Compositions and methods for treating and preventing pulmonary disease

Abstract

Compositions and methods for treating and preventing pulmonary disease are provided herein. In particular, SP-A peptides and their uses in treating and preventing pulmonary disease (e.g., asthma or COPD) are provided herein. The compositions may include a peptide having an amino acid sequence of KEQCVE (SEQ ID NO: 9). The peptide compositions may also be used to treat COVID-19.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Application No. 17 / 409,642, filed August 23, 2021, the specification of which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant Nos. R0I HL125602 and U19 AI125357 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0003] 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, such as inflammatory pulmonary diseases (e.g., asthma). [Background technology]

[0004] Asthma is the most common respiratory disease in both children and adults, affecting 10% of the population worldwide and 25 million people in the United States alone. Asthma is a chronic syndrome characterized by airway hyperresponsiveness, inflammation, and intermittent respiratory symptoms. The medical burden of asthma is significant, accounting for $81 billion in annual expenditures in the United States when direct medical care costs and lost productivity are considered. Despite the significant costs and increasing prevalence, asthma remains poorly understood and difficult to manage due to the heterogeneity of the disease.

[0005] A major cause of morbidity and mortality in asthma is acute exacerbations, which can lead to airway damage, remodeling, decline in lung function, and death. Most exacerbations are caused by respiratory infections (e.g., rhinovirus or Mycoplasma pneumoniae), and the response to these infections is complex and involves both the innate and adaptive immune systems. In severe asthma patients, exacerbations are associated with accelerated decline in lung function. This vicious cycle may promote an exacerbation-prone phenotype of asthma, as reduced lung function is a risk factor for severe exacerbations. Thus, understanding the mechanisms that drive asthma exacerbations has been a significant barrier to progress in understanding asthma pathobiology. The host response in asthma exacerbations is complex and involves both the innate and adaptive immune systems. Of the available treatments, there are currently no innate immune modulators for the treatment of asthma, and those directed at the adaptive immune system have not controlled the disease. Thus, there is a great need to develop new therapeutic approaches that target the natural response in the treatment of asthma and other inflammatory lung diseases. Summary of the Invention

[0006] The goal of the present invention is to provide compositions and methods that allow the treatment and prevention of pulmonary diseases, such as inflammatory pulmonary diseases, such as asthma, as specified in the independent claims. Embodiments of the invention are set out in the dependent claims. The embodiments of the invention may be freely combined with one another if they are not mutually exclusive.

[0007] Surfactant protein A (SP-A) is a secreted lipoprotein complex. SP-A is produced and secreted by several types of lung cells (alveolar type II cells, airway club cells, and submucosal gland cells) and is an innate immune modulator that acts as a first line of defense against inhalation insults (e.g., infectious and / or environmental insults) throughout the upper and lower airways. It acts as a regulator of pathogen phagocytosis and inflammatory processes in the lung. Mature SP-A is a hetero-oligomeric product derived from the SP-A1 and SP-A2 genes.

[0008] The experiments described herein demonstrated that in asthma patients, the SP-A2 Gln223Lys (i.e., Q223K) allele within the SP-A wild-type sequence (SEQ ID NO: 1) is associated with reduced lung function, impaired asthma control, and increased BAL and serum eosinophilia. Thus, SP-A is a key regulator of eosinophil degranulation and survival, as well as mucin secretion and type 2 inflammation, which may significantly affect asthma severity. In vitro studies using isolated eosinophils, SP-A-deficient mice, and SP-A containing specific SP-A allele oligomers of interest (e.g., purified peptides described herein, including SEQ ID NO: 9) have found that SP-A directly stimulates eosinophil apoptosis, that this effect can be reproduced by specific SP-A peptides, and that SP-A allele variants differentially modulate eosinophil responses. [ka]

[0009] Without wishing to limit the present invention to any theory or mechanism, SP-A encounters eosinophils in the bronchoalveolar compartment and is believed to be a critical regulator of their apoptosis during the resolution phase of the inflammatory process.As described herein, SP-A plays a role in directly inducing the apoptotic signaling pathway of eosinophils, which results in attenuation of allergic phenotypes such as mucin production and eosinophilia.SP-A attenuates IL-13-induced mucin and IL-6 in airway epithelial cells obtained from asthmatic subjects with allergic asthma or type 2 asthma.

[0010] The invention may feature compositions and methods for treating and preventing pulmonary diseases (e.g., inflammatory pulmonary diseases). In particular, SP-A peptides and their uses in treating and preventing pulmonary diseases (e.g., asthma) are provided herein. In some embodiments, the invention features a method of treating inflammatory pulmonary diseases in a subject in need of such treatment. The method may include administering to the subject a therapeutically effective amount of any one of the compositions (e.g., purified peptides) as described herein. The invention may further feature a purified peptide comprising the amino acid sequence of KEQCVE (SEQ ID NO: 9) for use in a method of treating inflammatory pulmonary diseases in a subject in need of such treatment.

[0011] In some embodiments, the invention features methods and compositions (e.g., pharmaceutical compositions) for enhancing SP-A activity in a cell. The compositions (e.g., pharmaceutical compositions) can include any one of the purified peptides as described herein and a pharmaceutical carrier. In some embodiments, the compositions are in preparation for aerosolization. The methods can include delivering any one of the compositions described herein to a cell (e.g., a lung cell).

[0012] Further embodiments provide a system 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.

[0013] One of the unique and inventive technical features of the present invention is the use of an amino acid sequence peptide that includes KEQCVE (SEQ ID NO: 9) (i.e., a 6-mer). Without wishing to limit the present invention to any theory or mechanism, it is believed that the technical feature of the present invention advantageously provides for inhalation delivery. Additionally, the inclusion of a 6-mer (i.e., KEQCVE (SEQ ID NO: 9)) is necessary for the activity of larger peptidomimetics (i.e., 10-mer or 20-mer). Additionally, smaller (i.e., 6-mer) peptides reach deeper into the lungs (i.e., the peptides can get it further into the lungs) compared to larger peptides. Furthermore, the peptides described herein allow for the replacement and / or augmentation of SP-A in the lungs. None of the currently known prior references and works have the unique and inventive technical features of the present invention.

[0014] Of the available treatments, there are currently no innate immune modulators for the treatment of asthma, and those directed at the adaptive immune system do not control the disease. Thus, there is a great need to develop new therapeutics directed at the innate response in the treatment of asthma and other inflammatory lung diseases. The inventors have discovered small molecules that mimic the effects of surfactant protein A (SP-A). SP-A is a natural component of the lung lining fluid and acts as the first line of defense. Some asthma patients have no SP-A or have damaged SP-A. Full-length SP-A delivered directly to the lung is not feasible due to its large size and complex structure. The inventors first developed a series of 10-20 amino acid peptides derived from the lectin domain of SP-A2 to determine the specific region of activity. The findings described herein demonstrate that 10-20 amino acid SP-A peptides reduce airway narrowing, a cardinal feature of asthma, in two different preclinical mouse models of asthma.

[0015] Moreover, the inventive technical features of the present invention contributed to surprising results. For example, the peptidomimetic containing the 6mer (i.e., KEQCVE (SEQ ID NO: 9)) affects (i.e., reduces) both eosinophil viability and STAT signaling, which may ultimately be beneficial for asthma patients. Additionally, the peptidomimetic containing the 6mer (i.e., KEQCVE (SEQ ID NO: 9)) is active in both males and females in preclinical animal studies. Furthermore, the peptidomimetic containing the 6mer (i.e., KEQCVE (SEQ ID NO: 9)) works in two phases: (1) an acute phase in which it reduces airway mucus production and hyperresponsiveness to methacholine, and (2) a late phase in which it clears inflammatory eosinophils and neutrophils from the lungs. Finally, the peptidomimetic containing the 6mer (i.e., KEQCVE (SEQ ID NO: 9)) works over a longer period of time; for example, a single dose may remain effective for 7-10 days. In human airway cells, the peptidomimetic also reduces mucosal and proinflammatory mediators.

[0016] Any feature or combination of features described herein is included within the scope of the present invention, unless the features included in any such combination are mutually inconsistent, as may be apparent from the context, the specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.

[0017] The features and advantages of the present invention will become apparent from consideration of the following detailed description taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0018] [Figure 1]Figure 1 provides a non-limiting overview of how 10-mer (i.e., SEQ ID NO: 4) and / or 20-mer (i.e., SEQ ID NO: 8) SP-A peptides, as well as other SP-A peptides described herein, reduce airway narrowing, a cardinal feature of asthma, in two different preclinical mouse models of asthma. As shown in Figure 1, the mechanism of protective effects discovered herein is due to 1) direct interaction with eosinophils, the key inflammatory cells in asthma, to induce apoptosis and promote airway clearance, and 2) direct interaction with epithelial cells lining the lungs and participating in the inflammatory process, to inhibit mucin production.

[0019] [Figure 2A] FIG. 2A shows that genetic variations in SP-A2 are associated with changes in lung function and asthma control. Percent predicted FEV1 and Asthma Control Questionnaire scores in a cohort of 53 asthmatic subjects stratified by alleles of rs1965708 (Gln223Lys) of the SP-A2 gene. Asthmatic subjects with the 223K / K genotype demonstrate significantly worse asthma control (right panel) and significantly lower lung function (left panel) compared with 223Q / K heterozygotes and major allele (homozygotes for 223Q / Q) genotypes. *p<0.05 compared with Q / Q. [Figure 2B] FIG. 2B shows that genetic variations in SP-A2 are associated with changes in lung function and asthma control. Percent predicted FEV1 and Asthma Control Questionnaire scores in a cohort of 53 asthmatic subjects stratified by alleles of rs1965708 (Gln223Lys) of the SP-A2 gene. Asthmatic subjects with the 223K / K genotype demonstrate significantly worse asthma control (right panel) and significantly lower lung function (left panel) compared with 223Q / K heterozygotes and major allele (homozygotes for 223Q / Q) genotypes. *p<0.05 compared with Q / Q.

[0020] [Figure 3A]Figure 3A shows that genetic variation in SP-A2 determines the degree of protection against IL-13-induced inflammation in humanized SP-A transgenic mice. BAL cells from mice challenged with IL-13 are shown. BAL cells consisted of macrophages, neutrophils, and eosinophils. [Figure 3B] Figure 3B shows that genetic variation in SP-A2 determines the degree of protection against IL-13-induced inflammation in humanized SP-A transgenic mice. Macrophages are shown. [Figure 3C] Figure 3C shows that genetic variation in SP-A2 determines the degree of protection against IL-13-induced inflammation in humanized SP-A transgenic mice. [Figure 3D] Figure 3D shows that genetic variation in SP-A2 determines the degree of protection against IL-13-induced inflammation in humanized SP-A transgenic mice. [Figure 3E] FIG. 3E shows that genetic variation in SP-A2 determines the degree of protection against IL-13-induced inflammation in humanized SP-A transgenic mice. PAS-scored lung histology of mice challenged with IL-13 is shown. N=12 WT; 15 SP-A- / -; 8 SP-A223Q / Q; 12 SP-A223K / K per group (three separate experimental replicates). *p<0.05, **p<0.01, ***p<0.001. ****p<0.0001 by one-way Anova with Dunnett's test for multiple comparisons. [Figure 3F] Figure 3F shows that genetic variation in SP-A2 determines the degree of protection against IL-13-induced inflammation in humanized SP-A transgenic mice. Representative PAS images of each genotype treated with IL-13 are shown. [Figure 3G](G) Genetic variation in SP-A2 determines the degree of protection against IL-13-induced inflammation in humanized SP-A transgenic mice. Western blots and densitometry of Stat3 phosphorylation from representative lung samples are shown. *p<0.05 by one-way anova with Tukey's multiple comparisons. [Figure 3H] (H) Genetic variation of SP-A2 determines the degree of protection against IL-13-induced inflammation in humanized SP-A transgenic mice. Western blots and Stat3 phosphorylation by densitometry from representative lung samples are shown. *p<0.05 by one-way anova with Tukey's multiple comparisons.

[0021] [Figure 4A] FIG. 4A shows that genetic variation in SP-A2 determines the degree of protection against IL-13-induced inflammation in bronchial epithelial cells from asthmatic participants. Expression of MUC5AC RNA in bronchial epithelial cells (n=3 normal, n=3 asthma) grown at air-liquid interface (ALI) and treated with IL-13 for 5 days in the presence or absence of full-length recombinant SP-A2(223K) (20 μg / ml) or SP-A2(223Q) (20 μg / ml) added 30 min prior to challenge is shown. After normalization to housekeeper genes, data are presented as fold over IL-13 unchallenged controls for each patient set with standard deviation shown. Mean fold change and standard deviation are shown. *p<0.05. [Figure 4B] Figure 4B shows that genetic variation in SP-A2 determines the degree of protection against IL-13-induced inflammation of bronchial epithelial cells from asthmatic participants. Genetic variants of SP-A2 that differ only at position 223 (Q and K) were examined for relative binding to IL-13 compared to the extracted human oligomeric control SP-A.

[0022] [Figure 5A]FIG 5A shows that HDM-challenged SP-A deficient mice treated with SP-A peptide have reduced hallmarks of inflammation. Shown are SP-A deficient mice challenged intranasally with HDM on days 0, 7, and 14. On day 15, mice were divided into groups and given either vehicle or SP-A peptide (10mer (i.e., SEQ ID NO: 4) or 20mer (i.e., SEQ ID NO: 8)) by oropharyngeal instillation at a concentration of 25 μg / ml (approximately 1 mg / kg body weight). [Figure 5B] FIG 5B shows that HDM-challenged SP-A-deficient mice treated with SP-A peptide had reduced hallmarks of inflammation. On day 19, mice were sacrificed and eosinophils in BAL were assessed. n=10, 10, **p<0.01, ***p<0.001 by one-way anova for multiple comparisons. [Figure 5C] FIG. 5C shows that HDM-challenged SP-A-deficient mice treated with SP-A peptide had reduced hallmarks of inflammation. On day 19, mice were sacrificed and mucin production was assessed. n=10, 10, **p<0.01, ***p<0.001 by one-way anova for multiple comparisons. [Figure 5D] FIG. 5D shows that HDM-challenged SP-A-deficient mice treated with SP-A peptide had reduced hallmarks of inflammation. On day 19, mice were sacrificed and lung histology sections were evaluated. n=10, 10, **p<0.01, ***p<0.001 by one-way Anova for multiple comparisons.

[0023] [Figure 6A] FIG. 6A shows that the truncated 10AA peptide reduces mucin production in a mouse HDM model. [Figure 6B] FIG. 6B shows that the truncated 10AA peptide reduces eosinophilia in a mouse HDM model. [Figure 6C] FIG. 6C shows that the truncated 10AA peptide reduces mucin (Muc5AC RNA) in human primary cells.

[0024] [Figure 7A] Figure 7A shows that HDM-challenged WT mice treated with SP-A peptide have reduced susceptibility to methacholine challenge. Figure 7A shows WT male mice intranasally challenged with HDM on days 0, 7, and 14. On days 1, 8, and 15, mice were divided into groups and received either vehicle or SP-A peptide (10-mer (i.e., SEQ ID NO: 4), 25 μg / ml, approximately 1 mg / kg body weight) by oropharyngeal instillation. On day 19, pulmonary function testing was performed during methacholine challenge while mice were anesthetized. [Figure 7B] FIG. 7B shows that HDM-challenged WT mice treated with SP-A peptide have reduced susceptibility to methacholine challenge, showing total airway resistance (Rrs). Data graphed are mean + / - SEM. n=12, 12, *p<0.05, **p<0.01 by t-test at each indicated dose. [Figure 7C] FIG. 7C shows that HDM-challenged WT mice treated with SP-A peptide have reduced susceptibility to methacholine challenge, showing Newtonian resistance (Rn). Graphed data are mean + / - SEM. n=12, 12, *p<0.05, **p<0.01 by t-test at each indicated dose. [Figure 7D] FIG. 7D shows that HDM-challenged WT mice treated with SP-A peptide have reduced susceptibility to methacholine challenge, showing total airway elastance (Ers). Data graphed are mean + / - SEM. n=12, 12, *p<0.05, **p<0.01 by t-test at each indicated dose. [Figure 7E] FIG. 7E shows that HDM-challenged WT mice treated with SP-A peptide have reduced susceptibility to methacholine challenge, and FIG. 7E shows tissue damping assessed by flexivent. Graphed data are mean + / - SEM. n=12, 12, *p<0.05, **p<0.01 by t-test at each indicated dose.

[0025] [Figure 8A] FIG. 8A shows that the SP-A peptide protects against airway hyperresponsiveness (AHR) in the IL-13 model. [Figure 8B] FIG. 8B shows that the SP-A peptide protects against airway hyperresponsiveness (AHR) in the IL-13 model.

[0026] [Figure 9] FIG. 9 shows that IL-13 challenged mice treated with SP-A 10mer peptide (i.e., SEQ ID NO: 4) have improved lung function. WT male mice were challenged with vehicle (saline) or IL-13 (3.9 μg) by oropharyngeal delivery on three consecutive days. Two hours after each IL-13 challenge, mice received either vehicle (saline) or SP-A 10mer peptide (25 μg / ml; approx. 1 mg / kg body weight) by oropharyngeal delivery. Pulmonary function tests were performed on day 4 in a Flexivent device with negative pressure driven forced expiratory (NPFE) extension (SCIREQ). IL-13 challenge significantly increased Newtonian resistance (Rn) at 0.05 seconds and reduced forced expiratory volume (FEV). Treatment with SP-A 10mer peptide protected against IL-13-induced increases in Rn and decreases in FEV. Graphed are the mean + / - SEM of the means and are shown from n=2 independent experiments. *p<0.05, ***p<0.001 by ANOVA for multiple comparisons.

[0027] [Figure 10] Figure 10 shows that primary human lung epithelial cells treated with SP-A peptide reduced IL-13-induced MUC5AC gene expression. Primary human bronchial epithelial cells from normal and asthmatic participants were incubated with either the 20mer (i.e., SEQ ID NO: 8) or 10mer (i.e., SEQ ID NO: 8) SP-A peptide (20 μg / ml) for 30 minutes, followed by stimulation with IL-13 (10 ng / ml) for 5 days. In summary (asthmatic and normal cells), MUC5AC gene expression was significantly reduced in the SP-A 20mer treatment group compared to IL-13 alone (p=0.004).

[0028] [Figure 11A] FIG. 11A shows the assessment of BALF eosinophilia over time, illustrating the OVA model of allergic airways. [Figure 11B] FIG. 11B shows the assessment of BALF eosinophilia over time, showing the cell distribution in BALF 24 hours, 3 days, and 5 days after the final challenge. [Figure 11C] FIG. 11C shows the assessment of BALF eosinophilia over time, showing the set change in eosinophil frequency over time. [Figure 11D] Figure 11D shows the assessment of BALF eosinophilia over time, differences in mean values ​​at 24 hours and 5 days, unpaired Student's t-test. One-way ANOVA with Bonferroni correction for multiple comparisons, *p<0.05, **p<0.01. Data (mean ± SEM) are from at least two independent experiments with n=3-5 mice / group.

[0029] [Figure 12A] FIG. 12A shows the assessment of tissue eosinophilia over time and shows representative brightfield images of eosinophils (arrows point to representative eosinophils) in lung tissue by Sirius red staining (upper panel: 40x magnification; lower panel: 100x magnification). [Figure 12B] FIG. 12B shows the assessment of tissue eosinophilia over time, with quantification of eosinophil counts on day 5. [Figure 12C] FIG. 12C shows the assessment of tissue eosinophilia over time, showing the net change in eosinophil frequency over time. [Figure 12D] FIG. 12D shows the assessment of tissue eosinophilia over time, showing the difference in mean values ​​at 24 hours and 5 days, unpaired Student's t-test, #p<0.05, **p<0.01. [Figure 12E]Figure 12E shows the assessment of tissue eosinophilia over time, showing eosinophil-associated ribonuclease (EAR) mRNA in lung tissue 5 days after the final challenge. One-way ANOVA with Bonferroni's correction for multiple comparisons, *p<0.05. Data (mean ± SEM) are from at least two independent experiments with n=3-5 mice / group.

[0030] [Figure 13A] FIG. 13A shows an assessment of the ability of SP-A to induce eosinophil apoptosis in mouse and human eosinophils in vitro, showing the time course of viability as assessed by trypan blue. [Figure 13B] FIG. 13B shows an evaluation of the ability of SP-A to induce eosinophil apoptosis in mouse and human eosinophils in vitro, showing real-time cell analyzer (RTCA) tracking and dose response of in vitro stimulation of mouse eosinophils with SP-A, AUC=area under the curve. [Figure 13C] FIG. 13C shows an evaluation of the ability of SP-A to induce eosinophil apoptosis in mouse and human eosinophils in vitro, and is a representative flow diagram of human eosinophil apoptosis and cell death by Annexin V and PI and quantification after 16 hours of incubation with SP-A. Viability = Annexin V-, PI-, early apoptosis = Annexin V+, PI-, late apoptosis / death = Annexin V+, PI+. [Figure 13D] Figure 13D shows an assessment of the ability of SP-A to induce eosinophil apoptosis in mouse and human eosinophils in vitro, showing densitometry of caspase-3 by Western blot of mouse eosinophils normalized to untreated controls. ANOVA with correction for multiple comparisons, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data (mean ± SEM) are from at least two independent experiments with n=2-3 replicates / treatment.

[0031] [Figure 14A]FIG. 14A shows evaluation of the effect of exogenous SP-A administration on eosinophils in SP-A-deficient mice after OVA challenge, and shows a schematic diagram of OVA challenge and SP-A rescue. [Figure 14B] FIG. 14B shows the evaluation of the effect of exogenous SP-A administration on eosinophils in SP-A-deficient mice after OVA challenge, showing a representative flow diagram of eosinophil apoptosis and cell death by annexin V and PI. [Figure 14C] FIG 14C shows the evaluation of the effect of exogenous SP-A administration on eosinophils in SP-A-deficient mice after OVA challenge, showing total viable eosinophil counts in BALF 5 days after the final challenge. *p<0.05. Data (mean±SEM) are representative of two independent experiments with n=5 mice / group.

[0032] [Figure 15A] FIG. 15A shows an analysis of inflammation in SP-A2 humanized mice in the Ova model. Mice were sensitized and challenged in the Ova model and assessed for BAL cellularity 24 hours post-challenge (left panel) and mucin production 7 days post-challenge (right panel). The presence of human SP-A 223Q in mice provided more protection as judged by less eosinophilia and mucin production compared to SP-A- / - mice. SP-A 223Q expressing mice had similar BAL eosinophilia and mucin production compared to WT control mice (with normal mouse SP-A) after Ova challenge. [Figure 15B]FIG. 15B shows an analysis of inflammation in SP-A2 humanized mice in the Ova model. Mice were sensitized and challenged in the Ova model and assessed for BAL cellularity 24 hours post-challenge (left panel) and mucin production 7 days post-challenge (right panel). The presence of human SP-A 223Q in mice provided more protection as judged by less eosinophilia and mucin production compared to SP-A- / - mice. SP-A 223Q expressing mice had similar BAL eosinophilia and mucin production compared to WT control mice (with normal mouse SP-A) after Ova challenge.

[0033] [Figure 16A] FIG. 16A shows an analysis of inflammation in mice receiving "therapeutic" SP-A peptides in the HDM model. Mice were sensitized and challenged in the HDM model by standard methods. 24 hours after the last challenge, mice received either vehicle, a 20mer or a 10mer encompassing the active site containing 223Q. To evaluate the role of SP-A on allergic airway resolution, BAL cellularity (left panel) and mucin production (right panel) were evaluated 7 days after challenge. SP-A KO mice had significantly enhanced BAL eosinophilia compared to WT mice. Both 223Q and 223K mice are somewhat protected in the acute phase of this model. [Figure 16B] FIG. 16B shows an analysis of inflammation in mice receiving "therapeutic" SP-A peptides in the HDM model. Mice were sensitized and challenged in the HDM model by standard methods. 24 hours after the last challenge, mice received either vehicle, a 20mer or a 10mer encompassing the active site containing 223Q. To evaluate the role of SP-A on allergic airway resolution, BAL cellularity (left panel) and mucin production (right panel) were evaluated 7 days after challenge. SP-A KO mice had significantly enhanced BAL eosinophilia compared to WT mice. Both 223Q and 223K mice are somewhat protected in the acute phase of this model.

[0034] [Figure 17] Figure 17 shows that the SP-A223Q 10mer peptide significantly suppresses the expression of MUCSAC in human airway epithelial cells after exposure to IL-13. Airway epithelial cells cultured at the air-liquid interface from two asthmatic participants were exposed to IL-13 alone or to IL-13 plus the SP-A2 peptide containing Gln at position 223 of the lectin domain (223Q). Full-length oligomeric SP-A homozygous at position 223 Q / Q was used as a positive control. After 48 hours of incubation, the expression of MUCSAC was measured by RT-PCR.

[0035] [Figure 18] Figure 18 shows that SP-A is significantly decreased in obese asthma patients, a potential target population for SP-A peptide therapy.

[0036] [Figure 19] FIG. 19 shows that peptides and lead peptidomimetics described herein (i.e., SEQ ID NO: 4 and SEQ ID NO: 8 (peptides); and SEQ ID NO: 12 (peptidomimetic)) reduce a key signaling pathway activated in asthma-STAT3 in human airway epithelial cells taken from asthma patients. Activation of Stat3 signaling leads to airway inflammation and mucus production that exacerbates asthma. Reducing this key signaling pathway by 40-50% and / or 80% here with top candidates (e.g., SEQ ID NO: 12) would lead to significant protection of the lungs of asthma patients by reducing asthma symptoms. Specifically, the bottom graph shows that the peptidomimetic lead (i.e., SEQ ID NO: 12) reduces IL-13 stimulated Stat-3 signaling in airway epithelial cells from asthma patients. Bronchial epithelial cells were cultured at air-liquid interface and allowed to differentiate for 14 days. Cells were basolaterally treated with increasing doses of the peptidomimetic Lead 867 (i.e., SEQ ID NO: 12) for 1 h prior to basolateral stimulation with IL-13 (50 ng / ml) for 30 min. Total cell lysates were analyzed by Western blot for phosphorylation of STAT3 relative to total STAT3 and β-actin.

[0037] [Figure 20] Figure 20 shows that peptidomimetic leads (e.g., SEQ ID NO: 25 or C892) reduce IL-6 stimulated STAT-3 signaling in HEK reporter cells. Cells were pretreated with C892 at increasing concentrations for 30 min before stimulation with IL-6 (16 ng / ml) for 18 h. STAT-3 activation, measured by cell fluorescence, was read after 1 min exposure to substrate on a Clariostar instrument. n=3 replicates per condition.

[0038] [Figure 21] Figure 21 shows that peptidomimetic leads (e.g., SEQ ID NO: 12 or C867) reduce the expression of MUC5AC in airway epithelial cells from four participants with type 2 asthma. Cells obtained by bronchoscopy were cultured at the air-liquid interface for 14 days. On day 14, the cells differentiate, express cilia, and produce mucus. Some cells were pretreated with C867 (13.08 μM) for 1 hour and then stimulated with IL-13 (10 ng / ml) for 5 days, which is a significant stimulus for the expression of mucus and mucin genes. The expression of the MUC5AC gene was significantly reduced by 42% (p=0.01).

[0039] [Figure 22] Figure 22 shows that peptidomimetic leads (e.g., SEQ ID NO: 25 or C892) reduce bronchoconstriction in response to methacholine challenge in an asthma model. WT C57BL / 6 female mice were treated with HDM 0, 7, 14 and 24 hours after each challenge, with some mice receiving lead compound (from Table 1). AHR to methacholine was performed on day 16. *p<0.05 by ANOVA, HDM vs. saline; HDM+lead compound was not different from saline control.

[0040] [Diagram 23]Figure 23 shows that the peptidomimetic Lead reduces IL-6 stimulated STAT-3 signaling in HEK reporter cells compared to full length SP-A. Cells were pretreated with either full length SP-A or Lead peptide at increasing concentrations for 30 min prior to stimulation with IL-6 (16 ng / ml) for 18 h. STAT-3 activation, measured by cell fluorescence, was read after 1 min exposure to substrate on a Clariostar instrument. n=3 replicates per condition.

[0041] [Figure 24] Figure 24 shows the structure of SEQ ID NO:23 containing a small oligoethylene glycol linker, Pego. Specifically, SEQ ID NO:23 contains three Pego units (i.e., Pego3), each with three ethylene glycols connected via diglyme diacid of MW 230 per unit. At the C-terminus, here there is a lipid attached, Hdc.

[0042] [Diagram 25] Figure 25 shows SP-A binding to HEK293T lysates overexpressing ACE2. SP-A coated plates were incubated with lysates from 293T cells overexpressing ACE2 at various concentrations in the presence (solid line) or absence (dashed line) of Ca2+. Binding was detected with human anti-ACE2 by absorbance at a wavelength of 450 nm and read by a plate reader. Non-transfected 293T cell lysates (dashed line) were tested as a negative control. n=3 experiments.

[0043] [Figure 26] Figure 26 shows that the SP-A 20mer peptide competes for binding to ACE-overexpressing cells. Full length SP-A or SP-A 20mer competed (inhibited) ACE2 binding to plate-bound SP-A. The 20mer scrambled (SCR) peptide had little or no effect. n=3 experiments.

[0044] [Figure 27A] Figure 27A shows that full-length SP-A reduces binding of spike protein to cells overexpressing ACE2. [Figure 27B] Figure 27B shows that full-length SP-A reduces binding of spike protein to cells overexpressing ACE2.

[0045] [Figure 28] FIG. 28 shows that SP-A reduced the transduction of S1 protein-pseudotyped lentiviral particles into cells overexpressing ACE2 in vitro.

[0046] [Figure 29] Figure 29 shows that SP-A attenuates SARS-CoV-2 infection in alveolar organoids. Pretreatment of alveolar organoids with SP-A significantly reduced the expression of SARS-CoV-2 N gene. Data are presented as fold change ± SEM relative to CoV2 samples. No expression of N gene was detected in MOCK samples. Data were analyzed using one-way ANOVA with Tukey's post-hoc test****p<0.0001. n=3 technical replicates.

[0047] [Diagram 30] FIG. 30 shows that the SP-A 20mer peptide inhibits ACE-2 / spike-mediated pseudovirus entry.

[0048] Definition: The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues, including natural or 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 modified proteins, such as deletions, additions, and substitutions of single or multiple amino acid residues relative to the native sequence, 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 eliminate a cleavage site. These modifications may be deliberate, such as by site-directed mutagenesis, or may be accidental, such as by mutation of the host producing the protein or errors due to polymerase chain reaction (PCR) amplification.

[0049] As used herein, the term "peptide" refers to a short polymer of amino acids linked together 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 may contain natural amino acids, unnatural amino acids, amino acid analogs, and / or modified amino acids. Peptides may be subsequences of naturally occurring proteins or unnatural (synthetic) sequences.

[0050] As used herein, the term "wild type" refers to a non-mutated version of a gene, allele, genotype, polypeptide, or phenotype, or any fragment thereof. It may be naturally occurring or recombinantly produced. As used herein, the term "variant" refers to 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.

[0051] The term "peptide mimetic" or "peptidomimetic" refers to a peptide-like molecule that mimics a sequence derived from a protein or peptide. A peptide mimetic or peptidomimetic may contain amino acids and / or non-amino acid components. Examples of peptidomimetics include chemically modified peptides, peptoids (wherein side chains are attached to nitrogen atoms of the peptide backbone rather than the alpha carbon), and P-peptides (wherein the amino group is attached to the beta carbon rather than the alpha carbon).

[0052] As used herein, a "conservative" amino acid substitution refers to the replacement of an amino acid in a peptide or polypeptide with another amino acid having similar chemical properties (e.g., 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).

[0053] Naturally occurring residues can be divided into classes based on common side chain properties, e.g., 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.

[0054] 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 reverse or inverted forms of amino acid moieties. The embodiments herein may, in some embodiments, be limited to natural amino acids, non-natural amino acids, and / or amino acid analogs. Non-conservative substitutions may include exchanging a member of one class for a member of another class.

[0055] 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 window of comparison (e.g., the length of the longer sequence, the length of the shorter sequence, a designated window, etc.), (2) determining the number of positions that contain identical (or similar) monomers (e.g., the same amino acid is present in both sequences, similar amino acids are present in both sequences) to obtain the number of matched positions, (3) dividing the number of matched positions by the total number of positions within the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a 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 long and have identical amino acids at all positions except one, then peptide A and peptide B have 95% sequence identity. If the amino acids at the non-identical positions share the same biophysical characteristics (e.g., both were acidic), then peptide A and peptide B have 100% sequence similarity. As another example, if peptide C is 20 amino acids long and peptide D is 15 amino acids long, and 14 of the 15 amino acids of peptide D are identical to those of a portion of peptide C, then 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, any gap in the aligned sequences is treated as a mismatch at that position.

[0056] "Subject," "individual," "host," "animal," and "patient" are used interchangeably herein to refer to mammals, including, but not limited to, rodents, apes, humans, cats, dogs, horses, cows, pigs, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets.

[0057] As used herein, the terms "administration" and "administering" refer to the act of giving a drug, prodrug, or other agent or therapeutic treatment (e.g., SP-A peptide) to a subject or to cells, tissues, and organs in vivo, in vitro, or ex vivo. Exemplary routes of administration to the human body can be through the subarachnoid space of the brain or spinal cord (subarachnoid space), eye (ophthalmic), mouth (oral), skin (topical or transdermal), nose (intranasal), lungs (inhalation), oral mucosa (buccal), ear, rectum, vagina, injection (e.g., intravenous, subcutaneous, intratumoral, intraperitoneal, etc.), and the like.

[0058] As used herein, the terms "co-administration" and "co-administering" refer to the administration of at least two agents (e.g., multiple SP-A peptides or an SP-A peptide and another therapeutic agent) or therapies to a subject. In some embodiments, the 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 at a lower dosage than would be appropriate for their administration alone. Thus, co-administration is particularly desirable in embodiments where the co-administration of agents or therapies reduces the required dosage of a potentially harmful (e.g., toxic) agent(s) and / or where the co-administration of two or more agents results in sensitization of the subject to the beneficial effects of one of the agents via the co-administration of the other agent.

[0059] "Treatment", as used herein, encompasses any administration or application of a therapeutic agent for a disease in a mammal, including a human, and includes inhibiting, halting the development of, or relieving the disease, for example, by causing regression or by restoring or repairing a lost, deficient, or defective function; or by stimulating an inefficient process.

[0060] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional 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 components of the formulation. A pharmaceutically acceptable carrier is appropriate for the formulation used. For example, if the therapeutic agent is administered orally, the carrier can be a gel capsule. If the therapeutic agent is administered subcutaneously, the carrier is ideally not irritating to the skin and does not cause injection site reactions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] Before the present compounds, compositions, and / or methods are disclosed and described, it is to be understood that this invention is not limited to specific synthetic methods or to particular compositions, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0062] 1-30, the present invention features 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, such as inflammatory pulmonary diseases (e.g., asthma).

[0063] The present invention features compositions and methods for treating and preventing pulmonary diseases (e.g., inflammatory pulmonary diseases) using peptides whose sequences are derived from the active region of endogenous human SP-A and include the major Q allele at position 223 of the SP-A2 peptide. For example, in some embodiments, compositions are provided that include a peptide comprising, consisting essentially of, or consisting of an amino acid sequence selected from, e.g., SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or a peptide having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity thereto. In some embodiments, the peptide binds to a receptor selected from, e.g., FC (CD16 / 32), sarp alpha, TLR-2, EGFR, or MYADM (myeloid-associated differentiation marker).

[0064] In some embodiments, the invention may feature a method of treating an inflammatory lung disease (e.g., asthma) in a subject in need of such treatment. The method may include administering to the subject a therapeutically effective amount of a purified peptide as described herein (e.g., a purified peptide comprising the amino acid sequence of KEQCVE (SEQ ID NO: 9)). In other embodiments, the invention features a purified peptide comprising the amino acid sequence of KEQCVE (SEQ ID NO: 9) for use in a method of treating an inflammatory lung disease in a subject in need of such treatment.

[0065] The invention may also feature methods and compositions (e.g., pharmaceutical compositions) for enhancing SP-A activity in a cell. The compositions may include any of the purified peptides as described herein and a pharmaceutical carrier. In some embodiments, the compositions (e.g., pharmaceutical compositions) include a purified peptide comprising the amino acid sequence of KEQCVE (SEQ ID NO: 9) and a pharmaceutical carrier. In other embodiments, the compositions (e.g., pharmaceutical compositions) include a purified peptide comprising the amino acid sequence of KEQCVE (Xaa) nIn a further embodiment, the composition (e.g., pharmaceutical composition) comprises a purified peptide comprising an amino acid sequence of (SEQ ID NO: 10) and a pharmaceutical carrier; wherein n ranges from 4 to 16 amino acids and Xaa is any natural or unnatural amino acid. n KEQCVE(Xaa) n (SEQ ID NO:20) and a pharmaceutical carrier; where n ranges from 1 to 16 amino acids and Xaa is any natural or unnatural amino acid. In some embodiments, the composition is in preparation for aerosolization. A method for enhancing SP-A activity in a cell can include delivering a composition comprising a purified peptide as described herein (e.g., a peptide comprising the amino acid sequence of KEQCVE (SEQ ID NO:9)) to a cell (e.g., a lung cell).

[0066] In some embodiments, n is in the range of 0 to 20 amino acids. In some embodiments, n is in the range of 0 to 15 amino acids. In some embodiments, n is in the range of 0 to 10 amino acids. In some embodiments, n is in the range of 0 to 5 amino acids. In some embodiments, n is in the range of 0 to 1 amino acid. In some embodiments, n is in the range of 1 to 20 amino acids. In some embodiments, n is in the range of 1 to 15 amino acids. In some embodiments, n is in the range of 1 to 10 amino acids. In some embodiments, n is in the range of 1 to 5 amino acids. In some embodiments, n is in the range of 4 to 20 amino acids. In some embodiments, n is in the range of 4 to 15 amino acids. In some embodiments, n is in the range of 4 to 10 amino acids. In some embodiments, n is in the range of 4 to 5 amino acids. In some embodiments, n is in the range of 5 to 20 amino acids. In some embodiments, n is in the range of 5 to 15 amino acids. In some embodiments, n is in the range of 5 to 10 amino acids. In some embodiments, n is in the range of 10 to 20 amino acids. In some embodiments, n is in the range of 10 to 15 amino acids. In some embodiments, n ranges from 15 to 20 amino acids.

[0067] Table 1 provides non-limiting examples of purified peptides that can be used in accordance with the compositions and methods described herein. [Table 1]

[0068] In some embodiments, the aforementioned peptides (i.e., peptides listed in Table 1) comprise, consist essentially of, or consist of an amino acid sequence at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%) identical to the provided peptides. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a pharma- ceutically acceptable carrier. In some embodiments, the composition is formulated for pulmonary delivery. In other embodiments, the aforementioned peptides (i.e., peptides listed in Table 1) may comprise, consist essentially of, or consist of an amino acid sequence at least 95%, 90%, 85%, 83%, 80%, 75%, or 70% identical to the provided peptides.

[0069] With reference to Table 1, Nle refers to norleucine, an amino acid with the formula CH3(CH2)3CH(NH2)CO2H (or its systematic name is 2-aminohexanoic acid), and Pego3 refers to PEGylation, a process of biochemical modification of biologically active molecules with polyethylene glycol (PEG), which imparts several desirable properties to proteins / peptides, antibodies, and vesicles that may be used for therapy or genetic modification of cells. PEGylation is routinely achieved by incubation of a reactive derivative of PEG with the target molecule. The covalent attachment of PEG to a drug or therapeutic protein can "mask" the agent from the host's immune system (reducing immunogenicity and antigenicity) and increase its hydrodynamic size (size in solution), extending its circulation time by reducing renal clearance. PEGylation can also provide water solubility to hydrophobic drugs and proteins. With its proven pharmacological benefits and acceptability, PEGylation technology is the basis of a growing multi-billion dollar industry. Additionally, acetyl (Ac) modifications can be used to closely match those of the native protein and stabilize the peptidomimetic against enzymatic degradation by exopeptidases.

[0070] Compound C892 (i.e., SEQ ID NO:25) is a stable equivalent analog of C867 (i.e., SEQ ID NO:12), and both show nearly identical activity in all assays tested. Compounds C939 (i.e., SEQ ID NO:23) and C940 (i.e., SEQ ID NO:24) are an additional step beyond compound C892 (i.e., SEQ ID NO:17), now involving PEGylation and lipidation (Hdc). Lipidation of peptides has proven to be the most robust strategy for generating new peptide leads, as this modification increases the peptide's in vivo stability and reduces renal clearance.

[0071] In some embodiments, the peptides described herein are modified by the addition of an amine or acid group to the C-terminus and acetylation or addition of histidine (H) to the N-terminus. In other embodiments, the peptides described herein are modified by the addition of an acid to the N-terminus. Non-limiting examples of acids that can be used to modify the N-terminus of the peptides described herein can include, but are not limited to, a hydroxyl group (-OH), a carboxyl / carboxylic acid group (COOH), or a combination thereof. In further embodiments, the peptides described herein are modified at the lipid group and the C-terminus and / or N-terminus.

[0072] In some embodiments, the cells are lung cells. In some embodiments, the compositions are for pulmonary delivery. In some embodiments, the invention features a system including a pharmaceutical composition as described herein and a device for pulmonary delivery of the composition. In some embodiments, the device is a metered dose inhaler or nebulizer. In some embodiments, the compositions described herein are for treating or preventing asthma, COPD, or COVID-19.

[0073] The invention features a method that includes delivering a composition comprising a peptide as described herein to a cell (e.g., a lung cell) of a subject. In some embodiments, the peptide comprises an amino acid sequence of KEQCVE (SEQ ID NO: 9). In other embodiments, the peptide comprises an amino acid sequence of KEQCVE(Xaa)n (SEQ ID NO: 10). In further embodiments, the peptide comprises an amino acid sequence of (Xaa)nKEQCVE(Xaa)n (SEQ ID NO: 20). Delivering the peptide to a cell (e.g., a lung cell) of a subject can enhance SP-A activity in the cell and / or treat or prevent asthma, COPD, or COVID-19 in a subject (i.e., a patient) or participant. In some embodiments, the composition is delivered to the cell (e.g., a lung cell) via a metered dose inhaler or nebulizer.

[0074] In some embodiments, the compositions described herein reduce mucin production and / or reduce eosinophilia in cells or lung tissue. In some embodiments, the subject is obese. In some embodiments, the peptide binds to a receptor such as FC (CD16 / 32), sarp alpha, TLR-2, or EGFR.

[0075] 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 (e.g., at positions involved in SP-A signaling or positions not involved in SP-A signaling) compared to the peptides described herein.

[0076] The embodiments are not limited to specific substitutions. In some embodiments, the peptides described herein are further modified (e.g., substitution, deletion, or addition of standard amino acids; chemical modification, etc.). Modifications understood in the art include N-terminal modifications, C-terminal modifications (to protect the peptide from proteolysis), alkylation of amide groups, and hydrocarbon "stapling" (e.g., to stabilize an alpha-helical conformation). In some embodiments, the peptides described herein can 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, for example, in the receptor binding site, 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 a C1-C4 alkyl. Additionally, one or more side or terminal groups may be protected by protecting groups known to the ordinary skilled peptide chemist. The α-carbon of an amino acid may be mono- or dimethylated.

[0077] In some embodiments, one or more intrapeptide disulfide bonds are introduced (e.g., between two cysteines in the peptide). The presence of intrapeptide disulfide bonds may stabilize the peptide.

[0078] Any of the embodiments described herein may include peptidomimetics that correspond to the peptides described herein, with various modifications that are 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, non-natural amino acids (or naturally occurring amino acids other than the standard 20 amino acids) are substituted to achieve desired properties.

[0079] In some embodiments, residues that have a positively charged side chain under physiological conditions, or where a positively charged side chain is desired, are lysine, homolysine, δ-hydroxylysine, homoarginine, 2,4-diaminobutyric acid, 3-homoarginine, D-arginine, arginal (wherein the -COOH of arginine is replaced by -CHO), 2-amino-3-guanidinopropionic acid, nitroarginine (N(G)-nitroarginine), nitrosoarginine (N(G)-nitrosoarginine), methylarginine (N-methylarginine), ε-N-methyllysine, allo-hydroxylysine, 2,3-diaminopropionic acid, 2,2′-diaminopimelic acid, ornithine, symmetric dimethylarginine, asymmetric dimethylarginine, 2,6-diaminohexynoic acid ... acid), p-aminobenzoic acid, and 3-aminotyrosine, as well as histidine, 1-methylhistidine, and 3-methylhistidine.

[0080] Neutral residues are those that have a side chain that is uncharged 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. In some embodiments, residues that have a side chain that is 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-dinitrotyrosine and 3-homoserine.

[0081] Residues having a non-polar, hydrophobic side chain are uncharged residues under physiological conditions, preferably having a hydropathic index greater than 0, particularly greater than 3. In some embodiments, the non-polar, hydrophobic side chain is 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 a non-polar, hydrophobic side chain, or residues where a non-polar, 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.

[0082] In some embodiments, the peptides and polypeptides are isolated and / or purified (or substantially isolated and / or substantially purified). Thus, in such embodiments, the peptides and / or polypeptides are provided in a substantially isolated form. In some embodiments, the peptides and / or polypeptides are isolated from other peptides and / or polypeptides, for example, as a result of solid-phase peptide synthesis. Alternatively, the peptides and / or polypeptides may 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 the peptides and / or polypeptides. In some embodiments, the invention provides for the preparation of the peptides and / or polypeptides in several formulations, depending on the desired use. For example, when the polypeptide is substantially isolated (or even 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 may contain protective agents, such as buffers, preservatives, cryoprotectants (e.g., sugars such as trehalose). The form of such preparations may be a solution, a gel, etc. In some embodiments, the peptides and / or polypeptides are prepared in a lyophilized form. Moreover, such preparations may contain other desired agents, such as small molecules or other peptides, polypeptides, or proteins, and indeed such preparations may be provided that contain mixtures of peptides and / or polypeptides of the different embodiments described herein.

[0083] In some embodiments, peptidomimetic versions of the peptide sequences described herein or variants thereof are provided herein. In some embodiments, a peptidomimetic is characterized as an entity that retains the polarity (or non-polarity, hydrophobicity, etc.), three-dimensional size, and functionality (biological activity) of its peptide equivalent, but in which all or part of the peptide bond has been replaced (e.g., by a more stable linkage). In some embodiments, "stable" refers to being more resistant to chemical degradation or enzymatic degradation by hydrolases. In some embodiments, the bond that replaces the amide bond (e.g., an amide bond surrogate) preserves some properties of the amide bond (e.g., conformation, steric bulk, electrostatic characteristics, 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), retroinverse amide (Chorev, M. and Goodman, M., Acc. Chem. Res., 1993, 26, 266; incorporated herein by reference in its entirety), thioamide (Sherman D.B. and Spatola, A.F.J. Am. Chem. Soc., 1990, 112, 433; incorporated herein by reference in its entirety), thioester, phosphonate, ketomethylene (Hoffman, R.V. and Kim, H.O.J. 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).

[0084] Similar to the replacement of amide bonds, peptidomimetics can involve replacing larger structural moieties with di- or tripeptidomimetic structures, in which case mimetic moieties containing peptide bonds, such as azole-derived mimetics, can be used as dipeptide substitutes. Suitable peptidomimetics include reduced peptides in which the amide bonds have been reduced to methylene amines by treatment with a reducing agent (e.g., hydride reagents such as borane or lithium aluminum hydride). Such reduction has the added benefit of increasing the overall cationic character of the molecule.

[0085] Peptides and polypeptides encompassing a substantially alpha helical peptide region disclosed herein may be further derivatized by chemical modifications such as amidation, glycosylation, acylation, sulfation, phosphorylation, acetylation, and cyclization. Such chemical modifications may be effected by chemical or biochemical methodologies, as well as by in vivo processes, or any combination thereof.

[0086] Other peptidomimetics include, for example, peptoids formed by stepwise synthesis of amide-functionalized polyglycines. Some peptidomimetics backbones may be readily accessible from their peptide precursors, for example fully methylated peptides. A suitable method is described in Ostresh, JM et al., Proc. Natl. Acad. Sci. USA (1994) 91, 11138-11142, the entirety of which is incorporated herein by reference.

[0087] The peptides and polypeptides described herein can be prepared as salts with various 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 can be formed by conventional means, such as by reacting the free acid or base form of the product with one or more equivalents of a suitable base or acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water that is subsequently removed in vacuum or by freeze-drying, or by exchanging an ion of an existing salt with another ion on a suitable ion exchange resin.

[0088] The peptides and polypeptides described herein may be formulated as their pharma- ceutically acceptable salts and / or complexes. Pharmaceutically acceptable salts include acid addition salts, such as sulfate, hydrochloride, phosphate, sulfamate, acetate, citrate, lactate, tartrate, succinate, oxalate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate. Pharmaceutically acceptable salts may 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 can 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 then removed in vacuo or by freeze-drying, or by exchanging an ion of an existing salt for another ion on a suitable ion exchange resin.

[0089] The peptides and polypeptides described herein can be formulated as pharmaceutical compositions for use in conjunction with the methods of the present disclosure. The compositions disclosed herein can be conveniently provided in the form of formulations suitable for parenteral administration, including subcutaneous, intramuscular, and intravenous administration, nasal administration, pulmonary administration, or oral administration. Such peptide and polypeptide formulations suitable for each route of administration are described in standard formulation treatises, for example, Remington's Pharmaceutical Sciences by EW Martin. See also Wang, YJ and Hanson, MA, "Parenteral Formulations of Proteins and Peptides: Stability and Stabilizers," Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42:2S (1988).

[0090] The pharmaceutical composition may be administered in a form formulated with a pharma- ceutically acceptable carrier and any excipients, adjuvants, etc., according to good pharmaceutical practice. The peptide-based pharmaceutical composition may be in the form of a solid, semi-solid or liquid dosage form: for example, powder, solution, elixir, syrup, suspension, cream, drop, paste and spray. As one skilled in the art will recognize, the route of administration (e.g., pill, injection, etc.) selected will determine the composition form. In general, it is preferred to use unit dosage forms to achieve easy and accurate administration of the active pharmaceutical peptide or polypeptide. Generally, the therapeutically effective pharmaceutical compound is 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 a desired unit dose. In some embodiments, the pharmaceutical composition may be administered in single or multiple doses. The particular route of administration and dosage regimen will be determined by the skilled artisan corresponding to the condition of the individual being 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 pharma- ceutically acceptable carriers, adjuvants or vehicles are provided in unit dosage form for administration to a subject. The amount of active ingredient that can be combined with such materials to produce a single dosage form can vary depending on various factors as described above. Various materials available in the pharmaceutical art can be used as carriers, adjuvants and vehicles in the compositions of the present invention. Injectable preparations, for example, oily solutions, suspensions or emulsions, can be formulated as known in the art using suitable dispersing or wetting agents and suspending agents as necessary. Sterile injectable preparations can use non-toxic parenterally acceptable diluents or solvents, such as sterile non-pyrogenic water or 1,3-butanediol. Among other acceptable vehicles and solvents that can be used are 5% Dextrose Injection, Ringer's Injection and Isotonic Sodium Chloride Injection (as described in USP / NF). In addition, sterile fixed oils can be conventionally used as solvents or suspending media. For this purpose any bland fixed oil can be employed including synthetic mono-, di-, or triglycerides.Fatty acids such as oleic acid can also be used in the preparation of injectable compositions.

[0091] Certain peptides and polypeptides described herein may be substantially insoluble in water and sparingly soluble in most pharma- ceutically acceptable protic solvents and vegetable oils. In certain embodiments, cyclodextrins may be added as aqueous solubility enhancers. Cyclodextrins include methyl, dimethyl, hydroxypropyl, hydroxyethyl, glucosyl, maltosyl, and maltotriosyl derivatives of alpha-, beta-, and gamma-cyclodextrin. An exemplary cyclodextrin solubility enhancer is hydroxypropyl-beta-cyclodextrin (HPBCD), which may be added to any of the above compositions to further improve the aqueous solubility characteristics of the peptide or polypeptide. In one embodiment, the composition comprises 0.1%-20% HPBCD, 1%-15% HPBCD, or 2.5%-10% HPBCD. The amount of solubility enhancer used may depend on the amount of the peptide or polypeptide of the present disclosure in the composition. In certain embodiments, the peptides may be formulated in a non-aqueous, polar, aprotic solvent such as DMSO, dimethylformamide (DMF) or N-methylpyrrolidone (NMP).

[0092] In some cases, it may be 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 advantageous to administer the additional agent separately from the polypeptide. For use, the pharmaceutical compositions of peptides and polypeptides described herein may be provided in a unit dosage form that contains an effective amount of the peptide or polypeptide for single administration. Unit dosage forms useful for subcutaneous administration include pre-filled syringes and injectors.

[0093] In certain embodiments, the polypeptide is administered in an amount expressed as the daily equivalent 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, 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 still further embodiments, the polypeptide is administered in an amount expressed as a daily equivalent of 1-10 mg per day, including 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, regardless of frequency of administration. 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 daily ("BID"). In typical embodiments, the polypeptide is administered for at least 3 months, at least 6 months, at least 12 months, or more. In some embodiments, the polypeptide is administered for at least 18 months, 2 years, 3 years, or more.

[0094] Any carrier that can deliver the active peptide or polypeptide (e.g., without destroying the peptide or polypeptide in the carrier) is a suitable carrier, and such carriers are well known in the art. In some embodiments, the composition is formulated for administration by any suitable route, including, but not limited to, oral (e.g., in tablet, capsule, granule, or powder form, etc.), sublingual, buccal, parenteral (e.g., by subcutaneous, intravenous, intramuscular, intradermal, or intrasternal injection or infusion (e.g., as a sterile injectable aqueous or non-aqueous solution or suspension, etc.)), nasal (including, for example, administration to the nasal membranes by inhalation spray), topical (e.g., in the form of a cream or ointment), transdermal (e.g., by a transdermal patch), rectal (e.g., in the form of a suppository), etc.

[0095] In one embodiment, the pharmaceutical composition of the present invention is suitable for inhalation administration.The pharmaceutical composition suitable for inhalation administration can typically be in the form of aerosol or powder.Such compositions are generally administered using well-known delivery devices, such as nebulizer inhaler, metered dose inhaler (MDI), dry powder inhaler (DPI) or similar delivery devices.

[0096] In certain embodiments of the present invention, the pharmaceutical composition comprising the active agent is administered by inhalation using a nebulizer inhaler. Such nebulizer devices typically generate a stream of high velocity air that sprays the pharmaceutical composition comprising the active agent as a mist that is carried to the respiratory tract of the patient. Thus, when formulated for use in a nebulizer inhaler, the active agent is typically 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 micronized particles of inhalable size, where micronization is typically 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, by PARI GmbH (Starnberg, German). Other nebulizer devices include the Respimat (Boehringer Ingelheim) and those disclosed, for example, in U.S. Pat. No. 6,123,068 by Lloyd et al. and WO 97 / 12687 (Eicher et al.). Exemplary pharmaceutical compositions for use in a nebulizer inhaler include an isotonic aqueous solution containing the SP-A peptide or a pharma- ceutically acceptable salt or solvate or stereoisomer thereof.

[0097] In another specific embodiment of the present invention, the pharmaceutical composition comprising the 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. A representative pharmaceutical composition for use in a dry powder inhaler comprises dry lactose having a particle size between about 1 μm and about 100 μm, and micronized particles of SP-A peptide or a pharma-ceutically acceptable salt or solvate or stereoisomer thereof.

[0098] Such dry powder formulations can be made, for example, by combining lactose with the active agent and then dry blending the components. 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 an inhalation cartridge or capsule for use with a dry powder delivery device. Examples of dry powder inhaler delivery devices include Diskhaler (GlaxoSmithKline, Research Triangle Park, NC) (see, e.g., U.S. Pat. No. 5,035,237 by Newell et al.); Diskus (GlaxoSmithKline) (see, e.g., U.S. Pat. No. 6,378,519 by Davies et al.); Turbuhaler (AstraZeneca, Wilmington, Del.) (see, e.g., U.S. Pat. No. 4,524,769 by Wetterlin); Rotahaler (GlaxoSmithKline) (see, e.g., U.S. Pat. No. 4,353,365 by 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.

[0099] In yet another specific embodiment of the present invention, a pharmaceutical composition comprising an active agent is administered by inhalation using a metered dose inhaler. Such metered dose inhalers typically use compressed propellant gas to release a measured amount of the active agent or its pharma- ceutically acceptable salts or solvates or stereoisomers. Thus, a pharmaceutical composition administered using a metered dose inhaler typically comprises a solution or suspension of the active agent in a liquefied propellant. Any suitable liquefied propellant may be used, including chlorofluorocarbons, e.g., CCl.sub.3F, and hydrofluoroalkanes (HFAs), e.g., 1,1,1,2-tetrafluoroethane (HFA134a) and 1,1,1,2,3,3,3-heptafluoro-n-propane (HFA227). Due to concerns regarding chlorofluorocarbons affecting the ozone layer, formulations containing HFAs are generally preferred. Additional optional components of HFA formulations include cosolvents, e.g., ethanol or pentane, and surfactants, e.g., sorbitan trioleate, oleic acid, lecithin, and glycerin. See, e.g., U.S. Patent No. 5,225,183 to Purewal et al., EP 0717987A2 (Minnesota Mining and Manufacturing Company), and WO 92 / 22286 (Minnesota Mining and Manufacturing Company). A representative 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 pharma- ceutically acceptable salt or solvate or stereoisomer thereof; about 0% to about 20% by weight of ethanol; and about 0% to about 5% by weight of surfactant; the remainder being HFA propellant.

[0100] Such compositions are typically prepared by adding chilled 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 combined with a propellant. The formulation is then loaded into an aerosol canister that forms part of a metered dose inhaler device. Examples of metered dose inhaler devices that have been specifically developed for use with HFA propellants are provided in U.S. Patent No. 6,006,745 by Marecki and U.S. Patent No. 6,143,277 by Ashurst. Alternatively, suspension formulations can be prepared by spray drying a coating of surfactant on micronized particles of the active agent. See, for example, WO 99 / 53901 (Glaxo Group Ltd.) and WO 00 / 61108 (Glaxo Group Ltd.), the disclosures of which are incorporated herein by reference in their entirety.

[0101] Additional examples of processes for preparing inhalable particles, and formulations and devices suitable for inhalation administration, are disclosed in 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), the disclosures of which are incorporated herein by reference in their entireties.

[0102] In some embodiments, the peptide / polypeptide is provided in a pharmaceutical composition and / or is co-administered (simultaneously or sequentially) with one or more additional therapeutic agents. Such additional agents may be for treating or preventing pulmonary inflammation (e.g., asthma). Additional agents include, but are not limited to, short-acting beta 2-adrenergic receptor agonists (SABA), such as salbutamol (albuterol USAN); long-acting beta agonists (LABA), such as salmeterol and formoterol; anticholinergics, such as ipratropium bromide, inhaled epinephrine, inhaled corticosteroids, such as budesonide, fluticasone, mometasone or ciclesonide, systemic corticosteroids, such as prednisone or methylprednisolone; leukotriene receptor antagonists (e.g., montelukast and zafirlukast); or combinations thereof.

[0103] Without wishing to limit the invention to any theory of mechanism, it is believed that the purified peptides described herein co-administered (simultaneously or sequentially) with one or more additional therapeutic agents (e.g., steroids) will allow for lower doses of steroids to treat severe asthma. Thus, combination therapy may have a steroid-sparing effect postulated by a different mechanism of action compared to inhaled corticosteroids alone or in combination therapy (ICS / LABA).

[0104] In some embodiments, provided herein are methods of 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, patients who are obese or non-obese may benefit. In some embodiments, the subject is identified as having an SP-A genotype (e.g., a genotype described herein) that is associated with asthma or an increased risk of severe asthma.

[0105] In some embodiments, pharmaceutical compositions comprising at least one SP-A peptide or polypeptide as described herein are delivered to such patients in an amount and location sufficient to treat the symptoms. In some embodiments, the peptides and / or polypeptides (or pharmaceutical compositions comprising same) can be delivered to the patient systemically or locally, and it will be within the ordinary skill of the medical professional treating such patients to ascertain the most appropriate delivery route, time course, and dosage for treatment. It will be understood that the applied method of treating the patient will most preferably substantially alleviate or even eliminate such symptoms. However, as with many medical treatments, the application of the method of the invention is considered successful if the symptoms of the disease or disorder in the patient are appreciably reduced during, after, or otherwise as a result of the method of the invention.

[0106] The present disclosure is not limited to the treatment of asthma. Any inflammatory condition known in the art or otherwise contemplated herein may be treated according to the presently disclosed and claimed inventive concept(s). Non-limiting examples of disease conditions having inflammation associated therewith include infection-related or non-infectious inflammatory conditions in the lungs (e.g., asthma, sepsis, chronic obstructive pulmonary disease (COPD), COVID-19, lung infections, 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); and the like. EXAMPLES

[0107] The following are non-limiting examples of the present invention. It should be understood that the examples are not intended to limit the present invention in any way. Equivalents or substitutes are within the scope of the present invention.

[0108] Example 1: Example 1 demonstrates that the genotype of SP-A at position 223 affects lung function and asthma control. Previous studies have shown that SP-A from asthmatic subjects is dysfunctional in regulating inflammatory symptoms such as IL-8 and MUCSAC production. To determine whether the genotype of SP-A affects lung function, lung physiology, and asthma control, SP-A was genotyped and measured in 53 mild to moderate asthmatic patients not receiving controller therapy. Figure 15A shows that among the 53 asthmatic patients screened, those homozygous for the minor allele (SP-A2 K223K) had lower lung function (FEV1%) than subjects with Q223Q or Q223K genotypes. Moreover, this cohort of asthmatic subjects demonstrates worse asthma control (Asthma Control Questionnaire; ACQ) than asthmatic patients with AC or CC genotypes (Figure 15B).

[0109] Additionally, SP-A humanized mice exhibit distinct phenotypes in allergy models. To more mechanistically study the impact of SP-A genetic variation at position 223Q / K in asthma, we generated SP-A humanized mice expressing either SP-A223Q (major allele) or SP-A223K (minor allele). It was found that the SP-A223Q allele conferred more protection compared to the SP-A223K allele when challenged in the ova model of allergic airway disease. Figure 15A shows that SP-A223Q mice had significantly reduced eosinophilia compared to SP-A-deficient mice 24 hours after challenge and significantly reduced mucin production (PAS score) 7 days after challenge (Figure 15B).

[0110] SP-A peptide encompassing the 223Q active site attenuates airway eosinophil and mucin production in an allergy model. Because asthmatic subjects carrying the 223K (minor) allele had worse asthma control and lung function compared to individuals with the 223Q (major) allele, and similar findings were observed in SP-A humanized mice in an allergy model, experiments were performed to find the active region of SP-A. The active peptide was determined to be a 10AA peptide that contains the 223Q site and is located in the carbohydrate recognition domain of endogenous SP-A. As shown in Figures 16A and 16B, SP-A-deficient mice were challenged in the HDM model and given either 20AA SP-A (PAGRGKEQCVEMYTDGQWND (SEQ ID NO: 8)) or 10AA SP-A (KEQCVEMYTD (SEQ ID NO: 4)) 24 hours after the last challenge, and compared to mice that received vehicle treatment. Mice that received the SP-A peptide had significantly lower eosinophilia in the lavage compartment (FIG. 16A) and less mucin production (FIG. 16B) compared to vehicle-treated mice.

[0111] The SP-A peptide (10AA) encompassing the 223Q active site attenuates the phenotype in primary human airway epithelial cells from asthma patients. Experiments were performed in which airway epithelial cells from two subjects with asthma not receiving controller therapy were cultured at the air-liquid interface for two weeks. In separate conditions, cells were exposed to 20 μg / ml of each peptide for 30 minutes, followed by 50 ng / ml of IL-13 and incubated for 48 hours. Cells were placed in Trizol and MUCSAC was measured by RT-PCR. Figure 17 shows the dramatic reduction in expression of MUCSAC with each peptide to negative control levels. The 10AA length is particularly useful as a therapeutic agent because its size allows it to be packaged into an inhaler type of device for delivery to the airways. This experiment demonstrates that the 223Q peptide has efficacy in suppressing the expression of mucin genes in human airway epithelial cells in the context of IL-13 exposure.

[0112] In summary, these experiments demonstrated that the SP-A 223 genotype affects lung function and asthma control; SP-A humanized mice exhibit distinct phenotypes in allergy models that depend on the 223Q / K site, an SP-A peptide encompassing the 223Q active site attenuates the phenotype in allergy models, and an SP-A peptide (10AA) encompassing the 223Q active site attenuates Muc5AC in primary human airway epithelial cells from asthma patients.

[0113] With reference to Figures 5A-5D, WT C57BL / 6 mice were sensitized and challenged in the House Dust Mite (HDM) model according to standard methods on days 0, 7, and 14 (black arrows). 24 hours after the final challenge, mice received either scrambled vehicle or a 20-mer SP-A peptide encompassing the active site containing 223Q (dashed arrow) (physiologic dose of 25 μg / mouse delivered in 40 μl sterile saline) by oropharyngeal instillation. Lung histological sections were analyzed for mucin production assessed by PAS staining / scoring to determine whether the SP-A peptide could protect against HDM-induced airway mucin production. Similar protective effects were observed on days 5 and 7 after SP-A peptide treatment. Peptide sequence: PAGRGKEQCVEMYTDGQWND (SEQ ID NO: 8).

[0114] With reference to Figures 6A and 6B, WT C57BL / 6 mice were sensitized and challenged in the house dust mite (HDM) model according to standard methods described in the previous paragraph (see Figure 5A). 24 hours after the last challenge, mice received either scrambled vehicle, a 20-mer encompassing the active site containing 223Q, or a 10-mer SP-A peptide (dashed arrow) (physiological dose of 25 μg / mouse delivered in 40 μl of sterile saline) by oropharyngeal instillation. In Figure 6A, lung tissue sections were analyzed for mucin production assessed by PAS staining / scoring to determine whether the SP-A peptide could protect against HDM-induced airway mucin production. Similar protective effects were observed at days 5 and 7 after SP-A peptide treatment. In Figure 6B, bronchoalveolar lavage fluid samples were analyzed for eosinophilia to determine whether SP-A peptide could protect against HDM-induced airway eosinophilia by reducing eosinophil viability. Viability was assessed by trypan blue exclusion versus cell counts. Peptide sequences: 20mer PAGRGKEQCVEMYTDGQWND (SEQ ID NO: 8), peptide 1 PAGRGKEQCV (SEQ ID NO: 2), peptide 2 EMYTDGQWND (SEQ ID NO: 3), peptide 3 KEQCVEMYTD (SEQ ID NO: 4).

[0115] With reference to FIG. 6C, human bronchial epithelial cells obtained from asthmatic participants, fully phenotyped by bronchoscopy, were grown at the ALI for 2 weeks prior to the experiment. For challenge, each SP-A test peptide (50 μg / ml) was added to the apical compartment at least 30 minutes prior to IL-13 challenge. Muc5AC was analyzed by RT-PCR from cell lysates and analyzed as a multiple of control samples. Peptide sequences: 20mer PAGRGKEQCVEMYTDGQWND (SEQ ID NO: 8), peptide 1 PAGRGKEQCV (SEQ ID NO: 2), peptide 2 EMYTDGQWND (SEQ ID NO: 3), peptide 3 KEQCVEMYTD (SEQ ID NO: 4) FL=full length oligomeric SP-A extracted from lavage fluid of an individual with pulmonary alveolar proteinosis.

[0116] With reference to FIG. 18, expression of SP-A was analyzed by Western blot from bronchoalveolar lavage fluids of lean, overweight and obese individuals with and without asthma.

[0117] Figures 5A-5D, 6A-6C, 15A-15B, and 18 show that SP-A223Q humanized mice produce less mucus than 223K mice in an allergy model (Ova model; Figures 15A-15B), that SP-A223Q humanized mice produce less mucus than 223K mice in an allergy model (HDM (house dust mite) model; Figures 5A-5D), that the truncated 10AA peptide reduces mucin production in a mouse HDM model (Figure 6A), that the truncated 10AA peptide reduces eosinophilia in a mouse HDM model (Figure 6B), that the truncated 10AA peptide reduces mucin (Muc5AC RNA) in human primary cells (Figure 6C), and that SP-A is significantly reduced in obesity (Figure 18).

[0118] Example 2: Example 2 describes the use of the HDM sensitization and challenge model in 10-12 adult primates selected by pre-screening for baseline sensitivity to methacholine challenge to test SP-A therapeutic peptides in a crossover study design.

[0119] First, HDM allergen is administered to all 10 primates by subcutaneous injection every other week for 10 weeks, at which point the animals are tested for HDM skin reactivity. HDM mask exposure is then administered every other week for a total of 8 weeks. After this HDM challenge period, airway hyperresponsiveness is assessed in all 12 primates, and lavage fluids and biopsy specimens are collected for analysis.

[0120] After the first round of analysis to evaluate the level of response each primate has to the HDM model, 10 primates are divided into two study groups in a randomized double-blind crossover design: Group 1 (n=6) receives SP-A peptide, followed by washout and then placebo; Group 2 (n=6) receives placebo first, followed by washout and then SP-A peptide. SP-A peptide and placebo are administered intranasally every other week for 4 weeks, while the primates still receive HDM mask treatment every other week. Administration of SP-A and placebo is approximately 24 hours after HDM mask exposure.

[0121] At the end of the first study period, 4 weeks, the primates are analyzed for airway hyperresponsiveness. Bronchoscopy is performed for lavage fluid and endobronchial biopsies. After a 4-week washout, the primates are challenged again with HDM mask treatment every other week for study period 2. As above, after 4 weeks of HDM mask treatment, group 1 (n=6) receives placebo and group 2 (n=6) receives SP-A peptide every other week. After completion of study period 2, all primates are analyzed for airway hyperresponsiveness and bronchoscopy is performed for lavage fluid and endobronchial biopsies.

[0122] Statistical Analysis: Primary outcome variables include airway hyperresponsiveness, lavage fluid and tissue eosinophilia, and tissue mucin production. These variables are analyzed using a two-period crossover analysis of variance model. Carry-over effects are tested at the 10% alpha level, whereas period and treatment effects are tested at the 5% alpha level. Data are expressed as mean ± SEM.

[0123] It is expected that the SP-A peptide can alleviate asthma-related phenotypes in primate HDM allergy models. So far, it has been shown that a single administration of the peptide in mice 24 hours after the last HDM challenge (which is the peak of inflammation) can significantly reduce mucin production and eosinophilia in the lavage compartment and lung tissue. It is expected that administration of the peptide every other week over the course of 4 weeks will significantly reduce eosinophilia and mucin production compared to placebo.

[0124] Example 3: Example 3 demonstrates that SP-A peptide protects against AHR in the HDM (house dust mite) model. As shown in FIG. 10, WT C57BL / 6 mice were sensitized and challenged in the HDM model according to standard methods (arrows). 24 hours after each challenge, mice received either scrambled vehicle or a 10-mer SP-A peptide (KEQCVEMYTD (SEQ ID NO: 4)) encompassing an active site containing 223Q (arrow) (physiologic dose of 25 mg / mouse delivered in 40 ml sterile saline) by oropharyngeal instillation. Pulmonary function tests were performed 3-5 days after HDM challenge to determine whether the SP-A peptide could protect against methacholine-induced airway hyperresponsiveness (AHR). Mice receiving SP-A peptide after HDM challenge had attenuated total resistance (Rrs) and central airway resistance (Rn) compared to mice challenged with HDM that received vehicle treatment. Similar protective effects were observed on days 3 and 5 after SP-A peptide treatment.

[0125] Example 4: Example 4 demonstrates that SP-A peptide protects against airway hyperresponsiveness (AHR) in an IL-13 model. WT C57BL / 6 mice were challenged with 3.9ug of IL-13 (arrow) once a day for three consecutive days by oropharyngeal instillation. As shown in Figures 7A-7E, 2 hours after each challenge, mice received either scrambled vehicle or a 10-mer SP-A peptide (KEQCVEMYTD (SEQ ID NO: 4)) encompassing an active site containing 223Q (arrow) (physiologic dose of 25mg / mouse delivered in 40ml of sterile saline) by oropharyngeal instillation. Pulmonary function tests were performed 24 hours after IL-13 challenge to determine whether SP-A peptide could protect against methacholine-induced airway hyperresponsiveness (AHR). Mice that received SP-A peptide after HDM challenge had attenuated total resistance (Rrs) and central airway resistance (Rn) compared with mice challenged with HDM that received vehicle treatment.

[0126] Example 5: Example 5 demonstrates that SP-A peptide protects against SARS-CoV-2 infection. As shown in FIG. 25, full-length SP-A binds to ACE2 in a dose-dependent and CaCl2-dependent manner. A 96-well plate assay was devised in which wells were coated with full-length human SP-A (500 ng / well) extracted from BAL obtained from patients with pulmonary alveolar proteinosis. Lysates from HEK293T cells transfected with an ACE2 expression plasmid were then added at various concentrations. After extensive washing, anti-human ACE2 antibodies and a development substrate were added to detect ACE2. The absorbance at a wavelength of 450 nm was read by a plate reader. Lysates overexpressing ACE2 bound to SP-A in a dose-dependent manner (FIG. 25, solid line). Since SP-A generally binds pathogens in a calcium-dependent manner via the carbohydrate recognition domain (CRD), we next determined whether SP-A binding to ACE2 was calcium-dependent. In the presence of the chelator EDTA, there was no detectable SP-A binding to ACE2 (Figure 25, dashed line), suggesting that binding occurs via the lectin domain of SPA. When lysates not overexpressing ACE2 were used as negative controls, no SP-A binding was detected (Figure 25, dashed line).

[0127] Referring to FIG. 26, SP-A peptide mimetics from the CRD compete with full-length SP-A for ACE2 binding. Both full-length SPA and SP-A 20-mer peptides competed (i.e., inhibited) ACE binding to plate-bound SP-A, whereas the scrambled 20-mer peptide had no effect (FIG. 26). In the general population, certain variants at position 223 of SP-A2 are associated with a pulmonary phenotype. In particular, SP-A containing a lysine at position 223 (223K) preferentially binds to the respiratory pathogen Mycoplasma pneumoniae compared to when a glutamine is present at this position (223Q). Additionally, peptides derived from this region have activity in various models of infection and inflammation as previously described herein. For these studies, full-length SP-A or SPA 20-mer peptides were evaluated to determine whether the peptides competed (i.e., inhibited) ACE2 binding to plate-bound SP-A. Both full-length SP-A and the 20-mer containing 223K competed effectively for binding, whereas the 20-mer scrambled (SCR) peptide had little or no effect, and the 20-mer containing 223Q had a moderate effect (n=3 experiments).

[0128] To evaluate the effect of SP-A on the attachment of SARS-CoV2 S protein, an S1 protein binding assay was used. Cells were incubated with recombinant His-tagged S1 subunit containing the SARS-CoV-2 receptor binding domain followed by Alexa Fluor-conjugated anti-His antibody. S1 protein binding to cells was evaluated by flow cytometry. The ability of this assay to specifically detect ACE2-mediated cellular binding of S1 protein was validated using HEK293T cells untransfected or stably transfected with human ACE2 (ACE2 / HEK293T). S1 binding was detected in less than 1% of untransfected HEK293T cells and ACE2 / HEK293T cells without S1 protein incubated with anti-His-AF (Figure 27A), whereas approximately 30% of ACE2 / HEK293T cells bound S1 protein in the absence of SP-A (Figure 27A, box). Addition of full-length SP-A resulted in a dose-dependent reduction in S1 binding, with the highest concentration of SP-A reducing binding by approximately 70% (FIG. 27B).

[0129] To investigate whether SP-A inhibition of HEK293T ACE-overexpressing cells and SARS-CoV-2 S1 protein attachment to HEK293T ACE-overexpressing cells also reduced S protein-mediated SARS-CoV-2 entry into these cells, we directly measured the entry of replication-deficient SARSCoV-2 S1 protein-pseudotyped lentiviral particles into cells preincubated with SP-A or PBS. Lentiviral particles carried a luciferase reporter gene that was transcribed and translated by the transduced cells and were pseudotyped with SARS-CoV-2 S protein or the G glycoprotein of pantropic VSV (positive transduction control). VSV-G-pseudotyped particles and SARS-CoV-2-pseudotyped particles efficiently transduced HEK293T ACE cells. SP-A had little or no effect on VSV G-LUC transfection, but SP-A dose-dependently reduced the transduction efficiency of SARSCoV-2 S protein-pseudotyped lentiviral particles (Figure 28).

[0130] Referring now to FIG. 29, SP-A attenuates the expression of SARS-CoV-2 N1 gene in a live infection model. Three-dimensional (3D) alveolar organoid cultures were established by resuspending fractionated HTII-280+ distal epithelial cells together with MRC5 human lung fibroblasts in a 50:50 (v / v) ratio of matrigel and Pneumacult ALI medium in transwell inserts in a 24-well format by common methods. Cultures were used for SARS-Co-V2 infection after 15-20 days. Prior to infection, matrigel was dissolved by adding 500 μL of dispase (500 μg / ml) to the apical and basal chambers of the inserts and incubating at 37°C for 1 h. Cultures were harvested, washed with ice-cold PBS, and gently dispersed with a P1000 tip by pipetting up and down three times so that the organoids "burst" and expose the apical surface of the cells. Organoids were treated by resuspending in 100 μL of medium containing SP-A (50 μg / ml) per well. After 3 h pretreatment, SARS-CoV-2 inoculum (1×10^4 TCID50 per well) was added to the cultures in 2 ml conical tubes and incubated for 2 h at 37°C (5% CO2). Every 15 min, the tubes were mixed gently to encourage viral adsorption to the cells. The inoculum was then replaced with fresh Pneumacult ALI medium and the cultures were transferred to the apical chamber of a 100 μL volume insert containing 500 μL of medium in the basal chamber. Cultures were incubated at 37°C (5% CO2) and harvested at 2 dpi. SP-A was maintained in the medium during the post-infection culture period. Organoids without drug treatment in the presence (CoV-2) or absence (mock) of viral infection were included as controls. Viral infection / replication was assessed by performing RT-qPCR for relative expression of the nucleoprotein (N) gene using 2019-nCoV_N1 primers obtained from the Center for Disease Control resources for research labs.

[0131] Figure 30 shows that SP-A 223Q and 223K 20mer peptides reduced transduction of S1 protein pseudotyped lentiviral particles into cells overexpressing ACE2 in vitro. Using the same system detailed in Figure 28 above, we next investigated whether 20mer peptides derived from SP-A behave similarly to full-length SP-A in their ability to inhibit S protein-mediated SARS-CoV-2 entry into these cells. Again, we directly measured the entry of replication-deficient SARSCoV-2 S1 protein-pseudotyped lentiviral particles into cells preincubated with SP-A, SP-A 223Q and 223K peptides or PBS. Lentiviral particles carried a luciferase reporter gene that was transcribed and translated by the transduced cells and were pseudotyped with SARS-CoV-2 S protein or the G glycoprotein of pantropic VSV (positive transduction control). VSV-G- and SARS-CoV-2-pseudotyped particles efficiently transduced HEK293T ACE cells. The SP-A 223Q and 223K peptides dose-dependently reduced the transduction efficiency of SARSCoV-2 S protein-pseudotyped lentiviruses to a greater extent than full-length SP-A at each given concentration (Figure 30).

[0132] As used herein, the term "about" refers to ±10% of the referenced number.

[0133] Although preferred embodiments of the present invention have been shown and described, it will be readily apparent to one skilled in the art that modifications may be made without departing from the scope of the appended claims. Thus, the scope of the present invention should be limited only by the appended claims. In some embodiments, the figures presented in this patent application are drawn to scale, including angles, dimensional proportions, etc. In some embodiments, the figures are merely representative, and the claims are not limited by the dimensions of the figures. In some embodiments, the description of the invention described herein using the phrase "comprising" includes embodiments that may be described as "consisting essentially of" or "consisting of," and thus satisfies the written description requirement for claiming one or more embodiments of the invention using the phrase "consisting essentially of" or "consisting of."

Claims

1. A pharmaceutical composition comprising a purified peptide having the amino acid sequence of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:

24.

2. The pharmaceutical composition described in claim 1, wherein the pharmaceutical composition is in a preparation for aerosolization or subcutaneous injection.

3. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is administered by nasal inhalation, subcutaneously, or orally.

4. A pharmaceutical composition described in any one of claims 1 to 3, wherein the pharmaceutical composition comprises the purified peptide together with one or more additional therapeutic agents.

5. The pharmaceutical composition described in claim 4, wherein the one or more therapeutic agents are administered simultaneously or sequentially with the purified peptide.

6. The pharmaceutical composition of claim 4, wherein the one or more therapeutic agents include a short-acting beta2-adrenergic receptor agonist (SABA), a long-acting beta agonist (LABA), an anticholinergic, a leukotriene receptor antagonist, or a combination thereof.

7. The pharmaceutical composition of claim 6, wherein the anticholinergic drug comprises ipratropium bromide, an inhaled corticosteroid, or a systemic corticosteroid.

8. The pharmaceutical composition described in claim 7, wherein the inhaled corticosteroid or systemic corticosteroid comprises budesonide, fluticasone, mometasone or ciclesonide.

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