FOR THE TREATMENT OF ALLERGIC ASTHMA

Crogg-Reference to Related Applications

This application claims the benefit of the filing date of U.S. application Serial No. 62/373,646, filed on August 11, 2016, the disclosure of which is incorporated by reference herein.

Background

Asthma is a common lung disease affecting over 300 million people worldwide and is associated with increased reactive oxygen species (ROS), eosinophilic airway inflammation, bronchoconstriction and mucus production (Barnes, 2008). The prevalence of asthma is between 6-9% of Americans.

Asthma leads to significant morbidity and mortality with estimated healthcare and lost opportunity costs in the billions of dollars annually in the United States.

Exposure of the respiratory epithelium to allergens is the initiating event in allergic asthma, which is characterized by excessive pulmonary inflammation, airway hyperreactivity, and mucus production. Improved therapies are necessary to reduce suffering and lost productivity in asthma patients. Hypertrophy, hyperreactivity, pathological remodeling, airway obstruction and inflammation are well established smooth muscle phenotypes in asthma patients. Therapies have focused on 'upstream' targets, such as G-protein-coupled receptors (e.g., histamine, adrenergic leukotriene), glucocorticoid receptors and reactive oxygen species that activate signaling pathways important for selective smooth muscle responses in asthma. However, these therapies have not reversed the increase in asthma-related morbidity or mortality. Moreover, despite contemporary stepwise treatment approaches, 5-10% of the estimated 26 million Americans with asthma do not achieve adequate symptom control.

Summary

In one embodiment, a population of cationic nanoparticles is provided, the surface of which are coated with one or more mucoadhesives. In one embodiment, the mucoadhesive comprises chitosan, polylysine, polyethylene glycol) (PEG), polyvinyl alcohol) (PVA), poly (vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA or Carbopol ^® ), poly(hydroxyethyl methacrylate) (PHEMA), hydroxy ethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methl cellulose (HPMC), methylcellulose, or sodium

carboxymethyl cellulose. ID one embodiment, the mucoadhesive comprises chitosan. In one embodiment, the mucoadhesive comprises polylysine. In one embodiment, the mucoadhesive comprises polyamidoamine (PAMAM). In one embodiment, the nanoparticle is formed of polyethylene glycol, polyQactic acid), poly(glycolic acid), copolymers of lactic and glycolic acid, copolymers of lactic and glycolic acid with polyethylene glycol, poly(E-caprolactone), poly(3- hydroxybutyrate), poly(p-dioxanone), polypropylene fumarate,

poly(orthoesters), polyol/diketene acetals addition polymers, poly(sebacic anhydride) (PSA), poly(carboxybiscarboxyphenoxyphenoxy hexone (PCPP) poly[bis (p-carboxypheonoxy) methane] (PCPM), copolymers of SA, CPP and CPM, poly(amino acids), poly(pseudo amino acids), polyphosphazenes, derivatives of poly[(dichloro)phosphazenes] and poly[(organo) phosphazenes], poly-hydroxybutyric acid, or S-caproic acid, polylactide-co-glycolide, polylactic acid, orpolyethylene glycol. In one embodiment, the nanoparticle is formed of polylactide, polyglycolide, poly(lactic-co-glycolic acid), polysulfenamide, polyanhydride, or polycaprolactone. In one embodiment, a population of cationic nanoparticles, the surface of which particles is coated with one or more mucoadhesives, comprises a diagnostic or therapeutic agent, e.g., an inhibitor of CaMKn. In one embodiment, the uncoated particles are about SO to about 200 nm, about 75 nm to about 125 nm, or about 100 nm to about 175 nm, in diameter. In one embodiment, the coated particles are about 200 to about 325 nm, about 225 nm to about 275 nm or about 250 nm to about 300 nm, in diameter. In one embodiment, the particle is formed of only one polymer, e.g., a cationic polymer. In one embodiment, the particle does not include a targeting molecule, e.g., a targeting peptide such as a mitocondrial targeting peptide (the particles are untargeted coated NPs). In one embodiment, the zeta potential of the coated particles is about 30 to 80 mV, e.g., 30 to 50 mV, 35 to 45 mV, 35 to 60 mV, or 60 to 80 mV. In one embodiment, the coating comprises low molecular weight chitosan of about 50,000 to 310,000 Da, e.g., 50,000 to

190,000 Da, and a viscosity of about 20 to about 300 cP Da, e.g., 50 to 300 cP, 25 to 250 cP or 50 to 190 cP.

Ca ²⁺ /calmodulin-dependent protein kinase (CaMKH) is expressed and activated in the bronchial epithelium of asthmatic patients, increases mucous accumulation, pulmonary eosinophila, and activates hypertrophic and

proinflammatory gene programs in smooth muscle, which contribute to bronchial hyper-reactivity. A cationic nanoparticle (NP)-based system is described herein for delivery of molecules, e.g., inhibitors of CaMKII such as the potent and specific CaMKII inhibitor peptide, CaMKIIN, to airways. As disclosed herein, CaMKHN-loaded NPs abrogated the severity of allergic asthma in a murine model. In particular, poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) were directly delivered to the lung via oropharyngeal instillation (OP). Further, chitosan coating of the CaMKIIN-loaded PLGA-NPs increased uptake in lung cells compared to uncoated NPs and led to reduced core features of allergic asthma including inflammation, mucus production, and airway hyper reactivity. These findings provide the basis for drug delivery therapies, particularly for site-specific treatment of pulmonary diseases such as asthma.

In one embodiment, the CaMKII inhibitor comprises staurosporine, fasudil, autocamtide-2-Related Inhibitory Peptide, 1-Naphthyl PP1, CaM Kinase Π (290-309), CaMKIIN, KRPPKLGQIGRSKRWIEDDRIDDVLK (SEQ ID NO:7), K-252a, KN-62, lavendustin C, 12(S>HPETE, K-252b, HA-1077 dihydrochloride, Arcyriaflavin A, or CaM Kinase Π inhibitor. In one

embodiment, the inhibitor comprises KN-93, KN-92, HMN-709, KN-62, KN-04, Scios-lSb, Bosutinib, Sanofi-32, Dainippon A: 8p, Dainippon B:2S, or

Rimacalib; SMP-114. In one embodiment, the uncoated particles are about SO to about 200 nm, about 75 nm to about 125 nm, or about 100 nm to about 175 nm, in diameter. In one embodiment, the coated particles are about 200 to about 325 nm, about 225 nm to about 275 nm or about 250 nm to about 300 nm, in diameter. In one embodiment, the particle is formed of only one polymer, e.g., a cationic polymer. In one embodiment, the particle does not include a targeting molecule, e.g., a targeting peptide such as a mitocondrial targeting peptide (the particles are untargeted coated NPs). In one embodiment, the zeta potential of the coated particles is about 30 to 80 mV, e.g., 30 to 50 mV, 35 to 45 mV, 35 to 60 mV, or 60 to 80 mV.

Further provided is a method to prevent, inhibit or treat a disease. The method includes administering to the mammal an effective amount of a composition comprising the population of coated nanoparticles. In one embodiment, the mammal is a human. In one embodiment, the disease is a pulmonary disease such as asthma, e.g., allergic asthma. In one embodiment, the mammal has allergic asthma. In one embodiment, the composition is administered to the lungs, e.g., via inhalation. In one embodiment, the amount inhibits airway resistance or airway hyperresponsiveness. In one embodiment, the coating comprises low molecular weight chitosan of about 50,000 to 310,000 Da, e.g., 50,000 to 190,000 Da, and a viscosity of about 20 to about 300 cP Da, e.g., 50 to 300 cP, 25 to 250 cP or 50 to 190 cP. Brief Description of Figures

Figures 1A-D. Chitosan coating of PLGANPs increases size, zeta potential, and cellular uptake by human airway epithelial cells. A)

Representative SEM image of chitosan-coated PLGANPs. B) Cumulative CaMKIIN release from PLGA NPs incubated at 37 °C and agitated at 300 rpm (n = 3). C) Size (unstippled) and zeta potential (stippled) of PLGA NPs with and without chitosan-coating determined by DLS. D) Cellular uptake of CaMKIIN loaded PLGA NPs with and without chitosan in HAECs as measured by flow cytometry. Control: cells incubated with culture medium alone (*P < 0.05, **P < 0.01, ****P < 0.0001).

Figures 2A-F. Chitosan-coated NPs localize in the lungs of mice and cause no significant toxicity in vivo. A) Representative images of control (PBS) and uncoated and coated NPs (50 ug of NPs) loaded with fluorescent dye in various organ systems 1, 24, and 48 h after oropharyngeal administration. B) Graphical analysis of NPs measured in lungs of mice at 1, 24, and 48 h (n = 3-5 mice/group). C) Timeline of OVA sensitization (IP) and challenge and NP treatment (oropharyngeal (OP); inhalation (INH) of ovalbumin and alum (OVA/ALUM)). Animals were euthanized and samples taken on day 18. (D,E) Normalized (to saline control group) bilirubin content (D) and AST activity (E) in serum of OVA sensitized mice after treatment with normal saline (N.S.), saline + CaMKUN-loaded NPs (N.S.+CN), OVA alone (OVA), OVA + empty NPs (OVA+E), and OVA + CaMKIIN NPs (OVA-fCN) on day 18. F) Percent weight change during sensitization and NP treatment protocol. Data were calculated relative to body weight on day 0. For toxicity studies, all NPs were coated with chitosan. Figures 3A-D. Cationic CaMKIIN-loaded nanoparticles (NPs) reduce airway hyperreactivity (AHR). A) Mice were sensitized to OVA alone or in the presence of 25 ng of soluble CaMKHN peptide. The dose of soluble CaMKIIN peptide was calculated based on the total amount of CaMKIIN peptide present in 50 ug of NPs (SO ug of NPs loaded with 0.S ug oflCaMKIIN/mg of NP = 25 ng total CaMKIIN). Control mice were sensitized to saline. B) AHR of OVA alone or OVA in the presence of empty chitosan-coated NPs (25, SO, and 100 ug of blank NPs). NS = not significant. C) AHR of saline, OVA alone, or OVA with chitosan-coated NPs (25, SO, and 100 ug of CN) loaded with CaMKIIN. D) AHR in OVA-sensitized mice exposed to SO ug of chitosan-coated empty NPs (OVA-E) or SO ug of chitosan-coated NPs loaded with 0.S ug/mg CaMKIIN (OVA-CN). Additional controls were not sensitized to OVA and given chitosan- coated NPs loaded with CaMKIIN (N.S.-CN). Data are means ± SEM; *P < 0.0S vs saline; $P < 0.05 100 ug of CNvs OVA; #P < 0.05 versus OVA+NP or OVA alone (n = 5-9 mice).

Figures 4A-J. Airway inflammation, cytokine expression, and mucus production are decreased by cationic CaMKIIN-loaded NPs. A) Total cell counts in bronchoalveolar lavage fluid (BALF). B) Eosinophil counts in BALF (n = 7-12 mice). C) Quantification of eosinophils/10 um2 area (40 x magnification). D) H&E staining of lung sections. E) Perivascular cuffing score (10x

magnification). F) qRT-PCR for eotaxin in lung homogenates. G) HL-5 protein levels in lung homogenates by ELISA. (Η,Ι) Representative images of PAS staining (H, 20 x magnification) and (I) quantification (3-5 sections per mouse). J) qRT-PCR for MUC5AC in lungs of allergen challenged mice. Data shown are mean ± SEM (n = 3 for saline groups and n = 6-8 for all OVA-challenged groups). *P < 0.05 OVA alone vs saline or as indicated by brackets. For (G), p < 0.09 N.S.

Figures SA-B. CaMKIIN-loaded NPs reduce OVA-mediated cell ROS in murine tracheal epithelial cells. (A) Representative images and quantification of cytoplasmic ROS production in primary murine tracheal epithelial cells

(MTBEC) isolated from OVA-challenged, WT mice after exposure to chitosan- coated empty NPs (50ug, OVA-E; n=3) or CaMKIIN-NPs (50μ¾ OVA-CN, n=3). ROS production was determined with. Data were quantified for 3-5 images per treatment. Scale bars are ΙΟΟμιη. Data are shown as mean ± SEM. Student's 2 tailed t test was used. * p < 0.05 OVA-E vs. OVA-CN.

Figure 6. OVA-induced IL-4 mKNA expression is reduced in the presence of CaMKIIN-NPs. IL-4 mRNA expression analyzed by qRT-PCR from WT mice exposed to OVA following exposure to chitosan-coated empty NPs (50ug, OVA-E; n=3) or CaMKIIN-NPs (50ug, OVA-CN, n=7). Data are shown as mean ± SEM. Student's 2 tailed t test was used. * p < 0.05 OVA-E vs. OVA- CN.

Figures 7A-C. Dose-dependent modulation of IL- 13 -mediated expression of Th2 cytokines in murine tracheal epithelial cells. Murine tracheal bronchial epithelial cells (MTBEC) were isolated from B6D2 mice. Cells were grown until confluent and exposed to 25, 50 or 100 μg chitosan-coated blank (E) or

CaMKIIN-loaded (CN) NP in the presence of lOng/ml IL-13 (14 days). mRNA expression was examined by qRT-PCR for (A) eotaxin, (B) IL-5. For (C), mRNA expression of

MUC5AC was examined in MTBEC exposed to chitosan-coated empty NPs (50ug, OVA-E) or CaMKIIN-NPs (50ug, OVA-CN). Data were compared between empty NP and corresponding dose of CaMKHN-NP and quantified as fold change compared to blank NP. Analysis of experiments was analyzed using two-way ANOVA and post hoc comparisons tested using Tukey correction. Data are shown as mean ± SEM. N = 3 independent experiments. * p < 0.05 blank NP vs. CaMKHNNP at same dose. # p < 0.05 between all doses of CaMKIIN-NP (e.g., 25 ug vs 50 ug or 100 ug; 50 ug vs. 25 ug or 100 μ& 100 ug vs. 25 ug or 5 Oug).

Detailed Description

Millions of adults and children suffer from asthma in the United States alone. Because there is not a complete cure for the disease, there is a need for the development of safe and effective treatments that allow asthma suffers to manage their symptoms as necessary. Polymeric nanoparticles (NPs) are an excellent candidate for the formulation of asthma therapies as they are able to encapsulate and release the therapeutic agents over time, reducing the number of administrations needed and can be easily coated with mucoadhesive agents in order to be retained in the airways of the lungs when delivered via nebulization. Thus, those who suffer from pulmonary diseases (such as asthma or Chronic Obstructive Pulmonary Diseases (COPD)) may benefit from mucoadhesive NPs as this system can provide local delivery and sustained release of drug to the lungs. Targeting of therapeutic agents to the lungs of patients with asthma may improve efficacy of treatments and minimize side effects.

NP delivery systems allow for local delivery of drugs while offering additional advantages such as sustained release of therapeutic molecules over a desired amount of time, ability to deliver both water-soluble and lipophilic drugs, and the need for fewer administered doses and decreased enzymatic degradation of drug. PolyQactic-co-glycolic acid) (PLGA) is a biodegradable polymer that is FDA approved for use in a wide variety of biomedical applications and can be utilized to fabricate NPs in which therapeutically active molecules are entrapped. In the case of pulmonary drug delivery, it has been shown that drug-loaded PLGA NPs offer superior therapeutic effects over delivery of soluble drug alone. The increase in therapeutic effects for PLGA NP systems can be attributed to sustained release of the drug over time and a longer residence time of NPs in the lungs compared to drug alone.

In one embodiment, a NP-based formulation for asthma therapy was developed that is capable of local and sustained release of drug in the lungs. For example, poly (lactic-co-glycolic acid) (PLGA), a biodegradable polymer, was used to formulate NP carriers for drugs such as therapeutic peptides. Once loaded with drug, the PLGA NPs were coated with chitosan, a natural polymer, to form an outer mucoadhesive layer. The NP formulations were administered to asthmatic mice and tested for lung function. As described herein, after treatment, mice treated with NPs formulations containing a therapeutic peptide exhibited significantly less asthmatic symptoms compared to a control group in which no treatment was administered. Thus, the advantages of this approach over conventional treatments include prolonged retention of drug in the lungs, a decrease in the number of daily doses required and the ability to package and transport poorly soluble drugs efficiently to the large and small airways of the lungs.

Thus, the presently disclosed compositions and methods may be utilized for treating or preventing diseases, such as pulmonary diseases and disorders, e.g., asthma and asthma-related conditions, and the symptoms thereof. In one embodiment, the methods typically comprise administering to a subject in need thereof an effective amount of a composition comprising a nanoparticle comprising a biodegradable polymer coated with a mucoadhesive, which nanoparticle comprises a therapeutic agent, e.g., a compound that modulates the activity of CaMKII (e.g., a CaMKH inhibitor). As used herein, an "effective amount" refers to an amount of a given composition that is necessary or sufficient to bring about a desired effect.

A "patient in need thereof" may include a patient in need of treatment or prevention with respect to any disease or condition. In one embodiment, the disease or condition is associated with calcium/calmodulin dependent protein kinase IL Examples of such diseases or conditions may include, but are not limited to pulmonary diseases or disorders such as asthma and asthma-related conditions. A "patient in need thereof' may include a patient undergoing therapy to treat a pulmonary disease or disorder such as asthma or an asthma-related condition.

As used herein, the terms "treatment," "treat" or "treating" refer to therapy or prophylaxis of diseases, including pulmonary diseases, disorders, and the symptoms thereof in a subject in need thereof. Therapy or prophylaxis typically results in beneficial or desirable clinical effects, such as alleviation of symptoms, diminishment of extent of disease, stabilization (i e., not worsening) of the state of the disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total and, whether detectable or undetectable). "Treatment" can also mean prolonging survival as compared to expected survival if a patient were not to receive treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

As used herein, the term "subject" means one in need of treatment or prevention of a disease, e.g., pulmonary diseases and disorders, such as asthma and asthma-related conditions, or the symptoms thereof. The term "subject" may be used interchangeably herein with the term "patient" or "individual" and may include an "animal" and in particular a "mammal." Mammalian subjects may include humans and other primates, domestic animals, farm animals, and companion animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and the like.

In one embodiment, pulmonary diseases and disorders treated or prevented by the disclosed methods may include asthma or asthma-related conditions. The term "asthma" is a condition in which the inside of the airways which carry air to the lungs become inflamed, resulting in narrowing of the airways and obstruction to air now. Asthma-related conditions may include, but are not limited to, fibrosis in epithelial organs, acute lung injury, rhinitis, anaphylaxis, sinusitis, hay fever, allergies, vocal cord dysfunction, and gastroesophageal reflux disease. Pulmonary diseases and disorders treated or prevented by the disclosed methods further may include chronic obstructive pulmonary disease (COPD), which may include chronic bronchitis and emphysema. In some embodiments, the presently disclosed methods may be utilized to treat or prevent symptoms of pulmonary diseases or disorders.

Symptoms of pulmonary diseases or disorders may include, but are not limited to, recurrent episodes of shortness of breath (i .e., dyspnea), wheezing, chest tightness, and cough.

As used herein, "CaMKE" refers to the enzyme "calcium/calmodulin dependent protein kinase H" In humans, there are four separate, highly homologous genes for CaMKII called alpha, beta, delta, or gamma (or α, β, δ and γ). Multiple isoforms of these genes are expressed through alternative splicing mechanisms. Representative sequences for the isoforms of these genes have been submitted to public depositories such as GenBank and include:

GenBank Accession No. NP— 741960. CaMKII alpha isoform 2; GenBank Accession No. NP—057065, CaMKII alpha isoform 1; GenBank Accession No. NP-742079, CaMKII beta isoform 6: GenBank Accession No. NP-742080, CaMKII beta isoform 7: GenBank Accession No. NP-742077. CaMKII beta isoform 4: GenBank Accession No. NP_001211, CaMKII beta isoform 1; GenBank Accession No. NP-742081. CaMKII beta isoform 8: GenBank Accession No. NP-742078. CaMKII beta isoform S : GenBank Accession No. NP-742076. CaMKII beta isoform 3: GenBank Accession No. NP-742075. CaMKII beta isoform 2; GenBank Accession No. NP-001212, CaMKII delta isoform 3 : GenBank Accession No. NP-742126, CaMKII delta isoform 2: GenBank Accession No. NP— 742125. CaMKII isoform 1; GenBank Accession No. NP-742113, CaMKH isoform 1; GenBank Accession No. NP-JOO 1020609, CaMKII delta isoform 2; NP-751910, CaMKII gamma isoform 3; GenBank Accession No. NP_ 751913, CaMKII gamma isoform 6: GenBank Accession No. NP— 751913. CaMKH gamma isoform 6; GenBank Accession No. NP_ 7S1911, CaMKII gamma isoform 1; GenBank Accession No. NP-7S1909, CaMKII gamma isoform 2; GenBank Accession No. NP— 751909, CaMKII gamma isoform 2; GenBank Accession No. NP— 001213, CaMKII gamma isoform 4; all of which GenBank entries are incorporated herein by reference in their entireties.

In one embodiment of the disclosed methods, a modulator of CaMKII activity is administered to a subject in need thereof. A modulator of CaMKII activity may include an inhibitor of CaMKII activity. An inhibitor of CaMKII may be any compound, composition, or agent that inhibits, either directly or indirectly, the activity or expression (e.g., the amount or the disease-causing effect) of one or more isoforms of CaMKII (i.e., one or more or the alpha, beta, delta, or gamma isoforms of CaMKII, and preferably at least the delta isoform of CaMKII). For example, a CaMKII inhibitor may be an agent that reduces an activity of CaMKII or that reduces the amount of expression of CaMKII, or both. CaMKII activity in a subject or the amount of CaMKII expression in a subject can be readily determined based on detection or measurement of a functional response. CaMKII inhibition may be reversible or irreversible.

A CaMKII inhibitor that is administered in the method may inhibit CaMKII directly (e.g., by directly inhibiting the kinase activity of CaMKII) or indirectly (e.g., by inhibiting activation of CaMKII). In some embodiments of the methods for treating or preventing pulmonary diseases or disorders in a patient, the methods include administering to the patient a therapeutic agent that inhibits oxidation of CaMKII. For example, the therapeutic agent may inhibit oxidation of CaMKII at methionine residues present at amino acid positions 281 and 282. Agents that inhibit oxidation of CaMKII may include agents that inhibit NADPH oxidase and may include, but are not limited to apocynin [4-hydroxy-3- methoxy-acetophenone], diphenylene iodoniumchloride (DPI), staurosporine, phenyl arsine oxide (PAO), 4-(2-Aminoethyl)-benzenesulfonyl fluoride

(AEBSF) and related compounds (see Viatchuk et al ., 1997, the content of which is incorporated herein by reference in its entirety), gp9 lds-tat. PR-39, VAS2870 [3-bezyl-7-(2-benzoxazolyl)thio-l,2,3-triazolo(4,5^)pyrimidi ne], and S17834 [6,8-diallyl 5,7-dihydroxy 2-(2-al1yl 3-hydroxy 4-methoxyphenyl)l-H benzo(b)pyran-4-one] .

Inhibitors of CaMKII are known in the art. (see, e.g., U.S. Pat. No.

7,320,959, the content of which is incorporated by reference in its entirety, particular the patent disclosure related to CaMKII inhibitors). A CaMKII inhibitor can be a peptide or non-pepude agent, including, for example, a nucleic acid that encodes a peptide inhibitor. Moreover, the agent can be an anti sense nucleic acid that inhibits expression of CaMKII (e.g., in lung tissue). CaMKII inhibitors may include the compound known as KN-93 or related compounds, analogs, or derivatives thereof having CaMKII inhibitory activity. Referring to the PubChem Database provided by the National Center for Biotechnology Information (NCBI) of the National Institute of Health (NM) at its website, CaMKII inhibitors contemplated herein may include the compounds referenced by compound identification (CID) Nos. 5312122, 16760530, 6419757, which entries are incorporated herein by reference in their entireties. Compounds related to KN-93, analogs, or derivatives thereof may include, for example, compounds referenced by compound identification (CID) Nos. 3837, 6419758, 18412788, 16760530, 9983993, 5353702, 3836, 24906277, 16219540, and 8122359, which entries are incorporated herein by reference in their entireties.

Inhibitors of CaMKII may include aryl-indolyl maleimide compounds. (See. e.g., Levy et al .(2008) and Lu et al. (2008): the contents of which are incorporated by reference in their entireties). Suitable aryl-indolyl maleimide compounds for use in the disclosed methods for treating or preventing pulmonary diseases or disorders may include, but are not limited to, the following compounds in Tables 1-14, and analogs and derivatives thereof having CaMKII inhibitory activity (in particular those having CaMKIK inhibitory activity):

Table 1

Table 2

Table 3

Table 4 Tables

Compound R ¹ R ² ICso(uM)

1* v ^H 0.38 (n=l)

13a ) CHj 3.81 ± 0.25 (n = 2)

K

14b » H 0.36 ± 0.01 (n = 2)

16b 2.32 (n=l)

14g H 1.35 ± 0.64 (n = 2)

16g C¾ 15.02 (n=l)

14t * H 0.034 ± 0.01 (n = 3)

16t >20(n=l)

A> r 1

^~ TaP \ CH ₂ CH ₂ CH ₂ NH ₂ 0.38 (n=l)

7g ¹³ f \_X ^H 119 (n=l)

8

7b J* CH2CH2CH2NH2 0.034 ±0.009

7e \ CH3 ( ^{n = 3} )

/ JjL^ 0.87 (n=l)

7c % CH2CH2CH2NH2 1.35 ±0.64

7f ^C¾ (n = 2)

7h ^H >20(n = l)

>20 (n = 1)

7d % ClfcCHaCHzNHa 0.18 ± 0.06 (n =

7i /^V"" ¹ * ^H 3)3.53(n=l)

In one embodiment, the inhibitor is a protein or peptide such as

CaMKHN. CaMKIIN or CaM-ΚΠΝ designates small endogenous proteins that inhbit CaMKH with high affinity. CaM=KIIN3 (79 amino acids) and CaM- ΚΠΝα (78 amino acids). The a and β in their names are unrelated to the CaMKn isoform, as either of these inhibits all CaMKII isoforms with ICso of SO nM (Chang et al., 2001). Identification of the core inhibitory domain of CaMKIIN led to generation of a 28 amino acid peptide inhibitor termed

CaMKIINtide that was subsequently shortened and modified to improve potency. CaMIINtide has been modified to increase potency. In one series of optimizations, a shorter sequence of 21 amino acids (CN21a) was found to retain the potency of CaMKIINtide. CN19o (Coultrap and Bayer, 2011) inhibited CaMKIIa with ICso < 0.4 nM and improved selectivity for tested kinases. A similar study generated a smaller optimized 17 amino acid peptide, <3Ν17β, with ICso of 30 nM and little inhibition of CaMKI or CaMKTV (Gomez-Monterrey et al, 2013). In one embodiment of the presently disclosed therapeutic methods, a coated nanop article comprising a therapeutic agent may be administered as part of a pharmaceutical composition. The term "pharmaceutical composition" may be utilized herein interchangeably with the term "therapeutic formulation." Therapeutic formulations used in accordance with the present methods may be prepared for storage by mixing a compound, e.g., CaMKII inhibitor, having a desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), for example in the form of lyophilized formulations or aqueous solutions. In addition to the pharmacologically active compounds in the compositions used in the therapeutic methods disclosed herein may contain one or more suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations that can be used pharmaceutically.

In the present methods, a coated nanoparticle comprising a compound, e.g., a CaMKII inhibitor, may be administered together with a pharmaceutically acceptable carrier. A "pharmaceutically acceptable" carrier typically is not biologically or otherwise undesirable, i.e., the carrier may be administered to a subject, along with nanoparticle without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition. In some embodiments, the carrier may be selected to minimize any degradation of the therapeutic agent, e.g., CaMKII inhibitor, or any of the other components of the pharmaceutical composition or to minimize any adverse side effects in the subject.

In the present methods, a coated nanoparticle comprising a therapeutic agent of interest may be administered in any suitable manner. In some embodiments, the compound of interest is present in a pharmaceutical composition that is administered orally, parenterally (e.g., intravenously, intramuscularly, intrathecally, or intraarterially), transdermally, extracorporeally, topically, intranasally, or via an inhalant. As used herein, "intranasal"

administration may include delivery of a pharmaceutical composition into the nose and nasal passages through one or both of the nares and may include delivery via a spraying mechanism or droplet mechanism, or via aerosolization of the therapeutic agent. The pharmaceutical composition may be delivered to the lower respiratory tract (e.g., trachea, bronchi and lungs) via intubation.

For aerosol administration, a coated nanoparticle comprising a compound of interest, e.g., a CaMKII inhibitor, may be supplied in finely divided form along with a surfactant and propel I ant. Typical percentages of therapeutic agents in aerosol formulation may be 0.01%-20% by weight, preferably 1-10%. The surfactant is non-toxic and in one embodiment is soluble in the propel I ant.

Surfactants may include esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. The surfactant may constitute 0.1%-20% by weight of the composition, preferably 0.25-5%. The balance of the composition is ordinarily propel 1 ant. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.

Suitable formulations for parenteral administration in the methods disclosed herein include aqueous solutions of the coated nanop articles comprising a therapeutic agent in water-soluble form, for example water-soluble salts and alkaline solutions. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, for example sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers.

Sustained-release preparations of a coated nanoparticles comprising a therapeutic agent for use in the present methods may be prepared as known in the art. Suitable examples of sustained-release preparations include

semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent, e.g., CaMKII inhibitor, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl- methacrylate), or pol y(vi nyl alcohol)), polylactides, non-degradable ethylene- vinyl acetate, and poly-D-(-)-3-hydroxybutyric acid.

Formulations to be used for in vivo administration in the disclosed methods typically are sterile. Sterile compositions may be prepared, for example, by filtration through sterile filtration membranes. The exact amount of the compositions delivered in the disclosed methods may vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the condition being treated, the particular composition used (e.g., with respect to concentration of therapeutic agent in the composition), its mode of administration, and the like. A therapeutic agent such as a CaMKII inhibitor may be administered in a dose of from about 0.0S mg to about 5.0 mg per kilogram of body weight of the subject. A therapeutic agent, e.g., a CaMKII inhibitor, alternatively, may be administered in a dose of from about 0.3 mg to about 3.0 mg per kilogram of body weight of the subject.

In some embodiments of the disclosed methods, a therapeutic agent may be administered to the patient (e.g., as an aerosol) in a dosage of between about 1 mg/mL and about 500 mg/mL. For example, a therapeutic agent may be administered in a dosage of about 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL, 50 mg/mL, 55 mg/mL, 60 mg/mL, 65 mg/mL, 70 mg/mL, 75 mg/mL, 80 mg/mL, 85 mg/mL, 90 mg/mL, 95 mg/mL, 100 mg/mL, 105 mg/mL, 110 mg/mL, 115 mg/mL, 120 mg/mL, 125 mg/mL, 130 mg/mL, 135 mg/mL, 140 mg/mL, 145 mg/mL, 150 mg/mL, 155 mg/mL, 160 mg/mL, 165 mg/mL, 170 mg/mL, 175 mg/mL, 180 mg/mL, 185 mg/mL, 190 mg/mL, 195 mg/mL, 200 mg/mL, 205 mg/mL, 210 mg/mL, 215 mg/mL, 220 mg/mL, 225 mg/mL, 230 mg/mL, 235 mg/mL, 240 mg/mL, 245 mg/mL, 250 mg/mL, 255 mg/mL, 260 mg/mL, 265 mg/mL, 270 mg/mL, 275 mg/mL. 280 mg/mL, 285 mg/mL, 290 mg/mL, 295 mg/mL, 300 mg/mL, 305 mg/mL, 310 mg/mL. 315 mg/mL, 320 mg/mL. 325 mg/mL, 330 mg/mL, 335 mg/mL, 340 mg/mL, 345 mg/mL. 350 mg/mL, 355 mg/mL. 360 mg/mL. 365 mg/mL, 370 mg/mL, 375 mg/mL, 380 mg/mL. 385 mg/mL, 390 mg/mL, 395 mg/mL or 400 mg/mL.

In the methods, a therapeutic agent may be administered according to a wide variety of dosing schedules. For example, a therapeutic agent such as a CaMKII inhibitor may be administered once daily for a predetermined amount of time (e.g., four to eight weeks, or more), or according to a weekly schedule (e.g., one day per week, two days per-week, three days per week, four days per week, five days per week, six days per week or seven days per week) for a predetermined amount of time (e.g., four to eight weeks, or more). The disclosed cationic biodegradable nanoparticles may include or may be formed from biodegradable polymeric molecules, which in some

embodiments may include dendrimers. Suitable dendrimers may include, but are not limited to, polyamidoamine (PAMAM) dendrimers. Polyamidoamine dendrimers suitable for preparing the presently disclosed nanoparticles may include 3rd-, 4th-, 5th-, or at least 6th-generation dendrimers.

The disclosed cationic biodegradable nanoparticles may include or may be formed from other biodegradable polymeric molecules which may include, but are not limited to polylactic acid (PLA), polyglycolic acid (PGA), co- polymers of PLA and PGA (i.e., polyactic-co-glycolic acid (PLGA)), poly-ε- caprolactone (PCL), polyethylene glycol (PEG), pol y (3 -hydroxybutyrate), poly(p-di oxanone), polypropylene fumarate, poly(orthoesters), polyol/diketene acetals addition polymers, poly-alkyl-cyano-acrylates (PAC), po1y(sebacic anhydride) (PSA), poly(carboxybiscarboxyphenoxyphenoxy hexone (PCPP) polyfbis (p-carboxypheonoxy)methane](PCPM), copolymers of PSA, PCPP and PCPM, poly(amino acids), poly(pseudo amino acids), polyphosphazenes, derivatives of poly[(dichloro)phosphazenes] and poly[(organo)phosphazenes], poly-hydroxybutyric acid, or S-caproic acid, elastin, or gelatin. (See, e.g., Kumari et al., Colloids and Surfaces B: Biointerfaces 75 (2010) 1-18; and U.S. Pat. Nos. 6,913,767; 6,884,435; 6,565,777; 6,534,092; 6,528,087; 6,379,704; 6,309,569; 6,264,987; 6,210,707; 6,090,925; 6,022,564; 5,981,719; 5,871,747; 5,723,269; 5,603,960; and 5,578,709; and U.S. Published Application No.

2007/0081972; and International Application Publication Nos. WO

2012/115806; and WO 2012/054425; the contents of which are incorporated herein by reference in their entireties).

The disclosed cationic biodegradable nanoparticles may be prepared by methods known in the art. (See, e.g., Nagavarma et al., Asian J. of Pharma. And Clin. Res., Vol 5, Suppl 3, 2012, pages 16-23; Cismaru et al., Rev. Roum.

Chim., 2010, 55(8), 433-442; and Internationa] Application Publication Nos. WO 2012/115806; and WO 2012/054425; the contents of which are incorporated herein by reference in their entireties). Suitable methods for preparing the nanoparticles may include methods that utilize a dispersion of a preformed polymer, which may include but are not limited to solvent evaporation, nanoprecipitation, emul sifi cati on/ solvent diffusion, salting out, dialysis, and supercritical fluid technology. In some embodiments, the nanoparticles may be prepared by forming a double emulsion (e.g., water-in-oil-in-water) and subsequently performing solvent-evaporation. The nanoparticles obtained by the disclosed methods may be subjected to further processing steps such as washing and lyophilization, as desired. Optionally, the nanoparticles may be combined with a preservative (e.g., trehalose).

Typically, the nanoparticles have a mean effective diameter of less than 1 micron, and preferably the nanoparticles have a mean effective diameter of between about 25 nm and about 500 nm, e.g., between about SO nm and about 2S0 nm, or about 100 nm to about ISO nm. The size of the particles (e.g., mean effective diameter) may be assessed by known methods in the art, which may include but are not limited to transmission electron microscopy (TEM), scanning electron microscopy (SEM), Atomic Force Microscopy (AFM), Photon

Correlation Spectroscopy (PCS), Nanoparticle Surface Area Monitor (NSAM), Condensation Particle Counter (CPC), Differential Mobility Analyzer (DMA), Scanning Mobility Particle Sizer (SMPS), Nanoparticle Tracking Analysis (NTA), X-Ray Diffraction (XRD), Aerosol Time of Flight Mass Spectroscopy (ATFMS), and Aerosol Particle Mass Analyzer (APM).

The disclosed cationic biodegradable nanoparticles may have a zeta- potential that facilitates uptake by a target cell . Typically, the nanoparticles have a zeta-potential greater than 0. In some embodiments, the nanoparticles have a zeta-potential between about S mV to about 45 mV, between about IS mV to about 35 mV, or between about 20 mV and about 40 mV. Zeta-potential may be determined via characteristics that include electrophoretic mobility or dynamic electrophoretic mobility. Electrokinetic phenomena and electroacoustic phenomena may be utilized to calculate zeta-potential.

The invention will be described by the following non-limiting example.

Example»

Methods

PLGANP Fabrication

PLGA NPs were prepared using the well-established double emulsion solvent evaporation method. PLGA (SO mg, Resomer RGS03, viscosity

0.32-0.44 dlVg, MW 24,000-38,000, Boehringer Ingelheim KG) and amine- end-capped PLGA (SO mg, MW 10,000-20,000, PolyScitech) were dissolved in a mixture of 2.35 mL of ethyl acetate (EA) and 0.250 mL of dimethyl sulfoxide (DMSO). Sixty milliliters of 2.5% (w/v) poly(vinyl alcohol) (PVA, Mowiol 8-88, 87-9089% hydrolyzed, MW 67,000, Sigma-Aldrich) in 10 mM phosphate buffered saline (PBS) was prepared, and 9 mL was added to a 20 mL scintillation vial along with 1 mL of EA. Fluorescently labeled CaMKHN peptide (sequence: H-KRP PKL GQI GRA KRV VIE DDR K (HF488>NH2; HF488: HiLyte Fluor 488 acid) (AnaSpec Inc.) was dissolved in a solution of water containing 1% PVA (w/v) at a concentration of 5 mg/mL.

To prepare the nanoparticles, the organic and aqueous phases were emulsified using a probe sonicator (Fisher Scientific). First, 125 uL of the CaMKHN solution was sonicated into the polymer/EA/DMSO solution at 40% amplitude for 60 s. Next, this emulsion was sonicated into 9 mL of 2.5% PVA solution containing 1 mL of EA. Finally, the emulsion was poured into the remaining 51 mL of 2.5% PVA solution. The particle suspension was stirred using a magnetic stir bar for 30 min, followed by centrifugation at 4500 * g for 5 min to pellet larger unwanted particles. The supernatant was removed and centrifuged at 10,000 x g for 30 min to form a pellet of NPs. NPs were then washed by discarding the supernatant and replacing it with water, followed by centrifugation at 10,000 * g. The particles were washed twice to remove residual surfactant. After washing, the particles were frozen at -80 °C overnight and lyophilized (Labconco). NPs were loaded with a near-infrared fluorescent dye (XenoLightTM DiR, PerkinElmer) to evaluate biodistribution. Particles were prepared according to the method described above except only a single emulsion was used. Due to the poor aqueous solubility of the dye, it was added directly to 100 mg of PLGA dissolved in 2.5 mL of EA. This solution was sonicated into 9 mL of 2.5% PVA solution containing 1 mL of EA. Then the emulsion was poured into 51 mL of 2.5% PVA, and the particle suspension was stirred using a magnetic stir bar for 30 min. The same collection, washing, and storage procedures as described above were used.

Chitosan Purification

Chitosan (low molecular weight, deacetyiation degree 96.1%, Sigma- Aldrich) was purified according to a previously established method.19 Chitosan (2 g) was dissolved in 200 mL of 1% (v/v) acetic acid then filtered (Whatman 541 filter paper). The filtrate was titrated with 1 N NaOH until the pH was approximately 8.S to precipitate the chitosan. The precipitate was removed via filtration and resuspended in 500 mL of buffer (0.1 M sodium bicarbonate, pH 8.3). Next, 2.5 g of sodium dodecyl sulfate (SDS) and 3.72 g of

ethylenediaminetetraacetic acid (EDTA) were added to the solution and stirred using a magnetic stir bar for 30 min. The insoluble chitosan was filtered, rinsed, and dialyzed (Snakeskin) in nanopure water for 24 h. During dialysis, the water was changed after 10 h and every hour afterward. The chitosan was collected from the dialysis tubing, frozen at -80 °C overnight, and lyophilized.

Chitosan Coating of NPs

Dry NPs were suspended in 0.5 mL of chitosan (3 mg/mL) dissolved in 1% (v/v) acetic acid. Subsequent to complete resuspension, NPs were centrifuged at 10,000 x g for 20 minutes. The supernatant was removed and replaced with 1 mL water.

Determination of Particle Morphology. Size and Zeta Potential

Physical characterization of the NPs was performed using a scanning electron microscope (SEM, Hitachi S4800). Dry NPs were dispersed in water. A small drop of NP suspension was placed on a silicon wafer fixed to an aluminum stub. After all water had evaporated, the sample was sputter-coated (Emitech Sputter Coater K550, Quorum Technologies) with a mixture of gold and palladium before imaging. Hydrodynamic diameter, zeta potential, and polydispersity index (PDI) of the NPs were determined in water using dynamic light scattering (DLS, Zeta SizerNanoZS, Malvern Instruments). CaMKHN Loading and Release. To assess CaMKUN loading, PLGA NPs were dissolved in 0.3 N NaOH (1 mg/100 uL). Once all NPs were degraded, the solution was neutralized to pH 7 using 1 N HC1. The concentration of fluorescently labeled CaMKIIN in the sample was determined by linear regression using standard CaMKUN solutions ranging from 0.4 to 50 ug/mL (diluted in PBS). The standards and samples were analyzed simultaneously in a 96-well plate using a SpectraMax Plus 384 microplate reader (Molecular Devices) with an excitation wavelength of 500 nm and emission collected at 530 nm. The background signal was determined using PBS. The encapsulation efficiency (equation below) was calculated according to Joshi et al. To measure CaMKIIN release, 29.4 mg of NPs was suspended in 0.S mL of PBS and agitated at 300 rpm and 37 °C. At time points from 30 min to 48 h, the sample was centrifuged at 15,000 x g to pellet NPs. The supernatant was collected and NPs resusp ended in 0.S mL of PBS. Samples were stored at -20 °C until the time of analysis. The amount of CaMKIIN released at each time point was determined using a SpectraMax Plus 384 microplate reader

(Molecular Devices) with 500 nm excitation and 530 nm emission as described for analysis of CaMKIIN loading.

In Vitro Cellular Uptake

Human airway epithelial cells (HAECs) were cultured in keratinocyte serum-free medium supplemented with 1% penicillin/streptomycin (Gibco) on collagen (rat tail, type 1, Sigma-Aldrich) at 37 °C and 5% C02. 183 21 Next, HAECs were plated in a 6-well plate at a density of 2 x 105 cells per well and incubated at 37 °C and 5% C02 for 24 h. After changing the medium, chitosan- coated and uncoated CaMKHN-loaded PLGA NPs (300 ug) were added to the wells and incubated for 24 h. The cells were collected from the wells by trypsinization and centrifuged at 230 * g for 5 min. The supernatant was removed and replaced with 0.5 mL of fresh medium, and cells were stored on ice until analysis. The amount of fiuorescently labeled CaMKIIN associated with the cells was assessed by flow cytometry (F AC Scan, Becton Dickinson

Immunocytometry Systems). The excitation wave-length was 488 nm, and the emission was collected at 530 nm using a 30 nm bandpass filter. The mean fluorescence intensity for 10,000 cells was determined for each sample.

Primary Bronchial Murine Epithelial Cell Culture and Treatment.

Primary murine tracheal epithelial cells (MTBEC) were isolated from

B62 mice (obtained from Charles River Laboratories) as previously described. 22 For analysis of expression of Th2 cytokines, cells were plated onto collagen- coated (BD Biosciences) coverslips and maintained in MTEC Plus culture medium as described previously. 22 Cells were grown until confluent and then exposed to chitosan-coated empty (E) or CaMKIIN (CN) loaded NPs (25, 50, or 100 ug) with recombinant murine IL-13 (10 ng/mL, R&D Systems) for 14 days. RNA was isolated as stated below.

Animals Six to ten week old C57B1/6J female and male mice (equal proportions) were obtained from Charles Rivers Laboratories International, Inc. All animal studies complied with NIH guidelines and were approved by the University of Iowa Institutional Animal Care and Use Committee.

OVA Sensitization. Challenge and NP Delivery

Mice were sensitized by intraperitoneal injection (IP) of 10 ug of OVA (Sigma) mixed with 1 mg of alum (or saline alone for control) on days 0 and 7. Mice were subsequently challenged by nebulization of OVA (1% solution in 0.9% saline, 40 min challenge) or saline on days 14-17. Prior to OVA challenge by inspiration of soluble OVA, on days 14 and 16, oropharyngeal (OP) delivery of chitosan-coated NPs was performed as described previously with some modifications.23,24 Briefly, mice were anesthetized with 2% isoflurane vapor in oxygen and then suspended by cranial incisors on a thin rubberband from a ring stand. To visualize the base of the tongue and the pharynx, the nares were pinched with curved forceps and the tongue gently extracted from the mouth using blunt forceps. NPs (25, 50, or 100 uL corresponding to 25, 50, or 100 ug of NP) were placed in the posterior pharynx with a micropipettor. Respiration was monitored to ensure the suspension was fully delivered before the tongue and nares were released. Airway reactivity to methacholine was determined 24 h after the last OVA challenge (day 18). In control experiments, 25 ng of

CaMKHN peptide alone was administered. Based on a loading of 0.6 (±0.02) ug of CaMKIIN per mg of NPs, this dose corresponds to the delivery of 50 ug of CaMKIIN-loaded NPs. Biodistribution. Near infrared dye-loaded nanoparticles were administered to mice via OP delivery. Particles were chitosan coated as described above or uncoated and instilled by OP delivery. For controls, mice were instilled with PBS alone. At 1, 24, and 48 h time points the fluorescence intensity of the organs was measured using a Xenogen In Vivo Imaging System (IVIS-200).

Assessment of Airway Hvperreactivitv (AHR>

AHR in response to methacholine was measured on a flexiVent small- animal ventilator (Scireq) using a single compartment model, which determines the dynamic resistance of the respiratory system (R), as described in Sanders et al. (2013).

Bronchoalveolar Lavage After the assessment of AHR, mice were euthanized, the trachea was cannulated, and two PBS washings were collected for analysis of total and differential counts in the bronchoalveolar lavage fluid (BALF). BALF cellular differential was determined on 250 uL of cytospins stained with Diff-Quik (Dade Behring).

Quantification of ROS in Murine Bronchial Epithelial Cells.

ROS were measured from freshly isolated MTBEC from mice exposed to OVA in the presence of blank (E) or CaMKHN (CN) loaded, chitosan-coated NP using dihydroe-thidium red (5 mM, Invitrogen).22 The cellular staining was confirmed by colocalizing with CellTracker Green (50 nM, Thermo Fisher Scientific). Cells were imaged using a LSM 510 confocal microscope (Carl Zeiss) and analyzed with ImageJ software (ImageJ64, version 1.48, National Institutes of Health). All images were taken at the same time and used the same imaging settings. Data are presented as fold change compared to blank (OVA+E) NPs.

After the assessment of AHR, mice were euthanized, the trachea was cannulated, and two PBS washings were collected for analysis of total and differential counts in the bronchoalveolar lavage fluid (BALF). BALF cellular differential was determined on 250 uL cytospins stained with Diff-Quik (Dade Behring).

Liver Toxicity and Weight Change in Mice

Commercially available kits were used to evaluate bilirubin

concentration (Sigma) and AST activity (Sigma) in the blood serum of mice. For bilirubin analysis, all experimental groups were normalized to saline control. The weight of individual mice was recorded before they were given any treatments and at the end of the treatment regimen (i.e., immediately before assessment of AHR) to determine percent weight change.

Lung Histology.

Lungs were fixed with 4% paraformaldehyde and then processed by paraffin embedding. Tissue sections (5 um) were cut and stained using hematoxylin and eosin (H&E) or Alcian Blue/periodic acid-Schiff (PAS) to determine mucin distribution. Images were acquired using a Leica light microscope. Eosinophilia from H&E sections was determined using the 40* objective; 4-5 random digital images per group were taken within areas of overt peri-bronchiolar inflammation. Total eosinophil cell counts were determined using ImageJ software (ImageJ64, version 1.48, National Institutes of Health) and expressed as number of cells per 10 um2. Severity of perivascular inflammation was quantified by a four-point scoring system where 0 = absence of cell cuffs, 1 = rare to few scattered perivascular inflammatory cell cuffs, 2 = multifocal to moderate numbers of perivascular inflammatory cell cuffs, and 3 = large number of diffuse perivascular inflammatory cell cuffs. 29025 For mucin measurements, PAS-stained slides were imaged (20 ^χ objective) and then ImageJ software was used to determine the percentage of positively stained area per total area.

IL-S Cytokine Determination

IL-5 was analyzed in lung homogenates by cytokine-specific ELISA Duo Set kit (R&D Systems) and normalized to total protein content (DC Assay, Bio- Rad) according to the manufacturer's instructions.

Statistical Analysis

Data are shown as means ± SEM. Analysis of experiments was performed using two-tailed students t test, two-way ANOVA or one-way ANOVA, and post hoc comparisons tested using Tukey correction. The

GraphPad Prism statistical software program was used for the analyses. *P < 0.0S; **P < 0.01; ***P < 0.001 were regarded as statistically significant. Results of experiments are compared between OVA-challenged mice or OVA-challenge in the presence of empty NPs.

Quantitative real-time PCR

Total RNA was isolated using the Qiagen RNeasy column- based kits. Complementary DNA was prepared using the Superscript ΙΠ reverse

transcription system (Invitrogen) with random nanomer primers. Expression of mRNA was quantified with the iQ LightCycler (Bio-Rad) and SYBR Green dye system and normalized to acidic ribosomal phosphoprotein 1 (Arp) mRNA

Pnmers

Arp forward - TCA TCC AGC TGT TTG ACA A (SEQ ID NO: 1), Arp reverse - ATT GCG GAC ACC CTG TAG GAA G (SEQ ID NO:2). Muc5ac forward- GTG GTG GAA ACT GAC ATT GG (SEQ ID NO:3), Muc5ac reverse - CAT CAA AGT TCC CAC ACA GG (SEQ ID NO:4). Eotaxin forward - CAC TTC CTT CAC CTC CCA GGT GC (SEQ ID N0:5), Eotaxin reverse- CCC ACT TCT TCT TGG GGT CAG CA (SEQ ID NO:6).

Labelled CaMKIIN peptide sequence

H - KRP PKL GQI GRA KRV VIE DDR K(HF488) - N¾ (SEQ JD NO:7) HF488: HiLyte™ Fluor 488 acid; which is based on

1 mseilpysed kmgrfgadpe gsdlsfscrl qdtnsffagn qakrppklgq igrakrwie 61 ddriddvlkg mgekppsgv (SEQ ID NO:8).

Other peptide inhibitors include but are not limited to KRP PKL GQI GRS KRV VIE DDR K (SEQ ID NO:9), P PKL GQI GRA KRV VIE DDR K (SEQ ID NO: 10), P PKL GQI GRs KRV VIE DDR K (SEQ JD NO: 11), KRP PKL GQI GRA KRV VIE D (SEQ JD NO: 12), KRP PKL GQI GRS KRV VIE D (SEQ JD NO: 13), KRP PKL GQI GRA KRV VI (SEQ ID NO: 14) or KRP PKL GQI GRS KRV VI (SEQ JD NO: 15).

Results and Discussion

Exposure of the respiratory epithelium to allergens is the initiating event in allergic asthma. Accordingly, delivery of therapeutic agents specifically to the respiratory epithelium is likely to quell core asthmatic phenotypes. Site-specific delivery of therapeutics to the airway has the potential to improve efficacy and lower drug toxicity (Heda et al., 2016). Nanoparticle drug delivery systems allow for local delivery and offer additional advantages such as sustained release of therapeutic molecules over a desired amount of time, ability to deliver a high concentration of drug (especially for poorly soluble drugs), fewer doses needed and decreased enzymatic degradation of drug (Panyam et al., 2002). PolyQactic- co-grycolic acid) (PLGA) is a biodegradable polymer that is FDA approved for use in a wide variety of biomedical applications that can be utilized to fabricate NPs and entrap therapeutically active molecules (Heda et al., 2016; Mahapatro and Singh, 2011).

CaMKn is activated by ROS (ox-CaMKH) (Erickson et al., 2008). Ox- CaMKn is increased in airway epithelium from asthmatic patients after allergen exposure and correlates with asthma severity (Sanders et al., 2013). Inhibition of CaMKn in the lungs of mice protected against allergen-induced phenotypes (Sanders et al., 2013). Inhibitory peptides such as CaMKIIN are notable for lacking activity against other calmodulin kinases or protein kinase C (Chang et al., 1998) and provide a potential approach for highly specific CaMKII inhibition. CaMKIIN-loaded PLGANPs were examined as an inhalable therapeutic tool for allergic airway disease. PLGA NPs were loaded with the 21 amino acid peptide CaMKIIN and conjugated with a HiLyte™ Fluor 488 moiety for ease of detection. Afterward, NPs were coated with chitosan and imaged using scanning electron microscopy (SEM). The NPs were found to be smooth in morphology and spherical in shape (Figure 1 A). The loading of CaMKIIN in the PLGA NPs was 0.S (± 0.02) ug CaMKIIN per mg of NPs. Because of the high water solubility of the peptide, the encapsulation efficiency was around 3%. An in vitro release study demonstrated that about 50% of CaMKIIN was released within the first 30 minutes, with an additional 15% release by 48 hours (Figure IB).

Chitosan is a natural, cationic polymer with known mucoadhesive properties and has been shown to promote adsorption, uptake and retention of therapeutic agents into lung epithelial cells (Area et al., 2009; Lee et al., 2013) in a mechanism that may include glycoprotein-mediated endocytosis (Artursson et al., 1994). To enhance the formulation of CaMKIIN delivery to the lungs, a chitosan layer was self-assembled onto the surface of the PLGA NPs via electrostatic interactions. To confirm the adsorption of chitosan on the surface of the NPs, dynamic light scattering (DLS) was used to measure size and zeta potential. In the absence of chitosan, the average hydrodynamic diameter of the PLGA NPs was 160 nm and the average zeta potential was 4 m V (Figure 1 C). After coating of the NPs with the chitosan layer, the average hydrodynamic diameter increased to 230 nm and the zeta potential to approximately 40 mV (Figure 1C). This confirmed that the surface of the NPs was modified by chitosan. Furthermore, the polydispersity index (PDI) of uncoated and chitosan- coated NPs were 0.2 (±0.1) and 0.22 (±0.09), respectively, indicating that there was no aggregation after coating the NPs with chitosan.

To determine the functional properties of chitosan -coated CaMKIIN- loaded PLGA NPs, cellular uptake was assessed in primary human airway epithelial cells (HAECs) by flow cytometry. There was a significant (4-fold) increase in uptake of chitosan-coated NPs in HAECs compared to uncoated NPs and the control (Figure ID). Because of the drastic enhancement of uptake in HAECs, chitosan-coated NPs were utilized for subsequent in vivo testing of allergic asthma. To assess specific delivery of NPs to the lungs, a biodistribution study was performed in male and female C57B1/6J mice using chitosan-coated and uncoated PLGA NPs loaded with a near-infrared dye (thought to be retained within the PLGA matrix) and administered by oropharyngeal instillation (OP), which would directly deliver NPs to the lung. After 1 hour, both coated and uncoated NPs were robustly detected in the lungs. Other organs had no appreciable increase in signal over PBS controls (Figures 2A-B). The fluorescent signal in the lungs of mice treated with chitosan-coated NPs was significantly lower at 1 hour post instillation compared to the lungs of mice treated with uncoated NPs (Figure 2B). However, there was higher reproducibility across the replicate measurements in the chitosan-coated group compared to noncoated NPs in which there was a large variability (1 hour, Figure 2B). In addition, at 24 and 48 hours after nanoparticle instillation, the near-IR dye signal was significantly reduced in mice treated with uncoated NP compared to 1 h. In contrast, mice exposed to chitosan-coated NPs showed steady-state lung retention between 1, 24, and 48 h (Figures 2A-B).

To assess if CaMKIIN-loaded, chitosan-coated PLGA NPs are potential therapeutic agents for asthma, an established murine model of allergic asthma (Sanders et al., 2013) was used that employed sensitization to ovalbumin (OVA, Figure 2C). It is known that after delivery to the lungs, NPs can be detected in other organs such as the liver, heart, spleen, gastrointestinal tract, and brain. Therefore, toxicity was measured after euthanizing animals on day 18, using serum biomarkers including bilirubin and aspartate transaminase (AST).

Administration of chitosan-coated CaM-KUN-loaded PLGA nanoparticles, as diagrammed in Figure 2C, had no significant effect on total bilirubin (Figure 2D), serum AST activity (Figure 2E), or animal weight (Figure 2F). It was hypothesized that chitosan-coated, CaMKIIN-loaded NPs delivered directly to the lungs would be able to ablate OVA-mediated, CaMKII- induced asthma phenotypes better than soluble CaMKIIN delivered to the lungs. This hypothesis was tested by instilling mice with CaMKIIN peptide without NP encapsulation prior to assessing OVA-mediated airway hyperreactivity (AHR). After challenge with methacholine, both groups (OVA alone and OVA + CaMKIIN peptide) had significantly higher AHR compared to the saline control (Figure 3 A). These results suggest that encapsulating the peptide into PLGA-NPs followed adding a chitosan-coating to the surface of NPs may be sufficient for cellular uptake (as discussed previously and shown in Figure ID) as well as for modification of asthma phenotypes in an experimental murine model of allergic asthma

The effect of three doses (25, SO, or 100 ug) of blank NPs on OVA- mediated, methacholine induced AHR was assessed. The results showed that exposure of mice to blank NP at any concentration had a similar outcome as OVA alone (Figure 3B), suggesting there were no dose-dependent effects. Next, mice were given the same doses of NPs used in Figure 3B but this time loaded with a set concentration of CaMKHN peptide (0.S ug CaMKUN/mg NP, see Methods and Materials). The low dose of CaMKDN-loaded NPs (25 μg) did not alleviate OVA-mediated AHR, while the high dose (100 ug) caused a significant increase in OVA-mediated AHR compared to OVA alone (Figure 3C) at the highest methacholine concentration. This suggested there was a no observable effect level (NOEL) at the low dose and an adverse effect at the highest dose. However, the intermediate dose (50 μg) was effective at reducing OVA-induced AHR (Figure 3D). These data also emphasize that NP-based approaches can be adapted to deliver to other peptide-based therapies in asthma and other lung diseases. Exposure of mice to SO ug of CaMKDN-loaded NPs (OVA +CN) significantly reduced resistance, whereas empty NPs (OVA+E) did not protect against OVA-mediated AHR (Figure 3D). Administration of CaMKDN-loaded NPs to nonasthmatic control mice (Saline-CN) did not alter airway resistance (Figure 3D). As the 50 ug dose was effective at preventing increased airway resistance in OVA challenged mice after methacholine exposure and did not induce notable toxicity (Figure 2G), this dose was utilized for subsequent experiments.

In vivo studies investigating reactive oxygen species (ROS) production in models of cardiovascular disease have shown a role for CaMKII. Previous studies have demonstrated inhibition of CaMKII in transgenic mice reduces cytoplasmic ROS in airway epithelium and mucus production and inhibits allergen-mediated AHR. Recently, in a murine model of asthma using oxidant- resistant CaMKII MMW5 where the protein could not be activated, compared to wild type mice, MMW6 mice also had reduced asthmatic phenotypes as well as reduced levels of ROS. Therefore, CaMKII is thought to be a key mediator of ROS production. To test whether inhibition of CaMKU in bronchial epithelial cells decreases ROS production, primary murine tracheal bronchial epithelial cells (MTBEC) were isolated from OVA-challenged mice treated with empty NP (SO ug, OVA-E) or CaMKIIN-loaded NP (SO ug, OVA-CN). Staining for cytoplasmic superoxide production (DHE) showed significantly higher signal in mice treated with blank-NPs compared to mice treated with CaMKUN-NPs (Figure S). These results further support a role for CaMKU in ROS production and suggest CaMKII activity is indeed inhibited by CaMKIIN-loaded, chitosan- coatedNPs.

Asthma is characterized by excessive airway inflammation and accumulation of eosinophils. It was determined whether chitosan-coated NPs loaded with CaMKUN could attenuate lung eosinophilic inflammation induced by allergen challenge. Sensitization to OVA significantly increased total cell counts (Figure 4A) and eosinophils in bronchoalveolar lavage (BAL) fluid (Figure 4B). Although empty-NPs alone (OVA+E) significantly reduced OVA- mediated eosinophilic inflammation in the BAL, OVA-challenged mice exposed to CaMKIIN-loaded NPs had a further reduction in cell count and BAL- eosinophils (Figures 4A-B). This is interesting, and shows similar results to a previous study in which chitosan displayed immunosuppressive properties in OVA and prostate specific antigen (PSA) tumor models. Similarly, histologic analysis of lung sections demonstrated that, following allergen challenge, eosinophil infiltration into the airway was significantly abrogated by CaMKHN- loaded NPs compared to OVA-alone or OVA-exposure with empty NPs (Figures 4C-D). Further assessment of allergen-mediated inflammation was determined by quantification of perivascular cuffs present in lung tissue sections. There was a reduction in perivascular cuffing in the lungs of mice exposed to CaMKIIN- loaded NPs compared to OVA-alone or mice challenged with OVA and exposed to empty NPs (Figure 4E).

Eotaxin, an eosinophil chemoattractant, is induced in different allergy models. CaMKIIN-loaded NPs eliminated eotaxin mRNA expression following OVA challenge, in contrast to control or empty NP-treated mice where eotaxin mRNA was significantly increased (Figure 4F). Eotaxin cooperates with other interleukins, including IL-S, to promote tissue eosinophilia. Compared to saline- treated mice, animals exposed to saline in conjunction with CaMKIIN-loaded NPs (N.S.+CN) showed a trend toward an increase in lung-derived IL-5 protein; however, OVA alone or in the presence of empty NPs had significantly increased cytokine levels, while CaMKUN-loaded NPs reduced IL-5 protein (Figure 4G). Finally, analysis of another prominent type Π cytokine involved in asthma phenotypes, IL-4, in OVA-challenged mice treated with CaMKUN-NPs had significantly lower mRNA expression compared to mice treated with empty NPs (Figure 6). These results provide compelling evidence that delivery of chitosan-coated CaMKUN-loaded PLGA NPs reduce key features of allergic asthma, including AHR, eosinophilic airway inflammation, and production of inflammatory cytokines.

Another key feature of allergic asthma is increased mucus production in the lungs, and previous work has implicated CaMKII in this process.

Consistently, mucin expression and MUC5AC mRNA gene expression were significantly reduced in CaMKHN NP-treated mice compared to empty NP- treated mice or untreated mice following OVA challenge (Figures 4H-J).

Although mice instilled with empty NPs also had a significant reduction in MUC5AC mRNA, mice instilled with CaMKUN-loaded NPs had a significantly greater MUCS AC mRNA reduction relative to untreated mice challenged with OVA (Figure 4J). Although these data show the potential benefit of CaMKDN- loaded NPs as a treatment option for preventing asthmatic exacerbations in patients, it was further assessed whether the dose-dependent phenomenon in CaMKIIN-NP effects seen in Figure 3C could be recapitulated ex vivo in bronchial epithelial cells. MTBEC isolated from wild type mice and treated with increasing concentrations of NPs in the presence of a potent type Π cytokine, IL- 13, was similar to the in vivo assessment of AHR (Figure 3C); compared to blank-NPs, the intermediate dose of CaMKUN-loaded NPs (SO μg) significantly reduced IL-13- mediated effects on mRNA expression of eotaxin, IL-5, and MUCSAC (SO ug, Figure 7). Interestingly, the low (25 ug) dose had no effect, while the high (100 ug) dose chitosan-coated CaMKUN-NPs increased IL-13- induced effects on mRNA expression (Figure 7), similar to our AHR assessment in Figure 3C. This study demonstrates that, while CaMKII acts as a key mediator of the asthma disease phenotypes, more in depth analysis of the pharmacological and toxicological profile of CaMKUN is needed to further investigate chitosan- coated, CaMKUN-NPs as a viable treatment option for patients with asthma. In summary, cationic NPs were shown to be effective vehicles for drug delivery to the lung. Surface modification of PLGA NPs with chitosan enhanced the uptake of the encapsulated therapeutic agent in primary airway epithelial cells compared to uncoated NPs, with a favorable in vivo safety profile. In addition, we provide evidence for a novel pepti de-based formulation for CaMKII inhibition in the lungs. The translational potential of these findings is high given that CaMKII inhibitors and use of nanotechnology to improve retention of therapeutic agents are currently under development. As such, utilization of NPs for drug delivery to the lungs could offer a more efficacious and safer treatment option for asthmatic patients.

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All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification, this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details herein may be varied considerably without departing from the basic principles of the invention.