Compositions of Antiviral Peptides and Methods of Use Thereof

Abstract

Provided are broad-spectrum antiviral peptides and compositions comprising a therapeutically effective amount of an antiviral peptide and a pharmaceutically acceptable carrier. The antiviral compositions exhibit a strong broad-spectrum antiviral effect without generating viral resistance. The antiviral compositions can be used to treat diseases caused by viral infections, particularly respiratory viruses such as enveloped coronaviruses (SARS-CoV-2, SARS-CoV, and MERS-CoV), pandemic A (H1N1)pdm09 virus, avian influenza A (H7N9) virus, and non-enveloped rhinoviruses.

Description

[0001] This international patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 991,407, filed on March 18, 2020, the entire content of which is incorporated herein by reference for all purposes. Field of the Invention

[0002] The present invention generally relates to broad-spectrum antiviral peptides and methods of treating antiviral infections. Background of the Invention

[0004] Due to the lack of pre-existing immunity, novel respiratory viruses typically cause severe respiratory infections and spread rapidly. In the past two decades, three highly pathogenic coronaviruses have crossed the species barrier and caused human diseases, including the bat-associated Severe Acute Respiratory Syndrome (SARS) coronavirus (CoV) (SARS-CoV) in 2003 1,2 , the Middle East Respiratory Syndrome coronavirus (MERS-CoV) since 2012 3,4 and the most recent 2019 novel coronavirus (SARS-CoV-2) 5 . In addition, the 2009 pandemic influenza A (H1N1)pdm09 virus caused the first influenza pandemic of the 21st century 6,7 . Due to the lack of effective antiviral drugs, especially against coronaviruses, these respiratory viruses are associated with significant morbidity and mortality. In addition, these emerging respiratory viruses have also caused severe economic and social disruptions.

[0005] The outbreak of COVID-19 clearly illustrates the importance of broad-spectrum antiviral drugs. Currently, there is no specific drug treatment for this novel virus. An effective broad-spectrum antiviral agent would improve patient outcomes and potentially reduce community and hospital transmission even before the emergence of a novel virus and the identification of a specific antiviral drug. The "one drug for one disease" approach to antiviral drugs has been successful for HIV, hepatitis C virus, and influenza virus 9 . However, there is an urgent need for broad-spectrum antiviral drugs to combat emerging and re-emerging novel virus outbreaks, such as SARS-CoV-2, before the identification of a novel virus or the availability of a specific antiviral drug. Summary of the Invention

[0007] One object of the present invention is to provide a broad-spectrum antiviral agent.

[0008] Another object of the present invention is to provide a composition of a broad-spectrum antiviral agent.

[0009] Yet another object of the present invention is to provide a method of treating a viral infection in a subject in need thereof.

[0010] Antiviral agents, compositions containing the antiviral agents, and methods of using the same are provided. The antiviral agents include P9R (SEQ ID NO:2) or P9R-like peptides derived from P9R, which are characterized by their "inhibition of endosomal acidification" and "peptide-virus binding", such as as determined by in vitro endosomal acidification and peptide-virus binding assays. In a preferred embodiment, the antiviral agent is P9R. The antiviral composition includes a therapeutically effective amount of the antiviral agent.

[0011] The antiviral composition can be administered to a subject in need thereof to treat symptoms associated with a viral infection. Preferably, the subject is infected with a respiratory virus, and more preferably, the subject is infected with a pH-dependent virus that requires endosomal acidification for virus-host membrane fusion. Examples include, but are not limited to, enveloped coronaviruses (SARS-CoV-2, SARS-CoV, and MERS-CoV), pandemic A (H1N1)pdm09 virus, avian influenza A (H7N9) virus, and non-enveloped rhinovirus. Brief Description of the Drawings

[0013] Figure 1A Shows the peptide sequences (P9 (SEQ ID NO:1); P9R (SEQ ID NO:2); PA1 (SEQ ID NO:3); and P9RS (SEQ ID NO:4)) and the positive charges analyzed by PepCalc of InnovaGen. Figure 1B-1H Shows that P9R inhibits viral replication of 2019 novel coronavirus (SARS-CoV-2), MERS-CoV, SARS-CoV, H1N1 virus, H7N9 virus, rhinovirus, and parainfluenza virus type 3 in cells. The virus was pre-mixed with different concentrations of P9R or P9 and then used to infect cells. The antiviral efficiency was evaluated by plaque reduction assay. The infection (%) was calculated by dividing the number of plaques of peptide-treated virus by the number of plaques of BSA-treated virus. Figure 1I The potent antiviral activity of P9R against the virus was shown by measuring the viral RNA copies in the supernatant 24 h post-infection when the virus was treated with P9R or BSA (50 - 100 μg mL -1 ). Figure 1J Shows the cytotoxicity of P9R in MDCK, Vero E6, and A549 cells. When the IC 50 of P9R was compared with the IC 50 of P9, * indicates P < 0.05, and ** indicates P < 0.01. The P value was calculated by two-tailed Student's t-test. The data are presented as the mean ± SD of at least three independent experiments.

[0014] Figure 2AShows the quantification of red fluorescence of endosomal acidification in MDCK cells treated with peptides. Red fluorescence intensity was calculated from 10 random microscopic fields. Figure 2B Shows the antiviral activities of P9R, P9RS, and PA1 against SARS-CoV-2 and A(H1N1)pdm09 viruses measured by plaque reduction assay at 25 μg mL -1 The plaque number (%) of peptide-treated virus was normalized to BSA-treated virus. Figure 2C Shows the binding of P9R and PA1 to SARS-CoV-2 and A(H1N1)pdm09 viruses. Virus-binding peptides were detected by ELISA and RT-qPCR. **Indicates P < 0.01 when compared with P9R. P values were calculated by two-tailed Student's t-test.

[0015] Figure 3A Shows that PA1 can reduce the binding of P9R to SARS-CoV-2 and A(H1N1)pdm09. Viruses were pretreated with PA1 or BSA, and then the binding of the treated viruses to the designated peptides was measured by RT-qPCR. **Indicates P < 0.01 when compared with viruses treated with BSA. (c) P9R can inhibit the release of viral RNP into the nucleus. H1N1 viruses were pretreated with BSA, P9R, or bafilomycin A1 (BA1), and then the treated viruses were used to infect MDCK cells. Images of viral NP (green) and nuclei (blue) were taken 3.5 h after infection. Figure 3B-3D Includes data showing that P9R can bind extensively to MERS-CoV, H7N9 virus, and rhinovirus. The relative RNA copies of virus bound to the peptide were normalized to virus bound to P9R. *Indicates P < 0.05 and **Indicates P < 0.01 when compared with P9R. P values were calculated by two-tailed Student's t-test. Figure 3E Shows peptides that bind to viruses and viral proteins. For peptides binding to SARS-CoV (left panel). SARS-CoV was incubated with the designated peptides on an ELISA plate for 1 h. Unbound virus was washed away and the bound SRAR-CoV was quantified by RT-qPCR. The relative RNA copies (%) were normalized to the RNA copies of virus bound to P9R. Peptides binding to H1N1 HA1 protein (middle panel). Peptides binding to MERS-CoV S protein (left panel). Peptides were coated on an ELISA plate. The binding of H1N1 HA and MERS-CoV S proteins to the peptides was measured by ELISA assay. *Indicates P < 0.05 and **Indicates P < 0.01 when compared with P9R. P values were calculated by two-tailed Student's t-test.

[0016] Figure 4AIt was shown that the therapeutic efficacy of P9R (50 μg / dose) against mice infected with A(H1N1) virus was the same as that of zanamivir (50 μg / dose). At 6 h post-infection, PBS, zanamivir, PA1, P9R or P9 was intranasally inoculated into mice, and two additional doses were administered to the mice within the next day. Each group included five mice. Figure 4B It shows the change in body weight of infected mice corresponding to ( Figure 4A ). Figure 4C It shows the effect of low-dose P9R on mice infected with A(H1N1)pdm09 virus compared with P9. At 6 h post-infection, PBS (n = 10), P9-25 (25.0 μg / dose, n = 5), P9-12.5 (12.5 μg / dose, n = 5), P9R-25 (25.0 μg / dose, n = 10) and P9R-12.5 (12.5 μg / dose, n = 10) were intranasally inoculated into mice and two additional doses were administered to the mice within the next day. Figure 4D It shows the change in body weight of infected mice corresponding to ( Figure 4C ). The P value was calculated by Gehan-Breslow-Wilcoxon test.

[0017] Figure 5A It shows the protocol of the drug resistance assay of zanamivir and P9R. The A(H1N1) virus was passaged in the presence of zanamivir and P9R at the specified concentrations. ND, not detected, because highly resistant H1N1 virus against zanamivir was generated before P16. Figure 5B It shows that zanamivir inhibits the parental A(H1N1) virus (P0). The IC 50 of zanamivir against parental H1N1 is 35 nM. Figure 5C It shows the antiviral efficiency of zanamivir against passaged A(H1N1) virus in the presence of zanamivir. Figure 5D It shows the antiviral efficiency of P9R against passaged A(H1N1) virus in the presence of P9R. The passaged virus was pre-mixed with zanamivir (nM) or P9R (μg mL -1 ) for infection. The supernatant was collected at 24 h post-infection. The virus titer in the supernatant was determined by RT-qPCR. The relative replication (%) was normalized to the corresponding passaged virus without treatment. The data are presented as the mean ± SD of three independent experiments. DETAILED DESCRIPTION OF THE INVENTION

[0019] I. Definitions

[0020] As used herein, "aerosol" refers to any preparation of fine mist-like particles, which can be a solution or a suspension, whether or not it is generated using a propellant.

[0021] "Emulsion" is a composition containing immiscible components homogeneously mixed together.

[0022] As used herein, "hydrophilic" refers to a substance having strongly polar groups that readily interact with water.

[0023] As used herein, "hydrophobic" refers to a substance lacking an affinity for water, tending to repel and not absorb water, and not dissolving in or mixing with water.

[0024] As used herein, "lipophilic" refers to a compound having an affinity for lipids.

[0025] As used herein, "parenteral administration" means administration by any method other than via the digestive tract or non-invasive topical or regional routes.

[0026] As used herein, a "patient" or "subject" to be treated refers to a human or non-human animal.

[0027] As used herein, "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that, within the scope of reasonable medical judgment, are suitable for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0028] As used herein, "pharmaceutically acceptable salt" refers to a derivative of a compound as defined herein, wherein the parent compound is modified by preparing its acidic or basic salt.

[0029] As used herein, "therapeutically effective" or "effective amount" means that the amount of the composition used is an amount sufficient to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires reduction or alteration and does not necessarily have to eliminate. As used herein, the terms "therapeutically effective amount", "therapeutic amount", and "pharmaceutically effective amount" are synonyms. A person skilled in the art can readily determine an appropriate therapeutic amount.

[0030] "Subject" or "patient" refers to a human, a primate, a non-human primate, a laboratory animal, a livestock animal, a domestic animal, or a domestic pet.

[0031] II. Compositions

[0032] The composition of the present invention includes a potent antiviral peptide P9R (NGAICWGPCPTAFRQIGNCGRFRVRCCRIR; SEQ ID NO: 2), which is derived from murine β - defensin - 4 and P9 (NGAICWGPCPTAFRQIGNCGHFKVRCCKIR; SEQ ID NO: 1). Mechanistic studies have shown that the positively charged P9R inhibits viral - host endosomal acidification by binding to different viruses and then prevents the endosomal release of pH - dependent viruses, thereby broadly inhibiting viral replication. We used P9R (which not only binds to viruses but also inhibits endosomal acidification), PA1 (which only binds to viruses), and P9RS (which only inhibits endosomal acidification) to identify and confirm a new antiviral mechanism of basic peptides. The antiviral activity of basic peptides can be enhanced by increasing the positive charge of the peptide and requires both virus binding and inhibition of endosomal acidification.

[0033] A. Antiviral Peptides

[0034] The antiviral peptides of the present disclosure preferably consist of the P9R sequence. However, the antiviral peptide can include peptides derived from P9R, provided that the amino acids at positions 21, 23, and 28 are positively charged amino acids. Thus, the peptide can have the same amino acid sequence as P9R, where the arginines at positions 21, 23, and 28 are replaced by positively charged amino acids such as lysine or histidine. However, it is important that any modification of the P9R structure ensures that the resulting peptide retains the inhibition of endosomal acidification and maintains the same level of virus binding as P9R. Thus, useful P9R - derived peptides (herein, P9R - like peptides) have the properties of "inhibition of endosomal acidification" and "virus binding". As shown in the examples of this application, changing the amino acids in P9R and testing for the required activities ("inhibition of endosomal acidification" and "virus binding") are within the capabilities of those of ordinary skill in the art.

[0035] The "virus binding assay" includes the following steps: Dissolve the peptide (0.1 μg per well) in H2O and coat it onto an ELISA plate, then incubate overnight at 4°C. Then, add 2% BSA to the blocked plate and incubate overnight at 4°C. For virus - peptide binding, dilute the virus in phosphate - buffered saline and then add it to the ELISA plate to bind to the coated peptide at room temperature for 1 h. After washing away the unbound virus, lyse the bound virus with RLT buffer of the RNeasy Mini Kit (Qiagen, Cat# 74106) for viral RNA extraction. Measure the viral RNA copies of the bound virus by RT - qPCR.

[0036] The "endosomal acidification assay" is as described above but slightly modified and can include the detection of endosomal acidification with a pH - sensitive dye (pHrodo Red dextran, Invitrogen, Cat# P10361) according to the manufacturer's instructions.14 First, MDCK cells were treated with BSA (25.0 μg mL -1 ), P9 (25.0 μg mL -1 ), P9R (25.0 μg mL -1 ), PA1 (25.0 μg mL -1 ), or P9RS (25.0 μg mL -1 ) at 4 °C for 15 min. Second, a pH-sensitive dye and DAPI at 100 μg mL -1 were added to the MDCK cells and then incubated at 4 °C for 15 min. Before taking images, the cells were further incubated at 37 °C for 15 min and then washed twice with PBS. Finally, PBS was added to the cells and images were immediately taken using a confocal microscope (e.g., Carl Zeiss LSM 700, Germany).

[0037] Thus, the P9R-derived peptide should have an overall net positive charge of at least 5, preferably at least 5.6, and preferably does not include the amino acid modifications as shown in P9RS (SEQ ID NO: 4). Also preferably, the P9R-derived peptide does not include the introduction of additional amino acid residues at the C-terminal arginine.

[0038] The amino acid substitutions in P9R to obtain P9R-like peptides preferably include conservative amino acid substitutions.

[0039] Examples of conservative amino acid substitutions include those in which the substitution is within one of the following five groups: 1) small aliphatic, nonpolar or micropolar residues (Ala, Ser, Thr, Pro, Gly); 2) polar, negatively charged residues and their amides (Asp, Asn, Glu, Gln); polar, positively charged residues (His, Arg, Lys); large aliphatic, nonpolar residues (Met, Leu, Ile, Val, Cys); and large aromatic residues (Phe, Tyr, Trp). Examples of non-conservative amino acid substitutions are those in which: 1) a hydrophilic residue, such as a seryl or threonyl residue, is replaced (or replaced by) a hydrophobic residue, such as a leucyl, isoleucyl, phenylalanyl, valyl or alanyl residue; 2) a cysteine or proline residue is replaced (or replaced by) any other residue; 3) a residue with a positively charged side chain, such as a lysyl, arginyl or histidyl residue, is replaced (or replaced by) a negatively charged residue, such as a glutamyl or aspartyl residue; or 4) a residue with a bulky side chain, such as a phenylalanine residue, is replaced (or replaced by) a residue without a side chain, such as a glycine residue.

[0040] However, it should be understood that any amino acid or amino acid analogue can be used for substitution at the amino acid position. For example, any naturally occurring amino acid (e.g., alanine, aspartic acid, asparagine, arginine, cysteine, glycine, glutamic acid, glutamine, histidine, leucine, valine, isoleucine, lysine, methionine, proline, threonine, serine, phenylalanine, tryptophan or tyrosine) can be used for substitution at the position.

[0041] B. Formulations

[0042] The peptides described herein can be formulated for enteral, parenteral or pulmonary administration. In a preferred embodiment, the peptide is formulated for pulmonary administration.

[0043] P9R (or a peptide derived therefrom) can be combined with one or more pharmaceutically acceptable carriers and / or excipients that are considered safe and effective, and can be administered to an individual without causing undesirable biological side effects or unwanted interactions. A carrier is all components present in a pharmaceutical formulation other than the one or more active ingredients.

[0044] P9R (or a peptide derived therefrom) disclosed herein can also be formulated to be used as a disinfectant, for example, in a hospital environment.

[0045] 1. Pulmonary formulations

[0046] In one embodiment, P9R (or a P9R-like peptide) is formulated for pulmonary delivery, such as intranasal administration or oral inhalation.

[0047] The respiratory tract is the structure involved in gas exchange between the atmosphere and the bloodstream. The lungs are branched structures that ultimately end in alveoli where gas exchange occurs. The alveolar surface area is the largest in the respiratory system and is where drug absorption takes place. The alveoli are covered by a thin epithelium without cilia or a mucus coating and secrete surfactant phospholipids. The respiratory tract encompasses the upper airway, including the oropharynx and larynx, and then the lower airway, which includes the trachea and then branches into bronchi and bronchioles. The upper airway and the lower airway are referred to as conducting airways. Then the terminal bronchioles divide into respiratory bronchioles, which then lead to the final respiratory zone, the alveoli or the deep lung. The deep lung or alveoli are the main target for inhaled therapeutic aerosols for systemic drug delivery.

[0048] Therapeutic compositions including pulmonary administration of low molecular weight drugs, such as β-androgen antagonists for the treatment of asthma, have been observed. Other therapeutically active agents that are active in the lungs have been systemically administered and targeted for absorption via the lungs. Nasal delivery is considered a promising therapeutic administration technique for the following reasons: Due to the coverage of many microvilli on the epithelial surface, the nose has a large surface area available for drug absorption, the subepithelial layer is highly vascularized, and venous blood from the nose passes directly into the systemic circulation and thus avoids drug loss due to first-pass metabolism in the liver. It offers lower doses, faster attainment of therapeutic blood levels, faster onset of pharmacological activity, fewer side effects, high total blood flow per cm 3 and is highly porous endothelial basement membrane and easily accessible.

[0049] Vectors for pulmonary formulations can be divided into vectors for dry powder formulations and vectors administered as solutions. Aerosols for delivering therapeutic agents to the respiratory tract are known in the art. Aerosols can be produced using standard techniques such as ultrasonic or high-pressure treatment. For administration via the upper respiratory tract, the formulation can be formulated as a solution, such as water or isotonic saline, buffered or unbuffered, or as a suspension for intranasal administration as drops or sprays. Preferably, such solutions or suspensions are isotonic with respect to nasal secretions and have approximately the same pH, for example in the range of about pH 4.0 to about pH 7.4, or pH 6.0 to pH 7.0. The buffer should be physiologically compatible and include, by way of example only, phosphate buffer. For example, a representative nasal decongestant is described as buffered to a pH of about 6.2. A person skilled in the art can readily determine the appropriate saline content and pH of a harmless aqueous solution for nasal and / or upper respiratory tract administration.

[0050] Preferably, the aqueous solution is water, a physiologically acceptable aqueous solution containing salts and / or buffers, such as phosphate buffered saline (PBS), or any other acceptable aqueous solution for administration to animals or humans. Such solutions are well known to those skilled in the art and include but are not limited to distilled water, deionized water, pure water or ultrapure water, saline, phosphate buffered saline (PBS). Other suitable aqueous vehicles include but are not limited to Ringer’s solution and isotonic sodium chloride. Aqueous suspensions can include suspending agents such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone, and tragacanth gum, and wetting agents such as lecithin. Preservatives suitable for aqueous suspensions include ethylparaben and propylparaben.

[0051] As solvents for low toxicity organic (i.e., non-aqueous) Class 3 residual solvents, solvents such as ethanol, acetone, ethyl acetate, tetrahydrofuran, diethyl ether, and propanol can be used in the formulation. The solvent is selected based on its ability to readily atomize the formulation. The solvent should not react detrimentally with P9R (or P9R-like peptides). A suitable solvent should be used to dissolve the compound or form a suspension of P9R (or P9R-like peptides). The solvent should have sufficient volatility to enable the formation of an aerosol of the solution or suspension. Additional solvents or aerosolizing agents, such as Freon, can be added as desired to increase the volatility of the solution or suspension.

[0052] In one embodiment, the composition can contain trace amounts of polymers, surfactants, or other excipients well known to those skilled in the art. As used herein, "trace amounts" means that there are no excipients that may affect or mediate the uptake of P9R (or P9R-like peptides) in the lung, and the excipients present are in amounts that do not have an adverse effect on the uptake of P9R (or P9R-like peptides) in the lung.

[0053] Due to its hydrophobic nature, dry lipid powders can be directly dispersed in ethanol. For lipids stored in organic solvents such as chloroform, an aliquot of the solution is placed in a vial and the chloroform is evaporated under a nitrogen stream to form a dry film on the surface of the glass vial. The film readily swells when reconstituted with ethanol. To fully disperse the lipid molecules in the organic solvent, the suspension is sonicated. A non-aqueous suspension of lipids can also be prepared in absolute ethanol using a reusable PARI LC Jet+ nebulizer (PARI Respiratory Equipment, Monterey, CA).

[0054] Dry powder formulations ("DPF") with large particle sizes have improved flow characteristics, such as less aggregation, easier aerosolization, and potentially less phagocytosis. Dry powder aerosols for inhalation therapy are typically produced with an average diameter mainly in the range of less than 5 microns, although the preferred aerodynamic diameter range is between 1 and 10 microns. Large "carrier" particles (drug-free) are co-delivered with the therapeutic aerosol to assist in achieving efficient atomization and other possible benefits.

[0055] Polymeric particles can be prepared using single emulsion and double emulsion solvent evaporation, spray drying, solvent extraction, solvent evaporation, phase separation, simple coacervation and complex coacervation, interfacial polymerization, and other methods well known to those of ordinary skill in the art. Particles can be prepared using methods known in the art for preparing microspheres or microcapsules. Preferred manufacturing methods are by spray drying and freeze drying, which require the use of a solution containing a surfactant, spraying to form droplets of the desired size, and removing the solvent.

[0056] Particles can be manufactured with appropriate materials, surface roughness, diameter, and tapped density for local delivery to selected regions of the respiratory tract, such as the deep lung or the upper respiratory tract. For example, higher density or larger particles can be used for upper airway delivery. Similarly, mixtures of different sized particles provided with the same or different EGS can be administered in a single dose to target different regions of the lung.

[0057] Formulations for pulmonary delivery include unilamellar phospholipid vesicles, liposomes, or lipoprotein particles. Formulations containing nucleic acids and methods for preparing such formulations are well known to those of ordinary skill in the art. Liposomes are formed from commercially available phospholipids provided by various suppliers including Avanti Polar Lipids, Inc. (Birmingham, Ala.). In one embodiment, the liposome can include ligand molecules specific for receptors on the surface of the target cell to direct the liposome to the target cell.

[0058] 2. Parenteral Formulations

[0059] The P9R (or P9R-like peptide) described herein can be formulated for parenteral administration.

[0060] For example, parenteral administration can include administering to a patient intravenously, intradermally, intraarterially, intraperitoneally, intracranially, intraarticularly, intraprostatically, intrathoracically, intratracheally, intravitreally, intratumorally, intramuscularly, subcutaneously, subconjunctivally, intravesicularly, intrapericardially, intraumbilically, by injection, and by infusion.

[0061] Parenteral formulations can be prepared as aqueous compositions using techniques known in the art. Generally, such compositions can be prepared as injectable formulations, such as solutions or suspensions, solid forms suitable for preparing solutions or suspensions upon addition of a reconstitution medium prior to injection, emulsions such as water-in-oil (w / o) emulsions, oil-in-water (o / w) emulsions, and their microemulsions, liposomes, or emulsomes.

[0062] The carrier can be a solvent or dispersion medium that contains, for example, water, ethanol, one or more polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol), oils such as vegetable oils (such as peanut oil, corn oil, sesame oil, etc.), and combinations thereof. Appropriate fluidity can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and / or by using surfactants. In many cases, it will preferably include isotonic agents such as sugars or sodium chloride.

[0063] Solutions and dispersions of the active compound as the free acid or base or a pharmaceutically acceptable salt thereof can be prepared in water or another solvent or dispersion medium which is suitably admixed with one or more pharmaceutically acceptable excipients, including but not limited to surfactants, dispersants, emulsifiers, pH regulators, viscosity regulators and combinations thereof.

[0064] Suitable surfactants can be anionic, cationic, amphoteric or nonionic surfactants. Suitable anionic surfactants include but are not limited to those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium salts of long-chain alkyl sulfonates and alkylaryl sulfonates such as sodium dodecylbenzenesulfonate; dialkyl sulfosuccinates (such as sodium dodecylbenzenesulfonate); dialkyl sulfosuccinates (such as bis-(2-ethylhexyl)-sulfosuccinate); and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include but are not limited to quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimide, stearyldimethylbenzylammonium chloride, polyoxyethylene and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol monomyristate, glycerol monostearate, glyceryl stearate, polyglyceryl 4-oleate, sorbitan acylates, sucrose acylates, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbate, polyoxyethylene octylphenyl ether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, 401, stearoyl monoisopropanolamide and polyoxyethylene hydrogenated tallowamide. Examples of amphoteric surfactants include sodium N-dodecyl-β-alaninate, sodium N-lauryl-β-imino dipropionate, myristoyl amphoacetate, lauryl betaine and lauryl sulfobetaine.

[0065] The formulation may contain preservatives to prevent the growth of microorganisms. Suitable preservatives include but are not limited to parabens, chlorobutanol, phenol, sorbic acid and thimerosal. The formulation may also contain antioxidants to prevent degradation of the active agent.

[0066] The formulation is typically buffered to a pH of 3 - 8 for parenteral administration upon reconstitution. Suitable buffers include but are not limited to phosphate buffer, acetate buffer and citrate buffer.

[0067] Water-soluble polymers are often used in formulations for parenteral administration. Suitable water-soluble polymers include but are not limited to polyvinylpyrrolidone, dextran, carboxymethylcellulose and polyethylene glycol.

[0068] A sterile injectable solution can be prepared by incorporating the desired amount of P9R (or P9R-like peptide) with one or more of the excipients listed above (as required) into a suitable solvent or dispersion medium, followed by filtration sterilization. Usually, the dispersion is prepared by incorporating the various sterile active ingredients into a sterile vehicle that contains the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredients from its previously sterile-filtered solution. The powder can be prepared in such a way that the particles are essentially porous, which can increase the dissolution of the particles. Methods for preparing porous particles are well known in the art.

[0069] (a) Controlled release formulations

[0070] The parenteral formulations described herein can be formulated for controlled release, including immediate release, delayed release, extended release, pulsed release, and combinations thereof.

[0071] i. Nanoparticles and microparticles

[0072] For parenteral administration, P9R (or P9R-like peptide) and optionally one or more additional active agents can be incorporated into microparticles, nanoparticles, or combinations thereof to provide controlled release of the disclosed P9R (or P9R-like peptide) and / or one or more additional active agents. In embodiments where the formulation contains two or more drugs, the drugs can be formulated for the same type of controlled release (e.g., delayed, extended, immediate, or pulsed) or the drugs can be formulated independently for different types of release (e.g., immediate and delayed, immediate and extended, delayed and extended, delayed and pulsed, etc.).

[0073] For example, P9R (or P9R-like peptide) and / or one or more additional active agents can be incorporated into polymeric microparticles, thereby providing controlled release of the drug. The release of the drug is controlled by diffusion of the drug out of the microparticles and / or degradation of the polymeric particles by hydrolysis and / or enzymatic degradation. Suitable polymers include ethyl cellulose and other natural or synthetic cellulose derivatives.

[0074] Polymers that slowly dissolve in an aqueous environment and form gels, such as hydroxypropyl methylcellulose or polyethylene oxide, can also be suitable as materials for drug-containing microparticles. Other polymers include, but are not limited to, polyanhydrides, poly(ester anhydrides), polyhydroxy acids, such as polylactide (PLA), polyglycolide (PGA), poly(lactide-glycolide) (PLGA), poly-3-hydroxybutyrate (PHB) and its copolymers, poly-4-hydroxybutyrate (P4HB) and its copolymers, polycaprolactone and its copolymers, and combinations thereof.

[0075] Alternatively, the drug can be incorporated into microparticles prepared from materials that are insoluble or slowly soluble in aqueous solution but capable of degrading within the GI tract, and the methods of degradation include enzymatic degradation, surfactant action of bile acids, and / or mechanical erosion. As used herein, the term "slowly soluble in water" refers to materials that do not dissolve in water within a 30-minute period. Preferred examples include fats, fatty substances, waxes, waxy substances, and mixtures thereof. Suitable fats and fatty substances include fatty alcohols (such as lauryl alcohol, myristyl alcohol, stearyl alcohol, cetyl alcohol, or cetearyl alcohol), fatty acids and derivatives, including but not limited to fatty acid esters, fatty acid glycerides (monoglycerides, diglycerides, and triglycerides), and hydrogenated fats. Specific examples include but are not limited to hydrogenated vegetable oils, hydrogenated cottonseed oil, hydrogenated castor oil, hydrogenated oils available under the trade name and hydrogenated oils, stearic acid, cocoa butter, and stearyl alcohol. Suitable waxes and waxy materials include natural or synthetic waxes, hydrocarbons, and common waxes. Specific examples of waxes include beeswax, glycowax, castor wax, carnauba wax, paraffin wax, and candelilla wax. As used herein, waxy materials are defined as any materials that are generally solid at room temperature and have a melting point of about 30 to 300 °C.

[0076] In some cases, it may be desirable to alter the rate of water penetration into the microparticles. To this end, rate control (wicking) agents can be formulated with the fats or waxes listed above. Examples of rate control materials include certain starch derivatives (such as waxy maltodextrin and drum-dried corn starch), cellulose derivatives (such as hydroxypropyl methylcellulose, hydroxypropyl cellulose, methylcellulose, and carboxymethylcellulose), alginic acid, lactose, and talc. Additionally, pharmaceutically acceptable surfactants (such as lecithin) can be added to facilitate the degradation of such microparticles.

[0077] Water-insoluble proteins, such as zein, can also be used as materials for forming drug-containing microparticles. Additionally, water-soluble proteins, polysaccharides, and combinations thereof can be formulated with the drug into microparticles and subsequently crosslinked to form an insoluble network. For example, cyclodextrin can be complexed with individual drug molecules and subsequently crosslinked.

[0078] ii. Methods for preparing nanoparticles and microparticles

[0079] Drug encapsulation or incorporation into a carrier material to produce drug-containing microparticles can be achieved by known pharmaceutical formulation techniques. In the case of formulation in a fat, wax, or wax-like material, the carrier material is typically heated above its melting temperature and the drug is added to form a mixture comprising drug particles suspended in the carrier material, drug dissolved in the carrier material, or a mixture thereof. The microparticles can then be formulated by several methods, including but not limited to processes of coagulation, extrusion, spray cooling, or aqueous dispersion. In a preferred process, the wax is heated above its melting temperature, the drug is added, and as the mixture cools, the molten wax-drug mixture is coagulated with continuous stirring. Alternatively, the molten wax-drug mixture can be extruded and rounded to form pellets or beads. These methods are known in the art.

[0080] For some carrier materials, it may be desirable to use solvent evaporation techniques to produce drug-containing microparticles. In this case the drug and carrier material are co-dissolved in a co-solvent, and microparticles can then be produced by several techniques, including but not limited to forming an emulsion in water or other suitable medium, spray drying, or evaporating the solvent from the bulk solution and milling the resulting material.

[0081] In some embodiments, the drug in particulate form is homogeneously dispersed in a material that is insoluble or slowly soluble in water. To minimize the size of the drug particles in the composition, the drug powder itself can be milled prior to formulation to produce fine particles. Jet milling, which is known in the pharmaceutical art, can be used for this purpose. In some embodiments, the drug in particulate form is homogeneously dispersed in a wax or wax-like material by heating the wax or wax-like material above its melting point and adding the drug particles while stirring the mixture. In this case, a pharmaceutically acceptable surfactant can be added to the mixture to facilitate dispersion of the drug particles.

[0082] The particles can also be coated with one or more modified release coatings. Fatty acid solid esters that are hydrolyzed by lipase can be sprayed on the microparticles or drug particles. Zein is an example of a protein that is naturally insoluble in water. It can be coated on the drug-containing microparticles or drug particles by spraying or wet granulation techniques. In addition to naturally water-insoluble substances, some substrates of digestive enzymes can be treated by crosslinking procedures, resulting in the formation of an insoluble network. Many protein crosslinking methods initiated by both chemical and physical methods have been reported. One of the most commonly used methods to obtain crosslinking is to use chemical crosslinking agents. Examples of chemical crosslinking agents include aldehydes (glutaraldehyde and formaldehyde), epoxides, carbodiimides, and genipin. In addition to these crosslinking agents, oxidized sugars and natural sugars are also used to crosslink gelatin. Crosslinking can also be accomplished by enzymatic methods; for example, transglutaminase has been approved as a GRAS substance for crosslinking seafood products. Finally, crosslinking can be initiated by physical methods such as heat treatment, ultraviolet irradiation, and gamma irradiation.

[0083] To produce a coating layer of crosslinked protein around the drug-containing microparticles or drug particles, a water-soluble protein can be sprayed on the microparticles and then crosslinked by one of the above methods. Alternatively, the drug-containing microparticles can be microencapsulated within the protein by coacervation-phase separation (e.g., by adding salt) and then crosslinked. Some suitable proteins for this purpose include gelatin, albumin, casein, and gluten.

[0084] Polysaccharides can also be crosslinked to form water-insoluble networks. For many polysaccharides, this can be achieved by reaction with calcium salts or polyvalent cations, which crosslink the major polymer chains. Pectin, alginate, dextran, amylose, and guar gum are crosslinked in the presence of polyvalent cations. Complexes can also be formed between polysaccharides with opposite charges; for example, pectin and chitosan can be complexed via electrostatic interactions.

[0085] (b) Injectable / Implantable Preparations

[0086] The P9R (or P9R-like peptide) described herein can be incorporated into injectable / implantable solid or semi-solid grafts, such as polymeric implants. In one embodiment, the P9R (or P9R-like peptide) is incorporated into a polymer that is liquid or paste at room temperature but exhibits an increase in viscosity upon contact with an aqueous medium such as physiological fluids to form a semi-solid or solid material. Exemplary polymers include, but are not limited to, polyhydroxyalkanoate polyesters, which are derived from the copolymerization of at least one unsaturated hydroxy fatty acid with a hydroxyalkanoic acid. The polymer can be melted, mixed with the active substance, and cast or injection molded into a device. Such melt fabrication requires that the melting point of the polymer be lower than the temperature at which the substance to be delivered and the polymer degrade or become reactive. The device can also be prepared by solvent casting, where the polymer is dissolved in a solvent and the drug is dissolved or dispersed in the polymer solution, and then the solvent is evaporated. The solvent method requires that the polymer be soluble in an organic solvent. Another method is compression molding of a mixed powder of the polymer and drug or polymer particles loaded with the active agent.

[0087] Alternatively, the P9R (or P9R-like peptide) can be incorporated into a polymer matrix and molded, compressed, or extruded into a device that is solid at room temperature. For example, the P9R (or P9R-like peptide) can be incorporated into a biodegradable polymer, such as polyanhydrides, polyhydroxyalkanoates (PHAs), PLA, PGA, PLGA, polycaprolactone, polyesters, polyamides, polyorthoesters, polyphosphazenes, proteins, and polysaccharides such as collagen, hyaluronic acid, albumin, and gelatin and their combinations, and compressed into a solid device, such as a disc, or extruded into a device, such as a rod.

[0088] The release of the peptide from the implant can be altered by the selection of the polymer, the molecular weight of the polymer, and / or the modification of the polymer for enhanced degradation (such as pore formation and / or incorporation of hydrolyzable bonds). Methods for improving the properties of biodegradable polymers to alter the release profile of compounds from implants are well known in the art.

[0089] 3. Enteral Formulations

[0090] Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups, and lozenges. Tablets can be prepared using compression or molding techniques well known in the art. Hard or soft gelatin capsule shells can be prepared using techniques well known in the art, which can encapsulate liquid, solid, and semi-solid filling materials. In embodiments where the formulation is for oral administration involving trans-gastrointestinal delivery, the formulation is preferably coated to protect the peptide from gastrointestinal enzymes.

[0091] The formulation can be prepared using pharmaceutically acceptable carriers. As commonly used herein, "carrier" includes, but is not limited to, diluents, preservatives, binders, lubricants, disintegrants, swelling agents, fillers, stabilizers, and combinations thereof.

[0092] The carrier also includes all components of the coating composition, which may include plasticizers, pigments, colorants, stabilizers, and glidants.

[0093] Examples of suitable coating materials include, but are not limited to, cellulose polymers (such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate), polyvinyl acetate phthalate, acrylic polymers and copolymers, and methacrylic resins commercially available under the trade name (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

[0094] In addition, the coating material may contain conventional carriers such as plasticizers, pigments, colorants, glidants, stabilizers, pore formers, and surfactants.

[0095] A "diluent", also known as a "filler", is generally necessary to increase the volume of a solid dosage form in order to provide a suitable size for tablet compression or the formation of beads and granules. Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starch, pregelatinized starch, silica, titanium dioxide, magnesium aluminum silicate, and powdered sugar.

[0096] An "adhesive" is used to impart cohesive properties to a solid dosage form formulation, thereby ensuring that the tablet or beads or granules remain intact after dosage form formation. Suitable adhesive materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextran, lactose, and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as gum arabic, tragacanth, sodium alginate, cellulose (including hydroxypropyl methylcellulose, hydroxypropyl cellulose, ethyl cellulose, and veegum), and synthetic polymers such as acrylic and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid / polymethacrylic acid, and polyvinylpyrrolidone.

[0097] A "lubricant" is used to facilitate tablet manufacture. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glyceryl behenate, polyethylene glycol, talc, and mineral oil.

[0098] "Disintegrants" are used to promote the disintegration or "breakdown" of a dosage form after administration and generally include, but are not limited to, starches, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethyl cellulose, hydroxypropyl cellulose, pregelatinized starch, clays, celluloses, alginine, gums or crosslinked polymers such as crosslinked PVP (XL from GAF Chemical Corp). XL.

[0099] "Stabilizers" are used to inhibit or retard drug decomposition reactions, which include, for example, oxidation reactions. Suitable stabilizers include, but are not limited to, antioxidants, butylated hydroxytoluene (BHT), ascorbic acid, its salts and esters, vitamin E, tocopherols and their salts, sulfites such as sodium metabisulfite, cysteine and its derivatives, citric acid, propyl gallate and butylated hydroxyanisole (BHA).

[0100] (a) Controlled-release enteral formulations

[0101] Oral dosage forms such as capsules, tablets, solutions and suspensions can be formulated for controlled release. For example, P9R (or P9R-like peptides) and optionally one or more additional active agents can be formulated into nanoparticles, microparticles and combinations thereof and encapsulated in soft gelatin or hard gelatin or non-gelatin capsules or dispersed in a dispersion medium to form an oral suspension or syrup. The particles can be formed from the drug and a controlled-release polymer or matrix. Alternatively, the drug particles can be coated with one or more controlled-release coatings before incorporation into the finished dosage form.

[0102] In another embodiment, P9R (or P9R-like peptides) and optionally one or more additional active agents are dispersed in a matrix material that gels or emulsifies upon contact with an aqueous medium such as physiological fluid. In the case of a gel, the matrix embedding the active agent swells and, over time, the active agent is slowly released by diffusion and / or degradation of the matrix material. Such matrices can be formulated as the filling material for tablets or hard and soft capsules.

[0103] In yet another embodiment, P9R (or P9R-like peptides) and optionally one or more additional active agents are formulated into a solid oral dosage form such as a tablet or capsule and the solid dosage form is coated with one or more controlled-release coatings such as delayed-release coatings or extended-release coatings. One or more of the coatings can also contain P9R (or P9R-like peptides) and / or additional active agents.

[0104] i. Extended-release dosage forms

[0105] Extended-release formulations are generally prepared as diffusion or osmotic systems known in the art. Diffusion systems typically consist of two types of devices, a reservoir and a matrix, and are well-known and described in the art. Matrix devices are generally prepared by compressing a drug with a slowly dissolving polymeric carrier into tablet form. Three main materials used for preparing matrix devices are insoluble plastics, hydrophilic polymers, and fatty compounds. Plastic matrices include, but are not limited to, methyl acrylate-methyl methacrylate, polyvinyl chloride, and polyethylene. Hydrophilic polymers include, but are not limited to, cellulose polymers such as methyl and ethyl cellulose, hydroxyalkyl celluloses such as hydroxypropyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, and 934, polyethylene oxide, and mixtures thereof. Fatty compounds include, but are not limited to, various waxes such as carnauba wax and glyceryl tristearate, and wax-like substances, including hydrogenated castor oil or hydrogenated vegetable oil or mixtures thereof.

[0106] In certain preferred embodiments, the plastic material is a pharmaceutically acceptable acrylic polymer, including, but not limited to, copolymers of acrylic acid and methacrylic acid, methyl methacrylate, methyl methacrylate copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymers, poly(acrylic acid), poly(methacrylic acid), alkylamine methacrylate copolymers, poly(methyl methacrylate), poly(methacrylic acid)(anhydride), polymethacrylate, polyacrylamide, poly(methacrylic anhydride), and glycidyl methacrylate copolymers.

[0107] In certain preferred embodiments, the acrylic polymer consists of one or more ammonium methacrylate copolymers. Ammonium methacrylate copolymers are well-known in the art and are described in NF XVII as fully polymerized copolymers of acrylates and methacrylates having a low content of quaternary ammonium groups.

[0108] In a preferred embodiment, the acrylic polymer is an acrylic resin lacquer, such as an acrylic resin lacquer commercially available from Rohm Pharma under the trade name EUDRAGIT In a further preferred embodiment, the acrylic polymer comprises a mixture of two acrylic resin lacquers, commercially available from Rohm Pharma under the trade names RL30D and RS30D, respectively. RL30D and RS30D are copolymers of acrylates and methacrylates having a low content of quaternary ammonium groups. In RL30D, the molar ratio of ammonium groups to the remaining neutral (meth)acrylate is 1:20, and in In RS30D, it is 1:40. The average molecular weight is about 150,000. S-100 and L-100 are also preferred. The code names RL (high permeability) and RS (low permeability) refer to the permeability characteristics of these reagents. The RL / RS mixture is insoluble in water and digestive fluids. However, the formed multi-particle system containing them is swellable and permeable in aqueous solutions and digestive fluids.

[0109] In order to ultimately obtain a sustained-release formulation with a desired dissolution profile, polymers such as RL / RS can be mixed together in any desired ratio. For example, it can be from 100% RL, 50% RL and 50% EUDRAGIT RS, and 10% RL and 90% RS to obtain a desired sustained-release multi-particle system. Those skilled in the art will recognize that other acrylic polymers can also be used, such as, for example L.

[0110] Alternatively, the extended-release formulation can be prepared using an osmotic system or by applying a semi-permeable coating to the dosage form. In the latter case, the desired drug release profile can be achieved by combining low-permeable and high-permeable coatings in appropriate proportions.

[0111] The devices with different drug release mechanisms described above can be combined into a final dosage form containing single or multiple units. Examples of multiple units include, but are not limited to, multi-layer tablets and capsules containing tablets, beads or granules. An immediate-release portion can be added to the extended-release system by applying an immediate-release layer on top of the extended-release core using a coating or compression process, or in a multi-unit system (such as a capsule containing extended or immediate-release beads).

[0112] Extended-release tablets containing hydrophilic polymers are prepared by techniques well known in the art, such as direct compression, wet granulation or dry granulation. Their formulations typically contain polymers, diluents, binders and lubricants as well as active pharmaceutical ingredients. Common diluents include inert powdered substances such as starch, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, cereal flours and similar edible powders. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or calcium sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders include substances such as starch, gelatin and sugars such as lactose, fructose and glucose. Natural and synthetic gums can also be used, including gum arabic, alginates, methylcellulose and polyvinylpyrrolidone. Polyethylene glycol, hydrophilic polymers, ethylcellulose and waxes can also be used as binders. Lubricants are required in tablet formulations to prevent the tablets and punches from sticking in the die. Lubricants are selected from smooth solids such as talc, magnesium stearate and calcium stearate, stearic acid and hydrogenated vegetable oils.

[0113] Extended-release tablets containing wax materials are generally prepared using methods known in the art, such as direct mixing methods, coagulation methods and aqueous dispersion methods. In the coagulation method, the drug is mixed with the wax material and spray coagulated or coagulated and screened and processed.

[0114] ii. Delayed-release dosage forms

[0115] Delayed-release formulations can be created by coating solid dosage forms with a polymeric film that is insoluble in the acidic environment of the stomach and soluble in the neutral environment of the small intestine.

[0116] For example, delayed release dosage units can be prepared by coating a drug or a drug-containing composition with a selected coating material. For example, the drug-containing composition can be a tablet for incorporation into a capsule, a tablet serving as the core of a "coated core" dosage form, or a plurality of drug-containing beads, granules, or particulates for incorporation into a tablet or capsule. Preferred coating materials include bioerodible, gradually hydrolyzable, gradually water-soluble, and / or enzymatically degradable polymers, and can be conventional "enteric" polymers. As will be understood by those skilled in the art, enteric polymers become soluble in the higher pH environment of the lower gastrointestinal tract or erode slowly as the dosage form passes through the gastrointestinal tract, while enzymatically degradable polymers are degraded by bacterial enzymes present in the lower gastrointestinal tract, particularly in the colon. Suitable coating materials for causing delayed release include, but are not limited to, cellulose polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, hypromellose acetate succinate, hydroxypropyl methyl cellulose phthalate, methyl cellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate, and sodium carboxymethyl cellulose; acrylic polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate, and / or ethyl methacrylate, and other methacrylic resins commercially available under the trade name (Rohm Pharma; Westerstadt, Germany), including L30D-55 and L100-55 (soluble at pH 5.5 and above), L-100 (soluble at pH 6.0 and above), S (soluble at pH 7.0 and above due to a higher degree of esterification), and NE, RL, and RS (water-insoluble polymers with different degrees of permeability and swellability); vinyl polymers and copolymers such as polyvinylpyrrolidone, vinyl acetate, vinyl acetate phthalate, vinyl acetate crotonic acid copolymer, and ethylene-vinyl acetate copolymer; enzymatically degradable polymers such as azo polymers, pectin, chitosan, amylose, and guar gum; zein and shellac. Combinations of different coating materials can also be used. Multilayer coatings can also be applied using different polymers.

[0117] By evaluating the individual release profiles of tablets, beads, and granules prepared with different amounts of various coating materials, those skilled in the art can readily determine the preferred coating weights of specific coating materials. The combination of materials, methods, and application forms results in the desired release characteristics, which can only be determined from clinical studies.

[0118] The coating composition may contain conventional additives such as plasticizers, pigments, colorants, stabilizers, glidants, etc. A plasticizer is usually present to reduce the brittleness of the coating and will typically account for about 10 wt.% to 50 wt.% relative to the dry weight of the polymer. Examples of typical plasticizers include polyethylene glycol, propylene glycol, glyceryl triacetate, dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dibutyl sebacate, triethyl citrate, tributyl citrate, triethyl acetylcitrate, castor oil, and acetylated monoglycerides. Stabilizers are preferably used to stabilize the particles in the dispersion. Typical stabilizers are nonionic emulsifiers such as sorbitan esters, polysorbates, and polyvinylpyrrolidone. Glidants are recommended to reduce the sticky effect during film formation and drying and will typically account for about 25 wt.% to 100 wt.% of the polymer weight in the coating solution. An effective glidant is talc. Other glidants such as magnesium stearate and glycerol monostearate can also be used. Pigments such as titanium dioxide can also be used. A small amount of defoamer such as silicone (e.g., dimethyl silicone oil) can also be added to the coating composition.

[0119] III. Method of Use

[0120] The method of the present disclosure is based on studies showing that P9R exhibits very broad-spectrum antiviral activity against enveloped SARS-CoV-2, MERS-CoV, SARS-CoV, A(H1N1)pdm09, A(H7N9) viruses, and non-enveloped rhinoviruses. When administered in vivo, P9R efficiently protects against viral attack, as exemplified by its protection of mice (after in vivo administration) from lethal A(H1N1)pdm09 virus attack. Even after the A(H1N1)pdm09 virus was passaged 40 times in the presence of P9R, P9R did not cause the emergence of drug-resistant viruses. Mechanistic studies have shown that the antiviral activity of P9R depends on direct binding to the virus and inhibition of virus-host endosomal acidification, which provides a new concept that basic peptides binding to the virus can broadly inhibit pH-dependent viruses.

[0121] Accordingly, a method for treating a subject infected with a virus is provided, the method comprising administering to the subject a formulation containing an effective amount of the disclosed peptide to ameliorate one or more symptoms associated with the viral infection. In a preferred embodiment, the treatment is effective in inhibiting viral replication in the subject. An effective amount of the peptide can be administered to the subject by treating the subject with the disclosed peptide: enterally, by pulmonary or nasal administration, or parenterally (intravenously, intradermally, intraarterially, intraperitoneally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intravitreally, intratumorally, intramuscularly, subcutaneously, subconjunctivally, intracapsularly, intrapericardially, intraumbilically, by injection, and by infusion).

[0122] The virus is preferably a respiratory virus, and more preferably a pH-dependent respiratory virus. Respiratory viruses are the most frequent disease pathogens in humans, having a major impact on morbidity and mortality worldwide, especially in children. Approximately one-fifth of all child deaths worldwide are associated with acute respiratory infections (ARI), particularly among the poor in tropical regions where the case-to-death ratio of ARI can be significantly higher than in the temperate regions of the world. Eight human respiratory viruses are prevalent in all age groups and are recognized to be adapted for efficient human-to-human transmission, including HRSV (human respiratory syncytial virus), HPIV (human parainfluenza virus), HRV (human rhinovirus), ADV (adenovirus), HCoV (human coronavirus) (HCoV-NL63, HCoV-HKU1), SARS-CoV, HMPV (human metapneumovirus), HPIV (human parainfluenza virus), and HBoV (human bocavirus). It is thought that HRSV internalization is pH-independent and may occur in the plasma or endosomal membrane.

[0123] Exemplary viral infections that can be treated with the formulations of the present disclosure include, but are not limited to, Zika virus, enterovirus-A71, Ebola virus, influenza virus, HRSV, HPIV, HRV, ADV, HPIV, HCoV, SARS-CoV-2, MERS-CoV, SARS-CoV, A(H1N1)pdm09, A(H7N9) virus, and non-enveloped rhinovirus.

[0124] The following non-limiting examples further illustrate the disclosed and claimed compositions and methods. Examples

[0125] Materials and Methods

[0126] Cells and Virus Cultures

[0127] Madin Darby canine kidney (MDCK, CCL-34), Vero E6 (CRL-1586), RD (CCL136), LLC-MK2 (CCL-7), A549 (CCL-185) cells obtained from ATCC (Manassas, VA, USA) were cultured in Dulbecco's Modified Eagle Medium (DMEM) or MEM supplemented with 10% fetal bovine serum (FBS), 100 IU / mL -1 penicillin and 100 μg / mL -1 streptomycin. The virus strains used in this study included the 2019 novel coronavirus (SARS-CoV-2) 43, SARS-CoV, MERS-CoV (hCoV-EMC / 2012), A / Hong Kong / 415742 / 2009, A / Hong Kong / 415742Md / 2009 (H1N1) (a highly virulent mouse-adapted strain), A / Anhui / 1 / 2013 (H7N9) 13 , rhinovirus 44 and human parainfluenza virus 3 (ATCC-C243). For in vitro experiments, the viruses were cultured in MDCK, Vero E6, RD, and LLC-MK2 cells. For animal experiments, the H1N1 virus was cultured in eggs as previously described 45 .

[0128] Design and synthesis of peptides

[0129] As shown in Figure 1a, P9, P9R, PA1, and P9RS were designed and synthesized by ChinaPeptide (Shanghai, China). The purity of all peptides was >95%. The purity and quality of each peptide were verified by HPLC and mass spectrometry.

[0130] Plaque reduction assay

[0131] The antiviral activity of the peptides was measured by plaque reduction assay as we previously described 14 . Briefly, the peptides were dissolved in 30 mM phosphate buffer containing 24.6 mM Na2HPO4 and 5.6 mM KH2PO4, pH 7.4. At room temperature, the peptides or bovine serum albumin (BSA, 0.4 - 50.0 μg mL -1 ) were pre-mixed with 50 PFU of coronavirus (SARS-CoV-2, MERS-CoV, and SARS-CoV), influenza virus (H1N1 virus and H7N9 virus), rhinovirus, or parainfluenza virus 3 in phosphate buffer. After incubation for 1 h, the peptide-virus mixture was transferred to Vero E6 (for coronavirus), MDCK (for influenza virus), RD (for rhinovirus), or LLC-MK2 (for parainfluenza virus). At 1 h post-infection, the infectious medium was removed and 1% low melting point agar was added to the cells. The cells were fixed with 4% formalin at 2 - 4 days post-infection. Crystal blue (0.1%) was added for staining, and the number of plaques was counted.

[0132] Antiviral multi-cycle growth assay

[0133] Coronavirus (SARS-CoV-2, MERS-CoV, and SARS-CoV), influenza virus (H1N1 and H7N9 viruses), and rhinovirus (0.005 MOI) were mixed with P9R or BSA (50 - 100 μg mL -1) Pre-mix in phosphate buffer for 1 h. After incubation, inoculate coronaviruses onto Vero E6. Inoculate influenza viruses onto MDCK cells. Inoculate rhinoviruses onto RD cells. After 1 h of infection, remove the infectious medium and add fresh medium supplemented with P9R or BSA (50 - 100 μg mL -1 ) to the infected cells for virus and cell culture. At 24 - 30 h post-infection, collect the cell supernatants for detection of viral RNA copies.

[0134] Cytotoxicity assay

[0135] As we described previously 13 , use the tetrazolium-based colorimetric MTT assay to detect the 50% cytotoxic concentration (CC 50 ) to determine the cytotoxicity of the peptides. Briefly, seed cells at an initial density of 2×10 4 cells per well in 96-well cell culture plates containing MEM or DMEM supplemented with 10% FBS and incubate overnight. Remove the cell medium, then add DMEM supplemented with various concentrations of the peptide and 1% FBS to each well. After incubation at 37 °C for 24 h, add MTT solution (5 mg mL -1 , 10 μL per well) to each well and incubate at 37 °C for 4 h. Then, add 100 μL of 10% SDS in 0.01 M HCl to each well. After further incubation and shaking overnight at room temperature, read the plates at OD570 using a VictorTM X3 Multilabel Reader (PerkinElmer, USA). Cell culture wells without peptides are used as experimental controls and medium alone serves as a blank control.

[0136] Peptide-virus binding assay

[0137] Coat the ELISA plates with the peptide (0.1 μg per well) dissolved in H2O and incubate overnight at 4 °C. Then, block the plates overnight at 4 °C with 2% BSA. For virus binding to the peptide, dilute the virus in phosphate buffer and then add it to the ELISA plates to bind to the coated peptide at room temperature for 1 h. After washing away the unbound virus, lyse the bound virus with RLT buffer from the RNeasy Mini Kit (Qiagen, Cat#74106) for viral RNA extraction. Measure the viral RNA copies of the bound virus by RT-qPCR.

[0138] ELISA assay

[0139] Perform the ELISA assay as described previously 14。Coat the ELISA plate with the peptide dissolved in H2O (0.1 μg per well) and incubate overnight at 4 °C. Then, block the plate overnight at 4 °C with 2% BSA. For HA and S binding, incubate 150 ng of HA1 or S in Solution I buffer (Sino Biological Inc., Cat # 11055-V08H4) with the peptide for 1 h at 37 °C. Determine the binding ability of the peptide to HA1 or S protein by incubating with rabbit anti-His-HRP (Invitrogen, Cat # R93125, 1:2,000) for 30 min at room temperature. Develop the reaction by adding 50 μL of TMB single solution (Life Technologies, Cat # 002023) for 15 min at 37 °C and terminate with 50 μL of 1 M H2SO4. Obtain the readings at 450 nm in an ELISA plate reader (Victor 1420 Multilabel Counter; PerkinElmer).

[0140] Viral RNA extraction and RT-qPCR

[0141] Extract viral RNA using the Viral RNA Mini Kit (QIAGEN, Cat # 52906, USA) according to the manufacturer's instructions. Perform real-time RT-qPCR as previously described by us 14 . Use the PCR system 9700 (Applied Biosystems, USA) to reverse-transcribe the extracted RNA into cDNA using the PrimeScript II 1 st Strand cDNA synthesis Kit (Takara, Cat#6210A). Then use the 480 SYBR Green I Master (Roach, USA) to amplify the cDNA with specific primers (Table 1) to detect SARS-CoV-2, MERS-CoV, SARS-CoV, H1N1, H7N9, and rhinovirus.

[0142] Table 1. RT-qPCR primers

[0143]

[0144] For quantification, prepare 10-fold serial dilutions of a standard plasmid equivalent to 10 1 to 10 6 copies per reaction to generate a calibration curve. Use Real-time qPCR experiments were performed using a 96system (Roche, USA).

[0145] Endosomal acidification assay

[0146] As described previously but with slight modifications 14 , endosomal acidification was detected using a pH-sensitive dye (pHrodo Red dextran, Invitrogen, Cat # P10361) according to the manufacturer's instructions. First, MDCK cells were treated with BSA (25.0 μg mL -1 ), P9 (25.0 μg mL -1 ), P9R (25.0 μg mL -1 ), PA1 (25.0 μg mL -1 ), or P9RS (25.0 μg mL -1 ) at 4 °C for 15 min. Second, 100 μg mL -1 of the pH-sensitive dye and DAPI were added to the MDCK cells, and then incubated at 4 °C for 15 min. Before taking images, the cells were further incubated at 37 °C for 15 min and then washed twice with PBS. Finally, PBS was added to the cells and images were immediately taken using a confocal microscope (Carl Zeiss LSM 700, Germany).

[0147] Co-localization assay of peptide and virus binding in cells

[0148] H1N1 virus was labeled with green Dio dye (Invitrogen, Cat # 3898) according to the manufacturer's instructions. The DIO-labeled virus was treated with TAMRA-labeled P9R and TAMRA-labeled P9RS at room temperature for 1 h. Pre-cooled MDCK cells were infected with the virus treated with the peptide on ice for 15 min and then transferred to 37 °C for incubation for 15 min. The cells were washed twice with PBS and then fixed with 4% formalin for 1 h. The cell nuclei were stained with DAPI to take images using a confocal microscope (Carl Zeiss LSM 700, Germany).

[0149] Nucleoprotein (NP) immunofluorescence assay

[0150] NP staining was performed as described previously 14 . MDCK cells were seeded on cell culture slides and infected with a virus at an MOI of 1 in the presence of BSA (25.0 μg mL -1 ), bafilomycin A1 (50.0 nM), or P9R (25.0 μg mL -1)The pretreated A(H1N1)pdm09 virus infects MDCK cells. 3.5 h after infection, the cells are fixed with 4% formalin for 1 h, and then permeabilized with 0.2% Triton X-100 in PBS for 5 min. The cells are washed with PBS and then blocked with 5% BSA for 1 h at room temperature. The cells are incubated with mouse IgG anti-NP (Millipore, Cat # 2817019, 1:600) for 1 h at room temperature, then washed with PBS for the next incubation with goat anti-mouse IgG Alexa-488 (Life Technologies, Cat#1752514, 1:600) for 1 h at room temperature. Finally, the cells are washed with PBS and stained with DAPI. Images are taken by confocal microscopy (Carl Zeiss LSM 700, Germany).

[0151] NMR Structure Analysis of P9R

[0152] 1 mg mL -1 (0.29 mM) of P9R in 0.5 mL freshly prepared solvent is used for NMR studies. Data are collected in H2O / D2O (19:1 v / v) and 99.996% D2O with trimethylsilylpropionic acid as the internal reference. All NMR spectra are obtained at 25 °C on a Bruker AVANCE III 600 MHz spectrometer (Bruker BioSpin, Germany) or a Bruker AVANCE III 700 MHz spectrometer. 2D 1 H- 1 H correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), and nuclear Overhauser effect spectroscopy (NOESY) spectra are recorded for resonance assignment. Proton-proton distance constraints are derived from 2D NOESY spectra with mixing times of 300 ms and 500 ms, using an automated NOE assignment strategy followed by manual inspection. Using CCPNMR Analysis 2.4.2 46 Extract the NOE intensities and chemical shifts and serve as the input for the Aria program. Using the program DANGLE 47 Predict the dihedral angles from the chemical shifts. Using the Aria 2.3 program 48Iteratively calculate the NMR solution structure of P9R. In each of the eight iterative cycles of the combined automated NOE assignment and structure calculation algorithm, one hundred random conformers were annealed using distance restraints. The final upper distance constraints output from the last iterative cycle were subjected to a comprehensive manual cross-check and a final water-solvent structure refinement cycle. Ten of the lowest energy conformers were retained from these refined 100 structures for statistical analysis. The convergence of the calculated structures was evaluated using root mean square deviation (RMSD) analysis. The backbone dihedral angles of the final converged structures were evaluated by using PROCHECK-NMR 49 to represent the Ramachandran dihedral angle pattern The distribution of. The three-dimensional structure and electrostatic surface potential of P9R were visualized using UCSF Chimera 1.13.1 50 Antiviral analysis of P9R in mice

[0153] BALB / c female mice aged 10 - 12 weeks were housed in a biosafety level 2 laboratory and allowed free access to standard pellet feed and water. All experimental protocols were in accordance with the standard operating procedures of the approved biosafety level 2 animal facility and were approved by the Committee on the Use of Live Animals in Teaching and Research of the University of Hong Kong

[0154] H1N1 virus adapted to mice was used for lethal challenge of mice. To evaluate the therapeutic effect, mice were challenged with 3LD 45 of the virus and then inoculated intranasally with PBS, P9, P9R, PA1, or zanamivir 6 h after virus inoculation. On the following day, two additional doses were given to the H1N1-challenged mice. Survival and general condition were monitored for 16 days or until death 50 Statistical analysis

[0155] The survival and statistical significance of mice were analyzed using GraphPad Prism 5. The statistical significance of other results was calculated by two-tailed Student's t-test using Stata statistical software. Results with P < 0.05 were considered significant

[0156] Results

[0157] The murine β-defensin-derived peptide P9R can broadly inhibit coronaviruses and other respiratory viruses

[0158] Proton influx into endosomes via the vacuolar membrane proton pump V-ATPase affects endosomal acidification

[0159] 27 ​. In theory, basic peptides with a strong net positive charge neutralize protons in endosomes, thereby inhibiting endosomal acidification. Therefore, to improve our previous antiviral peptide P9 13 , the weakly positively charged amino acids (histidine and lysine) were replaced with arginine at positions 21 (H→R), 23 (K→R), and 28 (K→R) Figure 1A ) to increase the net positive charge of P9 (+4.7) to the charge of P9R (+5.6). In the plaque reduction assay, the IC 50 of P9R against SARS-CoV-2 was significantly lower than that of P9 (0.9 μg mL -1 vs 2.4 μg mL -1 , P<0.01) ( Figure 1B ). In addition, P9R showed significantly stronger inhibitory effects against MERS-CoV, A(H1N1)pdm09 virus, A(H7N9) virus, and rhinovirus than P9 (Figure 1c-1g). However, the IC 50 of P9R and P9 against parainfluenza virus 3 was much higher (>25.0 μg mL -1 ), probably because endosomal acidification is not required in the viral life cycle of parainfluenza virus 3 ( Figure 1H ) 28 . In the multi-cycle growth assay, P9R inhibited the viral replication of SARS-CoV-2, MERS-CoV, and SARS-CoV by 1000-fold ( Figure 1I ). For A(H1N1)pdm09 virus, A(H7N9) virus, and rhinovirus, P9R could inhibit the viral replication by >20-fold ( Figure 1I ). In addition, for MDCK, VeroE6, and A549 cells, the CC 50 of P9R >300 μg mL -1 ( Figure 1J ). These results indicate that P9R with more positive charges can inhibit the novel coronavirus SARS-CoV-2 and other enveloped and non-enveloped respiratory viruses more efficiently than P9.

[0160] The degree of positive charge is crucial for the inhibition of endosomal acidification and antiviral activity

[0161] To determine whether the net charge of the peptide affects the inhibition of endosomal acidification, the endosomal acidification assay found that P9R (+5.6) could inhibit endosomal acidification in living cells more significantly than P9 (data not shown, and Figure 2A ), which is consistent with the stronger antiviral activity of P9R than P9. In addition, the peptide PA1 with less positive charge (+1.7), which has the same amino acid sequence as P9 except for 3 additional acidic amino acids at the C-terminus, could not inhibit endosomal acidification (data not shown, and Figure 2B) and lose antiviral activity ( Figure 2B ). Thus, the degree of net positive charge is related to the degree of inhibition of endosomal acidification and antiviral activity.

[0162] Inhibiting host endosomal acidification alone is not sufficient for positively charged peptides to inhibit virus replication

[0163] To determine whether antiviral activity depends solely on the positive charge of the peptide, a peptide P9RS(+5.6) with the same positive charge as P9R(+5.6) was designed, but P9RS differed from P9R in 11 out of 30 amino acids. P9RS potently inhibited host endosomal acidification to a similar extent as P9R in live cells (data not shown, and Figure 2A ). However, in plaque reduction assays, the number of plaques of SARS-CoV-2 and A(H1N1)pdm09 viruses was not significantly reduced when the virus was treated with even 25 μg mL -1 of P9RS ( Figure 2B ).

[0164] To investigate why P9RS failed to inhibit virus replication despite potently inhibiting host endosomal acidification, the binding between the peptide and the virus was studied. Using ELISA-RT-qPCR assays, P9R and PA1 could efficiently bind to SARS-CoV-2 and A(H1N1)pdm09 viruses, but P9RS did not bind to SARS-CoV-2 and A(H1N1)pdm09 viruses ( Figure 2C ). The observation that P9R rather than P9RS binds to the virus was further confirmed in H1N1-infected cells by confocal microscopy (data not shown). Thus, the antiviral activity of the positively charged peptide P9R requires direct interaction of the peptide with the virus. In contrast, P9RS, which does not have the ability to bind to the virus, cannot inhibit virus replication even though it carries the same positive charge as P9R and inhibits host endosomal acidification.

[0165] The broad-spectrum antiviral activity of P9R depends on targeting the virus to inhibit virus-host endosomal acidification

[0166] The above experiments showed that P9R and P9RS could inhibit virus-free endosomal acidification (data not shown, and Figure 2A ). However, in the absence of binding to the virus, P9RS could not inhibit virus replication. To illustrate this result, other studies showed that P9R and bafilomycin A1 could potently inhibit virus-host endosomal acidification in infected live cells, but P9RS could not inhibit virus-host endosomal acidification in infected live cells (Figure 3a), even though both P9R and P9RS could inhibit endosomal acidification of virus-free endosomes (data not shown). The potent inhibition of virus-host endosomal acidification by P9R may be due to the binding of P9R to the virus (data not shown, andFigure 2C ) and then inhibit virus-host endosomal acidification (data not shown). Due to the lack of binding ability to the virus (data not shown, and Figure 2E), P9RS cannot efficiently enter the endosome with the virus to inhibit virus-host endosomal acidification, which may be because the presence of the virus in the endosome prevents unbound P9RS from entering the endosome. Without the virus in the endosome, there is empty space in the virus-free endosome to allow P9RS to freely enter the endosome to prevent endosomal acidification (data not shown). It should be noted that PA1, which is similar to the P9R sequence, can efficiently bind to SARS-CoV-2 and A(H1N1)pdm09 viruses ( Figure 2C ), but it significantly loses its antiviral activity against SARS-CoV-2 and A(H1N1)pdm09 viruses ( Figure 2B ). When the virus is pretreated with PA1, the binding of P9R to SARS-CoV-2 and A(H1N1)pdm09 viruses can be significantly reduced ( Figure 3A ). This indicates that PA1 has the same binding site as P9R on the virus particle, but the binding of the peptide alone to the virus cannot explain the antiviral activity. The binding of P9R to the virus is the first step in exerting antiviral activity. After binding to the virus (data not shown), P9R can efficiently inhibit virus-host endosomal acidification (data not shown), and then inhibit virus replication by blocking the release of RNP (data not shown).

[0167] To further confirm that the broad-spectrum antiviral activity of P9R is due to the extensive binding of P9R to different viruses and viral proteins, additional studies have shown that P9R, rather than P9RS, can also bind to MERS-CoV, A(H7N9) virus, rhinovirus, SARS-CoV, and viral proteins ( Figure 3B-3D and Figure 3E ). This result further confirms that positively charged P9R can inhibit pH-dependent endosomal viruses if it can bind to the virus.

[0168] Next, NMR spectroscopy was used to study to determine the structure of P9R. The results showed that the solution structure of P9R is flexible, with short variable helical patches and a positively charged peptide surface (data not shown). Without being bound by theory, P9R can bind widely to different viruses because these short α-helical patches with flexible linkers may allow it to adjust its structure to fit the binding pockets of different viral proteins. In summary, this study demonstrated a new antiviral mechanism, that is, positively charged P9R needs to target the virus and then prevent virus-host endosomal acidification to inhibit the replication of pH-dependent viruses.

[0169] The efficacy of P9R treatment in vivo

[0170] Studies have shown that the high antiviral activity of P9R in vitro depends on its binding to the virus and the positive charge of P9R to inhibit virus-host endosomal acidification. To further investigate the antiviral activity of P9R in vivo, A(H1N1)pdm09-infected mice were treated with two additional doses one day after infection at 6 h post-infection. In this model, 80% of the P9R-treated mice survived, which was significantly better than the PBS-treated group and the PA1-treated group ( Figure 4A ). The protection of P9R against infected mice was the same as that of the zanamivir-treated group (80%) and better than that of P9. From day 4 to day 10 post-infection, the weight loss in the P9R group was significantly less than that in the PBS-treated group and the PA1-treated group ( Figure 4B ). The low-dose protection (25 μg / dose and 12.5 μg / dose) of P9R against infected mice and the reduced weight loss further indicated that P9R could significantly protect mice compared with the PBS-treated group ( Figure 4C and 4D ). The antiviral activity of P9R in vivo was better than that of P9 ( Figure 4C , P < 0.05 for 12.5 μg / dose), which was consistent with the significantly better antiviral activity of P9R than that of P9 in vitro.

[0171] No resistant virus to P9R emerged after continuous passage of the virus in the presence of P9R

[0172] The emergence of resistant mutants occurred occasionally 14 , especially with the new polymerase inhibitor baloxavir 29 . To determine whether P9R treatment induced virus resistance, the A(H1N1)pdm09 virus was continuously passaged 40 times in MDCK cells in the presence of P9R ( Figure 5A ). The A(H1N1)pdm09 virus was continuously passaged in the presence of zanamivir as a control for the resistance assay ( Figure 5A ). The IC 50 of zanamivir against the parental A(H1N1)pdm09 virus (P0) was 35 nM ( Figure 5B ). After 10 virus passages in the presence of zanamivir (100 nM) and another 5 virus passages in the presence of zanamivir (1000 nM), 2000 nM and 8000 nM zanamivir could not inhibit P10 and P15 virus replication, respectively ( Figure 5C ). These indicated that significant virus resistance to zanamivir occurred after 10 virus passages in the presence of zanamivir. However, for P9R, even after 40 passages of the A(H1N1)pdm09 virus in the presence of P9R (5.0 μg mL -1 P9R was used for the first 10 generations, 50.0 μg mL -1 P9R was used for the remaining 30 generations), P9R (5.0 μgmL-1 ) can efficiently inhibit the replication of P30 and P40 viruses Figure 5D ). No significantly drug-resistant viruses against P9R were detected. These results suggest that the possibility of P9R causing drug-resistant viruses is very low.

[0173] Discussion

[0174] In this study, a broad-spectrum antiviral peptide P9R with potent antiviral activity against enveloped coronaviruses (SARS-CoV-2, SARS-CoV, and MERS-CoV), influenza viruses, and non-enveloped rhinoviruses was identified. First, the study showed that the antiviral activity of P9R could be significantly enhanced by increasing the net positive charge, thus more efficiently inhibiting endosomal acidification. Second, mechanistic studies further demonstrated a novel antiviral mechanism, that is, the positively charged P9R could bind to different respiratory viruses to inhibit virus-host endosomal acidification. PA1 (which only binds to viruses) or P9RS (which only inhibits endosomal acidification) did not show antiviral activity. Mechanistic studies indicated that the positively charged P9R widely inhibited virus replication by binding to different viruses and then inhibiting virus-host endosomal acidification to prevent the endosomal release of pH-dependent viruses. P9R (which not only binds to viruses but also inhibits endosomal acidification), PA1 (which only binds to viruses), and P9RS (which only inhibits endosomal acidification) were used to identify and confirm the novel antiviral mechanism of basic peptides. Third, the in vivo antiviral activity of P9R was demonstrated by protecting mice from lethal influenza virus challenge. The antiviral activity of basic peptides could be enhanced by increasing the positive charge of the peptides and required both virus binding and inhibition of endosomal acidification. Fourth, the susceptibility of continuously passaged viruses (40 passages) to P9R did not decrease.

[0175] Endosomal acidification is a key step in the life cycle of many pH-dependent viruses and is one of the broad-spectrum antiviral targets 9 . In this study, as the positive charge in P9R increased, it could more efficiently inhibit pH-dependent viruses than P9. More positive charges in P9R allowed the peptide to more efficiently neutralize protons in endosomes, thus inhibiting endosomal acidification. In previous studies, it has been demonstrated that the clinically approved anti-malaria drug chloroquine with endosomal acidification inhibitory activity inhibits enterovirus-A7 30 , Zika virus 31 and SARS-CoV-2 32 . The anti-parasitic drug niclosamide can also inhibit influenza viruses, rhinoviruses, and dengue viruses by interfering with endosomal acidification 33,34 . However, the researchers demonstrated that chloroquine lacked protection against influenza viruses and Ebola viruses in vivo 35,36。Unlike these drugs that interfere with host endosomal acidification without targeting the virus, P9R inhibits virus replication by binding to the virus and subsequently inhibiting virus-host endosomal acidification, which allows P9R to selectively and efficiently inhibit endosomal viruses. The protection of P9R against A(H1N1)-infected mice further confirmed the antiviral efficiency in vivo.

[0176] The antiviral activity of P9R requires virus binding and inhibition of endosomal acidification. PA1 with fewer positive charges cannot inhibit SARS-CoV-2 and H1N1 viruses, even though it has a similar virus-binding ability and binding site to P9R (Figure 3b). When multiple substitutions were made on P9R to generate P9RS, even though P9RS has the same positive charge as P9R and efficiently inhibits host endosomal acidification, P9RS lost its binding ability and antiviral activity against all tested viruses. Although not bound by theory, the broad binding mechanism of P9R to different viral proteins may be due to the flexible structure of the positively charged surface of P9R (Figure 3h). The flexible structure may allow P9R to change its structure to adapt to the target protein for broad-specificity binding 37 , 38 , and the positive charge of P9R may play a role in binding to the negatively charged virus surface 39,40 . The five cysteines in P9R may also affect structure-based binding, as previous studies have shown that cysteine substitution may affect the structure and activity of defensin peptides 41,42 .

[0177] In addition, compared with zanamivir, which caused significant virus resistance after 10 virus passages in the presence of zanamivir, even when the A(H1N1)pdm09 virus was passaged 40 times in the presence of P9R, P9R showed a very low risk of causing drug-resistant viruses.

[0178] In summary, most newly emerging highly pathogenic viruses are endosomal pH-dependent viruses. The emergence and re-emergence of viral outbreaks remind us of the urgent need for broad-spectrum antiviral drugs. This study provides one such broad-spectrum antiviral drug.

[0179] The disclosed compositions and methods can be further understood by the numbered paragraphs below.

[0180] 1. An antiviral agent comprising P9R (SEQ ID NO:2) or a P9R-like peptide derived from P9R.

[0181] 2. The antiviral agent according to paragraph 1, wherein the P9R-like peptide is characterized in that the P9R-like peptide inhibits endosomal acidification and retains virus binding, as determined by in vitro endosomal acidification, optionally compared to a control, and a peptide-virus binding assay.

[0182] 3. The antiviral agent described in paragraph 1 or 2, which consists of P9R (SEQ ID NO: 2).

[0183] 4. The antiviral agent described in any one of paragraphs 1-3, wherein the antiviral agent has a net positive charge of at least 5.

[0184] 5. The antiviral agent described in any one of paragraphs 1-4, wherein the antiviral agent has a net positive charge of about 5.6.

[0185] 6. The antiviral agent described in any one of paragraphs 1-5, wherein the antiviral agent has a net positive charge of 5.6.

[0186] 7. A composition comprising a therapeutically effective amount of the antiviral agent described in any one of paragraphs 1-6 and a pharmaceutically acceptable carrier.

[0187] 8. The composition described in paragraph 7, wherein the antiviral agent inhibits viral replication in a subject.

[0188] 9. The composition described in paragraph 7 or 8, wherein the composition is in unit dosage form.

[0189] 10. The composition described in paragraph 9, wherein the unit dosage form is selected from tablets or capsules.

[0190] 11. The composition described in paragraph 7, which is in a form suitable for intranasal or pulmonary delivery.

[0191] 12. The composition described in paragraph 9, wherein the unit dosage form is injectable, and the composition further comprises a pharmaceutically acceptable carrier for injection into a human.

[0192] 13. A method of treating a viral infection in a subject in need thereof, the method comprising administering to the subject an effective amount of the antiviral agent described in any one of paragraphs 1-6 or the composition described in any one of paragraphs 7-12.

[0193] 14. The method described in paragraph 13, wherein the infection is caused by a respiratory virus.

[0194] 15. The method described in paragraph 13 or 14, wherein the infection is caused by a pH-dependent virus that requires endosomal acidification for virus-host membrane fusion.

[0195] 16. The method described in any one of paragraphs 13-15, wherein the composition is administered parenterally or orally.

[0196] 17. The method described in any one of paragraphs 13-15, wherein the composition is administered intranasally or by pulmonary administration.

[0197] 18. The method of any of paragraphs 13-17, wherein the infection is caused by Zika virus, enterovirus-A7, Ebola virus, influenza virus, SARS-CoV-2, SARS-CoV, MERS-CoV, A(H1N1)pdm09 virus, avian influenza A(H7N9) virus, and non-enveloped rhinovirus.

[0198] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0199] It should be understood that the methods and compositions of the present disclosure are not limited to the specific methods, protocols and reagents described, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims.

[0200] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0201] Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "include" and "comprising", mean "including but not limited to", and are not intended to exclude, for example, other additives, components, integers or steps.

[0202] “Optional” or “optionally” means that the subsequently described event, circumstance or material may or may not occur or exist, and that the description includes instances where the event, circumstance or material occurs or exists and instances where it does not.

[0203] Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, it is specifically contemplated and considered disclosed that ranges from one particular value and / or to another particular value are also disclosed, unless the context clearly dictates otherwise. Similarly, when values are expressed as approximations by use of the antecedent “about,” it will be understood that the particular value forms another specifically contemplated embodiment that should be considered disclosed, unless the context clearly dictates otherwise. It will further be understood that the endpoints of each of the ranges are significant both relative to the other endpoint and independently of the other endpoint, unless the context clearly dictates otherwise. It should be understood that all individual values and sub-ranges of values that are included in the explicitly disclosed ranges are also specifically contemplated and should be considered disclosed, unless the context clearly dictates otherwise. Finally, it should be understood that all ranges recite the recited range and the set of individual numbers from (and including) the first endpoint to (and including) the second endpoint. In the latter case, it should be understood that any individual number may be selected as a form of the quantity, value, or feature that the range refers to. In this way, the range describes a set of numbers or values from (and including) the first endpoint to (and including) the second endpoint, from which a single member of the set (i.e., a single number) may be selected as the quantity, value, or feature that the range refers to. The foregoing applies regardless of whether some or all of these embodiments are explicitly disclosed in a particular instance.

[0204] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed methods and compositions pertain. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present methods and compositions, the particularly useful methods, devices, and materials are as described. Publications and the materials cited therein are specifically incorporated herein by reference. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of references states the author's assertions, and the applicant reserves the right to challenge the accuracy and relevance of the cited documents. It will be clearly understood that, although many publications are referred to herein, such references do not constitute an admission that any of these documents forms a part of the common general knowledge in the art.

[0205] Although descriptions of materials, compositions, components, steps, techniques, etc. may include many options and alternatives, this should not be construed as and is not an admission that such options and alternatives are equivalent to each other, or particularly, obvious alternatives. Thus, for example, a list of different parts does not indicate that the listed parts are obvious to each other, nor is it an admission of equivalence or obviousness.

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[0255] 50. Pettersen, E.F. et al. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem 25, 1605 - 1612 (2004). Sequence Listing <110> The University of Hong Kong <120> Compositions of Antiviral Peptides and Methods of Use Thereof <130> IP00937 <150> US 62 / 991,407 <151> 2020-03-18 <160> 17 <170> PatentIn version 3.5 <210> 1 <211> 30 <212> PRT <213> Artificial sequence <220> <223> P9 peptide <400> 1 Asn Gly Ala Ile Cys Trp Gly Pro Cys Pro Thr Ala Phe Arg Gln Ile 1 5 10 15 Gly Asn Cys Gly His Phe Lys Val Arg Cys Cys Lys Ile Arg 20 25 30 <210> 2 <211> 30 <212> PRT <213> Artificial sequence <220> <223> P9R peptide <400> 2 Asn Gly Ala Ile Cys Trp Gly Pro Cys Pro Thr Ala Phe Arg Gln Ile 1 5 10 15 Gly Asn Cys Gly Arg Phe Arg Val Arg Cys Cys Arg Ile Arg 20 25 30 <210> 3 <211> 33 <212> PRT <213> Artificial sequence <220> <223> PA1 peptide <400> 3 Asn Gly Ala Ile Cys Trp Gly Pro Cys Pro Thr Ala Phe Arg Gln Ile 1 5 10 15 Gly Asn Cys Gly His Phe Lys Val Arg Cys Cys Lys Ile Arg Asp Glu 20 25 30 Asp <210> 4 <211> 30 <212> PRT <213> Artificial sequence <220> <223> P9RS peptide <400> 4 Asn Gly Ala His Ser Trp His Pro Asn Glu Thr His Phe Arg Gln Ile 1 5 10 15 His Asn Ser Gly Arg His Arg Val Arg Ser His Arg Ile Arg 20 25 30 <210> 5 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primer sequence of SARS-CoV-2 <400> 5 cctactaaat taaatgatct ctgctttact 30 <210> 6 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer sequence of SARS-CoV-2 <400> 6 caagctataa cgcagcctgt a 21 <210> 7 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Primer sequence of MERS-CoV <400> 7 caaaaccttc cctaagaagg aaaag 25 <210> 8 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer sequence for MERS-CoV <400> 8 gctcctttgg aggttcagac at 22 <210> 9 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primer sequence for SARS-CoV <400> 9 accagaatgg aggacgcaat 20 <210> 10 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Primer sequence for SARS-CoV <400> 10 gctgtgaacc aagacgcagt attat 25 <210> 11 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer sequence for H1N1 <400> 11 cttctaaccg aggtcgaaac g 21 <210> 12 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer sequence for H1N1 <400> 12 ggcattttgg acaaakcgtc ta 22 <210> 13 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer sequence for H7N9 <400> 13 cttctaaccg aggtcgaaac g 21 <210> 14 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer sequence for H7N9 <400> 14 ggcattttgg acaaakcgtc ta 22 <210> 15 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Primer sequence for rhinovirus <400> 15 agccygcgtg gckgcc 16 <210> 16 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Primer sequence for rhinovirus <400> 16 agccygcgtg gtgccc 16 <210> 17 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Probe sequence for rhinovirus <400> 17 tccggcccct gaatgyggct aa 22

Claims

1. An antiviral agent comprising P9R having an amino acid sequence as shown in SEQ ID NO:

2.

2. The antiviral agent according to claim 1, which consists of P9R having an amino acid sequence as shown in SEQ ID NO:

2.

3. The antiviral agent according to claim 1 or 2, wherein the antiviral agent has a net positive charge of at least 5.

4. The antiviral agent according to claim 3, wherein the antiviral agent has a net positive charge of 5.

6.

5. A composition comprising a therapeutically effective amount of the antiviral agent according to any one of claims 1 - 4 and a pharmaceutically acceptable carrier.

6. The composition according to claim 5, wherein the composition is in unit dosage form.

7. The composition according to claim 6, wherein the unit dosage form is selected from tablets or capsules.

8. The composition according to claim 5, which is in a form suitable for intranasal or pulmonary delivery.

9. The composition according to claim 6, wherein the unit dosage form is injectable, and the composition further comprises a pharmaceutically acceptable carrier for injection into a human.

10. Use of an effective amount of the antiviral agent according to any one of claims 1 - 4 or the composition according to any one of claims 5 - 9 in the preparation of a medicament for treating a viral infection in a subject in need thereof, wherein the infection is caused by SARS-CoV-2, SARS-CoV, MERS-CoV, A H1N1 pdm09 virus, avian influenza A H7N9 virus, and an unenveloped rhinovirus.

11. The use according to claim 10, wherein the composition is administered parenterally or orally.

12. The use according to claim 10, wherein the composition is administered intranasally or by pulmonary administration.

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