Methods for treating inflammatory conditions of lungs

Abstract

To provide methods for treating inflammatory conditions of lungs.SOLUTION: The present invention provides methods for treatment or prevention of asthma, mild intermittent asthma, mild persistent asthma, moderate persistent asthma, severe persistent asthma, eosinophilic asthma, neutrophilic asthma, steroid refractory asthma, status asthmaticus, pneumonia, bronchiectasis, COPD, sarcoidosis and lung cancer, based on reducing airway hyperresponsiveness, increasing pulmonary compliance, reducing lung inflammation, reducing inflammatory cell count in bronchoalveolar fluid and reducing cytokine production, by administration of a mammalian α- and / or β-defensin.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method of treating or preventing pulmonary inflammatory diseases including asthma, mild intermittent asthma, mild persistent asthma, moderate persistent asthma, severe persistent asthma, eosinophilic asthma, neutrophilic asthma, steroid resistant asthma, persistent asthma, pneumonia, bronchiectasis, COPD and lung cancer, based on the administration of mammalian α-defensins and / or β-defensins to normalize cytokine production and prevent cytokine storm, thereby reducing airway hyperresponsiveness, increasing lung compliance, decreasing lung inflammation, decreasing perivascular or peribronchial inflammation, decreasing inflammatory cell counts in bronchoalveolar lavage fluid, and restoring balance to the immune system. [Background technology]

[0002] Asthma is a heterogeneous inflammatory disorder of the airways characterized by chronic inflammation, airway hyperresponsiveness and recurrent symptoms of wheezing, coughing and shortness of breath. Asthma is a major public health problem affecting 300 million people worldwide and has seen a substantial increase in prevalence over the past 30 years, especially in Western countries (Cosmi et al., 2011). However, the pathogenetic mechanisms remain unknown. Combination therapy with steroids and long-acting beta-agonists is the mainstay of asthma treatment. Although these therapies efficiently suppress acute inflammatory symptoms and cytokine release, no prevention or cure for the disease exists so far.

[0003] Mild to moderate allergic asthma is generally characterized by acute or chronic airway inflammation consisting of activated Th2 lymphocyte and eosinophil infiltration associated with IgE production, mucus-secreting cells, hyperplasia and remodeling of the airway wall, and airway hyperresponsiveness (AHR). AHR is characterized by increased responsiveness to nonspecific contractile stimuli such as methacholine and narrowing of the airways (Hansbro et al., 2011). Th2 cells contribute to various pathological features of the disease, especially through their secretion of the cytokines IL-3, IL-4, IL-5, IL-9, and IL-13.

[0004] Severe asthma, neutrophilic asthma or steroid-resistant asthma, has pathological features distinct from mild to moderate allergic asthma and is characterized by a mixed Th2 / Th1 phenotype to which Th17 cells may contribute. Tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-17 and IL-27 are increased, which may lead to an influx of neutrophils (but not eosinophils) or airway infiltration of mixed granulocytes characteristic of this subtype of asthma. Patients with this subtype of asthma are resistant to glucocorticoid treatment, and both bacterial and viral infections have been implicated in the induction and progression of the disease (Hansbro et al., 2004). Patients with asthma and patients with atopic dermatitis are also more prone to developing infections, e.g., pneumonia, compared to non-atopic individuals.

[0005] The concept of treating asthma by targeting single cytokines, e.g., anti-IL-4; anti-IL-5; anti-TNF-α, has met with limited success, and indeed steroid therapy, the current mainstay of therapy, is thought to act by suppressing a broad range of proinflammatory pathways (Hansbro et al., 2011).

[0006] Chronic obstructive pulmonary disease (COPD) COPD is a major public health problem that is predicted to become the fourth leading cause of death worldwide by 2020. Although persistent inhalation of toxic particles and gases is the main risk factor, and smoking is the best example of this type of risk, only 15% of smokers develop COPD (Fletcher and Peto, 1977). Although smokers have a dysfunctional immune system (Bouloukaki et al., 2011), the onset of COPD and increased disease severity gradually exacerbate the inflammatory cell burden (Hogg et al., 2004).

[0007] Microbiome The infant microbiota is initially homogeneous across various body sites, which changes over the following days and weeks to site-specific communities. The lung microbiome of healthy adults is dominated by the phyla Bacteroidetes, Firmicutes and Proteobacteria, with the main microbiota consisting of Pseudomonas, Streptococcus, Prevotella, Fusobacterium, Veillonella, Haemophilus, Neisseria and Porphyromonas (Charlson et al., 2011). Among asthmatics, an increased frequency of Proteobacteria (especially Haemophilus, Moraxella and Neisseria) and Firmicutes (especially Lactobacillus species) and a decreased frequency of Bacteroidetes (especially Prevotella) was observed compared to controls (Hilty et al., 2010). Similarly, epidemiological data indicate that the gut microbiota differs between asthmatic and non-asthmatic infants (Penders et al., 2007).

[0008] Defensin Defensins represent one of the potent innate host defenses that act to maintain a healthy microbiome and prevent the development of potential pathogens (Wehkamp et al., 2002 and Salzman et al., 2007). Defensins are peptides with antimicrobial and anti-inflammatory activity against gram-positive and gram-negative bacteria, fungi and archaea. Defensins, especially hBD2, have shown therapeutic potential in the treatment of inflammatory bowel disease (WO2010 / 007166; WO2013 / 007596).

[0009] In conclusion, there is a need for new methods of treating subjects suffering from pulmonary inflammatory diseases, particularly therapeutic agents that can be administered via the airways for patients who are able to self-administer, e.g., inhale, the drugs, and treatments by other routes of administration for patients who are unable to inhale the drugs efficiently. Summary of the Invention

[0010] The inventors have surprisingly demonstrated that mammalian defensins have the ability to reduce airway hyperresponsiveness (AHR) and increase airway compliance (Cdyn); reduce lung inflammation; reduce neutrophil, eosinophil and macrophage numbers in bronchoalveolar lavage fluid (BALF) and reduce IFN-γ, TNF-α, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10 and IL-13 in lung cells. The inventors have also demonstrated efficacy in reducing tissue inflammatory parameters in asthma models, such as reducing perivascular and peribronchial inflammation.

[0011] The data indicate that administration of mammalian defensins results in normalization or reduction of key features of asthma, COPD and sarcoidosis, and is therefore useful in treating or preventing pulmonary inflammatory diseases including asthma, mild intermittent asthma, mild persistent asthma, moderate persistent asthma, severe persistent asthma, eosinophilic asthma, neutrophilic asthma, steroid-resistant asthma, persistent asthma, pneumonia, bronchiectasis, chronic obstructive pulmonary disease (COPD) and sarcoidosis.

[0012] Surprisingly, oral and intranasal administration of defensin has been demonstrated to be equally effective in at least some parameters tested in a mouse model of house dust mite allergy.This opens the possibility of treating inflammatory lung disease by oral administration to subjects who have difficulty inhaling drugs.As demonstrated in the examples, in a steroid-insensitive mouse model of asthma in which mice are sensitized to ovalbumin (OVA) and infected with C. muridarum, and in a steroid-sensitive mouse model in which mice are immunized with house dust mite (HDM) + Freund's adjuvant and challenged with HDM, administration of human β-defensin 2 (hBD2) can reduce AHR, increase Cdyn, reduce lung tissue inflammation, reduce the number of inflammatory cells in BALF, and reduce inflammatory cytokine production. Without treatment with hBD2, animals develop asthma characterized by a dramatic increase in AHR, a decrease in Cdyn, inflammatory histological changes in lung tissue, an increase in leukocyte numbers, particularly neutrophils, eosinophils and macrophages, and increased concentrations of inflammatory cytokines.

[0013] Given these findings, the inventors also contemplate the broader general use of defensins in the treatment of pulmonary inflammation. Examples of conditions that can cause pulmonary inflammation include lung cancer, sarcoidosis, and various types of medical treatments that affect host defense, including, but not limited to, antimicrobial treatments, chemotherapy, immunotherapy, immunosuppressive therapy, and radiation therapy.

[0014] The inventors have experimentally demonstrated that defensins restore balance to the immune system by completely normalizing cytokine levels, thereby preventing cytokine storms, as opposed to current asthma treatments, for example, with interleukin antibodies that knock out certain cytokines, or with systemic immunosuppression that results in a systemic suppression of the innate immune system, thus making defensins a promising alternative to current treatment methods.

[0015] Furthermore, the inventors have demonstrated that defensins can exert their effects in the lungs not only when administered directly to the lungs, but more importantly, and surprisingly, also when administered simply orally to the digestive tract.

[0016] Oral administration in the treatment of asthma attacks as well as for maintenance therapy would make life easier for asthma sufferers worldwide.

[0017] Thus, in one aspect, a method is provided for treating and / or preventing inflammatory diseases of the respiratory system, including restoring balance to the immune system, normalizing cytokine production and preventing cytokine storm, comprising oral or pulmonary administration of at least one defensin, preferably in a subject suffering from asthma.

[0018] In another aspect, a method is provided for treating and / or preventing a disease selected from the group consisting of mild intermittent asthma, mild persistent asthma, moderate persistent asthma, severe persistent asthma, eosinophilic asthma, neutrophilic asthma, steroid-resistant asthma, persistent asthma, bronchiectasis, bronchitis, COPD, sarcoidosis, pneumonia and emphysema, pulmonary fibrosis, preferably steroid-resistant asthma, comprising administration of at least one defensin.

[0019] Further provided are methods for treating and / or preventing lung cancer in a subject, comprising administering at least one type of defensin to the subject, and methods for reducing pulmonary tissue inflammation, perivascular and bronchovascular inflammation, inflammatory cell numbers in bronchoalveolar lavage fluid and / or inflammatory cytokine production in lung tissue homogenate in a subject in need thereof, comprising administering at least one type of defensin to the subject.

[0020] In yet another aspect, methods are provided for reducing lung tissue inflammation, the number of inflammatory cells in bronchoalveolar lavage fluid, restoring balance to the immune system by normalizing inflammatory cytokine production in lung tissue homogenates, and preventing / treating cytokine storm in a subject in need thereof, comprising administering at least one type of defensin to the subject.

[0021] In another aspect, a method is provided for increasing lung compliance, reducing airway hyperresponsiveness and / or increasing peak flow in a subject in need thereof, comprising administering at least one defensin to the subject.

[0022] A method for increasing forced expiratory volume in 1 second (FEV1) and / or maximum expiratory flow rate (PEFR) or decreasing PEFR variability in a subject in need thereof, comprising administering to the subject at least one type of defensin.

[0023] By administering at least one defensin to a subject, gene richness, number of phyla can be increased, butyrate and / or tryptophan production can be increased, and acetate production from the lung microbiota can be decreased in a subject in need thereof.

[0024] Further provided is a method for maintaining and / or stabilizing normal lung microflora, increasing the occurrence and abundance of important commensal bacteria and short chain fatty acid and / or butyrate and / or tryptophan producing strains in a subject in need thereof, comprising administering at least one defensin to the subject.

[0025] In other aspects, the disclosure relates to defensin polypeptides for use in treatment methods according to any of the methods described herein and the use of defensin polypeptides for the preparation of a medicament for the treatment of a disorder defined herein. [Brief description of the drawings]

[0026] [Figure 1] Overview of the experimental set-up to investigate the effects of mammalian α-defensins and / or β-defensins in a murine steroid-insensitive model of asthma (Essilfie et al., 2015), in which mice are sensitized with ovalbumin and infected with C. muridarum. [Diagram 2] Outline of the experimental set-up to investigate the effect of mammalian β-defensins in a murine steroid-sensitive model of asthma, where mice are immunized with house dust mite (HDM) plus Freund's adjuvant and challenged with HDM. [Figure 3a] Clustal W (2.1) multiple sequence alignment of human β-defensins 1-4. In the Clustal W alignment, * indicates a position with a single perfectly conserved residue, : indicates that one of the following "strong" groups is perfectly conserved: S,T,A; N,E,Q,K; N,H,Q,K; N,D,E,Q; Q,H,R,K; M,I,L,V; M,I,L,F; H,Y; F,Y,W, . indicates that one of the following "weaker" groups is perfectly conserved: C,S,A; A,T,V; S,A,G; S,T,N,K; S,T,P,A; S,G,N,D; S,N,D,E,Q,K; N,D,E,Q,H,K; N,E,Q,H,R,K; V,L,I,M; H,F,Y. [Figure 3b] Clustal alignment of HD5 and HD6. [Figure 4] Airway hyperresponsiveness following intranasal administration of hBD2 in an ovalbumin / C. muridarum mouse steroid-insensitive asthma model. The y-axis indicates Rn - airway-specific resistance units (tidal volume of 8 mL / kg at a respiratory rate of 450 breaths / min). **** indicates statistically significant differences with a p-value of p<0.05 using the Mann-Whitney test. [Figure 5a-5b]Airway hyperresponsiveness following intranasal (Fig. 5a) and oral (Fig. 5b) administration of hBD2 in a house dust mite mouse steroid-sensitive asthma model. Saline is a non-challenged control. HDM / vehicle is a vehicle-treated house dust mite-challenged control. "hBD2 IN 1.2mpk" is hBD2 administered intranasally at 1.2mg / kg. 5mpk is 5mg / kg. Symbols in Fig. 5a: ●-vehicle IN; ■-hBD2 IN 1.2mpk; ▲-saline; ◆-hBD2 IN 5mpk. Symbols in Fig. 5b: ●-HDM / vehicle IN; ■-saline; ▲-HDM / dexamethasone; ◆-HDM / hBD2 1.2mg / kg po [Figure 6a-6b] Lung compliance after intranasal (Fig. 6a) and oral (Fig. 6b) administration of hBD2 in a house dust mite mouse steroid-sensitive asthma model. Symbols in Fig. 6a: ●-vehicle IN; ■-hBD2 IN 1.2 mpk; ▲-saline; ◆-hBD2 IN 5 mpk. Symbols in Fig. 6b: ●-HDM / vehicle IN; ■-saline; ▲-HDM / dexamethasone; ◆-HDM / hBD2 1.2 mg / kg po [Figure 7] Total and differential cell counts in BALF following intranasal administration of hBD2 in an ovalbumin / C. muridarum mouse steroid-insensitive asthma model. Figure symbols: A: SPG / Sal B: SPG / Ova C: SPG / Ova / Dex D: Cmu / Sal E: Cmu / Ova F: Cmu / Ova / Dex G: Cmu / Ova / hBD2 H: Cmu / Ova / hBD2 / Dex [Table 1] [Figure 8a-8b]Total and differential cell counts in BALF after intranasal and oral administration of hBD2, respectively, in the house dust mite mouse steroid-sensitive asthma model. Figure 8a shows animals orally treated with hBD2. Figure 8b shows results from animals intranasally treated with hBD2. Results are shown as mean ± SEM. *: p<0.05 (significant difference vs. vehicle group, unpaired test). Figure symbols: saline IN, unchallenged untreated control. HDM / vehicle, untreated but challenged animals with HDM. HDM, house dust mite challenged animals. PO, oral administration, IN, intranasal administration. Columns marked with * are statistically significantly different from vehicle treated controls. [Figure 9-18] Figure 9 shows cytokine concentrations of IFN-γ (Figure 9), TNF-α (Figure 10), IL-1β (Figure 11), IL-4 (Figure 12), IL-5 (Figure 13), IL-6 (Figure 14), IL-8 (Figure 15), IL-9 (Figure 16), IL-10 (Figure 17) and IL-13 (Figure 18) after intranasal and oral administration, respectively, of hBD2 in a house dust mite mouse steroid-sensitive asthma model. Each figure has data from the intranasal group on the left and the oral group on the right. Results are shown as mean ± SEM in pg / mL. *: p<0.05 (significant difference vs. vehicle group, Mann-Whitney test). [Figure 19] Lung histology images from H&E / PAS specimens after intranasal and oral administration of hBD2 in a house dust mite mouse steroid-sensitive asthma model. Top left panel: untreated and unchallenged control; top right panel: untreated and HDM-challenged control; bottom left panel: HDM-challenged and orally treated with hBD2; bottom right panel: HDM-challenged and intranasally treated with hBD2. 50x magnification. [Figure 20] Severity of lung inflammation after intranasal and oral administration of hBD2 in a house dust mite mouse steroid-sensitive asthma model. *: p<0.05 (significant difference vs. vehicle group, Mann-Whitney test); #: p<0.05 (significant difference vs. vehicle group, Wilcoxon signed-rank test). [Figure 21]Perivascular and peribronchial inflammation after intranasal and oral administration of hBD2, respectively, in a house dust mite mouse steroid-sensitive asthma model. ◆: eosinophils; □: monocytes. *: p<0.05 (significant difference vs. vehicle group for perivascular infiltration of eosinophils, Mann-Whitney test); #: p<0.05 (significant difference vs. vehicle group for perivascular / peribronchial infiltration of eosinophils and monocytes, Wilcoxon signed-rank test). [Figure 22] Pharmacokinetic data following oral administration of 4 mg / kg hBD2 to female NMRI mice. The Y-axis represents hBD2 in μg / g tissue. Results are presented as group mean ± SEM. [Diagram 23] Pharmacokinetic data of hBD2 after subcutaneous and intravenous administration of 1 mg / kg, respectively. The Y-axis shows hBD2 in μg / mL. Different curves represent different experiments and assays (HPLC and ELISA). [Figure 24] Pharmacokinetic data of "hBD2-albumin N-terminal fusion" after subcutaneous and intravenous administration of 16.5 mg / kg, respectively. The Y-axis shows the concentration of the fusion protein in μg / mL. Results are the mean / sampling time ± SD of 4 mice. [Diagram 25] Pharmacokinetic data of "hBD2-Albumin C-Terminal Fusion" after subcutaneous and intravenous administration of 16.5 mg / kg, respectively. The Y-axis shows the concentration of the fusion protein in μg / mL. Results are the mean / sampling time ±SD of 4 mice. [Figure 26] Course of Disease Activity Index scores during an experiment with intravenous administration of "hBD2-albumin C-terminal fusion". Results are shown as mean values ​​± standard error of the mean of 9-10 animals per group. Significant differences from values ​​of the control (vehicle) group on the given dates are indicated as *: P<0.05; **: P<0.01; ***: P<0.001 (Kruskal-Wallis test for non-parametric data). Albucult® is a recombinant albumin available from NovoZymes A / S. If no compound is indicated on the graph, the compound is hBD2-albumin C-terminal fusion. [Figure 27]Histology scores of the proximal colon for "hBD2-albumin C-terminal fusion". Histology scores of proximal colon samples. Results are shown as mean ± standard error of the mean for n=9-10 animals per group. Significant differences from values ​​of the control (vehicle) group on the given dates are shown as ***: P<0,001; *: P<0,05 (Kruskal-Wallis test + Dunn's post-hoc test for non-parametric data). Albucult® is a recombinant albumin available from NovoZymes A / S. If no compound is listed on the graph, the compound is an hBD2-albumin C-terminal fusion. [Figure 28] FIG. 13: Outline of the experimental setup to investigate the effect of mammalian defensins for the prevention of asthma in a mouse steroid-sensitive model, where mice are immunized with house dust mite (HDM) plus Freund's adjuvant and challenged with HDM. [Figure 29] Airway hyperresponsiveness following prophylactic intranasal and oral administration of hBD2 in a house dust mite mouse steroid-sensitive asthma model. [Diagram 30] Lung compliance following prophylactic intranasal and oral administration of hBD2, respectively, in a house dust mite mouse steroid-sensitive asthma model. [Diagram 31] Neutrophil counts in BALF after prophylactic oral administration of hBD2 in a house dust mite mouse steroid-sensitive asthma model. *: p<0.05 (significant difference from the vehicle group, Mann-Whitney test). [Figure 32-37] Cytokine concentrations (pg / mL) of TNF-α (Figure 32), IL-4 (Figure 33), IL-5 (Figure 34), IL-6 (Figure 35), IL-9 (Figure 36) and IL-13 (Figure 37) in lung homogenates after prophylactic oral administration of hBD2 in a house dust mite mouse steroid-sensitive asthma model. Results are shown as mean ± SEM. *: p<0.05 (significant difference vs. vehicle group, Mann-Whitney test). [Figure 38-39] Overview of the experimental setup to investigate the effect of mammalian defensins (HD5, hBD2 and HD5+hBD2) on the composition of the microbiota in a high-fat diet mouse model. [Diagram 40] Genus analysis of microbial abundance after oral prophylactic treatment with HD5, hBD2 and HD5+hBD2 in a high-fat diet mouse model. [Diagram 41] Abundance of allobaculum in the small intestine after prophylactic oral administration of HD5 and hBD2 in a high-fat diet mouse model. [Diagram 42] FIG. 1 shows the abundance of Lactobacillaceae in the large intestine after prophylactic treatment with oral administration of hBD2 in a high fat mouse model. [Diagram 43] FIG. 1 shows the relative abundance of Barnesiella in the large intestine after 4 weeks (left panel) and 10 weeks (right panel) of prophylactic treatment with oral administration of hBD2 in a high fat diet mouse model. [Diagram 44] FIG. 1 shows the relative abundance of Alloprevotella in the large intestine after therapeutic intervention with oral administration of HD5 and hBD2 in a high-fat diet mouse model. [Diagram 45] FIG. 1 shows the relative abundance of Bifidobacteriaceae in the small intestine (left panel) and large intestine (right panel) after therapeutic intervention with oral administration of HD5 or hBD2 in a high-fat diet mouse model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] definition Defensin: As used herein, the term "defensin" refers to a polypeptide that belongs to the defensin family of antimicrobial peptides. Defensins represent one of the potent natural host defenses that maintain a healthy microbiome and prevent the development of potential pathogens (Wehkamp et al., 2002 and Salzman et al., 2007). Defensins are peptides that have antimicrobial and anti-inflammatory activity against gram-positive bacteria, gram-negative bacteria, fungi and archaea.

[0028] Human defensins are small cationic peptides classified into α-defensins and β-defensins based on the topology of their three intramolecular cysteine ​​disulfide bonds. α-defensins can be further subdivided into those expressed in neutrophil granules (HNP1-4) and those expressed by Paneth cells of the small intestinal crypts (HD5 and HD6 or DEFA5 and DEFA6). β-defensins (DEFBn) are mainly produced by epithelial cells in various tissues and organs, including the skin, eye, middle ear, mouth, trachea, lung, digestive tract, urogenital system, kidney, vagina, liver, pancreas and mammary gland. Examples of defensins include human intestinal alpha-defensin 5 (HD5; SEQ ID NO:5), human intestinal alpha-defensin 6 (HD6; SEQ ID NO:6), human neutrophil peptide 1 (HNP-1), human neutrophil peptide 2 (HNP-2), human neutrophil peptide 3 (HNP-3); also human beta-defensin 1 (hBD1; SEQ ID NO:1); human beta-defensin 2 (hBD2; SEQ ID NO:2); human beta-defensin 3 (hBD3; SEQ ID NO:3); human beta-defensin 4 (hBD4; SEQ ID NO:4); and truncated human beta-defensin 2 (SEQ ID NO:7), all of which belong to the alpha-defensin class.

[0029] WO2013 / 026794 demonstrates that truncated and untruncated hBD2 have similar biological activity.

[0030] Defensins are expressed as precursors and processed by cleavage of the signal peptide and possibly also by cleavage of the propeptide before secretion into the extracellular space. The best-characterized members of the human β-defensin family are hBD1-4. Some human defensins (e.g., hBD1) are constitutively produced, whereas others (e.g., hBD2, hBD3 and hBD4) are induced by inflammatory cytokines or exogenous microbial products. The above specific sequences represent predicted mature bioactive defensins. It will be understood by those skilled in the art that processing may vary between cells and that the resulting secreted mature peptides may differ from the predicted sequence by one or two C- or N-terminal amino acids yet still retain biological activity.

[0031] Identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "identity". The degree of identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) as implemented in the Needle program of the EMBOSS package (Rice et al., 2000, http: / / emboss.org), preferably version 3.0.0 or later. Optional parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of the Needle-labeled "longest identity" (obtained with the -nobreif option) is used as the percent identity, calculated as (identical residues x 100) / (alignment length - total number of gaps in the alignment).

[0032] Normal microbiota: As used herein, the term "normal microbiota" refers to a microbiota that is not in symbiotic balance.Normal microbiota is characterized by having a large abundance of genes.Normal gut microbiota is characterized by containing bacteria belonging to the genera Bacteroides, Faecalis Bacterium, Roseburia, Blautia, Ruminococcus, Coprococcus, Bifidobacterium, Methanobrevibacter, Lactobacillus, Coprococcus, Clostridium, Akkermansia, and Eubacterium.

[0033] The normal lung microbiota is characterized by the inclusion of bacteria belonging to the genera Bacteroides, Firmicutes and Proteobacteria, with the predominant microbiota being composed of Pseudomonas, Streptococcus, Prevotella, Fusobacterium, Veillonella, Haemophilus, Neisseria and Porphyromonas.

[0034] Treatment: As used herein, the terms "treatment" and "treating" refer to the management and care of a patient to combat a condition, disease or disorder. The term is intended to include the full range of treatments for a given condition suffered by a patient, including the administration of an active compound to reduce or alleviate a condition or complication; to slow the progression of a condition, disease or disorder; to cure or eliminate a condition, disease or disorder; and / or to prevent a condition, disease or disorder, and wherein "preventing" or "prevention" should be understood to refer to the management and care of a patient to prevent or reduce the risk of developing a condition or complication by preventing or reducing the active compound. The patient to be treated is preferably a mammal, particularly a human. The patient to be treated can be of various ages.

[0035] Patient: A patient is a subject who has been diagnosed with a particular disorder, such as a pulmonary inflammatory disorder, or who has particular symptoms indicative of a disorder, such as a pulmonary inflammatory disorder.

[0036] Mammalian α-Defensins and Mammalian β-Defensins The present disclosure relates to the use of mammalian α-defensins and / or β-defensins, such as human β-defensins, more preferably human defensins, in particular in the treatment of asthma, mild intermittent asthma, mild persistent asthma, moderate persistent asthma, severe persistent asthma, eosinophilic asthma, neutrophilic asthma, steroid-resistant asthma, persistent asthma, bronchiectasis and COPD.

[0037] In one embodiment, the mammalian α-defensin and / or β-defensin has a degree of identity of at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95% to any of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7. In other embodiments, the defensin differs from one of SEQ ID NOs: 1-7 by less than 10 amino acids, such as less than 8, such as less than 5, such as less than 4, such as less than 3, such as less than 2 amino acids.

[0038] In a preferred embodiment, the human α-defensin consists of α-defensin 5 (SEQ ID NO:5) and / or α-defensin 6 (SEQ ID NO:6). In a preferred embodiment, the mammalian β-defensin consists of human β-defensin 1 (SEQ ID NO:1), human β-defensin 2 (SEQ ID NO:2), human β-defensin 3 (SEQ ID NO:3), human β-defensin 4 (SEQ ID NO:4) or truncated human β-defensin 2 (SEQ ID NO:7).

[0039] In a preferred embodiment, the human α-defensin has a degree of identity of at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95% with the amino acid sequence of SEQ ID NO: 5. In a preferred embodiment, the human mammalian α-defensin consists of α-defensin 5 (SEQ ID NO: 5). In a preferred embodiment, the human β-defensin has a degree of identity of at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95% with the amino acid sequence of SEQ ID NO: 2. In a preferred embodiment, the human β-defensin consists of human β-defensin 2 (SEQ ID NO: 2) or truncated human β-defensin 2 (SEQ ID NO: 7).

[0040] In yet another embodiment, the mammalian α-defensin consists of human α-defensin and / or mouse α-defensin, and functionally equivalent variants thereof. Preferably, the mammalian α-defensin consists of human α-defensin 5, human α-defensin 6, and functionally equivalent variants thereof. More preferably, the mammalian α-defensin consists of human α-defensin 5, and functionally equivalent variants or orthologues thereof.

[0041] In yet further embodiments, the mammalian β-defensin consists of human β-defensin and / or mouse β-defensin, and functionally equivalent variants thereof. Preferably, the mammalian β-defensin consists of human β-defensin 1, human β-defensin 2, human β-defensin 3, human β-defensin 4, and functionally equivalent variants thereof. More preferably, the mammalian β-defensin consists of human β-defensin 2, and functionally equivalent variants or orthologues thereof.

[0042] In one embodiment, the methods comprise administering to a subject in need of such treatment an effective amount of at least one mammalian α-defensin.

[0043] A "functionally equivalent variant" of a mammalian (e.g., human) α-defensin or β-defensin is a modified mammalian (e.g., human) α-defensin or β-defensin that exhibits approximately the same effect on the microflora in the lung or intestine as the original mammalian (e.g., human) α-defensin and / or β-defensin. A functionally equivalent variant of a mammalian (e.g., human) defensin may contain 1 to 5 amino acid modifications, preferably 1 to 4 amino acid modifications, more preferably 1 to 3 amino acid modifications, most preferably 1 to 2 amino acid modifications, in particular 1 amino acid modification, compared to a mammalian (e.g., human) defensin amino acid sequence (e.g., any of SEQ ID NOs: 1 to 7). Preferably, for mammalian β-defensins, compared to human β-defensin 2 or truncated human β-defensin 2 having SEQ ID NO: 2 (SEQ ID NO: 7), and for α-defensins, compared to HD5 (SEQ ID NO: 5).

[0044] As used herein, the term "modification" refers to any chemical modification of a mammalian (e.g., human) defensin. The one or more modifications may be one or more amino acid substitutions, deletions and / or insertions, as well as one or more amino acid side chain exchanges; or the use of non-natural amino acids with similar properties in the amino acid sequence. In particular, the one or more modifications may be amidation, such as C-terminal amidation. Preferably, the amino acid modifications are minor, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the polypeptide; single deletions; small amino- or carboxyl-terminal extensions; or slight extensions that facilitate purification by altering the net charge or by another function, such as polyhistidine tags, antigenic epitopes or binding domains. In one embodiment, a small extension, such as a polyhistidine tag, antigenic epitope or binding domain, is attached to a mammalian (e.g., human) α-defensin or β-defensin by a small linker peptide of up to about 20-25 residues, which linker may include a restriction enzyme cleavage site.

[0045] The Clustal W alignment in Figure 3 can be used to predict which amino acid residues can be substituted without substantially affecting the biological activity of the protein. Sequences were aligned using Clustal W2.1 (http: / / www.geno,me.jp / tools / clustalw / ) and the following settings: Gap Open Penalty: 10, Gap Extension Penalty: 0,05, Weight Transition: NO, Hydrophilic residues in protein: GPSNDQE, Hydrophilic gaps: Yes, Weight matrix: BLOSUM (for PROTEIN). Substitutions within the following groups (Clustal W, "strong" conserved groups) are considered conservative substitutions: S, T, A; N, E, Q, K; N, H, Q, K; N, D, E, Q; Q, H, R, K; M, I, L, V; M, I, L, F; H, Y; F, Y, W. Substitutions within the following groups (Clustal W, "weak" conserved groups) are considered semi-conservative substitutions: C,S,A; A,T,V; S,A,G; S,T,N,K; S,T,P,A; S,G,N,D; S,N,D,E,Q,K; N,D,E,Q,H,K; N,E,Q,H,R,K; V,L,I,M; H,F,Y.

[0046] Examples of conservative substitutions are substitutions made within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally change a specific activity are known in the art and are described, for example, by Neurath and Hill (1979). The most commonly occurring exchanges are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0047] In addition to the 20 standard amino acids, non-standard amino acids (e.g., 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) may be substituted for amino acid residues in wild-type polypeptides. A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and unnatural amino acids may be substituted for amino acid residues. "Unnatural amino acids" are modified after protein synthesis and / or have a different chemical structure in their side chains than those of the standard amino acids. Unnatural amino acids can be chemically synthesized and are preferably commercially available, and include pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3-methylproline and 4-methylproline, and 3,3-dimethylproline.

[0048] Essential amino acids in mammalian α-defensins and / or β-defensins can be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resulting mutant molecules are tested for biological activity (i.e., activity against airway hyperresponsiveness or inhibition of cytokine, e.g., TNF-α activity) to identify amino acid residues that are important for the activity of the molecule. See also Hilton et al., 1996, J.Biol.Chem.271:4699-4708. The identity of essential amino acids can also be inferred from analysis of the identity of mammalian α-defensins and / or β-defensins with related polypeptides (see Clustal W alignment in Figure 3).

[0049] Single or multiple amino acid substitutions can be made and tested using known methods of mutagenesis, recombination and / or shuffling, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; WO95 / 17413; or WO95 / 22625, followed by associated screening methods. Other methods that may be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochem. 30:10832-10837; US 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127). When the outcome of a given substitution cannot be predicted with certainty, derivatives may be readily analyzed according to the methods previously described herein to determine the presence or absence of biological activity.

[0050] Long-acting defensins The half-life of an α-defensin or β-defensin may be extended by fusing or conjugating the α-defensin or β-defensin with another moiety or molecule, i.e., constructing a long-acting, biologically active α-defensin or β-defensin linked to a pharmacologically acceptable molecule, which provides an in vivo plasma half-life of the α-defensin or β-defensin that is significantly increased compared to the in vivo plasma half-life of an unconjugated α-defensin or β-defensin administered in the same manner as the conjugated α-defensin or β-defensin.

[0051] Long-acting bioactive α-defensins or β-defensins include mammalian α-defensins or analogs thereof or mammalian β-defensins or analogs thereof linked to a pharmacologically acceptable molecule selected from neonatal Fc receptor (FcRn), transferrin, albumin (HAS), XTEN®; or PEG, homoamino acid polymers (HAP), proline-alanine-serine polymers (PAS), or elastin-like peptides (ELP), hyaluronic acid, negatively charged highly sialylated peptides such as the carboxy terminal peptide (CTP) of the chorionic gonadotropin (CG) β chain, human IgG, and molecules having a bond to CH3(CH2)nCO- (where n is 8 to 22).

[0052] The α- or β-defensin analogs may be of non-mammalian origin and may be selected from small organic molecules, peptides, polypeptides and proteins.

[0053] The α-defensin or β-defensin agonist may be linked to a pharmacologically acceptable molecule by various methods described in the prior art, including but not limited to chemical bonding with a bifunctional linker, genetic techniques by linking the N-terminus or C-terminus of a defensin, such as an α-defensin or β-defensin, to a pharmacologically acceptable molecule, such as an albumin or an albumin analogue. In particular, the N-terminus of an albumin, such as human albumin, or an albumin analogue may be linked to the C-terminus of an α-defensin or β-defensin, or the N-terminus of an α-defensin or β-defensin; or the C-terminus of an albumin, such as human albumin, may be linked to the C-terminus of an α-defensin or β-defensin, or the N-terminus of an α-defensin or β-defensin. A linker sequence may be inserted between the albumin and the α-defensin chain or the β-defensin chain.

[0054] The α- or β-defensin agonist may be linked to the pharma- ceutical acceptable molecule via a stable linker or a less stable linker.Several linkers are known in the art, including bifunctional PEG molecules (see, for example, Paige et.al., Pharmaceutical Research, vol.12, no.12, 1995), hydrolyzable linkers (see, for example, Shechter et al., Bioconjugate Chem. 2005, 16:913-920, and International Journal of Peptide Research and Therapeutics, Vol.13, Nos.1-2, June 2007, and WO2009095479), PDPH and EMCH (see, for example, WO2010092135). In certain cases where chemical conjugation (linking of two or more molecules) of an α-defensin or β-defensin agonist to a pharmacologically acceptable molecule strongly reduces functional α-defensin or β-defensin activity, it may be preferable to use a more labile linker capable of releasing the functional α- or β-defensin agonist.

[0055] Half-life extension may also be achieved by acylation of the peptide backbone with a spacer, such as a γ-L-glutamyl spacer to lysine, and a C-18 fatty diacid chain. The fatty diacid side chain and spacer mediate tight but reversible binding to albumin, slowing release from the injection site and reducing renal clearance.

[0056] Methods and Uses Human β-defensin 2 has been found to be able to restore balance to the immune system and prevent cytokine storm by reducing airway hyperresponsiveness; increasing lung compliance; reducing pulmonary inflammation; reducing neutrophil, eosinophil and macrophage numbers in the BALF, and normalizing IFN-γ, TNF-α, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10 and IL-13 concentrations, thereby demonstrating potent activity as an agent for the prevention or treatment of pulmonary inflammatory diseases, such as asthma and COPD.

[0057] Surprisingly, it has been found that parenteral and oral administration of defensins is effective in treating pulmonary inflammatory diseases.This is unexpected because hBD2 is known not to be absorbed from the intestine, as demonstrated in Example 3 of the present disclosure.The advantage of this finding is that patients with reduced lung capacity can orally take defensin drugs.It is also expected that critically ill patients, such as mechanically ventilated patients and patients with persistent asthma, can be treated by parenteral administration of at least one defensin.

[0058] Therefore, in one aspect, the present disclosure relates to the method of treating inflammatory lung disease by parenteral or oral administration of at least one defensin.Preferably, the administration is oral.Oral and parenteral administration are beneficial for patients with impaired breathing or patients receiving artificial respiration therapy.

[0059] In another aspect, a method is provided for treating mild intermittent asthma, mild persistent asthma, moderate persistent asthma, severe persistent asthma, eosinophilic asthma, neutrophilic asthma, steroid-resistant asthma, persistent asthma, pneumonia, bronchiectasis, COPD, sarcoidosis, pulmonary fibrosis, or lung cancer by administering an effective amount of a mammalian defensin to a subject in need of such treatment. These diseases can be treated by intrapulmonary, oral, or parenteral administration. Preferably, administration is intrapulmonary or oral.

[0060] The provided methods can treat or prevent pulmonary inflammation by reducing leukocyte migration, such as neutrophils, eosinophils and macrophages in BALF. Administration of hBD2 has been shown to be effective in reducing neutrophils and macrophages in BALF in particular.

[0061] The method can restore balance to the immune system by normalizing cytokine production, for example, IFN-γ, TNF-α, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10 and IL-13 in lung tissue homogenate, thus preventing or treating cytokine storm in subjects suffering from one of the above-mentioned diseases, as described herein.Preferably, the treatment method results in a decrease in cytokine production, where the cytokine is IFN-γ, TNF-α, IL-4, IL-5, IL-6, IL-9, IL-10 or IL-13.In particular, the method can reduce the amount of IFN-γ, TNF-α, IL-4, IL-5, IL-9 and IL-13.The amount of cytokine can be determined in lung biopsy or BALF.

[0062] Additionally, the method, as described herein, may reduce airway hyperresponsiveness and increase lung compliance in a subject suffering from one of the above-mentioned diseases.

[0063] The provided methods can prevent or treat lung inflammation in a subject suffering from one of the above-mentioned diseases as described herein by altering the bacterial phenotype and the structure and composition of the pulmonary microbiota or the pulmonary metabolome through changes in transcription levels.

[0064] Without wishing to be bound by theory, the effects observed using oral administration may be due to changes in the gut microbiota and gut metabolome, which may affect the lung via the so-called gut-lung axis.

[0065] Chronic lung disorders such as asthma, COPD and cystic fibrosis all show components of intestinal disease symptoms, indicating that there is an important crosstalk between these two mucosal sites in the human body, and that various respiratory diseases are associated with dysbiosis of the gut microbiota as well as the airway microbiota (Marsland et al., 2015). Caesarean section birth is associated with a predisposition to asthma during childhood, while decreasing the diversity and altering the composition of the gut microbiota in early life (Jakobsson et al., 2014). Early life exposure to environmental microbes has been found to be protective against asthma (Ege et al., 2011), while early life and prenatal antibiotic exposure increases the risk of allergic asthma (Marra et al., 2009). The inverse correlation between childhood infections and the development of asthma and allergies has been recognized for many years, giving rise to the “hygiene hypothesis” that reduced exposure to infection during early life leads to impaired immune tolerance and increased autoimmune conditions (Wills-Karp et al, 2001).

[0066] A complementary hypothesis is that variations in the composition of the gastrointestinal microbiota resulting from antibiotic use and unhealthy diets (low fiber, high carbohydrate) in Westernized regions interfere with gastrointestinal microbiome-mediated mechanisms of mucosal immune tolerance.

[0067] Commensal microbes modulate innate and adaptive immune responses, primarily by producing small molecules that mediate host-microbe interactions and influence activation thresholds to pathogenic stimuli (Donia and Fishback, 2015). While the epithelial barrier ensures that microbes are primarily confined to the gastrointestinal tract, microbial metabolites can penetrate the epithelial barrier, allowing them to enter and accumulate in the host circulatory system where they are detected by immune cells (Dorrestein et al., 2014). Trompette et al. (2013) demonstrated in mice that fermentable fiber in the diet alters the composition of the gastrointestinal as well as pulmonary microbiota, particularly the ratio of Firmicutes to Bacteroidetes, the latter of which causes an increase in local and systemic short-chain fatty acid levels, which in turn affect the generation and function of dendritic cells, thereby shaping the lung immune environment and influencing the severity of allergic inflammation. Schirmer et al. further demonstrated in the Human Genome Project that interindividual variation in cytokine responses is associated with specific microbes and microbial functions. The majority of associations detected were both cytokine- and stimulus-specific, suggesting that the immune system recognizes and interacts with microbes and their products with high specificity, and that these microbial factors are associated with specific immune phenotypes.TNF-α and IFN-γ production capacity appeared to be more strongly influenced by the microbiome, while other cytokines such as IL-1β, IL-6, and Th17-derived IL-17 and IL-22 showed fewer but more specific associations with the gut microbiota.

[0068] A further aspect provides a method for preventing or treating mild intermittent asthma, mild persistent asthma, moderate persistent asthma, severe persistent asthma, eosinophilic asthma, neutrophilic asthma, steroid-resistant asthma, persistent asthma, pneumonia, bronchiectasis, COPD or lung cancer by orally administering an effective amount of α-defensin and / or β-defensin and / or administering an effective amount of β-defensin to a subject in need of such treatment. In a preferred embodiment, the asthma is steroid-resistant asthma. In one embodiment, administration is oral, buccal, sublingual, rectal, intravaginal, intratracheal, intrapulmonary, intranasal, intracranial, subcutaneous, intravenous, dermal or transdermal. Preferably, administration is oral or intrapulmonary. Oral administration may be beneficial for patients with impaired breathing or those receiving mechanical ventilation.

[0069] Further provided are methods for the treatment of asthma, mild intermittent asthma, mild persistent asthma, moderate persistent asthma, severe persistent asthma, eosinophilic asthma, neutrophilic asthma, steroid-resistant asthma, persistent asthma, bronchiectasis and COPD by administering an effective amount of an α-defensin and / or a β-defensin parenterally, for example subcutaneously or intravenously, to a subject in need of such treatment.

[0070] The methods of treatment described herein can be administered by administering a composition comprising at least one mammalian α-defensin and / or β-defensin in combination with any of glucocorticoids, β-agonists, leukotriene receptor antagonists, theophylline, antibiotics, rifaximin, chemotherapy or immunotherapy, immunosuppressants, prebiotics, probiotics, tryptophan, short chain fatty acids, HNP-1, HNP-2, HNP-3, HNP-4, cathelicidin, lactoferrin, lactoferricin, lysozyme, fecal transplants, or combinations thereof. The defensins described herein can be used to alleviate one or more symptoms of antibiotics, chemotherapy, radiation therapy, immunotherapy, or immunosuppressive therapy. The defensins can be administered individually or together with one or more of these therapies. The defensins can be administered together with other agents that can be used to treat asthma.

[0071] Importantly, the methods of the disclosure can be used for the treatment, prevention or normalization of a dysbiotic imbalance in the lungs of subjects who have undergone and / or are undergoing antibiotic treatment or chemotherapy or immunotherapy or immunosuppressant therapy or radiation therapy or another treatment that has a negative impact on the lung microbiota or gut microbiota.

[0072] Normalizing the pulmonary microbiota may include activating the population of lung bacteria belonging to the genera Bacteroides, Firmicutes and Proteobacteria, with the main microbiota consisting of Pseudomonas, Streptococcus, Prevotella, Fusobacterium, Veillonella, Haemophilus, Neisseria and Porphyromonas.

[0073] Normalizing the lung microbiota may involve shifting the metabolome to one that produces relatively more tryptophan and / or butyrate and relatively less acetate.

[0074] The subject to be treated may experience asthma symptoms, such as daily symptoms, persistent symptoms, etc., <2 times per week.

[0075] The subjects to be treated may present with asthma attacks of different intensities, such as attacks that affect activity, attacks that limit physical activity, and / or may present with >2 symptoms per week lasting several days, >2 nocturnal asthma symptoms per month, such as frequent nocturnal symptoms.

[0076] In one embodiment, the subject being treated has a forced expiratory volume in 1 second (FEV1) of <80% of predicted value, for example, an FEV1 of 60-80%, an FEV1 of <60%.

[0077] Additionally, the subject may have a peak expiratory flow rate (PEFR) with a variability of >20%, for example, a PEFR variability of 20-30%, a PEFR variability of >30%, a PEFR variability of >60%, etc.

[0078] A subject in need of treatment provided by the methods of the present disclosure may exhibit one or more of the following symptoms prior to treatment: Mild intermittent: <2 symptoms per week Asymptomatic interictal periods and normal peak expiratory flow (PEFR) Brief bouts of varying intensity <2 nocturnal symptoms per month Forced expiratory flow in 1 second (FEV1) or PEFR >80% predicted <20% PEFR variation Mild persistent: Symptoms >2 times per week but <1 time per day Exacerbations that may affect activity >2 nighttime episodes per month FEV1 >80% of predicted PEFR fluctuations between 20% and 30% Moderate persistent: daily symptoms Daily use of short-acting beta-agonists Seizures that affect activity Exacerbations that occur >2 times per week and may last for several days >1 nocturnal symptom per week FEV1 >60% but <80% of predicted value >30% PEFR change Severe persistent: Persistent symptoms Limited physical activity Frequent exacerbations Frequent nocturnal symptoms FEV1 <60% of predicted >60% PEFR change

[0079] In one embodiment, the treatment results in the improvement of at least one symptom, such that the treated subject changes from severe persistent to moderate persistent, mild persistent, or mild intermittent. The treatment may result in the improvement of symptoms, such that the treated subject changes from moderate persistent to mild persistent or mild intermittent, or from mild persistent to mild intermittent.

[0080] In another embodiment, the defensin is used to treat lung inflammation associated with lung cancer. The lung cancer may be small cell lung cancer or non-small cell lung cancer (NSCLC). The NSCLC may be selected from adenocarcinoma, squamous cell carcinoma, large cell carcinoma and bronchioloalveolar carcinoma. It is expected that administration of one or more defensins to a lung cancer patient can ameliorate one or more inflammatory symptoms of the disease or one or more side effects of cancer treatment. The side effects of cancer treatment can be side effects of radiation, chemotherapy, immunotherapy or surgery. It is expected that administration of at least one α-defensin and / or β-defensin may also reduce tumor cell proliferation by improving the host defense of the lung.

[0081] In vitro synthesis Mammalian α-defensins and mammalian β-defensins can be produced by in vitro synthesis using conventional methods known in the art. Various commercial synthesis equipment is available, such as automated synthesizers from Applied Biosystems Inc., Beckman Inc., and others. By using synthesizers, natural amino acids can be substituted with unnatural amino acids, particularly D-isomers (or D-forms), such as D-alanine and D-isoleucine, diastereoisomers, side chains with different lengths or functionality, and the like. The specific sequence and preparation method are determined by convenience, economics, required purity, and the like. Chemical linkages can be imparted to various peptides or proteins that contain functionalities convenient for conjugation, such as amino groups for amide or substituted amine formation, e.g., reductive amination, thiol groups for thioether or disulfide formation, carboxyl groups for amide formation, and the like. If necessary, various groups can be introduced into the peptide during synthesis or expression, which allow for linkage to other molecules or to surfaces. Thus, cysteine ​​can be used to provide thioethers, histidine for binding to metal ion complexes, carboxyl groups to form amides or esters, amino groups to form amides, and so on.

[0082] Mammalian α-defensins and mammalian β-defensins, or their functional equivalents, may also be isolated and purified according to conventional methods of recombinant synthesis. Recombinant synthesis may be carried out using appropriate expression vectors and eukaryotic expression systems. A solution may be prepared from the expression host and medium, and the defensins present may be purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques. A method for recombinant expression of human β-defensin 2 in E. coli is disclosed in WO2010 / 007166 (Novozymes).

[0083] Mammalian α-defensins and β-defensins may also be induced by administration of the corresponding mRNA.

[0084] Dosage Mammalian α-defensins and mammalian β-defensins, such as human α-defensins and human β-defensins, are preferably used in pharmaceutical compositions in an amount effective to treat pulmonary inflammation in general, or in an amount effective to treat mild intermittent asthma, mild persistent asthma, moderate persistent asthma, severe persistent asthma, eosinophilic asthma, neutrophilic asthma, steroid-resistant asthma, persistent asthma, bronchiectasis, COPD or lung cancer, preferably with acceptable toxicity to the patient. Mammalian α-defensins and mammalian β-defensins, such as human α-defensins and human β-defensins, are also preferably used in pharmaceutical compositions in an amount effective to maintain the composition of the normal microflora of the lungs and / or intestine, or to treat or normalize a symbiotic imbalanced microflora of the lungs and / or intestine, preferably with acceptable toxicity to the patient in need of treatment.

[0085] In such treatments, the appropriate dosage will of course vary depending on, for example, the chemical nature and the pharmacokinetic data of the compound used, the individual host, the mode of administration and the nature and severity of the condition to be treated.

[0086] In general, however, a desirable daily dosage of human α-defensin for satisfactory results in a mammal, e.g., a human, is preferably from about 0.1 mg HD5 / kg body weight to about 10 mg HD5 / kg body weight, more preferably from about 0.5 mg HD5 / kg body weight to about 10 mg HD5 / kg body weight, such as from 1 mg HD5 / kg body weight to 10 mg HD5 / kg body weight, more preferably from about 1.2 mg HD5 / kg body weight to about 10 mg HD5 / kg body weight, preferably from about 1.2 mg HD5 / kg body weight to about 5 mg HD5 / kg body weight, even more preferably 1.2 mg HD5 / kg body weight, e.g., administered in divided doses up to 1, 2 or 3 times daily.

[0087] In one embodiment, a desirable daily dosage of human β-defensin is preferably from about 0.1 mg hBD2 / kg body weight to about 10 mg hBD2 / kg body weight, more preferably from about 0.5 mg hBD2 / kg body weight to about 10 mg hBD2 / kg body weight, such as from 1 mg hBD2 / kg body weight to 10 mg hBD2 / kg body weight, more preferably from about 1.2 mg hBD2 / kg body weight to about 10 mg hBD2 / kg body weight, preferably from about 1.2 mg hBD2 / kg body weight to about 5 mg hBD2 / kg body weight, even more preferably 1.2 mg hBD2 / kg body weight, e.g., administered in divided doses up to 1, 2 or 3 times daily.

[0088] When two different defensins are administered in a single dose, the dose may contain equal or nearly equal amounts of the two defensins, determined on a weight or molar basis. The ratio may also vary such that the ratio of α-defensin to β-defensin varies from 10:1 to 1:10, such as 5:1 to 1:5, for example, from 2:1 to 1:2, determined on a weight or molar basis.

[0089] The compounds of the preferred embodiments may be administered to larger mammals, such as humans, in similar dosages and by similar modes of administration as those conventionally used.

[0090] In one embodiment, a method is provided in which the daily dosage is 0.1-10 mg defensin / kg, such as 0.5-5 mg defensin / kg, for example 1-2 mg defensin / kg, for example 1.2 mg defensin / kg per day, as described herein.

[0091] In certain embodiments, pharmaceutical compositions of preferred embodiments may contain mammalian α-defensin and / or mammalian β-defensin, such as human α-defensin and / or human β-defensin, in an amount of about 0.5 mg or less to about 1500 mg or more, preferably about 0.5, 0.6, 0.7, 0.8 or 0.9 mg to about 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 or 1,000 mg, more preferably about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 mg to about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mg per unit dosage form. In certain embodiments, however, dosages lower or higher than those above may be preferred. Suitable concentrations and dosages can be readily determined by one of skill in the art. In certain embodiments, the pharmaceutical compositions of the preferred embodiments comprise a mammalian α-defensin, such as a human α-defensin. In certain embodiments, the pharmaceutical compositions of the preferred embodiments comprise a mammalian β-defensin, such as a human β-defensin. In further embodiments, the pharmaceutical compositions of the preferred embodiments comprise a mammalian α-defensin and a mammalian β-defensin, such as a human α-defensin and a human β-defensin, wherein the α-defensin and the β-defensin are present in equal amounts on a molar or mg / mL basis.

[0092] In one embodiment, the mammalian α-defensin and / or β-defensin is administered at least once a day, such as at least twice a day, for example at least three times a day or continuously.

[0093] In one embodiment, the mammalian α-defensin and / or β-defensin is administered intravenously by continuous infusion or intrapulmonary by continuous mechanical ventilation in patients on an external ventilator.

[0094] Formulation for oral or parenteral administration Mammalian α-defensin or β-defensin can be used therapeutically in compositions formulated for administration by any common route.In one embodiment, the administration of at least one mammalian β-defensin according to the method of the present disclosure is generally intranasal.Intranasal administration is standard for pulmonary drug delivery.

[0095] In one embodiment, administration of the at least one mammalian α-defensin and / or the at least one mammalian β-defensin, according to the methods of the present disclosure, is oral.

[0096] In one embodiment, administration of the at least one mammalian α-defensin and / or the at least one mammalian β-defensin, according to the methods of the present disclosure, is subcutaneous or intravenous.

[0097] In some embodiments, the compositions of the preferred embodiments may be formulated as lyophilisates and lyophilisates using appropriate excipients that provide stability after rehydration. Pharmaceutical compositions comprising mammalian α-defensins and / or mammalian β-defensins, such as human α-defensins and / or human β-defensins, can be manufactured by conventional methods, for example by mixing, granulating, coating, dissolving or lyophilising processes. In a preferred embodiment, pharmaceutical compositions comprising mammalian α-defensins and / or mammalian β-defensins are formulated as sterile, isotonic solutions.

[0098] Pharmaceutically acceptable carriers and / or diluents are known to those skilled in the art. For compositions formulated as liquid solutions, acceptable carriers and / or diluents should include sterile water, including physiological saline, and the compositions may optionally contain antioxidants, buffers, bacteriostats, and other common additives.

[0099] The compounds of the present disclosure can be formulated into a wide variety of preparations for oral administration.Solid preparations can include powder, tablet, drop, capsule, cachet, lozenge and dispersible granule.Other dosage forms suitable for oral administration can include liquid preparations including emulsion, syrup, elixir, aqueous solution, aqueous suspension, toothpaste, toothpaste gel, chewing gum, or solid preparations intended to be converted into liquid preparations such as solution, suspension and emulsion immediately before use.

[0100] The compounds of the present disclosure can be formulated into a wide variety of dosage forms for intranasal, subcutaneous or intravenous administration.The formulations can contain (in addition to mammalian α-defensins and / or mammalian β-defensins and any other active ingredients) carriers, bulking agents, disintegrants, flow regulators, sugars and sweeteners, flavorings, preservatives, stabilizers, wetting agents, emulsifiers, solubilizers, salts for adjusting osmotic pressure, buffers, diluents, dispersing and surfactants, binders, lubricants, and / or other pharmaceutical excipients known in the art.Those skilled in the art can further formulate mammalian α-defensins and mammalian β-defensins in a suitable manner and according to accepted practice, such as those described in Remington's Pharmaceutical Sciences, Gennaro (1990).

[0101] Mammalian alpha-defensins and mammalian beta-defensins, such as human alpha-defensins and human beta-defensins, can be used alone or in combination therapy with one or more other pharmaceutical compounds or drug substances, for example, with glucocorticoids, beta-agonists, leukotriene receptor antagonists, theophylline, antibiotics, chemotherapy or immunotherapy or combinations thereof, and / or with one or more pharmacologically acceptable excipients.

[0102] Airway administration Airway administration may be used to administer the composition of the present disclosure.Intrapulmonary administration refers to local administration to the lung.As used herein, the term "intratracheal administration, intrabronchial administration or intraalveolar administration" includes all such forms of administration, in which defensin is applied to the trachea, bronchus or alveolus, respectively, as an aerosolized or nebulized solution or suspension, or inhalation powder or gel, whether by instillation of defensin solution, with or without added stabilizers or other excipients, by application of defensin in powder form, or by inhalation of defensin to allow defensin to reach the appropriate part of the airway.

[0103] Methods of intrabronchial / alveolar administration include, but are not limited to, bronchoalveolar lavage (BAL) by methods known to those skilled in the art using lavage fluid in which the defensin is dissolved as a physiologically acceptable composition, or indeed any other effective form of intrabronchial administration, including the use of inhalation powders containing the defensin in dry form, with or without excipients, or direct application of the defensin in solution or suspension form or powder form during bronchoscopy. Methods of intratracheal administration include, but are not limited to, blind tracheal lavage with a similar solution of dissolved defensin or defensin suspension, or inhalation of aerosolized droplets containing dissolved defensin or defensin suspension obtained by using any nebulizer device suitable for this purpose.

[0104] In other embodiments, intratracheal, intrabronchial or intraalveolar administration does not involve inhalation of a product, but involves instillation or application of a solution of a defensin or a powder or gel containing a defensin into the trachea or lower respiratory tract.

[0105] Other preferred methods of administration may include the use of the following devices: 1. Pressurized nebulizers that use a compressed air / oxygen mixture 2.Ultrasonic atomizer 3. Electronic micro-pump sprayer 4. Metered dose inhaler (MDI) 5. Dry Powder Inhalation System (DPI)

[0106] The aerosol may be delivered a) via a face mask or b) via an endotracheal tube in intubated patients during mechanical ventilation (devices 1, 2 and 3). Devices 4 and 5 can also be used by patients without assistance, provided that the patient is able to activate the nebulizer device themselves.

[0107] Preferred concentrations of the solution containing the defensin and / or functional homologue or variant of the defensin range from about 0.1 μg to 1000 μg per ml of solution, such as from about 0.1 μg to 250 μg per ml of solution.

[0108] Pharmaceutical Compositions for Pulmonary Administration A pharmaceutical composition or formulation for use in the present disclosure comprises defensin in combination with, and preferably dissolved in, a pharma- ceutically acceptable carrier, preferably an aqueous carrier or diluent, or delivered to the lower respiratory tract as a pegylated preparation, or as a liposomal or nanoparticle preparation administered as an aerosol by inhalation, or as a lavage solution administered via a bronchoscope as a bronchoalveolar lavage or as a blind intratracheal wash or lavage. A variety of aqueous carriers can be used, including, but not limited to, 0.9% saline, buffered saline, physiologically compatible buffers, and the like. The composition may be sterilized by conventional techniques known to those skilled in the art. The resulting aqueous solution can be packaged for use, or filtered and lyophilized under aseptic conditions, with the lyophilized preparation being dissolved in a sterile aqueous solution prior to administration.

[0109] In one embodiment, the lyophilized defensin preparation may be prepackaged, for example in a single dose unit, hi an even more preferred embodiment, the single dose unit is tailored to the patient.

[0110] The compositions may contain pharmacologically acceptable auxiliary agents or adjuvants, including, but not limited to, pH adjusting and buffering agents and / or isotonicity agents, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, and the like.

[0111] The formulations may contain pharmacologically acceptable carriers and excipients, including microspheres, liposomes, microcapsules, nanoparticles, etc. Conventional liposomes are usually composed of phospholipids (neutral or negatively charged) and / or cholesterol. Liposomes are vesicular structures based on lipid bilayers that enclose aqueous compartments. Liposomes can vary in their physicochemical properties, such as size, lipid composition, surface charge and number of phospholipid bilayers, and fluidity.The lipids most frequently used in liposome formation are: 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DO ...dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphate (monosodium salt) (DMPA), 1,2-dipalmitoyl-sn-glycero-3-phosphate (monosodium salt) (DPPA), 1,2-dioleoyl-sn-glycero 1,2-Dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)](sodium salt)(DMPG), 1,2-Dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)](sodium salt)(DPPG), 1,2-Dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)](sodium salt)(DOPG), 1,2-Dimyristoyl-sn-glycero -3-[phospho-L-serine] (sodium salt) (DMPS), 1,2-dipalmitoyl-sn-glycero-3-[phospho-L-serine) (sodium salt) (DPPS), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (sodium salt) (DOPS), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl) (sodium salt) and 1,1',2,2'-tetramyristoylcardiolipin (ammonium salt). Formulations consisting of DPPC in combination with another lipid or liposomal modifier are preferred, for example in combination with cholesterol and / or phosphatidylcholine.

[0112] Long-circulating liposomes are characterized by their ability to extravasate at body sites where the permeability of the vascular wall is increased. The most common method of producing long-circulating liposomes is to covalently attach the hydrophilic polymer polyethylene glycol (PEG) to the outer surface of the liposome. Some of the preferred lipids are: 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (ammonium salt), 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP).

[0113] Lipids that may be applicable to the liposomes are supplied by Avanti, Polar Lipids, Inc, Alabaster, Ala. Additionally, the liposomal suspension may contain lipid protective agents that protect lipids from damage due to free radicals and lipid peroxidation upon storage. Lipid-soluble free radical quenchers such as α-tocopherol and water-soluble iron-specific chelators such as ferrioxamine are preferred.

[0114] A variety of methods can be used to prepare liposomes, as described, for example, in Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. No. 4,235,871, U.S. Pat. No. 4,501,728, and U.S. Pat. No. 4,837,028, all of which are incorporated herein by reference. Another method produces multilamellar vesicles of heterogeneous size. In this method, the vesicle-forming lipids are dissolved in a suitable organic solvent or solvent system and dried under vacuum or inert gas to form a thin lipid film. If desired, this film may be dissolved again in a suitable solvent, such as tertiary butanol, and then lyophilized to form a more homogenous lipid mixture in a powder-like form that is more easily hydrated. The film is covered with an aqueous solution of the targeting agent and targeting moiety, and is allowed to hydrate, usually over a period of 15 to 60 minutes with stirring. The size distribution of the resulting multilamellar vesicles can be shifted towards smaller sizes by hydrating the lipids under more vigorous shaking conditions or by adding solubilizing detergents such as deoxycholate.

[0115] Micelles are formed in aqueous solution by surfactants, molecules that contain a hydrophobic portion and one or more ionic or strongly hydrophilic groups.

[0116] Common surfactants known to those skilled in the art can be used in the micelles of the present invention. Suitable surfactants include sodium laurate, sodium oleate, sodium lauryl sulfate, octaoxyethylene glycol monododecyl ether, octoxynol 9 and PLURONIC F-127 (Wyandotte Chemicals Corp.). Preferred surfactants are non-ionic polyoxyethylene and polyoxypropylene surfactants compatible with intravenous injection, such as TWEEN-80, PLURONIC F-68, n-octyl-β-D-glucopyranoside, etc. Additionally, phospholipids such as those described for use in the manufacture of liposomes may be used for micelle formation.

[0117] Working Example Example 1 Determining and evaluating the efficacy of mammalian β-defensins in a steroid-insensitive neutrophilic allergic airway disease model of infection-induced severe asthma in mice (Figure 1)

[0118] material and method Treatment Method: Six- to eight-week-old female BALB / c mice were sensitized intraperitoneally (IP) with 50 μg ovalbumin in 200 μL of 0.9% saline containing 1 mg alum adjuvant. Mice were challenged intranasally (IN) with ovalbumin (10 μg in 50 μL of sterile saline) on days 12–13 and 33–34. On day 14, mice were inoculated IN with the natural mouse pathogen Chlamydia muridarum (Cmu:100 inclusion-forming units, ATCCVR-123, 30 μL of sucrose phosphate glutamate buffer (SPG)). Dexamethasone (DEX) was administered IN (2 mg / kg; 50 μL of phosphate-buffered saline (PBS)) on days 32–34 along with the ovalbumin challenge. hBD2 was administered IN on days 30, 32 and 34 (5 mg / kg; 50 μL phosphate buffered saline).

[0119] Agents administered to mice by intranasal delivery are expected to reach the lungs via the airways and is an art-accepted model of intrapulmonary administration.

[0120] test: Airway Inflammation: Differential leukocyte counts were obtained from May-Grünwald-Giemsa stained BALF cells using light microscopy.

[0121] Pulmonary function: AHR was measured in anesthetized cannulated mice using the Scireq Flexivent FX1 system. Data are expressed as airway resistance at 10 mg / kg methacholine and as a dose-response curve.

[0122] result Airway inflammation: Mice sensitized with ovalbumin and infected with C. muridarum developed highly statistically significant increases in total leukocytes, macrophages, lymphocytes, neutrophils, and to a lesser extent eosinophils. Groups treated with hBD2 IN showed complete normalization of neutrophil counts and to a lesser extent lymphocytes, while macrophages and eosinophils were unchanged. Groups treated with hBD2 plus DEX IN showed complete normalization of eosinophils, but no other additive effects were observed (Figure 7).

[0123] Pulmonary function: Mice sensitized with ovalbumin (Ova) had a higher AHR compared to saline (Sal) (non-asthmatic) controls. This difference was highly statistically significant. The INhBD2-treated group (Cmu / Ova / hBD2) showed a fully normalized AHR comparable to the saline control group. The group treated with hBD2 plus DEX IN showed a fully normalized AHR comparable to the saline control group, although DEX did not appear to have an additive effect (Figure 4).

[0124] Conclusion: This example demonstrates that intranasally administered hBD2 is able to completely normalize airway hyperresponsiveness and neutrophil counts in BALF in a known steroid-resistant animal model of asthma.

[0125] Example 2 Determination and evaluation of efficacy of mammalian β-defensins administered orally versus IN in a murine house dust mite / Freund's complete adjuvant-induced model of allergic asthma (Figure 2)

[0126] material and method Treatment Methods: 7-10 week old female BALB / c mice were randomly assigned to seven test groups 1 day prior to the start of the study and sensitized subcutaneously (SC) to house dust mites (100 μg HDM in 200 μL saline and Freund's complete adjuvant in 0.9% saline). Mice were then challenged intranasally (IN) with HDM on day 14 (25 μg HDM in 50 μL saline). Dexamethasone was administered orally on day 14 (1 mg / kg BID; 50 μL phosphate buffered saline (PBS)). hBD2 was administered IN or orally on day 14 (1.7 mg / kg TID IN; 0.4 mg / kg TID IN; 0.4 mg / kg TID orally, 50 μL phosphate buffered saline). The first dose was administered 60 min prior to challenge, with subsequent doses administered approximately 6 h apart.

[0127] test: Airway inflammation: 48 hours after challenge, bronchoalveolar lavage was performed and the lungs were washed with three volumes of cold PBS (0.4; 0.3 and 0.3 mL (total 1 mL)). Total and differential white blood cell counts were determined with an automated hematology analyzer, Sysmex XT-2000iV.

[0128] Pulmonary function: Starting 48 hours after HDM challenge, lung resistance and compliance measurements were performed in anesthetized, cannulated mice after methacholine challenge (MCH1, 3.125 mg / mL; MCH2, 6.25 mg / mL; MCH3, 12.5 mg / mL and MCH4, 25 mg / mL) using a Buxco Finepointe RC system from DSI. Data are presented as airway resistance at 10 mg / kg methacholine and as dose-response curves.

[0129] Lung harvest for cytokine analysis: After completion of all BALs, lungs were removed from the thorax, flash frozen in liquid nitrogen and stored frozen at -80 degrees Celsius until analysis of cytokine concentrations of IL-1β, TNF-α, IL-6, IL-10 and IFNγ by ELISA.

[0130] result Increased lung resistance and decreased lung compliance were observed in HDM-challenged and vehicle-treated animals compared to saline-challenged (non-asthmatic) mice. Inflammatory responses in both vehicle-treated groups (oral and intranasal) were induced by a single HDM challenge 14 days after sensitization with HDM and adjuvants. The inflammatory response was characterized by a statistically significant increase (p<0.05) in total cell, eosinophil, neutrophil, macrophage and lymphocyte counts in BALF compared to saline-challenged controls. Also, concentration analysis of five cytokines, IL-1β, TNF-α, IL-6, IL-10 and IFN-γ, in lung tissue homogenates revealed significantly higher levels in HDM-challenged animals compared to saline-challenged controls.

[0131] Dexamethasone treatment significantly suppressed the total cell and eosinophil counts in BALF, but not the neutrophil, macrophage, and lymphocyte counts. In agreement with the cellular data, dexamethasone did not affect the levels of IL-1β, TNF-α, IL-6, IL-10, and IFN-γ in lung tissue homogenates compared to HDM / vehicle controls. However, dexamethasone did affect AHR measures related to eosinophil counts. The results obtained indicate that this model is to some extent steroid-resistant.

[0132] Test article hBD2 effectively suppressed the increase in airway resistance (Figures 5a and 5b) and the decrease in lung compliance (Figures 6a and 6b) compared to HDM-challenged vehicle-treated animals after both three oral and intranasal applications on day 14. More pronounced effects were observed in some measured parameters after intranasal application, e.g., in cellular influx in the BALF, where both doses (0.4 mg / kg / day TID and 1.7 mg / kg / day TID) significantly suppressed neutrophil counts. On the other hand, the steroid standard dexamethasone failed to suppress neutrophil counts. Similar significant effects were observed in IL-6, IL-10 and IFN-γ cytokine levels in lung tissue homogenates for both routes of administration (Figures 9, 14, 17). Orally administered hBD2 significantly reduced TNF-α (Figure 10), whereas intranasally administered hBD2 was not significantly different from the control. Figure 11 shows the effect on IL-1β.

[0133] Conclusion: All the obtained results show a clear anti-inflammatory effect of hBD2 in the house dust mite / Freund's complete adjuvant-induced mouse model of allergic asthma. Surprisingly, orally administered hBD2 was also effective in treating asthma and reducing inflammation in asthmatic mice.

[0134] Example 3 Determination and evaluation of efficacy of mammalian β-defensins administered orally versus IN in a murine house dust mite / Freund's complete adjuvant-induced model of allergic asthma (Figure 2)

[0135] material and method Treatment Methods: 7-10 week old female BALB / c mice randomly assigned to 4 test groups 1 day prior to study initiation were sensitized subcutaneously (SC) to house dust mites (100 μg HDM in 200 μL saline and Freund's complete adjuvant in 0.9% saline). Mice were challenged intranasally (IN) with HDM on day 14 (25 μg HDM in 50 μL saline). hBD2 was administered IN or orally on day 14 (0.4 mg / kg TID IN; 0.4 mg / kg TID orally, 50 μL phosphate buffered saline). The first dose was administered 60 min prior to challenge, with subsequent doses administered approximately 6 h apart.

[0136] test: Lung tissue harvest: Lungs were removed from the thorax, flash frozen in liquid nitrogen and stored frozen at -80 degrees Celsius until analysis of cytokine concentrations by ELISA for IL-4, IL-5, IL-8 (KC), IL-9 and IL-13.

[0137] Lungs were inflated in situ in 10% buffered formalin, removed from the thorax, individually placed in 10% buffered formalin, en bloc embedded in paraffin, sectioned, and stained with H&E / PAS.

[0138] blood sampling All terminal blood samples were collected by venous blood draw. Blood was collected into lithium heparin tubes, placed on ice, and immediately centrifuged at 4° C. Plasma was separated and stored at −80° C. until future SCFA analysis.

[0139] Lung tissue sampling The lungs were exposed and removed by gentle incision of the chest and by cutting off both sides of the sternum and ribs. Lungs from the first six animals per group were removed from the thorax, snap frozen in liquid nitrogen, and stored frozen at -80°C until analysis of cytokine concentrations by ELISA.

[0140] Lungs from eight other animals per group were inflated in situ with 10% buffered formalin, removed from the thorax, individually placed in 10% buffered formalin, bulk embedded in paraffin, sectioned, and stained with H&E / PAS. Paraffin blocks were retained for IHC analysis.

[0141] reading Histopathology (H&E;PAS) (N=8 / group; total N=32) Cytokines (IL-4, IL-5, KC, IL-9, and IL-13) in lung tissue homogenates (N=6 / group; total N=24)

[0142] histopathology Cellular influx (mononuclear cells, eosinophils, neutrophils) was assessed semiquantitatively on H&E stained slides for peribronchial / bronchiolar and perivascular spaces separately as follows: 0 None 1 A few scattered inflammatory cells 2. Larger Aggregations 3. Significant cell accumulation A total inflammation score was calculated as the sum of the scores of all individuals. Goblet cell metaplasia was assessed separately at the level of large and small airways on PAS-stained slides as follows: 0 No mucus-containing cells along the basement membrane 1 A few positive cells with less than 75% stained cytoplasm along the basement membrane 2 A few positive cells with more than 75% stained cytoplasm along the basement membrane 3 Multiple positive cells with less than 75% stained cytoplasm along the basement membrane 4 Multiple positive cells with more than 75% stained cytoplasm along the basement membrane

[0143] Statistical evaluation Data was processed using MS Excel. Statistical analysis was performed using GraphPad Prism software (version 5.04). Differences between groups were considered statistically significant when p<0.05.

[0144] Statistical analysis of selected histological score values ​​(data) was performed using median and non-parametric Mann-Whitney test methods.

[0145] result An inflammatory response was elicited in mice in both vehicle-treated groups (oral and intranasal) by a single HDM challenge 14 days after sensitization with HDM and adjuvant, characterized by a statistically significant increase in the concentrations of five cytokines, IL-4, IL-5, IL-8, IL-9 and IL-13, in lung tissue homogenates in HDM-challenged animals compared with saline-challenged controls, and by severe histological inflammatory changes in lung tissue.

[0146] Test article hBD2 effectively suppressed the increase in tissue inflammation in lung tissue compared to HDM-challenged vehicle-treated animals after both three oral and intranasal applications on day 14 (Figures 19, 20, 21). Similar significant effects were observed on IL-4, IL-5, IL-8, IL-9 and IL-13 cytokine levels in lung tissue homogenates by both routes of administration (Figures 12, 13, 15, 16, 18).

[0147] Conclusion: All the obtained results show a pronounced anti-inflammatory effect of hBD2 in the house dust mite / Freund's complete adjuvant-induced mouse model of allergic asthma, which was obtained using both intranasal and oral administration of hBD2.

[0148] Example 4 Pharmacokinetic study to establish the pharmacokinetic profile of hBD2 following a single oral gavage of 4 mg / kg in NMRI mice

[0149] material and method Treatment method: 21 female NMRI mice were administered 5ml / kg by oral gavage using a gavage tube and 1ml syringe according to individual body weight obtained on the day of administration. Urine was collected at random time points by gentle massage of the abdominal groin. The first blood sample was obtained using the submandibular gland collection method. The second blood sample was collected from mice anesthetized with isoflurane. Intestinal samples were obtained after euthanasia. The abdomen of each mouse was incised and three segments of the intestinal tract were collected.

[0150] result hBD2 was not detectable by HPLC in either serum or urine samples as all values ​​were below the detection level of <10 pg / ml, indicating that hBD2 is not systemically available in mice after oral dosing at 4 mg / kg (Figure 22).

[0151] Example 5 Examination and comparison of the pharmacokinetic profiles of hBD2 fused to the C-terminus (molecular weight 71.336 Da) or N-terminus (molecular weight 71.666 Da) of human serum albumin following subcutaneous or intravenous administration of a molar equivalent of 1 mg / kg hBD2 (molecular weight 66,437 Da) to female NMRI mice

[0152] material and method Treatment method: Animals were administered 10 ml / kg of a stock concentration of 1.65 mg / ml according to individual body weight (300 μL for a 30 gram mouse). The first blood sample was obtained using the submandibular gland collection method and the second blood sample was obtained after anesthesia and euthanasia with soflurane.

[0153] result hBD2 showed a half-life of 1 hour and the two fusion proteins showed a half-life of 12 hours. The AUCs changed dramatically. Renal clearance also changed from 10 ml / min for hBD2 to 0.5-2.2 ml / min for the two fusion molecules (Figures 23, 24, 25).

[0154] This example demonstrates that the half-life of hBD2 can be significantly extended by C- or N-terminal conjugation to albumin.

[0155] Example 6 Determination and evaluation of the anti-inflammatory effect of "hBD2-albumin N-terminal fusion" in an acute colitis model induced by dextran sulfate sodium (DSS) administration for 10 days in mice

[0156] material and method Treatment method: "hBD2-Albumin N-Term" was administered intravenously or subcutaneously via the tail vein using a sterile 25G needle at a dose volume of 10 ml / kg body weight. Animals received one dose daily for 10 consecutive days. Dexamethasone (DEX), the active control, was given subcutaneously at a dose of 1 mg / kg in a dose volume of 10 ml / kg body weight once daily.

[0157] result Treatment with "hBD2-Albumin N-Term" resulted in a significant suppression of the Disease Activity Index (DAI) when administered daily via the intravenous route at a dose of 1.65 mg / kg (p<0.05). Furthermore, a significant suppression of the DAI score on day 10 was observed when "hBD2-Albumin N-Term" was administered daily subcutaneously at doses of 1.65 mg / kg and 125 mg / kg, respectively (p<0.05). Administration of dextran sulfate sodium resulted in significant inflammation and damage to colonic tissue as evidenced by histological examination. Treatment with "hBD2-albumin N-term" did not result in any statistically significant reduction in this tissue damage, while the active control DEX similarly failed to significantly reduce tissue injury. The results further showed that animals treated with "hBD2-albumin N-term" showed a significant weight gain on day 7 despite a transient weight loss on days 2 and 3. In contrast, animals treated with DEX showed a highly significant weight loss from day 5 onwards (p<0.01).

[0158] This example demonstrates that hBD2-albumin N-terminal fusions are biologically active in animal models of inflammatory disease.

[0159] Example 7 Determination and evaluation of the anti-inflammatory effect of "hBD2-albumin C-terminal fusion" in an acute colitis model induced by dextran sulfate sodium (DSS) administration for 10 days in mice

[0160] material and method Treatment method: "hBD2-Albumin C-Term" was administered intravenously or subcutaneously via the tail vein using a sterile 25G needle at a dose volume of 10 ml / kg body weight. Animals received one dose daily for 10 consecutive days. The active control, prednisolone (Pred), was given orally by gavage at a dose of 1 mg / kg once daily at a dose volume of 10 ml / kg body weight.

[0161] result Treatment with "hBD2-albumin C-term" resulted in significant suppression of DAI when administered daily via the intravenous route at a dose of 1.6 mg / kg (p<0.05). Furthermore, "hBD2-albumin C-term" resulted in significant suppression of DAI when administered via the intravenous route at a dose of 1.6 mg / kg every other day on days 0, 2, 4, 6, 8 and 10 (p<0.05) (Figure 26). Daily treatment with Pred resulted in significant suppression of DAI on day 9 (p<0.05). Administration of dextran sulfate sodium resulted in significant inflammation and damage of colonic tissue, as evident from histological examination. Treatment with "hBD2-albumin C-term" at a dose of 1.6 mg / kg resulted in a statistically significant reduction in this tissue damage (p<0.05). Similarly, daily treatment with "hBD2-albumin C-term" at doses of 1.6 mg / kg and 16.5 mg / kg on days 0, 2, 4, 6, 8 and 10 resulted in a significant reduction in colonic tissue damage (p<0.01) (Figure 27). Treatment with the active control Pred failed to significantly reduce tissue injury in the proximal colon, but reduced damage in the distal colon (p<0.01). The results further showed a significant increase in body weight in animals treated with "hBD2-albumin C-term" (p<0.05).

[0162] This example demonstrates that hBD2-albumin C-terminal fusions are biologically active in animal models of inflammatory disease.

[0163] Example 8 [Table 2]

[0164] Example 9 Determining and evaluating the efficacy of oral versus IN prophylaxis with mammalian β-defensins in a murine house dust mite-induced model of allergic asthma

[0165] material and method Treatment Method: 7-10 week old female BALB / c mice were randomly assigned to 5 test groups 1 day before the start of the study and subcutaneously (SC) sensitized to house dust mites (100 μg HDM in 200 μL saline and Freund's complete adjuvant in 0.9% saline). Mice were treated orally and intranasally with hBD2 at a dose of 1.2 mg / kg / day (0.4 mg / kg TID), respectively, 3 consecutive times daily at approximately 6 hour intervals starting on the morning of day 12. The final dose was administered 1 hour prior to challenge on day 14. The total number of doses was 8 doses or the total dose of hBD2 was 2 mg / kg. Mice were then challenged intranasally (IN) with HDM on day 14 (25 μg HDM in 50 μL saline).

[0166] test: Airway inflammation: 48 hours after challenge, bronchoalveolar lavage was performed and the lungs were washed with three volumes of cold PBS (0.4; 0.3 and 0.3 mL (total 1 mL)). Total and differential white blood cell counts were determined with an automated hematology analyzer, Sysmex XT-2000iV.

[0167] Pulmonary function: Starting 48 hours after HDM challenge, lung resistance and compliance measurements were performed after methacholine challenge (MCH1, 3.125 mg / mL; MCH2, 6.25 mg / mL; MCH3, 12.5 mg / mL and MCH4, 25 mg / mL) in anesthetized, cannulated mice using a Buxco Finepointe RC system from DSI. Data are presented as airway resistance at 10 mg / kg methacholine and as dose-response curves.

[0168] Lung harvest for cytokine analysis: After completion of all BALs, lungs were removed from the thorax, flash frozen in liquid nitrogen and stored frozen at -80 degrees Celsius until analysis of cytokine concentrations in lung homogenates by ELISA for TNF-α, IL-4, IL-5, IL-6, IL-9, IL-13 and IL-33.

[0169] result Increased lung resistance and decreased lung compliance were observed in HDM-challenged vehicle-treated animals compared to saline-challenged (non-asthmatic) mice. Inflammatory responses in both vehicle-treated groups (oral and intranasal) were induced by a single HDM challenge 14 days after HDM sensitization. The inflammatory response was characterized by a statistically significant increase (p<0.05) in total cell, eosinophil, neutrophil, macrophage and lymphocyte counts in BALF compared to saline-challenged controls. Concentration analysis of seven cytokines, TNF-α, IL-4, IL-5, IL-6, IL-9, IL-13 and IL-33, in lung tissue homogenates revealed significantly higher levels in HDM-challenged animals compared to saline-challenged controls.

[0170] hBD2 administered on days 12-14 effectively preserved normal lung function and suppressed the increase in airway resistance (Figure 29) and the decrease in lung compliance (Figure 30) compared to HDM-loaded vehicle-treated animals, both after oral application three times a day and after intranasal application (total of 2.0 mg / kg for eight doses). An effect on cellular influx in the BALF was observed after oral application, which significantly suppressed neutrophil numbers (Figure 31), whereas other immune cells migrated to the BALF as is usually observed in asthma. However, importantly, the cytokine storm and the basis of asthma attacks, which are often observed in asthma, were prevented by a complete normalization of cytokine concentrations in lung tissue homogenates, especially after oral administration of hBD2. Cytokine levels of TNF-α, IL-4, IL-5, IL-6, IL-9 and IL-13 after oral administration are shown in Figures 32-37. There was a trend towards decreased TNF-α, IL-4, IL-5, IL-6, IL-9 and IL-13 following intranasal administration of hBD2, but this was not statistically significantly different from controls.

[0171] Conclusion: All obtained results show a clear protective, preventive and anti-inflammatory effect of hBD2 in a house dust mite-induced mouse model of allergic asthma.

[0172] Example 10 Protecting and preserving the gut microbiota through prophylactic treatment with defensins Mice: Mice were housed in groups of 4 cages, 3 mice each. Food intake was recorded daily just before lights off at 6 pm. Experimental procedures were performed on individual mice in a rotating order by group and cage. Mice were maintained at room temperature under standard conditions in SPF with a 12-hour light-dark cycle. Treatment methods are described in Figure 38.

[0173] Diet: For dosing purposes, an average body weight of 25 grams per mouse was assumed. Mice eat approximately 3 g of food per mouse per day.

[0174] Treatment: Mice were fed either a high-fat diet (HFD) or a low-fat (LF) control diet. The HFD contained four subgroups: 1. hBD2, 1. HD5, 1. hBD2 / HD5, 1. Standard HFD without added defensins. Defensin concentration was 1.2 mg hBD2 / kg mouse per day. HD5 was given at equimolar concentrations to hBD2. The combination group received 50% hBD2 + 50% HD5 so the total amount of defensins was equivalent to the remaining study groups.

[0175] test: Microbial analysis was performed to investigate the gut microbiota. Longitudinal 16S characterization was performed on a total of 240 samples, four sets of samples from 60 mice. Each mouse was sampled before the diet change, one week after the diet change, four weeks after the diet change, and at termination, thus ensuring a complete characterization of the fecal microbiota as a result of defensin treatment.

[0176] result microbiome hBD2 mainly affected microbial occurrence, whereas HD5 and hBD2+HD5 mainly affected microbial abundance. Figure 40 shows the relative abundance of species in the different treatment groups and illustrates the profound effect of hBD2 and HD5 on the gut microbiota. A statistically significant increase in the abundance of Allobaculum was observed in the small intestine after HD5 prophylaxis (p<0.02; Figure 41). Allobaculum is a species that produces short-chain fatty acids. Short-chain fatty acids play an important role in regulating the homeostasis of colonic Treg cells mediated by GPCR43. A statistically significant increase in the abundance of Barnesiella was observed in the large intestine after hBD2 prophylaxis (p<0.03; Figure 43). Barnesiella is a bacterium that can eliminate antibiotic-resistant pathogenic bacteria that can be observed in hospitalized patients and prevent their intestinal dominance. The abundance of Barnesiella corresponds to the abundance of several immunoregulatory cells. The higher the level of Barnesiella in the colon, the more marginal zone B cells and invariant natural killer T cells were counted in the spleen and liver. In the development of colitis in IL10- / - mice, higher levels of Barnesiella phylotypes correlated with lower disease activity levels. A trend toward decreased abundance of Lactobacillaceae was observed in the colon after prophylactic treatment with hBD2 (p=0.1; FIG. 42).

[0177] Conclusion: Lung microbiota as well as microbiota seem to play an important role in asthma (the latter by the gut-lung axis). The significant impact of defensins on the occurrence and abundance of important commensal bacteria and on colonic T cell homeostasis could explain the pulmonary effects observed in allergic asthmatic mice after oral treatment with defensins as well as the difference between the pulmonary effects observed after oral versus intranasal administration. This Example 10 shows that both α- and β-defensins, especially HD5 and hBD2, have a significant impact on the composition of the microbiota in terms of the number of species and total number of bacteria present, thus appearing to protect and preserve a healthy microbiota. More specifically, defensins seem to promote bacteria that produce short-chain fatty acids (SCFAs), which play a key role in colonic Treg cell homeostasis.

[0178] Example 11 Treatment of gut microbial imbalance by intervention with defensins

[0179] Mice and diets This study reveals the effect of hBD2 and HD5 on the microbiota in diet-induced obese mice. The intervention was preceded by a 13-week acclimation period during which the mice were fed a very high-fat diet (60% of energy from fat). Only mice that met the criterion of a minimum weight gain of 12 g during the acclimation period (approximately 50% of initial weight) were included in the final analysis. Mice that did not meet this criterion were kept in their own cage as hierarchical "keepers". They were subjected to all experimental tests but were excluded from the analysis.

[0180] Treatment method: All mice were MR scanned before the intervention. Cages of mice were assigned to experimental groups based on their fat mass. All subsequent measurements were paired with data from the same mice before the intervention. Experiments on a LFD (low fat diet) reference group were performed in parallel. As controls for the intervention, two additional groups were included: 1. Very high fat diet and 1. Low fat diet. Experimental mice were kept on a very high fat diet during the intervention. Mice received the test diet for 10 weeks. They were co-housed with 4 mice per cage, 3 cages per group, throughout the entire duration of the experiment. All tests were performed over 3 days, with one cage per group per day. The treatment method is shown in Figure 39.

[0181] Study: A study microbial analysis was performed to investigate the gut microbiota. A longitudinal 16S characterization was performed on 4 sets of samples from 60 mice, a total of 240 samples. Each mouse was sampled before diet change, 1 week after diet change, 4 weeks after diet change, and at termination, thus ensuring a complete characterization of the fecal microbiota as a result of defensin treatment.

[0182] result microbiome Both defensins showed a significant effect on the abundance and absence of bacteria. HD5 statistically significantly increased the abundance of Alloprevotella in the large intestine (p<0.02) (Figure 44), while hBD2 had no effect on the abundance of Alloprevotella. hBD2 dramatically and statistically significantly increased the relative abundance of Bifidobacteriaceae in both the small and large intestines (p<0.0001 and p<0.04, respectively; Figure 45). HD5 tended to increase the abundance of Bifidobacteriaceae in the small intestine (Figure 45).

[0183] The lung microbiota as well as the gut microbiota appear to play important roles in asthma (the latter via the gut-lung axis). The profound impact of defensins on the occurrence and abundance of key commensal bacteria and on colonic T cell homeostasis could explain the pulmonary effects observed in allergic asthmatic mice after oral treatment with defensins as well as the differences between the pulmonary effects observed after oral versus intranasal administration.

[0184] Conclusion: This Example 11 shows that both α- and β-defensins, especially hBD2 and HD5, have a significant effect on the composition of the microbiota in terms of the number of species and total number of bacteria present, thus appearing to protect and maintain a healthy microbiota. More specifically, defensins appear to promote bacteria that produce short chain fatty acids (SCFAs), which play an important role in the homeostasis of colonic Treg cells.

[0185] References Bouloukaki, I. et al., 2011. Sputum and nasal lavage lung-specific biomarkers before and after smoking cessation. BMC Pulmonary Medicine 11: 35 Charlson, E.S. et al. 2011. Topographical continuity of bacterial populations in the healthy human respiratory tract. Am J Respir Crit Care Med 184: 957-963. Cosmi, L et al., 2011. TH17 cells: new players in asthma pathogenesis. Allergy 66: 989-998. Donia, M.S. and Fischbach, M.A. 2015. Small molecules from the human microbiota. Science 349, 1254766 Dorrestein, P.C. et al., 2014. Finding the missing links among metabolites, microbes, and the host. Immunity 40: 824-832. Ege, M.J. et al., 2011. Exposure to environmental microorganisms and childhood asthma. NEJM 364: 701-709. Essilfie, A. et al., 2015. Macrolide therapy suppresses key features of experimental steroid-sensitive and steroid-insensitive asthma. Thorax 70: 458-467. Fletcher, C. and Peto, R. 1977. The natural history of chronic airflow obstruction. BMJ: 1: 1645-1648. Hansbro, P.M. et al, 2004. Role of atypical bacterial infection on the lung in predisposition / protection of asthma. Pharmacol Ther 101: 193-210 Hansbro, P.M. et al., 2011. Cytokine / anti-cytokine therapy - novel treatments for asthma? BJP 163: 81-95. Hilty, M. et al., 2010. Disordered microbial communities in asthmatic airways. PLoS ONE5, e8578 Hogg, J.C. et al. 2004. The nature of small-airway obstruction in chronic obstructive pulmonary disease. NEJM: 350: 2645-2653. Jakobsson, H.E. et al. 2014. Decreased gut microbiota diversity, delayed Bacteroidetes colonization and reduced Th1 responses in infants delivered by Caesarian section. Gut 63: 559-566. Marra, F. et al., 2009. Antibiotic use in children is associated with increased risk of asthma. Pediatrics 123: 1003-1010. Marsland, B.J. et al., 2015. The gut-lung axis in respiratory disease. Annals ATS 12: S150-156 Penders, J. et al., 2007. The role of the intestinal microbiota in the development of atopic disorders. Allergy 2007: 1223-1236 Salzman NH, Underwood MA and Bevins CL, 2007. Paneth cells, defensins, and the commensal microbiota: a hypothesis on intimate interplay at the intestinal mucosa. Semin Immunol 19(2):70-83. Schirmer, M. et al., 2016. Linking the human gut microbiome to inflammatory cytokine production capacity. Cell 167: 1125-1136 Trompette, A. et al., 2013. Gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis. Nature Medicine 20: 159-168. Wehkamp J, et al., 2002. Innate immunity and colonic inflammation: enhanced expression of epithelial alpha-defensins. Dig Dis Sci. 47(6):1349-55. Wills-Karp, M. et al., 2001. The germless theory of allergic disease: revisiting the hygiene Hypothesis. Nat Rev Immunol (1): 69-75. WO 2010 / 007166 WO 92 / 06204 WO 95 / 17413 WO 95 / 22625 US 5,223,409 WO 2013 / 007596

Claims

1. A pharmaceutical composition comprising at least one defensin selected from the group consisting of hBD2 (sequence number 2) and truncated hBD2 (sequence number 7), for use in the treatment and / or prevention of asthma, chronic obstructive pulmonary disorder (COPD), bronchiectasis, and emphysema in a subject by oral or intrapulmonary administration of the pharmaceutical composition.

2. 2. The composition of claim 1, wherein the asthma is selected from the group consisting of mild intermittent asthma, mild persistent asthma, moderate persistent asthma, severe persistent asthma, eosinophilic asthma, neutrophilic asthma, steroid-resistant asthma, and persistent asthma.

3. The treatment and / or prevention may comprise: a. restoring balance to the immune system by reducing lung tissue inflammation, inflammatory cell counts in bronchoalveolar lavage fluid, normalizing inflammatory cytokine production in lung tissue homogenates and preventing / treating cytokine storm; b. Increasing lung compliance, decreasing airway hyperresponsiveness, and / or increasing peak flow in a subject in need thereof; c. increasing gene richness, number of phyla, increasing butyrate production and / or tryptophan production, decreasing acetate production of and / or from the lung microbiota; d. Maintaining and / or stabilizing the normal microflora in the lungs; and e. increasing forced expiratory volume in 1 second (FEV1) and / or peak expiratory flow rate (PEFR) or decreasing PEFR variability; The composition of claim 1 or 2, comprising one or more selected from the group consisting of:

4. The composition according to any one of claims 1 to 3, wherein the composition comprises two types of defensins.

5. The composition of any one of claims 1 to 4, wherein the defensin further comprises at least one additional moiety selected from the group consisting of a cell penetrating peptide (CPP), an albumin binding moiety (ABM), a detectable moiety (Z), and a half-life extending peptide.

6. 6. The composition of any one of claims 1 to 5, wherein the defensin further comprises a half-life extending peptide such as an additional moiety selected from the group consisting of neonatal Fc receptor (FcRn), transferrin, albumin (HAS), XTEN or PEG, homoamino acid polymers (HAP), proline-alanine-serine polymers (PAS), or elastin-like peptides (ELPs), hyaluronic acid, negatively charged sialylated peptides such as the carboxy terminal peptide (CTP) of the chorionic gonadotropin (CG) β chain, human IgG, and molecules capable of binding to CH3(CH2)nCO-, where n is 8 to 22.

7. The subject to be treated may have one or more of the following: a. >2 asthma symptoms per week, including daily symptoms, including persistent symptoms; b. Asthma attacks of different intensities, such as attacks that affect activity, attacks that limit physical activity, c. Nocturnal asthma symptoms >2 times per month, such as frequent nocturnal symptoms, such as >2 symptoms per week lasting several days; d. Forced expiratory volume in 1 second (FEV1) of <80%, such as FEV1 of <60%, such as FEV1 of 60-80%, and e. Peak expiratory flow rate (PEFR) with >20% variation, such as PEFR variation of 20-30%, such as PEFR variation of >30%, such as PEFR variation of >60%; The composition according to any one of claims 1 to 6, wherein

8. 8. The composition of any one of claims 1 to 7, wherein the defensin is administered in combination with a glucocorticoid, a beta agonist, a leukotriene receptor antagonist, theophylline, an antibiotic, rifaximin, an immunosuppressant, chemotherapy or immunotherapy, a prebiotic, a probiotic, tryptophan, a short chain fatty acid, HNP-1, HNP-2, HNP-3, HNP-4, cathelicidin, lactoferrin, lactoferricin, lysozyme, fecal transplant, or any combination thereof.

9. 9. The composition of any one of claims 1 to 8, wherein the daily dosage is 0.1 to 10 mg defensin / kg, such as 0.5 to 5 mg defensin / kg, such as 1 to 2 mg defensin / kg per day, such as 1.2 mg defensin / kg per day.

10. 10. The composition of any one of claims 1 to 9, wherein the defensin is administered at least once a day, such as at least twice a day, such as at least three times a day, such as at least four times a day, such as at least five times a day.

11. The composition according to any one of claims 1 to 10, wherein the administration is oral administration.

12. The composition of any one of claims 1 to 11, wherein the administration is by pulmonary administration, such as intratracheal, intrabronchial, or bronchoalveolar epithelial administration, for example by inhaler, nebulizer, or vaporizer.

13. A composition comprising at least one defensin selected from the group consisting of hBD2 (sequence number 2) and truncated hBD2 (sequence number 7), for use in the treatment and / or prevention of COPD in a subject by oral or pulmonary administration of the composition.

14. A composition comprising at least one defensin selected from the group consisting of hBD2 (sequence number 2) and truncated hBD2 (sequence number 7), for use in reducing the risk of developing COPD in a subject by oral or pulmonary administration of the composition.

Images