WO2018222581A1

WO2018222581A1 - Methods and formulations for administering beta glucan

Info

Publication number: WO2018222581A1
Application number: PCT/US2018/034862
Authority: WO
Inventors: Jesse D. Keicher
Original assignee: Resinnate Therapeutics Llc
Priority date: 2017-05-29
Filing date: 2018-05-29
Publication date: 2018-12-06

Abstract

The present invention is directed to aerosolized b-glucan, aerosol formulations comprising b-glucan and methods for the treatment of chronic lung infection and/or pulmonary fibrosis in a subject wherein excess M2 polarized macrophages contribute to pulmonary disorders

Description

METHODS AND FORMULATIONS FOR ADMINISTERING BETA GLUCAN

FIELD OF THE INVENTION

The present invention is directed to b-glucan aerosol compositions and formulations and methods of administering b-glucan by aerosolization to the upper and lower respiratory tract of a subject in an amount effective to treat, acute and or chronic pulmonary infections and pulmonary fibrotic diseases in which excess M2 polarized macrophage- have been shown to play a role, in part, in the progression or worsening of the pulmonary disease or disorder.

BACKGROUND

Beta-glucan

Beta glucans (also referred to herein as "β-glucans" or "b-glucans") are a heterogeneous group of natural polysaccharides, consisting of D-glucose monomers linked by a β- glycosidic bond. They function as important structural elements of the cell wall and also serve as energy storage in bacteria, fungi including yeast, algae, and plants (e.g. barley and wheat), while they are absent in vertebrate and invertebrate tissue. The individual glucose subunits are primarily linked either by (1,3)-β, (1,4)-β, or (1,6)-β glycosidic bonds. In most cases, β-glucans exhibit a uniformly constructed backbone of various lengths with side-chains of D-glucose attached by (1,4)-β, or (1,6)-β bindings.

Many, but not all, β-glucans are able to modulate immune functions. These properties mainly depend on the primary chemical structure of the β-glucans. Cellulose for example, a (l,4)^-linked glucan, does not exhibit immune modulatory effects. In contrast, β- glucans derived from fungi and yeast, which consist of a (l,3)^-linked backbone with small numbers of (l,6)^-linked side chains, are essentially known for their "immune modulating" effects [Bohn and BeMiller, Carbohydr Polym 1995;28(1):3-14].

Biologically active β-Glucans may consist of a backbone of β(l→3)-linked β-D- glucopyranosyl units with, for example, β(1→6) linked and/or β(1→4) linked side chains of varying distribution and length. Beta-glucan, poly-(l,6)^-D-glucopyranosyl-(l,3)^- D-glucopyranose ("PGG-glucan") is one example. Since their discovery in the late 1980's beta-glucans have been studied extensively. [Soltanian et al., Crit Rev Microbiol 2009;35: 109-138, Lehtovaarra et al., J Agric Food Chem 2011;59:6813-6828; Thompson et al., Expert Rev Anti Infect Ther 2010;8:339-352]. β-glucans are classic pathogen- associated molecular patterns (PAMP) and are recognized by particular cells of the innate immune system of vertebrates, as well as invertebrates. [Janeway, C.A. Jr. Immunol. Today 1992; 13 : 11-16]. Therapeutic applications that have been investigated include antitumor, infectious disease, serum cholesterol reduction, and wound healing.

In addition to enhancing antimicrobial activity, certain beta-glucans, are capable of "priming" the host immune response when administered at therapeutic doses without directly stimulating the production and secretion of proinflammatory cytokines at clinically significant levels. For example, PGG-glucans have been shown to enhance antimicrobial activity without significant induction of pro-inflammatory cytokines such as T Fa or IL-Ιβ [Michalek et al., J Leukoc.Biol 1998;64:337-344]; U.S. Patent No. 5,783,569; Babineau, et al., Ann. Surg. 1994;220:601-609, Babineau, et al., Arch Surg 1994b; 129: 1204-1210]. Similarly, it is reported that some heavy molecular weight beta glucans have been shown to enhance antimicrobial activity without significant induction of pro-inflammatory cytokines such as TNFa or IL-1B [U.S. Patent No. 7,022,685]. Moreover, freely soluble, non-immobilized beta-glucans show less induction

inflammatory cytokines, such as TNFa. [Michalek et al., J Leukoc.Biol 1998;64:337- 344].

PGG-glucan

PGG-glucan, poly-(l,6)-P-D-glucopyranosyl-(l,3)-P-D-glucopyranose, is generally produced as a highly purified pharmaceutical grade, water-soluble, glucose polymer. Specifically, PGG-glucan is the alkali soluble fraction of whole beta glucan particles (WGP) derived from yeast Saccharomyces cerevisiae. One example of PGG-glucan is Betafectin® (Alpha-Beta Technology, Worcester, MA), having a weight-average molecular weight value of around 150 kD, predominantly compromised of triple helices, derived from yeast Saccharomyces cerevisiae (R4 deposit made in connection with U.S. Pat. No. 4,810,646; Agricultural Research Service No. NRRL Y- 15903 and R4 Ad). A second example of PGG-glucan is Imprime-PGG® (Biothera Pharmaceutical Inc. Eagan, MN, USA) also having an average molecular weight value around 150 kD [Halstenson et al., Invest New Drugs 2016;34:202-215]. PGG-glucan® is composed of p-(l-3)-linked glucose backbone with P-(l-6)-linked β-(1-3) branches. Its preparation is described in U.S. Patent No. 5,622,939 and U.S. Patent No. 5,817,643. PGG-glucan may be formed from starting material that includes glucan particles, for example, whole glucan particles described by U.S. Patent No. 4,810,646, U.S. Patent No. 4,992,540, U.S. Patent No. 5,082,936 and/or U.S. Patent No. 5,028,703, which are hereby incorporated by reference. In another example, PGG-glucan may be formed from whole glucan particles such as WGP 3-6® from Biothera Pharmaceutical Inc. (Eagan, MN, USA).

When PGG-glucan is introduced into an animal host, it is identified as a pathogen and is taken up by macrophages and processed into small bioactive fragments. These bioactive fragments are then secreted and recognized by the β2 integrin complement receptor 3(CR3; CDl lb/CD18). CR3 is expressed on innate cells such as neutrophils, monocytes, macrophages, dendritic cells (DC), and natural killer (NK) cells, as well as lung epithelial cells and at lower levels on adaptive immune cells such as CD8 (killer T-cells) and CD4 (helper T-cells) [Muto et al., J Clin Immunol 1993; 13 : 175-184]. Collectively, host cell recognition triggers a cascade of responses that fully activates innate and adaptive host defenses for a coordinated attack against infection. Unlike other beta-glucan

preparations, PGG-glucan does not induce pro-inflammatory cytokines such as IL-Ιβ and TNF in vitro or in vivo [U.S. Patent No. 5,783,569; Babineau, et al., Ann. Surg.

1994;220:601-609, Babineau, et al., Arch Surg 1994b;129: 1204-1210]. Despite the absences of inducing pro-inflammatory cytokines such as IL-Ιβ and TNFc^ it primes or enhances immune cells antimicrobial activity against bacteria and other stimuli [Bleicher and Mackin. J. Biotechnol. Healthcare 1995;2:207-222; Adams et al., J. Leukoc. Biol. 1997;62:865-873].

PGG-glucan enhances non-specific resistance to infectious challenge. Improved survival rates and decreased bacterial burden have been demonstrated in vivo against multiple pathogens including, Gram -positive bacteria (e.g., Streptococcus pneumonia and planktonic or MDR Staphylococcus aureus), Gram-negative bacteria (e.g., Escherichia coli), mycobacteria, polymicrobial peritonitis, Plasmodium berghei, viruses (e.g., influenza), and fungi (e.g., C. albicans) [DiLuzio et al., Cancer Immunol Immunother . 1979;6:73-79; Onderdonk et al., Infect. Immun. 1992;60: 1642-1647; Tzianabos et al., Ann. N. Y. Acad. Sci. 1996;797:285-287; Cisneroset al., Infect. Immun. 1996;64:2201- 2205; Liang et al., Int J Immunopharmacol. 1998;20(11):595-614; Hetland, 1998, 2000; Kernodle et al., Antimicrob. Agents Chemother. 1988;42:545-549; Kaiser et al.,

Antimicrob Agents Chemother. 1998;42(9):2449-51]. For example, in a rat model of polymicrobial peritonitis, PGG-glucan reduced mortality from 75% down to only 8%. Furthermore, this protective effect could be transferred to naive mice with spleen cells, spleen lysates, peripheral blood leukocytes or serum from PGG-glucan treated animals [Cisneros et al., Infect. Immun. 1996;64:2201-2205]. In this same animal model PGG- glucan enhanced antimicrobial therapy and significantly reduced bioburden over the use of antibiotics alone.

PGG-glucan as anti-infective prophylactic agents in clinical studies

PGG-glucan has been systemically administered to over 1000 subjects in Phase I to Phase III clinical trials for the prevention of infection in patients undergoing high-risk surgery [Babineu et al., Arch Surg 1994; 129: 1204-1210; Babineu et al., Ann. Surg.

1994;220:601-609, Dellinger et al., Arch Surg. 1999; 134(9):977-83]. In the clinic, ex- vivo testing of patients' peripheral blood against S. aureus and E. coli with or without prior intravenous administration of PGG-glucan confirmed that while effectively enhancing innate cell effector responses of microbial killing and phagocytosis, PGG- glucan does not induce the production of systemic pro-inflammatory cytokines such as TNFa or IL-Ι β. [Babineu et al., Arch Surg 1994; 129: 1204-1210]. In this clinical study, multiday day intravenous dosing of PGG-glucan to healthy adults was safe and well tolerated up to 2.25 mg/kg [Babineau, T et al., Ann. Surg. 1994;220:601-609]. Clinical assessments of physical conditions, vital signs and electrocardiograms showed no clinically significant abnormalities, including the absence of fever, nausea, myalgia or bone pain. These observations suggest that the β-glucan preparations of PGG-glucan are free from the pyrogenic and inflammatory effects common to many PAMPs as well as many other preparations of b-glucan. PGG-glucan preparations showed the potential to "prime" the immune system for enhanced antimicrobial activity without directly stimulating the release of potentially harmful pro-inflammatory cytokines.

In a Phase III study for the prevention of infection and sepsis in high risk, non-colorectal surgery, PGG-glucan decreased serious postoperative infection and death by 39%

(p<0.02). An even more pronounced decrease in serious postoperative infection and death was observed in subjects who were malnourished, 44% (p < .001). All doses were well tolerated and serious adverse events were not statistically different between the PGG- glucan treated groups and placebo, confirming the safety of systemically delivered PGG- glucan at therapeutic doses [Dellinger et al., Arch Surg. 1999; 134(9):977-983].

Soluble beta glucans, with properties similar to PGG-glucan, can also be derived from other sources from other (e.g. other fungi, bacteria, and plants). Moreover, synthetic beta glucans are also known in the art, such as the oligosaccharides disclosed in U.S. Patent 7,365,191. Methods for extracting beta glucans and purifying beta glucans to varying degrees are known in the art. [Lee et al., Biosci Biotechnol & Biochem 2001; 65. 837- 841].

Administration of beta-glucans

Due to the poor oral bioavailability of beta-glucans in general, the clinical use of PGG- glucan has been limited to intravenous administration. For example, Rice and colleagues measured the oral bioavailability in mice for several isolated and purified β-glucan preparations and determined oral bioavailability to range from 0.5 to 5% (Lehne et al. Clin Exp Immunol. 2006; 143(1): 65-69; Rice et al. J Pharmacol Exp Ther

2005;314(3): 1079-1086]. Very low oral bioavailability for β-glucans in general, has been corroborated by others and published elsewhere [Sandvik et al. Clin Exp Immunol 2007; 148: 168-177]. In a Phase I study, Lehne and colleagues administered β-glucans orally to healthy individuals at doses up to 400mg/day, however, repeated measurements for the presence of β-glucans in serum revealed no systemic absorption [Lehne et al., 2006; 143 :65-69].

Intravenous formulations may be advantageous for some applications, however, there are several drawbacks with intravenous glucan delivery for the treatment of pulmonary infections. PGG-glucan elicits it function by binding to the β2 integrin complement receptor 3 (CR3; CD 1 lb/CD 18), present on innate immune cells such as macrophages, monocytes, dendritic cells, neutrophils, and NK cells which prime host cellular defense mechanisms for enhanced defense against infection. In patients with underlying respiratory diseases, infections such as chronic Pseudomonas aeruginosa grow in the lower respiratory tract of the endobronchial space. For intravenous administration of PGG-glucan to be effective in these patients, therapeutic, effective concentrations of PGG-glucan must be achieved on the luminal side of the lung within the endobronchial secretions to bind CR3 and activate resident innate immune cells, primarily resident alveolar macrophages and neutrophils to facilitate the clearance of infection. However, like the gut mucosa, absorption into the lumen of the lung is restricted due to

physiochemical properties limiting the effectiveness of intravenous administration. Hydrophilic molecules and molecules greater than 25 kD are known to have poor penetration across mucosal barriers. PGG-glucan is both highly water soluble

(hydrophilic) and has a molecular weight much greater than 25 kD (about 95 to 250 kD). Furthermore, in patients with underlying chronic lung disease, for example CF, a thick dehydrated mucus layer presents another challenge to achieve effective concentrations of PGG-glucan on the luminal side of the lung within the endobronchial space after intravenous administration. [Olsson et al., Controlled Pulmonary Drug Delivery Chp. 2 "Pulmonary Drug Metabolism, Clearance, and Absorption" pp 21-50, Springer, New York, 2011]. Thus, both oral and intravenous routes of PGG-glucan administration are not ideal methods to safely achieve therapeutic concentrations in the lung.

Immune response and infection in chronic lung disease

When the immune system detects a pathogen in the lung, epithelial cells and resident alveolar macrophages in the lung respond to the infectious exposure by releasing proinflammatory mediators that recruit additional inflammatory immune cells to the lungs. In healthy adults, theses proinflammatory mediators trigger cellular effector responses such as microbial killing and phagocytosis from innate cells such as neutrophils and macrophages which are effective in defending against opportunistic infections such as P. aeruginosa. Alveolar macrophages are the main phagocyte population in the lumen of the lungs of healthy adults and are the first responders to invading pathogens.

In addition to host defense, macrophages play a critical role in the homeostasis and repair of nearly every tissue in the body and perform myriad tissue-specific functions.

Depending on the microenvironment and stimulus, macrophages can be polarized into different states with classically activated macrophages (Ml macrophages) or alternatively activated macrophages (M2 macrophages) representing the two polar extremes. Ml macrophages are important for elimination of infectious pathogens, while M2

macrophages have anti-inflammatory properties and are involved in resolution of inflammation and tissue repair. In addition, classically activated macrophages (Ml macrophages) promote Thl type T-cell differentiation and proliferation, characterized by IFN-γ production and induction of antimicrobial effector T-cell responses. In contrast, M2 macrophages release mediators that promote Th2 type T-cell differentiation and proliferation. Th2 pattern cells are prone to produce anti-inflammatory mediators such as IL-10 and support the resolution of inflammation and support the process of tissue repair but are also responsible for promoting fibrosis as well as allergic type inflammation such as the expression of IL-4, IL-5, IL-9, and IL-13 [Berger, £Μ/2000;321(7258):424; Duffield et al., Annu Rev Pathol. 2013;24(8): 241-276].

Immune response and fibrosis in chronic lung disease

In many subjects with chronic lung disease, the immune system is ineffective at defending against pulmonary infection, despite a robust inflammatory response and leukocyte recruitment to the lungs. Regardless of the cause of the lung disease (e.g. genetic factors, such as the CFTR genetic mutation in cystic fibrosis, or extrinsic factors, as with persistent inflammation), host defense is compromised and cellular inflammatory responses to infectious stimuli are dysregulated. Consequently, these subjects have increase susceptibility to recurrent episodes of respiratory infections. Due to

compromised host defense mechanisms in the lungs, recurrent infections eventually become chronic in nature. Chronic respiratory infections punctuate and accelerate the pathological manifestations of diseases. Moreover, certain bacteria such as Streptococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), non-tuberculosis mycobacteria (NTM), Burkholderia cepacia complex (mostly B. multivorans and B. cenocepacia), Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, and or

Pseudomonas aeruginosa have developed ways of surviving and thriving in the lungs of subjects with inflammatory lung diseases.

In subjects with CF, it has been shown that macrophages are less capable of microbial killing and the clearance of infections. Furthermore, the inflammatory response of macrophages to infectious stimuli are dysregulated and inappropriate for effective microbial killing. Ineffective macrophage efferocytosis, result in the accumulation of cellular debris such as the large number of dead or dying neutrophils that act as danger associated molecular patterns (DAMPs) and serve as additional sources of inflammation. Decreased airway surface liquid and a thick sticky dehydrated mucus layer, a

consequence of the CFTR mutation in the lung epithelium, is yet another source of inflammation. The persistent release of cytokines from the lung epithelium and resident immune cells in the lung induce a local cytokine milieu that favors alternatively activated macrophages (M2 macrophages). This in turn, compromises their capacity and efficacy in performing various antimicrobial functions such as host defense against infection and the removal of dead and dying cells and other tissue debris.

In CF, the contribution of macrophage polarization to the lung disease is still unclear, however, several descriptive studies suggest that macrophages from patients with chronic P. aeruogenosa infection transition from classically activated Ml macrophages, to that of alternatively activated M2 macrophages with concomitant Th2 type T-cell responses (Hartl D et al., J Allergy Clin Immunol 2006; 117:204-211 2006, Grasemann H et al, Am J Respir Crit

Care Med 2005; 172: 1523-1528, Murphy BS et al., J Cyst Fibros 2010;9 314-322 2010),

Persistent lung inflammation comprising a milieu of cytokines promote the differentiation of resident and alveolar macrophages to an alternatively activated M2 state. This in turn, promotes anti-inflammatory mediators in attempt to resolve inflammation and promote tissue-repair. Consequently however, macrophages that are persistently in an alternatively activated M2 macrophages promote excessive tissue repair leading to tissue fibrosis and are ineffective in protecting the host from infection or the removal of dead and dying neutrophils and other tissue debris.

In support of this theory, Murphy et al, for example, investigated the activation states of macrophages from P. aeruginosa infected patients and showed that lung macrophages were more M2 polarized in patients infected with P. aeruginosa compared to non- infected CF patients. Furthermore, compared to non-infected CF patients, macrophages from CF patients with P. aeruginosa were less effective in ability to properly regulate the inflammatory response and to recognize and clear bacteria. [Murphy et al., Journal of Cystic Fibrosis 2010;9:314-322]. In addition to their role in innate immunity,

macrophages also influence the type of adaptive T-cell response. Ml polarized macrophages promote the activation and proliferation of antimicrobial Thl type T-cell and T-cell responses, necessary for microbial clearance. M2 polarized macrophages promote the activation and proliferation of Th2 type T-cells and T-cell responses, that facilitate the resolution of inflammation and tissue repair. Haiti and colleagues reported that BAL fluid from patients with CF infected with P. aeruginosa had higher IL-4 and IL-13 concentrations, indicative of a Th2 type immune response and lower levels of IFNy (associated with Thl), compared with uninfected CF patients. Furthermore, they found that BAL fluid levels of IL-4 and IL-13 (Th2 cytokines) correlated inversely with forced expiratory volume in one second, FEV1 values. FEV1 is a test performed to evaluate lung function. It measures the amount of air a person can forcefully exhale in one second. Patients with greater FEV1 scores have better lung function and clinical outcomes. FEV1 values are the primary endpoint used in clinical studies of CF medications seeking FDA approval. [Haiti et al, J Allergy Clin Immunol 2006; 117(1):204-211]. Additional studies have shown that, in response to P. aeruginosa outer membrane proteins, monocytes from P. aeruginosa infected CF patients produce more IL-4 and less IFNy than monocytes from non-infected patients [Moser et al., APMIS 2000; 108:329-335]. Taken together, the local cytokine milieu of the lung in CF patients is skewed to favor macrophage antiinflammatory activities and tissue repair responses. In contrast, their abilities to recognize and effectively kill and clear microbial pathogens are compromised, contributing to the persistence of P. aeruginosa. Once established in the lungs of infected CF patients, P. aeruginosa is virtually impossible to eradicate, despite the use aggressive antimicrobial therapy.

Macrophage transition between activation states is a normal process during infection and infection resolution. When persistent however, alternatively activated macrophages (M2 macrophages) and their associated Th2 T-cell responses can lead to excessive tissue repair processes that can lead to tissue fibrosis. Fibrosis is a pathological feature of most chronic inflammatory diseases. Fibrosis is characterized by the overgrowth, hardening, and/or scarring of various tissues and is attributed to excess deposition of extracellular matrix components including collagen (T.A. Wynn J Pathol. 2008;4(2): 199-210).

Macrophages, specifically M2 macrophage and Th2 T-cells play predominant roles in the fibrotic processes. Fibrosis is the final, common pathological outcome of many chronic inflammatory diseases. Although collagen deposition is an indispensable and, typically, reversible part of wound healing, normal tissue repair can evolve into a progressively irreversible fibrotic response if the tissue injury is severe or repetitive or if the wound- healing response itself becomes dysregulated (TA Wynn, Nat Med. 2012; 18(7): 1028- 104). In certain fibrotic diseases, there is an excessive accumulation of fibrous connective tissue (components of the extracellular matrix (ECM) such as collagen and fibronectin), in and around inflamed or damaged tissue, which can lead to permanent scarring, organ malfunction and, ultimately, death in diseases, not limited to, cystic fibrosis (CF), interstitial lung disease (ILD), idiopathic pulmonary fibrosis (IPF), usual interstitial pneumonia (UIP), acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), as well as other chronic inflammatory diseases. Alternative activation of macrophages (M2 macrophages), are associated with the maintenance and progression of ILD, notably IPF/UIP [Murray L et al., Plos One 2010;5(3) e9683]. They are also the predominant phenotype of activated macrophage found in the lungs of IPF/UIP patients [Hancock A, et al., Am J Respir Cell Mol Biol 1998;18:60-65].

Furthermore, macrophage phenotype also plays a predominant role in the pathogenesis of pulmonary alveolar proteinosis (PAP). PAP is an autoimmune disease characterized by high levels of autoantibody to GM-CSF that ultimately results in the accumulation of excess lung surfactant in the alveoli. This excess lung surfactant in the alveoli results in varying degrees of respiratory insufficiency and myeloid cell dysfunction which in turn, increases risk of infection [Carey and Trapnell, Clin Immunol. 2010; 135(2):223-235]. Pulmonary surfactant is a natural substance comprised of lipids and proteins that is produced in alveolar epithelial cells-type II (AEC-II) and is vital to lung structure and function. It acts at the alveolar air-liquid-tissue interface by reducing surface tension and preventing alveolar collapse. Surfactant also contributes to pulmonary host defense by opsonization of microbial pathogens and by direct microbial killing. The critical role of GM-CSF in PAP was discovered when it was observed that mice deficient in

granulocyte/macrophage-stimulating factor (GM-CSF) were found to develop a phenotype virtually identical to PAP in humans [Carey B et al. Clin Immunol. 2010 May; 135(2): 223-235.; Dranoff et al., Science 1994;264:713-6; Stanley et al., Proc Natl Acad Sci USA 1994;91 :5592-6]. Results also identified a critical role for GM-CSF in the regulation of surfactant homeostasis, alveolar macrophage maturation and function, lung host defense, and innate immunity. Granulocyte macrophage colony-stimulating factor (GM-CSF) is associated with Ml or classically activated macrophage and is required for maturation of alveolar macrophages, a critical innate cell involved in the monitoring and regulation of the alveolar surfactant layer. Proper regulation of the alveolar surfactant layer is critical to maintaining a low surface tension and enabling the opening of alveoli with each breath. The lack of functionally active GM-CSF correlates with highly elevated concentrations of M-CSF in the lungs of PAP patients and in GM-CSF KO mice. M-CSF has been associated with M2 or alternative macrophage activation, and in models of pulmonary fibrosis, M-CSF also contributes to tissue resorption and fibrosis Several studies suggest that in the absence of GM-CSF, excess M-CSF in PAP may redirect alveolar macrophage activation state toward M2 which is ineffective in monitoring and regulating alveolar surfactant layer (Bonfield T et al., J Leukoc

Biol. 2006;9(1): 133-9).

Cystic Fibrosis

Cystic fibrosis is an autosomal recessive disease caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene. The gene defect in CF results in a myriad of medical problems for the patient, the most meddlesome clinical feature, chronic pulmonary infection with Pseudomonas aeruginosa. Ultimately, 80 to 95% of patients with CF succumb to respiratory failure brought on by chronic bacterial infection and concomitant airway inflammation (J Lyczak Clin Microbiol Rev. 2002;(2): 194-222). The CFTR mutation in the airway epithelial cells of CF patients limits the transport of Cl- ion across the airway epithelium and into the lumen. The resulting osmotic imbalance causes the volume of airway surface liquid to be reduced resulting in a concentrated viscous and sticky mucus layer that impairs normal mucocilliary clearance (MCC) and traps bacteria and provides a favorable environment for infection. MCC plays an important role in removing inhaled bacteria from the airways. Persistent infection and concomitant inflammation skews macrophage phenotype towards M2 with results in the unregulated accumulation of profibrotic components leading to the destruction of alveolar architecture and gradually reducing lung compliance and compromising gas exchange. Novel methods to eradicate chronic Pseudomonas aeruginosa remains a major unmet medical need.

In CF, the CFTR mutation in the airway epithelial cells and dehydrated mucus promote inflammation that recruit a massive influx of neutrophils into the airways which, unlike healthy adults where in 80% of the cells are macrophages, in CF, neutrophils make up about 80%) of the cells in the lumen of the lung. Normally, recruited neutrophils are highly phagocytic and cooperate with lung macrophages to fight and clear infection. However, in lung disease where chronic lung infections are present, such as P.

aeruginosa in CF subjects, neutrophil responses are dysregulated and ineffective.

Excessive neutrophil degranulation which occurs in CF causing lung tissue damage and the excess release of proteases impede microbial killing and clearance of resident phagocytes, mostly macrophages. Furthermore, excess ROS/RNS release destroys lung tissue promoting excessive tissue repair functions that support fibrosis and loss of lung function (Rada B et al., Pathogens. 2017;6(1): 10).

Current treatment of fibrosis

Only a few treatments have been FDA approved to slow the progression of fibrotic diseases. Nintedanib (Ofev®): Nintedanib is an anti-fibrotic drug that is approved to treat IPF in the United States. In clinical trials, nintedanib has been shown to slow the decline in lung function in mild-to-moderate IPF. It is taken by mouth twice a day. Pirfenidone (Esbriet®, Pirfenex®, Pirespa®) is an anti-fibrotic and anti-inflammatory drug approved to treat IPF in the United States, EU, Canada, and Asia. In clinical trials, pirfenidone has been shown to slow progression of mild-to-moderate IPF. It is taken by mouth three times a day. Every individual diagnosed with pulmonary fibrosis has a unique experience with the disease and there is no "standard" or expected clinical course. Furthermore, these drugs are only effective in slowing the progression of the disease. A number of attempts have been made to treat fibrosis with immunosuppressants such as

corticosteroids but with limited success. Currently, there are no effective therapies that inhibit or reverse the fibrotic process.

Pseudomonas aeruginosa and other pathogens in CF patients

Perhaps the most serious clinical outcome of the CFTR defect is the eventual succumbing to chronic pulmonary infection with Pseudomonas aeruginosa. The virulence factors of P. aeruginosa including its ability to acquire a mucoid phenotype and utilize quorum- sensing signals to form biofilms, render this microbe resistant to antibiotic killing in the contexts of a host immune defenses system that is compromised in subjects with CF and other chronic lung diseases. Moreover, multidrug-resistant P. aeruginosa (defined as resistance to all drugs in more than one antimicrobial drug classes e.g. fluoroquinolones; beta-lactam antibiotics; and aminoglycosides) are associated with greater lung disease severity, more rapid decline in FEV1, increased use of intravenous antibiotics, and increased hospital costs and frequency of medical visits (Stefani S et al., Int J Med Microbiol. 2017:307(6):353-362; Nathwani D et al., Antimicrob Resist Infect Control. 2014;3 :32; Morales E et al., BMC Health Serv Res. 2012;12: 122; Aloush V et al., Antimicrob. Agents Chemother. 2006;50(l):43-48). The presence of antibiotic-resistant P. aeruginosa not only limits the potential antimicrobial treatment options but can also preclude patients from eligibility for lung transplantation and other potentially life-saving modalities. An additional concern is that the introduction of new broad-spectrum antimicrobial agents to treat resistant P. aeruginosa could be contributing to the emergence of other, intrinsically antibiotic resistant pathogens that may be associated with increased morbidity in CF (i.e. methicillin-resistant Staphylococcus aureus (MRSA), non-tuberculous mycobacteria (NTM), as well as B. cepacia complex, S. maltophilia, and A. xylosoxidans)

In addition to P. aeruginosa, emerging antibiotic resistant pathogens such as methicillin- resistant S. aureus (MRSA), Burkholderia cepacia complex (BCC), and non-tuberculous mycobacteria (NTM) are increasingly being detected in the sputum of CF patients.

Similar to P. aeruginosa, CF patients with chronic MRSA infection have more hospitalizations, faster decline in lung function, and reduced life expectancy.

Furthermore, Burkholderia bacteria, often detected as a co-infection with P. aeruginosa, can result in "cepacia syndrome," characterized by a rapid decline in lung function, necrotizing pneumonia, bacteremia, and sepsis (Kooi C et al., Microbiology (2009), 155, 2818-2825). Pulmonary disease caused by NTM requires lengthy and complicated treatment regimens with daily oral and intravenous antibiotics recommended for a period of 16-17 months [Floto AR et al. Thorax 2016;71 :88-90]. Furthermore, Burkholderia cepacia complex, Pseudomonas aeruginosa, and Staphylococcus aureus have all been shown to spread between patients with CF and as a result, hospitalized CF patients must be quarantined from one another [Holby and Koch. Thorax 1990;45:881-884]. Like Pseudomonas aeruginosa, recurrent and chronic infection with these pathogens is a major contributor to the accelerated progression of disease pathology and mortality. Several other bacterial species including Stenotrophomonas maltophilia, Alcaligenes

xylosoxidans, Haemophilus influenzae, Stenotrophomonas maltophilia, and Aspergillus fumigatus, also contribute to morbidity and mortality [Ftoiby and Pressler. Eur Respir Mon 2006;35: 66-7].

Current treatment of infection (P. aeruginosa and other pathogens) in CF patients

Management of chronic infection of pathogens such as Pseudomonas aeruginosa in patients with cystic fibrosis largely consists of chronic administration of inhaled antibiotics that reduce microbial burden, reduce exacerbations, and increase lung function [Shteinberg and Elborn, Adv Ther. 2015;32(1): 1-9]. Tobramycin and aztreonam are FDA-approved for the management of P. aeruginosa in cystic fibrosis patients. The EMA has also approved colistin (polymyxin E) in addition to tobramycin and aztreonam. Regimens typically consist of a single antibiotic administered 2 to 3 times daily with on/ off cycles every 28-days. Continuous antibiotic treatment is also an option and consists of cycling every 28 days between two antibiotics which are administered 2 to 3 times daily [Flume PA, et al., Am J Respir Crit Care Med 2007; 176:957-969; Am J Respir Crit Care Med. 2007;176(10):957-969].

Early and intensive therapy of airway infections with either oral or aerosolized antibiotic drug treatment is considered to be a major reason for the ongoing rise in life expectancy. Although drug treatment with aerosolized antibiotics has several therapeutic benefits over oral treatment, administration of these drugs with currently available nebulizer equipment takes up a significant amount of time daily, which is a major burden that negatively influences the quality of life and lowers adherence to therapy.

Additionally, there are several FDA-approved expectorant agents for CF that work by reducing the viscosity of the mucus layer. Expectorants are meant to increase the volume of airway water or secretion in order to increase the effectiveness of sputum removal through cough. Two examples of expectorant agents include inhaled hypertonic saline and mannitol dry powder (Bronchitol™).

Mucolytics are medications that change the biophysical properties of secretions by degrading the mucin polymers, DNA, fibrin, or F-actin in airway secretions. For example, recombinant human DNase I (rhDNase) or Dorinase alpha (Pulmozyme) has been shown to de-polymerise DNA and thereby reduce the in vitro viscoelasticity of sputum in patients with cystic fibrosis allowing for the removal of sputum by chough. [Shah PL et al, Thorax, 1996;51(2)): 119-125]. Once established, Pseudomonas aeruginosa it is virtually impossible to eradicate despite advances in treatments, as well as the aggressive use of antibiotics. Chronic Pseudomonas aeruginosa infection is a major contributor to irreversible lung tissue damage and progression of the disease.

Greater than 90% of CF patients die prematurely due to respiratory failure because of chronic infection, principally with Pseudomonas aeruginosa [Doring et al., Eur Respir J 2000; 16:749-767]. There is a great need for improved treatments for patients with chronic lung infections, particularly, treatments for the eradication of chronic

Pseudomonas aeruginosa infection in patients with CF.

SUMMARY OF THE INVENTION The present invention is directed to aerosolized b-glucan, aerosol formulations comprising b-glucan and methods for the treatment of chronic lung infection and/or pulmonary fibrosis in a subject wherein excess M2 polarized macrophages contribute to pulmonary disorders, in particular, for patient suffering from a disease selected from the group consisting of cystic fibrosis (CF), chronic obstructive pulmonary disorder (COPD), bronchiectasis (BE), interstitial lung disease (ILD), idiopathic pulmonary fibrosis (TPF), usual interstitial pneumonia (UIP), acute respiratory distress syndrome (ARDS), or pulmonary alveolar proteinosis (PAP).

In one embodiment of the invention an aerosolized, non-inflammatory b-glucan is provided, preferably the beta-glucan is a freely soluble non-immobilized beta-glucan, most preferably PGG-glucan.

In another embodiment of the invention, an aerosol formulation in dry powder form is provided, the formulation comprising a non-inflammatory b-glucan composition is provided, preferably comprising a composition of PGG-glucan.

In another embodiment of the invention, the aerosol formulation comprising b-glucan dry powder is reconstituted into solution containing some amount of NaCl for inhalation by nebulization.

In another embodiment of the invention, the aerosol formulation comprising b-glucan dry powder composition is reconstituted in deionized water for inhalation by nebulization.

In another embodiment of the invention, the aerosol formulation comprises between about O. lmg to about 250 mg of PGG-glucan, or preferably between about O. lmg to about 50 mg of PGG-glucan.

In another embodiment of the invention the aerosol formulation comprising b-glucan has a pH that is neutral or acidic, preferably having a pH range between 7.0 and 5.5, or between 6.5 to 6.0, preferably near 7.0.

In another embodiment of the invention the aerosol formulation comprises b-glucan in an isotonic or hypotonic aqueous solution. In another embodiment of the invention the aerosol formulation comprising b-glucan is in aqueous solution containing between 0.1% (w/v) to 0.9% (w/v) NaCl, having an osmolarity of between about 34 to about 310.

In another embodiment of the invention the aerosol formulation comprising b-glucan is a dry powder containing between 0.225%) (w/v) and 0.45% (w/v) NaCl and having an osmolarity between about 77.5 and 155 when reconstituted for use in 1 mL to 5 mL and preferably, in 3mL to 5 mL water with or without pH buffer and/or preservatives.

In another embodiment of the invention the aerosol formulation comprising b-glucan is administered to the endobronchial space of a subject in need thereof, using a jet or ultrasonic nebulizer able to produce aerosol particle sizes which are predominantly between 0.3 and 5 microns, preferably between 0.5 to 5 microns, and more preferably between 1.0 and 5.0 microns.

In another embodiment of the invention the aerosol formulation comprising b-glucan is delivered by a nebulizer with a delivery efficiency of 40%, more preferably 60%> or greater to the endobronchial space of a subject.

In another embodiment of the invention an aerosol formulation comprising b-glucan is provided wherein the aerosol formulation of b-glucan is a spray-dry powder amenable to inhalation using a dry powder inhaler (DPI).

In another embodiment of the invention an aerosol formulation comprising b-glucan is provided wherein the b-glucan is PGG-glucan and is present in the spray-dry powder composition in an amount between 10%> and 90% by weight of the composition, or between 10% to 80%, preferably 10% to 70%, and more preferably 30% to 70%.

In another embodiment of the invention an aerosol formulation comprises b-glucan in a spray-dry powder composition comprising a bulking agent suitable for medicinal formulations delivered by DPI. Preferably the bulking agent is a mono, di or tri- saccharide selected from the group consisting of: lactose, mannitol, or trehalose, more preferably the saccharide is trehalose and b-glucan is PGG-glucan. In another embodiment of the invention an aerosol formulation comprises b-glucan in a spray-dry powder composition comprising the bulking agent present in an amount between 10% and 90% by weight of the composition, or between 10%> to 80%>, preferably 10% to 70%, and more preferably 30% to 70% by weight.

In another embodiment of the invention an aerosol formulation comprises b-glucan in a spray-dry powder composition further comprising a salt preferably selected from the group consisting of NaCl, NaBr, or Nal, more preferably NaCl wherein the salt is present in an amount less than 25%, preferably less than 10%> by weight.

In another embodiment of the invention an aerosol formulation comprises b-glucan in a spray-dry powder composition comprising a pH buffer selected from the group including, but not limited to, phosphate, acetate, citrate, TRIS, arginine, and histidine, preferably phosphate buffer, in an amount less than 25%, preferably less than 15%> by weight.

In another embodiment of the invention an aerosol formulation comprises b-glucan in a spray-dry powder composition comprising one or more pH adjusters. Suitable pH adjusters for the present invention include, but are not limited to, hydrochloric acid, sodium hydroxide, lactic acid, tartaric acid, succinic acid, and any combination thereof.

In another embodiment of the invention, an aerosol formulation comprises b-glucan in a spray-dry powder composition comprising a hydrophobic amino acid, wherein the b- glucan is preferably PGG glucan.

In another embodiment of the invention, an aerosol formulation comprises b-glucan in a spray-dry powder composition comprising a hydrophobic amino acid selected from the group consisting of: tryptophan, tyrosine, leucine, trileucine, or phenylalanine, preferably leucine wherein the hydrophobic amino acid is present in an amount less than 50%> or less than 30%) by weight of the composition, preferably the hydrophobic amino acid is present in an amount between about 1%> to 20% by weight of the composition.

In another embodiment of the invention an aerosol formulation comprises b-glucan in spray-dry powder composition wherein at least 90% of the of the spray-dry powder composition comprise of particles with a mass median aerodynamic diameter (MMAD) between about 0.3 microns and 10 microns, preferably between about 0.3 microns and 5 microns, more preferably between 0.5 microns to 5 microns, or between 1 micron to 5 microns.

In another embodiment of the invention an aerosol formulation comprises b-glucan in a spray-dry powder wherein the dry powder is encapsulated wherein the capsule is preferably comprising hydroxypropyl methylcellulose.

In another embodiment of the invention, an aerosol formulation comprises b-glucan in a dry powder composition having low water or low moisture content, preferably below about 10% by weight water, preferably below about 7% by weight water, and more preferably below about 5% by weight water.

In another embodiment of the invention an aerosol formulation comprises b-glucan in a spray-dry powder composition that is predominantly amorphous.

In another embodiment of the invention an aerosol formulation comprises b-glucan in a spray-dry powder composition wherein the pH of the spray-dry powder reconstituted in 3 mL to 5 mL of water is between 5.5 and 7

In another embodiment of the invention an aerosol formulation comprises b-glucan in a spray-dry powder composition wherein the salinity of the spray-dry powder reconstituted in 3 mL to 5 mL contains between 0.1% (w/v) to 0.9% (w/v) NaCl, having an osmolarity of between about 34 to about 310, preferably containing between 0.225% (w/v) and 0.45%) (w/v) NaCl and having an osmolarity between about 77.5 and 155.

In another embodiment of the invention an aerosol formulation comprises b-glucan in a spray-dry powder composition comprising dry powder that is substantially amorphous.

In another embodiment of the invention an aerosol formulation comprises b-glucan in a spray-dry powder composition wherein the pH of the spray-dry powder reconstituted in 3 mL to 5 mL of water is between 5.5 and 7, preferably about 7.0.

In another embodiment of the invention an aerosol formulation comprises b-glucan in a spray-dry powder having a tap density of greater than about 0.4 g/cm3.

In another embodiment of the invention, an aerosol formulation comprising b-glucan in a spray-dry powder composition is delivered to the endobronchial space of a subject in need thereof using a dry powder inhaler (DPI), preferably a DPI that provides a delivery efficiency of 40% or more, and more preferably a delivery efficiency of 60% or more.

In another embodiment of the invention, an aerosol formulation comprises b-glucan in a spray-dry powder composition comprising one or more pH adjusters including, but not limited to, hydrochloric acid, sodium hydroxide, lactic acid, tartaric acid, succinic acid, or any combination thereof.

In another embodiment of the invention a method for treatment of lung disease, preferably a chronic lung infection and/or pulmonary fibrosis and/or excess M2 polarized macrophage-mediated lung disease in a subject in need thereof is provided, the method comprising administering to the subject aerosolized b-glucan.

In another embodiment of the invention, a method for treatment of lung disease, preferably, a chronic lung disease in a subject in need thereof is provided, the method comprising administering to the subject aerosolized b-glucan, wherein the subject is suffering from a lung disease selected from the group consisting of: cystic fibrosis (CF), chronic obstructive pulmonary disorder (COPD), bronchiectasis (BE), interstitial lung disease (ILD), idiopathic pulmonary fibrosis (IPF), usual interstitial pneumonia (UIP), acute respiratory distress syndrome (ARDS), or pulmonary alveolar proteinosis (PAP).

In another embodiment of the invention, a method for treatment of lung disease in a subject, preferably a fibrotic lung disease, is provided, the method comprising

administering to the subject an aerosolized b-glucan, wherein the disease is selected from the group consisting of preferably, cystic fibrosis (CF), chronic obstructive pulmonary disorder (COPD), bronchiectasis (BE), interstitial lung disease (ILD), idiopathic pulmonary fibrosis (IPF), usual interstitial pneumonia (UIP), and acute respiratory distress syndrome (ARDS), or pulmonary alveolar proteinosis (PAP).

In another embodiment of the invention, a method for treatment of pulmonary alveolar proteinosis (PAP) in a subject in need thereof is provided, the method comprising administering to the subject aerosolized b-glucan.

In another embodiment of the invention, a method for treatment of lung disease, preferably infection in a subject in need thereof, is provided, the method comprising administering to the subject aerosolized b-glucan, wherein the disease is selected form the group consisting of cystic fibrosis (CF), chronic obstructive pulmonary disorder, (COPD) and bronchiectasis (BE), interstitial lung disease (ILD), idiopathic pulmonary fibrosis (IPF), usual interstitial pneumonia (UIP), acute respiratory distress syndrome (ARDS), or pulmonary alveolar proteinosis (PAP).

In another embodiment of the invention, a method for treatment of lung infection is provided, preferably P. aeruginosa infection in a subject in need thereof, preferably a subject suffering from cystic fibrosis (CF).

In another embodiment of the invention a method for treatment of pulmonary bacterial infection in a subject, preferably a subject with chronic lung disease, in need thereof is provided, the method comprising administering to the subject an aerosolized b-glucan, and preferably continuing treatment with continuous or episodic administration to prevent, reduce the frequency, magnitude, and or duration of infection or for the clearance or eradication of pulmonary bacterial infection.

In another embodiment of the invention, a combination therapy and method for treating pulmonary infection is provided wherein aerosolized b-glucan is administered as adjunct treatment to antibiotic therapy, preferably standard of care antibiotic therapy indicated for the specific bacterial infection or to reduce the use of standard of care antibiotic therapy, and more preferably where the antibiotic therapy is selected from the group of tobramycin, aztreonam, colistin, and vancomycin and the bacteria is preferentially selected from the group consisting of P. aeruogenosa or MRS A.

In another embodiment of the invention a method for treating pulmonary disease is provided wherein aerosolized b-glucan is administered to a subject once weekly, twice weekly, three times weekly, every other day, daily, or twice daily, preferably once or twice weekly, more preferably, once daily.

In another embodiment of the invention, a method for treating pulmonary disease wherein excess M2 polarized macrophages contributes in part to the worsening of the disease is provided wherein aerosolized b-glucan is administered to a subject suffering from an underlying chronic lung disease, and particularly a chronic lung disease selected from the group consisting of cystic fibrosis (CF), chronic obstructive pulmonary disorder, (COPD) and bronchiectasis (BE), interstitial lung disease (ILD), idiopathic pulmonary fibrosis (IPF), usual interstitial pneumonia (UIP), acute respiratory distress syndrome (ARDS) and pulmonary alveolar proteinosis (PAP).

In another embodiment of the invention, a composition for treating pulmonary disease preferably a disease wherein excess M2 polarized macrophages contributes in part to the worsening of the disease, is provided, the composition comprising a GM-CSF and beta- glucan, preferably a non-inflammatory beta-glucan such as PGG-glucan.

In another embodiment of the invention, a method for enhancing a therapy for treating a pulmonary disease, (for example infection, fibrosis and PAP), preferably a disease in which excess M2 polarized macrophages contributes to the worsening of the disease, is provided, wherein the therapy includes administering a composition comprising GM-CSF to a patient, the method of improving the efficacy of the therapy comprising coadministering a therapeutic amount of beta-glucan to with composition, preferably a noninflammatory beta-glucan such as PGG-glucan.

In another embodiment of the invention, a method for enhancing a therapy for treating a pulmonary disease (for example infection, fibrosis and PAP), preferably a disease in which excess M2 polarized macrophages contributes to the worsening of the disease, is provided, wherein the therapy includes administering a composition comprising GM-CSF to a patient, the method of improving the efficacy of the therapy comprising replacing all or portion of the GM-CSF in the composition with a therapeutic amount of beta-glucan, preferably a non-inflammatory beta-glucan such as PGG-glucan.

In another embodiment of the invention, a method for treating pulmonary disease is provided wherein the method comprises administering aerosolized b-glucan to a subject to treat or prevent fibrosis and/or to treat or prevent bacterial infections from the group consisting of Pseudomonas aeruginosa, chronic mucoid Pseudomonas aeruginosa, multidrug resistant (MDR) Pseudomonas aeruginosa, Streptococcus aureus, methicillin- resistant Staphylococcus aureus (MRSA), non-tuberculosis mycobacteria (NTM), Burkholderia cepacia complex (Bcc), Stenotrophomonas maltophilia, or Alcaligenes xylosoxidans. In another embodiment of the invention a method for preventing and or treating pulmonary infection is provided, the method comprising administering an aerosolized beta-glucan with or without standard of care antibiotics to a subject on mechanical ventilation.

In another embodiment of the invention a method for preventing or treating pulmonary infection is provided, the method comprising administering an aerosolized beta-glucan to a subject to prevent or treat acute pulmonary bacterial infection caused by Pseudomonas aeruginosa, preferably to a subject with CF, COPD, BE or other chronic lung disease, more preferably in a subject with CF with or without concomitant use of standard of care antibiotics.

In another embodiment of the invention a method for treating pulmonary disease is provided, the method comprising administering an aerosolized beta-glucan to a subject to treat fibrosis and/or to treat a chronic pulmonary bacterial infection caused by mucoid or MDR Pseudomonas aeruginosa, preferably in a subject with CF, COPD, BE or other chronic lung disease, more preferably in a subject with CF in combination with standard of care antibiotics.

In another embodiment of the invention a method for preventing pulmonary infection is provided, the method comprising administering an aerosolized beta-glucan to a subject prophylactically to prevent a respiratory infection, preferably acute Pseudomonas aeruginosa infection to a subject with CF, COPD, BE or other chronic lung disease, more preferably in a subject with CF.

In another embodiment of the invention a method of containing, reducing or preventing the spread of respiratory infection between humans is provided, the method comprising administering to the human subject with CF, COPD, BE or other chronic lung disease an aerosolized b-glucan, preferably wherein the infection is one of the group consisting of Burkholderia cepacia complex, Pseudomonas aeruginosa, or Staphylococcus aureus with or without concomitant standard of care antibiotics.

In another embodiment of the invention, a method is provided for treating a subject already infected with methicillin-resistant Staphylococcus aureus (MRSA), non- tuberculosis mycobacteria (NTM), Burkholderia cepacia complex (Bcc), Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, or Pseudomonas aeruginosa, preferably Pseudomonas aeruginosa, the method comprising administering to the subject an aerosolized b-glucan, preferably a subject with CF, COPD or BE or other chronic lung disease administered with or without standard of care antibiotics.

In another embodiment of the invention, a method is provided for decreasing the period of infectivity of an infected subject, preferably in a subject with CF, COPD, BE or other chronic lung disease, even more preferably, in a subject with CF infected with

Pseudomonas aeruginosa, the method comprising administering to the subject an aerosolized b-glucan administered with or without standard of care antibiotics.

In another embodiment of the invention, a method is provided for containing the infectivity of an infected subject, preferably in a subject with CF, COPD, BE or other chronic lung disease, even more preferably, in a subject with CF infected with

In another embodiment of the invention, a method to improve mucus rheological properties in a patient is provided, the method comprising administering to the subject an aerosolized b-glucan preferably via nebulization or dry powder inhaler to the

endobronchial space in order to facilitate an increase in air surface liquid and rehydration of the mucus layer facilitating clearance of mucus and trapped bacteria from the lungs through cough and/or improved mucocilliary clearance (MCC).

In one embodiment of the current invention, a method of reducing free radical damage caused by excessive and aberrant host cellular responses such as the excessive release of reactive oxygen and nitrogen species (ROS and RNS) is provided, the method comprising administering to the subject an aerosolized b-glucan via nebulization or dry powder inhaler to the endobronchial space to act as an antioxidant and free radical scavenger to reduce damage to epithelial lung tissues.

In another embodiment of the invention, a method to prevent or reduce the re-growth or re-population of antibiotic resistant or non-metabolically active antibiotic resistant bacteria during antimicrobial off cycles (twenty-eight days without antibiotics, e.g.

inhaled tobramycin ) is provided, the method comprising administering aerosolized b- glucan to a subject in need thereof during antimicrobial off cycles to prevent or reduce the re-growth or re-population of bacteria, wherein the bacteria is preferentially methicillin-resistant Staphylococcus aureus (MRSA), non-tuberculosis mycobacteria (NTM), Burkholderia cepacia complex (Bcc), Stenotrophomonas maltophilia,

Alcaligenes xylosoxidans, or Pseudomonas aeruginosa, preferably, Pseudomonas aeruginosa.

In another embodiment of the invention, a formulation comprises from about 0.1-10 mg of PGG-glucan dissolved in about 1 mL to about 5 mL of a 0.1% to 0.9% saline solution having an osmolality between about 34 and 310 mOsm/L, and pH between about 5.5 and 7.0 and wherein the formulation is administered by aerosolization using a jet or ultrasonic nebulizer able to produce particles with a mass medium average diameter between about 0.3 and 5 microns, preferably between about 0.5 and 5 microns, more preferably between about 1 and 5 microns.

In another embodiment of the invention a formulation comprises from about 10-100 mg of PGG-glucan dissolved in about 1 mL to about 5 mL of a 0.1% to 0.9% saline solution having an osmolality between about 34 and 310 mOsm/L, and pH between about 5.5 and 7.0 and wherein the formulation is administered by aerosolization using a jet or ultrasonic nebulizer able to produce particles with a mass medium average diameter between about 0.3 and 5 microns, preferably between about 0.5 and 5 microns, more preferably between about 1 and 5 microns.

In another embodiment of the invention a formulation comprises from about 100-250 mg of PGG-glucan dissolved in about 1 mL to about 5 mL of a 0.1% to 0.9% saline solution having an osmolality between about 34 and 310 mOsm/L, and pH between about 5.5 and 7.0 and wherein the formulation is administered by aerosolization using a jet or ultrasonic nebulizer able to produce particles with a mass medium average diameter between about 0.3 and 5 microns, preferably between about 0.5 and 5 microns, more preferably between about 1 and 5 microns. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the Structure of PGG Glucan (Gawronski et al., Biopolymers 1999, 50(6) 569-781)

Figure 2 shows conductive and respiratory zones of the lungs. (Hoiby et al., BMC Medicine 2011, 9:32)

Figure 3 shows results from studies on the effect of PGG-glucan on macrophage differentiation when PGG is administered early in the differentiation process.

Figure 4 shows results from studies on the effect of PGG-glucan on macrophage differentiation when PGG is administered during the differentiation process

Figure 5 shows results from studies on the effect of PGG-glucan on macrophage differentiation when PGG is administered later in the differentiation process.

DETAILED DESCRIPTION

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the immunogenic compositions. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings.

As used herein:

The terms "beta-glucan," "β-glucan," "b-glucan," or "beta glucan" with respect to the present invention refer to a polysaccharide having a (l,3)-P-linked backbone and at least one (1,6)-P"linked side chain.

The term "aerosol" or "aerosolized" refers to a fine suspension of solid particles or liquid droplets dispersed in air or gas or the state of being in such a suspension, and

compositions formulated for delivery as a fine suspension.

The term "PGG-glucan" refers to the beta glucan poly-(l-6)- -D-glucopyranosyl-(l-3)- - D-glucopyranose preferably derived from the yeast Saccharomyces cerevisiae, such as IMPRIME PGG®, (Biothera, Eagan, MN) or PGG-glucan® or Betafectin®, (Alpha-Beta Technology, Worcester, MA; Biothera, Eagan, MN). Also see Figure 1. The term "non-inflammatory beta-glucan" refers to a composition of b-glucan so long as the composition is capable of "priming" the host immune response without directly stimulating the production and secretion of proinflammatory cytokines IL-Ι β and TNFa in PBMCs or whole blood.

The term "subject" or "host" or "patient" and the like, is a vertebrate, preferably an animal or mammal, more preferably a human patient. Mammals include, but are not limited to murines, simians, human patients, farm animals, and pets.

The terms "to treat," "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The treatment may be therapeutic in terms of a partial or complete cure for a disease or disorder and/or adverse effect attributable to the disease or disorder. "To treat," "treatment," "treating," and the like as used herein, covers any treatment of a disease or disorder in a mammal, preferably a human, and includes: preventing the disease; decreasing the risk of death due to the disease; inhibiting the disease or disorder, i.e., arresting its development (e.g., reducing the rate of disease progression); and relieving the disease, i.e., causing regression of the disease, including inhibition and/or alleviation of a clinical symptoms associated with the disease "To treat," "treating," "treatment," and the like as used herein, also refers to the

"containment" of the disease, thus reducing the spread of said disease or condition, preferably respiratory infection, between mammals, preferably between humans.

Therapeutic benefits of the present invention include, but are not necessarily limited to, reduction of risk of onset or severity of disease or conditions associated with infection. Thus, treatment of a disease or disorder as used herein refers separately to (though is not limited to) the eradication, containment, and or a reduction in the duration of infectivity of an acute and/or chronic infection.

The term "therapeutic amount" or "therapeutically effective dose" of a drug substance composition, product, or the like, refers to an amount of any active agent defined herein that is administered to a subject, which provides for the desired effect or benefit to the subject without undue adverse events or side effects such as local or systemic toxicity, proportionate with a reasonable benefit/risk ratio when used as described in the present invention. For the described beta-glucan compositions and/or formulations, desired effects or benefits include priming or enhancing of the immune system including a reduction, prevention, and or the treatment of respiratory tract infections in a subject, preferably a human. The precise "therapeutic amount" or "therapeutically effective dose" may vary with such factors as the type and severity of the disease or condition being treated, the specific composition to be used, the health, size and weight of the treated subject, preferably a human, the nature and amount of any concurrent therapy, the treatment duration and regimen, dosage, dose form, and the amount of excipients contained in the composition.

The term "immune response," "host immune response" or "host defense" includes all of the specific and non-specific processes and mechanisms involved in how the body defends, tolerates, and repairs itself against bacteria, viruses, fungi, parasites, allergens and all substances, insults, challenges, disorders biological and/or physical invasions of the body that are harmful to the body.

The term "enhancing," as in "enhancing the immune response," "enhancing the host immune response" or "enhancing host defense" means to promote a functional change to the immune system of an animal, preferably a mammal, and any of its specific and nonspecific processes and mechanisms involved, in particular in how the mammal's body defends, tolerates, and repairs itself against bacteria, viruses, fungi, parasites, and all substances, insults, challenges, disorders, biological and/or physical invasions of the body that are harmful to the body which provides a benefit to the mammal, preferably a human.

The term "prime" or "priming" as used herein refers to initiating some or all the processes, mechanisms, and function of mammalian innate immune cells that follow exposure to a pathogen or component of a pathogen, such as a pathogen associated molecular pattern (PAMP), such that the effector or defense functions of innate immune cells, such as phagocytosis or production of ROS/RNS are enhanced upon exposure to a secondary related or unrelated stimulus such as an infection, compared to the effector responses unprimed innate immune cells.

The term "PAMP" refers to "pathogen-associated molecular patterns," which are molecules associated with groups of pathogens that are recognized by cells of the innate immune system. These molecules can be referred to as small molecular motifs conserved within a class of microbes.

The terms "ROS" and "RNS" refers to reactive oxygen species and reactive nitrogen species respectively.

The term "disease" is defined as the significant impairment in the function of a tissue, an organ, or a system of a subject.

The term "eradication," "eradicate," or "eradicated" and the like, as well as the term "clearance," "clear," "cleared," and the like, as used herein refer to the absence of microbial infection or absence of positive culture of microbial infection as detected by colony culture, for example, essentially no growth of P. aeruginosa measured in sputum, culture, BALF, or nasopharyngeal swab, and the like, having previously been P.

aeruginosa culture positive.

The terms "contain" or "containment" as used herein, refers to the act of keeping something from spreading, preferably with respect to the spread of infection from one individual to another, more preferably the spread of infected from an infected subject to a subject previously uninfected.

The term "infection" as used herein refers to the presence of bacteria, virus or fungi as detected by standard methods known in the art, e.g. colony culture for bacteria by isolation of the bacterium in culture.

The term "chronic infection" as used herein refers to the persistence of infection, such as shown by positive culture for a bacterial infection, despite the use of standard

antimicrobial therapy.

The term "acute infection" as used herein refers infections that are not chronic in nature and can be cleared or eradicated with standard antimicrobial therapy,

The term "MRS A" or "methicillin-resistant S. aureus" as used herein, refers to methicillin-resistant Staphylococcus aureus

The term "NTM" as used herein, refers to "non-tuberculosis mycobacteria" The term "Bcc," "BCC," or "B. cepacia complex" as used herein, refers to Burkholderia cepacia complex (Bcc).

The term "MDR" as used herein, refers to multi-drug resistance, preferably with respect to bacteria.

The term "MDR Pseudomonas aeruginosa " as used herein, refers to any strain of Pseudomonas aeruginosa that is resistant to all drugs in more than one antimicrobial drug class

The term "mucoid Pseudomonas aeruginosa" as used herein refers to any strain of Pseudomonas aeruginosa that synthesize an exopolysaccharide alginate which allows the formation of protective biofilm microcolonies that provide increased resistance to opsonization, phagocytosis, and destruction by antimicrobial agents.

The term "CF" as used herein, refers to cystic fibrosis.

The term "COPD" as used herein, refers to chronic obstructive pulmonary disorder.

The term "BE" as used herein, refers to bronchiectasis.

The term "IPF" as used herein, refers to idiopathic pulmonary fibrosis.

The term "UPF" as used herein, refers to usual pulmonary fibrosis.

The term "ILD" as used herein, refers to interstitial lung disease.

The term "UIP" as used herein, refers to usual interstitial pneumonia.

The term "ARDS" as used herein, refers to acute respiratory distress syndrome.

The term "PAP" as used herein, refers to pulmonary alveolar proteinosis.

The term "MTB", "TB", or "M. tuberculosis," as used herein, refers to "M. tuberculosis," "MTB," or means bacterium Mycobacterium tuberculosis or tuberculum bacilli.

The term "MMAD" as used herein, means mass medium average diameter or mass median average diameter wherein 50% of the particles by mass are larger and 50% are smaller. The term "0.1% saline" as used herein, means a water solution containing 0.1%

(weight/volume) NaCl.

The term "predominantly" as used herein means at least 70%, and with respect to particle size distribution, the term means at least 70% of the particles so described are the given particle size or are within the given range, for example, the phrase "wherein the particle size of the particles is predominantly between 1 and 5 microns," means at least 70% of the particles are between land 5 micron in size.

The term "1 normal saline" as used herein, means water solution containing 0.9%

(weight/volume) sodium chloride (NaCl).

The term "ELF" as used herein refers to epithelial lining fluid.

The term "pharmaceutically acceptable solution" as used herein means an aqueous solution which may contain physiologically acceptable salts, excipients, diluents, buffers, and the like. Preferably, the physiologically acceptable solution will contain a NaCl content anywhere between about 0.1% NaCl (weight/volume) to 1 normal saline, i.e., 0.9% NaCl (weight/volume), an osmolarity between about 34 and 310 mOsm/L, and pH between about 5.5 and 7.0.

The term "pharmaceutically acceptable dry powder" means a dry powder having particle size MMAD predominantly between 0.3 and 5 microns and which may contain physiologically acceptable salts, excipients, diluents, buffers, etc. Preferably, the physiologically acceptable dry powder will comprise a NaCl content anywhere between about 0.1%) NaCl (weight/volume) to 1 normal saline, i.e., 0.9% NaCl (weight/volume), an osmolarity between about 34 and 310 mOsm/L, and pH between about 5.5 and 7.0 when reconstituted with about 1 to 5 mL of water.

The term "formulation" means a specific composition formulated for a specific use, such as an aqueous beta-glucan formulation for aerosolization via nebulization or as a dry powder beta-glucan formulation for aerosolization via dry powder inhaler (DPI). The term "beta-glucan compositions" as used herein, means a beta-glucan in combination with other components, such as salts, excipients, diluents, osmotic solutions, preferably isotonic or hypotonic solutions, buffers, and the like.

The term "pharmaceutically acceptable carrier" refers to any substance that is combined with or may come in contact with the compositions of the present invention. These "pharmaceutically acceptable carriers" are generally regarded as safe (GRAS) for use in humans and are used to effectively deliver the composition to the subject. These "pharmaceutically acceptable carriers" and are also referred to as vehicles, carriers, or carrier systems and can be composed of for example, amino acid and or the saccharide class of excipients including glucose, sorbitol, lactose, sucrose, mannitol, or trehalose.

The term "dry" as in "dry powder" used herein, refers to a composition with a moisture content such that the particles are readily dispersible in an inhalation device to form an aerosol. Preferably, the moisture content may be below about 10% by weight water, below about 7% by weight water, below about 5% by weight water or below about 3% by weight water.

The term "powder" as in "dry powder" used herein, refers to a composition that consists of finely dispersed solid particles that are capable of being readily dispersed in an inhalation device and subsequently inhaled by a subject so that the particles reach the lungs to permit penetration into the upper and lower airways. Thus, the powder is said to be "respirable." Preferably, the powder composition consists predominantly of finely dispersed solid particles having a MMAD predominantly between about 0.3 to 5 microns with moisture content below about 10% by weight water and a tap density greater than about 0.4 g/cm, greater than about 0.45 g/cm or greater than about 0.3 g/cm.

The term "cytokine" as used herein can refer to cytokines, chemokines, interleukins and the like.

The term "standard of care" means current, up to date, and clinically accepted pharmacotherapy used in treating or managing the d sease or infection.

In the present description, any concentration range, percentage range, ratio range or other integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. As used herein, "about" or "comprising essentially of means +/- 15%. The use of alternative (i.e., or) should be understood to mean one, both, or any combination thereof of the alternatives. As used herein, the use of an indefinite article, such as "a" or "an," should be understood to refer to the singular and the plural of a noun or noun phrase. In addition, it should be understood that the individual compositions, formulations, or components, or groups of components, formulations, or compounds, derived from the various components or combinations of the composition described herein, are disclosed by the present application to the same extent as if each composition or component or group of compositions or components was set forth individually. Thus, selection of particular components or compositions is within the scope of the present invention.

Beta-glucan aerosol

Aerosolization of β-glucan, and in particular a β-glucan that does not directly stimulate the production of proinflammatory cytokines such TNFa and IL-lb such as PGG-glucan allows for the delivery of high concentrations of the drug directly to the site of action in the airways while maximizing safety and tolerability which limit the dose and this clinical efficacy of other PAMPs in general. Furthermore, the physical properties of b- glucan typically limit penetration across the mucosal surface of the lungs which minimize systemic absorption and any potential negative off target effects. Aerosolization and inhalation of β-glucans such as PGG-glucan represents a safer, more effective method of activating the immune system and host defense for the prevention and treatment of respiratory infections and/or lung fibrosis than systemic delivery of the drug especially when used in conjunction with standard of care antibiotics. PGG-glucan aerosol formulation is particularly suitable for delivery of therapeutic concentrations delivered directly to the lungs to treat and prevent chronic and acute lung infections in humans, preferably infected subjects with underlying lung disease. PGG-glucan aerosol formulation is also particularly suitable for delivery directly to the lungs to treat fibrotic lung diseases. Safe and effective delivery of b-glucan to the endobronchial space via a nebulizer requires a physiologically compatible formulation that can be nebulized to generate small and uniform particle size aerosols with a mass median average diameter (MMAD) between 0.3 to 5 microns. Aerosols containing particles larger than 5 microns are deposited in the upper airways and back of the throat while particles less than 0.3 microns are not deposited, and are removed from the lungs upon exhalation, both decreasing the amount of medicament delivered to the treatment site in the lower respiratory tract.

Hypertonic aerosols can cause bronchospasm that is uncomfortable for the patient and limits drug deposition. Aerosolized solutions that are well tolerated have an osmolality that is isotonic or hypotonic. Solution pH is also an important factor in the tolerability of the aerosol. Acceptable pH ranges for aerosols range from 5.5 to 7.

Liquid and dry powder formulas and or compositions are aerosolized for administration directly to the mucosal layer of the endobronchial space of patients with CF to rehydrate the thick sticky mucus layer and improve mucocilliary clearance, mucus expectoration, and the removal of trapped bacteria while simultaneously improving host immune cell microbial killing and clearance mechanisms.

Given the chronic inflammation, infection, and subsequent weakened innate and adaptive host defenses associated with CF, BE, and COPD or other chronic lung disease, it is believed that administration of PGG-glucan aerosol to patients with chronic lung disease will enhance and/or restore normal immune defenses to treat fibrosis and/or facilitate the clearance of infection or to increase the efficacy of existing antimicrobial therapy for acute, chronic, and difficult to treat respiratory infections, such as P. aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA), non-tuberculous mycobacteria (NTM), as well as B. cepacia complex, S. maltophilia, and A. xylosoxidans, preferably, P. aeruginosa in subjects with CF.

Administering the PGG-glucan as an inhaled aerosol enables the targeted delivery of high concentrations of PGG-glucan to resident innate immune cells (i.e., achieve lung concentrations which would otherwise be toxic if administered systemically), present in the endobronchial secretions of the endobronchial space of airways for the treatment of P. aeruginosa including, chronic and MDR ⁵. aeruginosa, methicillin-resistant S. aureus (MRSA), Burkholderia, and non-tuberculous mycobacteria (NTM) infections in patients in need thereof. Such targeted delivery may be of particular benefit to patients with CF, COPD, BE or other chronic lung disease, where targeted delivery of high concentrations of PGG-glucan to the endobronchial secretions of the endobronchial space of airways provides greater ease and shorter delivery times, and convenience, including at home self-administration (compared to hospitalized systemic administration), thus improving patient adherence to therapy and thus better clinical outcomes. This may be particularly useful in such a heavily treatment-burdened patient population, such as cystic fibrosis patients. Administration of aerosolized b-glucan is for the prevention, treatment and preferably eradication of acute, chronic, and/or MDR forms of P. aeruginosa infection, and particularly in patients suffering from lung disease, such as CF.

In one embodiment the treatment consists of aerosolized PGG-glucan, that is delivered directly to site of action in the upper and lower respiratory tract, capable of augmenting or restoring normal immune cellular defense mechanisms to work in concert with standard of care antimicrobials and improve overall microbial clearance, decrease exacerbations, and/ or improve lung function (FEV1). As PGG-glucan is a noninflammatory b-glucan, the use of PGG-glucan potentiates immune host defense mechanisms without inducing potentially harmful pro-inflammatory mediators such as TNFa and IL-Ιβ. Such therapy would preferably comprise inhalation of an aerosolized PGG-glucan drug formulation at therapeutically effective amounts directly to the endobronchial space of infected airways to safely achieve therapeutic concentrations in the endobronchial space which otherwise may be unattainable with systemic

administration at non-toxic dose levels.

Beta-glucan treatment has the potential to decrease exacerbations which require hospitalizations and need of intravenous antipseudomonal antibiotics. Any reduction in antibiotic use will reduce the risk for resistant bacteria strains. Use of beta-glucan in combination with antibiotics effectively reduces the emergence of resistant strains of bacteria.

In view of the heavy treatment burden that is associated with chronic lung disease, particularly CF, co-formulation of PGG-glucan with standard of care antibiotics such as tobramycin for administration, would significantly reduce the burden of treatment for the patient suffering from infection, such as chronic P. aeruginosa. Decreased burden of treatment facilitates an increase in patient adherence, which in turn has a direct effect on the severity of the disease, frequency of exacerbations, need for hospitalization, and overall health care costs. Thus, PGG-glucan aerosol is administered directly to the lung alone or as adjunct therapy to antibiotics for the treatment of P. aeruginosa.

Some factors that can influence the products efficacy are: the specific formulation including the b-glucan solution concentration, viscosity, pH, salinity, particle size, nebulizer or inhaler and proper administration technique. In addition, using a

physiologically suitable formulation to reduce bronchospasm facilitates medication retention in the endobronchial space. Moreover, inhaled therapeutics are often formulated to be paired with a specific device which has been optimized for the delivery of that specific therapeutic formulation.

Pulmonary drug delivery

The lungs consist of a respiratory zone and a respectively smaller conductive zone. The respiratory zone includes respiratory bronchioles, alveolar ducts and alveolar sacs while the smaller conducting zone includes the trachea, the bronchi and the terminal bronchioles (Fig. 1) [Ftoiby et al. Future Microbiol 2010;5: 1663-1674; Ftoiby N. Microbe 2006; 1 :571-577; West J. Pulmonary Physiology and Pathophysiology Philadelphia: Lippincott Williams & Wilkins; 2001.]. Because chronic respiratory infections, including P aeruginosa, Burkholderia cepacian complex (mostly B.multivorans and B. cenocepacia) and Achromobacter xylosoxidans grow in both the respiratory and the conductive zones of the lungs [Farrell et al., Pediatr Pulmonol 2003;36:230-240; Koch C. Pediatr Pulmonol 2002;34:232-236; Bjarnsholt et al., Pediatr Pulmonol 2009;44:547- 558], the size of aerosolized liquid droplets or dry powder particles are an important factor in terms of where they are distributed and consequently on efficacy. The optimal particle size ranges from 0.3-5 microns to distribute throughout the lung including the alveoli [Heijerman et al., J Cystic Fibros 2009;8:295-315]. Larger particles will be deposited in the trachea while smaller particles are not deposited and are removed from the lungs upon exhalation. Inhaled medications have been available for many years for the treatment of lung diseases and are widely accepted as being the optimal route of administration of first-line therapy for various lung diseases including asthma, chronic obstructive pulmonary diseases, and cystic fibrosis. The advantages of inhaled over oral delivery are many and include, the ability to deliver high drug concentrations directly to the disease site, a reduced risk of systemic side-effects, a rapid clinical response, lower therapeutic dose requirements, and the ability to bypass gastrointestinal absorption issues and first-pass metabolism in the liver. Pulmonary delivery is also independent of dietary

complications, extracellular enzymes, and inter-patient metabolic differences affecting gastrointestinal absorption.

Pulmonary drug delivery is accomplished via a number of various atomized delivery systems including nebulizers, metered-dose inhalers (MDI), and dry powder inhalers (DPIs). Respirable particles may be made by several methods including nebulization, micronization, precipitation, freeze drying, and spray drying. Spray drying is a high- throughput process with the ability to produce solid particles in a more controlled manner, such as directing particle size and size distribution, and particle morphology, which are important particle features for pulmonary dry powder drug delivery by inhalation.

In the case where the lungs are the target organ or a route of administration, the appropriate amount of drug must be deposited past the oropharyngeal region to achieve therapeutic effectiveness. The site of deposition (i.e., central or peripheral airways), and whether the distribution of the inhaled drug is uniform or non-uniform may also play a role in an inhaled drug's effectiveness. For inhaled therapy that targets cells of the innate and adaptive immune system, the deposition should be evenly distributed throughout the lung, since immune cells, such as, lymphocytes, macrophages, neutrophils, and dendritic cells, are present throughout the airways and the alveolar tissue. To achieve effective deposition throughout the lung, particle sizes in the range of 0.3-5 micron in diameter are suggested. See Figure 2. (from Hoiby et al., BMC Medicine 2011, 9:32). To achieve deposition in the lower respiratory tract and small peripheral airways, particles sizes of less than 1 micron in diameter are suggested. Particle sizes in excess of 5 micron are frequently deposited in the oropharyngeal upper respiratory region [Labiris and Dolovich, Br (2003) J Clin Pharmacol. 56(6): 588-599 and 600-612; Tena and Clara (2012) Arch Bronconeumol. 48(7): 240-24].

The essential particle properties for targeted delivery to smaller airways and deep lung region include particles with relatively narrow unimodal sizing distribution between 0.3 and 5 micron, spherical geometry with relatively smooth surface morphology, and low water content. However, PGG-glucan is composed of rod shaped triple helices of varying length with a range of aggregation from 1 to about 25, an average aggregation number of 9 and a wide non-unimodal sizing distribution. Furthermore, PGG-glucan has a weight-average molecular weight value around 150 kD [Gawronski et al., Biopolymers. 1999;50(6):569-781]. Thus, the compound PGG-glucan does not have the essential particle properties for targeted delivery such as the efficient deposition throughout the lung, especially with respect to delivery to the lower airways and alveoli.

In one embodiment, a spray dried powder formulation of PGG-glucan may have optimal size range between 0.3 to 5 micron, relatively narrow unimodal sizing distribution, spherical particles with relatively smooth surface morphology, and low water content for targeted deposition throughout the upper and lower respiratory tract, including delivery to smaller airways and deep lung region.

Spray drying (or spray-drying) as used herein, is a process in which a homogeneous aqueous formulation comprising PGG-glucan that is introduced via a nozzle (e. g., a two fluid nozzle), spinning disc or an equivalent device into a hot gas stream to atomize the solution to form fine droplets. The aqueous mixture may be a solution, suspension, slurry, or the like, but needs to be homogeneous to ensure uniform distribution of the

components in the mixture and ultimately the powdered composition. Preferably the aqueous mixture is a solution. The solvent, generally water, rapidly evaporates from the droplets producing a fine dry powder having particles with MMAD predominantly between 1 to 5 μπι. The spray drying is done under conditions that result in substantially amorphous powder of homogeneous constitution having a particle size that is respirable, and low moisture content and flow characteristics that allow for ready aerosolization. Preferably the particle size of the resulting powder is such that more than 90% of the particles have a MMAD of less than 10 microns. Preferably the particle size of the resulting powder is predominantly between 1 and 5 microns.

In order to improve current P. aeruginosa infection management, with treatments that improve P. aeruginosa infection clearance and infectivity containment, in one

embodiment of the invention b-glucan is delivered via nebulization or dry powder inhalation to the sight of action no more than once daily and that can be administered by the patient in under 10 mins, preferably under 5 mins.

One embodiment of the present invention generally relates to aqueous PGG-glucan compositions and methods for administering and preparing such compositions. A composition of the present invention may be administered to a subject via pulmonary administration in an amount effective to eradicate, treat, prevent, contain, or shorten the duration of infectivity of a bacterial infection in the subject. Administration of an effective amount of a composition of the present invention may be particularly useful in treating methicillin-resistant Staphylococcus aureus (MRSA), non-tuberculosis mycobacteria (NTM), Burkholderia cepacia complex (Bcc), Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, or Pseudomonas aeruginosa including chronic mucoid and multi-drug resistant (MDR) Pseudomonas aeruginosa, preferably,

Pseudomonas aeruginosa including chronic mucoid and multi-drug resistant (MDR) Pseudomonas aeruginosa alone or as adjunct therapy, preferably as adjunct therapy to standard of care antimicrobial treatment in a subject in need thereof, including subjects with CF, COPD, BE or other chronic lung disease, preferably CF.

In one embodiment, a formulation comprising about 0.1 to 250 mg PGG-glucan is reconstituted in 1 to 5 mL of about 0.1% to 0.9% NaCl solution having an osmolarity between about 34 and 310 mOsm/L, pH between about 5.5 and 7.0, and aerosolized by a jet or ultrasonic nebulizer, such as a PARI, Sinustar™, or Omron, with a mass median average diameter (MMAD) between about 0.3 and 5 micron to the endobronchial space to achieve a concentration between about 1 and 1000 ug/mL in the endobronchial secretions or epithelial lining fluid (ELF) is administered to a subject with CF, COPD, BE or other chronic lung disease, preferably, in a subject with CF, anywhere from 2 times daily to once weekly, preferably, once daily, suffering from methicillin-resistant Staphylococcus aureus (MRSA), non-tuberculous mycobacteria (NTM), B. cepacia complex, S. maltophilia, A. xylosoxidans, or P. aeruginosa, preferably, P. aeruginosa including chronic mucoid and multi-drug resistant (MDR) Pseudomonas aeruginosa, for a treatment duration ranging from continuous chronic treatment to that of 1, 2, 3, 4, 5, 6, 7, or 8 week on/off cycles for more effective management of infection including decreased exacerbation rate, hospitalization, need for intravenous antibiotics and the like or until the infection is eradicated. In a preferred embodiment of said method, said formulation is administered to a subject with CF, COPD, BE or other chronic lung disease, preferably, in a subject with CF, suffering from P. aeruginosa, methicillin- resistant Staphylococcus aureus (MRSA), non-tuberculous mycobacteria (NTM), B. cepacia complex, S. maltophilia, or A. xylosoxidans, preferably, P. aeruginosa as adjunct therapy to standard of care antibiotics. In another particular embodiment of said method, said formulation is administered to a subject with CF suffering from P. aeruginosa as adjunct therapy to tobramycin, colistin, and or cayston (aztreonam), preferably tobramycin. In said method, tobramycin colistin, and or cayston (aztreonam) may be administered via any approved administration route, i.e., aerosolized via nebulization or dry-power inhaler, oral or intravenously, preferably, administration is by oral inhalation via dry-power inhaler, more preferably, tobramycin in administered is by oral inhalation via dry-power inhaler,

DPIs have unique advantages including high dose delivery, higher chemical stability relative to the liquid state, and the potential to tailor particle properties in the solid state. The performance of DPI formulations is influenced by several particle properties, including size, size distribution, morphology, and particle surface properties. Particle surface properties including surface morphology and inter-particulate forces such as van der Waals, electrostatic, and capillary forces (Li et al. (2014) J Aerosol Med. Pulmonary DrugDeliv. 27(2): 81-93; Hickey and Mansour (2009) In: AT Florence, and J Siepmann, (eds) Modern Pharmaceutics. Taylor & Francis, New York; pp. 191-219).

A dry powder composition of the present disclosure may be delivered to a subject by any means so long as the solid particles of the dry powder composition are capable of being inhaled by a subject so that the particles reach the lungs to permit penetration into the upper and lower airways. In certain embodiments, a dry powder composition of the present disclosure may be delivered to a subject by placing the dry powder within a suitable dosage receptacle in a sufficient amount. Suitable dosage receptacles include those used in reservoir devices (e.g., devices that contain more than one dose in which the device itself meters the dose) or factory- metered dose devices (e.g., devices in which each dose is contained in either a single unit or multiple units). In one example, a suitable reservoir device may have a dosage receptacle that fits within a suitable inhalation device to allow for the aerosolization of the dry powder composition by dispersion into a gas stream to form an aerosol and then delivering the aerosol so produced from a mouthpiece attached for subsequent inhalation by a subject in need of treatment. Such a dosage receptacle includes any container enclosing the composition known in the art such as gelatin, hydroxypropyl methyl cellulose or plastic capsules with a removable portion or body that can be cut or pierced that allows dispersal of the dry powder composition (e.g., via a gas stream directed into the container and via centrifugal force).

A PGG-glucan (or more generally a non-inflammatory b-glucan) spray dry powder composition herein may be administered with any conventional inhaler device and according to conventional procedures by any inhaled route to the upper and or lower respiratory tract. For example, a liquid preparation of PGG-glucan may be aerosolized for inhalation through the nasal cavity to the upper respiratory tract. Alternatively, a liquid preparation of PGG-glucan may be aerosolized for inhalation to the lungs. These preparations may be administered with single dose or use delivery devices or delivery devises capable of multiple doses.

The PGG-glucan spray dry powder composition may be administered by intranasal or oral inhalation administration. In one example, PGG-glucan composition is delivered to the lower respiratory tract to a subject through oral inhalation as a dry powder formulation. In another example, PGG-glucan composition is delivered to the upper respiratory tract of a subject through nasal inhalation as a dry powder formulation.

Appropriate dosage forms for such administration, such as a liquid aerosol formulation or by a powdered metered dose inhaler, may be prepared by conventional techniques. In another embodiment of the present invention generally relates to dry powder PGG- glucan compositions and methods for administering and preparing such compositions. In some embodiments, a composition of the present disclosure may be administered to a subject via pulmonary administration in an amount effective to treat fibrosis and/or eradicate, treat, prevent, contain, or shorten the duration of infectivity of a bacterial infection in the subject. Administration of an effective amount of a composition of the present disclosure may be particularly useful in treating methicillin-resistant

Staphylococcus aureus (MRSA), non-tuberculosis mycobacteria (NTM), Burkholderia cepacia complex (Bcc), Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, or Pseudomonas aeruginosa including chronic mucoid and multi-drug resistant (MDR) Pseudomonas aeruginosa preferably Pseudomonas aeruginosa including chronic mucoid and multi-drug resistant (MDR) Pseudomonas aeruginosa alone or as adjunct therapy, preferably as adjunct therapy to standard of care antimicrobial treatment in a subject in need thereof, including subjects with CF, COPD, BE or other chronic lung disease, preferably CF.

In one embodiment, the PGG-glucan spray dry powder composition may be comprised of PGG-glucan and at least one water-dispersible or water-soluble pharmaceutically acceptable carrier to form a solid dispersion or a solid solution. Preferably, a solid dispersion is formed between water-dispersible or water-soluble pharmaceutically acceptable carrier and at least one pharmaceutically active ingredient. In a preferred embodiment, the solid dispersion or solid solution is obtained by using known techniques (such as high shear mixing, spray drying or fluid bed granulation). Within the meaning of the present invention, a solid dispersion denotes a dispersion of one or more compound(s) in an inert carrier at solid state. The term "solid solution" denotes a solid-state solution of one or more solutes in a solvent, wherein the mixture remains in a single homogenous phase. The water-dispersible or water-soluble carrier is selected from the group consisting of modified or unmodified carbohydrates, preferably monomeric, oligomeric or polymeric carbohydrates, preferably modified or unmodified monomeric, oligomeric or polymeric monosaccharides, or straight or branched oligosaccharides or

polysaccharides; wax; gum; organic or inorganic acids or bases, or a salt thereof;

surfactants; synthetic polymers; modified or unmodified silica; mineral pharmaceutical excipients or a combination thereof. More specifically, the carrier is selected from the group consisting of sucrose, maltose, lactose, glucose, mannose, mannitol, sorbitol, xylitol, erythritol, lactitol, maltitol, a starch or modified starch, such as pregelatinized starch, corn starch, potato starch, or maize starch; an alginate, gelatin, carrageenan, dextran, maltodextran, dextrates, dextrin, polydextrose, or tragacanth; acacia, guar gum, xanthan gum; cellulose such as carboxymethylcellulose, methylcellulose, sodium carboxymethyl cellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, powdered cellulose, or microcrystalline cellulose; polyacrylic acid, modified orunmodified alginate or chitosan, gelatin, pectin, polyethylene, glycolpolyethylene glycol, polyvinylpyrrolidone, polyvinylalcohol, polyoxyethylene copolymers, polyoxypropylene copolymers, orpolyethyleneoxide;

arginine, meglumine, lysine, monoethanolamine, diethanolamine, triethanolamine, propanolamine, dipropanolamine, thiamine, sodium salicylate, or a mixture thereof. In a preferred embodiment, the PGG-glucan spray dry powder composition according to any of the preceding items, wherein the carrier is selected from the group consisting of lactose, sucrose, maltose, mannitol, sorbitol, xylitol, erythritol, lactitol, maltitol, starch and cellulose. In another more preferred embodiment, the carrier is selected from the group consisting trehalose.

Dry powder, including co-formulated spray-dry powder compositions for delivery to the nasal mucosa or lung mucosa by inhalation may, for example, be presented in capsules and cartridges consisting of, for example HPMC or gelatin. Each capsule or cartridge may generally contain between lmg to 250 mgs of the spray dry PGG-glucan

composition of the present invention. Alternatively, the compositions of the invention may be presented without excipients. The delivery device for the administration of the invention to the subject includes for example, a dry-powder inhaler (DPI), a reservoir dry powder inhaler (RDPI), a metered dose inhaler (MDI), or a multi-dose dry powder inhaler (MDPI).

Compositions for administration by inhalation have controlled particle size range, which can be achieved by optimization of the spray-drying conditions, for example. The optimum particle size for inhalation into the mid and lower lung is usually 0.3-5 μπι, preferably 0.3-5 μηι, while particles having a size above 20 μηι, preferably 20-100 μιη, are preferred for upper respiratory tract delivery via intranasal administration. The desired fraction, appropriately sized may be separated out by air classification or sieving.

Intranasal sprays may be formulated with aqueous or non-aqueous vehicles or carriers with the addition of thickening and or thinning agents, emulsifiers, salts or buffer salts or acid or alkali to adjust the pH, viscosity, osmolarity, and the like. For examples of carriers, stabilizers and adjuvants, see Remington's Pharmaceutical Sciences, edited by E. W. Martin (Mack Publishing Company, 18th ed., 1990). The compositions also can include stabilizers and preservatives.

Solutions for inhalation by nebulization may be formulated with an aqueous vehicle with the addition of agents such as acid or alkali, buffer salts, isotonicity adjusting agents.

Intranasal sprays and or solutions for administration via oral inhalation may

advantageously target the organ or tissue of interest and in the process, reduce the efficacious dose required for delivery to the patient. For example, intranasal sprays may advantageously target the upper respiratory mucosa, while oral inhalation may advantageously target the lung and lower respiratory mucosa. Furthermore,

administration by oral inhalation may reduce the systemic exposure of the compound and in the process, avoid potential systemic toxicity or other undesired systemic effects of the compound.

EXAMPLES

Highly purified, soluble beta glucan (PGG-glucan) was tested to determine its potential to redirect M2 macrophages away from an M2 phenotype, towards a more Ml -like phenotype. Water soluble PGG-glucan used in the examples below was prepared from whole glucan particles, WGP 3-6®, from Biothera Pharmaceutical Inc. (Eagan, MN, USA) using a sequence of acid and alkaline treatments. The resulting soluble glucan was further processed by a series of ultrafiltration steps to isolate the b-glucan fraction having an average molecular weight of about 150 kD. For use in the examples below, the water soluble PGG-glucan was concentrated to about lmg/mL, and equilibrated in sterile isotonic saline by diafiltration. The experiments were performed on bone marrow derived macrophages (BMDM) from wild-type mice, or in the case of the second study, BMDM from wild type and CFTR-/- mice, and macrophages were grown in cRPMI media containing L929 (M-CSF).

BMDM cultures were grown in cRPMI media from L929 cells which contain M-CSF, which directs monocyte differentiation toward an M2 phenotype. Media was used to grow all the cells (2ml media in each 6-well plate (5ml PSG, 50ml FBS, and 500ml RPMI, filter the media; then, 185ml of cRPMI add 46ml of L929).

Macrophages from wild type CF mice were: i) left unstimulated ("US"),

ii) treated with PGG-glucan (listed as "RES" or "RES 001" in the Figures), iii) treated with Granulocyte Macrophage Colony Stimulating Factor (listed as "GM" or "GM-CSF"in the Figures), or

iv) treated with both GM-C SF+ PGG-glucan.

In contrast M-CSF in the culture media, GM-CSF in the culture media directs monocyte differentiation toward an Ml phenotype. Where PGG-glucan is used, cells are incubated with PGG-glucan (25ug/ml in PBS) 80 ul for 1 hour followed by wash out and return to cRPMI media one day prior to the addition of GM-CSF. Where GM-CSF is used, cells are incubated with GM-CSF 20ul.

Five different target genes were used to determine specific Ml or M2 phenotypes. Ml phenotype was demonstrated by the expression of Ml associated genes: TLR-2, MARCO and TNF-a. M2 phenotype was demonstrated by the expression M2 associated genes IL- 10 and MMP12. TNF- a was not measured in the second study.

Example 1 "prior to wtBMDM M2-macrophage differentiation"

In one study, PGG-glucan was added early in the differentiation process, "pre- differentiation" as outlined below. Results are shown in Figure 3.

Lane 1 US (Bone marrow cells with the media that has L929)

Lane 2 PGG-glucan stim. on 4^th day

th

Lane 3 GM-CSF stim. on 5 day Lane 4 GM-CSF stim. 5 day and PGG-gluan stim. 4 day

Harvested these cells -11^th day

Example 2 "during wtBMDM and CFTR-/- BMDM M2-macrophage differentiation" In another study PGG was added "during differentiation," as outlined below. Results are shown in Figure 4.

Lane 1 US

th

Lane 2 GM-CSF stim. 5 day

Lane 3 PGG-glucan stim. 4^th day

Lane 4 GM-CSF stim. 5^th day, PGG-glucan stim. 7^th day

Harvested these cells -11^th day

Example 3 "post wtBMDM M2-macrophage differentiation"

In a third study PGG-glucan was added later in the differentiation process,

"postdifferentiation" as shown below. Results are shown in Figure 5.

Lane 1 US

Lane 2 PGG-glucan stim. lO^day

th th

Lane 3 GM-CSF stim. 5 day & 7 day

th th th

Lane 4 GM-CSF stim. 5 and 7 day, & PGG-glucan stim. on 10 day

th

Harvested these cells - 12 day Results

Redirection by PGG-glucan during the course of M2 differentiation of wild-type BMDM. With respect to the effect on BMDM from wild type mice in Figures 1, 2 and 3, lane 1 (US) show the delta ct values that represent M-CSF's capacity to direct BMDM toward M2 macrophage differentiation. The delta ct values for GM-CSF +PGG-Glucan (Lane 4) suggest that PGG-glucan is redirecting macrophage phenotype from M2 to Ml and that the effect or magnitude of Ml macrophage redirection resulting directly from the addition of b-glucan is more pronounced the earlier it is added during the course of M-CSF mediated differentiation of BMDM to M2 macrophages. Furthermore, literature strongly supports the hypothesGMis that effect or magnitude of Ml macrophage redirection resulting directly from the addition of b-glucan would be further pronounced with longer b-glucan the incubation times (up to 24hours), wherein in this preliminary study only 1- hour incubation times were used.

Redirection by PGG-glucan during the course of M2 differentiation of CFTR-/- BMDM With respect to the effect on BMDM from CF type mice (CFTR-/-) in Figure 4, lane 1 (US) show the delta ct values that represent M-CSF's capacity to direct CF BMDM toward M2 macrophage differentiation. The delta ct values for GM-CSF+PGG-Glucan (lane 4) are similar to that of wtBMDM and likewise suggest that PGG-glucan is redirecting macrophage phenotype from M2 to Ml . Furthermore we would expect that the effect or magnitude of Ml macrophage redirection resulting directly from the addition of b-glucan more pronounced the earlier it is added during the course of M-CSF mediated differentiation of CFTR-/-C BMDM to M2 macrophages. Furthermore, literature strongly supports the hypothesis that effect or magnitude of Ml macrophage redirection resulting directly from the addition of b-glucan would be further pronounced with longer b-glucan the incubation times (up to 24 hours), wherein in this preliminary study only 1-hour incubation times were used. Ultimately, the data suggests that the CF mutation in BMDM does not affect b-glucan' s ability to redirect CF BMDM toward a more Ml-like state despite culture in M2 directing M-CSF containing media.

Claims

1. A pharmaceutical aerosol formulation comprising a b-glucan.

2. An aerosol formulation of the previous claim wherein the b-glucan is a noninflammatory b-glucan.

3. An aerosol formulation of any of the previous claims, wherein the b-glucan is a PGG-glucan composition.

4. An aerosol formulation of any of the previous claims, wherein the b-glucan is b- glucan dry powder reconstituted in saline for inhalation by nebulization.

5. An aerosol formulation of any of the previous claims, wherein the b-glucan is b- glucan dry powder composition reconstituted in deionized water for inhalation by nebulization.

6. An aerosol formulation of any of the previous claims, wherein the aerosol

formulation comprises between about O. lmg to about 250 mg of PGG-glucan, or preferably between about O. lmg to about 50 mg of PGG-glucan.

7. An aerosol formulation of any of the previous claims, wherein the aerosol

formulation comprising b-glucan has a pH that is neutral or acidic, preferably having a pH range between 7.0 and 5.5, or between 6.5 to 6.0.

8. An aerosol formulation of any of the preceding claims wherein the aerosol

formulation comprises b-glucan in an isotonic or hypotonic aqueous solution.

9. An aerosol formulation of any of the previous claims, wherein the aerosol

formulation comprising b-glucan is in aqueous solution containing between 0.1% (w/v) to 0.9% (w/v) NaCl, having an osmolality of between about 34 to about 310, preferably containing between about 0.225% (w/v) and about 0.45% (w/v) NaCl and having an osmolality between about 77.5 and about 155.

10. An aerosol formulation of any of the previous claims, wherein the aerosol

formulation comprising b-glucan is a dry powder that is reconstituted for nebulization in about ImL to about 5 mL and preferably in 3mL to 5 mL in water or saline with or without pH buffer.

11. An aerosol formulation of any of the previous claims, wherein the aerosol

formulation comprising b-glucan is administered to the endobronchial space of a subject in need thereof, using a jet or ultrasonic nebulizer able to produce aerosol particle sizes which are predominantly between 0.3 and 5 microns, preferably between 0.5 to 5 microns, and more preferably between 1.0 and 5.0 microns.

12. An aerosol formulation of any of the previous claims, wherein the aerosol

formulation comprising b-glucan is delivered by a nebulizer with a delivery efficiency of 40% or more and preferably 60% or more to the endobronchial space of a subject.

13. An aerosol formulation of any of the previous claims, wherein the aerosol

formulation comprising b-glucan is delivered by a nebulizer with a delivery efficiency capable of delivering a dose of b-glucan that results in at least 1 ug/mL, preferably, at least 10 ug/mL or up to 500 ug/mL concentrations in the subject's epithelial lining fluid.

14. An aerosol formulation of any of the previous claims 1-3, wherein the aerosol formulation comprising b-glucan is a spray-dry powder formulated for inhalation using a dry powder inhaler (DPI).

15. An aerosol formulation of any of the previous claims 1-3, and 14 wherein the b- glucan, preferably PGG-glucan, is present in a spray-dry powder composition, in an amount by percent weight of the composition between 10% and 90% or between 10% to 80%, or between 10% to 70%, or between 30% to 70%.

16. An aerosol formulation of any of the previous claims 1-3 and 14-15, wherein the aerosol formulation comprises b-glucan in a spray-dry powder composition and a bulking agent suitable for medicinal formulations delivered by DPI, preferably wherein the carbohydrate bulking agent is selected from the group consisting of: lactose, mannitol, or trehalose, more preferably trehalose, and wherein the b- glucan is preferably PGG-glucan.

17. An aerosol formulation of any of the previous claims 1-3 and 14-16, wherein the aerosol formulation comprises b-glucan in a spray-dry powder composition comprising the bulking agent present in an amount between 10% and 90% by weight of the composition, or between 10% to 80%, preferably 10% to 70%, and more preferably 30% to 70% by weight.

18. An aerosol formulation of any of the previous claims 1-3 and 14-17, wherein the aerosol formulation comprises b-glucan in a spray-dry powder composition further comprising a salt preferably selected from the group consisting of NaCl, NaBr, and Nal, more preferably NaCl and where salt is present in an amount less than 25% and preferably less than 10% by weight.

19. An aerosol formulation of any of the previous claims 1-3 and 14-18, wherein the aerosol formulation comprises b-glucan in a spray-dry powder composition comprising a pH buffer selected from the group including but not limited to, phosphate, acetate, citrate, TRIS, arginine, and histidine, preferably phosphate buffer, in an amount less than 25% and preferably less than 15% by weight.

20. An aerosol formulation of any of the previous claims 1-3 and 14-19, wherein the aerosol formulation comprises b-glucan in a spray-dry powder composition comprising a hydrophobic amino acid, wherein the b-glucan is preferably PGG glucan.

21. An aerosol formulation of any of the previous claims 1-3 and 14-20, wherein the aerosol formulation comprises b-glucan in a spray-dry powder composition comprising a hydrophobic amino acid selected from the group consisting of: tryptophan, tyrosine, leucine, trileucine, and phenylalanine, preferably leucine, and wherein the hydrophobic amino acid is present in an amount less than 25% by weight of the composition, or less than 20% or preferably in an amount between about 1% and 10% by weight of the composition.

22. An aerosol formulation of any of the previous claims 1-3 and 14-21, wherein the aerosol formulation comprises b-glucan in spray-dry powder composition particles wherein at least 90% of the particles have a mass median aerodynamic diameter of between about 0.3 microns and 10 microns, preferably between about 0.3 microns and 5 microns, more preferably between 0.5 microns to 5 microns, or even more preferably, between 1 micron to 5 microns.

23. An aerosol formulation of any of the previous claims 1-3 and 14-22, wherein the aerosol formulation comprises b-glucan as a spray-dry powder composition wherein the dry powder is encapsulated in a capsule, preferably comprising hydroxypropyl methylcellulose.

24. An aerosol formulation of any of the previous claims 1-3 and 14-23, wherein the aerosol formulation comprises b-glucan as a spray dry powder composition having low water or low moisture content, preferably below about 10% water by weight, preferably below about 7% water by weight, and more preferably below about 5% by weight water.

25. An aerosol formulation of any of the previous claims 1-3 and 14-24, wherein the aerosol formulation comprises b-glucan as a spray-dry powder composition wherein the composition is predominantly an amorphous solid.

26. An aerosol formulation of any of the previous claims 1-3 and 14-25, wherein the aerosol formulation comprises b-glucan in a spray-dry powder composition wherein the pH of the spray-dry powder when reconstituted in 3 mL to 5 mL of water is between a pH of 5.5 to 7.0

27. An aerosol formulation of any of the previous claims 1-3 and 14-26, wherein the aerosol formulation comprises b-glucan in a spray-dry powder composition wherein the salinity of the spray-dry powder when reconstituted in 3 mL to 5 mL of water contains between 0.1% (w/v) to 0.9% (w/v) NaCl, having an osmolarity of between about 34 to about 310, preferably containing between 0.225% (w/v) to 0.45%) (w/v) NaCl and having an osmolarity between about 77.5 and 155.

28. An aerosol formulation of any of the previous claims 1-3 and 14-27, wherein the aerosol formulation comprises b-glucan as a spray-dry powder composition comprising predominantly amorphous dry powder.

29. An aerosol formulation of any of the previous claims 1-3 and 14-28, wherein the aerosol formulation comprises b-glucan as a spray-dry powder composition wherein the pH of the spray-dry powder reconstituted in 3 mL to 5 mL of water is between a pH of 5.5 to 7.0, preferably about 7.0.

30. An aerosol formulation of any of the previous claims 1-3 and 14-29, wherein the aerosol formulation comprises b-glucan as a spray-dry powder having a tap density of greater than about 0.4 g/cm3.

31. An aerosol formulation of any of the previous claims 1-3 and 14-30, wherein the aerosol formulation comprising b-glucan as a spray-dry powder composition is delivered to the endobronchial space of a subject in need thereof using a dry powder inhaler (DPI), preferably a DPI that provides a delivery efficiency of 40% or more, more preferably, a delivery efficiency of 60%> or greater.

32. An aerosol formulation of any of the previous claims 1-3 and 14-31, wherein the aerosol formulation comprises b-glucan as a spray-dry powder composition comprising one or more pH adjusters including, but not limited to, hydrochloric acid, sodium hydroxide, lactic acid, tartaric acid, succinic acid, and/or any combination thereof.

33. A method for the treatment of lung disease, preferably a chronic lung infection and/or fibrosis in a subject, the method comprising administering to the subject an aerosolized b-glucan according to any of the previous claims.

34. A method for the treatment of pulmonary bacterial infection in a subject in need thereof according to the previous claim 33, the method comprising administering to the subject an aerosolized b-glucan, and preferably continuing treatment with continuous or episodic administration to reduce the frequency, magnitude, and or duration of infection or until the pulmonary bacterial infection is eradicated.

35. A method of adjunct treatment to antibiotic therapy comprising administering aerosolized beta-glucan according to any of the previous claims 33 and 34 to a patient in need of antibiotic therapy, preferably the antibiotic therapy is standard of care antibiotic therapy used for the specific bacterial species wherein the use of aerosolized beta-glucan according to any of the previous claims 33 and 34 allow for reduced antibiotic use including, dose, frequency of administration, or reduced duration of administration, and the standard of care antibiotic therapy is preferably selected from the group of tobramycin, aztreonam, colistin, and vancomycin.

36. A method according to any of the previous claims 33-35 wherein the aerosolized b-glucan is administered to a subject once weekly, twice weekly, three times weekly, every other day, daily, or twice daily, more preferably once or twice weekly.

37. A method according to any of the previous claims 33-36 wherein the aerosolized b-glucan is administered to a subject suffering from an underlying chronic lung disease, and particularly a chronic lung disease selected from the group consisting of cystic fibrosis (CF), chronic obstructive pulmonary disorder, (COPD) and bronchiectasis (BE), interstitial lung disease (ILD), idiopathic pulmonary fibrosis (IPF), usual interstitial pneumonia (UIP), acute respiratory distress syndrome (ARDS) and pulmonary alveolar proteinosis (PAP).

38. A method according to any of the previous claims 33-37 wherein the aerosolized b-glucan is administered to a subject to treat, reduce, prevent or slow the progression of fibrosis and/or to treat and/or eradicate bacterial infections selected from the group consisting of Pseudomonas aeruginosa, chronic mucoid

Pseudomonas aeruginosa, multi-drug resistant (MDR) Pseudomonas aeruginosa, Streptococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), non- tuberculosis mycobacteria (NTM), Burkholderia cepacia complex (Bcc),

Stenotrophomonas maltophilia, and Alcaligenes xylosoxidans, more preferably Pseudomonas aeruginosa, chronic mucoid Pseudomonas aeruginosa, multi-drug resistant (MDR) Pseudomonas aeruginosa.

39. A method according to any of the previous claims 33-38 wherein the aerosolized b-glucan is administered to a subject receiving mechanical ventilation.

40. A method according to any of the previous claims 33-39 wherein the aerosolized b-glucan is administered to a subject to treat fibrosis and/or acute pulmonary bacterial infection caused by Pseudomonas aeruginosa, preferably to a subject with CF, COPD, BE or other chronic lung diseases, more preferably in a subject with CF.

41. A method according to any of the previous claims 33-40 wherein the aerosolized b-glucan is administered to a subject to treat fibrosis and/or a chronic pulmonary bacterial infection caused by mucoid or MDR forms of Pseudomonas aeruginosa, preferably in a subject with CF, COPD, BE or other chronic lung disease, more preferably in a subject with CF.

42. A method according to any of the previous claims 33-39 wherein the aerosolized b-glucan is administered to a subject to treat fibrosis, preferably in a subject with (COPD), interstitial lung disease (ILD), idiopathic pulmonary fibrosis (IPF), usual interstitial pneumonia (UIP), acute respiratory distress syndrome (ARDS) and pulmonary alveolar proteinosis (PAP).

43. A method according to any of the previous claims 33-39 and 42 wherein the

aerosolized b-glucan is administered to a subject to treat the excessive accumulation of lung surfactant in the alveoli, preferably in a subject with pulmonary alveolar proteinosis (PAP).

44. A method according to any of the previous claims wherein the aerosolized b- glucan is administered to a subject to prevent a respiratory infection, preferably to a subject with CF, COPD, BE or other chronic lung diseases, more preferably in a subject with CF.

45. A method of containing, reducing or preventing the spread of respiratory infection between humans comprising administering to the human subject with CF, COPD, BE or other chronic lung disease an aerosolized b-glucan, according to any of the previous claims preferably wherein the infection is one of the group consisting of Burkholderia cepacia complex, Pseudomonas aeruginosa, or Staphylococcus aureus, more preferably Pseudomonas aeruginosa.

46. A method for treating a subject infected with methicillin-resistant Staphylococcus aureus (MRSA), non-tuberculosis mycobacteria (NTM), Burkholderia cepacia complex (Bcc), Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, or Pseudomonas aeruginosa, preferably Pseudomonas aeruginosa, the method comprising administering to the subject an aerosolized b-glucan according to any of the previous claims, preferably a subject with CF, COPD or BE or other chronic lung diseases.

47. A method for decreasing the period of microbial infectivity of an infected subject, preferably in a subject with CF, COPD, BE or other chronic lung disease, even more preferably, in a subject with CF infected with Pseudomonas aeruginosa, the method comprising administering to the subject an aerosolized b-glucan according to any of the previous claims.

48. A method for containing the infectivity of an infected subject, preferably in a subject with CF, COPD, BE or other chronic lung disease, even more preferably, in a subject with CF infected with Pseudomonas aeruginosa, the method comprising administering to the subject an aerosolized b-glucan according to any of the previous claims.

49. A method to improve mucus rheological properties in a subject, the method

comprising administering to the subject an aerosolized b-glucan according to any of the previous claims preferably via nebulization or dry powder inhaler to the endobronchial space.

50. A method of reducing free radical damage caused by host cellular release of reactive oxygen and nitrogen species (ROS and RNS), the method comprising administering to the subject an aerosolized b-glucan according to any of the previous claims via nebulization or dry powder inhaler to the endobronchial space.

51. A method to prevent or reduce the re-growth or re-population of antibiotic

resistant microbial infection, the method comprising administering aerosolized b- glucan according to any of the previous claims to a subject in need thereof during antimicrobial off cycles to prevent or reduce the re-growth or re-population of antibiotic resistant or non-metabolically active bacteria, preferably bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), non-tuberculosis mycobacteria (NTM), Burkholderia cepacia complex (Bcc), Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, Pseudomonas aeruginosa, preferably, Pseudomonas aeruginosa.

52. A formulation or method according to any of the previous claims wherein the formulation comprises from about 0.1-250 mg of PGG-glucan dissolved in about 1 mL to about 5 mL of a 0.1% to 0.9% saline solution having an osmolality between about 34 and 310 mOsm/L, and pH between about 5.5 and 7.0 and wherein the formulation is administered by aerosolization using a jet or ultrasonic nebulizer able to produce particles with a mass medium average diameter between about 0.3 and 5 microns.

53. A formulation or method according to any of the previous claims wherein the formulation comprises from about 0.1-100 mg of PGG-glucan dissolved in about 1 mL to about 5 mL of a 0.1% to 0.9% saline solution having an osmolality between about 34 and 310 mOsm/L, and pH between about 5.5 and 7.0 and wherein the formulation is administered by aerosolization using a jet or ultrasonic nebulizer able to produce particles with a mass medium average diameter between about 0.3 and 5 microns.

54. A formulation or method according to any of the previous claims wherein the formulation comprises from about 0.1-10 mg of PGG-glucan dissolved in about 1 mL to about 5 mL of a 0.1% to 0.9% saline solution having an osmolarity between about 34 and 310 mOsm/L, and pH between about 5.5 and 7.0 and wherein the formulation is administered by aerosolization using a jet or ultrasonic nebulizer able to produce particles with a mass medium average diameter between about 0.3 and 5 microns.

55. A formulation or method according to any of the previous claims wherein the formulation comprises from about 0.1-1.0 mg of PGG-glucan dissolved in about 1 mL to about 5 mL of a 0.1% to 0.9% saline solution having an osmolarity between about 34 and 310 mOsm/L, and pH between about 5.5 and 7.0 and wherein the formulation is administered by aerosolization using a jet or ultrasonic nebulizer able to produce particles with a mass medium average diameter between about 0.3 and 5 microns.

56. A pharmaceutically acceptable solution comprising beta-glucan.

57. A pharmaceutically acceptable dry powder formulation comprising beta-glucan.

58. An aerosol formulation comprising a beta-glucan in aqueous solution with an osmolarity value between about 77.5 and 155.

59. An aerosol formulation comprising a beta-glucan and a bulking agent suitable for medicinal formulations amenable to delivery to the endobronchial space when administered by dry powder inhaler (DPI).

60. A method of increasing pulmonary Ml macrophage activity in the lumen or in lung tissue of a patient comprising administering aerosolized beta-glucan to a patient via inhalation to achieved clinically effective concentrations.

61. A method of reducing pulmonary fibrotic activity in a patient comprising

administering aerosolized beta-glucan to the patient via inhalation.

62. A method of redirecting resident lung and/or alveolar macrophage polarization towards a more Ml like phenotype for more effective antimicrobial activity in a subject comprising administering aerosolized beta-glucan to the subject via inhalation.

63. A method of redirecting resident lung and/or alveolar macrophage polarization towards a more Ml like phenotype to reduce fibrotic activity in a subject comprising administering aerosolized beta-glucan to the subject via inhalation.

64. A method of enhancing resident lung and/or alveolar macrophage antimicrobial responses in a subject comprising administering aerosolized beta-glucan to the subject via inhalation.

65. A method of reducing pulmonary bacterial load in a subject comprising

administering aerosolized beta-glucan to the subject via inhalation.

66. A method of facilitating improved mucocilliary clearance (MCC) in a subject comprising administering aerosolized beta-glucan to the subject via inhalation.

67. A method of enhancing pulmonary antimicrobial therapy in a subject and

significantly reducing pulmonary bacterial load over the use of antibiotics alone comprising administering aerosolized beta-glucan to the subject via inhalation.

68. A method of priming the immune system for enhanced immune defense against infection in a subject comprising administering aerosolized beta-glucan to the subject via inhalation.

69. A method of potentiating immune host defense mechanisms in a subject without inducing potentially harmful pro-inflammatory responses such as TNFa and IL- 1β comprising administering aerosolized beta-glucan to the subject via inhalation.

70. A method of reducing, inhibiting, or reversing the pulmonary fibrotic process in a subject comprising administering aerosolized beta-glucan to the subject via inhalation.

71. A method of enhancing pulmonary microbial clearance in a subject comprising administering aerosolized beta-glucan to the subject via inhalation.

72. A method of reducing reactive oxygen and/or nitrogen free radical damage to lung tissue in a subject comprising administering aerosolized beta-glucan to the subject via inhalation.

73. A method of delivering a therapeutically effective dose of beta-glucan to the

endobronchial space of a subject comprising administering aerosolized beta- glucan to the subject via inhalation.

74. A method of delivering therapeutically effective dose of beta glucan to endobronchial space of a subject comprising administering aerosolized beta- glucan in dry powder formulation to the subject via inhalation.

75. A method of decreasing pulmonary fibroblast proliferation in a subject

comprising administering aerosolized beta-glucan to the subject via inhalation.

76. A method of decreasing pulmonary collagen production in a subject comprising administering aerosolized beta-glucan to the subject via inhalation.

77. A method of modulating matrix metalloproteinase activity in a subject to favor antifibrotic processes comprising administering aerosolized beta-glucan to the subject via inhalation.

78. A method of reducing excess pulmonary surfactant and increasing alveolar oxygen uptake from the blood in a subject comprising administering aerosolized beta-glucan to the subject via inhalation.

79. A composition for treating pulmonary disease comprising GM-CSF and beta- glucan.

80. The composition of claim 79 wherein the beta-gluan is PGG-glucan.