccharides, disaccharides and polysaccharides. For example, monosaccharides such as dextrose (anhydrous and monohydrate), galactose, mannitol, D-mannose, sorbitol, sorbose and the like; disaccharides such as lactose, maltose, sucrose, trehalose, and the like; tri saccharides such as raffinose and the like; and other carbohydrates such as starches (hydroxy ethyl starch), cyclodextrins and maltodextrins. Other excipients suitable for use with the present invention, including amino acids, are known in the art such as those disclosed in WO 95 / 31479, WO 96 / 32096, and WO 96 / 32149. Mixtures of carbohydrates and amino acids are further held to be within the scope of the present invention. The inclusion of both inorganic (e.g., sodium chloride, etc.), organic acids and their salts (e.g., carboxylic acids and their salts such as sodium citrate, sodium ascorbate, magnesium gluconate, sodium gluconate, tromethamine hydrochloride, etc.) and buffers is also contemplated.

[0185] The dispersions or powders of the invention may be used in conjunction with metered dose inhalers (MDIs), dry powder inhalers (DPIs), atomizers, nebulizers or liquid dose instillation (LDI) techniques to provide for effective drug delivery.

[0186] The medicament is formulated in a way such that it readily disperses into discrete particles with a mass median aerodynamic diameter (MMAD) of the powders will characteristically range from about 0.5-100 micronsD. In some embodiments, the medicament is formulated in a way such that it readily disperses into discrete particles with a MMAD of the powders will characteristically range from about 0.5-90, 0.5-80, 0.5-70, 0.5- 60, 0.5-50, 0.5-40, 0.5-30, 0.5-20, 0.5-10, or 0.5-5 microns. In some embodiments, the medicament is formulated in a way such that it readily disperses into discrete particles with a MMAD of the powders will characteristically range from about 5-100, 10-100, 20-100, 30- 100, 40-100, 50-100, 60-100, 70-100, 80-100, or 90-100 microns. In some embodiments where the formulation is designed for nasal administration, the medicament is formulated in a way such that it readily disperses into discrete particles with a MMAD of the powders will preferably range from about 10-40 microns.

[0187] As discussed above, the stabilized dispersions disclosed herein may also be administered to the nasal or pulmonary air passages of a patient via aerosolization, such as with a metered dose inhaler. MDIs are well known in the art and could easily be employed for administration of the claimed dispersions without undue experimentation. Breath activated MDIs, as well as those comprising other types of improvements which have been, or will be, developed are also compatible with the stabilized dispersions and present invention and, as such, are contemplated as being within the scope thereof. However, it should be emphasized that, in preferred embodiments, the stabilized dispersions may be administered with an MDI using a number of different routes including, but not limited to, topical, nasal, pulmonary or oral. Those skilled in the art will appreciate that such routes are well known and that the dosing and administration procedures may be easily derived for the stabilized dispersions of the present invention.

[0188] Along with the aforementioned embodiments, the stabilized dispersions of the present invention may also be used in conjunction with nebulizers as disclosed in PCT WO 99 / 16420, the disclosure of which is hereby incorporated in its entirety by reference, in order to provide an aerosolized medicament that may be administered to the pulmonary air passages of a patient in need thereof. Nebulizers are well known in the art and could easily be employed for administration of the claimed dispersions without undue experimentation. Breath activated nebulizers, as well as those comprising other types of improvements which have been, or will be, developed are also compatible with the stabilized dispersions and present invention and are contemplated as being within the scope thereof.

[0189] Along with DPIs, MDIs and nebulizers, it will be appreciated that the stabilized dispersions of the present invention may be used in conjunction with liquid dose instillation or LDI techniques as disclosed in, for example, WO 99 / 16421 hereby incorporated in its entirety by reference. Liquid dose instillation involves the direct administration of a stabilized dispersion to the lung. In this regard, direct pulmonary administration of bioactive peptide conjugates is particularly effective in the treatment of disorders especially where poor vascular circulation of diseased portions of a lung reduces the effectiveness of intravenous drug delivery. With respect to LDI the stabilized dispersions are preferably used in conjunction with partial liquid ventilation or total liquid ventilation. Moreover, the present invention may further comprise introducing a therapeutically beneficial amount of a physiologically acceptable gas (such as nitric oxide or oxygen) into the pharmaceutical microdispersion prior to, during or following administration.

[0190] Combination Therapies

[0191] The peptide conjugate or composition described herein can be co-administered with other active agents and therapies.

[0192] In some embodiments, the other active agent includes, but is not limited to, antibodies against a paramyxovirus such as HPIV-3. For example, the protective antibodies PI3-E12 against HPIV-3 are described in Boonyaratanakornkit et al., “Protective antibodies against human parainfluenza virus type 3 infection”, mAbs. Volume 13, 2021, Issue 1, https: / / doi.org / 10.1080 / 19420862.2021.1912884.

[0193] In some embodiments, the other active agent includes, but is not limited to, antibodies against SARS-CoV-2. Suitable antibodies are described in, for example, US 2022 / 0017604, US 2022 / 0017614; US 2021 / 0403550, US 2021 / 0395345, US 2021 / 0403537; US 2021 / 0388066, US 2021 / 0388065, US 2021 / 0347859, or US 2021 / 0309733, which are incorporated herein by reference. In some embodiments, the antibody is a monoclonal antibody such as casirivimab, imdevimab, bamlanivimab, or etesevimab. In some embodiments, the antibody is a monoclonal antibody therapy such as casirivimab plus imdevimab, bamlanivimab, or bamlanivimab plus etesevimab. The active agents and compositions of the present invention are also intended for use with general care provided patients with viral infections, including parenteral fluids (including dextrose saline and Ringer's lactate) and nutrition, antibiotic (including metronidazole and cephalosporin antibiotics, such as ceftriaxone and cefuroxime) and / or antiviral prophylaxis, fever (e.g., acetaminophen) and pain medication, antiemetic (such as metoclopramide) and / or antidiarrheal agents, vitamin and mineral supplements (including Vitamin K and zinc sulfate), anti-inflammatory agents (such as ibuprofen), pain medications, and medications for other common diseases in the patient population, such as artemether, artesunate-lumefantrine combination therapy), quinolone antibiotics, such as ciprofloxacin, macrolide antibiotics, such as azithromycin, cephalosporin antibiotics, such as ceftriaxone, or aminopenicillins, such as ampicillin), or shigellosis.

[0194] The combination therapy may be administered as a simultaneous or sequential regimen. When administered sequentially, the combination may be administered in two or more administrations.

[0195] Co-administration of a peptide conjugate of the invention with one or more other active therapeutic agents generally refers to simultaneous or sequential administration of a peptide conjugate of the invention and one or more other active therapeutic agents, such that therapeutically effective amounts of the peptide conjugate of the invention and one or more other active therapeutic agents are both present in the body of the patient.

[0196] Co-administration includes administration of unit dosages of the peptide conjugates of the invention before or after administration of unit dosages of one or more other active therapeutic agents, for example, administration of the peptide conjugates of the invention within seconds, minutes, or hours of the administration of one or more other active therapeutic agents and / or as part of the same treatment regimen. For example, a unit dose of a peptide conjugate of the invention can be administered first, followed within seconds or minutes or days by administration of a unit dose of one or more other active therapeutic agents. Alternatively, a unit dose of one or more other therapeutic agents can be administered first, followed by administration of a unit dose of a peptide conjugate of the invention within seconds or minutes or days. In some cases, it may be desirable to administer a unit dose of a peptide conjugate of the invention first, followed, after a period of hours (e.g., 1-12 hours), by administration of a unit dose of one or more other active therapeutic agents. In other cases, it may be desirable to administer a unit dose of one or more other active therapeutic agents first, followed, after a period of hours (e.g., 1-12 hours), by administration of a unit dose of a peptide conjugate of the invention. The combination therapy may provide "synergy" and "synergistic," i.e., the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the peptide conjugates separately.

[0197] As used herein, the words “a” and “an” are meant to include one or more unless otherwise specified. For example, the term “an agent” encompasses both a single agent and a combination of two or more agents.

[0198] The term "treating" or "treatment" as used herein covers the treatment of the disease or condition of interest (e.g., a respiratory infection) in a mammal, preferably a human, having the disease or condition of interest, and includes, for example: preventing or delaying the onset of the disease or condition from occurring in a mammal, in particular, when such mammal is at risk of developing the disease but has not yet become symptomatic and / or been diagnosed as having it; inhibiting the disease or condition, i.e., arresting its development; relieving the disease or condition, i.e., causing regression of the disease or condition; and / or stabilizing the disease or condition. Treatment includes ameliorating or lessening the severity of symptoms of the disease or condition, and / or inhibition of further progression or worsening of those symptoms. Treatment also includes shortening the time course and / or severity of a disease or condition compared to the expected or historical time course and / or severity of the disease.

[0199] As used herein the terms "preventing," means causing the clinical symptoms of a disease or condition not to develop and includes inhibiting the onset of a viral infection in a subject that may be exposed to or predisposed to the viral infection but does not yet experience or display symptoms of the infection.

[0200] An “effective amount” or a “therapeutically effective amount” of a peptide conjugate or composition described herein refers to an amount of the peptide conjugate that is sufficient to achieve a specific effect or result, and / or prevents or treats the disease or condition and / or the symptoms therefore, for example, alleviating, in whole or in part, symptoms associated with the disorder or condition, or halts or slows further progression or worsening of those symptoms, or prevents or provides prophylaxis for the disorder or condition. The “effective amount” and “therapeutically effective amount” includes specifically an anti-viral amount of a peptide conjugate of the invention (alone or in combination with another active agent) or the composition described herein. Examples

[0201] This example describes and summarizes the exploratory formulation and stability studies performed on peptide conjugates DCOY101, DCOY102, and / or DCOY103. These peptide conjugates are pan-coronavirus fusion inhibitors targeted for use in a liquid nasal spray for intranasal administration at a desired concentration of 10 mg / mL to facilitate a clinical unit dose in the range of 1 to 5 mg. DCOY101, DCOY102, and DCOY103 have previously been shown to be difficult to formulate in aqueous solution without co-solvents, such as phosphate buffered saline or water. First, it was imperative to identify a method to dissolve these peptide conjugates into solution that ensures short-term stability and is compatible for nasal route administration. These were thus conducted with the goals of (1) developing a liquid formulation compatible for all pan-coronavirus peptide conjugates for intranasal administration, (2) identifying suitable excipients to enhance storage stability and intranasal delivery of DCOY102, and (3) elucidating degradation pathway(s) of DCOY101, DCOY102, and DCOY103. A series of studies were performed to assess solubility and stability to achieve these goals. Results identify a FIH liquid formulation for lead candidate DCOY102 at 10 mg / mL.

[0202] Example 1: Procedure For Liquid Formulation of Peptide Conjugates

[0203] Described below is the general procedure used to formulate 10 mg / mL peptide conjugate solutions. This general procedure was used to formulate all samples in this report:

[0204] 1. Peptide conjugates retain moisture when stored at -30°C. To reduce moisture before formulating, lyophilize the dry powder for 24 hrs.

[0205] 2. Initial concentration of peptide conjugates is 50 mg / mL. Weigh out desired mass of peptide conjugate and hydrate with 100% propylene glycol. Note: propylene glycol is viscous. Confirm pipette contains appropriate volume. a. Vortex for 30 seconds b. Sonicate for 5 minutes c. Warm to 37°C for 5 minutes d. Cycle from 2a - 2c until solution is optically clear without precipitates. i. Initially, the solution is cloudy upon addition of propylene glycol. Solution should become optically clear within 2-5 cycles.

[0206] 3. Perform 1 :5 dilution of the concentrated stock (50 mg / mL) in 100% propylene glycol (Formulated in Step 2) to final concentration of 10 mg / mL and 20% v / v propylene glycol. a. Suggested dilution buffer: 0. IX PBS i. Note: Do not exceed 0. IX PBS concentration. Avoid high salt content aqueous buffers. b. First, add the dilution buffer into a new tube. Then, aliquot the propylene glycol stock solution into the dilution buffer. Pipette to mix.

[0207] 4. Solution will be optically clear. Vortex for 10-30 seconds to ensure no obvious phase separation and a uniform distribution of peptide conjugate within the solution.

[0208] 5. Adjust the solution to pH 5.0 - 7.0 using HCl / NaOH. a. Initial pH of solution is typically between pH 2.5 - 3.0. b. When adding NaOH, the solution may transiently turn cloudy at pH 4.0. c. Guidance for volume of IN NaOH required: For 300 pL solution, add approximately 3.5 pL of IN NaOH.

[0209] Final composition of formulation:

[0210] 10 mg / mL peptide conjugate, 20% v / v propylene glycol in 0. IX PBS at pH 5.0 - 7.0.

[0211] Example 2: Liquid Formulation Development of Peptide Conjugates

[0212] For translation as an aqueous-based intranasal spray for clinical use, peptide conjugates must be formulated in a liquid, biocompatible formulation. DCOY101, DCOY102, and DCOY103 are difficult to dissolve into aqueous solutions due to the cholesterol hydrophobicity. Here, we developed and identified a platform formulation for DCOY101, DCOY102, and DCOY103 at a clinical dosage strength of 10 mg / mL.

[0213] Propylene as Co-solvent for DCOY101, DCOY102, & DCOY103

[0214] DCOY101, DCOY102, and DCOY103 are peptide conjugates that are poorly soluble in aqueous solution. Here, propylene glycol, a polar solvent used to enhance the solubility of hydrophobic compounds, was evaluated for use as a co-solvent for DCOY101, DCOY102, and DCOY103. Maximum solubility in propylene glycol was determined for each compound to support continued formulation development.

[0215] Experimental Design

[0216] DCOY101, DCOY102, and DCOY103 were evaluated at 100, 200, and 250 mg / mL in propylene glycol. Samples were diluted with propylene glycol to determine estimated maximum solubility concentration only if over the concentration limit. All samples were assessed for solution clarity via visual appearance. Results

[0217] DCOY101, DCOY102, and DCOY103 were initially evaluated at 100 mg / mL in propylene glycol. All compounds were soluble after 3 cycles of: vortex, sonication for 5 min, and warming to 37°C for 5 min. All samples were optically clear solutions.

[0218] DCOY101, DCOY102, and DCOY103 were then evaluated at 200 mg / mL in propylene glycol. DCOY102 and DCOY103 were soluble after 3 cycles of: vortex, sonication for 5 min, and warming to 37°C for 5 min, and resulted in optically clear solutions. DCOY101 was insoluble after 4 cycles of: vortex, sonication for 5 min, and warming to 37°C for 5 min. DCOY101 resulted in clear solution with insoluble large, white chunks that indicated the solution was above its maximum solubility concentration. DCOY101 was diluted to 184 mg / mL, which resulted in an optically clear solution.

[0219] Only DCOY102 and DCOY103 were evaluated at 250 mg / mL in propylene glycol. DCOY102 and DCOY103 were soluble after 2 cycles of: vortex, sonication for 5 min, and warming to 37°C for 5 min. Although the samples resulted in optically clear solutions, pipetting could not occur due to crystallization deeming this concentration unusable. DCOY102 and DCOY103 were diluted with propylene glycol to 232 and 231 mg / mL, respectively, and resulted in optically clear solutions that were usable.

[0220] Maximum solubility results are summarized below in Table 1.

[0221] Table 1. Maximum solubility results in propylene glycol for DCOY101, DCOY102, and DCOY103.

[0222] Discussion

[0223] Propylene glycol is a suitable co-solvent to use to formulate peptide conjugates for nasal administration. DCOY101, DCOY102, and DCOY103 can be concentrated to at least 180 mg / mL in propylene glycol neat solution. It is vital for these solutions to undergo cyclic vortex, sonication, and warming to 37°C to dissolve the peptide conjugate in propylene glycol. pH Assessment on Liquid Formulation of 10 mg / mL Peptide Conjugate

[0224] Peptide conjugates were formulated in aqueous solution using propylene glycol as a cosolvent. Formulation limits for intranasal administration include (1) propylene glycol <20% v / v and (2) pH ranging from 5.0 - 7.0. Here, the effects of pH on the solubility and short-term stability of DCOY101, DCOY102, and DCOY103 was evaluated to identify the optimal pH for each compound to support continued formulation development.

[0225] Experimental Design

[0226] DCOY101, DCOY102, and DCOY103 (10 mg / mL) were evaluated at pH ranging from 2.0 - 8.0 in 20% v / v propylene glycol in 0.1X PBS. Formulations are summarized in Table 2. All formulations were assessed for solution clarity via visual appearance and optical density (ODeoo), purity (LC-MS), concentration (LC-MS; 220 nm), and pH for one week at room temperature (20 - 22°C).

[0227] Table 2. Formulations evaluated for pH. All formulations were formulated in 20% (v / v) propylene glycol in 0. IX PBS.

[0228] Results

[0229] Data tables summarizing the results of this study are in Tables 3-5.

[0230] Table 3. pH effects on stability for DCOY102 with pH ranging 2.0 - 8.0 (Fl - F4) analyzed for visual appearance, optical density (ODeoo), pH, concentration, and purity (%) at t = 0, 1, 4, & 7 days. a Visual Appearance Key : 0 = optically clear, 1 = 1-2 particulates, 2 = many particulates, 3 = slightly cloudy, 4 = very cloudy, 5 =opaque NT = not tested

[0231] N / A = not applicable

[0232] Table 4. pH effects on stability for DCOY103 with pH ranging 2.0 - 8.0 (F5 - F8) analyzed for visual appearance, optical density (ODeoo), pH, concentration, and purity (%) at t = 0, 1, 4, & 7 days. a Visual Appearance Key : 0 = optically clear, 1 = 1-2 particulates, 2 = many particulates,

[0233] 3 = slightly cloudy, 4 = very cloudy, 5 =opaque

[0234] NT = not tested

[0235] N / A = not applicable

[0236] Table 5. pH effects on stability for DCOY103 with pH ranging 2.0 - 8.0 (F9 - F12) analyzed for visual appearance, optical density (ODeoo), pH, concentration, and purity (%) at t = 0, 1, 4, & 7 days. a Visual Appearance Key : 0 = optically clear, 1 = 1-2 particulates, 2 = many particulates, 3 = slightly cloudy, 4 = very cloudy, 5 =opaque

[0237] NT = not tested

[0238] N / A = not applicable

[0239] Clarity of Solution: Formulations at pH 2.0, 6.0, and 8.0 remained soluble and optically clear throughout the duration of the study. However, formulations at pH 4.0 (F2, F6, F10) were cloudy upon titration to pH 4.0 and remained cloudy over one week with compound precipitating out of solution over time. Optical density measurements are graphed in Figure 9. pH Stability: Formulations at pH 2.0, 4.0, and 6.0 were pH-stable throughout the duration of the study. For formulations at pH 8.0 (F4, F8, F12), there was an apparent pH drift over the course of one week, which may be due to absorption of carbon dioxide and conversion to carbonic acid. More specifically, DCOY102 (F8) showed the largest pH drift of -1.2 at 7 days. See change in pH in Figure 1 (top row .

[0240] Concentration: All formulations remained < ± 3 mg / mL from starting concentration. Any concentration drifts may be due to (1) insufficient mixing prior to testing causing increased or decreased concentration or (2) evaporation effects throughout the duration of the study causing increased concentration. See change in concentration in Figure 1 (middle row .

[0241] Purity of Compounds: Formulations that changed > ±2% over time were DCOY102 (Fl) and DCOY 103 (F5) at pH 2.0 & DCOY103 (F8) at pH 8.0. The other formulations not listed remained within ±2% purity change over one week. See changes in purity in Figure 1 (bottom row}. It is to be noted that there was an additional peak arising in all pH 4.0, 6.0, and 8.0 formulations (not pH 2.0 formulations) between 1.8 - 2.0 min at 0.9%, 1.8%, and 2.7% on average, respectively. This peak is also detectable in 254 nm and 280 nm UV trace indicating that it is an aromatic compound. The peak mass was consistently detected in the LC-MS as 427.2 m / z which may be the cholesterol moiety hydrolysis (428.7 g / mol) from the addition of sodium hydroxide during pH titration from initial pH 3.0.

[0242] Discussion

[0243] Importantly, all peptide conjugates tested were stable at pH 6.0 for one week at room temperature (20-22°C), a pH level that is within the acceptable range for intranasal administration. Interestingly, all compounds at pH 4.0 resulted in cloudy solutions and precipitated over time; DCOY101, DCOY102, and DCOY103 have estimated pl of 4.2 thereby showing that these peptide conjugates are unstable in solution where the pH is close to the pl.

[0244] A new impurity emerges in pH 4.0, 6.0, and 8.0 formulations but not in pH 2.0 samples. The amount of impurity increases with increasing pH. This may be due to pH titration with IN NaOH during formulating the samples. Samples at pH 4.0 required less volume of IN NaOH compared to pH 8.0, for instance. Whereas samples at pH 2.0 did not require addition of NaOH thereby did not have the impurity. The impurity emerges in low abundance and does not influence purity of DCOY101, DCOY102, or DCOY103 over time. Further investigation with acid vs. base hydrolysis is performed in Section 5.1 and 5.2 to support this hypothesis.

[0245] The current stable formulation for all peptide conjugates tested is: 10 mg / mL peptide conjugate, 20% (v / v) propylene glycol in 0.1X PBS at pH 6.0. However, this formulation is hypotonic (approximately 28 mOsm / L); intranasal administration requires isotonic solutions (270 - 310 mOsm / L).

[0246] Isotonic Liquid Formulation of 10 mg / mL Peptide Conjugate

[0247] For intranasal administration, it is a requirement that the osmolality is within an acceptable range centered on isotonic that avoids adverse reactions at the administration site due to hypotonic or hypertonic effects. Thereby, the current formulation was supplemented with D-mannitol and / or sodium chloride (NaCl) to increase the tonicity of the formulation from 28 mOsm / L to approximately 280 mOsm / L. Here, DCOY101, DCOY102, and DCOY103 were evaluated for solubility and stability in five isotonic solutions. The optimal isotonic formulations were identified, serving as a basis for the liquid formulation development for the clinic.

[0248] Experimental Design

[0249] DCOY101, DCOY102, and DCOY103 (10 mg / mL) were evaluated in 280 mOsm / L solutions with tonicity contribution from NaCl only, D-mannitol only, 1 : 1 NaCl / D-mannitol, 1 :3 NaCl:D-mannitol, or 3 / 1 NaCl:D-mannitol. All formulations contained 10 mg / mL peptide conjugate, 20% v / v propylene glycol in 0. IX PBS at pH 6.0. Formulations are summarized in Table 6. All formulations were assessed for solution clarity via visual appearance and optical density (ODeoo), purity (LC-MS), concentration (LC-MS; 220 nm), and pH for one week at room temperature (20 - 22°C).

[0250] Table 6. Formulations evaluated for preferred excipient for isotonicity. All formulations were formulated in 20% (v / v) propylene glycol in 0. IX PBS at pH 6.0.

[0251] Results

[0252] Data tables summarizing the results of this study were shown in Tables 7-9.

[0253] Table 7. Isotonic formulations with DCOY101 (Fl - F5) analyzed for visual appearance, optical density (ODeoo), pH, concentration, and purity (%) at t = 0, 3, & 7 days. a Visual Appearance Key : 0 = optically clear, 1 = 1-2 particulates, 2 = many particulates, 3 = slightly cloudy, 4 = very cloudy, 5 =opaque

[0254] NT = not tested N / A = not applicable

[0255] Table 8. Isotonic formulations with DCOY102 (F6 - F10) analyzed for visual appearance, optical density (ODeoo), pH, concentration, and purity (%) at t = 0, 3, & 7 days. a Visual Appearance Key : 0 = optically clear, = 1-2 particulates, 2 = many particulates, 3 = slightly cloudy, 4 = very cloudy, 5 =opaque

[0256] Failed formulations (F6, F8, F10) NT = not tested

[0257] N / A = not applicable

[0258] Table 9. Isotonic formulations with DCOY103 (Fl 1 - F15) analyzed for visual appearance, optical density (ODeoo), pH, concentration, and purity (%) at t = 0, 3, & 7 days. a Visual Appearance Key : 0 = optically clear, 1 = 1-2 particulates, 2 = many particulates,

[0259] 3 = slightly cloudy, 4 = very cloudy, 5 =opaque

[0260] Failed formulations (Fl 1, F13-F15)

[0261] NT = not tested

[0262] N / A = not applicable

[0263] Clarity of Solution: For DCOY101, all formulations (F1-F5) were optically clear for 7 days as confirmed by optical density. For DCOY102, formulations with D-mannitol only (F7) and 1 :3 NaCl:D-mannitol (F9) were optically clear for 7 days as confirmed by optical density. Other formulations (F6, F8, F10) resulted in slightly cloudy solutions indicating insolubility; these were failed formulations that slowly crashed out of solution over time and were not assessed for purity. For DCOY103, D-mannitol only formulation (F12) was optically clear for 7 days as confirmed by optical density. Other formulations (Fl 1, F13-F15) resulted in opaque solutions indicating insolubility; these were failed formulations that slowly crashed out of solution over time and were not assessed for purity. Images of all formulations at t = 0 are shown in Figure 10. pH Stability: All formulations (F1-F15) were pH-stable at 6.0 ± 0.2 for 7 days; this includes failed formulations.

[0264] Concentration: All formulations (Fl -Fl 5) concentration remained constant for 7 days.

[0265] Purity of Compounds: For DCOY101, all formulations (F1-F5) remained between ±3% purity from t = 0 for 7 days. For DCOY102, formulations with D-mannitol only (F7) and 25 / 75 NaCl / D-mannitol (F9) remained between ±2% purity from t = 0 for 7 days. The other formulations (F6, F8, F10) were not tested due to failure of visual appearance. For DCOY103, D-mannitol only formulation (F12) remained between ±1% purity from t = 0 for 7 days. The other formulations (Fl 1, F13-F15) were not tested due to failure of visual appearance. See changes in purity in Figure 2. Discussion

[0266] All isotonic solutions tested with DCOY101 are suitable formulations. The concentrations of D-mannitol or NaCl did not influence stability.

[0267] DCOY102 was only stable to precipitation in isotonic formulations with the addition of 280 mM D-mannitol or 210 mM D-mannitol / 34 mM NaCl. These formulations are lead candidates for clinical formulations for DCOY102 and are further analyzed for stability below. The instability of DCOY102 in the other isotonic formulations indicates that DCOY102 is insoluble at high NaCl salt concentrations. Even though F9 had an additional 34 mM NaCl added for tonicity, no precipitates were present over time. This indicates that there is a unique balance between NaCl and D-mannitol in the presence of DCOY102 before precipitation occurs. DCOY103 was stable in isotonic formulation with the addition of 280 mM D-mannitol over one week at room temperature. This indicates that DCOY103 is very sensitive to the addition of NaCl seeing that all formulations that had NaCl added to the formulation as a tonicity agent precipitated indicating a failed formulation. However, these failed formulations did become clearer over time indicating some increase in solubility with potential degraded products (no purity or concentration tests were performed with failed formulations, so degradation would need to be confirmed analytically).

[0268] The behaviors of DCOY101, DCOY102, and DCOY103 vary in the presence of ±D- mannitol and ±NaCl in isotonic formulations. DCOY101 is the most robust compound while DCOY102 and DCOY103 are more sensitive under these conditions. These differences can be contributed to the linker chemistry since DCOY101, DCOY102, and DCOY103 have the same peptide sequences but different linkers.

[0269] The platform liquid formulation that can be used for DCOY101, DCOY102, and DCOY103 for intranasal administration is as follows: 10 mg / mL peptide conjugate, 20% v / v propylene glycol, 268 - 313 mM D-mannitol in 0.1X PBS, pH 5.5 - 7.0.

[0270] DCOY102 Stability in Isotonic Liquid Formulation

[0271] As shown above, DCOY102 was stable in two leading isotonic formulations: the addition of 280 mM D-mannitol and the addition of 210 mM D-mannitol / 34 mM NaCl over one week at room temperature. The study in the previous section “Isotonic Liquid Formulation of 10 mg / mL Peptide Conjugate” was extended for these selected formulations to further analyze stability. Experimental Design

[0272] DCOY102 (10 mg / mL) evaluation in 280 mOsm / L solutions with tonicity contribution from D-mannitol only (F7) and 1 :3 NaCl:D-mannitol (F9) was extended. Formulations are summarized in Table 3. Here, the stability analysis was continued for the selected formulations for purity (LC-MS) and concentration (LC-MS; 220 nm) up to 28 days at room temperature (20 - 22°C).

[0273] Results

[0274] Data tables summarizing the results of this study were shown in Table 10.

[0275] Table 10. Isotonic formulations with DCOY102 (F7 & F9) analyzed for visual appearance, concentration, and purity (%) at t = 0, 3, 7, 14, 21, & 28 days. a Visual Appearance Key : 0 = optically clear, 1 = 1-2 particulates, 2 = many particulates, 3 = slightly cloudy, 4 = very cloudy, 5 =opaque

[0276] Clarity of Solution: All formulations (F7, F9) were optically clear up to 28 days.

[0277] Concentration: All formulations (F7, F9) concentration remained constant up to 28 days.

[0278] Purity of Compounds: All formulations (F7, F9) remained between ±2% purity up to 28 days. See changes in purity in Figure 3.

[0279] Discussion

[0280] DCOY102 was stable with the addition of 280 mM D-mannitol or 210 mM D- mannitol / 34 mM NaCl isotonic solutions up to 1 month at room temperature (20 - 22°C). Further investigation of stability at storage, accelerated, excursion and stress conditions over >1 month should be tested to understand shelf-life stability.

[0281] The platform formulation (see formulation F7 in Table 3) was chosen to investigate DCOY102 stability with temperature cycling stability in the Section below, “DCOY102 Temperature Cycling Stability”, and with additional excipients in Example 3.

[0282] DCOY102 (10 mg / mL) Stability in Isotonic Liquid Formulation at Various Storage Conditions

[0283] In Section “DCOY102 Stability in Isotonic Liquid Formulation”, DCOY102 was stable in the platform liquid formulation with the addition of 280 mM D-mannitol solution up to 1 month at room temperature (20 - 22°C). Further investigation of stability at storage conditions was evaluated to support preclinical studies and shelf-life of the liquid formulation.

[0284] Experimental Design

[0285] Stability studies included DCOY102 at room temperature (20-22°C), 2-8°C, -30°C, and -80°C. All formulations contained 10 mg / mL peptide conjugate, 20% v / v propylene glycol, 280 mM D-mannitol in 0.1X PBS at pH 6.0. Formulations are summarized in Table 11. All formulations were assessed for solution clarity via visual appearance and purity (LC-MS) for 6 months.

[0286] Table 11. Formulations evaluated for storage conditions for 6 months. All formulations were formulated as 20% (v / v) propylene glycol, 280 mM D-mannitol in 0. IX PBS.

[0287] Results

[0288] Data table summarizing the results of this study shown in Table 12. Table 12. Stability of 10 mg / mL DCOY102 in 20% v / v propylene glycol, 280 mM D- mannitol in 0.1X PBS at pH 6.0 analyzed for visual appearance and purity (%) at t = 0, 0.5, 1, 3, and 6 months. particulates, 3 = slightly cloudy, 4 = very cloudy, 5 =opaque b Visual Appearance results for diluted solution NT = not tested Clarity of solution: All formulations (F1-F4) were optically clear solution up to 3 months at 2-8°, -30°C, and -80°C storage conditions. At 6 months, Fl resulted in clear viscose gel-like material indicating failed formulation, F2 & F4 were optically clear, and F3 was optically clear but with settled precipitates visualized as white opaque substance on the bottom of the vial (vortexed into solution before analysis).

[0289] Purity of Compound: Formulations (F2-F4) remained between ±2% purity up to 6 months at 2-8°, -30°C, and -80°C storage conditions. Formulation Fl remained between ±2% purity up to 1 month at 25°C, then the purity was > -2% purity at t = 3m with a failed solution at t = 6m so it did not undergo analysis.

[0290] Discussion

[0291] DCOY102 (10 mg / mL) in 20% (v / v) propylene glycol, 280 mM D-mannitol in 0.1X PBS at pH 6.0 is stable up to 1 month at room temperature, up to 3 months at -30°C, and up to 6 months at 2-8°C or -80°C. This study suggests the feasibility of short-term storage and shipment conditions at refrigerated or frozen temperatures to support preclinical studies and shelf-life for the liquid formulation.

[0292] DCOY101 and DCOY102 (20 mg / mL) Stability in Isotonic Liquid Formulation at Various Storage Conditions

[0293] Select in vitro and in vivo studies require higher dosing concentrations of API. Previous study in Section “Propylene as Co-Solvent for DCOY101, DCOY102, & DCOY103” supports liquid formulation at 20 mg / mL peptide conjugate. DCOY101 and DCOY102 were formulated at 20 mg / mL in the platform liquid formulation to support shipment and storage conditions.

[0294] Experimental Design

[0295] Stability studies included DCOY101 and DCOY102 at room temperature (20-22°C) and 2-8°C. Procedure for liquid formulation is described in Section “Procedure for liquid Formulation of Peptide Conjugates” with amendment of initial stock concentration of peptide conjugates at 100 mg / mL in 100% (v / v) propylene glycol. All formulations contained 20 mg / mL peptide conjugate, 20% v / v propylene glycol, 280 mM D-mannitol in 0. IX PBS at pH 6.0. Formulations are summarized in Table 13. All formulations were assessed for solution clarity via visual appearance and purity (LC-MS) for 8 weeks with an additional timepoint at 13 months. Table 13. Formulations were evaluated for storage conditions for up to 8 weeks or 13 months. All formulations were formulated as 20% (v / v) propylene glycol, 280 mM D- mannitol in 0.1 X PBS. Results

[0296] Data table summarizing the results of this study shown in Table 14.

[0297] Table 14. Stability of 20 mg / mL DCOY101 or DCOY102 in 20% v / v propylene glycol, 280 mM D-mannitol in 0.1X PBS at pH 6.0 analyzed for visual appearance and purity (%) at t = 0, 0.3, 1, 2, 3, 4, and 8 weeks with ± 13 months. a Visual Appearance Key: 0 = optically clear, 1 = 1-2 particulates, 2 = many particulates, 3 = slightly cloudy, 4 = very cloudy, 5 =opaque b Visual Appearance results for diluted solution

[0298] NT = not tested, N / A = not applicable

[0299] Clarity of solution: Formulations at 25°C room temp (Fl, F3) were optically clear solutions up to 4 weeks and formed clear viscose gel-like material when checked at 8 weeks indicating a failed formulation. Formulations at 2-8°C in the refrigerator (F2, F4) were optically clear solutions up to 13 months.

[0300] Purity of Compound: All formulations remained between ±5% purity up to 4 weeks at room temperature (Fl, F3) and 13 months at 2-8°C (F2, F4). Room temperature formulations (Fl, F3) resulted in failed solutions at t = 8 weeks, therefore they did not undergo analysis.

[0301] Discussion

[0302] Both DCOY101 and DCOY102 (20 mg / mL) in 20% (v / v) propylene glycol, 280 mM D-mannitol in 0.1X PBS at pH 6.0 are stable up to 1 month at room temperature and up to 13 months at 2-8°C. This study suggests feasibility of short-term storage and shipment conditions at high concentration of DCOY101 or DCOY102 at refrigerated temperatures to support preclinical studies and shelf-life for the liquid formulation.

[0303] DCOY102 Temperature Cycling Stability

[0304] For the pan-coronavirus program, preclinical PREP and PEP dosing schemes include multi-day dosing between 3 - 5 days of treatment. Compound stability is important to understand throughout the duration of the study. Here, the stability of DCOY102 was evaluated in the platform formulation as it was subjected to temperature cycling to mimic treatment schemes for preclinical studies if storage and handling procedures suggest compound to be stored in the fridge with treatment dosing at room temperature.

[0305] Experimental Design

[0306] Liquid formulation of DCOY102 will undergo 8X temperature cycles with storage at 2- 8°C and warming to room temperature (20-22°C) to mimic day of treatment use. DCOY102 formulation is described in the Section above, “Isotonic Liquid Formulation of 10 mg / mL Peptide Conjugate”. The samples at t = 0 and select temperature cycles (2X, 4X, 6X, and 8X) was assessed for solution clarity via visual appearance and optical density (ODeoo), purity (LC-MS), concentration (LC-MS; 220 nm), and pH over a total of 8X temperature cycles.

[0307] Results

[0308] Data tables summarizing the results of this study were shown in Table 15.

[0309] Table 15. Temperature cycling of liquid formulation DCOY102 analyzed for visual appearance, optical density (ODeoo), pH, concentration, and purity (%) at t = 0 and 2X, 4X, 6X, 8X temperature cycles. a Visual Appearance Key : 0 = optically clear, 1 = 1-2 particulates, 2 = many particulates, 3 = slightly cloudy, 4 = very cloudy, 5 =opaque

[0310] Clarity of solution: The formulation was optically clear after undergoing 8X temperature cycles. Optical density measurements reported parallel results throughout the entirety of the study. pH Stability: The pH of the formulation remained at pH 6.0 after undergoing 8X temperature cycles.

[0311] Concentration: The formulation concentration remained constant after undergoing 8X temperature cycles. Purity of Compound: The formulation remained between ±2% purity after all 8X temperature cycles compared to t = 0.

[0312] Discussion

[0313] DCOY102 is stable after undergoing 8X temperature cycles from fridge to room temperature in the chosen isotonic formulation. DCOY102 can be stored in the fridge at 2- 8°C and warmed to room temperature for use of up to 8 cycles without concern of stability.

[0314] DCOY102 (50 mg / mL) Concentrated Stock Stability in Propylene Glycol

[0315] For manufacturing the liquid formulation of peptide conjugates, a concentrated stock solution in propylene glycol is required to formulate (see Example 1 and Example 2, “Propylene as Co-solvent for DCOY101, DCOY102, & DCOY103”) prior to dilution in buffer solution due to their insolubility in aqueous solution alone (not shown). Peptide conjugates must be dissolved in propylene glycol at 50 mg / mL. Then, the concentrated stock solution undergoes 1 :5 dilution for 10 mg / mL dosage in 20% propylene glycol (the desired amount for intranasal administration) as general procedure is described in Example 1. It is important to understand the storage stability of the starting material at the research-scale to support manufacturing formulation at large-scale. Here, DCOY102 concentrated stock will be staged in various storage conditions for short-term stability.

[0316] Experimental Design

[0317] Stability studies included DCOY102 (50 mg / mL; 20 pL) in 100% propylene glycol at room temperature (20-22°C), 2-8°C, -30°C, and -80°C. All formulations were assessed for solution clarity via visual appearance and purity (LC-MS) for 6 months. Formulations are summarized in Table 16. All formulations were diluted in water to test purity (10 mg / mL DCOY102, 20% v / v propylene glycol in water).

[0318] Table 16. Formulations evaluated for concentrated stock DCOY102 storage stability. All formulations were formulated in 100% v / v propylene glycol.

[0319] Data tables summarizing the results of this study were shown in Table 13. Results

[0320] Table 17. Stability of 50 mg / mL DCOY102 stock solution in propylene glycol analyzed for visual appearance and purity (%) at t = 0, 0.5, 1, 3, and 6 months. a Visual Appearance Key : 0 = optically clear, 1 = 1-2 particulates, 2 = many particulates, 3 = slightly cloudy, 4 = very cloudy, 5 =opaque b Visual Appearance results for diluted solution NT = not tested

[0321] Clarity of solution: All formulations (F2-F4) were optically clear and resulted in optically clear diluted solution up to 3 months at 2-8°, -30°C, and -80°C storage conditions. Formulation at 25°C (Fl) was optically clear and resulted in optically clear diluted solution up to 1 month. At 3 months, Fl gelated proving difficult to get into solution.

[0322] Purity of Compound: All formulations (F2-F4) remained between ±2% purity up to 3 months at 2-8°, -30°C, and -80°C storage conditions. Formulation Fl remained between ±2% purity up to 1 month at 25°C. Discussion

[0323] The concentrated stock solution of DCOY102 (50 mg / mL) in 100% propylene glycol is stable up to 1 month at room temperature and up to 3 months at 2-8°C, -30°C, and -80°C. This permits batch formulating of the concentrated stock solution and storage for later use to support manufacturing of the liquid formulation across a range of production scales.

[0324] Example 3: Excipient Stability for The Liquid Formulation of DcoylOl

[0325] In Example 2, “Isotonic Liquid Formulation of 10 mg / mL Peptide Conjugate”, a platform formulation for peptide conjugates was identified. Here, further investigation with the platform formulation and DCOY102 was conducted utilizing additional excipients (preservatives etc.) under consideration for inclusion in clinical formulations. Excipients were tested for compatibility with the platform formulation and DCOY102. Excipients tested were surfactants, preservatives, and mucoadhesives to either increase shelf-life stability, preserve the compound, increase solution viscosity optimize the spray particle size and increase contact time with mucosa via intranasal administration, respectively.

[0326] Surfactant Ranging Study using Polysorbate-20

[0327] Polysorbate-20 (PS-20) is a non-ionic surfactant to stabilize drug formulations for increased shelf-life stability. The FDA inactive ingredient guidance limit for nasal route dosage of PS-20 is 2.5% w / w. Here, the percentage of PS-20 was varied to determine starting percentage(s) of PS-20 to eventually test for long-term stability if compatible in the two isotonic formulations for DCOY102 determined in Example 2, “DCOY102 Stability in Isotonic Liquid Formulation”.

[0328] Experimental Design

[0329] PS-20% was ranged from 0 - 0.2% w / v in two isotonic formulations containing D- mannitol only or D-mannitol / NaCl. All formulations contained 10 mg / mL peptide conjugate, 20% v / v propylene glycol in 0.1X PBS at pH 6.0. Formulations are summarized in Table 18. All formulations were assessed for solution clarity via visual appearance and optical density (ODeoo), purity (LC), concentration (LC; 220 nm), and pH for one week at room temperature (20 - 22°C). Table 18. Formulations evaluated for PS-20% ranging study at t = 0, 4, & 7 days in room temperature. All formulations were formulated in 20% v / v propylene glycol in 0. IX PBS at pH 6.0. aNaCl in addition the contribution of the NaCl (13.7 mM) in the 0.1X PBS solution.

[0330] Results

[0331] Data tables summarizing the results of this study were shown in Table 19. Table 19. DCOY102 stability of isotonic formulations in the presence of polysorbate- 20%. Analysis includes visual appearance, optical density (ODeoo), pH, and purity (%) at t = 0, 4, & 7 days in room temperature (20-22°C).

[0332] a Visual Appearance Key : 0 = optically clear, 1 = 1-2 particulates, 2 = many particulates,

[0333] 3 = slightly cloudy, 4 = very cloudy, 5 =opaque

[0334] NT = not tested

[0335] N / A = not applicable

[0336] Clarity of Solution: Formulations in D-mannitol only with PS-20 (F1-F4) were optically clear solutions from 0 - 0.2% PS-20. Optical density measurements reported parallel results throughout the entirety of the study (1 week). Formulations in D-mannitol / NaCl mixture with PS-20 (F5-F7) were optically clear solutions for 0, 0.01, and 0.1% PS-20; whereas formulation at 0.2% PS-20 (F8) resulted in a very cloudy solution. However, optical density measurements showed that both 0.1% PS-20 (F7) and 0.2% PS-20 (F8) were cloudy solutions at t = 0 resulting in failed formulations. Over 3 days, the solutions turned optically clear confirmed by optical density measurements until the end of the study. pH Stability: All formulations (F1-F8) maintained a stable pH at 6.0 ± 0.1 throughout the entirety of the study (1 week).

[0337] Purity of Compound: All formulations (F1-F8) remained within ± 2.0% purity throughout the entirety of the study (1 week).

[0338] Discussion

[0339] DCOY102 isotonic formulation with D-mannitol addition only showed stability over 1 week from 0.01 - 0.2% w / v PS-20. However, formulations with D-mannitol / NaCl mixture only showed stability over 1 week at 0.01% PS-20 at room temperature. Although 0.1 - 0.2% PS-20 resulted in cloudy solutions in the D-mannitol / NaCl mixture initially, this did not influence purity. Results suggest adding PS-20 < 0.2% for D-mannitol only formulation and < 0.01% for D-mannitol / NaCl mixture formulation. Overall, this study also suggests that the addition of NaCl might cause precipitation over time if in the presence of PS-20. Formulation with D-mannitol only (platform formulation) was utilized for subsequent excipient studies. Further investigation with in vitro cell-based or model tissue testing needs to be performed to understand how PS-20 may influence the potency of DCOY102.

[0340] Preservative Ranging Study using Benzalkonium Chloride and Phenethyl Alcohol

[0341] Commonly used preservatives in intranasal formulations are benzalkonium chloride and phenethyl alcohol. The FDA inactive ingredient guidance limit for nasal route dosage of benzalkonium chloride and phenethyl alcohol is 0.12% w / w and 0.25% w / w, respectively. Here, benzalkonium chloride and phenethyl alcohol were tested at high and low percentages with DCOY102 formulation to identify preservative(s) that are compatibility with the platform formulation for intranasal administration.

[0342] Experimental Design

[0343] Benzalkonium chloride and phenethyl alcohol were tested at 0.01% and 0.10%. All formulations contained 10 mg / mL DCOY102, 20% v / v propylene glycol, 280 mM D- mannitol in 0.1X PBS at pH 6.0. Formulations are summarized in Table 20. All formulations were assessed for solution clarity via visual appearance and optical density (ODeoo), purity (LC-MS), concentration (LC-MS; 220 nm), and pH for one week at room temperature (20 - 22°C).

[0344] Table 20. Formulations evaluated for preservative study with benzalkonium chloride and phenethyl alcohol at t = 0, 2, & 7 days in room temperature. All formulations were formulated in 20% v / v propylene glycol, 280 mM D-mannitol in 0.1X PBS at pH 6.0 in addition to %preservative.

[0345] Results

[0346] Data tables summarizing the results of this study were shown in Table 21.

[0347] Table 21. DCOY102 stability in the presence of benzalkonium chloride and phenethyl alcohol. Analysis includes visual appearance, optical density (ODeoo), pH, purity (%), and concentration at t = 0, 2, & 7 days in room temperature (20-22°C). a Visual Appearance Key : 0 = optically clear, 1 = 1-2 particulates, 2 = many particulates,

[0348] 3 = slightly cloudy, 4 = very cloudy, 5 =opaque

[0349] NT = not tested

[0350] N / A = not applicable

[0351] Clarity of Solution: All formulations remained optically clear solutions throughout the entirety of the study. Optical density measurements reported parallel results throughout the entirety of the study (1 week). pH Stability. All formulations maintained a stable pH at 6.0 ± 0.2 throughout the entirety of the study (1 week).

[0352] Purity of Compound: Benzalkonium chloride formulations 0.01% w / v (Fl) and 0.10% w / v (F2) showed decreased purity at t = 0 when compared to formulation without benzalkonium chloride (use F7 t = 0 in Section 3.3). By the end of the study, formulations Fl and F2 decreased purity by 6.5% and 28.7%, respectively. Conversely, phenethyl alcohol formulations 0.01% v / v (F5) and 0.10% v / v (F6) maintained stable purity ±2% throughout the entirety of the study. See changes in purity in Figure 4.

[0353] Concentration: All formulations maintained initial concentration over the entirety of the study (1 week).

[0354] Discussion

[0355] DCOY102 is sensitive to benzalkonium chloride content. At even low percentage of 0.01% w / v, DCOY102 purity decreased >5% over 1 week. Conversely, DCOY102 remains stable using phenethyl alcohol at 0.01 & 0.2% v / v over 1 week. This positions phenethyl alcohol as the better candidate for preservative addition for the formulation of DCOY102.

[0356] Preservative Ranging Study using Methylparaben and Propylparaben

[0357] Commonly used preservatives in intranasal formulations are methylparaben and propylparaben. The FDA inactive ingredient guidance limit for nasal route dosage of methylparaben and propylparaben is 0.7% w / w and 0.3% w / w, respectively. Here, methylparaben and propylparaben were tested at high and low percentages with DCOY102 formulation to identify preservative(s) that are compatibility with intranasal administration.

[0358] Experimental Design

[0359] Methylparaben was tested at 0.01% w / v and 0.30%w / v, and propylparaben was tested at 0.01% w / v only due to solubility limitations. All formulations contained 10 mg / mL DCOY102, 20% v / v propylene glycol, 280 mM D-mannitol in 0.1X PBS at pH 6.0. Formulations are summarized in Table 22. All formulations were assessed for solution clarity via visual appearance and optical density (ODeoo), purity (LC-MS), concentration (LC-MS; 220 nm), and pH for one week at room temperature (20 - 22°C).

[0360] Table 22. Formulations evaluated for preservative study with methylparaben and propylparaben at t = 0, 4, & 7 days in room temperature. All formulations were formulated in 20% v / v propylene glycol, 280 mM D-mannitol in 0.1X PBS at pH 6.0 in addition to %preservative.

[0361] Results

[0362] Data tables summarizing the results of this study were shown in Table 23.

[0363] Table 23. DCOY102 stability in the presence of methylparaben and propylparaben.

[0364] Analysis includes visual appearance, optical density (ODeoo), pH, and purity (%) at t = 0, 4, & 7 days in room temperature (20-22°C). a Visual Appearance Key : 0 = optically clear, 1 = 1-2 particulates, 2 = many particulates,

[0365] 3 = slightly cloudy, 4 = very cloudy, 5 =opaque

[0366] NT = not tested

[0367] N / A = not applicable

[0368] Clarity of Solution'. All formulations (F1-F3) remained optically clear solutions throughout the entirety of the study (1 week). Optical density measurements reported parallel results. pH Stability. All formulations (F1-F3) maintained a stable pH at 6.0 ± 0.2 throughout the entirety of the study (1 week).

[0369] Purity of Compound. All formulations showed stable purity ±2% throughout the entirety of the study (1 week). See changes in purity in Figure 5.

[0370] Discussion

[0371] DCOY102 remains stable using methylparaben at 0.01% and 0.3% w / v, and propylparaben at 0.01% w / v over 1 week. Both parabens are candidate preservatives to add to the formulation for DCOY102.

[0372] Mucoadhesive Ranging Study using CMC-Na and HPMC

[0373] Mucoadhesives are added into formulations for intranasal route administration to increase residence time at the nasal cavity mucosal membranes as well as to optimize solution viscosity and resultant droplet size. Commonly used and water soluble mucoadhesives include carboxymethyl cellulose sodium (CMC -Na) and hydroxypropyl methylcellulose (HPMC). The FDA inactive ingredient guidance limit for nasal route dosage of CMC-Na is 2% w / w which was used as a reference for the HPMC limit during study design. Here, CMC- Na and HPMC will be tested at high and low percentages to identify mucoadhesive(s) that are compatible with DCOY102.

[0374] Experimental Design

[0375] CMC-Na and HPMC were tested at 0.01% w / v and 0.10% w / v. All formulations contained 10 mg / mL DCOY102, 20% v / v propylene glycol, 280 mM D-mannitol in 0.1X PBS at pH 6.0. Formulations are summarized in Table 20. All formulations were assessed for solution clarity via visual appearance and optical density (ODeoo), purity (LC-MS), concentration (LC-MS; 220 nm), and pH for one week at room temperature (20 - 22°C).

[0376] Table 24. Formulations evaluated for mucoadhesive study with CMC-Na and HPMC at t = 0, 4, & 7 days in room temperature. All formulations were formulated in 20% v / v propylene glycol, 280 mM D-mannitol in 0. IX PBS at pH 6.0 in addition to %mucoadhesive.

[0377] Results

[0378] Data tables summarizing the results of this study were shown in Table 25.

[0379] Table 25. DCOY102 stability in the presence of mucoadhesives, CMC-Na and HPMC. Analysis includes visual appearance, optical density (ODeoo), pH, and purity (%) at t = 0, 4, & 7 days in room temperature (20-22°C). a Visual Appearance Key : 0 = optically clear, 1 = 1-2 particulates, 2 = many particulates, 3 = slightly cloudy, 4 = very cloudy, 5 =opaque NT = not tested

[0380] N / A = not applicable

[0381] Clarity of Solution'. All formulations remained optically clear solutions throughout the entirety of the study. Optical density measurements reported parallel results throughout the entirety of the study (1 week). pH Stability. All formulations maintained a stable pH at 6.0 ± 0.2 throughout the entirety of the study except for 0.01% w / v HPMC (F3) which drifted +0.6 after 7 days.

[0382] Purity of Compound. All formulations showed stable purity ±2% throughout the entirety of the study except 0.01% w / v CMC-Na (Fl), dipping at -4% at day 4 but rising back within the ±2% at 7 days. This could be an outlier / artifact at day 4, but replicates would need to be tested to confirm this.

[0383] Discussion

[0384] DCOY102 is stable using mucoadhesives CMC-Na & HPMC between 0.01 - 0.1% w / v over one week. Both CMC-Na and HPMC are candidate excipients to add if mucoadhesion and / or viscosity enhancements desired in the formulation for DCOY102.

[0385] Example 4: Forced Degradation of Peptide Conjugates

[0386] Forced degradation studies were performed on peptide conjugates DCOY101, DCOY102, and DCOY103 to establish and compare their degradation pathways and to determine their intrinsic stability. Three forced degradation conditions were evaluated: (1) hydrolysis by acid, (2) hydrolysis by base, and (3) oxidation by peroxide.

[0387] Hydrolysis by Acid using Hydrochloric Acid

[0388] Experimental Design DCOY101, DCOY102, and DCOY103 (1 mg / mL) were tested for hydrolysis using 0.1M and 1.0M hydrochloric acid (HC1). Formulations are summarized in Table 22. All formulations were assessed for solution clarity via visual appearance and purity & impurities (LC-MS) over one week at timepoints t = 0, 1 or 2, and 7 days at room temperature (20 - 22°C). Table 26. Forced degradation for hydrolysis by acid for DCOY101, DCOY102, and DCOY103 at t = 0, 1 or 2, & 7 days in room temperature. All formulations used 20% v / v propylene glycol with aqueous HC1 solution. Results

[0389] Data tables summarizing the results of this study were shown in Table 27.

[0390] Table 27. Forced degradation by hydrolysis of 1 mg / mL DCOY101, DCOY102, and DCOY103 using 0.1M and 1.0M HC1. Samples were analyzed for visual appearance and purity (%) at t = 0, 1 or 2, & 7 days. a Visual Appearance Key : 0 = optically clear, 1 = 1-2 particulates, 2 = many particulates, 3 = slightly cloudy, 4 = very cloudy, 5 =opaque

[0391] Clarity of Solution: In the presence of 1.0M HC1, DCOY102 (F2) and DCOY103 (Fl 4) were slightly cloudy and became optically clear by the end of the study, whereas DCOY101 (F9) was optically clear throughout the entirety of the study. All formulations at 0. IM HC1 were optically clear throughout the entirety of the study, except DCOY102 (Fl) which became slightly cloudy.

[0392] Purity of Compound: For DCOY101, both concentrations of HC1 at 0. IM (F8) and 1.0M (F9) degraded the compound producing 18.5% and 79.4% total impurities, respectively. Samples slightly decreased in total impurities throughout the entirety of the study, except for 1.0M HC1 sample (F9) at 2 days which may be an outlier. Replicates must be performed to confirm this (see Figure 6, left). For DCOY102, sample stressed with 0.1M HC1 (Fl) only resulted in 5.2% total impurities which is comparable to the control (see Table 5; F7 95.2% purity at t = 0) and remained within range of control throughout the entirety of the study. Increased HC1 concentration to 1.0M, samples (F2) resulted in 49.2% total impurities, reaching 100% degradation at the end of the study (see Figure 6, middle). For DCOY103, both concentrations of HC1 at 0.1M (F13) and 1.0M (F14) degraded the compound producing 25.2% and 81.3% total impurities, respectively. Samples decreased in total impurities to 7.6% and 49.9% by the end of the study (see Figure 6, right).

[0393] Discussion

[0394] DCOY102 is resistant to hydrolysis at low HC1 concentration, while the other peptide conjugates DCOY101 and DCOY103 show approximately 20% degradation. All peptide conjugates are sensitive to high HC1 concentration, however, DCOY102 is slightly more resistant by degrading only 50% upon contact of 1.0M HC1 compared to DCOY101 and DCOY103 with 80% degradation.

[0395] Hydrolysis by acid is a degradation pathway for all peptide conjugates with varying degree and rate of degradation: DCOY102 >1.0M and DCOY101 / DCOY103 at > 0.1M. These peptide conjugates differ by linker chemistry which may play a role in hydrolytic resistance for DCOY102. Further investigation on elucidating the degradation product structures is required to confirm. For pH titration to formulations containing peptide conjugates, final concentration of HC1 should be < 0.1M to avoid unwanted hydrolysis.

[0396] Hydrolysis using Sodium Hydroxide

[0397] Experimental Design

[0398] DCOY101, DCOY102, and DCOY103 (1 mg / mL) were tested for hydrolysis using 0.1M and 1.0M sodium hydroxide (NaOH). Formulations are summarized in Table 28. All formulations were assessed for solution clarity via visual appearance and purity & impurities (LC-MS) over one week at timepoints t = 0, 1 or 2, and 7 days at room temperature (20 - 22°C).

[0399] Table 28. Forced degradation for hydrolysis by base for DCOY101, DCOY102, and DCOY103 at t = 0, 1 or 2, & 7 days in room temperature. All formulations used 20% v / v propylene glycol with aqueous NaOH solution. Results

[0400] Data tables summarizing the results of this study were shown in Table 29.

[0401] Table 29. Forced degradation by hydrolysis of 1 mg / mL DCOY101, DCOY102, and

[0402] DCOY103 using 0.1M and l.OM NaOH. Samples were analyzed for visual appearance and purity (%) at t = 0, 1 or 2, & 7 days. a Visual Appearance Key : 0 = optically clear, 1 = 1-2 particulates, 2 = many particulates, 3 = slightly cloudy, 4 = very cloudy, 5 =opaque

[0403] Clarity of Solution: In the presence of 1 ,0M NaOH, DCOY102 (F2) and DCOY103 (Fl 4) were slightly cloudy and became optically clear by the end of the study, whereas DCOY101 (F9) was optically clear throughout the entirety of the study. All formulations at O. lM NaOH were optically clear throughout the entirety of the study, except DCOY102 (Fl) which became slightly cloudy.

[0404] Purity of Compound: For DCOY101, samples stressed with 0.1M NaOH (F10) and 1 ,0M NaOH (F 11) degraded the compound producing 8.6% and 34.2% total impurities, respectively. After 1 day, samples quickly increased total impurities to 95.1 and 100% and was sustained throughout the study (see Figure 7, left). For DCOY102, samples stressed with 0.1M NaOH (F3) and 1.0M NaOH (F4) degraded the compound producing 6.4% and 39.2% total impurities, respectively. Samples increased total impurities to 100% and 96.5% by the end of the study (see Figure 7, middle). For DCOY103, samples stressed with 0. IM NaOH (F13) and l.OM NaOH (F14) degraded the compound producing 4.1% and 49.9% total impurities, respectively. After 1 day, samples quickly increased total impurities to 96.6 and 100% and were sustained throughout the study (see Figure 7, right).

[0405] Discussion

[0406] All peptide conjugates are sensitive to hydrolysis by base. DCOY102 is slightly less sensitive to hydrolysis with NaOH compared to DCOY101 and DCOY103. Complete degradation of DCOY101 and DCOY103 occurs within the first 24 hours of contact with >0.1M NaOH where DCOY102 reaches complete degradation by one week. These impurities with NaOH have been seen in Example 2, the “pH Assessment” section, confirming that those impurities are caused by pH titration with NaOH.

[0407] Hydrolysis by base is a degradation pathway for all peptide conjugates with varying rates of degradation. These peptide conjugates differ by linker chemistry which may play a role varying degradation kinetics for DCOY102.

[0408] Oxidation using Hydrogen Peroxide

[0409] Experimental Design

[0410] DCOY101, DCOY102, and DCOY103 (1 mg / mL) were tested for oxidation using 3.0% v / v hydrogen peroxide (H2O2). Formulations are summarized in Table 30. All formulations were assessed for solution clarity via visual appearance and purity & impurities (LC-MS) over one week at timepoints t = 0, 1 or 2, and 7 days at room temperature (20 - 22°C).

[0411] Table 30. Forced degradation study for oxidation for DCOY101, DCOY102, and DCOY103 at t = 0, 1 or 2, & 7 days in room temperature. All formulations used 20% v / v propylene glycol with aqueous hydrogen peroxide (H2O2) solution. Results

[0412] Data tables summarizing the results of this study were shown in Table 31.

[0413] Table 31. Forced degradation by oxidation of 1 mg / mL DCOY101, DCOY102, and DCOY103 using 3% hydrogen peroxide (H2O2). Samples were analyzed for visual appearance and purity (%) at t = 0, 1 or 2, & 7 days. a Visual Appearance Key : 0 = optically clear, 1 = 1-2 particulates, 2 = many particulates, 3 = slightly cloudy, 4 = very cloudy, 5 =opaque

[0414] Clarity of Solution: In the presence of 3.0% H2O2, DCOY101 (Fl 2) and DCOY102 (F6) samples were slightly cloudy. DCOY102 (F6) became optically clear by the end of the study, whereas DCOY101 (F12) had precipitated by the end of the study. DCOY103 (F17) was optically clear throughout the entirety of the study.

[0415] Purity of Compound: For DCOY101, samples stressed with 3% H2O2 (F 12) degraded the compound to 17.3% total impurities initially. Samples increased total impurities gradually to 62.3% by the end of the study (see Figure 8, left). For DCOY102, samples stressed with 3% H2O2 (F6) degraded the compound to 4.8% total impurities which is comparable to the control (see Table A5; F7 95.2% purity at t = 0) and remained within range of control for 2 days. Then, samples slightly increased total impurities to 19.1% by the end of the study (see Figure 8, middle). For DCOY103, samples stressed with 3% H2O2 (F17) degraded the compound to 13.7% total impurities initially. Samples increased total impurities gradually to 46.5% by the end of the study (see Figure 8, right).

[0416] Discussion

[0417] DCOY102 is less sensitive to oxidation with hydrogen peroxide compared to

[0418] DCOY101 and DCOY103. DCOY102 was uninfluenced by hydrogen peroxide for the first 48 hours, whereas DCOY101 and DCOY103 showed approximately 20-30% degradation. The rate of degradation caused by oxidation varies between the peptide conjugates.

[0419] Oxidation by 3.0% H2O2 is a degradation pathway for all peptide conjugates at varying degrees. These peptide conjugates differ by linker chemistry which may play a role varying degradation kinetics for DCOY102.

[0420] CONCLUSIONS

[0421] A series of exploratory stability studies were performed on peptide conjugates to develop a stable platform liquid formulation, to identify suitable excipients, and to elucidate their degradation pathway(s). The key results of these studies can be summarized as:

[0422] Propylene glycol can be used as co-solvent to develop formulations for DCOY101, DCOY102, and DCOY103.

[0423] DCOY102 and DCOY103 are soluble at a maximum concentration of 230 mg / mL in propylene glycol. This supports use as co-solvent for intranasal formulation development for <20% v / v propylene glycol at clinical dosage strength of 10 mg / mL.

[0424] Concentrated stock solution of DCOY102 (50 mg / mL) in propylene glycol is stable at room temperature, 2-8°C, -30°C, and -80°C for up to 1 month.

[0425] DCOY102 and DCOY103 can reach doses up to 45 mg / mL at 20% v / v propylene glycol.

[0426] A platform liquid formulation was identified and was stable for one week in ambient conditions: 10 mg / mL peptide conjugate, 20% v / v propylene glycol, 280 mM D-mannitol in 0.1X PBS, pH 6.0.

[0427] DCOY102 is stable for 1 month in the platform liquid formulation under ambient conditions and can undergo 8X temperature cycling from 2-8°C to room temperature without influencing stability.

[0428] Suggested preservatives for formulating with DCOY102 are phenethyl alcohol (<0.1% v / v), methylparaben (<0.3% w / v), or propylparaben (<0.01% w / v). DCOY102 was stable over 1 week at room temperature with these preservatives.

[0429] Benzalkonium chloride degrades DCOY102, and its use should be avoided during clinical formulation development.

[0430] Suggested mucoadhesives for formulating with DCOY102 are CMC -Na (<0.1% w / v) or HPMC (<0.1% w / v) as both were stable over 1 week at room temperature.

[0431] DCOY102 is more resistant to forced hydrolysis by acid at 0.1M HC1 compared to DCOY101 and DCOY103. All peptide conjugates are sensitive to hydrolysis at 1.0M HC1. All peptide conjugates tested are sensitive to forced hydrolysis by base at 0.1 and l.OM NaOH.

[0432] For pH titration during formulation with peptide conjugates, the final concentration of HC1 and NaOH should be < 0.1 M to avoid degradation. DCOY102 is resistant to forced oxidation at 3.0% H2O2 for 48 hours showing minimal degradation after one week, whereas DCOY101 and DCOY103 are sensitive to oxidation.

[0433] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

[0434] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. It will also be understood that none of the embodiments described herein are mutually exclusive and may be combined in various ways without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A pharmaceutical composition for the prevention or treatment of a condition or disease associated with a coronaviral infection, comprising an effective amount of peptide conjugate dissolved in a first solvent; wherein the peptide conjugate has the formula of (Peptide-Linker)n-B-Hydrophobic Moiety, wherein each Peptide is independently a therapeutic peptide or a targeting peptide, provided that at least one Peptide is a therapeutic peptide, each Linker is independently an optional bivalent linking moiety, B is a multivalent moiety, Hydrophobic Moiety is a lipid or derivative thereof, and n is an integer selected from 1, 2, 3 or more; the first solvent is selected from ethanol, glycerol, polyethylene glycol, propanediol (propane-1, 3-diol), butylene glycol (butane- 1,3 -diol), butane-2,3,-diol, butane- 1,2-diol, triacetin, dimethyl sulfoxide, isopropyl alcohol, propylene glycol, propylene glycol derivative, vegetable oil and any combination thereof; and the pharmaceutical composition has a pH of about 4.5 to about 7.8.

2. The pharmaceutical composition of claim 1, further comprising a second solvent selected from water, saline solution, and phosphate-buffered saline (PBS).

3. The pharmaceutical composition of claim 1 or claim 2, wherein the peptide conjugate has a concentration of about Img / mL to about 50mg / mL.

4. The pharmaceutical composition of any one of the preceding claims, wherein the first solvent is propylene glycol.

5. The pharmaceutical composition of any one of the preceding claims, wherein the first solvent and the second solvent have a volume ratio of 100:0 to 1 : 100.

6. The pharmaceutical composition of any one of the preceding claims, wherein the second solvent is phosphate-buffered saline (PBS).

7. The pharmaceutical composition of any one of claims 1-5, wherein the second solvent is water.

8. The pharmaceutical composition of any one of the preceding claims, wherein the first solvent is propylene glycol, and the pharmaceutical composition comprises propylene glycol at a concentration of no more than about 50%, about 40%, about 30%, or about 20% v / v.

9. The pharmaceutical composition of any one of the preceding claims, wherein the first solvent is propylene glycol, and the pharmaceutical composition comprises propylene glycol at a concentration of about 20% v / v.

10. The pharmaceutical composition of claim 6, wherein the PBS is diluted to a value between 0.01X and IX (e.g., about 0.5X, about 0.2X, about 0.1X, about 0.05X; preferably, the PBS is diluted to about 0. IX).

11. The pharmaceutical composition of any one of the preceding claims, further comprising one or more of an excipient, a surfactant, a preservative, an osmotic agent, and a mucoadhesive.

12. The pharmaceutical composition of claim 10, wherein the osmotic agent is D- mannitol, sodium chloride (NaCl), or a mixture of both.

13. The pharmaceutical composition of any one of the preceding claims, the peptide conjugate has a concentration of about 5mg / mL, about 6mg / mL, about 7mg / mL, about 8mg / mL, about 9mg / mL, about lOmg / mL, about l lmg / mL, about 12mg / mL, about 13mg / mL, about 14mg / mL, or about 15mg / mL.

14. The pharmaceutical composition of any one of the preceding claims, wherein the first solvent and the second solvent have a volume ratio of about 5: 100, about 10: 100, about 15: 100, about 20:100, about 25: 100, about 30: 100, about 35: 100, or about 40: 100.

15. The pharmaceutical composition of any one of the preceding claims, the pharmaceutical composition has a pH of about 5.0 to about 7.0, preferably about 6.0.

16. The pharmaceutical composition of any one of the preceding claims, wherein Hydrophobic Moiety is selected from a cholesterol, a cholesterol ester, a phospholipid, and a sphingolipid (preferably, a cholesterol).

17. The pharmaceutical composition of any one of the preceding claims, wherein the B moiety comprises one or more thioether groups, one or more diamino acids, or both one or more thioether groups and one or more diamino acids.

18. The pharmaceutical composition of claim 17, wherein the one or more amino acids selected from cysteine, lysine, arginine, ornithine, and diaminopimelic acid (DAP).

19. The pharmaceutical composition of any one of the preceding claims, wherein the B moiety further comprises -(CH2O)m-, -(0CH2)m-, -(CH2CH2O)m-, -(OCH2CH2)m-, -O-, -S-, - NH-, -CONH-, -NHCO-, -COO-, -OCO-, Ci-6-alkyl, substituted or unsubstituted 5- or 6- membered aryl, substituted or unsubstituted 5- or 6-membered heteroaryl, substituted or unsubstituted 5- or 6-membered cyclic alkyl, substituted or unsubstituted 5- or 6-membered cyclic heteroalkyl, or any combination thereof; m is an integer between 1 and 8.

20. The pharmaceutical composition of any one of the preceding claims, wherein each Linker is independently an amino acid linker that comprises one or more glycine (G), serine (S), alanine (A), or any combination thereof and has about 2 to about 20 amino acids in length.

21. The pharmaceutical composition of any one of the preceding claims, wherein the therapeutic peptide is a peptide inhibitor against a coronavirus selected from HCoV-OC43, HCoV-HKUl, HCoV-229E, HCoV-NL63, SARS-CoV, MERS-CoV, SARS-CoV-2, and any variant thereof; and the targeting peptide is a receptor binding domain (RBD) binding peptide or an ACE2 targeting peptide.

22. The pharmaceutical composition of any one of the preceding claims, wherein the therapeutic peptide is a HRC peptide of a coronavirus, or an analog thereof.

23. The pharmaceutical composition of any one of the preceding claims, wherein the therapeutic peptide is a peptide inhibitor against SARS-CoV-2 or any variant thereof.

24. The pharmaceutical composition of any one of the preceding claims, wherein the therapeutic peptide is selected from SEQ ID Nos. 1-5:Acn-DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL (SEQ ID NO. 1), wherein n is 0 or 1; dldGdSdldD NASVVNIQKEIDRLNEVAKNLNESLIDLQEL (SEQ ID NO. 2)DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGSGSG (SEQ ID NO. 3) dldGdSdldD NASVVNIQKEIDRLNEVAKNLNESLIDLQELGSGSG (SEQ ID NO.4) andH2N-DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGSGSG (SEQ ID NO.5).

25. The pharmaceutical composition of any one of the preceding claims, wherein the peptide conjugate is selected from DCOY101, DCOY102, and DCOY103 (preferably, DCOY101 or DCOY102; more preferably, DCOY102).

26. A pharmaceutical composition for the prevention or treatment of a condition or disease associated with a coronaviral infection (e.g., SARS-CoV-2 viral infection), comprising an effective amount of peptide conjugate dissolved in a first solvent; wherein the peptide conjugate is selected from DCOY101, DCOY102, and DCOY103; the first solvent is selected from ethanol, glycerol, polyethylene glycol, propanediol (propane-1, 3-diol), butylene glycol (butane- 1,3 -diol), butane-2,3,-diol, butane- 1,2-diol, triacetin, dimethyl sulfoxide, isopropyl alcohol, propylene glycol, propylene glycol derivative, vegetable oil and any combination thereof; and the pharmaceutical composition has a pH of about 4.5 to about 7.8.

27. A method of treating or preventing a condition or disease associated with a coronavirus in a subject in need, wherein the method comprises intranasally administering the pharmaceutical composition of any one of the preceding claims to the subject.

28. The method of claim 27, wherein the intranasal administration is via an intranasal spray, an inhaler, or a nebulizer.

Images