Compositions and methods for treating and preventing pulmonary disease

Abstract

Provided herein are compositions and methods for treating and preventing pulmonary diseases. In particular, provided herein are surfactant protein A (SP-A) peptide analogs (e.g., SP-A peptidomimetics) and their use in treating and preventing pulmonary diseases (e.g., asthma or COPD).

Description

Detailed Description of the Invention

[0001] [Technical field] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 006,831, filed April 8, 2020, the entire contents of which are incorporated herein by reference.

[0002] Provided herein are compositions and methods for treating and preventing pulmonary diseases. In particular, provided herein are surfactant protein A (SP-A) peptide analogs (e.g., SP-A peptidomimetics) and their use in treating and preventing pulmonary diseases (e.g., asthma or COPD). [Background technology] Asthma is the most common respiratory disease in both children and adults, presenting as a syndrome of nonspecific airway hyperresponsiveness, inflammation, and intermittent respiratory symptoms that affects 10% of the population (Bousquet et al. Bull World Health Organ. 2005;83(7):548-54. PubMed PMID:16175830; Mannino et al. Surveillance for asthma--United States, 1980-1999. MMWR Surveill Summ. 2002;51(1):1-13. PubMed PMID:12420904). It can be triggered by infection, environmental allergens, or other stimuli (Bousquet et al. Bull World Health Organ. 2005;83(7):548-54. PubMed PMID:16175830; Mannino et al. Surveillance for asthma--United States, 1980-1999. MMWR Surveill Summ. 2002;51(1):1-13. PubMed PMID:12420904).

[0003] Asthma remains poorly understood and often difficult to manage due to the heterogeneity of the disease. Acute exacerbations are a significant cause of asthma morbidity and mortality, and can lead to airway damage, remodeling, decreased lung function, and death. (Halwani R, et al. Curr Opin Pharmacol. 2010;10(3):236-45. PubMed PMID:20591736; Firszt R, Kraft M. Pharmacotherapy of severe asthma. Curr Opin Pharmacol. 2010;10(3):266-71. PubMed PMID:20462794) Most exacerbations are caused by respiratory infections such as rhinovirus or Mycoplasma pneumoniae. The response to infection is complex and involves both the innate and adaptive immune systems (Kim HY et al. The many paths to asthma: phenotype shaped by innate and adaptive immunity. Nat Immunol. 2010;11(7):577-84. PubMed PMID:20562844). Exacerbations are of particular concern in patients with more severe asthma, as hospitalizations due to acute exacerbations account for one-third of the $14.7 billion spent annually on asthma-related health care in the United States. Furthermore, exacerbations in this population are associated with an accelerated decline in lung function (Peat JK, Woolcock AJ, Cullen K. Rate of decline of lung function in subjects with asthma. Eur J Respir Dis. 1987;70(3):171-9. PubMed PMID:3569449; Peat JK, Woolcock AJ, Cullen K. Rate of decline of lung function in subjects with asthma. Eur J Respir Dis. 1987;70(3):171-9. PubMed PMID:3569449).Because reduced lung function is a risk factor for severe exacerbations (Osborne ML, Pedula KL, O'Hollaren M, Ettinger KM, Stibold T, Buist AS et al. Assessing future need for acute care in adult asthmatics: the Profile of Asthma Risk Study: a prospective health maintenance organization-based study. Chest. 2007;132(4):1151-61. PubMed PMID:17573515; Dougherty RH, Fahy JV. Acute exacerbations of asthma: epidemiology, biology and the exacerbation-prone phenotype. Clin Exp Allergy. 2009;39(2):193-202. PubMed PMID:19187331), this vicious cycle may promote an exacerbation-prone phenotype in asthma. Thus, understanding the mechanisms that trigger asthma exacerbations remains a key obstacle in understanding the pathobiology of asthma.

[0004] An intact immune system and host defenses are important for preventing asthma exacerbations. Surfactant is a lipoprotein complex that reduces surface tension at the air-liquid interface in the lung and participates in host defense (Han S, Mallampalli RK. The role of surfactant in lung disease and host defense against pulmonary infections. Annals of the American Thoracic Society. 2015;12(5):765-74. Epub 2015 / 03 / 06. doi:10.1513 / AnnalsATS.201411-507FR. PubMed PMID:25742123). The pulmonary surfactant system is a complex of extracellular lipids and proteins present at the air / tissue interface that regulates both the biophysical properties of the alveolar compartment and the organ's innate immune system. Surfactant protein A (SP-A) has been shown to promote important cellular functions that attenuate disease severity and exacerbations, such as promoting apoptosis of eosinophils, a key cell in asthma pathobiology, reducing mucin production by airway epithelial cells in the setting of interleukin (IL)-13 exposure, a cytokine essential for the allergic asthma phenotype, and decreasing IL-6 production, another cytokine important in type 2 or allergic inflammation.

[0005] Airway inflammation is a hallmark of asthma. Eosinophils are prominent in individuals with the type 2 inflammatory asthma phenotype and occur in large numbers in the circulation, sputum, and airway mucosa (see, e.g., Wenzel, SE, Nature Medicine, 2012. 18(5): p. 716-25). The accumulation and prolonged survival of eosinophils in the airways strongly correlates with increased asthma severity (see, e.g., Green, RH, et al., Lancet, 2002. 360(9347): pp. 1715-21; Duncan, CJ, et al., The European respiratory journal, 2003. 22(3): pp. 484-90; Gibson, PG, et al., Thorax, 2003. 58(2): pp. 116-21; Leitch, AE, et al., Mucosal immunology, 2008. 1(5): pp. 350-63), and their presence is driven by the type 2 cytokines interleukin (IL)-4, 5, and 13. Recent studies have shown that eosinophils are present in approximately 50% of lung tissue in patients with severe asthma (see, e.g., Wenzel, SE, et al., American Journal of Respiratory and Critical Care Medicine, 1999, 160(3):1001-8; Wenzel, SE, Asthma phenotypes: the evolution from clinical to molecular approaches. Nature medicine, 2012, 18(5):716-25). Furthermore, therapeutic strategies targeting eosinophil reduction have been shown to reduce asthma hospitalization rates and exacerbations (see, e.g., Green, RH, et al., Lancet, 2002, 360(9347):1715-21; Jayaram, L., et al., The European Respiratory Journal, 2006, 27(3):483-94). Clearance and rapid removal of apoptotic cells is a critical process leading to resolution of inflammation and relief of asthma symptoms.Inefficient apoptotic cell clearance leads to secondary necrosis or cell lysis, release of cellular contents that can damage tissue, and prolonged inflammation and the duration of asthma symptoms. Furthermore, the severity of asthma is strongly correlated with prolonged eosinophil survival (see, e.g., Duncan, CJ, et al., The European Respiratory Journal, 2003. 22(3): pp. 484-90; Fitzpatrick, AM, et al., The Journal of Allergy and Clinical Immunology, 2008. 121(6): pp. 1372-8, 1378 e1-3; Leitch, AE, et al., Relevance of granulocyte apoptosis to resolution of inflammation at the respiratory mucosa. Mucosal Immunology, 2008. 1(5): pp. 350-63). Interestingly, inhaled beta-2 agonists, the mainstay of asthma treatment worldwide, have been shown to prolong eosinophil survival (see, e.g., Nielson, C.P. and N.E. Hadjokas, American Journal of Respiratory and Critical Care Medicine, 1998. 157(1):184-91), which may actually exacerbate asthma or at least contribute to the various responses seen with beta-2 agonists (see, e.g., Choudhry, S., et al., Pharmacogenetics and Genomics, 2010. 20(6):351-8).

[0006] Further treatment for asthma is needed.

[0007] The present invention addresses this need. [Summary of the Invention] Surfactant protein A (SP-A) is the most abundant protein component of the lipoprotein complex, pulmonary surfactant. In humans, full-length oligomeric SP-A is the product of the SP-A1 and SP-A2 genes. Although alveolar type II cells in the peripheral airways are the primary producers of SP-A, it is also synthesized by club cells and submucosal glands independently of pulmonary surfactant in the conducting airways (see Auten, R.L., et al., 1990 Am J Respir Cell Mol Biol 3:491-496; Goss, K.L., 1998 Am J Respir Cell Mol Biol 19:613-621). In the nasal mucosa, SP-A can be detected within the cytoplasm of ciliated epithelial cells, serous acini, and submucosal glands (see Kim, JK, et al., 2007 Am J Physiol Lung Cell Mol Physiol 292:L879-884; Wootten, CT, et al., 2006 Arch Otolaryngol Head Neck Surg 132:1001-1007; Woodworth, BA, et al., 2006 Am J Rhinol 20:461-465).

[0008] SP-A plays an important role in regulating type 2-associated allergen-induced inflammation. When SP-A-deficient mice were challenged with ovalbumin (OVA), they exhibited significantly increased levels of type 2-associated cytokines, IgE, and especially eosinophils compared to wild-type mice (see Pastva, AM, et al., 2011 J Immunol 186:2842-2849). Obese asthmatics with reduced levels of SP-A have more severe tissue eosinophilia, and treatment with exogenous SP-A has been shown to significantly reduce tissue eosinophilia in mouse models of asthma (see Lugogo, N., et al., 2017 J Allergy Clin Immunol.; Desai, D., et al., 2013 Am J Respir Crit Care Med 188:657-663; van der Wiel, E., et al., 2014 Am J Respir Crit Care Med 189:1281-1284). Furthermore, SP-A isolated from asthmatic patients failed to attenuate the production of airway epithelial IL-8 and Muc5ac in response to infection with Mycoplasma pneumoniae (Mp), a bacterium highly associated with asthma exacerbations, compared with SP-A isolated from non-asthmatic patients (see Wang, Y., et al., 2011 Am J Physiol Lung Cell Mol Physiol 301:L598-606).

[0009] A single nucleotide polymorphism in SP-A2 substituting glutamine (Q) with lysine (K) at position 223 has been shown to alter eosinophil regulation in allergic airway inflammation (see Dy, ABC, et al., 2019 J Immunol 203:1122-1130) (Gln223Lys) (223Q / K in the SP-A wild-type amino acid sequence shown in SEQ ID NO: 1). More specifically, SP-A2 with this Q-to-K amino acid substitution is unable to promote eosinophil apoptosis compared to SP-A2 containing a Q at position 223. Furthermore, the presence of a Q at this position has been shown to be protective against respiratory disorders (see Lofgren, J., et al., 2002 J Infect Dis 185:283-289; Marttila, R., et al., 2003 Ann Med 35:344-352). Such findings highlight the relevance of this active region within SP-A for achieving normal airway function.

[0010] [ka]

[0011] Eosinophils are well-known end-stage effector cells and are primarily responsible for the symptoms experienced in type 2 asthma. Inhaled corticosteroid therapy, which helps reduce eosinophil survival by inhibiting the production of eosinophil-specific chemokines (Stellato, C., et al., 1999 J Immunol 163:5624-5632) and cytokines (Schleimer, RP, and BS Bochner, 1994 J Allergy Clin Immunol 94:1202-1213), which promote eosinophil survival, is a highly effective treatment strategy for asthma symptoms and exacerbations. Therefore, it can be inferred that eosinophil apoptosis and subsequent clearance are important steps in resolving type 2-associated airway inflammation.

[0012] By inhibiting eosinophil survival, corticosteroids can be considered a therapeutic strategy for normalizing eosinophils. This strategy contrasts with eosinophil-depleting treatment strategies using biologics such as mepolizumab, reslizumab (anti-IL5 antibody), and benralizumab (anti-IL5Rα antibody), whose goal is to dramatically reduce circulating eosinophils and their maturation within the bone marrow (see Roufosse, F. 2018. Front Med (Lausanne) 5:49). Despite inhaled corticosteroid therapy being the mainstay of asthma treatment, steroid resistance remains a challenge, in addition to the known side effects associated with this type of long-term therapy. Biologics currently in clinical trials and on the market offer steroid-sparing benefits, but the long-term effects of eosinophil depletion remain unclear.

[0013] Preliminary data suggest that SP-A native peptides of 10 and 20 amino acids in length (10-mer and 20-mer) derived from the active site spanning position 223 can reduce airway hyperresponsiveness, airway mucus production, and eosinophilia in a preclinical mouse model of asthma (Figure 1).

[0014] Experiments conducted during the development of embodiments of the present invention have led to the development of small molecules derived from the SP-A active site with improved stability and bioavailability as therapeutics targeted at eosinophil normalization. Indeed, in these experiments, 10-mer and 20-mer SP-A peptides were screened alongside a series of peptidomimetics derived from full-length SP-A for direct pro-apoptotic function against eosinophils. Several potential peptidomimetics were identified that closely resembled full-length SP-A in terms of the cytotoxic effect of SP-A on eosinophils in vitro. These results represent proof of concept that small molecules derived from the SP-A active site have activity against eosinophils, paving the way for the development of a new class of therapeutics for allergic airway inflammation.

[0015] Accordingly, provided herein are compositions and methods for treating and preventing pulmonary diseases. In particular, provided herein are SP-A peptide analogs (e.g., SP-A peptidomimetics) and their use in treating and preventing pulmonary diseases (e.g., asthma or COPD).

[0016] For example, in some embodiments, Ac-KEQCVEMYTD-NH2 (SEQ ID NO: 2), Ac-WGKEQCVEMYTD-NH2 (SEQ ID NO: 3), (Ac-KEQCVEMYTD-NH2)2 (SEQ ID NO: 4), Ac-KEQCVEMYTD-acid (SEQ ID NO: 5), H-KEQCVEMYTD-acid (SEQ ID NO: 6), Ac-KEQCVE-Nle-YTD-NH2 (SEQ ID NO: 7), Ac-KEQSVEMYTD-NH2 (SEQ ID NO: 8) ), Ac-KEQAVEMYTD-NH2 (SEQ ID NO: 9), Ac-SDGTPVNYTNWYRGEPAGRGKEQ-NH2 (SEQ ID NO: 10), Ac-GDFRYSDGTPVNYTNWYRGE-NH2 (SEQ ID NO: 11), Ac-WGKEQAVE-Nle-YTD-NH2 (SEQ ID NO: 12), Ac-WGKEQCVE-Nle-YTD-NH2 (SEQ ID NO: 13), Ac-RGKEQCVE-Nle-YTD-NH2 (SEQ ID NO: 14),

[0017] [ka]

[0018] or peptide analogs having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to these peptides. Further embodiments include, for example, Ac-KEQCVEMYTD-NH2 (SEQ ID NO: 2), Ac-WGKEQCVEMYTD-NH2 (SEQ ID NO: 3), (Ac-KEQCVEMYTD-NH2)2 (SEQ ID NO: 4), Ac-KEQCVEMYTD-acid (SEQ ID NO: 5), H-KEQCVEMYTD-acid (SEQ ID NO: 6), Ac-KEQCVE-Nle-YTD-NH2 (SEQ ID NO: 7), Ac-KEQSVEMYTD-NH2 (SEQ ID NO: 8), A c-KEQAVEMYTD-NH2 (SEQ ID NO: 9), Ac-SDGTPVNYTNWYRGEPAGRGKEQ-NH2 (SEQ ID NO: 10), Ac-GDFRYSDGTPVNYTNWYRGE-NH2 (SEQ ID NO: 11), Ac-WGKEQAVE-Nle-YTD-NH2 (SEQ ID NO: 12), Ac-WGKEQCVE-Nle-YTD-NH2 (SEQ ID NO: 13), Ac-RGKEQCVE-Nle-YTD-NH2 (SEQ ID NO: 14),

[0019] [ka]

[0020] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition includes a pharmaceutically acceptable carrier. In some embodiments, the composition is formulated for pulmonary delivery.

[0021] Further embodiments provide systems comprising: a) any one of the compositions described herein; and b) a device for pulmonary delivery of the composition. In some embodiments, the device is a metered dose inhaler.

[0022] Further embodiments provide: a method of enhancing SP-A activity in a cell, comprising delivering any one of the compositions described herein to the cell. In some embodiments, the cell is a lung cell. In some embodiments, the cell is in vivo. In some embodiments, the composition reduces mucin production and / or alleviates eosinophilia in the lung. In some embodiments, the cell is in a subject diagnosed with asthma. In some embodiments, administration reduces or prevents a symptom or marker of asthma in the subject. In some embodiments, the subject is obese or non-obese. In some embodiments, the peptide binds to a receptor selected from, e.g., FC (CD16 / 32), Sirp-α, TLR-2, or EGFR.

[0023] Yet other embodiments provide: a method for treating or preventing a pulmonary disease (e.g., asthma or COPD) in a subject, comprising administering to the subject any one of the compositions described herein.

[0024] Yet other embodiments provide the use of any one of the compositions described herein to enhance SP-A activity in a cell. Other embodiments provide the use of any one of the compositions described herein to treat or prevent a pulmonary disease (e.g., asthma or COPD) in a subject.

[0025] Further embodiments are described herein. [Brief description of the drawing] Figure 1: Evaluation of SP-A-derived 10-mer native peptides in an in vivo mouse model of asthma. A) Schematic of the HDM allergen challenge experiment. B) Newtonian resistance (Rn) during methacholine challenge in wild-type mice 6 days after the final HDM challenge. C) Total eosinophil counts in BAL (left panel) and mucin production (right panel) by PAS scoring. Unpaired t-test. * p<0.05, ** p<0.01.

[0026] Figure 2: Cytotoxic effects of full-length SP-A and native peptides on eosinophils evaluated by RTCA. The normalized cell index and calculated area under the curve for each dose are shown for SP-A (A-B), 20-mer peptides (C-D), and 10-mer peptides (E-F).

[0027] [Figure 3A] Cytotoxic effects of candidate peptide mimetics on eosinophils assessed by RTCA using mass concentration. The normalized cell index for each dose is shown for 856, 867, 868, 870, 871, 882, 883, and 884.

[0028] [Figure 3B] Cytotoxic effects of candidate peptide mimetics on eosinophils assessed by RTCA using mass concentrations. The area under the curve calculated for each dose is shown for 856, 867, 868, 870, 871, 882, 883, and 884.

[0029] [Figure 4A] Evaluation of the cytotoxic effect of candidate peptide mimetics on eosinophils by RTCA using molar concentrations. The normalized cell index for each dose is shown for 888, 889, 891, 892, 893, and 894.

[0030] [Figure 4B] Evaluation of the cytotoxic effect of candidate peptide mimetics on eosinophils by RTCA using molar concentrations. The area under the curve calculated for each dose is shown for 888, 889, 891, 892, 893, and 894.

[0031] definition The terms "polypeptide" and "protein" are used interchangeably and refer to polymers of amino acid residues, including natural and non-natural amino acid residues, and are not limited to a minimum length. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition. Both full-length proteins and fragments thereof are encompassed within the definition. The term also includes post-translational modifications of the polypeptide, including, for example, glycosylation, sialylation, acetylation, and phosphorylation. Furthermore, "polypeptide" herein also refers to proteins that have been modified relative to the native sequence, such as by deletion, addition, or substitution of single or multiple amino acid residues, so long as the protein maintains the desired activity. For example, a serine residue may be substituted to remove a single reactive cysteine, or a disulfide bond may be eliminated, or a conservative amino acid substitution may be made to remove a cleavage site. These modifications may be intentional, such as through site-directed mutagenesis, or may be accidental, such as through host mutation, resulting in the generation of proteins or errors by polymerase chain reaction (PCR) amplification.

[0032] As used herein, the term "peptide" refers to a short polymer of amino acids linked by peptide bonds. In contrast to other amino acid polymers (e.g., proteins, polypeptides, etc.), peptides are about 50 amino acids or less in length. Peptides can include natural amino acids, unnatural amino acids, amino acid analogs, and / or modified amino acids. Peptides can be subsequences of natural proteins or unnatural (synthetic) sequences.

[0033] "Wild-type" refers to the non-mutated form of a gene, allele, genotype, polypeptide, or phenotype, or a fragment of any of these, which may be naturally occurring or recombinantly produced.

[0034] A "variant" is a nucleic acid molecule or polypeptide that differs from a reference nucleic acid molecule or polypeptide by single or multiple amino acid substitutions, deletions, and / or additions, and that substantially retains at least one biological activity of the reference nucleic acid molecule or polypeptide.

[0035] The term "pseudopeptide" or "peptidomimetic" refers to a peptide-like molecule that emulates a sequence derived from a protein or peptide. Peptide mimetics or peptidomimetics can contain amino acid and / or non-amino acid components. Examples of peptidomimetics include chemically modified peptides, peptoids (in which side chains are attached to nitrogen atoms of the peptide backbone rather than the alpha carbon), and beta peptides (in which the amino group is attached to the beta carbon rather than the alpha carbon).

[0036] As used herein, a "conservative" amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid that has similar chemical properties, such as size or charge. For purposes of this disclosure, each of the following eight groups contains amino acids that are conservative substitutions for one another: 1) Alanine (A) and Glycine (G); 2) aspartic acid (D) and glutamic acid (E); 3) asparagine (N) and glutamine (Q); 4) arginine (R) and lysine (K); 5) isoleucine (I), leucine (L), methionine (M), and valine (V); 6) phenylalanine (F), tyrosine (Y), and tryptophan (W); 7) serine (S) and threonine (T); and 8) Cysteine ​​(C) and methionine (M).

[0037] Naturally occurring residues may be grouped into classes based on common side chain properties, such as: polar positive (histidine (H), lysine (K), and arginine (R)); polar negative (aspartic acid (D), glutamic acid (E)); polar neutral (serine (S), threonine (T), asparagine (N), glutamine (Q)); nonpolar aliphatic (alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M)); nonpolar aromatic (phenylalanine (F), tyrosine (Y), tryptophan (W)); proline and glycine; and cysteine. As used herein, a "semi-conservative" amino acid substitution refers to the replacement of an amino acid in a peptide or polypeptide with another amino acid within the same class.

[0038] In some embodiments, unless otherwise specified, conservative or semi-conservative amino acid substitutions may also include non-naturally occurring amino acid residues that have similar chemical properties as the natural residues. These non-natural residues are typically incorporated by chemical peptide synthesis rather than synthesis in biological systems. These include, but are not limited to, peptidomimetics and other reversed or inverted forms of amino acid moieties. Embodiments herein are, in some embodiments, limited to natural amino acids, non-natural amino acids, and / or amino acid analogs.

[0039] Non-conservative substitutions may involve exchanging a member of one of these classes for another class.

[0040] As used herein, the term "sequence identity" refers to the extent to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have the same sequential composition of monomer subunits. The term "sequence similarity" refers to the extent to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) differ only by conservative and / or semi-conservative amino acid substitutions. "Percent sequence identity" (or "percent sequence similarity") is calculated by: (1) comparing two optimally aligned sequences over a comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, the length of the designated sequence), (2) determining the number of positions containing identical (or similar) monomers (e.g., the same amino acid is present in both sequences, the similar amino acid is present in both sequences) to obtain the number of matching positions, (3) dividing the number of matching positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, the designated window), and (4) multiplying the result by 100 to obtain the percent sequence identity or percent sequence similarity. For example, if peptides A and B are both 20 amino acids in length and have identical amino acids at all but one position, peptide A and peptide B have 95% sequence identity. If the amino acids at non-identical positions share the same biophysical characteristics (e.g., both are acidic), peptide A and peptide B have 100% sequence similarity. As another example, if peptide C is 20 amino acids in length and peptide D is 15 amino acids in length, and 14 of the 15 amino acids in peptide D are identical to some amino acids in peptide C, peptides C and D have 70% sequence identity, but peptide D has 93.3% sequence identity over the optimal comparison window of peptide C. For purposes of calculating "percent sequence identity" (or "percent sequence similarity") herein, a gap in the aligned sequences is treated as a mismatch at that position.

[0041] "Subject," "individual," "host," "animal," and "patient" are used interchangeably and refer to mammals, including, but not limited to, rodents, monkeys, humans, cats, dogs, horses, cattle, pigs, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets.

[0042] As used herein, the terms "administration" and "administering" refer to the act of providing a drug, prodrug, or other agent or therapeutic treatment (e.g., SP-A peptide) to a subject or to in vivo, in vitro, or ex vivo cells, tissues, and organs. Exemplary routes of administration to the human body can be via the subarachnoid region of the brain or spinal cord (intraceutical), eye (intral), mouth (oral), skin (topical or transdermal), nose (intranasal), lung (inhalation), oral mucosa (buccal), ear, rectum, vagina, by injection (e.g., intravenous, subcutaneous, intratumoral, intraperitoneal, etc.), etc.

[0043] As used herein, the terms "co-administration" and "co-administering" refer to the administration of at least two agents or therapies (e.g., multiple SP-A peptides or an SP-A peptide and another therapeutic agent) or therapies to a subject. In some embodiments, co-administration of two or more agents or therapies is simultaneous. In other embodiments, a first agent / therapy is administered before a second agent / therapy. Those skilled in the art will appreciate that the formulations and / or routes of administration of the various agents or therapies used may vary. Appropriate dosages for co-administration can be readily determined by those skilled in the art. In some embodiments, when agents or therapies are co-administered, each agent or therapy is administered in a lower dosage than would be appropriate for its administration alone. Thus, co-administration of agents or therapies is particularly desirable in embodiments where co-administration reduces the required dosage of potentially dangerous (e.g., toxic) agent(s) and / or where co-administration of two or more agents sensitizes the subject to the beneficial effects of one of the agents upon co-administration.

[0044] As used herein, "treatment" includes any administration or application of a therapeutic agent against a disease in a mammal, including a human, to inhibit the disease, prevent its onset, or alleviate the disease, e.g., induce regression, or restore or repair lost, deficient, or defective function; or stimulate an inefficient process.

[0045] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid, or carrier conventionally used in the art for use with a therapeutic agent for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to a recipient at the dosage and concentration used and is compatible with other ingredients of the formulation. A pharmaceutically acceptable carrier is appropriate for the formulation used. For example, if the therapeutic agent is administered orally, the carrier may be a gel capsule. If the therapeutic agent is administered subcutaneously, the carrier ideally is not irritating to the skin and does not cause injection site reactions. [Mode for Carrying Out the Invention] As described above, experiments conducted during the development of embodiments of the present invention have led to the development of small molecules derived from the SP-A active site with improved stability and bioavailability as therapeutics targeted at eosinophil normalization. Indeed, in such experiments, 10-mer and 20-mer SP-A peptides were screened along with a series of peptidomimetics derived from full-length SP-A for direct pro-apoptotic function against eosinophils. Regarding the cytotoxic effect of SP-A on eosinophils in vitro, several potential peptidomimetics were identified that closely resembled full-length SP-A. These results represent proof of concept that small molecules derived from the SP-A active site have activity against eosinophils, paving the way for the development of a new class of therapeutics for allergic airway inflammation.

[0046] Accordingly, provided herein are compositions and methods for treating and preventing pulmonary diseases. In particular, provided herein are SP-A peptides and their uses in treating and preventing pulmonary diseases (e.g., asthma).

[0047] In certain embodiments, the present invention provides for the treatment of asthma using peptide analogs whose sequences are derived from or match the active region of endogenous human SP-A, including the predominant Q allele at position 223 of the SP-A2 peptide. For example, in some embodiments, peptide analogs such as Ac-KEQCVEMYTD-NH2 (SEQ ID NO: 2), Ac-WGKEQCVEMYTD-NH2 (SEQ ID NO: 3), (Ac-KEQCVEMYTD-NH2)2 (SEQ ID NO: 4), Ac-KEQCVEMYTD-acid (SEQ ID NO: 5), H-KEQCVEMYTD-acid (SEQ ID NO: 6), Ac-KEQCVE-Nle-YTD-NH2 (SEQ ID NO: 7), Ac-KEQSVEMYTD-NH2 (SEQ ID NO: 8) ), Ac-KEQAVEMYTD-NH2 (SEQ ID NO: 9), Ac-SDGTPVNYTNWYRGEPAGRGKEQ-NH2 (SEQ ID NO: 10), Ac-GDFRYSDGTPVNYTNWYRGE-NH2 (SEQ ID NO: 11), Ac-WGKEQAVE-Nle-YTD-NH2 (SEQ ID NO: 12), Ac-WGKEQCVE-Nle-YTD-NH2 (SEQ ID NO: 13), Ac-RGKEQCVE-Nle-YTD-NH2 (SEQ ID NO: 14),

[0048] [ka]

[0049] or peptide analogs having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to these peptides. Further embodiments include, for example, Ac-KEQCVEMYTD-NH2 (SEQ ID NO: 2), Ac-WGKEQCVEMYTD-NH2 (SEQ ID NO: 3), (Ac-KEQCVEMYTD-NH2)2 (SEQ ID NO: 4), Ac-KEQCVEMYTD-acid (SEQ ID NO: 5), H-KEQCVEMYTD-acid (SEQ ID NO: 6), Ac-KEQCVE-Nle-YTD-NH2 (SEQ ID NO: 7), Ac-KEQSVEMYTD-NH2 (SEQ ID NO: 8), A c-KEQAVEMYTD-NH2 (SEQ ID NO: 9), Ac-SDGTPVNYTNWYRGEPAGRGKEQ-NH2 (SEQ ID NO: 10), Ac-GDFRYSDGTPVNYTNWYRGE-NH2 (SEQ ID NO: 11), Ac-WGKEQAVE-Nle-YTD-NH2 (SEQ ID NO: 12), Ac-WGKEQCVE-Nle-YTD-NH2 (SEQ ID NO: 13), Ac-RGKEQCVE-Nle-YTD-NH2 (SEQ ID NO: 14),

[0050] [ka]

[0051] In some embodiments, the peptide binds to a receptor selected from, for example, FC (CD16 / 32), Sirp-α, TLR-2, or EGFR.

[0052] The present invention further provides variants and mimetics of the SP-A peptides described herein. In some embodiments, the SP-A peptides include conservative, semi-conservative, and / or non-conservative substitutions relative to the peptides described herein (e.g., at positions involved in SP-A signaling or positions not involved in SP-A signaling).

[0053] Embodiments are not limited to specific substitutions. In some embodiments, the peptides described herein are further modified (e.g., by substitution, deletion, or addition of standard amino acids; chemical modification, etc.). Modifications understood in the art include N-terminal modifications, C-terminal modifications (which protect the peptide from proteolysis), alkylation of amide groups, and hydrocarbon "stapling" (e.g., to stabilize an α-helical structure). In some embodiments, the peptides described herein may be modified, for example, by conservative residue substitutions of charged residues (K to R, R to K, D to E, and E to D). In some embodiments, such conservative substitutions result in subtle changes to, for example, receptor binding sites, with the goal of improving specificity and / or biological activity. Modifications of the terminal carboxy group include, but are not limited to, amide, lower alkyl amide, constrained alkyl (e.g., branched, cyclic, fused, adamantyl) alkyl, dialkyl amide, and lower alkyl ester modifications. Lower alkyl is C1-C4 alkyl. Additionally, one or more side chains or terminal groups may be protected by protecting groups known to skilled peptide chemists. The α-carbon of the amino acid may be mono- or dimethylated.

[0054] In some embodiments, one or more intrapeptide disulfide bonds (e.g., between two cysteines within the peptide) are introduced. In some embodiments, the presence of intrapeptide disulfide bonds stabilizes the peptide.

[0055] In some embodiments, any of the embodiments described herein may include peptidomimetics that correspond to the peptides described herein with various modifications understood in the art. In some embodiments, residues in the peptide sequences described herein may be substituted with amino acids that have similar characteristics (e.g., hydrophobic to hydrophobic, neutral to neutral, etc.) or other desired characteristics (e.g., more acidic, more hydrophobic, less bulky, more bulky, etc.). In some embodiments, unnatural amino acids (or natural amino acids other than the standard 20 amino acids) are substituted to achieve desired properties.

[0056] In some embodiments, residues that have a positively charged side chain under physiological conditions, or where a positively charged side chain is desired, are selected from the group consisting of lysine, homolysine, δ-hydroxylysine, homoarginine, 2,4-diaminobutyric acid, 3-homoarginine, D-arginine, arginine (where COOH in arginine is replaced by —CHO), 2-amino-3-guanidinopropionic acid, nitroarginine (N(G)-nitroarginine), nitrosoarginine (N(G)-nitrosoarginine), and the like. Substitutions include, but are not limited to, residues such as arginine, methylarginine (N-methylarginine), ε-N-methyllysine, allo-hydroxylysine, 2,3-diaminopropionic acid, 2,2'-diaminopimelic acid, ornithine, sym-dimethylarginine, asym-dimethylarginine, 2,6-diaminohexynoic acid, p-aminobenzoic acid, and 3-aminotyrosine, as well as histidine, 1-methylhistidine, and 3-methylhistidine. Neutral residues are residues with uncharged side chains under physiological conditions. Polar residues preferably have at least one polar group in the side chain. In some embodiments, the polar group is selected from hydroxyl, sulfhydryl, amine, amide, and ester groups, or other groups that allow the formation of hydrogen bridges.

[0057] In some embodiments, residues having side chains that are neutral / polar under physiological conditions, or where a neutral side chain is desired, are substituted with residues including, but not limited to, asparagine, cysteine, glutamine, serine, threonine, tyrosine, citrulline, N-methylserine, homoserine, allo-threonine and 3,5-dinitro-tyrosine, and β-homoserine.

[0058] Residues having nonpolar hydrophobic side chains are uncharged residues under physiological conditions, preferably having a hydropathic index greater than 0, particularly greater than 3. In some embodiments, nonpolar hydrophobic side chains are selected from alkyl, alkylene, alkoxy, alkenoxy, alkylsulfanyl, and alkenylsulfanyl residues having 1 to 10, preferably 2 to 6, carbon atoms, or aryl residues having 5 to 12 carbon atoms. In some embodiments, residues having nonpolar hydrophobic side chains, or residues where a nonpolar hydrophobic side chain is desired, are substituted with residues including, but not limited to, leucine, isoleucine, valine, methionine, alanine, phenylalanine, N-methylleucine, tert-butylglycine, octylglycine, cyclohexylalanine, β-alanine, 1-aminocyclohexylcarboxylic acid, N-methylisoleucine, norleucine, norvaline, and N-methylvaline.

[0059] In some embodiments, peptides and polypeptides are isolated and / or purified (or substantially isolated and / or substantially purified). Thus, in such embodiments, peptides and / or polypeptides are provided in substantially isolated form. In some embodiments, peptides and / or polypeptides are isolated from other peptides and / or polypeptides, for example, as a result of solid-phase peptide synthesis. Alternatively, peptides and / or polypeptides can be substantially isolated from other proteins after cell lysis from recombinant production. Standard methods of protein purification (e.g., HPLC) can be used to substantially purify peptides and / or polypeptides. In some embodiments, the present invention provides preparations of peptides and / or polypeptides in a number of formulations, depending on the intended use. For example, when a polypeptide is substantially isolated (or almost completely isolated from other proteins), it can be formulated in a medium solution suitable for storage (e.g., under refrigerated or frozen conditions). Such preparations can contain protective agents, such as buffers, preservatives, and cryoprotectants (e.g., sugars such as trehalose). The form of such preparations can be a solution, a gel, or the like. In some embodiments, the peptides and / or polypeptides are prepared in lyophilized form. Additionally, such preparations may contain other desired agents, such as small molecules or other peptides, polypeptides, or proteins. Indeed, such preparations may be provided that contain mixtures of different embodiments of the peptides and / or polypeptides described herein.

[0060] In some embodiments, provided herein are peptidomimetic versions of the peptide sequences described herein, or variants thereof. In some embodiments, peptidomimetics are characterized by entities that retain the polarity (or non-polarity, hydrophobicity, etc.), three-dimensional size, and functionality (biological activity) of their peptide equivalents, except that all or some of the peptide bonds have been replaced (e.g., by more stable bonds). In some embodiments, "stable" refers to being more resistant to chemical or enzymatic degradation by hydrolases. In some embodiments, the bond that replaces the amide bond (e.g., an amide bond surrogate) retains some of the properties of the amide bond (e.g., conformation, steric bulk, electrostatic properties, hydrogen-bonding capacity, etc.). Chapter 14 of "Drug Design and Development," Krogsgaard, Larsen, Liljefors, and Madsen (Eds.) 1996, Horwood Acad. Publishers, provides a general discussion of techniques for the design and synthesis of peptidomimetics, and is incorporated herein by reference in its entirety.Suitable amide bond surrogates include: N-alkylated (Schmidt, R. et al., Int. J. Peptide Protein Res., 1995, 46, 47; incorporated herein by reference in its entirety), reverse-inverted amide (Chorev, M. and Goodman, M., Acc. Chem. Res., 1993, 26, 266; incorporated herein by reference in its entirety), thioamide (Sherman DB and Spatola, AFJ Am. Chem. Soc., 1990, 112, 433; incorporated herein by reference in its entirety), thioester, phosphonate, ketomethylene (Hoffman, RV and Kim, HOJ Org. Chem., 1995, 60, 5107; incorporated herein by reference in its entirety), hydroxymethylene, fluorovinyl (Allmendinger, T. et al., Tetrahydron Lett., 1990, 31, 7297; incorporated herein by reference in its entirety), vinyl, methyleneamino (Sasaki, Y and Abe, J. Chem. Pharm. Bull. 1997 45, 13; incorporated herein by reference in its entirety), methylenethio (Spatola, AF, Methods Neurosci., 1993, 13, 19; incorporated herein by reference in its entirety), alkane (Lavielle, S. et al., Int. J. Peptide Protein Res., 1993, 42, 270; incorporated herein by reference in its entirety), and sulfonamide (Luisi, G. et al. Tetrahedron Lett. 1993, 34, 2391; incorporated herein by reference in its entirety).

[0061] Similar to the replacement of amide bonds, peptidomimetics can also include the replacement of larger structural moieties with dipeptide or tripeptidomimetic structures, in which case mimetic moieties containing peptide bonds, such as azole-derived mimetics, can be used as dipeptide replacements. Suitable peptidomimetics include reduced peptides in which the amide bonds have been reduced to methylene amines by treatment with a reducing agent (e.g., borane or a hydride reagent, such as lithium aluminum hydride); such reduction has the added benefit of increasing the overall cationic character of the molecule.

[0062] Other peptidomimetics include, for example, peptoids formed by the stepwise synthesis of amide-functionalized polyglycines. Some peptidomimetic backbones can be readily obtained from their peptide precursors, such as multi-methylated peptides, and suitable methods are described in Ostresh, J. Met. al. in Proc. Natl. Acad. Sci. USA (1994) 91, 11138-11142; the entire contents of which are incorporated herein by reference.

[0063] Any carrier that can deliver the active peptide or polypeptide (e.g., without destroying the peptide or polypeptide within the carrier) is a suitable carrier, and such carriers are well known in the art. In some embodiments, the compositions are formulated for administration by any suitable route, including, but not limited to, oral (e.g., in tablet, capsule, granule, or powder form), sublingual, buccal, parenteral (subcutaneous, intravenous, intramuscular, intradermal, or intrasternal injection or infusion (e.g., as a sterile injectable aqueous or non-aqueous solution or suspension), nasal (including administration to the nasal membranes, such as by inhalation spray), topical (in the form of a cream or ointment), transdermal (such as by a transdermal patch), rectal (in the form of a suppository), etc.

[0064] Pharmaceutical compositions may be administered in a formulated form with a pharmaceutically acceptable carrier and optional excipients, adjuvants, etc., in accordance with good pharmaceutical practice. Peptide-based pharmaceutical compositions may be in the form of solid, semisolid, or liquid dosage forms, such as powders, solutions, elixirs, syrups, suspensions, creams, drops, pastes, and sprays. As those skilled in the art will recognize, the form of the composition will depend on the selected route of administration (e.g., pill, injection, etc.). Generally, unit dosage forms are preferred to achieve easy and accurate administration of the active pharmaceutical peptide or polypeptide. The therapeutically active pharmaceutical compound is generally present in such dosage forms at a concentration level ranging from about 0.5% to about 99% by weight of the total composition, for example, in an amount sufficient to provide the desired unit dose. In some embodiments, the pharmaceutical composition may be administered in a single dose or multiple doses. The particular route of administration and dosage regimen will be determined by those skilled in the art, taking into account the condition of the individual to be treated and the individual's response to treatment. In some embodiments, peptide-based pharmaceutical compositions comprising a peptide or polypeptide and one or more non-toxic pharmaceutically acceptable carriers, adjuvants, or vehicles are provided in a unit dosage form for administration to a subject. The amount of active ingredient that may be combined with such materials to produce a single dosage form varies depending on various factors, as described above. A variety of materials, available in the pharmaceutical arts, can be used as carriers, adjuvants, and vehicles in the compositions of the present invention. Injectable preparations, such as oily solutions, suspensions, or emulsions, can be formulated using suitable dispersing or wetting agents and suspending agents, as needed, as known in the art. For sterile injectable preparations, non-toxic, parenterally acceptable diluents or solvents, such as sterile, non-pyrogenic water or 1,3-butanediol, may be used. Other acceptable vehicles and solvents that may be used include 5% dextrose injection, Ringer's injection, and isotonic sodium chloride injection (as described in USP / NF). Additionally, sterile, fixed oils may be conventionally used as solvents or suspending media. For this purpose any bland fixed oil may be employed including synthetic mono-, di-, or triglycerides.Fatty acids, such as oleic acid, can also be used in the preparation of injectable compositions. The peptides and polypeptides disclosed herein, including the substantially α-helical peptide region, can be further derivatized by chemical alterations such as amidation, glycosylation, acylation, sulfation, phosphorylation, acetylation, and cyclization. Such chemical alterations can be imparted by chemical or biochemical methodologies, as well as by in vivo processes, or any combination thereof.

[0065] The peptides and polypeptides described herein may be prepared as salts with a variety of inorganic and organic acids and bases. Such salts include salts prepared with organic and inorganic acids, such as HCl, HBr, H2SO4, H3PO4, trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid, toluenesulfonic acid, maleic acid, fumaric acid, and camphorsulfonic acid. Salts prepared with bases include ammonium salts, alkali metal salts, such as sodium and potassium salts, alkaline earth salts, such as calcium and magnesium salts, and zinc salts. Salts may be formed by conventional means, such as reacting the free acid or base form of the product with one or more equivalents of the appropriate base or acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water, which is subsequently removed by vacuum or lyophilization, or by exchanging the existing salt ion for another ion on a suitable ion exchange resin.

[0066] The peptides and polypeptides described herein can be formulated as pharmaceutically acceptable salts and / or complexes thereof. Pharmaceutically acceptable salts include acid addition salts, including sulfate, hydrochloride, phosphate, sulfamate, acetate, citrate, lactate, tartrate, succinate, oxalate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate, and quinate. Pharmaceutically acceptable salts can be obtained from acids such as hydrochloric acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, and quinic acid. Such salts may be prepared, for example, by reacting the free acid or base form of the product with one or more equivalents of the appropriate base or acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water (which is subsequently removed by vacuum or lyophilization, or by exchanging the existing salt ion for another ion on a suitable ion exchange resin).

[0067] The peptides and polypeptides described herein may be formulated as pharmaceutical compositions for use in conjunction with the methods of the present disclosure. The compositions disclosed herein may conveniently be provided in formulations suitable for parenteral administration, including subcutaneous, intramuscular, and intravenous administration, intranasal administration, and pulmonary administration, or oral administration. Suitable formulations of peptides and polypeptides for each such route of administration are described in standard formulation treatises, e.g., Remington's Pharmaceutical Sciences by E.W. Martin. See also Wang, Y.J., and Hanson, M.A., "Parenteral Formulations of Proteins and Peptides: Stability and Stabilizers," Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42:2S (1988).

[0068] Certain peptides and polypeptides described herein may be substantially insoluble in water and sparingly soluble in most pharmaceutically acceptable protic solvents and vegetable oils. In certain embodiments, cyclodextrins may be added as water solubility enhancers. Cyclodextrins include methyl, dimethyl, hydroxypropyl, hydroxyethyl, glucosyl, maltosyl, and maltotriosyl derivatives of α-, β-, and γ-cyclodextrin. An exemplary cyclodextrin solubility enhancer is hydroxypropyl-β-cyclodextrin (HPBCD), which may be added to any of the above compositions to further enhance the water solubility properties of the peptide or polypeptide. In one embodiment, the composition contains 0.1% to 20% HPBCD, 1% to 15% HPBCD, or 2.5% to 10% HPBCD. The amount of solubility enhancer used depends on the amount of the peptide or polypeptide of the present disclosure in the composition. In certain embodiments, the peptides may be formulated in non-aqueous polar aprotic solvents such as DMSO, dimethylformamide (DMF) or N-methylpyrrolidone (NMP).

[0069] In some cases, it is convenient to provide the peptide or polypeptide and another active agent in a single composition or solution for administration together. In other cases, it may be more effective to administer the additional agent separately from the polypeptide. For use, the pharmaceutical compositions of the peptides and polypeptides described herein may be provided in a unit dosage form containing an effective amount of the peptide or polypeptide for a single administration. Unit dosage forms useful for subcutaneous administration include pre-filled syringes and injectors.

[0070] In certain embodiments, the polypeptide is administered in an amount of 50 micrograms ("mcg") per day, 60 mcg per day, 70 mcg per day, 75 mcg per day, 100 mcg per day, 150 mcg per day, 200 mcg per day, or 250 mcg per day, expressed as an equivalent daily dose regardless of frequency of administration. In some embodiments, the polypeptide is administered in an amount of 500 mcg per day, 750 mcg per day, or 1 milligram ("mg") per day. In further embodiments, the polypeptide is administered in an amount of 1-10 mg per day, 1 mg per day, 1.5 mg per day, 1.75 mg per day, 2 mg per day, 2.5 mg per day, 3 mg per day, 3.5 mg per day, 4 mg per day, 4.5 mg per day, 5 mg per day, 5.5 mg per day, 6 mg per day, 6.5 mg per day, 7 mg per day, 7.5 mg per day, 8 mg per day, 8.5 mg per day, 9 mg per day, 9.5 mg per day, or 10 mg per day, expressed as an equivalent daily dose regardless of frequency of administration.

[0071] In various embodiments, the polypeptide is administered on a monthly dosing schedule. In other embodiments, the polypeptide is administered every other week. In still other embodiments, the polypeptide is administered weekly. In certain embodiments, the polypeptide is administered daily ("QD"). In selected embodiments, the polypeptide is administered twice a day ("BID").

[0072] In general embodiments, the polypeptide is administered for at least 3 months, at least 6 months, at least 12 months, or more, hi some embodiments, the polypeptide is administered for at least 18 months, 2 years, 3 years, or more.

[0073] In one embodiment, the pharmaceutical composition of the present invention is suitable for inhalation administration.The pharmaceutical composition suitable for inhalation administration is generally in the form of aerosol or powder.This composition is generally administered using well-known delivery device, such as nebulizer inhaler, metered dose inhaler (MDI), dry powder inhaler (DPI) or similar delivery device.

[0074] In certain embodiments of the present invention, pharmaceutical compositions containing active agents are administered by inhalation using a nebulizer inhaler. Such nebulizer devices generally generate a high-velocity airflow that causes the pharmaceutical composition containing active agents to spray as a mist that is carried into the patient's respiratory tract. Therefore, when formulated for use in a nebulizer inhaler, the active agent is usually dissolved in a suitable carrier to form a solution. Alternatively, the active agent can be micronized and combined with a suitable carrier to form a suspension of finely divided particles of respirable size, where micronization is generally defined as about 90% or more of the particles having a diameter of less than about 10 μm. Suitable nebulizer devices are commercially available, for example, from PARI GmbH (Starnberg, Germany). Other nebulizer devices include Respimat (Boehringer Ingelheim) and the devices disclosed, for example, in U.S. Patent No. 6,123,068 to Lloyd et al. and WO97 / 12687 (Eicher et al.).

[0075] A representative pharmaceutical composition for use in a nebulizer inhaler comprises an isotonic aqueous solution comprising SP-A peptide or a pharmaceutically acceptable salt or solvate or stereoisomer thereof.

[0076] In another specific embodiment of the present invention, a pharmaceutical composition containing an active agent is administered by inhalation using a dry powder inhaler. Such dry powder inhalers typically administer the active agent as a free-flowing powder that is dispersed in the patient's airstream during inspiration. To achieve a free-flowing powder, the active agent is typically formulated with a suitable excipient, such as lactose or starch.

[0077] A typical pharmaceutical composition for use in a dry powder inhaler comprises dry lactose having a particle size of about 1 μm to about 100 μm and micronized particles of SP-A peptide, or a pharmaceutically acceptable salt or solvate or stereoisomer thereof.

[0078] Such dry powder formulations can be made, for example, by combining lactose with the active agent and then dry blending the ingredients. Alternatively, if desired, the active agent can be formulated without excipients. The pharmaceutical composition is then typically loaded into a dry powder dispenser or into inhalation cartridges or capsules for use with a dry powder delivery device.

[0079] Examples of dry powder inhalation delivery devices include Diskhaler (GlaxoSmithKline, Research Triangle Park, NC) (see, e.g., U.S. Pat. No. 5,035,237 to Newell et al.); D. Diskus (GlaxoSmithKline) (see, e.g., U.S. Pat. No. 6,378,519 to Davies et al.); Turbuhaler (AstraZeneca, Wilmington, Del.) (see, e.g., U.S. Pat. No. 4,524,769 to Wetterlin); Rotahaler (GlaxoSmithKline) (see, e.g., U.S. Pat. No. 4,353,365 to Hallworth et al.) and Handihaler (Boehringer Ingelheim). Further examples of suitable DPI devices are described in U.S. Pat. No. 5,415,162 to Casper et al., U.S. Pat. No. 5,239,993 to Evans, and U.S. Pat. No. 5,715,810 to Armstrong et al., and references cited therein.

[0080] In yet another specific embodiment of the present invention, a pharmaceutical composition containing an active agent is administered by inhalation using a metered-dose inhaler. Such metered-dose inhalers typically use compressed propellant gas to deliver a measured amount of the active agent, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof. Thus, pharmaceutical compositions administered using a metered-dose inhaler typically contain a solution or suspension of the active agent in a liquefied propellant. Any suitable liquefied propellant may be used, including chlorofluorocarbons such as CCl.sub.3F and hydrofluoroalkanes (HFAs) such as 1,1,1,2-tetrafluoroethane (HFA134a) and 1,1,1,2,3,3,3-heptafluoro-n-propane (HFA227). Due to concerns about the impact of chlorofluorocarbons on the ozone layer, formulations containing HFAs are generally preferred. Additional optional components of HFA formulations include cosolvents such as ethanol or pentane, and surfactants such as sorbitan trioleate, oleic acid, lecithin, and glycerin. See, for example, U.S. Patent No. 5,225,183 to Purewal et al., EP 0717987 A2 (Minnesota Mining and Manufacturing Company), and WO 92 / 22286 (Minnesota Mining and Manufacturing Company).

[0081] A typical pharmaceutical composition for use in a metered dose inhaler comprises about 0.01% to about 5% by weight of a compound of SP-A peptide, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof; about 0% to about 20% by weight of ethanol; and about 0% to about 5% by weight of a surfactant; the remainder being HFA propellant.

[0082] Such compositions are usually prepared by adding cooled or pressurized hydrofluoroalkane to a suitable container containing the active agent, ethanol (if present), and surfactant (if present). To prepare a suspension, the active agent is micronized and then mixed with a propellant. The formulation is then loaded into an aerosol canister that forms part of a metered-dose inhaler. Examples of metered-dose inhalers specifically developed for use with HFA propellants are provided in U.S. Patent No. 6,006,745 to Marecki and U.S. Patent No. 6,143,277 to Ashurst et al. Alternatively, suspension formulations can be prepared by spray-drying a surfactant coating on micronized particles of the active agent. See, for example, WO99 / 53901 (Glaxo Group Ltd.) and WO00 / 61108 (Glaxo Group Ltd.).

[0083] For additional examples of processes for preparing respirable particles, as well as formulations and devices suitable for inhalation administration, see U.S. Pat. No. 6,268,533 to Gao et al., U.S. Pat. No. 5,983,956 to Trofast, U.S. Pat. No. 5,874,063 to Briggner et al., and U.S. Pat. No. 6,221,398 to Jakupovic et al.; and WO 99 / 55319 (Glaxo Group Ltd.) and WO 00 / 30614 (AstraZeneca AB).

[0084] In some embodiments, the peptide / polypeptide is provided in a pharmaceutical composition and / or co-administered (concurrently or sequentially) with one or more additional therapeutic agents. Such additional agents may be for the treatment or prevention of pulmonary inflammation (e.g., asthma). Additional agents include, but are not limited to, short-acting beta-2 adrenergic agonists (SABAs), such as salbutamol (albuterol USAN); anticholinergics, such as ipratropium bromide, inhaled epinephrine, inhaled or systemic corticosteroids; leukotriene receptor antagonists (e.g., montelukast and zafirlukast); and combinations thereof.

[0085] In some embodiments, provided herein are methods for treating a patient suffering from (or at risk for) a pulmonary disease (e.g., asthma) and / or in need of treatment (or preventative therapy). In some embodiments, the subject is obese or non-obese. In some embodiments, the subject is identified as having an SP-A genotype (e.g., a genotype described herein) associated with asthma or an increased risk of severe asthma.

[0086] In some embodiments, a pharmaceutical composition comprising at least one SP-A peptide or polypeptide described herein is delivered to such a patient in an amount and location sufficient to treat the condition. In some embodiments, the peptide and / or polypeptide (or pharmaceutical composition comprising same) can be delivered to the patient systemically or locally, and ascertaining the most appropriate delivery route, time course, and dosage for treatment is within the ordinary skill of the medical professional treating such a patient. It is understood that the applied method of treating a patient most preferably substantially alleviates or even eliminates such symptoms; however, as with many medical treatments, application of the methods of the invention is considered successful if the symptoms of the disease or disorder in the patient appreciably subside during, after, or as a result of application of the methods of the invention.

[0087] The present disclosure is not limited to the treatment of asthma. Any inflammatory condition known in the art or contemplated herein may be treated according to the presently disclosed and claimed inventive concept(s). Non-limiting examples of disease states with associated inflammation include infection-related or non-infectious inflammatory conditions in the lungs (e.g., asthma, sepsis, chronic obstructive pulmonary disease (COPD), pulmonary infection, respiratory distress syndrome, bronchopulmonary dysplasia, etc.); infection-related or non-infectious inflammatory conditions in other organs (e.g., colitis, inflammatory bowel disease, diabetic nephropathy, hemorrhagic shock); inflammation-induced cancer (i.e., cancer progression in patients with colitis or inflammatory bowel disease), etc. [Example] Example I. This example describes the materials and methods utilized in Example II.

[0088] Eosinophil isolation IL-5 transgenic mice were euthanized, and blood was collected by cardiac puncture from the left ventricle. Red blood cells (RBCs) were lysed using erythrocyte lysis solution (Miltenyi Biotec, Auburn, CA). Eosinophils were isolated by negative selection using biotin-conjugated antibodies (CD45R, Thy1.2, F4 / 80) and magnetic beads, as previously described (see Dy, ABC, et al., 2019 J Immunol 203:1122-1130; Ledford, JG, et al., 2012 PLoS One 7:e32436). Purity of each preparation was confirmed to be greater than 95% using standard morphometric analysis of cytocentrifuge slides stained with the Easy III™ rapid differential staining kit (Azer Scientific, Morgantown, PA).

[0089] Generation of 10 and 20 amino acid peptides derived from full-length SP-A 10-mer and 20-mer amino acid peptides were custom synthesized (Genscript Biotech Corporation, Piscataway, NJ) and confirmed to be 98.8% and 98.0% pure, respectively. Each vial of lyophilized 10-mer peptide was reconstituted to an initial concentration of 2 mg / ml using sterile-filtered PBS (Gibco, Gaithersburg, MD), while each vial of lyophilized 20-mer peptide was reconstituted to an initial concentration of 2 mg / ml using molecular biology-grade HO (Corning, Tewksbury, MA). Solvent selection was based on the solubility report provided by Genscript.

[0090] Generation of peptide mimetics Peptide mimetics were synthesized by solid-phase synthesis at the Ligand Discovery Laboratory (The University of Arizona, Tucson, AZ). The peptidomimetics were designed to be small molecule derivatives mimicking the mature SP-A active site (KEQCVEMYTD) with improved stability and bioavailability. The products were purified by high-performance liquid chromatography (HPLC), and their structures were analyzed by nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS). Each vial of lyophilized peptidomimetic was reconstituted with molecular biology-grade HO (Corning, Tewksbury, MA) and a maximum final concentration of 10 mM DMSO (Sigma, St. Louis, MO) to an initial concentration of 1 mg / ml.

[0091] Assessment of eosinophil cytotoxicity by real-time impedance tracing High-throughput, real-time monitoring of eosinophil cell death was evaluated by measuring electrical impedance using the xCELLigence Real-Time Cell Analyzer (ACEA Biosciences, San Diego, CA) as previously described (see Dy, ABC, et al., 2019 J Immunol 203:1122-1130; Flynn, AN, et al., FASEB J 27:1498-1510; Zeng, C., et al., Environ Res 164:452-458). Initial background readings were obtained using medium alone in a 96-well gold electrode-coated plate (E-Plate, ACEA Biosciences) incubated at 37°C and 5% CO2. Eosinophils were added at 1 x 10 in a total volume of 100 μl. 6 Cells per well were seeded and allowed to rest for approximately 5 hours. Test compounds were added at various concentrations (1, 3, 10, and 30 μg / ml), and changes in electrical impedance were measured over time. Impedance measurements were calculated and presented as a normalized cell index (see Flynn, AN, et al., FASEB J 27:1498-1510; Zeng, C., et al., Environ Res 164:452-458), where a decrease in cell index corresponds to an increase in eosinophil cytotoxicity. Time-dependent impedance tracings were obtained from the average of three to four technical replicates. To quantify and compare cytotoxicity, the cell index values ​​were used to calculate the area under the curve (AUC). For each peptide mimetic, the molar equivalent concentration and the corresponding dose-response curve were used to determine the half-maximal effective concentration (EC). 50 ) value was generated.

[0092] statistical analysis All statistical analyses were performed using GraphPad Prism software. One-way analysis of variance was used to assess overall differences between samples, followed by multiple t-tests with Bonferroni correction for multiple comparisons.

[0093] Example II. The cytotoxic effect of SP-A-derived peptides was lower than that of full-length SP-A. First, we evaluated the direct effects of these 10- and 20-amino acid peptides (10mer and 20mer) on eosinophil viability by RTCA. Similar to previous results (see Dy, ABC, et al., 2019 J Immunol 203:1122-1130), full-length SP-A induced eosinophil cell death in a dose-dependent manner, where addition of 30 μg / ml SP-A resulted in a decrease in the cell index corresponding to a mean AUC of -13.89 (Figures 2A and 2B). Addition of 10- and 20-mer SP-A-derived peptides to eosinophils also resulted in increased cell death, as indicated by negative AUC values. The mean magnitudes of the highest AUCs corresponding to a peptide concentration of 30 μg / ml were -2.62 and -3.50, respectively (Figures 2D and 2F). Normalized cell index tracings showed a decrease in peptide activity after 24 hours, indicated by an increasing trend, for both the 10-mer and the 20-mer (Figures 2C, 2E).

[0094] Two candidate peptide mimetics mimicked the cytotoxic effect of SP-A at 3 μg / ml.

[0095] To improve the stability of SP-A-derived peptides, peptide mimetics were synthesized for testing. Fourteen peptide mimetics were initially screened. Peptide mimetics 856, 867, 868, 870, and 871 were modified from the original 10-mer native peptide residues by adding an amine or acid group to the C-terminus and acetylation or histidine to the N-terminus (Table 1). Peptide mimetics 882, 883, 884, 891, 892, 893, and 894 were modified from the original 10-mer native peptide residues by single amino acid substitutions (Table 1). Peptide mimetic 888 is a 23-amino acid sequence corresponding to positions 181–203 of SP-A2, while peptide mimetic 889 is a 20-amino acid sequence corresponding to positions 175–195 of SP-A2 (Table 1). The sequences and corresponding molecular weights of the peptide mimetics are summarized in Table 1.

[0096] [Table 1]

[0097] Using the same approach as in Figure 1, we present the mean normalized cell index and calculated AUC (Figures 3 and 4). The mass concentration range used for the peptidomimetics in Figure 3 is the range in which both full-length SP-A and the 10-mer and 20-mer peptides were found to be active (see Figure 2). However, to account for the size difference between full-length SP-A and the various peptide sequence lengths, the molar equivalent concentration range in which full-length SP-A was found to be active was used in subsequent screening analyses (Figure 4). Peptidomimetics 867 and 868 exhibited the most robust cytotoxic effects against eosinophils at 3 μg / ml, as measured by AUC (Figures 3A and 2B).

[0098] Calculated median effective concentration (EC 50 ) was lower than that of the natural 10-mer and 20-mer peptides. Full-length SP-A is a much larger molecule compared to both peptides and peptidomimetics. Due to the discrepancy in size of the compounds tested, the molar concentrations of all compounds were calculated based on their respective molecular weights to allow for proper comparison of dose-response curves (Table 1). EC of full-length SP-A 50 The EC of all tested peptidomimetics was 0.158 μM (Table 2). 50 The values ​​were lower than both the 10-mer and 20-mer peptides, with 892 and 894 showing the two lowest values ​​at 0.008 and 0.012 μM, respectively (Table 2).

[0099] [Table 2]

[0100] Consideration Recently, SP-A has been shown to promote eosinophil apoptosis, a mechanism that contributes to the clearance of eosinophils from the lung cavity after allergic challenge experiments (see Dy, ABC, et al., 2019 J Immunol 203:1122-1130). This activity of SP-A was also shown to be altered by a genetic mutation in SP-A2, substituting lysine for glutamine at position 223. This suggests that the active site within SP-A that promotes eosinophil apoptosis resides within this region, motivating further investigation. First, we synthesized peptides (10-mer and 20-mer) and peptidomimetics of this SP-A region with the aim of improving stability while maintaining biological activity. Second, we tested these synthesized small molecules for their ability to promote eosinophil cell death similar to full-length SP-A.

[0101] The experiments performed herein demonstrate that many of the synthesized small molecules were able to induce eosinophil cell death. Full-length SP-A served as a positive control, and the expected dose-dependent decrease in eosinophil viability, as measured by RTCA, was observed. Furthermore, normalized cell index traces showed an overall decreasing trend, suggesting a consistent and continuous effect in inducing cell death over 48 hours. 10-mer and 20-mer peptides derived from SP-A were similarly able to induce eosinophil cell death. However, when comparing these peptides at various concentrations with full-length SP-A, the extent of cell death induced by the peptides, as indicated by the magnitude of the calculated AUC, was less than that induced by full-length SP-A. The AUC value for the peptides at 30 μg / ml was comparable to that of 3 μg / ml full-length SP-A. Furthermore, the normalized cell index traces for the 10-mer and 20-mer peptides suggest less robust peptide activity compared to full-length SP-A, which may indicate limited function in vivo.

[0102] Considering these results, we next conducted experiments to choose to synthesize peptide mimetics to address this potential issue. Of the 14 peptide mimetics screened so far, several produced promising results that will be further evaluated. Based on the magnitude of the AUC at each dose, 867 and 868 showed the most comparable response to full-length SP-A, with their AUC at 3 μg / ml being comparable to that of full-length SP-A at 30 μg / ml (see Table 3). Due to the large difference in molecular size between full-length SP-A and the synthetic molecule, the median effective concentration (EC ), an important indicator of efficacy, was significantly lower. 50 ) calculations were based on molar equivalent concentrations. Peptide mimetics 892 and 894 had the two lowest EC of the 14 candidate molecules at 0.008 μM and 0.012 μM, respectively. 50 (See Table 3.) However, EC 50 Despite the low AUC, the magnitude of the calculated AUC for these peptidomimetics is much smaller than that for full-length SP-A, the 10-mer and 20-mer peptides, and several peptidomimetics (see Table 3). This suggests that although low concentrations of peptidomimetics 892 and 894 are required to exert some degree of cytotoxicity against eosinophils, the effect is not very robust.

[0103] In summary, our goal was to identify candidate peptide mimetics that recapitulate the cytotoxic activity of full-length SP-A against eosinophils, and therefore used EC as a measure of potency. 50 Not only was it important to derive a value, but it was equally essential to compare the magnitude of change in cell index as an indicator of efficacy. Therefore, four lead peptide mimetics (867, 868, 892, and 894 in Table 3) were identified.

[0104] In summary, the experiments performed herein provide evidence that small molecules derived from the active region of SP-A responsible for its proapoptotic activity can induce similar effects on eosinophils. Future preclinical studies include further optimization of candidate peptide mimetics, in vitro validation using human eosinophils by flow cytometry, and in vivo rescue experiments using animal models of asthma.

[0105] [Table 3]

[0106] Incorporation by Reference The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

[0107] equivalent The present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments, therefore, are to be considered in all respects as illustrative and not limiting of the invention described herein. The scope of the invention is, therefore, indicated by the appended claims, rather than the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein. [Brief explanation of the drawings]

[0108] [Figure 1] Evaluation of SP-A-derived 10-mer native peptides in an in vivo mouse model of asthma. A) Schematic of the HDM allergen challenge experiment. B) Newtonian resistance (Rn) during methacholine challenge in wild-type mice 6 days after the final HDM challenge. C) Total eosinophil counts in the BAL (left panel) and mucin production (right panel) by PAS scoring. Unpaired t-test, *p<0.05, **p<0.01. [Figure 2-1]RTCA evaluation of the cytotoxic effects of full-length SP-A and native peptides on eosinophils. The normalized cell index and calculated area under the curve for each dose are shown for SP-A (A-B), 20-mer peptides (C-D), and 10-mer peptides (E-F). [Figure 2-2] Continued from Figure 2-1. [Figure 3A-1] RTCA evaluation of the cytotoxic effect of candidate peptide mimetics on eosinophils using mass concentrations. The normalized cell index for each dose is shown for 856, 867, 868, 870, 871, 882, 883, and 884. [Figure 3A-2] Continued from Figure 3A-1. [Figure 3B] RTCA evaluation of the cytotoxic effect of candidate peptide mimetics on eosinophils using mass concentrations. The area under the curve calculated for each dose is shown for 856, 867, 868, 870, 871, 882, 883, and 884. [Figure 4A-1] RTCA evaluation of the cytotoxic effect of candidate peptide mimetics on eosinophils using molar concentrations. The normalized cell index for each dose is shown for 888, 889, 891, 892, 893, and 894. [Figure 4A-2] Continued from Figure 4A-1. [Figure 4B] RTCA evaluation of the cytotoxic effect of candidate peptide mimetics on eosinophils using molar concentrations. The area under the curve calculated for each dose is shown for 888, 889, 891, 892, 893, and 894.

Claims

1. Ac-KEQCVEMYTD-NH ２ (SEQ ID NO: 2), Ac-WGKEQCVEMYTD-NH ２ (SEQ ID NO: 3), (Ac-KEQCVEMYTD-NH ２ ) ２ (SEQ ID NO: 4), Ac-KEQCVEMYTD-acid (SEQ ID NO: 5), H-KEQCVEMYTD-acid (SEQ ID NO: 6), Ac-KEQCVE-Nle-YTD-NH ２ (SEQ ID NO: 7), Ac-KEQSVEMYTD-NH ２ (SEQ ID NO: 8), Ac-KEQAVEMYTD-NH ２ (SEQ ID NO: 9), Ac-SDGTPVNYTNWYRGEPAGRGKEQ-NH ２ (SEQ ID NO: 10), Ac-GDFRYSDGTPVNYTNWYRGE-NH ２ (SEQ ID NO: 11), Ac-WGKEQAVE-Nle-YTD-NH ２ (SEQ ID NO: 12), Ac-WGKEQCVE-Nle-YTD-NH ２ (SEQ ID NO: 13), Ac-RGKEQCVE-Nle-YTD-NH ２ (SEQ ID NO: 14), 【Chemistry 1】 A composition comprising a surfactant protein A (SP-A) peptide analog selected from the group consisting of:

2. The composition of claim 1 , wherein the composition is a pharmaceutical composition.

3. The composition of claim 2 , wherein the composition comprises a pharmaceutically acceptable carrier.

4. The composition of any one of claims 1 to 3, wherein the composition is formulated for pulmonary delivery.

5. a) a composition according to any one of claims 1 to 4; and b) A system comprising a device for pulmonary delivery of said composition.

6. 6. The system of claim 5, wherein the device is a metered dose inhaler.

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