Use of RADA16 to reduce mucosal inflammation

Abstract

The present invention provides methods for treating mucosal inflammation or vascular leakage in a subject. The methods involve administering to the subject a pharmaceutical composition comprising RADA16 self-assembling peptide.

Description

Attorney Docket No.:049648-629729 UF Ref. UFPT19408WO001 USE OF RADA16 TO REDUCE MUCOSAL INFLAMMATION CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of US Provisional Application No.63 / 647,514, filed May 14, 2024, which is incorporated by reference in its entirety for all purposes. STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with government support under Grant No(s). R01 AI134698, R01 AI144364, R01 DE031928, awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO A SEQUENCE LISTING

[0003] The application includes sequences in an electronic sequence listing named 629729SEQLST.XML of size 1.9KB, created May 12, 2025, which is incorporated by reference. BACKGROUND OF THE INVENTION

[0004] Chronic rhinosinusitis (CRS) affects up to 16% of the United States population with direct costs of nearly $22 billion per year (1). Its negative impact on quality of life exceeds other chronic conditions, such as heart failure and chronic obstructive pulmonary disease (2-4). CRS represents one disease that is composed of a wide variety of clinical phenotypes. CRS with nasal polyps (CRSwNP) is the most difficult form of the disease to treat. CRSwNP is characterized by a polarized type 2 microenvironment with elevated eosinophils, fibroblasts, CD4+Th2 cells, and cytokines such as interleukin-4 (IL-4), IL-5, IL-13, IL-25, IL-33 and thymic stromal lymphopoietin (TSLP) (5-11). The significance of these factors is that they drive many of the physical symptoms of CRSwNP including rhinorrhea, mucus production and tissue remodeling (12, 13). The most effective therapy for CRSwNP is systemic steroids to suppress this inflammatory response, but prolonged use is not practical due to adverse side effects. When this fails, greater than 50% of patients with CRSwNP go on to have multiple surgeries (14). Emerging therapies for CRSwNP are primarily systemic monoclonal antibodies targeting IgE orAttorney Docket No.:049648-629729 UF Ref. UFPT19408WO001 type 2 cytokines (15-18). These approaches are expensive and may have significant systemic side effects, thus the need for cost effective therapies with limited side effects is great.

[0005] Self-assembling peptide (SAP) RADA16, which forms a nanofiber hydrogel upon pH neutralization, has been used to block flow of blood at site of application in patients (19). BRIEF SUMMARY OF THE CLAIMED INVENTION

[0006] In one aspect, the invention provides a method of treating mucosal inflammation or vascular leakage in a subject, comprising administering to the subject a pharmaceutical composition comprising RADA16 self-assembling peptide.

[0007] In some methods, the mucosal inflammation is in an upper airway, a trachea, a lung, or a gastrointestinal tract. In some methods, the mucosal inflammation is in the upper airway. In some methods, the mucosal inflammation in the upper airway is in the nose, nasal passages, paranasal sinuses, pharynx, or larynx.

[0008] In some methods, the mucosal inflammation in the upper airway is associated with chronic rhinosinusitis without nasal polyps (CRSsNP), chronic rhinosinusitis with nasal polyps (CRSwNP), subsets of chronic rhinosinusitis with nasal polyps, upper airway disease treatment in cystic fibrosis (CF), allergic rhinitis, non-allergic rhinitis, chronic sinusitis, asthma, sinusitis, inflammatory-driven anosmia, inflammatory-driven hyposmia, or an allergy. In some methods, the subsets of chronic rhinosinusitis with nasal polyps are selected from the group consisting of allergic fungal rhinosinusitis (AFRS), aspirin-exacerbated respiratory disease (AERD), and common variable immunodeficiency (CIVD). In some methods, the allergic rhinitis, asthma, sinusitis, or allergy is associated with an inhaled allergen.

[0009] In some methods, the mucosal inflammation is in the trachea. In some methods, the mucosal inflammation is in the lung. In some methods, the mucosal inflammation is in the gastrointestinal tract. In some methods, the vascular leakage is in the lung.

[0010] In another aspect, the invention provides a method of treating chronic rhinosinusitis without nasal polyps (CRSsNP), chronic rhinosinusitis with nasal polyps (CRSwNP), subsets of chronic rhinosinusitis with nasal polyps, upper airway disease treatment in cystic fibrosis (CF),Attorney Docket No.:049648-629729 UF Ref. UFPT19408WO001 allergic rhinitis, non-allergic rhinitis, chronic sinusitis, asthma, sinusitis, inflammatory-driven anosmia, inflammatory-driven hyposmia, or an allergy in a subject, comprising administering to the subject a pharmaceutical composition comprising RADA16 self-assembling peptide.

[0011] In some methods, the subsets of chronic rhinosinusitis with nasal polyps are selected from the group consisting of allergic fungal rhinosinusitis (AFRS), aspirin-exacerbated respiratory disease (AERD), and common variable immunodeficiency (CIVD). In some methods, the allergic rhinitis, asthma, sinusitis, or an allergy is associated with an inhaled allergen.

[0012] In some methods, the pharmaceutical composition comprises RADA16 self-assembling peptide at a concentration of about 0.1% w / v to about 10.0% w / v. In some methods, the pharmaceutical composition comprises RADA16 self-assembling peptide at a concentration of about 0.3% w / v to about 8.0% w / v. In some methods, the pharmaceutical composition comprises RADA16 self-assembling peptide at a concentration of about 0.5% w / v to about 5.0% w / v. In some methods, the pharmaceutical composition comprises RADA16 self-assembling peptide at a concentration of about 1.0% w / v to about 3.0% w / v. In some methods, the pharmaceutical composition comprises RADA16 self-assembling peptide at a concentration of about 2.5% w / v.

[0013] In some methods, the pharmaceutical composition comprises PURAGEL hemostatic gel. In some methods, the pharmaceutical composition is administered topically.

[0014] In some methods, the subject is a mammal. In some methods, the mammal is a primate. In some methods, the mammal is a non-human primate. In some methods, the mammal is a human.

[0015] In some methods, the mammal is a rodent. In some methods, the rodent is a mouse, rat, guinea pig, hamster, or gerbil. In some methods, the mammal is selected from the group consisting of human, baboon, chimpanzee, monkey, cynomolgus, marmoset, rhesus, rodent, rabbit, cat, dog, horse, cow, sheep, goat, pig, ferret, guinea pig, hamster, and gerbil. In some methods, the subject is a non-human mammal, bird, reptile, amphibian, or fish.Attorney Docket No.:049648-629729 UF Ref. UFPT19408WO001

[0016] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 depicts an ex vivo lung perfusion system used to model biopsy bleeding.

[0018] Figure 2 depicts procedure of studies testing PuraGel in ex vivo pig lung perfusion system. 2A Biopsy site, Bronchial bifurcation, black circle refers to where the biopsy will be taken; 2B Evans Blue Addition, Same site as 2A showing appearance of the bifurcation after the addition of Evans Blue into the lung circulation; 2C, Biopsy, left and right panels showing biopsing of the site shown in 2A and B; 2D, left and right panels, Puragel Application, top arrows point to Puragel, bottom arrows point to Puragel catheter; 2E Puragel lavage, arrow points to Puragel.

[0019] Figure 3 depicts a schematic showing cultured nasal cells in a pseudostratified epithelium in culture plate. Top arrow points to apical treatment side where PuraGel or Chitogel was applied. Bottom arrow points to basolateral side.

[0020] Figure 4 depicts schematic showing effect of gap junctions breaking down in Chitogel treated cultured nasal cells in a pseudostratified epithelium. Left-side schematic depicts pseudostratified epithelium before Chitogel treatment. Right-side schematic depicts pseudostratified epithelium after Chitogel treatment. Basolateral media crosses to the apical side (upward arrows on right-side schematic).

[0021] Figures 5A-E depict results of studies showing PuraGel reduces basolateral inflammation 6 hours post treatment from control and CRSwNP epithelial cells.5A IL-86 Hours Post-Treatment, 5B C36 Hours Post-Treatment, 5C IL-66 Hours Post-Treatment, 5D C3a 6 Hours Post-Treatment, Black dot = control subject (n=5), Red dot = CRSwNP patient (n=5), p<0.05 is statistically significant. All data basolateral; 5E schematic of experimental set-up.

[0022] Figures 6A-E depict results of studies showing PuraGel reduces basolateral inflammation 24 hours post treatment from control and CRSwNP epithelial cells for mostAttorney Docket No.:049648-629729 UF Ref. UFPT19408WO001 cytokines.6A IL-824 Hours Post-Treatment, 6B C324 Hours Post-Treatment, 6C IL-624 Hours Post-Treatment, 6D C3a 24 Hours Post-Treatment, Black dot = control subject (n=5), Red dot = CRSwNP patient (n=5), p<0.05 is statistically significant. All data basolateral; 6E schematic of experimental set-up.

[0023] Figures7A-E depict results of studies showing PuraGel reduces most apical inflammation 24 hours post treatment from control and CRSwNP epithelial cells.7A IL-824 Hours Post-Treatment, 7B C324 Hours Post-Treatment, 7C IL-624 Hours Post-Treatment, 7D C3a 24 Hours Post-Treatment, Black dot = control subject (n=5), Red dot = CRSwNP patient (n=5), p<0.05 is statistically significant. All data apical rinse; 7E schematic of experimental set- up.

[0024] Figures 8A-C depict results of studies showing Basolateral bFGF, which stimulates fibroblast proliferation, is modestly reduced as 6 hours, unchanged at 24 hours post treatment. 8A bFGF 6 Hours Post-Treatment, 8B bFGF 24 Hours Post-Treatment. Black dot = control subject (n=5), Red dot = CRSwNP patient (n=5), p<0.05 is statistically significant. All data basolateral; 8C schematic of experimental set-up.

[0025] Figures 9A-B depict results of studies showing Apical treatment with PuraGel can modestly reduce total protein in the basolateral media.9A BCA (a read out of total bulk protein) 24 Hours Post-Treatment. Black dot = control subject (n=5), Red dot = CRSwNP patient (n=5), p<0.05 is statistically significant. All data basolateral; 9B schematic of experimental set-up.

[0026] Figure 10 depicts results of studies showing RADA16 reduced passage of dextran into the subepithelial space, even with antigen stimulation by Aspergillus Fumigates (Af) extract. N=4 patients with CRSwNP.

[0027] Figure 11 depicts results of studies showing RADA16 treatment for 2 weeks was safe to human sinonasal epithelial cells (HSENC). Samples are from humans sinonasal epithelial cells from= 3 control subjects + 3 CRSwNP patients (dots represent individual patient data); LDH assay (Lactate Dehydrogenase assay) is a method used to detect cell death or cytotoxicity. Values shown are mean + / - SD.Attorney Docket No.:049648-629729 UF Ref. UFPT19408WO001

[0028] Figure 12 depicts results of studies showing two weeks post-treatment RADA16 reduces apical levels of C3. Samples are from humans sinonasal epithelial cells from= 3 control subjects + 3 CRSwNP patients (dots represent individual patient data); Values shown are mean + / - SD.

[0029] Figures 13A-C depict results of studies showing C3 is linked to loss of sense of smell. 13A: C3, 13B C3a, 13C C3b. DEFINITIONS

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. The following references provide one of skill with a general definition of many of the terms used in this invention: Academic Press Dictionary of Science and Technology, Morris (Ed.), Academic Press (1sted., 1992); Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (Eds.), Oxford University Press (revised ed., 2000); Encyclopaedic Dictionary of Chemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionary of Microbiology and Molecular Biology, Singleton et al. (Eds.), John Wiley & Sons (3rded., 2002); Dictionary of Chemistry, Hunt (Ed.), Routledge (1sted., 1999); Dictionary of Pharmaceutical Medicine, Nahler (Ed.), Springer-Verlag Telos (1994); Dictionary of Organic Chemistry, Kumar and Anand (Eds.), Anmol Publications Pvt. Ltd. (2002); and A Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (Eds.), Oxford University Press (4thed., 2000). In addition, the following definitions are provided to assist the reader in the practice of the invention.

[0031] The term “patient” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.

[0032] The term “disease” refers to any abnormal condition that impairs physiological function. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition, or syndrome in which physiological function is impaired, irrespective of the nature of the etiology.Attorney Docket No.:049648-629729 UF Ref. UFPT19408WO001

[0033] The term “symptom” refers to a subjective evidence of a disease, as perceived by the subject. A "sign" refers to objective evidence of a disease as observed by a physician.

[0034] The term "individual" or “subject” refers to a human or a non-human animal. The non- human subject may be, for example, a non-human animal, and may be, for example, a non- human mammal, bird, reptile, amphibian, or fish.. The “mammal” may include any animal classified as such, including humans, non-human primates, primates, baboons, chimpanzees, monkeys, cynomolgus, marmoset, rhesus, rodents (e.g., mice, rats), rabbits, cats, dogs, horses, cows, sheep, goats, pigs, ferrets, guinea pigs, hamsters, gerbils etc.

[0035] The term “upper airway” or “upper respiratory tract” refers to the nose, nasal passages, paranasal sinuses, pharynx, or larynx.

[0036] The term “mucosal inflammation” refers to a heightened immune response that can be chronic or acute that take place in areas of the body, characterized by the presence of mucus which includes the upper and lower airways and digestive system.

[0037] Examples of a condition associated with mucosal inflammation in the upper airway include chronic rhinosinusitis without nasal polyps (CRSsNP), chronic rhinosinusitis with nasal polyps (CRSwNP), subsets of Chronic rhinosinusitis with nasal polyps, upper airway disease treatment in cystic fibrosis (CF), allergic rhinitis, non-allergic rhinitis, chronic sinusitis, asthma, sinusitis, inflammatory-driven anosmia, inflammatory-driven hyposmia, and an allergy. Subsets of Chronic rhinosinusitis with nasal polyps include allergic fungal rhinosinusitis (AFRS), aspirin-exacerbated respiratory disease (AERD), and common variable immunodeficiency (CIVD).

[0038] The term “apical” refers to a surface of an epithelial cell facing a body exterior or outside compartment of an organism or cavity of an internal organ, and refers to a top side for cells that contact cilia and where mucus is produced.

[0039] The term “basolateral” refers to a surface of an epithelial cell facing serosa and an inside compartment of an organism, and refers to an underside of cells which would be secreted into the sub epithelial space.Attorney Docket No.:049648-629729 UF Ref. UFPT19408WO001

[0040] The terms "therapeutically effective dose," or "therapeutically effective amount," refer to that amount of a compound that results in prevention, delay of onset of symptoms, or amelioration of symptoms of mucosal inflammation. A therapeutically effective amount will, for example, be sufficient to treat, prevent, reduce the severity, delay the onset, or reduce the risk of occurrence of one or more symptoms of mucosal inflammation. The effective amount can be determined by methods well known in the art and as described in subsequent sections of this description.

[0041] The terms "treatment," "therapeutic method," and their cognates refer to treatment and prophylactic / preventative measures. Those in need of treatment may include individuals already having a particular medical disorder as well as those who may ultimately acquire the disorder. The need for treatment is assessed, for example, by the presence of one or more risk factors associated with the development of a disorder, the presence or progression of a disorder, or likely receptiveness to treatment of a subject having the disorder. Treatment may include slowing or reversing the progression of a disorder.

[0042] The term “topical administration” refers to delivery of a compound directly to a site of inflammation or injury.

[0043] The term “inflammatory mediator” refers to a protein that can cause the immune system to enter an activated state.

[0044] Compositions or methods “comprising” or “including” one or more recited elements may include other elements not specifically recited. For example, a composition that “comprises” or “includes” RADA16 self-assembling peptide may contain RADA16 self-assembling peptide alone or in combination with other ingredients. When the disclosure refers to a feature comprising specified elements, the disclosure should alternative be understood as referring to the feature consisting essentially of or consisting of the specified elements. Moreover, elements that are shown or described as being combined with other elements, can, in various embodiments, exist as stand-alone elements.

[0045] Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range.Attorney Docket No.:049648-629729 UF Ref. UFPT19408WO001

[0046] Unless otherwise apparent from the context, the term “about” encompasses insubstantial variations, such as values within a standard margin of error of measurement (e.g., SEM or standard deviation) of a stated value. Unless otherwise apparent from the context, the term “about” encompasses values within ±5% or ±10% of a stated value.

[0047] Statistical significance means p^0.05.

[0048] The singular forms of the articles “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” can include a plurality of compounds, including mixtures thereof. DETAILED DESCRIPTION I. General

[0049] The present invention provides methods of treating mucosal inflammation and diseases associated with mucosal inflammation and methods of treating vascular leakage and diseases associated with vascular leakage in a subject using RADA16 self-assembling peptide. The present methods are useful in treating diseases associated with mucosal inflammation for example diseases of the upper airway, trachea, lungs, and gastrointestinal tract. The methods are useful in treatment of disorders associated with mucosal inflammation in the upper airway including, for example, chronic rhinosinusitis without nasal polyps (CRSsNP), chronic rhinosinusitis with nasal polyps (CRSwNP), subsets of chronic rhinosinusitis with nasal polyps (for example allergic fungal rhinosinusitis (AFRS), aspirin-exacerbated respiratory disease (AERD), or common variable immunodeficiency (CIVD)), upper airway disease treatment in cystic fibrosis (CF), allergic rhinitis, non-allergic rhinitis, chronic sinusitis, asthma, sinusitis, inflammatory-driven anosmia, inflammatory-driven hyposmia, and an allergy.

[0050] The methods are useful in reducing mucosal inflammation in patient or subject, for example by absorbing inflammatory mediators, cytokines, and / or proteins in inflamed mucosal tissue and / or by preventing apical inflammatory mediators, cytokines, and / or proteins from passing into the basolateral space of an inflamed mucosal tissue. Exemplary cytokines include IL-8, IL-6, C3, and C3a. Exemplary inflammatory mediators are all members of the interleukin family, all members of the chemokine family of proteins, all members of the complement family, all members of theAttorney Docket No.:049648-629729 UF Ref. UFPT19408WO001 mucin family and any other secreted immune modulatory product. RADA16 self-assembling peptide is useful in absorbing inflammatory mediators, cytokines, and / or proteins and reducing bioavailability of an inflammatory mediator, cytokine, and / or protein present in mucus, for example nasal mucus, that RADA16 self-assembling peptide comes in contact with. The methods are useful in treating mucosal inflammation and diseases associated with mucosal inflammation in humans and animals.

[0051] For example, the present invention provides a method of using RADA16 self-assembling peptide as a therapeutic agent in treating chronic rhinosinusitis with nasal polyps (CRSwNP) in a human or animal subject. In a CRSwNP epithelial cell-based assay, PURAGEL RADA16 self- assembling peptide was shown to be absorbent and to reduce basolateral inflammation and apical inflammation and to reduce cytokines IL-8, IL-6, C3, and C3a in both apical and basolateral spaces.

[0052] For example, the present invention provides a method of using RADA16 self-assembling peptide as a therapeutic agent to reduce inflammation caused by inhaled airway irritants or inhaled allergens in a human or animal subject. In a human sinonasal epithelial cell (HSNEC)- based assay, PURAGEL RADA16 self-assembling peptide was shown to reduce the passage of dextran into the subepithelial space, in cells stimulated with antigen Aspergillus Fumigates (Af) extract.

[0053] For example, the present invention provides a method for stopping vascular leakage following surgical biopsy in a human or an animal subject. In ex vivo studies examining pig lung biopsies, PURAGEL RADA16 self-assembling peptide was shown to be absorbent and to stop vascular leakage following surgical biopsy.

[0054] PURAGEL RADA16 self-assembling peptide when applied to the mucosal surfaces has anti-inflammatory properties by absorbing inflammatory mediators present in airway mucus. Surgical or medical management of these disease does not involve management of bleeding, but is largely focused on reduction of inflammation. Given its ability to reduce airway inflammation potential diseases of the upper airway include, but are not limited to: ^ Chronic rhinosinusitis without nasal polyps (CRSsNP)Attorney Docket No.:049648-629729 UF Ref. UFPT19408WO001 ^ Chronic rhinosinusitis with nasal polyps (CRSwNP) ^ Subsets of Chronic rhinosinusitis with nasal polyps o Allergic fungal rhinosinusitis (AFRS) o Aspirin-exacerbated respiratory disease (AERD) o Common variable immunodeficiency (CIVD) ^ Upper airway disease treatment in cystic fibrosis (CF) ^ Allergic rhinitis ^ Non-allergic rhinitis ^ Chronic sinusitis ^ Asthma ^ Sinusitis ^ Inflammatory-driven anosmia ^ Inflammatory-driven hyposmia; and ^ an allergy. II. RADA16 self-assembling peptide

[0055] RADA16 synthetic oligopeptide belongs to a group called Self-Assembling Peptides (SAPs). The peptide is a 16-unit oligopeptide termed RADA16 composed of four repeating amino acid sequences of four amino acids, Arginine (R) – Alanine (A) – Aspartic acid (D) – Alanine (A). The sequence of RADA16 is RADARADARADARADA (SEQ ID NO:1). Within the 16-amino acid monomer sequence, the alternating positively and negatively charged amino acids (arginine and aspartic acid), along with the non-polar alanines between the charged amino acids, create two distinct structural surfaces, one hydrophilic and the other hydrophobic. (19; World Wide Web at 3dmatrix.com / us / puragel / product / ).

[0056] The monomer building-blocks form β-sheet structures via non-covalent interactions. When exposed to physiological pH (blood or tissue), the β-sheet structures increase in number and the solution gels, resulting in the formation of a nanofibrous hydrogel.

[0057] Multiple formulations and concentrations of RADA16 self-assembling peptide are described in the literature (for example, PURAGEL hemostatic gel (2.5% w / v)), andAttorney Docket No.:049648-629729 UF Ref. UFPT19408WO001 PURAMATRIX synthetic peptide hydrogel (1% w / v) (Corning, Glendale, AZ, Product Number 354250). PURAGEL is also referred to here as “Puragel” and “PuraGel”..

[0058] RADA16 self-assembling peptide is distributed under trade names PURAGEL, PURASTAT (Cat.621-062), PURASINUS, and PURAMATRIX in the US by 3-D Matrix Medical Technology (Tokyo, Japan). PURAGEL is currently FDA approved in the US as a medical device for the treatment of epistaxis (severe nose bleeds). World Wide Web at 3dmatrix.com / us / .

[0059] PURAGEL achieves its hemostatic effects by forming a 3-dimensional hydrogel matrix barrier that blocks the flow of blood at the site of application.

[0060] PURAGEL, at a basic structural level, is composed of a matrix of nanofibrils formed from individual peptide monomers. These fibrils are 10-20 nm in diameter and are interwoven to create an ordered structure with 50-100 nm pore sizes. This woven network structure, or matrix barrier, is similar to the microarchitecture of endogenous extracellular matrix (ECM).

[0061] PURAGEL monomers form β-sheet structures via non-covalent interactions. These β- sheets form the fibril structures that constitute the nanofibrous hydrogel.

[0062] A 1% solution (w / v) of RADA16 self-assembling peptide is sold under the tradename PURAMATRIX for cell culture use by 3-D Matrix Medical Technology (Tokyo, Japan).

[0063] PURAGEL hemostatic gel is a 2.5% RADA16 self-assembling peptide (w / v) and 97.5% water (w / v) formulation supplied in a pre-filled syringe by 3-D Matrix Medical Technology (Cat. # 651-005). PURAGEL hemostatic gel may be applied directly from the syringe to cells in vitro or to a lung. PURAGEL hemostatic gel may be extruded from the supplied syringe either directly to a mucosal surface or through a catheter attached to the syringe and then spread across the mucosal surface using instruments found in the operating room.

[0064] PURAGEL (2.5% RADA16 self-assembling peptide w / v and 97.5% water w / v) forms a clear gel upon pH neutralization such as when exposed to physiological concentrations of salt or physiological fluids.Attorney Docket No.:049648-629729 UF Ref. UFPT19408WO001

[0065] RADA16 self-assembling peptide and its preparation are described in the art (20). In an example, RADA16 is prepared using the following equipment and steps.

[0066] RADA16 self-assembling peptide may be prepared by stepwise solid-phase synthesis, in manual and automatic modes.

[0067] Manual Synthesis: RADA16 is synthesized manually by solid-phase peptide synthesis by using 9-fluorenylmethoxycarbonyl (Fmoc) / tert-butyl (tBu) chemistry on an aminomethyl- ChemMatrix resin (21) (Matrix Innovation, scale 0.1 mmol, loading: 0.62 mmol / g, 35-100 mesh). Peptide elongation is performed in polypropylene syringes fitted with a polyethylene porous disk. Solvents and soluble reagents are removed by suction. The linker Rink-Amide (3 equiv., Iris Biotech) is anchored to the resin with N,N’-di-isopropylcarbodiimide (DIPCDI) / 1- hydroxybenzotriazole (HOBt)(3 equiv. each) in DMF for 12 h. At this point, a positive ninhydrin test indicates free amino groups, so the linker reaction is repeated, this time for 2 h. The Fmoc groups are removed by treatment with piperidine / DMF (1:4) in the presence of 0.1 m HOBt (1 x 2 min, 2 x 10 min), which is used to minimize aspartimide formation.(22) The following Fmoc- L-protected amino acid derivatives are used: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH and Fmoc- Asp(tBu)-OH. Chain extension is carried out by using 5 equiv. of the amino acid, at each coupling step, in the presence of 2-(6-chloro-1-H-benzotriazole-1-yl)-1,1,3,3- tetramethylaminium hexa-fluorophosphate (HCTU) (5 equiv.) and diisopropylethylamine (DIEA) (10 equiv.) for 1 h. In case of a positive ninhydrin test, a recoupling is performed, using a fivefold molar excess of the Fmoc-amino acid, activated essentially in situ by DIPCDI / 7-aza-1- hydroxybenzotriazole (HOAt) in DMF for 3 h. Incorporation of the following residues requires multiple recouplings: Ala2,Asp3, Ala4,Arg5,Ala6,Arg9,Ala10,Asp11, and Ala12. After Fmoc removal of the last Arg residue, acetylation is performed by treatment with Ac2O (10 equiv.) and DIEA (10 equiv.) for 30 min. All washings of the peptidyl-resin between Fmoc removal, amino acid coupling and the final acetylation are done with DMF. Once the fully protected peptide has been synthesized, it is subject to global deprotection (removal of the side-chain protecting groups and cleavage from the resin) by treatment with a mixture of TFA / triisopropylsilane (TIS) / H2O (95:2.5:2.5) for 4 h. The peptide is isolated by precipitation with cold diethyl ether, centrifuged, dissolved in H2O / MeCN / TFA (7:2.8:0.2, starting with neat TFA), and sequentially lyophilized. It is then characterized by HPLC [Column I; tR= 7.29 min, 75 % purity, gradient (%B): 5-25 in 15Attorney Docket No.:049648-629729 UF Ref. UFPT19408WO001 min] and MALDI-TOF MS shows the mass of the desired peptide {m / z calcd. for Ac-(RADA)4- NH2 (C66H113N29O25) 1711.8; found 1712.8 [M + H]+}. Other impurities are also detected from the HPLC analysis and characterized by MALDI-TOF MS: the peak that eluted at tR= 6.88 min (peak area: 6.84 % purity) corresponds to a byproduct that lacks the tripeptide RDA {m / z calcd. for Ac-(RADA)4-NH2 - RAD (C53H91N23O20) 1369.6; found 1370.7 [M + H]+}.

[0068] Automated Synthesis: The automated synthesis is conducted on the same resin used for the manual synthesis (aminomethyl-Chem-Matrix), with an ABI 433A synthesizer (Applied Biosystems, Foster City), on 0.1 mmol (peptide) scale, following standard Fmoc chemistry and using a 10-fold excess of Fmoc-protected l-amino acids and N-[(1H -benzotriazol-1-yl)- dimethylamino-methylene]-N-methylmethanaminium tetrafluoro-borate N-oxide (TBTU, 0.45 m) in the presence of HOBt as coupling reagents in DMF. Each deprotection step is carried out in 15 min and each coupling step in 35 min. Acetylation, cleavage and isolation of the completed peptide are done as described for the manual synthesis. The product is characterized by HPLC [Column I, tR = 7.06 min, 60 % purity, gradient (%B): 5-25 in 15 min] and by MALDI-TOF MS, which shows the mass of the desired peptide {m / z calcd. for Ac-(RADA)4-NH2 (C66H113N29O25) 1711.8; found 1712.6 [M + H]+}.

[0069] Various synthesis processes for RADA16 have previously been described in the scientific literature, including but not limited to Eur J Org Chem - 2013 - Paradís-Bas - RADA-16 A Tough Peptide Strategies for Synthesis and Purification.” (20). III. Methods of using RADA16 self-assembling peptide as a Therapeutic for treating Mucosal Inflammation and to Stop Vascular Leakage

[0070] The present invention relates to methods of using RADA16 self-assembling peptide for the treatment or prevention of mucosal inflammation, for example in diseases of the upper airway, trachea, lungs, and gastrointestinal tract. Exemplary disorders associated with mucosal inflammation in the upper airway include chronic rhinosinusitis without nasal polyps (CRSsNP), chronic rhinosinusitis with nasal polyps (CRSwNP), subsets of chronic rhinosinusitis with nasal polyps (for example allergic fungal rhinosinusitis (AFRS), aspirin-exacerbated respiratory disease (AERD), or common variable immunodeficiency (CIVD)), upper airway disease treatment in cystic fibrosis (CF), allergic rhinitis, non-allergic rhinitis, chronic sinusitis, asthma,Attorney Docket No.:049648-629729 UF Ref. UFPT19408WO001 sinusitis, inflammatory-driven anosmia, inflammatory-driven hyposmia, and an allergy. The allergic rhinitis, asthma, sinusitis, and / or allergy can be associated with an inhaled allergen. Exemplary inhaled allergens include, for example a fungus, for example Aspergillus fumigates; other species of mold; a house dust mite antigen; or a pollen such as grass or tree. The non- allergic rhinitis can be associated with occupational exposures or rhinitis caused by pollution. Inflammatory-driven loss of sense of smell or inflammation-driven loss of sense of smell includes inflammatory-driven anosmia and inflammatory-driven hyposmia.

[0071] RADA16 self-assembling peptide can block inhaled antigens from passing into the subepithelial space. RADA16 self-assembling peptide can be used to reduce allergic exposure and / or environmental exposure driven inflammation in the upper airway. RADA16 self- assembling peptide can be used to treat inflammation caused by inhaled allergens, such as allergic rhinitis.

[0072] RADA16 self-assembling peptide can rapidly absorb a wide variety of inflammatory mediators immediately following its application to the epithelium. RADA16 self-assembling peptide has sustained anti-inflammatory properties for larger sized immune mediators and proteins, such as complement. RADA16 self-assembling peptide can reduce inflammation caused by inhaled airway irritants by blocking them from coming in contact with epithelial cells. Blocking inhaled airway irritants from coming in contact with epithelial cells reduces epithelial cell release of inflammatory mediators in response to irritant exposure and reduces the passage of irritants from entering into the subepithelial space which can active resident immune cells, further driving inflammation.

[0073] The present invention relates to methods of using RADA16 self-assembling peptide for the treatment or prevention of vascular leakage, for example in diseases in which a surgical injury has created a damaged mucosal site.

[0074] RADA16 self-assembling peptide can be used, for example to treat or prevent biopsy- associated bleeding associated with taking a biopsy of a patient or subject. Taking a tissue biopsy sample from a patient or subject can be useful in diagnosing or treating a patient, however carries the risk of biopsy-associated bleeding. Superficial biopsies can lead to inadequate tissue for diagnosis. Current methods of reducing biopsy-associated bleeding include application ofAttorney Docket No.:049648-629729 UF Ref. UFPT19408WO001 pressure and absorbent material used for biopsy induced bleeding, for example putting gauze into a lung and holding it in place until the bleeding stops. The methods herein are useful in optimizing biopsy sampling to reduce biopsy-associated bleeding. For example the biopsy can be from a lung.

[0075] RADA16 self-assembling peptide can be used during surgical procedures, for example in, Video Assisted Thoracoscopic Surgery (VATS), to treat or prevent or reduce bleeding during the procedure. VATS is a relatively non-invasive procedure and is used to minimize surgical trauma. VATS is a common procedure with risk of incision site bleeding. Complications with bleeding during VATS often require traditional surgery, a more expansive procedure.

[0076] RADA16 self-assembling peptide can be used to treat or prevent or reduce post surgery bleeding complications, for example in a surgery procedure such as a cancerous lung resection or a non-cancerous lung resection, or at a vascular or a bronchial anastomosis site. Post surgery bleeding complications are the most common cause of unplanned return to the operation room. For example, RADA16 self-assembling peptide is useful, for example, to treat or prevent or reduce post surgery bleeding of post cancer resection incisions.

[0077] The subject of application of the present invention may be human or non-human. The non-human subject may be, for example, a non-human animal, and may be, for example, a non- human mammal, bird, reptile, amphibian, or fish. Examples of the non-human mammal include rodents (for example, mice and rats), dogs, cats, horses, pigs, cows, sheep, goats, primates, and the like. In addition, all aforementioned embodiments are applicable to domesticated, agricultural, or zoo-maintained mammals experiencing mucosal inflammation, as well as to humans. For example, RADA16 self-assembling peptide may be administered to humans, non- human primates, primates, baboons, chimpanzees, monkeys, cynomolgus, marmoset, rhesus, rodents (e.g., mice, rats), rabbits, cats, dogs, horses, cows, sheep, goats, pigs, ferrets, guinea pigs, hamsters, gerbils etc. RADA16 self-assembling peptide may be administered to house pets such as dogs, cats, rabbits, ferrets, guinea pigs, hamsters and gerbils, as well as to agricultural animals, such as horses, sheep, cows, and pigs, or to animals such as camel, cynomolgus, marmoset, rhesus and chimpanzee. RADA16 self-assembling peptide may be administered to a human.Attorney Docket No.:049648-629729 UF Ref. UFPT19408WO001

[0078] The upper airway (also known as upper respiratory tract) includes the nose and nasal passages, paranasal sinuses, the pharynx, and the portion of the larynx above the vocal folds (cords).

[0079] The method and the route of administration for the pharmaceutical composition useful in methods of the present invention may be topically administrable to the upper airway, trachea, lungs, and gastrointestinal tract.. For example, topical administration under endoscopic guidance, administration using the syringe holding PURAGEL, or by attaching a catheter, administration from a surgical incision site of the upper airway, and the like may be used. In one embodiment of the present invention, the pharmaceutical composition is administered in the form of a suppository.

[0080] The dosage and the frequency of administration of the pharmaceutical composition useful in methods of the present invention may be determined appropriately by a person having ordinary skill in the art (for example, a doctor) according to a target pathological condition.

[0081] The concentration of RADA16 self-assembling peptide in the pharmaceutical composition useful in methods of the present invention may be about 0.1% w / v to about 10.0% w / v, and may be about 0.3% w / v to about 8.0% w / v, about 0.5% w / v to about 5.0% w / v, or about 1.0% to about 3.0% w / v. In some embodiments the concentration of RADA16 self-assembling peptide in the pharmaceutical composition is about 2.5% w / v. In some embodiments the concentration of RADA16 self-assembling peptide in the pharmaceutical composition is 2.5% w / v. In some methods, the pharmaceutical composition comprising RADA16 self-assembling peptide is PURAGEL hemostatic gel. In some methods, PURAGEL hemostatic gel is administered to a patient or subject.

[0082] The pharmaceutical composition useful in methods of the present invention may further contain another drug effective at treating or preventing mucosal inflammation in the upper airway, trachea, lungs, and gastrointestinal tract and / or effective at treating or preventing vascular leakage, or may be used in combination with another drug effective at treating or preventing mucosal inflammation in the upper airway, trachea, lungs, and gastrointestinal tract and / or effective at treating or preventing vascular leakage. The aspect of combining use of the pharmaceutical composition and the other drug is not limited, and for example, may be an aspectAttorney Docket No.:049648-629729 UF Ref. UFPT19408WO001 in which separately prepared respective drugs are administered simultaneously to the subject, or may be an aspect in which the respective drugs are administered to the subject at different times.

[0083] For example, PURAGEL may be used to apply drugs for slow release to an affected surface, that would have otherwise been removed rapidly by mucociliary clearance, gravity and patient movement, thereby improving the efficacy of the drug due to sustained delivery.

[0084] When the pharmaceutical composition useful in methods of the present invention is used together with another drug, the ratio of the pharmaceutical composition to the other drug may be arbitrary.

[0085] RADA16 self-assembling peptide used in the present invention may be modified (or labeled) as long as main properties of the peptide intended by the present invention are not lost, and peptides modified (or labeled) in this manner are also included in the RADA16 self- assembling peptide used in methods of the present invention. The method for modifying (or labeling) the RADA16 self-assembling peptide used in the present invention may be arbitrary selected by a person having ordinary skill in the art, and may be, for example, an addition of a functional group or the like, an addition of a chemical substance, or addition of an additional protein or peptide. Examples of the addition of the functional group or the like may include acylation, acetylation, alkylation, amidation, biotinylation, formylation, carboxylation, glutamylation, glycosylation (addition of a sugar chain), glycylation, hydroxylation, isoprenylation, lipoylation, addition of a nucleotide or a derivative thereof, polyethylene glycol (PEG)ylation, and addition of a lipid chain. In addition, examples of the addition of the chemical substance may include addition of a suitable labeling agent such as a radioisotope (for example,123I,i31I,3H,i4C, and the like), an enzyme (for example, p-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malic acid dehydrogenase, and the like), a fluorescent substance (for example, fluorescamin, fluorescein isothiocyanate, and the like), a luminescent substance (for example, luminol, a luminol derivative, luciferin, lucigenin, and the like), an affinity tag (for example, biotin and the like). In addition, further examples of the addition of the protein or peptide may include ISGylation, SUMOylation, and ubiquitination.

[0086] All patent filings, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extentAttorney Docket No.:049648-629729 UF Ref. UFPT19408WO001 as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the invention can be used in combination with any other unless specifically indicated otherwise. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims. EXAMPLES

[0087] Example 1: Materials

[0088] RADA16 Self-assembling Peptide Formulation PURAGEL is a 2.5% w / v RADA16 self-assembling peptide and 97.5% w / v water formulation supplied in a pre-filled syringe by 3-D Matrix Medical Technology (Cat. # 651-005). PURAGEL was applied directly from the syringe to primary human sinonasal epithelial cells in vitro or to a lung. Chitogel (Medtronic, Minneapolis, MN, Cat. CSK-4).

[0089] Example 2: RADA16 Self-assembling Peptide is Absorbent in Pig Lung Biopsies

[0090] RADA16 self-assembling peptide was tested for absorbency in pig lung biopsies.

[0091] Modeling Biopsy Bleeding An ex vivo lung perfusion system was used for these studies. Ex vivo lung perfusion systems have been previously used (25, 26) clinically for optimized donor lung procurement and used experimentally (27, 28) for drug development, delivery, and lung procedure optimization. An experimental set-up for ex vivo lung perfusion is shown in Figure 1.Attorney Docket No.:049648-629729 UF Ref. UFPT19408WO001

[0092] Experimental Design • Pig lungs were procured and placed on the ex vivo perfusion machine, ventilated and perfused with blood like solution • To visualize biopsy induced vascular leak a blue dye (Evans Blue) was added to the perfusion solution • Biopsy was taken from a bronchiole bifurcation causing a small vascular leak. • Catheter was then inserted down the bronchoscope and Puragel applied to the biopsy site (Images detailing process are shown in Figure 2)

[0093] Figure 2 shows images of the procedure. Fig.2A shows Biopsy site, Bronchial bifurcation, black circle refers to where the biopsy will be taken. Fig.2B shows Evans Blue Addition, same site as 2A showing appearance of the bifurcation after the addition of Evans Blue into the lung circulation. Fig.2C shows biopsy, left and right panels showing biopsing of the site shown in 2A and B. Fig.2D shows Puragel application. Top arrows point to Puragel. Bottom arrows point to Puragel catheter. Fig.2E shows Puragel lavage. Arrow points to Puragel.

[0094] Results • Effective delivery via provided catheter • Gel set within seconds of interacting with the perfusion fluid leaking through the biopsy induced injury • Repeated fluid washes past the gel did not lead to gel dislodging • Gel integrity persisted for the 2 hours (length of the experiment) post application

[0095] Example 3: Primary human sinonasal epithelial cell culture model for topical drug delivery in patients with chronic rhinosinusitis with nasal polyposis

[0096] Primary human sinonasal epithelial cells are cultured using methods as described in Ref. 23.

[0097] Mucosal biopsy procurementAttorney Docket No.:049648-629729 UF Ref. UFPT19408WO001

[0098] Procurement of the sinus mucosal biopsies utilized to generate primary nasal epithelial cell cultures was approved by the Medical University of South Carolina Institutional Review Board. Control subjects were defined as patients free of rhinosinusitis and undergoing surgery for either cerebrospinal fluid leak repair or tumor removal. Established consensus diagnostic criteria were used to define patients as having CRSwNP (24). Exclusion criteria included the following: use of oral steroids or immunotherapy within the preceding four weeks, aspirin sensitivity (ASA triad), ciliary dysfunction, autoimmune disease, cystic fibrosis or any known immunodeficiency. All tissue from both groups was derived from Schneiderian mucosa within the middle meatus.

[0099] Primary nasal epithelial cell culture

[0100] Tissue explants were washed and digested in Pronase for 90 min at 37°C. Cell suspensions were separated from particulate matter by centrifugation and resuspended in basal epithelial growth medium (BEGM) (Lonza, Basel, Switzerland). Cells were plated for 2 h on standard tissue culture plates to remove contaminating fibroblasts. Cells were then expanded for three to five days on collagen-coated 75 cm2dishes (Corning Life Sciences, Corning, NY, USA). Once confluent, the HSNECs were trypsinized and re-seeded evenly on human collagen type IV- coated tissue 6-well culture plates (Corning Life Sciences). Air–liquid interface cultures were grown on 6-well Transwell permeable supports (Corning Life Sciences) in Dulbecco's modified Eagle media (DMEM) (Invitrogen, Carlsbad, CA, USA) until fully confluent and differentiated with for 21 days to allow for ciliation and mucus production.

[0101] Example 4: RADA16 Self-assembling Peptide is Absorbent and Reduces Inflammation in CRSwNP Epithelial Cells

[0102] RADA16 self-assembling peptide was tested for absorbency and ability to reduce inflammation in CRSwNP epithelial cells.

[0103] Study Objective • To determine if PuraGel possesses anti-inflammatory properties when applied to human sinonasal epithelial cells cultured in an air liquid interface model • Included 24 hour time points + apical rinse dataAttorney Docket No.:049648-629729 UF Ref. UFPT19408WO001

[0104] Study Design • Cell lines from 5 control subjects and 5 patients with chronic rhinosinusitis without nasal polyps (CRSwNP) were cultured • Cells were cultured for 21 days to allow for cilia development and mucus production • Cells were treated apically for 24 hours: • No treatment • 500 µl Chitogel • 500 µl PuraGel (RADA162.5%) • Basolateral media was collected 6 and 24 hours post treatment • 24 hour basolateral and apical rinse data • No 6 hour apical because we did not want to disturb the PuraGel • Also 6 hour post-op rinse would not occur in clinic

[0105] Figure 3 is a schematic showing cultured nasal cells in a pseudostratified epithelium in culture plate. Top arrow points to apical treatment side where PuraGel or Chitogel was applied. Bottom arrow points to basolateral side.

[0106] Model / data interpretation • Immune mediators produced in the air-liquid interface (ALI) system can be apical, basolateral or both. • There are also studies that have shown some cytokines are produced apically, then pass through to the basolateral space. • PuraGel appears to reduce inflammation in the basolateral space, because it prevents apical cytokines from being present to pass through into the basolateral space. • Because the apical side was rinsed with 1 mL PBS, those cytokine levels can be different than those in the basolateral space which contains 2 mL of media. • This model was not designed to provide a direct comparison of apical vs basolateral levels. • This model can only provide direct comparison of no treatment vs PuraGel.Attorney Docket No.:049648-629729 UF Ref. UFPT19408WO001 • Similarly, comparison between 6 vs 24 hours are not applicable since the longer time skews the data for 24 hour supernatants to have higher value.

[0107] Observations PuraGel had no visible impact on membrane integrity. Chitogel treatment caused damage to the cells, resulting in a loss of barrier function which manifest as media leeching to the apical side. Figure 4 shows effect of gap junctions breaking down in Chitogel treated cultured nasal cells in a pseudostratified epithelium in culture plate. Left-side schematic depicts pseudostratified epithelium before Chitogel treatment. Right-side schematic depicts pseudostratified epithelium after Chitogel treatment. Basolateral media crosses to the apical side (upward arrows on right- side schematic). Media should stay on the basolateral side, but instead comes up to the apical side. Because the media is mixed across sides, meaning volumes are no longer consistent, we cannot accurately measure the concentrations of cytokines in samples treated with Chitogel

[0108] Results shown in Figures 5-9 only include PuraGel-treated cells vs. untreated cells. Results for Chitogel-treated cells not shown.

[0109] Results: Figure 5 shows that PuraGel reduces basolateral inflammation 6 hours post treatment from control and CRSwNP epithelial cells Black dot = control subject (n=5) Red dot = CRSwNP patient (n=5) p<0.05 is statistically significant All data basolateral

[0110] Results: Figure 6 shows that PuraGel reduces basolateral inflammation 24 hours post treatment from control and CRSwNP epithelial cells for most cytokines All data basolateral Black dot = control subject (n=5) Red dot = CRSwNP patient (n=5) p<0.05 is statistically significantAttorney Docket No.:049648-629729 UF Ref. UFPT19408WO001

[0111] Results: Figure 7 shows that PuraGel reduces most apical inflammation 24 hours post treatment from control and CRSwNP epithelial cells

[0112] Notes on Figure 7: In the future we would rinse the apical side with saline after initial application. The decline here may also be because the PuraGel exposed to air, may have bound to proteins during the apical rinse. All data Apical Rinse Black dot = control subject (n=5) Red dot = CRSwNP patient (n=5) p<0.05 is statistically significant

[0113] Results: Figure 8 shows that Basolateral bFGF, which stimulates fibroblast proliferation, is modestly reduced as 6 hours, unchanged at 24 hours post treatment

[0114] Notes on Figure 8 • bFGF is mostly secreted basolateral • Its role is to activate fibroblast to proliferate which begins the early stages of wound healing • unchanged at 24 hours • All data basolateral • Black dot = control subject (n=5) • Red dot = CRSwNP patient (n=5) • p<0.05 is statistically significant

[0115] Results: Figure 9 shows that apical treatment with PuraGel can modestly reduce total protein in the basolateral media Black dot = control subject (n=5) Red dot = CRSwNP patient (n=5) p<0.05 is statistically significant

[0116] Notes on Figure 9 • This assay measures total proteinAttorney Docket No.:049648-629729 UF Ref. UFPT19408WO001 • The BCA assay is also used to measure cell / tissue injury which will be indicated by increase BCA • Apical data not applicable as PuraGel was detected, giving a false positive (same as Chitogel) • This suggests that PuraGel is safe since it did not induce an increase in total protein • This media contains fetal bovine serum, which is very high in protein. Future studies, would switch to serum free. To find any change in very surprising given the amount of total protein from the serum. • All data basolateral

[0117] Other observations from these experiments • At the 24 hour time point the PuraGel had changed from clear to pink, the same color as the media (what cells grown in). This is similar to the lung application in which Evans Blue Dye that had been perfused through the lung, when leaking from the injured lung, changed the color of the PuraGel to blue. It should be noted evens blue contains high levels of albumin; a protein also found in very high quantities of nasal mucus. Albumin is higher is CRSwNP nasal mucus than control subjects • There was a very modest increase in the size of PuraGel on the apical side at 24 hours, suggesting proteins had diffused into the Puragel • When PuraGel was placed on transwells with no cells / mucus, there was no change in the color of the PuraGel, suggesting no diffusion by the media alone.

[0118] Hypothesis and Results Summary • Based on the current data and literature, we hypothesize that when PuraGel is applied to mucosal surfaces, that through passive diffusion, proteins and cytokines transfer into the PuraGel. • The end result of this is a reduction in cytokines present in both apical and basolateral spaces. Because IL-8 is produced at all times in high levels, the fact that PuraGel had no impact at 24 hours, suggest that it may be most effect at initial application and less at later time points.Attorney Docket No.:049648-629729 UF Ref. UFPT19408WO001 • PuraGel did not reduce levels of bFGF at 24 hours, which is secreted mostly into the basolateral space, further supporting the hypothesis that it prevents apical cytokines from passing into the basolateral space.Size in kDA: C3185, C3a 9, Il-620.9, Il-88.4, bFGF 17.2

[0119] Applicability to Clinical Observations • Collectively, these data may explain clinical observations the PuraGel speeds wound healing, possibly by reducing inflammation without interfering with key wound healing events, such as stimulation of fibroblast proliferation. • These findings also suggest that PuraGel may have different effects when applied to mucosal sites vs skin or other non-mucosal tissues. • The increase in mucus production by CRSwNP vs control cells, may explain why the anti-inflammatory effects of PuraGel are greater when applied to these cells. This could also account for why PuraGel may have anti-inflammatory indications in the gastrointestinal setting, since there are also high levels of mucus present in the gut. The change mucus causes to PuraGel, may also explain why PuraGel can work as a hemostatic agent “filling the holes” to stop bleeding but not adsorbing blood. The high levels of airway mucus in the pig lung vs transwell culture system, may also explain why application in the lung was able to absorb Evans Blue in 2 hours vs CRSwNP model that took 24 hours.

[0120] Example 5 RADA16 reduced the passage of dextran into the subepithelial space, even with antigen stimulation by Aspergillus Fumigates (Af) extract.

[0121] RADA16 self-assembling peptide was tested for effects on passage of inhaled antigens into the subepithelial space. In patients with allergic rhinitis, asthma and sinusitis, the tight junctions between epithelial cells are weak. This allows inhaled allergens to come in contract with immune cells in the subepithelial space. The immune cells then become activated by the allergen and caused increased inflammation which causes the physical systems of airway inflammation (29).

[0122] RADA16 was tested for effects on passage of allergic antigens into the subepithelial space.Attorney Docket No.:049648-629729 UF Ref. UFPT19408WO001

[0123] Experimental Design • HSNEC were cultured for 21 days to become ciliated • The apical side was treated with either saline or Aspergillus Fumigates (Af) extract (from Greer Laboratories) • Af is one the most common fungi around the world • Af exposure causes epithelial cell membranes to become more “leaky” • All wells were treated apically with Dextran that was fluorescently labeled • Dextran is an inert particle that is used to measure epithelial cell membrane integrity. • Antigen passage was measured 1 hour after treatment in the basolateral space (30).

[0124] Results are shown in Figure 10. Af treatment increased the passage of dextran into the basolateral space. RADA16 reduced the passage of dextran into the subepithelial space, even with antigen stimulation by Af.

[0125] Example 6: Effect of two-week treatment with RADA16 on human sinonasal epithelial cells (HSENC)

[0126] Studies tested effect of two-week treatment with RADA16 on human sinonasal epithelial cells (HSENC). The goal was to determine if the anti-inflammatory properties of RADA16 sustained past 24 hours until 2 weeks. Cellular toxicity was also assessed.

[0127] Experimental Design Culture methods for human sinonasal epithelial cells (HSENC) were as in Example 3. HSNEC were treated apically with 500 μl of saline, RADA161% or RADA162.5%. RADA161% was tested to determine if whether a dose response was detected. RADA162.5% (tradename PuraGel). Cells were cultured under normal conditions for 2 weeks, after which time supernatants were collected and assayed for immune mediators.

[0128] Figure 11 shows that RADA16 treatment for 2 weeks was safe to HSENC. Samples were from humans sinonasal epithelial cells from= 3 control subjects + 3 CRSwNP patients (dots represent individual patient data). LDH assay (Lactate Dehydrogenase assay) was used to detectAttorney Docket No.:049648-629729 UF Ref. UFPT19408WO001 cell death or cytotoxicity. There were no significant changes in LDH secretion, suggesting both doses of RADA16 were safe in vitro for 2 weeks. Values shown are mean + / - SD.

[0129] Figure 12 shows that two weeks post-treatment RADA16 reduces apical levels of C3. Samples were from humans sinonasal epithelial cells from= 3 control subjects + 3 CRSwNP patients (dots represent individual patient data). A dose dependent response to the percent of RADA16 was observed. Values shown are mean + / - SD. C3 and its cleavage fragment C3a, have been associated with more severe CRSwNP (31) and with loss of sense of smell (Figure 13).

[0130] Apical rinse levels of IL-6 and IL-8 were not altered at 2 weeks post-treatment (data not shown). This may reflect that over time RADA16 retains large molecules as opposed to smaller ones. C3 is 185 kD, while IL-8 is 8 kD and IL-6 is 21-26 kD. Basolateral levels of C3, IL-6 and IL-8 were not altered at 2 weeks post-treatment with RADA161 or 2.5% (data not shown). These data suggest that direct contact with RADA16 is required for its anti-inflammatory / absorbent properties to be effective. It also suggests that it will not impact normal cellular communication to cells in the subepithelial spaces, similar to the bFGF data in Figure 8 and Example 4.Attorney Docket No.:049648-629729 UF Ref. UFPT19408WO001

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Thymic stromal lymphopoietin activity is increased in nasal polyps of patients with chronic rhinosinusitis. The Journal of allergy and clinical immunology.2013;132(3):593-600.e12. Epub 2013 / 05 / 22.. 12. Hammad H, Lambrecht BN. Dendritic cells and epithelial cells: linking innate and adaptive immunity in asthma. Nat Rev Immunol.2008;8(3):193-204.. 13. Wynn TA. Type 2 cytokines: mechanisms and therapeutic strategies. Nat Rev Immunol. 2015;15(5):271-82. Epub 2015 / 04 / 18. 14. Philpott C, Hopkins C, Erskine S, Kumar N, Robertson A, Farboud A, Ahmed S, Anari S, Cathcart R, Khalil H, Jervis P, Carrie S, Kara N, Prinsley P, Almeyda R, Mansell N, Sunkaraneni S, Salam M, Ray J, Panesaar J, Hobson J, Clark A, Morris S. The burden of revision sinonasalAttorney Docket No.:049648-629729 UF Ref. UFPT19408WO001 surgery in the UK-data from the Chronic Rhinosinusitis Epidemiology Study (CRES): a cross- sectional study. BMJ open.2015;5(4):e006680.. 15. Steinke JW, Kennedy JL. 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The Journal of allergy and clinical immunology.2006;118(5):1133-41. 19. Sankar, S. et al., Frontiers I Bioengineering and Biotechnology June 2021, Volume 9, Article 679525 20. Paradís-Bas, M., Tulla-Puche, J., Zompra, A.A. and Albericio, F. (2013), RADA-16: A Tough Peptide – Strategies for Synthesis and Purification. Eur. J. Org. Chem., 2013: 5871-5878. 21. S. Côté, WO 2005 / 012277, 2005. 22. M. Quibell, D. Owen, L. C. Packman, T. Johnson, J. Chem. Soc., Chem. Commun.1994, 2343–2344. 23. Benjamin S Bleier, Ryan M Mulligan, Rodney J Schlosser, Primary human sinonasal epithelial cell culture model for topical drug delivery in patients with chronic rhinosinusitis with nasal polyposis, Journal of Pharmacy and Pharmacology, Volume 64, Issue 3, March 2012, Pages 449–456. 24. Meltzer EO et al. Rhinosinusitis: developing guidance for clinical trials. J Allergy Clin Immunol 2006; 118 (Suppl.): S17–S61. 25. Reeb, J. et al., Expanding the lung donor pool: advancements and emerging pathways. Current Opinion in Organ Transplantation 20(5):p 498-505, October 2015. 26. Peel, J.K. et al., Evaluating the Impact of Ex Vivo Lung Perfusion on Organ Transplantation: A Retrospective Cohort Study. Annals of Surgery 278(2):p 288-296, August 2023. 27. Wong A, et al., Potential therapeutic targets for lung repair during human ex vivo lung perfusion. Eur Respir J.2020 Apr 9;55(4):1902222. 28. Nykänen, A.I. et al. Creating superior lungs for transplantation with next-generation gene therapy during ex vivo lung perfusion. The Journal of Heart and Lung Transplantation, Volume 43, Issue 5, 838 - 848 29. Wynne M, et al., Contribution of Epithelial Cell Dysfunction to the Pathogenesis of Chronic Rhinosinusitis with Nasal Polyps. American Journal of Rhinology & Allergy. 2019;33(6):782-790. 30. Leung C, et al., Structural and functional variations in human bronchial epithelial cells cultured in air-liquid interface using different growth media. Am J Physiol Lung Cell Mol Physiol.2020 May 1;318(5):L1063-L1073.Attorney Docket No.:049648-629729 UF Ref. UFPT19408WO001 31. Mulligan, J. K. et al. C3a receptor antagonism as a novel therapeutic target for chronic rhinosinusitis Mucosal Immunology, 2018 Volume 11, Issue 5, 1375 - 1385

[0132] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0133] All publications, databases, patents, and patent applications cited in this specification are herein incorporated by reference as if each was specifically and individually indicated to be incorporated by reference.

Claims

Attorney Docket No.:049648-629729 UF Ref. UFPT19408WO001 WHAT IS CLAIMED IS:

1. A method of treating mucosal inflammation or vascular leakage in a subject, comprising administering to the subject a pharmaceutical composition comprising RADA16 self-assembling peptide.

2. The method of claim 1, wherein the mucosal inflammation is in an upper airway, a trachea, a lung, or a gastrointestinal tract.

3. The method of claim 2, wherein the mucosal inflammation is in the upper airway.

4. The method of claim 3, wherein the mucosal inflammation in the upper airway is in the nose, nasal passages, paranasal sinuses, pharynx, or larynx.

5. The method of claim 2, wherein the mucosal inflammation in the upper airway is associated with chronic rhinosinusitis without nasal polyps (CRSsNP), chronic rhinosinusitis with nasal polyps (CRSwNP), subsets of chronic rhinosinusitis with nasal polyps, upper airway disease treatment in cystic fibrosis (CF), allergic rhinitis, non-allergic rhinitis, chronic sinusitis, asthma, sinusitis, inflammatory-driven anosmia, inflammatory-driven hyposmia, or an allergy.

6. The method of claim 5, wherein the subsets of chronic rhinosinusitis with nasal polyps are selected from the group consisting of allergic fungal rhinosinusitis (AFRS), aspirin-exacerbated respiratory disease (AERD), and common variable immunodeficiency (CIVD).

7. The method of claim 5, wherein the allergic rhinitis, asthma, sinusitis, or allergy is associated with an inhaled allergen.

8. The method of claim 2, wherein the mucosal inflammation is in the trachea.

9. The method of claim 2, wherein the mucosal inflammation is in the lung.Attorney Docket No.:049648-629729 UF Ref. UFPT19408WO001 10. The method of claim 2, wherein the mucosal inflammation is in the gastrointestinal tract.

11. The method of claim 1, wherein the vascular leakage is in the lung.

12. A method of treating chronic rhinosinusitis without nasal polyps (CRSsNP), chronic rhinosinusitis with nasal polyps (CRSwNP), subsets of chronic rhinosinusitis with nasal polyps, upper airway disease treatment in cystic fibrosis (CF), allergic rhinitis, non- allergic rhinitis, chronic sinusitis, asthma, sinusitis, inflammatory-driven anosmia, inflammatory- driven hyposmia, or an allergy in a subject, comprising administering to the subject a pharmaceutical composition comprising RADA16 self-assembling peptide.

13. The method of claim 12, wherein the subsets of chronic rhinosinusitis with nasal polyps are selected from the group consisting of allergic fungal rhinosinusitis (AFRS), aspirin-exacerbated respiratory disease (AERD), and common variable immunodeficiency (CIVD).

14. The method of claim 12, wherein the allergic rhinitis, asthma, sinusitis, or an allergy is associated with an inhaled allergen.

15. The method of any one of claims 1-14, wherein the pharmaceutical composition comprises RADA16 self-assembling peptide at a concentration of about 0.1% w / v to about 10.0% w / v.

16. The method of claim 15, wherein the pharmaceutical composition comprises RADA16 self-assembling peptide at a concentration of about 0.3% w / v to about 8.0% w / v.

17. The method of claim 16, wherein the pharmaceutical composition comprises RADA16 self-assembling peptide at a concentration of about 0.5% w / v to about 5.0% w / v.Attorney Docket No.:049648-629729 UF Ref. UFPT19408WO001 18. The method of claim 17, wherein the pharmaceutical composition comprises RADA16 self-assembling peptide at a concentration of about 1.0% w / v to about 3.0% w / v.

19. The method of claim 18, wherein the pharmaceutical composition comprises RADA16 self-assembling peptide at a concentration of about 2.5% w / v.

20. The method of any one of claims 1-19, wherein the pharmaceutical composition comprises PURAGEL hemostatic gel.

21. The method of any one of claims 1-20, wherein the pharmaceutical composition is administered topically.

22. The method of any one of claims 1-21, wherein the subject is a mammal.

23. The method of claim 22, wherein the mammal is a primate.

24. The method of claim 22, wherein the mammal is a non-human primate.

25. The method of claim 22, wherein the mammal is a human.

26. The method of claim 22, wherein the mammal is a rodent.

27. The method of claim 26, wherein the rodent is a mouse, rat, guinea pig, hamster, or gerbil.

28. The method of claim 22, wherein the mammal is selected from the group consisting of human, baboon, chimpanzee, monkey, cynomolgus, marmoset, rhesus, rodent, rabbit, cat, dog, horse, cow, sheep, goat, pig, ferret, guinea pig, hamster, and gerbil.

29. The method of any one of claims 1-21, wherein the subject is a non-human mammal, bird, reptile, amphibian, or fish.

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