WO2018031771A1 - Nanoparticules inhibitrice de camkii cationique pour le traitement de l'asthme allergique. - Google Patents

Nanoparticules inhibitrice de camkii cationique pour le traitement de l'asthme allergique. Download PDF

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WO2018031771A1
WO2018031771A1 PCT/US2017/046294 US2017046294W WO2018031771A1 WO 2018031771 A1 WO2018031771 A1 WO 2018031771A1 US 2017046294 W US2017046294 W US 2017046294W WO 2018031771 A1 WO2018031771 A1 WO 2018031771A1
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nps
poly
population
camkii
chitosan
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Aliasger K. Salem
Isabella GRUMBACH
Angie Sue THORN
John Daryl PASCHKE
Sara Carolyn SEBAG
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University Of Iowa Research Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/553—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one oxygen as ring hetero atoms, e.g. loxapine, staurosporine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/005—Enzyme inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11017—Ca2+/Calmodulin-dependent protein kinase (2.7.11.17)

Definitions

  • 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.
  • ROS reactive oxygen species
  • a population of cationic nanoparticles is provided, the surface of which are coated with one or more mucoadhesives.
  • 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
  • the mucoadhesive comprises chitosan. In one embodiment, the mucoadhesive comprises polylysine. In one embodiment, the mucoadhesive comprises polyamidoamine (PAMAM).
  • 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.
  • PSA poly(sebacic anhydride)
  • PCPP poly(carboxybiscarboxyphenoxyphenoxy hexone
  • PCPM poly[bis (p-carboxypheonoxy) methane]
  • the nanoparticle is formed of polylactide, polyglycolide, poly(lactic-co-glycolic acid), polysulfenamide, polyanhydride, or polycaprolactone.
  • 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.
  • 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.
  • 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.
  • the particle is formed of only one polymer, e.g., a cationic polymer.
  • 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).
  • 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.
  • 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
  • NP cationic nanoparticle
  • CaMKII such as the potent and specific CaMKII inhibitor peptide, CaMKIIN
  • CaMKHN-loaded NPs abrogated the severity of allergic asthma in a murine model.
  • PLGA poly(lactic-co-glycolic acid) nanoparticles (NPs) were directly delivered to the lung via oropharyngeal instillation (OP).
  • 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.
  • the CaMKII inhibitor comprises staurosporine, fasudil, autocamtide-2-Related Inhibitory Peptide, 1-Naphthyl PP1, CaM Kinase ⁇ (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 ⁇ inhibitor.
  • staurosporine fasudil
  • autocamtide-2-Related Inhibitory Peptide 1-Naphthyl PP1
  • CaM Kinase ⁇ 290-309
  • CaMKIIN CaMKIIN
  • KRPPKLGQIGRSKRWIEDDRIDDVLK SEQ ID NO:7
  • K-252a KN-62
  • 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
  • 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.
  • 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.
  • the particle is formed of only one polymer, e.g., a cationic polymer.
  • 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).
  • 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.
  • the method includes administering to the mammal an effective amount of a composition comprising the population of coated nanoparticles.
  • the mammal is a human.
  • the disease is a pulmonary disease such as asthma, e.g., allergic asthma.
  • the mammal has allergic asthma.
  • the composition is administered to the lungs, e.g., via inhalation.
  • the amount inhibits airway resistance or airway hyperresponsiveness.
  • 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.
  • FIGS 1A-D Chitosan coating of PLGANPs increases size, zeta potential, and cellular uptake by human airway epithelial cells.
  • FIGS 2A-F Chitosan-coated NPs localize in the lungs of mice and cause no significant toxicity in vivo.
  • B) Graphical analysis of NPs measured in lungs of mice at 1, 24, and 48 h (n 3-5 mice/group).
  • IP OVA sensitization
  • OP oropharyngeal
  • IH inhalation
  • OVA/ALUM ovalbumin and alum
  • 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 reduce airway hyperreactivity (AHR).
  • NS not significant.
  • FIGS 4A-J Airway inflammation, cytokine expression, and mucus production are decreased by cationic CaMKIIN-loaded NPs.
  • B) Eosinophil counts in BALF (n 7-12 mice).
  • F qRT-PCR for eotaxin in lung homogenates.
  • G HL-5 protein levels in lung homogenates by ELISA.
  • ⁇ , ⁇ Representative images of PAS staining (H, 20 x magnification) and (I) quantification (3-5 sections per mouse).
  • FIG. SA-B CaMKIIN-loaded NPs reduce OVA-mediated cell ROS in murine tracheal epithelial cells.
  • FIG. 1 Representative images and quantification of cytoplasmic ROS production in primary murine tracheal epithelial cells
  • MTBEC chitosan- coated empty NPs
  • ROS production was determined with.
  • Data were quantified for 3-5 images per treatment. Scale bars are ⁇ . Data are shown as mean ⁇ SEM. Student's 2 tailed t test was used. * p ⁇ 0.05 OVA-E vs. OVA-CN.
  • FIG. 6 OVA-induced IL-4 mKNA expression is reduced in the presence of CaMKIIN-NPs.
  • FIGS 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 ⁇ g chitosan-coated blank (E) or
  • NPs Polymeric nanoparticles
  • COPD Chronic Obstructive Pulmonary Diseases
  • 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.
  • PLGA NPs 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.
  • a NP-based formulation for asthma therapy was developed that is capable of local and sustained release of drug in the lungs.
  • poly (lactic-co-glycolic acid) (PLGA) a biodegradable polymer
  • PLGA poly (lactic-co-glycolic acid)
  • chitosan a natural polymer
  • the NP formulations were administered to asthmatic mice and tested for lung function.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the disease or condition is associated with calcium/calmodulin dependent protein kinase IL
  • 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.
  • treatment refers 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.
  • 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.
  • 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.
  • 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.
  • COPD chronic obstructive pulmonary disease
  • 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.
  • CaMKE refers to the enzyme "calcium/calmodulin dependent protein kinase H”
  • alpha, beta, delta, or gamma or ⁇ , ⁇ , ⁇ and ⁇ .
  • gamma or ⁇ , ⁇ , ⁇ and ⁇ .
  • 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 8 GenBank Accession No. NP-742078.
  • CaMKII beta isoform S GenBank Accession No.
  • CaMKII beta isoform 3 GenBank Accession No. NP-742075.
  • 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.
  • 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).
  • 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).
  • the methods include administering to the patient a therapeutic agent that inhibits oxidation of CaMKII.
  • 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
  • Inhibitors of CaMKII are known in the art. (see, e.g., U.S. Pat. No.
  • 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.
  • 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.
  • CID compound identification
  • 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.
  • 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):
  • the inhibitor is a protein or peptide such as
  • CaMKHN CaMKIIN or CaM- ⁇ designates small endogenous proteins that inhbit CaMKH with high affinity.
  • CaM KIIN3 (79 amino acids) and CaM- ⁇ (78 amino acids).
  • the a and ⁇ 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.
  • 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, ⁇ 3 ⁇ 17 ⁇ , with ICso of 30 nM and little inhibition of CaMKI or CaMKTV (Gomez-Monterrey et al, 2013).
  • a coated nanop article comprising a therapeutic agent may be administered as part of a pharmaceutical composition.
  • 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.
  • 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.
  • a coated nanoparticle comprising a compound e.g., a CaMKII inhibitor
  • 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.
  • 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.
  • a coated nanoparticle comprising a therapeutic agent of interest may be administered in any suitable manner.
  • 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.
  • parenterally e.g., intravenously, intramuscularly, intrathecally, or intraarterially
  • transdermally e.g., extracorporeally, topically, intranasally, or via an inhalant.
  • intranasal e.g., intravenously, intramuscularly, intrathecally, or intraarterially
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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
  • a therapeutic agent may be administered according to a wide variety of dosing schedules.
  • 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
  • Suitable dendrimers may include, but are not limited to, polyamidoamine (PAMAM) dendrimers.
  • PAMAM polyamidoamine
  • 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- ⁇ - 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),
  • 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.
  • 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.
  • 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.
  • the nanoparticles may be combined with a preservative (e.g., trehalose).
  • 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 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
  • PCS Correlation Spectroscopy
  • NSAM Nanoparticle Surface Area Monitor
  • CPC Condensation Particle Counter
  • DMA Differential Mobility Analyzer
  • SMPS Scanning Mobility Particle Sizer
  • NTA Nanoparticle Tracking Analysis
  • XRD X-Ray Diffraction
  • ATFMS Aerosol Time of Flight Mass Spectroscopy
  • API Aerosol Particle Mass Analyzer
  • the disclosed cationic biodegradable nanoparticles may have a zeta- potential that facilitates uptake by a target cell .
  • the nanoparticles have a zeta-potential greater than 0.
  • 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.
  • 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).
  • EA ethyl acetate
  • DMSO dimethyl sulfoxide
  • 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.
  • the organic and aqueous phases were emulsified using a probe sonicator (Fisher Scientific).
  • a probe sonicator (Fisher Scientific).
  • 125 uL of the CaMKHN solution was sonicated into the polymer/EA/DMSO solution at 40% amplitude for 60 s.
  • this emulsion was sonicated into 9 mL of 2.5% PVA solution containing 1 mL of EA.
  • 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.
  • 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.
  • 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.
  • EDTA ethylenediaminetetraacetic acid
  • 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.
  • 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.
  • NPs 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.
  • SEM scanning electron microscope
  • 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.
  • NPs 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
  • HAECs Human airway epithelial cells
  • keratinocyte serum-free medium supplemented with 1% penicillin/streptomycin (Gibco) on collagen (rat tail, type 1, Sigma-Aldrich) at 37 °C and 5% C02.
  • 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.
  • 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
  • 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.
  • MTBEC Primary murine tracheal epithelial cells
  • mice 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.
  • 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.
  • IP intraperitoneal injection
  • NPs 25, 50, or 100 uL corresponding to 25, 50, or 100 ug of NP
  • 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).
  • IVIS-200 Xenogen In Vivo Imaging System
  • 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).
  • 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).
  • 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.
  • 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).
  • kits were used to evaluate bilirubin
  • mice 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.
  • 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.
  • 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.
  • RNA was isolated using the Qiagen RNeasy column- based kits.
  • Complementary DNA was prepared using the Superscript ⁇ reverse
  • HF488 HiLyteTM Fluor 488 acid; which is based on
  • 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).
  • 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 HiLyteTM Fluor 488 moiety for ease of detection.
  • 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).
  • a chitosan layer was self-assembled onto the surface of the PLGA NPs via electrostatic interactions.
  • DLS dynamic light scattering
  • the average hydrodynamic diameter of the PLGA NPs was 160 nm and the average zeta potential was 4 m V ( Figure 1 C).
  • the average hydrodynamic diameter increased to 230 nm and the zeta potential to approximately 40 mV ( Figure 1C).
  • PDI polydispersity index
  • chitosan-coated CaMKIIN- loaded PLGA NPs 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.
  • HAECs primary human airway epithelial cells
  • chitosan-coated PLGA NPs are potential therapeutic agents for asthma
  • an established murine model of allergic asthma anders et al., 2013
  • OVA ovalbumin
  • 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).
  • MTBEC primary murine tracheal bronchial epithelial cells
  • 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).
  • Eotaxin an eosinophil chemoattractant
  • 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.
  • mice instilled with empty NPs also had a significant reduction in MUC5AC mRNA

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Abstract

L'invention porte sur une population de nanoparticules dégradables comprenant un revêtement mucoadhésif, avec un agent de diagnostic ou thérapeutique facultatif, ainsi que sur les méthodes d'utilisation des nanoparticules.
PCT/US2017/046294 2016-08-11 2017-08-10 Nanoparticules inhibitrice de camkii cationique pour le traitement de l'asthme allergique. WO2018031771A1 (fr)

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WO2021142245A1 (fr) * 2020-01-10 2021-07-15 Translate Bio, Inc. Composés, compositions pharmaceutiques et méthodes pour moduler l'expression de la muc5b dans des cellules et des tissus pulmonaires
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