Camkii inhibitor-loaded liposomes for pulmonary treatment

Abstract

Compositions and methods for treating chlorine gas-induced pulmonary injury are disclosed. The described approach targets calcium / calmodulin-dependent protein kinase II (CaMKII), an enzyme activated in lung cells upon chlorine exposure, which contributes to oxidative stress and inflammation. The compositions include one or more CaMKII inhibitors, such as CaMKIIN peptides, encapsulated in liposomes and / or nanoparticles designed for pulmonary delivery. The liposomes can be conjugated with mitochondrial targeting moieties, such as triphenyl phosphonium bromide (TPP), to enhance delivery to mitochondria, where CaMKII activation occurs. The method involves administering the composition via inhalation or other routes to mitigate lung damage, reduce oxidative stress, and improve pulmonary function. This approach provides a targeted and effective solution for managing chlorine¬ induced respiratory distress, addressing limitations of current therapies.

Description

[0001] CAMKII INHIBITOR-LOADED LIPOSOMES FOR PULMONARY TREATMENT

[0002] PRIORITY

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 703,682, filed October 4, 2024, the content of which is herein incorporated by reference in its entirety. GOVERNMENT GRANT SUPPORT

[0004] This invention was made with government support under R21 ES032937 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0005] INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0006] This application contains a Sequence Listing which has been submitted electronically in ST26 format and hereby incorporated by reference in its entirety. Said ST26 file, created on October 3, 2025, is named 875253WO1.xml and is 2,881 bytes in size.

[0007] BACKGROUND

[0008] Inhalation of toxic oxidizing agents such as chlorine gas represents a notable challenge in respiratory medicine, with exposures leading to acute and chronic pulmonary distress. When inhaled, these agents interact directly with airway epithelium and alveolar structures, triggering inflammation, bronchoconstriction, and fluid accumulation. Clinically, patients may present with coughing, chest tightness, dyspnea, and in severe cases, acute respiratory distress syndrome. Given the rapid onset of tissue injury and inflammation, interventions are designed to address both the immediate biochemical insults and the secondary inflammatory cascades that contribute to lung damage.

[0009] A variety of approaches have been explored to ameliorate the effects of oxidative lung injury, including antioxidants, anti-inflammatory agents, and bronchodilators. The primary aim of these therapies is to restore redox balance, reduce cytokine release, and preserve airway function. Targeted pulmonary delivery is often preferred to achieve high local concentrations while minimizing systemic exposure. Various delivery platforms - ranging from small-molecule aerosols to liposomal carriers - have been evaluated for their capacity to enhance drug residence time in the lung and improve uptake by injured epithelial cells.

[0010] Despite these efforts, the management of chlorine-induced lung injury remains suboptimal. Systemically administered agents often fail to achieve therapeutically relevant concentrations in the respiratory tract, and inhaled formulations can be rapidly cleared by mucociliary mechanisms or suffer from limited penetration into distal airways. Moreover, the alveolar-capillary barrier and rapid enzymatic degradation of many bioactive compounds further restrict effective dosing. As a result, oxidative stress and inflammatory mediators continue to drive tissue injury, and current treatment regimens lack the ability to arrest the underlying cellular pathways responsible for progressive dysfunction.

[0011] SUM MARY

[0012] One of the potential targets associated with oxidative stress is calcium / calmodulin dependent protein kinase II (CaMKII). Provided herein is the use of agents to target CaMKII, which is activated in lung cells upon chlorine gas exposure. Inhibitors of CaMKII that specifically inhibit CaMKII in the lung are provided as therapeutics for chlorine toxicity. In some aspects, the CaMKII inhibitor is formulated with one or more lipids and / or nanoparticles and administered to subject that has been exposed to chlorine.

[0013] In one embodiment, the disclosure includes a composition comprising one or more CaMKII inhibitor-loaded liposomes. The CaMKII inhibitor can be a CaMKIIN peptide, hesperadin, ALK inhibitor 2, resveratrol, quercetin, ubiquinol or any combination thereof. The liposomes further comprise one or more phospholipids, cholesterol, sphingolipids, PEGylated lipids, cationic lipids, anionic lipids, fusogenic lipids or pH-sensitive lipids, and may include a mitochondrial targeting moiety such as triphenyl phosphonium bromide, a mitochondrial targeting sequence, szeto-Schiller peptides, mitochondria-penetrating peptides, dequalinium, rhodamine-based compounds or F16 peptide. The composition can also contain a pharmaceutically acceptable excipient suitable for aerosol or inhalation administration, for example lactose, microcrystalline cellulose, mannitol, starch, dicalcium phosphate, povidone, hydroxypropyl methylcellulose, ethyl cellulose, polyethylene glycol, croscarmellose sodium, sodium starch glycolate, crospovidone, magnesium stearate, stearic acid, talc, colloidal silicon dioxide, polyvinyl alcohol, propylene glycol, triethyl citrate, sucralose, aspartame, saccharin, flavors, parabens, benzyl alcohol, potassium sorbate, citric acid, sodium hydroxide, hydrochloric acid, polysorbates, cremophor, BHA, BHT, ascorbic acid, lecithin, polysorbate 80, xanthan gum, carboxymethylcellulose, iron oxide pigments, FD&C dyes, sodium chloride, dextrose, phosphate or citrate buffers, or combinations thereof.

[0014] In another embodiment, the disclosure includes a liposomal pharmaceutical composition comprising a plurality of liposomes, each liposome having a bilayer formed from one or more phospholipids and cholesterol, a CaMKIIN peptide encapsulated within the aqueous interior of the liposome and a triphenyl phosphonium moiety covalently conjugated to at least one lipid of the bilayer. The phospholipids can include DPPC and / or DSPE-PEG2ooo, and the CaMKIIN peptide can comprise the amino acid sequence KRPPKLGQIGRAKRVVIEDDRIDDVLK (SEQ ID NO: 1).

[0015] In a further embodiment, the disclosure includes a method for treating chlorine gas toxicity and / or chlorine-induced pulmonary injury by administering to a subject in need thereof the above-described composition. Administration can occur after exposure to chlorine gas and may be performed by oral, sublingual, buccal, topical, transdermal, nasal, inhalation, intravenous, intramuscular, subcutaneous or intradermal routes, or any combination thereof, including oropharyngeal aspiration directly to the lungs.

[0016] BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments discussed herein.

[0018] FIG. 1. Ch Exposure Study design using C57BI6 / J, female mice. Mice are administered prophylactic pain medication pre-exposure to chlorine gas. 4hr- or 24hr-post exposure, mouse lung mechanics are evaluated, and animals are sacrificed and tissues / bronchoalveolar lavage fluid (BALF) harvested for processing and further evaluation.

[0019] FIGS. 2A-2D. The pulmonary resistance measured using FlexiVent system (A), total protein concentrations (B), TNF a levels (C), and IL-6 levels (D) in BALF of unexposed mice or exposed to different chlorine gas concentrations (values represent means ± SD). Statistical analysis was performed using one-way ANOVA, followed by Tukey’s post-hoc test. P-values of less than or equal to 0.05 were considered significant. *p <0.05, ** p <0.01 , ***p <0.001 , ****p <0.0001.

[0020] FIGS. 3A-3C. Total cell counts (A), neutrophil counts (B), and macrophage counts (C) in the BALF of unexposed mice or exposed to different chlorine gas concentrations (values represent means ± SD). Statistical analysis was performed using one-way ANOVA, followed by Tukey’s post-hoc test. P-values of less than or equal to 0.05 were considered significant. *p <0.05, ** p <0.01 , ***p <0.001 , ****p <0.0001.

[0021] FIGS. 4A-4B. Fluorescence microscope images of lung tissues (A) and the mean fluorescence intensity (MFI) (B) of Alexa Flour 568 represent the oxidized CaMKII in lung tissues of unexposed mice and exposed to 200 ppm or 250 ppm chlorine concentration for 20 min. Anti-oxidized CaMKII antibody was conjugated to Alexa Fluor 568 for visualization.

[0022] FIG. 5. CaMKIIN-loaded liposomes (LPs) preparation. A lipid solution in ethanol is coinjected onto a microfluidic chip with a CaMKIIN solution in PBS. Resulting formation of CaMKIIN-loaded liposomes are then introduced to a solution of EDC / NHS to incorporate a mitochondrial targeting moiety (TPP) to the loaded liposomes. Resulting liposomes are administered to mice via oropharyngeal aspiration post-exposure to chlorine gas.

[0023] FIG. 6. Confirmation of TPP conjugation. MFI of fluorescamine dye, an amine specific dye, after incubation with liposomes unconjugated (LPs-NH2) and conjugated with TPP (LPs- TPP) to evaluate the conjugation efficiency of the TPP to the LP surface. Values represent means ± SD, *p <0.05, ** p <0.01 , ***p <0.001 , ****p <0.0001.

[0024] FIG. 7. Characterization of CaMKIIN-loaded liposomes prepared using microfluidics system with TPP cross linked (values are means ± SD). Entrapment efficiency, drug loading, mean particle size, polydispersity index and Zeta potentials were calculated.

[0025] FIG. 8. In vitro cumulative release profile of CaMKIIN from LPs prepared using microfluidics system with TPP cross linked after the LP preparation. Values represent means ± SD, n=3.

[0026] FIG. 9. MTS-based cytotoxicity assay of HEK293T cells following 6h and 24 h incubation with blank LPs and different concentration of CaMKIIN-loaded LPs. Values represent means ± SD. *p<0.05.

[0027] FIG. 10. LPs administration to mice after chlorine exposure. Mice are exposed to chlorine gas for a single, 20-min interval, followed by administration of pain medication and oropharyngeal aspiration of the "treatment." Control mice receive saline, while experimental groups receive either a solution of CaMKIIN or CaMKIIN-loaded liposomes. At 24hr postexposure, animals are sacrificed and tissues / BALF harvested for further evaluation.

[0028] FIGS. 11A-11 B. Total cell count (A) and total protein concentrations measured using microBCA (B) in the BALF of exposed mice to 200 ppm for 20 min and received either saline or CaMKIIN treatment (values represent means ± SD). Statistical analysis was done using oneway ANOVA, followed by Tukey’s post-hoc test. *p<0.05.

[0029] FIGS. 12A-12B. IL-6 levels (A), and TNF a levels (B) in the BALF of mice exposed to 200 ppm for 20 min and received either saline or CaMKIIN treatment (values represent means ± SD). Statistical analysis was done using one-way ANOVA, followed by Tukey’s post-hoc test. *p<0.05.

[0030] FIGS. 13. Neutrophil and macrophage cell counts were collected from the BALF of mice exposed to 200 ppm for 20 min and received either saline or CaMKIIN treatment (values represent means ± SD). Statistical analysis was done using one-way ANOVA, followed by Tukey’s post-hoc test. *p<0.05.

[0031] FIG. 14. Simple Western™ analysis for oxidized CaMKII levels in lung tissues of unexposed and exposed mice to 200 ppm or 250 ppm chlorine concentration for 20 min presented as peak area normalized to total protein (values represent means ± SD). Statistical analysis was performed using one-way ANOVA, followed by Dunnett’s post-hoc test. P-values of less than or equal to 0.05 were considered significant.

[0032] FIG. 15. ROS generation after exposure of HBECs to either air or chlorine gas measured using chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA). The ROS generated represented as the DCF mean fluorescence intensity (values represent means ± SD). Statistical analysis was performed using one-way ANOVA, followed by Dunnett’s post-hoc test. P-values of less than or equal to 0.05 were considered significant.

[0033] FIGS. 16A-16C. The pulmonary mechanics parameters measured using FlexiVent system; resistance (A), (H%) elastic stiffness (B) and compliance (C) measured for mice exposed to 200 ppm of chlorine gas concentrations for 20 min and received either saline or CaMKIIN-LPs treatment. Values represent the mean ± SD.

[0034] DETAILED DESCRIPTION

[0035] Reference will now be made in detail to certain embodiments of the disclosed subject matter. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0036] The inhalation of toxic oxidizing agents, such as chlorine gas, presents a significant challenge in respiratory medicine, leading to acute and chronic pulmonary distress. Chlorine gas exposure causes oxidative stress and inflammation, resulting in damage to airway epithelial cells, fluid accumulation, and bronchoconstriction. Current therapeutic approaches, including antioxidants, anti-inflammatory agents, and bronchodilators, aim to restore redox balance, reduce cytokine release, and preserve airway function. However, these treatments are often suboptimal due to systemic side effects, rapid mucociliary clearance of inhaled formulations, limited penetration into distal airways, and the inability to effectively target the underlying cellular pathways driving progressive lung dysfunction. Furthermore, the alveolar-capillary barrier and enzymatic degradation of bioactive compounds further restrict the efficacy of existing therapies, leaving a significant gap in the management of chlorine-induced lung injury.

[0037] The present disclosure addresses these limitations by providing a novel therapeutic approach that targets calcium / calmodulin-dependent protein kinase II (CaMKII), an enzyme activated in lung cells upon chlorine gas exposure. The approach utilizes CaMKII inhibitors, such as CaMKIIN peptides, formulated within liposomal carriers to enhance pulmonary delivery and therapeutic efficacy. These liposomes are engineered using microfluidic techniques to achieve precise size control, high drug loading efficiency, and uniform distribution. Furthermore, the liposomes are conjugated with mitochondrial targeting moieties, such as triphenyl phosphonium bromide (TPP), to ensure targeted delivery to mitochondria, where CaMKII activation contributes to oxidative stress-induced damage. This targeted delivery system not only improves the bioavailability of the therapeutic agent in the lungs but also minimizes systemic exposure and off-target effects.

[0038] By addressing the root cause of chlorine-induced lung injury-CaMKII activation-this development offers a notable advancement over conventional treatments. The liposomal formulation ensures prolonged residence time in the lungs, efficient uptake by injured epithelial cells, and precise targeting of mitochondrial pathways. This approach provides a robust and effective solution for mitigating the detrimental effects of chlorine gas exposure, addressing an important unmet need in respiratory medicine.

[0039] Definitions

[0040] The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as Hawley's Condensed Chemical Dictionary 14th Edition, by R.J. Lewis, John Wiley & Sons, New York, N.Y., 2001.

[0041] References in the specification to "one embodiment," "an embodiment," etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described.

[0042] The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, so that a compound X includes a plurality of compounds X. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with any element described herein, and / or the recitation of claim elements or use of "negative" limitations.

[0043] The term "and / or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrase "one or more" is readily understood by one of skill in the art, particularly when read in context of its usage. For example, one or more substituents on a phenyl ring refers to one to five, or one to four, for example if the phenyl ring is di-substituted.

[0044] As used herein, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating a listing of items, “and / or” or “or” shall be interpreted as being inclusive, e.g., the inclusion of at least one, but also including more than one of a number of items, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. , “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of”.

[0045] As used herein, the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof, are intended to be inclusive similar to the term “comprising.”

[0046] The term "about" can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent. For integer ranges, the term "about" can include one or two integers greater than and / or less than a recited integer at each end of the range. Unless indicated otherwise herein, the term "about" is intended to include values, e.g., weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment. The term about can also modify the endpoints of a recited range as discuss above in this paragraph.

[0047] As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements.

[0048] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. A recited range (e.g., weight percentages or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to," "at least," "greater than," "less than," "more than," "or more," and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents.

[0049] One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group.

[0050] Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, for use in an explicit negative limitation.

[0051] The subject matter disclosed herein is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0052] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed subject matter belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the disclosed subject matter, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0053] It is appreciated that certain features of the disclosed subject matter, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed subject matter, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the disclosed subject matter and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0054] Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises, such as Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-lnterscience, New York, 1992 (with periodic updates). Methods for chemical synthesis of nucleic acids are discussed, for example, in Beaucage and Carruthers, Tetra. Letts. 22: 1859-1862, 1981 , and Matteucci et al., J. Am. Chem. Soc. 103:3185, 1981.

[0055] The term “nucleic acid” typically refers to polynucleotides. By “nucleic acid” is meant any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).

[0056] As used herein, the term “nucleic acid” encompasses RNA as well as single and double-stranded DNA and cDNA. Furthermore, the terms, “nucleic acid,” “DNA,” “RNA” and similar terms also include nucleic acid analogs, i.e. , analogs having otherthan a phosphodiester backbone. For example, the so-called “peptide nucleic acids,” which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5’-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5’-direction. The direction of 5’ to 3’ addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the “coding strand”; sequences on the DNA strand which are located 5’ to a reference point on the DNA are referred to as “upstream sequences”; sequences on the DNA strand which are 3’ to a reference point on the DNA are referred to as “downstream sequences.”

[0057] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns. As used herein, a “substantially homologous amino acid sequences” includes those amino acid sequences which have at least about 95% homology, at least about 96% homology, at least about 97% homology, at least about 98% homology, or at least about 99% or more homology to an amino acid sequence of a reference antibody chain. Amino acid sequence similarity or identity can be computed by using the BLASTP and TBLASTN programs which employ the BLAST (basic local alignment search tool) 2.0.14 algorithm. The default settings used for these programs are suitable for identifying substantially similar amino acid sequences for purposes of the present invention.

[0058] The term “amino acid” is used interchangeably with “amino acid residue,” and may refer to a free amino acid and to an amino acid residue of a peptide / protein. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide / protein.

[0059] The expression “amino acid” as used herein is meant to include both natural and synthetic amino acids, and both D and L amino acids. “Standard amino acid” means any of the twenty standard L-amino acids commonly found in naturally occurring peptides / proteins. “Nonstandard amino acid residue” means any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or derived from a natural source. As used herein, “synthetic amino acid” also encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and substitutions. Amino acids contained within the peptides / proteins of the present invention, and particularly at the carboxy- or amino-terminus, can be modified by methylation, amidation, acetylation or substitution with other chemical groups which can change the peptide’s / protein’s circulating half-life without adversely affecting their activity (e.g., peptidomimetic for making peptides protease resistant).

[0060] Amino acids have the following general structure:

[0061] Amino acids may be classified into seven groups on the basis of the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, an imino acid in which the side chain is fused to the amino group.

[0062] The nomenclature used to describe the peptide / protein compounds of the present invention follows the conventional practice wherein the amino group is presented to the left and the carboxy group to the right of each amino acid residue. In the formulae representing selected specific embodiments of the present invention, the amino-and carboxy-terminal groups, although not specifically shown, will be understood to be in the form they would assume at physiologic pH values, unless otherwise specified.

[0063] The term “basic” or “positively charged” amino acid as used herein, refers to amino acids in which the R groups have a net positive charge at pH 7.0, and include, but are not limited to, the standard amino acids lysine, arginine, and histidine.

[0064] As used herein, the term “conservative amino acid substitution” is defined herein as an amino acid exchange within one of the following five groups:

[0065] I. Small aliphatic, nonpolar or slightly polar residues:

[0066] Ala, Ser, Thr, Pro, Gly;

[0067] II. Polar, negatively charged residues and their amides:

[0068] Asp, Asn, Glu, Gin;

[0069] III. Polar, positively charged residues:

[0070] His, Arg, Lys;

[0071] IV. Large, aliphatic, nonpolar residues:

[0072] Met, Leu, lie, Vai, Cys

[0073] V. Large, aromatic residues:

[0074] Phe, Tyr, Trp.

[0075] "Encoded by" refers to a nucleic acid sequence which codes for a polypeptide or RNA sequence. For example, the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence. The RNA sequence or a portion thereof contains a nucleotide sequence of at least 3 to 5 nucleotides, including at least 8 to 10 nucleotides, such as at least 15 to 20 nucleotides.

[0076] As used herein, a “subject in need thereof” is a patient, animal, mammal, or human, who will benefit from the method of this invention.

[0077] The terms "treating," "treat" and "treatment" include (i) preventing a disease, pathologic or medical condition from occurring (e.g., prophylaxis); (ii) inhibiting the disease, pathologic or medical condition or arresting its development; (iii) relieving the disease, pathologic or medical condition; and / or (iv) diminishing symptoms associated with the disease, pathologic or medical condition. Thus, the terms "treat", "treatment", and "treating" can extend to prophylaxis and can include prevent, prevention, preventing, lowering, stopping or reversing the progression or severity of the condition or symptoms being treated. As such, the term "treatment" can include medical, therapeutic, and / or prophylactic administration, as appropriate.

[0078] The term “regulate” refers to either stimulating or inhibiting a function or activity of interest.

[0079] CaMKII / CaMKII inhibitors

[0080] As used herein, “CaMKII” refers to the enzyme “calcium / calmodulin dependent protein kinase II.” In humans, there are four separate, highly homologous genes for CaMKII called alpha, beta, delta, or gamma (or a, p, 5 and y). 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 5: 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, CaMKII isoform 1 ; GenBank Accession No. NP-001020609, CaMKII delta isoform 2; NP-751910, CaMKII gamma isoform 3; GenBank Accession No. NP-751913, CaMKII gamma isoform 6: GenBank Accession No. NP-751913. CaMKII gamma isoform 6; GenBank Accession No. NP-751911 , CaMKII gamma isoform 1 ; GenBank Accession No. NP-751909, 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.

[0081] In 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.

[0082] 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).

[0083] 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-peptide agent, including, for example, a nucleic acid that encodes a peptide inhibitor. Moreover, the agent can be an antisense 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 (NIH) 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.

[0084] 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).

[0085] Provided herein are inhibitors of CaMKII to treat chlorine gas toxicity. CaMKII inhibitors include, but are not limited to, staurosporine, fasudil, autocamtide-2-Related Inhibitory Peptide, 1-Naphthyl PP1 , CaM Kinase II (290-309), CaMKIIN, KRPPKLGQIGRSKRVVIEDDRIDDVLK, K-252a, KN-62, lavendustin C, 12(S)-HPETE, K-252b, HA-1077 dihydrochloride, Arcyriaflavin A, CaM Kinase II inhibitor, KN-62, KN-93, KN-92, AIP (Autocamtide-2-related inhibitory peptide), HMN-709, KN-04, Scios-15b, Sanofi-32, Dainipon: 8p; Dainippon B:25, CN21 peptide, tatCN21 , AC3-I peptide, STO-609, Bosutinib, Sunitinib, Berberine, Myricetin, Green tea polyphenols, RP5217, RP5063, Rimacalib (SMP-114) or combinations thereof.

[0086] In one embodiment, the inhibitor is a protein or peptide such as CaMKIIN. CaMKIIN or CaM-KIIN designates small endogenous proteins that inhibit CaMKII with high affinity, such as CaM-KIINp (79 amino acids) and CaM-KIINa (78 amino acids). The a and p in their names are unrelated to the CaMKII isoform, as either of these inhibits all CaMKII isoforms with ICso of 50 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 CaMKIla with IC50 < 0.4 nM and improved selectivity for tested kinases. A similar study generated a smaller optimized 17 amino acid peptide, CN17p, with IC50 of 30 nM and little inhibition of CaMKI or CaMKIV (Gomez-Monterrey et al., 2013).

[0087] CaMKII inhibitor peptide (CaMKIIN): KRPPKLGQIGRAKRVVIEDDR-K (SEQ ID NO: 2)

[0088] Hesperadin

[0089] IIIPAC Name: N-[(3Z)-2-Oxo-3-(phenyl{4-[(piperidin-1-yl)methyl]anilino}methylidene)-2,3- dihydro-1 H-indol-5-yl]ethanesulfonamide). Anaplastic lymphoma kinase (ALK) inhibitor 2

[0090] ILIPAC Name: 5-[(E)-2-(4-Hydroxyphenyl)ethen-1-yl]benzene-1 ,3-diol.

[0091] Quercetin

[0092]

[0093] Liposomes are vesicle structures usually composed of a bilayer membrane of amphipathic molecules such as, phospholipids, entrapping an aqueous core. Drugs are either encapsulated in the aqueous core or interdigitated in the bilayer membrane. Drugs interdigitated in the membrane transfer out of the liposome when it is diluted into the body. Drugs that are encapsulated in the aqueous core or held in complexes in the aqueous core are generally retained substantially longer than drugs in the bilayer. (D. Drummond et al., J. Pharm. Sci., (2008) 97(11):4696-4740, PMID 10581328).

[0094] Exemplary liposomal membranes useful in the current invention may be formed from a variety of vesicle-forming lipids, typically including dialiphatic chain lipids, such as phospholipids, diglycerides, dialiphatic glycolipids, single lipids such as sphingomyelin and glycosphingolipid, cholesterol and derivates thereof, and combinations thereof. As defined herein, phospholipids are amphiphilic agents having hydrophobic groups formed of long-chain alkyl chains, and a hydrophilic group containing a phosphate moiety. The group of phospholipids includes phosphatidic acid, phosphatidyl glycerols, phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols, phosphatidylserines, and mixtures thereof. Preferably, the phospholipids are chosen from 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), dimyristoyl-phosphatidylcholine (DMPC), hydrogenated soy phosphatidylcholine (HSPC), soy phosphatidylcholine (SPC), dimyristoylphosphatidylglycerol (DMPG), disrearoylphosphatidylglycerol (DSPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)distearoyl phosphatidylcholine (DSPC), egg yolk phosphatidylcholine (EYPC) or hydrogenated egg yolk phosphatidylcholine (HEPC), sterol modified lipids (SML), cationic lipids and inverse-zwitterlipids.

[0095] In accordance with the invention, liposomes can be prepared by any of the techniques now known or subsequently developed for preparing liposomes. For example, the liposomes can be formed by the conventional technique for preparing multilamellar lipid vesicles (MLVs), that is, by depositing one or more selected lipids on the inside walls of a suitable vessel by dissolving the lipids in chloroform and then evaporating the chloroform, and by then adding the aqueous solution which is to be encapsulated to the vessel, allowing the aqueous solution to hydrate the lipid, and swirling or vortexing the resulting lipid suspension. This process engenders a mixture including the desired liposomes. Alternatively, techniques used for producing large unilamellar lipid vesicles (LLIVs), such as reverse-phase evaporation, infusion procedures, and detergent dilution, can be used to produce the liposomes. A review of these and other methods for producing lipid vesicles can be found in the text Liposome Technology, Volume I, Gregory Gregoriadis Ed., ORC Press, Boca Raton, Fla., (1984), which is incorporated herein by reference. For example, the lipid-containing particles can be in the form of steroidal lipid vesicles, stable plurilamellar lipid vesicles (SPLVs), monophasic vesicles (MPVs), or lipid matrix carriers (LMCs). In the case of MLVs, if desired, the liposomes can be subjected to multiple (five or more) freeze-thaw cycles to enhance their trapped volumes and trapping efficiencies and to provide a more uniform interlamellar distribution of solute.

[0096] Following liposome preparation, the liposomes are optionally sized to achieve a desired size range and relatively narrow distribution of liposome sizes. A size range of about 20-200 nanometers allows the liposome suspension to be sterilized by filtration through a conventional filter, typically a 0.22 or 0.4 micron filter. The filter sterilization method can be carried out on a high through-put basis if the liposomes have been sized down to about 20-200 nanometers. Several techniques are available for sizing liposomes to a desired size. Sonicating a liposome suspension either by bath or probe sonication produces a progressive size reduction down to small unilamellar vesicles less than about 50 nanometer in size. Homogenization is another method which relies on shearing energy to fragment large liposomes into smaller ones. In a typical homogenization procedure, multilamellar vesicles are recirculated through a standard emulsion homogenizer until selected liposome sizes, typically between about 50 and 500 nanometers, are observed. In both methods, the particle size distribution can be monitored by conventional laser-beam particle size determination. Extrusion of liposome through a smallpore polycarbonate membrane or an asymmetric ceramic membrane is also an effective method for reducing liposome sizes to a relatively well-defined size distribution. Typically, the suspension is cycled through the membrane one or more times until the desired liposome size distribution is achieved. The liposomes may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in liposome size. Alternatively controlled size liposomes can be prepared using microfluidic techniques wherein the lipid in an organic solvent such as ethanol or ethanol-aprotic solvent mixtures is rapidly mixed with the aqueous medium, so that the organic solvent / water ratio is less than 30%, in a microchannel with dimensions less than 300 microns and preferable less than 150 microns in wide and 50 microns in height. The organic solvent is then removed from the liposomes by dialysis. Other useful sizing methods such as sonication, solvent vaporization or reverse phase evaporation are known to those of skill in the art.

[0097] Exemplary liposomes for use in various embodiments of the invention have a size of from about 30 nanometers to about 40 microns.

[0098] The internal aqueous medium, as referred to herein, typically is the original medium in which the liposomes were prepared, and which initially becomes encapsulated upon formation of the liposome. In accordance with the present invention, freshly prepared liposomes encapsulating the original aqueous medium can be used directly for active loading. Embodiments are also envisaged however wherein the liposomes, after preparation, are dehydrated, e.g. for storage. In such embodiments the present process may involve addition of the dehydrated liposomes directly to the external aqueous medium used to create the transmembrane gradients. However, it is also possible to hydrate the liposomes in another external medium first, as will be understood by those skilled in the art. Liposomes are optionally dehydrated under reduced pressure using standard freeze-drying equipment or equivalent apparatus. In various embodiments, the liposomes and their surrounding medium are frozen in liquid nitrogen before being dehydrated and placed under reduced pressure. To ensure that the liposomes will survive the dehydration process without losing a substantial portion of their internal contents, one or more protective sugars are typically employed to interact with the lipid vesicle membranes and keep them intact as the water in the system is removed. A variety of sugars can be used, including such sugars as trehalose, maltose, sucrose, glucose, lactose, and dextran. In general, disaccharide sugars have been found to work better than monosaccharide sugars, with the disaccharide sugars trehalose and sucrose being most effective. Other more complicated sugars can also be used. For example, aminoglycosides, including streptomycin and dihydrostreptomycin, have been found to protect liposomes during dehydration. Typically, one or more sugars are included as part of either the internal or external media of the lipid vesicles. Most preferably, the sugars are included in both the internal and external media so that they can interact with both the inside and outside surfaces of the liposomes' membranes. Inclusion in the internal medium is accomplished by adding the sugar or sugars to the buffer which becomes encapsulated in the lipid vesicles during the liposome formation process. In these embodiments the external medium used during the active loading process should also preferably include one or more of the protective sugars.

[0099] A variety of loading methods for encapsulating functional compounds, particularly drugs, in liposomes is available. Hydrophilic compounds for example can be encapsulated in liposomes by hydrating a mixture of the functional compounds and vesicle-forming lipids. This technique is called passive loading. The functional compound is encapsulated in the liposome as the nanoparticle is formed. The available lipid vesicle (liposome) production procedures are satisfactory for most applications where water-soluble drugs are encapsulated (G. Gregoriadis, Ed., Liposome Technology, (2006) Liposome Preparation and Related Techniques, 3rd Ed.)

[0100] Passive loading of lipophilic and to a lesser extent amphiphilic functional compounds is somewhat more efficient than hydrophilic functional compounds because they partition in both the lipid bilayer and the intraliposomal (internal) aqueous medium. Generally, the concentration of drug in the liposome equals that of the surrounding fluid and drug not entrapped in the internal aqueous medium is washed away after encapsulation. Drugs loaded into the bilayer are released from the liposome very rapidly when the liposome is injected into a subject. For sustained release of the drug in a patient it is preferable that the drug is encapsulated within the interior of the liposome.

[0101] Certain hydrophilic or amphiphilic compounds can be loaded into preformed liposomes using transmembrane pH- or ion-gradients (D. Zucker et al., Journal of Controlled Release (2009) 139:73-80). This technique is called active or remote loading. Compounds amenable to active loading should be able to change from an uncharged form, which can diffuse across the liposomal membrane, to a charged form that is not capable thereof. Typically, the functional compound is loaded by adding it to a suspension of liposomes prepared to have a lower inside / higher outside pH- or ion-gradient. Via active loading, a high functional-compound-to- lipid mass ratio and a high loading efficiency (up to 100%) can be achieved. Examples are active loading of anticancer drugs doxorubicin, daunorubicin, and vincristine (P. R. Cullis et al., Biochimica et Biophysica Acta, (1997) 1331 :187-211 , and references therein).

[0102] Biodegradable nanoparticles can also be employed in the compositions and methods of the invention. The 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.

[0103] The 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-e-caprolactone (PCL), polyethylene glycol (PEG), poly(3-hydroxybutyrate), poly(p-dioxanone), polypropylene fumarate, poly(orthoesters), polyol / diketene acetals addition polymers, poly-alkyl-cyano-acrylates (PAG), poly(sebacic anhydride) (PSA), poly(carboxybiscarboxyphenoxyphenoxy hexone (PCPP) poly[bis (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).

[0104] The 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 International 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, emulsification / 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).

[0105] 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 50 nm and about 250 nm, or about 100 nm to about 150 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).

[0106] The 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 5 mV to about 45 mV, between about 15 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.

[0107] A liposome or nanoparticle design may employ surface ligands, such as a targeting moiety, for attaching to desired cells or tissues.

[0108] Mitochondrial Targeting Moiety

[0109] In certain embodiments, one or more moieties that specifically target the liposome to a particular cell type, tissue or the like are incorporated into the membrane. Targeting of liposomes using a variety of targeting moieties (e.g., ligands, receptors and monoclonal antibodies) has been previously described. In some embodiments, a mitochondrial targeting moiety is employed.

[0110] Suitable examples of mitochondrial targeting moieties include, but are not limited to, triphenyl phosphonium bromide (TPP), mitochondrial targeting sequence (MTS), szeto-Schiller (SS) peptides, mitochondria-penetrating peptides (MPPs), dequalinium, rhodamine-based compounds, F16 peptide or a combination thereof.

[0111] Targeting mechanisms generally require that the targeting agents be positioned on the surface of the liposome in such a manner that the target moieties are available for interaction with the target, for example, a cell surface receptor. In an exemplary embodiment, the liposome is manufactured to include a connector portion incorporated into the membrane at the time of forming the membrane. An exemplary connector portion has a lipophilic portion which is firmly embedded and anchored in the membrane. An exemplary connector portion also includes a hydrophilic portion which is chemically available on the aqueous surface of the liposome. The hydrophilic portion is selected so that it will be chemically suitable to form a stable chemical bond with the targeting agent, which is added later. Techniques for incorporating a targeting moiety in the liposomal membrane are generally known in the art.

[0112] Pharmaceutical Compositions / Administration

[0113] A "pharmaceutical composition" refers to a chemical or biological composition suitable for administration to a subject (e.g., mammal). Such compositions may be specifically formulated for administration via one or more of a number of routes including, but not limited to, oral, sublingual, buccal, topical, transdermal, nasal, inhalation, oropharyngeal, intramuscular (IM), subcutaneous, intradermal or intraperitoneal. In addition, administration can by means of capsule, drops, foams, gel, gum, injection, liquid, patch, pill, porous pouch, powder, tablet, or other suitable means of administration.

[0114] For aerosol administration, a liposome or nanoparticle comprising a CaMKII inhibitor may be supplied in finely divided form along with a surfactant and propellant. Typical percentages of CaMKII inhibitors in aerosol formulation may be 0.01 %-20% by weight, including 1-10%. The surfactant is non-toxic and preferably is soluble in the propellant. 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 propellant. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.

[0115] Pharmaceutical compositions can be, and desirably are, sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, nanoparticle, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.

[0116] Pharmaceutically acceptable carriers have been amply described in a variety of publications, including, for example, A Gennaro (2000) "Remington: The Science and Practice of Pharmacy", 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A H. Kibbe et al., eds., 3rd ed. Amer. Pharmaceutical Assoc. Pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

[0117] 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 CaMKII inhibitor in the composition), its mode of administration, and the like. A CaMKII inhibitor may be administered in a dose of from about 0.02 mg to about 5.0 mg per kilogram of body weight of the subject. 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.

[0118] In some embodiments of the disclosed methods, a CaMKII inhibitor 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 CaMKII inhibitor 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,

[0119] In the methods, a CaMKII inhibitor may be administered according to a wide variety of dosing schedules. For example, 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).

[0120] The following Examples illustrate some of the materials, methods, and experiments that were used or performed in the development of the invention.

[0121] EXAMPLE I

[0122] Introduction

[0123] Chlorine (CI2), a highly reactive oxidizing agent extensively utilized in various industrial and domestic applications, poses significant health risks, both through accidental exposure and intentional use as a chemical weapon. Exposure to Ch gas can lead to respiratory symptoms and long-term health issues due to oxidative stress and inflammation, with no known antidote available. Inhalation of CI2 primarily impacts the respiratory tract, inducing a spectrum of acute symptoms ranging from throat and upper respiratory airway irritation to chest pain, chest tightness, dyspnea, coughing, and acute edema. Acute exposure to elevated CI2 concentrations results in the damage of airway epithelial cells and leads to detrimental effects on respiratory health, including airway remodeling and hyperreactivity. This can further result in severe outcomes including pulmonary obstruction, reactive airway dysfunction syndrome (RADS), acute respiratory distress syndrome, and fatalities.

[0124] Ca2+ / calmodulin-dependent protein kinase II (CaMKII) emerges as a therapeutic target post-chlorine exposure, given its implication in oxidative stress-related damage. Previous studies indicated that CaMKII activity has been increased under stress conditions such as asthma, cardiovascular disease, diabetes mellitus, acute ischemic stroke, and cancer. Recent research emphasizes CaMKH's significant contribution to pathological events within the pulmonary system, highlighting its relevance in both physiological and disease contexts. A gap in the knowledge that needs to be addressed is whether CaMKII activation in lung cells is associated with CI2 gas exposure.

[0125] This study explored the relationship between CaMKII activation and CI2 gas toxicity. The data disclosed shows CaMKII plays a key role in Ch-induced lung injury and is a target for treatment after Ch exposure. An upregulation in the activation of CaMKII, induced by generation of reactive oxygen species (ROS), was observed following exposure to Ch gas. Thus, CaMKII is a target for ameliorating chlorine-induced lung damage. CaMKII peptide inhibitor (CaMKIIN) mitigates CaMKII-related conditions. Herein, CaMKII activation is evaluated after Ch exposure and the efficacy of CaMKIIN-loaded liposomes in ameliorating chlorine-induced lung injury is demonstrated.

[0126] Methods

[0127] CaMKIIN-loaded liposome preparation

[0128] A liposomal solution (12 mM) in ethanol was prepared using Egg PC (62 mM), 1 ,2- Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG 2000) (8 mM), cholesterol (30 mM) and an aqueous / solvent flow ratio of 3:1 . The liposomal solution was injected onto a microfluidic chip simultaneously with a solution of CaMKIIN (in PBS). Resulting CaMKIIN-loaded liposomes were modified to include a mitochondrial targeting moiety known as triphenylphosphonium bromide (TPP). CaMKIIN-loaded liposomes underwent conjugation to TPP via a solution of 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, Thermo Scientific) and N- hydroxysuccinimide (NHS). The resulting CaMKIIN-loaded, TPP-conjugated liposomes were evaluated for crosslinking using fluorescamine and a fluorescence spectrometer.

[0129] Immunohistochemistry & fluorescence microscopy

[0130] Briefly, the right lobes of the lungs were first perfused and then preserved using 10% buffered formalin (Fisher Scientific) until processing. The tissues were then paraffin-embedded, sectioned (5 pm thickness) and sections collected on slides. The slides were deparaffinized and then blocked with 5% fat free milk and 5% normal goat serum at room temperature for 2 h. Then, the tissue sections were incubated with anti-oxidized CaMKII (Met281 / 282) rabbit polyclonal antibody (Sigma-Aldrich, St. Louis, MO) at 4°C overnight. After washing with phosphate buffer saline (PBS), the sections were decorated with goat anti-rabbit IgG-biotin conjugated secondary antibody followed by incubation with Streptavidin conjugated to Alexa Fluor 568 (Life Technologies). Finally, the slides were mounted using Vectashield with DAPI mounting media (Vector Laboratories, Newark, CA) and imaged using fluorescence microscope (Olympus BX-63). The main fluorescence intensity of the signal indicating the oxidized CaMKII will be measured using Imaged software.

[0131] Results and Discussion

[0132] A pilot study was conducted by exposing C57BI6 / J female mice to two concentrations of chlorine: either 200 ppm or 250 ppm for 20 mins. 4 h or 24 h following the exposure, pulmonary mechanics were assessed using airway hyper-reactivity test (after methacholine challenge) (FIG. 1). There was a dose-dependent increase in pulmonary responsiveness to methacholine reflected in all the parameters of lung function (FIG. 2A). Chlorine induced a significant airway hyperresponsiveness following methacholine challenge in both dose groups 24 h post-exposure. Total protein, tumor necrosis factor alpha (TNF-a), interleukin-6 (IL-6), (FIG. 2B, 2C, 2D) and total and differential cell count in the bronchoalveolar lavage fluid (BALF) were evaluated following the Ch exposure (FIG. 3A, 3B, 3C). There was a significant dosedependent increase in the BALF protein level, and cell counts 24 h following the chlorine exposure confirming the inflammation and injury that happened in the lung epithelium (FIG. 2B, 3A-3C). In addition, there was a significant increase in IL-6 and TNF-a level in the BALF of the mice group exposed to 250 ppm of chlorine concentration and euthanized 4 h after exposure (FIG. 2C, 2D). Furthermore, the level of oxidized CaMKII in the lung tissues following exposure was evaluated using immunofluorescence staining (FIG. 4).

[0133] Notably, the oxidation of CaMKII in the lung tissues significantly increased after Ch exposure with the group exposed to 250 ppm and euthanized 4 h after exposure showing a higher oxidation than the group exposed to the same concentration and euthanized 24 h after exposure (FIG. 4). These results support the hypothesis that Ch exposure triggers the activation of CaMKII through oxidation. To explore the hypothesis that inhibition of CaMKII activity can mitigate the Ch-induced lung injury in vivo, CaMKII inhibitor peptide (CaMKIIN) was loaded in liposomes (LPs) using microfluidics technique (FIG. 5) and then conjugated with a mitochondrial targeting moiety, triphenyl phosphonium bromide (TPP) (FIGS. 6,). The prepared CaMKIIN-loaded liposomes had a uniform size distribution (63.7 ± 7.5 nm diameter with PDI = 0.13± 0.07) with a positive charge (6.33 ± 0.36 mV) (FIG 7) and showed minimal cytotoxicity (FIG. 9). C57BI6 / J female mice (n= 6 per group) were exposed to 200ppm Ch for 20 mins, and immediately after the exposure, mice received either saline solution or a treatment by oropharyngeal aspiration (OA) (FIG. 10). The assigned treatments were LPs equivalent to either 25 ng or 150 ng CaMKIIN or 25 ng CaMKIIN solution in PBS. The total protein level and cell count were evaluated 24 h after treatment (FIG. 11 A, 11 B), as well as cytokine levels (FIG. 12A, 12B) and differential cell counts (FIG. 13) present in the BALF of experimental and control mice. These data did not reveal significant effects of CaMKIIN treatment on immune cell population or cytokine levels in mice exposed to chlorine gas.

[0134] EXAMPLE II

[0135] Simple Western™ Analysis

[0136] Lung tissues were homogenized in radioimmunoprecipitation assay lysis buffer (RIPA buffer, Sigma-Aldrich) with protease and phosphatase inhibitors (Thermo Fischer Scientific) to protect the proteins during extraction. After centrifugation of the tissue lysate to get rid of the cell debris, the supernatants were collected, and the protein concentrations were measured using the micro BCA™ Protein Assay kit (Thermo Fischer Scientific). Simple Western™ analysis (ProteinSimple, Bio-Techne Corporation, Minneapolis, MN) was performed according to the ProteinSimple user manual. Briefly, tissue lysate samples were mixed with a master mix (ProteinSimple, Bio-Techne Corporation, Minneapolis, MN), then heated at 95 °C for 5 minutes. The samples, blocking reagent, primary antibodies, HRP conjugated second antibodies, chemiluminescent substrate, separation and stacking matrices were dispensed to designated wells in a 384-well plate. All the steps including the sample loading, protein separation, immunoprobing, washing, detection and data analysis were fully automated. Target proteins were immunoprobed with primary antibodies (anti-oxidized CaMKII (Met281 / 282) rabbit polyclonal antibody (Sigma-Aldrich, St. Louis, MO)) followed by anti-rabbit HRP-conjugated secondary antibodies (ProteinSimple, Bio-Techne Corporation). Luminol and peroxide (ProteinSimple, Bio-Techne Corporation) were used to generate chemiluminescence which was captured by a CCD camera. The digital image was analyzed with Compass software (ProteinSimple, Bio-Techne Corporation), and the quantified data of the detected protein were reported. The detected proteins were normalized to GAPDH antibody as a house keeping gene or total protein normalization using the RePlex™ module.

[0137] The Simple Western™ analysis for oxidized CaMKII was performed on the lung tissue sections obtained from both unexposed and chlorine-exposed mice groups. This was done to assess the correlation between chlorine exposure and the activation of CaMKII in the lung tissues. Figure 14 demonstrates the expression of oxidized CaMKII in all chlorine-exposed groups compared to the unexposed group. These results which support the results we got using immunofluorescence assay confirm the hypothesis that chlorine exposure triggers the activation of CaMKII through oxidation at Met281 / 282.

[0138] Oxidative stress was evaluated in response to chlorine gas exposure using human bronchial epithelial cells (HBECs, ATCC). Intracellular ROS was assessed using chloromethyl -2', 7'- dichlorodihydrofluorescein diacetate (CM-H2DCFDA, Invitrogen) that passively penetrates cells and reacts with ROS to form the highly fluorescent 2', 7'-dichlorofluorescein compound (DCF). There was a dose-dependent increase in mean fluorescence intensity (MFI) of DCF after exposure that indicated the higher levels of ROS generated by the cells in response to chlorine exposure (Figure 15). These results confirm the oxidative stress after chlorine exposure and support the hypothesis that that chlorine exposure increase ROS generation which triggers the activation of CaMKII through oxidation at Met281 / 282.

[0139] A second efficacy study following the same procedure of the previous animal study but this time a higher dose of CaMKIIN-loaded LPs was examine (equivalent to 450 ng of CaMKIIN) and the mice were euthanized at 48- and 96-hours post-treatment. An enhanced lung function was observed in the mice treated with the higher dose of CaMKIIN-loaded LPs compared to the saline-treated group 96 hours after treatment. The CaMKIIN-treated LPs group showed a decreased lung resistance, decreased lung stiffness (Elastance, H) and enhanced lung compliance compared to the saline-treated group (Figures 16A-16C). Although other parameters including the total protein and total cell count in the BALF did not show a pronounce difference between CaMKIIN- or Saline- treated groups, the finding showed the efficacy of CaMKIIN in mitigating the lung damage after chlorine exposure and lead us to continue our experiments to define the optimum effective dose.

[0140] 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 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.

Claims

WHAT IS CLAIMED IS:Claims1. A composition comprising one or more CaMKII inhibitor-loaded liposomes.

2. The composition of claim 1 , wherein the CaMKII inhibitor comprises CaMKII inhibitor peptide (CaMKIIN), hesperadin, ALK inhibitor 2, resveratrol, quercetin, ubiquinol or a combination thereof3. The composition of claim 1 or 2, wherein the CaMKII inhibitor-loaded liposomes further comprise one or more phospholipids, cholesterol, sphingolipids, PEGylated lipids, cationic lipids, anionic lipids, fusogenic lipids, or pH-sensitive lipids.

4. The composition of any one of claims 1 to 3, wherein the CaMKII inhibitor-loaded liposomes further comprise a mitochondrial targeting moiety.

5. The composition of claim 4, wherein the mitochondrial targeting moiety comprises triphenyl phosphonium bromide (TPP), mitochondrial targeting sequence (MTS), szeto- Schiller (SS) peptides, mitochondria-penetrating peptides (MPPs), dequalinium, rhodaminebased compounds, F16 peptide, or a combination thereof.

6. A liposomal pharmaceutical composition comprising a plurality of liposomes, each liposome comprising: a bilayer formed from one or more phospholipids and cholesterol; a CaMKIIN peptide encapsulated within an aqueous interior of the liposome; and a triphenyl phosphonium moiety covalently conjugated to at least one lipid of the bilayer.

7. The composition of claim 6, wherein the one or more phospholipids comprise 1 ,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and / or 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000],8. The composition of claim 6 or 7, wherein the CaMKIIN peptide comprises the amino acid sequence KRPPKLGQIGRAKRVVIEDDRIDDVLK (SEQ ID NO: 1).

9. The composition of any one of claims 1 to 8, wherein the composition further comprises a pharmaceutically acceptable excipient.

10. The composition of any one of claims 1 to 9, wherein the pharmaceutically acceptable excipient is suitable for aerosol or inhalation administration.

11. The composition of claim 9 or 10, wherein the pharmaceutically acceptable excipient comprises lactose, microcrystalline cellulose, mannitol, starch, dicalcium phosphate,povidone (PVP), hydroxypropyl methylcellulose (HPMC), ethyl cellulose, polyethylene glycol (PEG), croscarmellose sodium, sodium starch glycolate, crospovidone, magnesium stearate, stearic acid, talc, colloidal silicon dioxide, polyvinyl alcohol (PVA), propylene glycol, triethyl citrate, sucralose, aspartame, saccharin, natural and artificial flavors, parabens, benzyl alcohol, potassium sorbate, citric acid, sodium hydroxide, hydrochloric acid, polysorbates, cremophor, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ascorbic acid, lecithin, polysorbate 80, xanthan gum, carboxymethylcellulose, iron oxide pigments, FD&C dyes, sodium chloride, dextrose, phosphate buffers, citrate buffers or a combination thereof.

12. A method to treat chlorine gas toxicity and / or chlorine gas-induced pulmonary injury comprising administering to a subject in need thereof the composition of any one of claims 1 to 11.

13. The method of claim 12, wherein the composition is administered to the subject after exposure to chlorine gas.

14. The method of claim 12 or 13, wherein the composition is administered by oral, sublingual, buccal, topical, transdermal, nasal, inhalation, intravenous, intramuscular, subcutaneous, intradermal or combination thereof.

15. The method of any one of claims 12 to 14, wherein administering comprises oropharyngeal aspiration of the composition.

16. The method of any one of claims 12 to 15, wherein the composition is administered to the lungs.

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