WO2025244639A1 - Methods for treating lung diseases and disorders - Google Patents

Abstract

The disclosure features methods that are useful for treatment of a subject having a lungdisease or disorder. In particular, methods for treating lung diseases or disorders involving the administration of N-cadherin antagonists are disclosed herein.

Description

METHODS FOR TREATING LUNG DISEASES AND DISORDERS

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos. AI001083-14 and AI151695-01 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE DISCLOSURE

Asthma is a chronic inflammatory lung disease that affects more than 300 million people worldwide. Severe asthma induces substantial mortality and chronic disability due to irreversible or “fixed” airway obstruction (FAO) resistant to currently available therapies including corticosteroids and -adrenergic agonist bronchodilators. Nearly 5-10% of patients with asthma have severe, uncontrolled disease, and a majority of this group has FAO. No therapeutics currently exist to broadly limit FAO in asthma. Accordingly, improved therapies for the treatment of asthma, including FAO in asthma, are urgently needed.

SUMMARY OF THE DISCLOSURE

As described below, the present disclosure features compositions and methods for the treatment of lung diseases or disorders, including those associated with fixed” airway obstruction (FAO) (e.g., asthma, chronic obstructive pulmonary disorder, bronchiolitis).

In an aspect, the present disclosure provides a method of inhibiting N-cadherin activity in an airway smooth muscle cell. The method involves contacting an airway smooth muscle cell with an effective amount of an agent having N-cadherin antagonist activity, thereby inhibiting N- cadherin activity in the airway smooth muscle cell.

In another aspect, the present disclosure provides a method of reducing contractile agent- induced airway smooth muscle cell contraction. The method involves contacting an airway smooth muscle cell with an agent having N-cadherin antagonist activity, where the airway smooth muscle cell will be contacted with a contractile agent, was concurrently contacted with a contractile agent, or has been contacted with a contractile agent. In another aspect, the present disclosure provides a method of inducing bronchodilation in an airway smooth muscle cell. The method involves contacting an airway smooth muscle cell with an agent having N-cadherin antagonist activity.

In another aspect, the present disclosure provides a method of inhibiting N-cadherin activity in an airway smooth muscle cell. The method involves contacting an airway smooth muscle cell with an N-cadherin antagonist that is a small molecule compound, linear peptide, a cyclic peptide, or an antibody or antigen binding portion thereof that binds N-cadherin competitively and reversibly inhibits homotypic intercellular interactions.

In another aspect, the present disclosure provides a method of treating a lung disorder in a subject. The method involves administering to the subject an agent having N-cadherin antagonist activity, where the subject has a lung disorder, where the lung disorder is asthma, bronchiolitis, bronchiectasis, bronchitis, or chronic obstructive pulmonary disease (COPD), and where the agent is a linear peptide, a cyclic peptide, or an antibody or antigen binding portion thereof, where the agent binds N-cadherin competitively and reversibly inhibits homotypic intercellular interactions.

In another aspect, the present disclosure provides a method of reducing or preventing airway hyperresponsiveness (AHR) in a subject. The method involves administering to the subject an N-cadherin antagonist, thereby reducing or preventing AHR in the subject.

In another aspect, the present disclosure provides a pharmaceutical composition including an N-cadherin antagonist and a pharmaceutically acceptable excipient, where the composition is formulated for inhalation.

In another aspect, the present disclosure provides a kit including an N-cadherin antagonist and instructions for using the N-cadherin antagonist or pharmaceutical composition in the method of any of the above aspects, or embodiments thereof.

In another aspect, the present disclosure provides a method of inducing bronchodilation in an airway smooth muscle cell. The method involves contacting an airway smooth muscle cell with an agent having N-cadherin antagonist activity and an agent having beta agonist activity.

In another aspect, the present disclosure provides a method of inducing bronchodilation in an airway smooth muscle cell. The method involves contacting an airway smooth muscle cell with an agent having N-cadherin antagonist activity and an agent having corticosteroid activity.

In another aspect, the present disclosure provides a pharmaceutical composition including an N-cadherin antagonist, a beta agonist, and a pharmaceutically acceptable excipient. In another aspect, the present disclosure provides a pharmaceutical composition including an N-cadherin antagonist, a corticosteroid, and a pharmaceutically acceptable excipient.

In any of the above aspects, or embodiments thereof, the N-cadherin antagonist is a linear peptide or a cyclic peptide comprising a His-Ala-Val amino acid sequence that binds N-cadherin competitively and reversibly inhibits homotypic intercellular interactions.

In any of the above aspects, or embodiments thereof, the airway smooth muscle cell is in vitro or in vivo.

In any of the above aspects, or embodiments thereof, the airway smooth muscle cell is in vivo, and the N-cadherein antagonist is administered via inhalation.

In any of the above aspects, or embodiments thereof, the method reduces contractile agent-induced airway smooth muscle cell contraction.

In any of the above aspects, or embodiments thereof, the contractile agent is a cholinergic agonist, histamine, prostaglandin, leukotriene, adenosine, NSAID, tachykinin, bradykinin, or endotoxin.

In any of the above aspects, or embodiments thereof, the contractile agent is acetylcholine, methacholine, carbachol, histamine, or bradykinin.

In any of the above aspects, or embodiments thereof, the method induces bronchodilation in the airway smooth muscle cell.

In any of the above aspects, or embodiments thereof, the agent is a linear or cyclic peptide including a His-Ala-Val amino acid sequence.

In any of the above aspects, or embodiments thereof, the peptide is a cyclic peptide including the following sequence:

, where XI and X2 are optional, and if present, are independently selected from the group consisting of amino acid residues linked by peptide bonds, and where Xi and X₂ independently range in size from 0 to 10 residues, such that the sum of residues contained within Xi and X₂ ranges from 1 to 12; wherein Y i and Y₂ are amino acid residues, and where a covalent bond is formed between residues Y i and Y₂; and wherein Zi and Z₂ are optional, and if present, are amino acid residues linked by peptide bonds.

In any of the above aspects, or embodiments thereof, the peptide includes the sequence AHAVSE or LRAHA VDVNG. In any of the above aspects, or embodiments thereof, the peptide is cyclized via a disulfide bond; an amide bond between terminal functional groups, and/or a thioether bond between terminal function groups, between residue side-chains, or between one terminal functional group and one residual side chain.

In any of the above aspects, or embodiments thereof, the N-cadherin antagonist is ADH-1 or LCRF006.

In any of the above aspects, or embodiments thereof, the subject has a fixed airway obstruction associated with the lung disorder.

In any of the above aspects, or embodiments thereof, the method reduces actin remodeling, reduces histamine-induced airway smooth muscle cell contraction, and/or induces bronchodilation in the airway smooth muscle cell.

In any of the above aspects, or embodiments thereof, the method further includes administering to the subject a second agent, where the second agent is a: leukotriene receptor antagonist; corticosteroid; theophylline; beta agonist; anti-cholinergic agent; or antiinflammatory agent.

In any of the above aspects, or embodiments thereof, the second agent is montelukast, zafirlukast, zileuton, prednisone, methylprednisone, fluticasone propionate, budesonide, ciclesonide, beclomethasone, mometasone, fluticasone furonate, theophylline, albuterol, levalbuterol, ipratropium, or tiotropium.

In any of the above aspects, or embodiments thereof, the N-cadherin antagonist is ADH-1 or LCRF006.

In any of the above aspects, or embodiments thereof, the pharmaceutical composition is formulated for inhalation.

Other features and advantages of the disclosure will be apparent from the detailed description, and from the claims.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof. In embodiments, the agent is an N-cadherin antagonist.

By “airway hyperresponsiveness” or “AHR” is meant a susceptibility of an airway (e.g., in a subject having a lung disease or disorder) to narrow excessively in response to stimuli that would produce little or no effect in a reference airway (e.g., an airway in a healthy subject). In embodiments, AHR is characterized by abnormal levels of bronchoconstriction, for example, in response to stimuli.

By "alteration" is meant a change (increase or decrease) in a clinical indication, or in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, a 25% change, a 40% change, and even a 50% or greater change in expression levels.

By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease. In embodiments, the disease is fixed airway obstruction (FAO) associated with asthma, or another lung disease or disorder.

By "analog" is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.

By “antagonist” is meant an agent that interferes with or inhibits the activity of another agent. An N-cadherin antagonist inhibits n-cadherin activity by at least about 10%, 20%, 30%, 50%, 75%, or more.

“Biological sample” as used herein means a biological material isolated from a subject. Exemplary biological samples include any tissue, cell, fluid, or other material obtained from or derived from the subject. In some embodiments, the subject is human. The biological sample may contain any biological material suitable for detecting the desired analytes (e.g., a mosaic chromosomal alteration), and may comprise cellular and/or non-cellular material obtained from the subject. The biological sample preferably comprises DNA. In particular embodiments, the biological sample is blood. Biological samples include tissue samples (e.g., cell samples, biopsy samples). Biological samples also include bodily fluids, including, but not limited to, cerebrospinal fluid, blood, lymph, blood serum, plasma, saliva, and urine. In some embodiments, the biological sample comprises blood, skin, sputum, gargles, bronchial washings, urine, semen, feces, cerebrospinal fluid, biopsies, or dried blood spots.

In this disclosure, "comprises," "comprising," "containing", and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of' or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments. Any embodiments specified as “comprising” a particular component(s) or element(s) are also contemplated as “consisting of’ or “consisting essentially of’ the particular component(s) or element(s) in some embodiments.

By “contractile agent” or “contractile mediator” is meant an agent capable of inducing contraction in a muscle cell (e.g., an airway smooth muscle cell). Non-limiting examples of contractile agents include: cholinergic agonists (e.g., acetylcholine, methacholine, carbachol), histamine, prostaglandins, leukotrienes, adenosine, NSAIDs, tachykinins, bradykinin, and endotoxin.

“Detect” refers to identifying the presence, absence or amount of the object to be detected.

By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. In embodiments, the disease is a lung disease or disorder (e.g., asthma, bronchiolitis, bronchiectasis, bronchitis, or chronic obstructive pulmonary disease (COPD)). In embodiments, the disease is “fixed” airway obstruction associated with one of the aforementioned conditions.

By "effective amount" is meant the amount of an agent required to ameliorate, reduce, or prevent the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present disclosure for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount. By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

By “Neural cadherin (N-cadherin) is meant a polypeptide having at least about 85% identity to NP_001295105.1 that mediates cell-cell interaction. An exemplary N-cadherin amino acid sequence is provided below.

1 mfllrryvci fteklknqae lyvfl svkfs ncngkrkvqy es sepadfkv dedgmvyavr

61 sfpl ssehak fliyaqdket qekwqvavkl sl kptltees vkesaeveei vfprqfskhs

121 ghlqrqkrdw vippinlpen srgpfpqelv rirsdrdknl slrysvtgpg adqpptgifi

181 inpi sgql sv tkpldreqia rfhlrahavd ingnqvenpi divinvidnm dnrpeflhqv

241 wngtvpegs k pgtyvmtvta idaddpnaln gmlryrivsq apstpspnmf tinnetgdii

301 tvaagldrek vqqytliiqa tdmegnptyg Isntatavit vtdvndnppe ftamtfygev

361 penrvdiiva nltvtdkdqp htpawnavyr isggdptgrf aiqtdpnsnd glvtwkpid

421 fetnrmfvlt vaaenqvpla kgiqhppqst atvsvtvidv nenpyfapnp kiirqeeglh

481 agtmlttfta qdpdrymqqn irytkl sdpa nwl kidpvng qittiavldr espnvknniy

541 natflasdng ippmsgtgtl qiylldindn apqvlpqeae tcetpdpnsi nitaldydid

601 pnagpfafdl pl spvti krn wtitrlngdf aqlnl kikfl eagiyevpii itdsgnppks

661 ni silrvkvc qcdsngdctd vdrivgaglg tgaiiaillc iiillilvlm fwwmkrrdk

721 erqakqllid peddvrdnil kydeegggee dqdydl sqlq qpdtvepdai kpvgirrmde

781 rpihaepqyp vrsaaphpgd igdfinegl k aadndptapp ydsllvfdye gsgstagsl s

841 slns ss sgge qdydylndwg prfkkladmy gggdd

By “N-cadherin activity” is meant mediating Ca2+-dependent cell-cell adhesion. In embodiments, cadherin molecules interact with actin and cytoskeletal associated proteins (e.g., actinin, catenins).

By “N-cadherin polynucleotide” is meant a nucleic acid molecule encoding an N- cadherin polypeptide. An exemplary N-cadherin polynucleotide sequence is provided at NCBI Accession No. NM_001308176.

1 caaaagaact ttatccctgc tttcattctg acatacctat tatgactttg ttcactaagc

61 agaaggaatt atgggaaatg gaaacttgat ggcatgtttt tattaaggcg ttatgtgtgt

121 atcttcactg agaaattaaa gaaccaagca gaattgtatg ttttcctttc agtgaagttt

181 agcaactgca atggaaaaag aaaagtacaa tatgagagca gtgagcctgc agattttaag

241 gtggatgaag atggcatggt gtatgccgtg agaagctttc cactctcttc tgagcatgcc

301 aagttcctga tatatgccca agacaaagag acccaggaaa agtggcaagt ggcagtaaaa

361 ttgagcctga agccaacctt aactgaggag tcagtgaagg agtcagcaga agttgaagaa

421 atagtgttcc caagacaatt cagtaagcac agtggccacc tacaaaggca gaagagagac

481 tgggtcatcc ctccaatcaa cttgccagaa aactccaggg gaccttttcc tcaagagctt 541 gtcaggatca ggtctgatag agataaaaac ctttcactgc ggtacagtgt aactgggcca 601 ggagctgacc agcctccaac tggtatcttc attatcaacc ccatctcggg tcagctgtcg 661 gtgacaaagc ccctggatcg cgagcagata gcccggtttc atttgagggc acatgcagta 721 gatattaatg gaaatcaagt ggagaacccc attgacattg tcatcaatgt tattgacatg 781 aatgacaaca gacctgagtt cttacaccag gtttggaatg ggacagttcc tgagggatca 841 aagcctggaa catatgtgat gaccgtaaca gcaattgatg ctgacgatcc caatgccctc 901 aatgggatgt tgaggtacag aatcgtgtct caggctccaa gcaccccttc acccaacatg 961 tttacaatca acaatgagac tggtgacatc atcacagtgg cagctggact tgatcgagaa 1021 aaagtgcaac agtatacgtt aataattcaa gctacagaca tggaaggcaa tcccacatat 1081 ggcctttcaa acacagccac ggccgtcatc acagtgacag atgtcaatga caatcctcca 1141 gagtttactg ccatgacgtt ttatggtgaa gttcctgaga acagggtaga catcatagta 1201 gctaatctaa ctgtgaccga taaggatcaa ccccatacac cagcctggaa cgcagtgtac 1261 agaatcagtg gcggagatcc tactggacgg ttcgccatcc agaccgaccc aaacagcaac 1321 gacgggttag tcaccgtggt caaaccaatc gactttgaaa caaataggat gtttgtcctt 1381 actgttgctg cagaaaatca agtgccatta gccaagggaa ttcagcaccc ccctcagtca 1441 actgcaaccg tgtctgttac agttattgac gtaaatgaaa acccttattt tgcccccaat 1501 cctaagatca ttcgccaaga agaagggctt catgccggta ccatgttgac aacattcact 1561 gctcaggacc cagatcgata tatgcagcaa aatattagat acactaaatt atctgatcct 1621 gccaattggc taaaaataga tcctgtgaat ggacaaataa ctacaattgc tgttttggac 1681 cgagaatcac caaatgtgaa aaacaatata tataatgcta ctttccttgc ttctgacaat 1741 ggaattcctc ctatgagtgg aacaggaacg ctgcagatct atttacttga tattaatgac 1801 aatgcccctc aagtgttacc tcaagaggca gagacttgcg aaactccaga ccccaattca 1861 attaatatta cagcacttga ttatgacatt gatccaaatg ctggaccatt tgcttttgat 1921 cttcctttat ctccagtgac tattaagaga aattggacca tcactcggct taatggtgat 1981 tttgctcagc ttaatttaaa gataaaattt cttgaagctg gtatctatga agttcccatc 2041 ataatcacag attcgggtaa tcctcccaaa tcaaatattt ccatcctgcg tgtgaaggtt 2101 tgccagtgtg actccaacgg ggactgcaca gatgtggaca ggattgtggg tgcggggctt 2161 ggcaccggtg ccatcattgc catcctgctc tgcatcatca tcctgcttat ccttgtgctg 2221 atgtttgtgg tatggatgaa acgccgggat aaagaacgcc aggccaaaca acttttaatt 2281 gatccagaag atgatgtaag agataatatt ttaaaatatg atgaagaagg tggaggagaa 2341 gaagaccagg actatgactt gagccagctg cagcagcctg acactgtgga gcctgatgcc 2401 atcaagcctg tgggaatccg acgaatggat gaaagaccca tccacgccga gccccagtat 2461 ccggtccgat ctgcagcccc acaccctgga gacattgggg acttcattaa tgagggcctt 2521 aaagcggctg acaatgaccc cacagctcca ccatatgact ccctgttagt gtttgactat 2581 gaaggcagtg gctccactgc tgggtccttg agctccctta attcctcaag tagtggtggt 2641 gagcaggact atgattacct gaacgactgg gggccacggt tcaagaaact tgctgacatg 2701 tatggtggag gtgatgactg aacttcaggg tgaacttggt ttttggacaa gtacaaacaa 2761 tttcaactga tattcccaaa aagcattcag aagctaggct ttaactttgt agtctactag 2821 cacagtgctt gctggaggct ttggcatagg ctgcaaacca atttgggctc agagggaata 2881 tcagtgatcc atactgtttg gaaaaacact gagctcagtt acacttgaat tttacagtac 2941 agaagcactg ggattttatg tgcctttttg tacctttttc agattggaat tagttttctg 3001 tttaaggctt taatggtact gatttctgaa acgataagta aaagacaaaa tattttgtgg

3061 tgggagcagt aagttaaacc atgatatgct tcaacacgct tttgttacat tgcatttgct

3121 tttattaaaa tacaaaatta aacaaacaaa aaaactcatg gagcgatttt attatcttgg

3181 gggatgagac catgagattg gaaaatgtac attacttcta gttttagact ttagtttgtt

3241 tttttttttt tcactaaaat cttaaaactt actcagctgg ttgcaaataa agggagtttt

3301 catatcacca atttgtagca aaattgaatt ttttcataaa ctagaatgtt agacacattt

3361 tggtcttaat ccatgtacac ttttttattt ctgtattttt ccacttcact gtaaaaatag

3421 tatgtgtaca taatgtttta ttggcatagt ctatggagaa gtgcagaaac ttcagaacat

3481 gtgtatgtat tatttggact atggattcag gttttttgca tgtttatatc tttcgttatg

3541 gataaagtat ttacaaaaca gtgacatttg attcaattgt tgagctgtag ttagaatact

3601 caatttttaa tttttttaat ttttttattt tttattttct ttttggtttg gggagggaga

3661 aaagttctta gcacaaatgt tttacataat ttgtaccaaa aaaaaaaaaa aaggaaagga

3721 aagaaagggg tggcctgaca ctggtggcac tactaagtgt gtgttttttt aaaaaaaaaa

3781 tggaaaaaaa aaagctttta aactggagag acttctgaca acagctttgc ctctgtattg

3841 tgtaccagaa tataaatgat acacctctga ccccagcgtt ctgaataaaa tgctaatttt

3901 ggatctgg

By “N-cadherin antagonist” is meant an agent that inhibits N-cadherin mediated cell adhesion.

As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.

By “prevent,” “preventing,” “prevention,” “prophylactic treatment”, and the like is meant reducing the probability of developing a disorder, disease, or condition in a subject, who does not have, but is at risk of or susceptible to developing the disorder, disease, or condition.

By “reduces” is meant a negative alteration of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

By “reference” is meant a standard or control condition. In embodiments, a reference subject is a healthy subject. In embodiments, a reference subject is an untreated subject having a lung disease or disorder.

By "subject" or “patient” is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline. In some embodiments, the subject is a human. The terms “subject” and “patient” are used interchangeably herein.

By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). In an embodiment, such a sequence is at least 60%, more preferably 80% or 85%, and 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BUAST, BESTFIT, GAP, or PIEEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e'³ and e'¹⁰⁰ indicating a closely related sequence.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated. In some embodiments, a treatment for FAO associated with asthma, or another lung disorder can include treating a subject to preventthe asthma or the lung disorder. .

Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGs. 1A-1F provide images and plots showing airway smooth muscle (ASM) N- cadherin expression in asthma. (FIG. 1A) N-cadherin detected in human ASM (HASM) lysates by immunoblotting (n=5 donors/group). (FIG. IB) N-cadherin/b-actin ratios; means ± SEM from n=12-13 donors/group, *P=0.03, unpaired / test. (FIG. 1C) N-cadherin in lysates from serum starved HASM cells left untreated or stimulated with TGFp (5 ng/mL), IL-33 (50 ng/mL), TSLP (10 ng/mL), PDGFp (10 ng/mL), or IL-4 plus IL-13 (100 ng/mL each) for 16 hours. (FIG. ID) N-cadherin/actin ratios. Means ± SEM from n=4-6 sets of cells. **P=0.002 vs. control, Kruskal-Wallis ANOVA, Dunn multiple comparisons. (FIGs. 1E-1F) Human (FIG. IE) or mouse (FIG. IF) lung sections immunostained with N-cadherin antibody and counterstained with hematoxylin. Scale bar=100 pm (E), 200 pm (F).

FIGs. 2A-2F provide images, plots, and graphs showing that ASM-derived N-cadherin is required for allergen induced AHR. (FIG. 2A) Relative Cdh2/Actb expression in lung RNA from control (CTRL=('z//72// or Cdh2fl/fl) and Cdh2fl/+-SMAA Cre mice. Means ± SEM of n=5-6 mice/group. **/’=0.002, unpaired / test. (FIGs. 2B-2C) N-cadherin protein expression in lung lysates from control or //?2///+-SM A A Cre mice detected by immunoblotting (FIG. 2B) and N-cadherin/b-acitn ratios determined by ImageJ analysis (FIG. 2C). Means ± SEM from n=7-8 mice/group. * =0.03, unpaired / test. (FIG. 2D) Lung resistance in naive or Af challenged mice. Means ± SEM from n=7-10 mice/group. **/’=0.003, ****7^?<0.0001 (CTRL vs. Cdh2fl/+- SMAA Cre, A -treated), 2-way ANOVA, Sidak multiple comparisons. (FIGs. 2E-2F) Airway smooth muscle thickness detected by immunostaining (green) and quantified by ImageJ (FIG. 2F). Means ± SEM from n=14-24 airway s/group. Scale bar=20 pm.

FIGs. 3A-3G provide images and plots showing that smooth muscle specific Cdh2 haploinsufficiency does not affect allergic lung inflammation. (FIG. 3A) Images of hematoxylin/eosin-stained lung sections from mice, representative of n=4-5 mice/group. Scale bar=100 pm. (FIGs. 3B-3C) BALF total leukocyte counts (FIG. 3B) and differential composition (FIG. 3C) in PBS or Af challenged mice. Means ± SEM from n=4-5 mice/group. (FIGs. 3D-3F) Allergy associated cytokines IL-4 (FIG. 3D), IL-5 (FIG. 3E), or IL- 13 (FIG. 3F) in BALF. Means ± SEM from 5-9 mice/group, n.s., 2-way ANOVA, Tukey multiple comparisons. (FIG. 3G) Images of PAS-stained lung sections from mice, representative of n=4- 5 mice/group. Scale bar=100 pm.

FIGs. 4A-4F provide an image and plots showing that N-cadherin disruption is bronchoprotective and bronchodilatory. (FIGs. 4A-4C) Percentage contraction (from baseline area) in PCLS from Cdh2fl/+-SMAA Cre mice (FIG. 4A) or pre-treated with vehicle or ADH-1 [pg/mL] (FIGs. 4B-4C) and stimulated with MCh (10 pM, 20 min). (FIG. 4A) Means ± SEM from n=27-32 airway s/group; **7^?=0.005, Mann-Whitney. (FIG. 4B) Means ± SEM from n=12- 22 airways/group; * =0.01, **** <0.0001 vs. control, 1-way ANOVA, Dunnett multiple comparisons. (FIG. 4C) Means ± SEM from n=12-34 airways/group. (FIGs. 4D-4E) PCLS contracted with MCh (10 pM, 20 min) and then treated with vehicle (0.5% DMSO) or ADH-1 (500 pg/mL) for an additional 10 minutes. Means ± SEM from n=l 1-29 airways/group *P=0.01 vs. MCh, 1-way ANOVA, Tukey multiple comparisons. (FIG. 4F) PCLS left untreated or treated with formoterol (100 ng/mL x 20 min). Airways were then treated with MCh (10 pM, 20 min), followed by addition of either vehicle (DMSO), formoterol, or ADH-1 (500 pg/mL). Means ± SEM from n=7-30 airways/group; *P=Q.Q 1, 1-way ANOVA, Tukey multiple comparisons.

FIGs. 5A-5D provide images and graphs showing that N-cadherin antagonist ADH-1 inhibits HASM contraction. (FIGs. 5A-5B) Representative cellular images (FIG. 5A) and corresponding traction maps (FIG. 5B) in HASM cells pre-treated with vehicle (Veh) or ADH-1 (500 pg/mL) for 24 hours and then stimulated with histamine (3 pM, 20 minutes). Scale bar=50 pm. Mean traction force is noted above each field. (FIG. 5C) Probability distribution of traction and its variance in vehicle (Veh) or ADH-1 -treated HASM cells stimulated with histamine. Each distribution is calculated over n=4 distinct fields comprising -400 cells per field in 2 independent experiments. (FIG. 5D) Time-course of contraction. Means ± SEM from n=4 distinct fields comprising -400 cells per field.

FIGs. 6A-6F provide graphs, plots, and images showing that ADH-1 inhibits HASM contraction independent of canonical excitation-contraction pathways. (FIGs. 6A-6B) Intracellular Ca2+ (relative fluorescence units, RFU) over time (FIG. 6A) and peak amounts (FIG. 6B) in HASM cells pre-treated with vehicle or ADH-1 (pg/mL) for 24 hours and stimulated with histamine (3 pM). Means ± SEM from n=4-12 biological replicates/group. (FIGs. 6C-6F) HASM cells pretreated with vehicle or ADH-1 (pg/mL) for 24 hours and stimulated with histamine (1 pM, FIG. 6C) or CCh (20 pM) (FIG. 6F) for 10 minutes. Representative blots (FIGs. 6C, 6F) and aggregated data (FIGs. 6D-6E) (means ± SEM) from n=3-6 donors/group.

FIGs. 7A-7E provide images and plots showing that ADH-1 prevents agonist-induced actin remodeling in HASM. (FIG. 7A) HASM cells pretreated with vehicle or ADH-1 (250 pg/mL) and left untreated or stimulated with histamine (10 pM) for 10 minutes, fixed and stained with phalloidin and DAPI. Scale bar=20 pm. (FIGs. 7B-7C) Total F-actin (FIG. 7B) and F-actin anisotropy (FIG. 7C) quantified; means ± SEM from n=17-22 separate fields/group. ***j>=0.0004, 2-way ANOVA, Sidak multiple comparisons. (FIG. 7D) Lung sections from control or C /12//+-SMAA Cre mice challenged with PBS or Af- stained with phalloidin, SMAA, and DAPI. (FIG. 7E) F-actin in bronchial smooth muscle; n=28-31 airway s/group. Scale bar=20 pm. **** <0.0001, 2-way ANOVA, Tukey multiple comparisons.

FIGs. 8A-8H provide a graph, images, and plots showing that ADH-1 reduces allergen induced AHR independent of allergic inflammation. (FIG. 8A) Lung resistance in ^ -challenged mice pre-treated with vehicle (Veh) or ADH-1 (200 mg/kg via i.p. injection). Means ± SEM from n=l 1-15 mice/group. * =0.01, ** =0.005, 2-way ANOVA, Benjamini, Krieger, Yekutieli corrected multiple comparisons. (FIG. 8B) Hematoxylin/eosin-stained lung sections from mice, representative of n=4-5 mice/group. Scale bar=100 pm. (FIGs. 8C-8D) BALF total leukocyte counts (FIG. 8C) and differential composition (FIG. 8D) in Af challenged mice pretreated with vehicle (Veh) or ADH-1. Means ± SEM from n=4-5 mice/group. (FIG. 8E) Images of PAS- stained lung sections from mice, representative of n=4-5 mice/group. Scale bar=100 pm. (FIGs. 8F-8H) Levels of IL-4 (FIG. 8F), IL-5 (FIG. 8G), or IL-13 (FIG. 8H) in BALF from Af challenged mice. Means ± SEM from 8-10 mice/group, n.s., 2-way ANOVA, Tukey multiple comparisons.

FIG. 9 provides a plot showing that N-cadherein deficiency or ADH-1 does not affect baseline airway luminal areas in PCLS. Means ± SEM from n=13-34 airway s/group.

FIGs. 10A-10D provide plots showing that ADH-1 does not induce cytotoxicity on HASM cells. (FIGs. 10A-10C) HASM cells were treated with ADH-1 (250 pg/mL for 24 hours) followed assessment of viability (FIG. 10A), cytotoxicity (FIG. 10B), or apoptosis (FIG. IOC) by multiplexed assay. (FIG. 10D) Viability in HASM cells treated with vehicle (2.5% DMSO) or various concentrations of ADH-1 (in 2.5% DMSO at 500 pg/mL) for 24 hours. DETAILED DESCRIPTION OF THE DISCLOSURE

The disclosure features compositions and methods for treatment of obstructive lung diseases or disorders (e.g., asthma, chronic obstructive pulmonary disorder, bronchiolitis) in a subject.

The disclosure is based at least in part upon the discovery that an antagonist of N- cadherin-mediated intercellular interactions (e.g., ADH-1) conferred bronchoprotective effects when administered in an asthma model. A key effector of the changes associated with asthma is exaggerated airway smooth muscle (ASM) cell contraction to spasmogens. Unfortunately, no drugs in clinical use effectively prevent ASM hyper-contraction in asthma across all severities. As reported in detail below, N-cadherin, a plasma membrane associated intercellular adhesion protein upregulated in ASM cells derived from donors with fatal asthma is required for the development of airway obstruction in mouse models of severe asthma induced by allergic airway inflammation. Pharmacological inhibition of N-cadherin by ADH-1 prevented bronchoconstriction and actively promoted bronchodilation of murine airways ex vivo. In human ASM cells, ADH-1 inhibited collective cell contraction at homeostasis and agonist induced ASM contraction by preventing actin stress fiber formation. These results indicate an intercellular communication pathway mediating ASM contraction and identify N-cadherin as a therapy for inhibiting bronchoconstriction in asthma.

Obstructive Lung Diseases and Disorders

Obstructive lung disease is a category of respiratory disease characterized by airway obstruction. Many obstructive diseases of the lung result from narrowing (obstruction) of the smaller bronchi and larger bronchioles, often because of excessive contraction of the smooth muscle itself. It is generally characterized by inflamed and easily collapsible airways, obstruction to airflow, problems exhaling, and frequent medical clinic visits and hospitalizations. Types of obstructive lung disease include asthma, bronchiolitis, bronchiectasis, bronchitis, and chronic obstructive pulmonary disease (COPD). Cystic fibrosis is also sometimes included in obstructive pulmonary disease.

Asthma

In asthma, episodic and reversible airway obstruction evokes shortness of breath, wheezing, and cough. While the pathogenesis of asthma involves diverse underlying pathways of inflammation, each culminates in augmented bronchoconstriction to spasmogens such as acetylcholine (ACh) — a phenotype termed “airway hyper-responsiveness (AHR)”. The most common inflammatory signature involves IgE-antibody responses to ubiquitous environmental allergens, which induces a type 2 (T2) immune response, recruitment of inflammatory cells to the lung (primarily type 2 CD4⁺ lymphocytes and eosinophils), and local accumulation of T2 cytokines including IL-4, 5, and 13.

Although the recent development of anti-inflammatory biologies targeting asthma- associated cytokines has transformed the care of patients with chronic asthma over the last decade, these agents have no defined role in FAO treatment. In established asthma, remodeling of airway structural cells, including the respiratory epithelium and ASM and surrounding extracellular matrix (ECM), arises from chronic inflammation and correlates with disease severity (Hsieh et al., Front Physiol 14, 1113100 (2023)), but its impact on ASM hypercontractility and FAO remain incompletely understood (Camoretti-Mercado et al., J Allergy Clin Immunol 147, 1983-1995 (2021)). ASM-targeted drugs in clinical use promote bronchorelaxation (e.g. -adrenergic agonists), and these are prone to tolerance and lack of efficacy in a T2 inflammatory milieu, which can increase mortality and morbidity (Nwaru et al., Eur Respir J 55, (2020)).

Bronchiectasis

Bronchiectasis refers to the abnormal, irreversible dilatation of the bronchi caused by destructive and inflammatory changes in the airway walls. Bronchiectasis has three major anatomical patterns: cylindrical bronchiectasis, varicose bronchiectasis and cystic bronchiectasis. Symptoms typically include a chronic cough with mucus production. Other symptoms include shortness of breath, coughing up blood, and chest pain.

Bronchiolitis

Bronchiolitis is inflammation of the small airways in the lungs. Acute bronchiolitis is caused by a viral lower respiratory tract infection, typically in young children. Acute bronchiolitis is characterized by obstruction of small airways caused by acute inflammation, edema and necrosis of the epithelial cells lining the small airways as well as increased mucus production. Respiratory syncytial virus (RSV) is responsible for most cases. However, other viruses, including human metapneumovirus (HMPV), influenza, rhinovirus, adenovirus and parainfluenza can all cause bronchiolitis.

Chronic Obstructive Pulmonary Disease (COPD)

COPD is a heterogeneous lung condition characterized by chronic respiratory symptoms (dyspnea, cough, sputum production and/or exacerbations) due to abnormalities of the airways (bronchitis, bronchiolitis) and/or alveoli (emphysema) that cause persistent, often progressive, airflow obstruction.

The main symptoms of COPD include shortness of breath and a cough, which may or may not produce mucus. COPD progressively worsens, with everyday activities such as walking or dressing becoming difficult. While COPD is incurable, it is preventable and treatable. The two most common types of COPD are emphysema and chronic bronchitis. Emphysema is defined as enlarged airspaces (alveoli) whose walls have broken down resulting in permanent damage to the lung tissue. Chronic bronchitis is defined as a productive cough that is present for at least three months each year for two years.

Cadherins

Cadherins are a family of widely expressed integral membrane proteins whose Camdependent homotypic interactions between adjacent cells mediate intercellular adhesion. Vascular endothelial (VE)-cadherin-mediated junctional contacts within the endothelium are essential for vascular barrier maintenance (Hellenthal et al., Cells 11, (2022)). In asthma, allergens elicit T2 airway inflammation by disrupting epithelial (E)-cadherin-mediated adherens junctions in respiratory epithelial cells (Bradbury et al., Inflammation 45, 1209-1223 (2022); Wiesner et al., Cell Host Microbe 27, 614-628 e616 (2020)).

Airway smooth muscle (ASM) cells primarily express neuronal (N)-cadherin but not E- cadherin. While global Cdh2 knockout in mice is embryonic lethal, tissue-specific deletion has revealed important functions of N-cadherin in muscle tissues (Radice, Prog Mol Biol Transl Sci 116, 263-289 (2013)). Mice with inducible N-cadherin deficiency in adult myocardium Chd ¹- aMHC-MerCre) are prone to developing cardiomyopathy and arrythmias associated with impaired adhesion between cardiomyocytes (Li et al., Circ Res 97, 474-481 (2005)). Although N-cadherin has been linked to the internal ASM cytoskeleton through documented interactions with actin-binding proteins including -catenin and a-actinin (Jansen et al., Am J Physiol Lung Cell Mol Physiol 299, L204-214 (2010); Wang et al., J Biol Chem 290, 8913-8924 (2015); Ouyang et al. Front Cell Dev Biol 10, 942058 (2022)), its role in AHR and irreversible or “fixed” airway obstruction (FAO) was previously unknown.

N-cadherin is a member of the Type I cadherin subfamily. It is a single pass transmembrane glycoprotein containing extracellular (EC), transmembrane and cytoplasmic (CP) domains. Type I cadherin monomers exist in different states in the plasma membrane and at intercellular adhesive contacts. The monomers apparently interact within the plane of the plasma membrane (referred to as cis interactions) and with identical monomers on the surface of apposing cells (referred to as trans interactions). These cis and trans interactions involve the cell adhesion recognition (CAR) sequence Histidine- Alanine-Valine (His79-Ala80-Val81) which is found towards the terminus of all Type I cadherin ECI domains.

Without intending to be bound by theory, the Examples provided herein revealed that N- cadherin is essential for the development of AHR in allergen-challenged mice through its effects on collective force transmission and actin remodeling in ASM. Pharmacological N-cadherin antagonism is bronchoprotective and elicits bronchodilation, highlighting a previously unrecognized therapeutic approach to FAO in severe asthma.

N-Cadherin Antagonists

Aspects and embodiments of the present disclosure provide methods involving the administration of agents that act as N-cadherin antagonists to ASM cells of a subject in need thereof. N-cadherin antagonists may also be administered in vitro or ex vivo to ASM cells in certain aspects and embodiments of the present disclosure.

In embodiments, the N-cadherin antagonists include polypeptides (e.g., monoclonal antibodies (Mabs), that specifically bind to N-cadherin, or an antigenic fragment thereof. Exemplary antibodies are known in the art and described, for example, in Tanaka H, et al. 2010. Monoclonal antibody targeting of N-cadherin inhibits prostate cancer growth, metastasis and castration resistance. Nat. Med. 16, 1414-1420.

In embodiments, an N-cadherin antagonist binds a cell adhesion recognition sequence comprising Histidine- Alanine-Valine (His79-Ala80-Val81). See, for example, Blaschuk, Philos Trans R Soc Lond B Biol Sci. 2015 Feb 5; 370(1661): 20140039.

In embodiments, the N-cadherin antagonists include agents that specifically bind to and/or interfere with N-cadherin binding activity. For example, polypeptides that are N-cadherin antagonists may include a Type I cadherin cell adhesion recognition (CAR) sequence (e.g., AHAVSE and/or LRAHAVDVNG).

In embodiments, the polypeptide is a cyclic peptide including the amino acid sequence: wherein Xi, and X2 are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations thereof in which the residues are linked by peptide bonds, and wherein Xi and X2 independently range in size from 0 to 10 residues, such that the sum of residues contained within Xi and X2 ranges from 1 to 12; wherein Y 1 and Y2 are independently selected from the group consisting of amino acid residues, and wherein a covalent bond is formed between residues Y 1 and Y2; and wherein Zi and Z2 are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations thereof in which the residues are linked by peptide bonds. Such cyclic peptides may comprise modifications such as an N-acetyl or N-alkoxybenzyl group and/or a C- terminal amide or ester group. Cyclic peptides may be cyclized via, for example, a disulfide bond; an amide bond between terminal functional groups, between residue side-chains or between one terminal functional group and one residue side chain; a thioether bond or 8181- ditryptophan, or a derivative thereof. In embodiments, the cyclic peptide may be one or more of: N— AC-CHAVC-NH₂ (ADH-1), N— AC-CHAVC-Y— NH₂. N— AC-YCHAVC-NH₂. N— Ac- CHA VDC-NH2, N— Ac-CHA VDIC-NH2. N— Ac-CHAVDINC-NH2. N— Ac-CHA VDINGC- NH₂, N— AC-CAHAVC-NH2. N— AC-CAHAVDC-NH2. N— AC-CAHAVDIC-NH2. N— AC- CRAHAVDC-NH2. N— Ac-CLRAHAVC-NH₂. N— Ac-CLRAHAVDC-NH₂. N— Ac- CSHAVC-NH2. N— AC-CFSHAVC-NH₂. N— AC-CLFSHAVC-NH₂. N— AC-CHAVSC-NH₂. N— AC-CSHAVSC-NH₂. N— AC-CSHAVSSC-NH₂. N— AC-CHAVSSC-NH₂. N— AC- KHAVD-NH2. N— AC-DHAVK-NH₂. N— AC-KHA VE-NH₂. N— AC-AHAVDI-NH₂. N— AC- SHAVDSS-NH2. N— Ac-KSHAVSSD-NH₂. N— Ac-CHAVC-S-NH₂. N— Ac— S-CHAVC- NH₂, N— AC-CHAVC-SS— NH₂. N— AC— S-CHAVC-S— NH₂. N— AC-CHAVC-T— NH₂. N— AC-CHAVC-E— NH2. N— AC-CHAVC-D— NHX N— Ac-CHA VYC-NH2. CH₃— SO2— HN- CHAVC-Y— NH>, CH₃— SO2— HN-CHAVC-NH2, HC(O)— NH-CHAVC-NH2, N— Ac- CHAVPen-NH2. N — Ac-PenHAVC-NH2 and N — Ac-CHA VPC-NH2. as well as derivatives thereof in which the N — Ac group is replaced by a different terminal group. Polypeptide antagonists of N-cadherin are discussed in detail, for example, in U.S. Patent. No. 6,610,821 Bl, a copy of which is hereby incorporated by reference. In other embodiments, U.S. Patent No. 8,603,986, which describes N-cadherin antagonists. Further discussion of N-cadherein antagonists suitable for use in aspects and embodiments of the present disclosure is found, for example, in U.S. Patent Application Pub. Nos. 2003/0109454 Al, 2004/0106545 Al, 2004/0175361 Al, 2005/0129676 Al, 2008/0081831 Al, 2009/0291967 Al, or U.S. Patent Nos. 6,031,072 A, 6,169,071 Bl, 6,203,788 Bl, 6,207,639 Bl, 6,333,307 Bl, 6,346,512 Bl, 6,417,325 Bl, 6,465,427 Bl, or 6,562,786 Bl, the entireties of each of which are hereby incorporated by reference. In embodiments, the peptide N-cadherin antagonist is ADH-1. ADH-1 is described, for example, by Blaschuk et al., Eur. J Pharmacol. 625, 195-198, 2009, which is incorporated herein by reference in its entirety . ADH-1 is a cyclic pentapeptide with CAS No. 229971-81-7 and having the following structure:

In another embodiment, the N-cadherin antagonist comprises the sequence H- SWTLYTPSGQSK-NH2, which is described, for example, by Devemy et al., Peptides 29, 1853— 1861, 2008.

Small molecule antagonists of N-cadherin are also contemplated for use in the methods of the present disclosure. For example, in embodiments, the small molecule N-cadherin antagonist is LCRF-0006. LCRF-0006 is a small molecule having the following structure: In still other embodiments, the N-cadherin antagonist is a synthetic linear peptide having an HAV motif. In one embodiment the peptide comprises or consists of the following sequence: N-Ac-LRAHA VDING-NH2.

Contractile Agents or Mediators

In aspects of this disclosure, compositions and methods are provided that reduce muscle cell contraction (e.g., in an airway smooth muscle cell) in response to a contractile agent or mediator. Contractile agents or mediators include, without limitation, cholinergic agonists (e.g., acetylcholine, methacholine, carbachol), histamine, prostaglandins, leukotrienes, adenosine, NSAIDs, tachykinins, bradykinin, and endotoxin.

Contractile agents are further discussed, for example, in Doeing et al., 2013, J. Appl. Physiol. 114:834-843, the entirety which is hereby incorporated by reference.

Methods of Treatment

In one aspect the disclosure provides a method for administering a treatment to prevent, reduce, or ameliorate a lung disease or disorder (e.g., asthma, bronchiolitis, bronchiectasis, bronchitis, or chronic obstructive pulmonary disease (COPD)) in a subject in need thereof. The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of an agent, or a composition to produce a desired effect (e.g., treatment of a lung disease or disorder). The agent can be an N-cadherin antagonist. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).

The therapeutic methods of the disclosure (which include prophylactic treatments) in general comprise administration of a therapeutically effective amount of an agent for treatment of a lung disease or disorder to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, or symptom thereof.

In another aspect, the present disclosure provides methods of reducing or preventing bronchoconstriction (e.g., in a subject). These methods involve contacting an airway smooth muscle cell (e.g., in a subject in need thereof) with an agent of the present disclosure (e.g., an N- cadherin antagonist). In another aspect, the present disclosure provides methods of inducing bronchodilation (e.g., in a subject). These methods involve contacting an airway smooth muscle cell (e.g., in a subject in need thereof) with an agent of the present disclosure (e.g., an N-cadherin antagonist).

In another aspect, the present disclosure provides methods of reducing or preventing airway hyperresponsiveness (AHR) (e.g., in a subject). These methods involve contacting an airway smooth muscle cell (e.g., in a subject in need thereof) with an agent of the present disclosure (e.g., an N-cadherin antagonist).

In another aspect, the present disclosure provides methods of preventing or reducing actin remodeling (e.g., in a subject). These methods involve contacting an airway smooth muscle cell (e.g., in a subject in need thereof) with an agent of the present disclosure (e.g., an N-cadherin antagonist).

The examples provided herein demonstrate that agents disclosed herein (e.g., N-cadherin antagonists) were able to confer bronchoprotective effects in a lung disease model. In embodiments, the methods provided herein are effective to ameliorate one or more symptoms of a lung disease or disorder, such as, but not limited to, bronchoconstriction, AHR, shortness of breath, wheezing, cough, chest pain, and/or airflow/airway obstruction. In embodiments, the methods of the present disclosure induce bronchodilation in an airway (e.g., of a subject in need thereof).

The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a pharmaceutical composition to produce a treatment or preventative effect. The therapeutic methods of the disclosure in general comprise administration of a therapeutically effective amount of a pharmaceutical composition to a subject in need thereof, including a mammal, particularly a human. Treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a lung disease, disorder, or symptom thereof. Determination of those subjects "at risk" can be made according to the judgment of a subject or a health care professional.

Pharmaceutical Compositions

Agents of the present disclosure (e.g., an N-cadherin antagonist), can be incorporated into a variety of formulations for therapeutic use (e.g., by administration) or in the manufacture of a medicament (e.g., for treating a lung disease or disorder) by combining the agents with appropriate pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms. Examples of such formulations include, without limitation, tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.

Pharmaceutical compositions provided herein can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include the steps of bringing the agent or agents provided herein (e.g., an N-cadherin antagonist), i.e., the “active ingredient”, into association with a carrier or excipient, and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping, and/or packaging the product into a desired single- or multi-dose unit.

Within the scope of this disclosure is a composition that contains a suitable carrier and one or more of the therapeutic agents described above. The composition can be a pharmaceutical composition that contains a pharmaceutically acceptable carrier, a dietary composition that contains a dietarily acceptable suitable carrier, or a cosmetic composition that contains a cosmetically acceptable carrier.

The term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo, or ex vivo. A “pharmaceutically acceptable carrier,” after administered to or upon a subject, does not cause undesirable physiological effects. The carrier in the pharmaceutical composition must be “acceptable” also in the sense that it is compatible with the active ingredient and can be capable of stabilizing it. One or more solubilizing agents can be utilized as pharmaceutical carriers for delivery of an active compound or agent. Examples of a pharmaceutically acceptable carrier include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a composition usable as a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, sodium lauryl sulfate, and D&C Yellow # 10.

Pharmaceutically acceptable salts are salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, or allergic response, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts of amines, carboxylic acids, and other types of compounds and agents, are well known in the art. For example, S.M. Berge, et al. describe pharmaceutically acceptable salts in detail in Pharmaceutical Sciences, 66: 1-19 (1977), incorporated herein by reference. The salts can be prepared in situ during the final isolation and purification of the agents of the disclosure, or separately by reacting a free base or free acid function with a suitable reagent, as described generally below. For example, a free base function can be reacted with a suitable acid. Furthermore, where the agents of the disclosure carry an acidic moiety, suitable pharmaceutically acceptable salts thereof may, include metal salts such as alkali metal salts, e.g. sodium or potassium salts; and alkaline earth metal salts, e.g. calcium or magnesium salts. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts, include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphor sulfonate, citrate, cyclopentane propionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl sulfonate and aryl sulfonate.

As described above, the pharmaceutical compositions of the present disclosure additionally include a pharmaceutically acceptable carrier, which, as used herein, includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, and lubricants, as suited to the particular dosage form desired. Remington’s Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the agents of the disclosure, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatine; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil, sesame oil; olive oil; com oil and soybean oil; glycols; such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; natural and synthetic phospholipids, such as soybean and egg yolk phosphatides, lecithin, hydrogenated soy lecithin, dimyristoyl lecithin, dipalmitoyl lecithin, distearoyl lecithin, dioleoyl lecithin, hydroxylated lecithin, lysophosphatidylcholine, cardiolipin, sphingomyelin, phosphatidylcholine, phosphatidyl ethanolamine, diastearoyl phosphatidylethanolamine (DSPE) and its pegylated esters, such as DSPE-PEG750 and, DSPE-PEG2000, phosphatidic acid, phosphatidyl glycerol and phosphatidyl serine. Commercial grades of lecithin which are preferred include those which are available under the trade name Phosal® or Phospholipon® and include Phosal 53 MCT, Phosal 50 PG, Phosal 75 SA, Phospholipon 90H, Phospholipon 90G and Phospholipon 90 NG; soy-phosphatidylcholine (SoyPC) and DSPE-PEG2000 are particularly preferred; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

A pharmaceutical composition of this disclosure can be administered parenterally, or through inhalation. The term “parenteral” as used herein refers to subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrastemal, intrathecal, intralesional, or intracranial injection, as well as any suitable infusion technique.

A sterile injectable composition can be a solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Such solutions include, but are not limited to, 1,3- butanediol, mannitol, water, Ringer’s solution, and isotonic sodium chloride solution. In addition, fixed oils are conventionally employed as a solvent or suspending medium (e.g., synthetic mono- or diglycerides). Fatty acid, such as, but not limited to, oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as, but not limited to, olive oil or castor oil, polyoxyethylated versions thereof. These oil solutions or suspensions also can contain a long chain alcohol diluent or dispersant such as, but not limited to, carboxymethyl cellulose, or similar dispersing agents. Other commonly used surfactants, such as, but not limited to, Tweens or Spans or other similar emulsifying agents or bioavailability enhancers, which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms also can be used for the purpose of formulation.

For administration by inhalation, the agents may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin, for use in an inhaler or insufflator may be formulated containing a powder mix of the agent and a suitable powder base such as lactose or starch.

Pharmaceutical compositions may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The agents may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

In some embodiments of any of the aspects, a pharmaceutical formulation is provided for inhalation or parenteral administration, in which case the formulation may contain alternative pharmaceutically acceptable carriers, vehicles, additives, etc. particularly suited to inhalation or parenteral drug administration.

In some embodiments of any of the aspects, the formulation comprising a compound/agent comprises one or more additional components, wherein the additional component is at least one of an osmolar component that provides an isotonic, or near isotonic solution compatible with human cells or blood, and a preservative.

In some embodiments of any of the aspects, the osmolar component is a salt, such as sodium chloride, or a sugar or a combination of two or more of these components. In some embodiments of any of the aspects, the sugar may be a monosaccharide such as dextrose, a disaccharide such as sucrose or lactose, a polysaccharide such as dextran 40, dextran 60, or starch, or a sugar alcohol such as mannitol. The osmolar component is readily selected by those skilled in the art.

In some embodiments of any of the aspects, the preservative is at least one of parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In some embodiments of any of the aspects, the formulation comprising an agent is in the form of a sustained release formulation and optionally, further comprises one or more additional components (e.g., an anti-inflammatory agent); and a preservative.

Combination Therapies

Optionally, an agent disclosed herein (e.g., an N-cadherin antagonist), may be administered together with any other standard treatment for an obstructive lung disease or disorder, including but not limited to, leukotriene receptor antagonists (e.g., montelukast, zafirlukast, zileuton), corticosteroids (e.g., prednisone, methylprednisone, fluticasone propionate, budesonide, ciclesonide, beclomethasone, mometasone, fluticasone furonate), theophylline, beta agonists (e.g., albuterol, levalbuterol), anti-cholinergic agents (e.g., ipratropium, tiotropium), other bronchodilators, and/or anti-inflammatory agents.

Accordingly, in an aspect, the present disclosure provides a pharmaceutical composition comprising an agent disclosed herein (e.g., an N-cadherin antagonist) and another agent for the treatment of an obstructive lung disease or disorder, including but not limited to, leukotriene receptor antagonists (e.g., montelukast, zafirlukast, zileuton), corticosteroids (e.g., prednisone, methylprednisone, fluticasone propionate, budesonide, ciclesonide, beclomethasone, mometasone, fluticasone furonate), theophylline, beta agonists (e.g., albuterol, levalbuterol), anti-cholinergic agents (e.g., ipratropium, tiotropium), other bronchodilators, and/or antiinflammatory agents. In embodiments, the agents may be formulated together or separately.

Without intending to be bound by theory, the examples provided herein demonstrate that the agents disclosed herein (e.g., N-cadherin antagonists) work to ameliorate symptoms of lung diseases and disorders without affecting conventional pathways for treating such symptoms (e.g., type 2 inflammation). Accordingly, in some emboidments, the agents provided herein (e.g., N- cadherin antagonists) have additive and/or synergistic effects when combined with conventional treatments for lung diseases or disorders which are directed to conventional pathways for treating symptoms of lung diseases and disorders (e.g., inflammation). In embodiments, a pharmaceutical composition of the present disclosure which comprises both an agent disclosed herein (e.g., an N-cadherin antagonist) and another agent for the treatment of an obstructive lung disease or disorder includes a lowered dosage of the other agent for the treatment of an obstructive lung disease or disorder, as compared to a reference dosage of the other agent when the other agent is administered alone. This reduced dosage is based, at least in part, on the synergistic and/or additive effects of the combination of agents. Kits

The disclosure provides kits for treating a lung disease or disorder. The kits may include a therapeutic composition comprising one or more agents for treating a lung disease or disorder. In some embodiments, the agent is an N-cadherin antagonist.

In some embodiments, the kit comprises a sterile container which contains a pharmaceutical composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

If desired, the kit further comprises instructions for administering a pharmaceutical composition to a subject in need thereof (e.g., a subject having a lung disease or disorder). In particular embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for reducing lung disease or disorder symptoms; precautions; warnings; indications; counter-indications; over dosage information; adverse reactions; animal pharmacology; clinical studies; instructions on how to assess subject risk for disease or disorder; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container. The instructions can be provided in digital form on a portable data storage medium (e.g., a compact disk or USB drive) or stored remotely on a server that can be accessed remotely.

The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the disclosure, and, as such, may be considered in making and practicing the disclosure. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure.

EXAMPLES Example 1: ASM N-cadherin expression in asthma

Patterns of N-cadherin expression in the lung were examined and increased expression of immunoreactive N-cadherin was detected in the smooth muscle (smooth muscle a-actin⁺) bundles surrounding the airways in human lung tissue. N-cadherein protein was detected in human airway smooth muscle (ASM) (HASM) cells (FIG. 1A) and N-cadherin protein expression was increased in human airway smooth muscle (ASM) (HASM) cells cultured from subjects with fatal asthma compared to non-diseased controls (FIGs. IB). To determine the mechanisms underlying these differences, HASM cells were treated with asthma-related cytokines, several of which (IL4/13, TGFP) were known to increase ASM contractility (Ojiaku et al., Am J Respir Cell Mol Biol 58, 575-584 (2018); Manson et al., J Allergy Clin Immunol 145, 808-817 e802 (2020)). Treatment of HASM with TGFP but not IL-33, thymic stromal lymphopoietin (TSLP), IL-4 plus IL- 13, or platelet-derived growth factor beta (PDGFP) increased N-cadherin expression (FIGs. 1C-1D). N-cadherin expression in situ was limited to the ASM bundles of human and mouse airways as assessed by immunohistochemistry (FIGs. 1E-1F).

Example 2: N-cadherin is required for the development of airway hyperresponsiveness (AHR) in experimental asthma

To study the impact of N-cadherin deficiency specifically on bronchial smooth muscle functions in vivo, Cdh2^{n n} mice were crossed with mice expressing Cre recombinase driven by the a smooth muscle actin (SMAA) promoter (Wong et al. J Allergy Clin Immunol 146, 1152- 1164 el l l3 (2020)). Homozygous Cdhd^d-SMAA Cre mice could not be generated, suggesting that N-cadherin expression is required for proper embryonic development. However, Cdh2 abundance (FIG. 2A) and N-cadherin expression (FIGs. 2B-2C) in lung tissue was reduced by ~50% in Cdh^⁺-SMAA Cre mice compared to controls (Cdh^ or Cdh2^ll These heterozygous smooth muscle-specific N-cadherin “knockdown” mice were viable and fertile, with no gross phenotypic abnormalities at homeostasis. To examine AHR in these mice, the mice were sensitized with allergen extracts of Aspergillus fiimigatus Af), a ubiquitous environmental mold associated with severe asthma (Cameron et al., Otolaryngol Clin North Am, (2023)), followed by consecutive respiratory challenges with PBS or Af and measurements of airway resistance in live animals after exposure to various doses of the ACh analogue methacholine (MCh). Airway resistance at homeostasis was comparable in N-cadherin knockdown mice and controls. Strikingly, however, ^/-challenged mice ('dh2'^{, ll}-SMAA Cre mice did not develop any AHR, with lung resistance values that were comparable to those in mice of either genotype challenged with PBS alone (FIG. 2D). The absence of AHR was not due to a defect in smooth muscle development as the thickness of the SMAA+ area around airways was equivalent in mice of either genotype (FIGs. 2E-2F).

Example 3: N-cadherin deficiency does not affect type 2 lung inflammation

To determine the mechanism(s) underlying the absent AHR in allergen-challenged N- cadherin knockdown mice, various parameters of lung inflammation wer examined. Histology of lungs from PBS challenged ('dh2^,1 -Si\/lAA Cre mice appeared normal. Peribronchial and perivascular accumulation of leukocytes (predominantly eosinophils) in ^/-challenged Cdh2ⁿ - SMAA-Cre mice was similar to controls (FIG. 3A). Likewise, total leukocyte counts (FIG. 3B) leukocyte composition (FIG. 3C), and airway levels of T2 cytokines (IL-4, IL-5, and IL- 13) (FIGs. 3D-3F) were similar in control and ('dh2^{,l ,} -Si\4AA Cre mice. Among several mechanisms, T2 cytokines promote airway obstruction by eliciting increasing mucin expression in bronchial epithelial cells. Mucin within the respiratory epithelium detected by Periodic Acid Schiff (PAS) staining was equivalent in control and Cdhd‘ -S!vlAA Cre mice (FIG. 3G). Overall, these findings suggested that N-cadherin-deficient mice were resistant to developing AHR independent of allergic airway inflammation.

Example 4: Role of N-cadherin in airway biomechanics

To evaluate the effects of N-cadherin deficiency on bronchial contraction directly, the narrowing of airways in precision cut lung slices (PCLS) isolated from naive mice ex vivo was examined. While baseline airway luminal area was similar in PCLS from control or Cdh2ⁿ - SMAA Cre mice (FIG. 9), in response to MCh, airways from N-cadherin knockdown mice contracted significantly less than those from controls (FIG. 4A). Likewise, treatment of PCLS with the clinically relevant pharmacological N-cadherin antagonist ADH-1, a cyclic peptide that binds N-cadherin competitively and reversibly inhibits homotypic intercellular interactions (Blaschuk, Front Cell Dev Biol 10, 866200 (2022)) did not affect baseline airway diameter (FIG. 9) but significantly curtailed MCh-induced contraction (FIG. 4B). As expected, ADH-1 had little to no impact on MCh-induced contraction in PCLS from ('dh2^l, -SA AA Cre mice (FIG. 4C) ADH-1 also acutely relaxed MCh-preconstricted airways (FIGs. 4D-4E), even in airways previously desensitized to b-agonist (formoterol) (FIG. 4F). These results suggested that N- cadherin antagonism had both bronchoprotective and bronchodilatory benefits.

Example 5: Role of N-cadherin on HASM contractility

The impact of ADH-1 on contraction of cultured HASM cells was investigated. Responses to histamine, an asthma-related mediator, were focused on, as histamine elicits more robust responses than MCh in unmanipulated primary HASM due to poor m3 muscarinic receptor expression. Use of traction force microscopy revealed that pretreatment of HASM with ADH-1 reduced the magnitude of the traction footprint (FIGs. 5A-5B) and variance in the traction distribution (FIG. 5C). Consistent with these findings, ADH-1 dose dependently inhibited histamine-induced contraction (FIG. 5D). ADH-1 treatment did not induce cytotoxicity or apoptosis or reduce overall viability of HASM cells even at high doses (FIGs. 10A-10D).

Example 6: N-cadherin inhibition prevents actin remodeling in HASM

Unexpectedly, ADH-1 had no impact on several canonical pathways of agonist-induced excitation-contraction that typically result in cell shortening, including acute intracellular Ca2+ flux (FIGs. 6A-6B), myosin light chain (MFC) phosphorylation, or RhoA activation (as indicated by phosphorylation of the RhoA-responsive effector myosin light chain phosphatase [MYPT1]) elicited by histamine (FIGs. 6C-6E) or CCh (FIG. 6F).

In contrast, N-cadherin blockade markedly reduced agonist-induced actin remodeling in terms of overall F-actin quantity (FIGs. 7A-7B) and alignment (anisotropy) (FIG. 7C). Consistent with these findings, F-actin was significantly lower in bronchial smooth muscle of allergen challenged Cdh2fl/+-SMAA Cre mice control mice than in controls (FIGs. 7D-7E).

Example 7: ADH-1 alleviates AHR in experimental asthma

To evaluate the potential of ADH-1 as a treatment for asthma, mice were treated with ADH-1 by i.p. injection and lung resistance was measured as previously described. Eike the genetic model, pretreatment with ADH-1 did not affect lung resistance in naive mice but significantly reduced MCh-induced airway obstruction in Af challenged Balb/c mice (FIG. 8A). ADH-1 treatment had no appreciable impact on inflammation including the histological pattern of peribronchial inflammation (FIG. 8B) total leukocyte numbers (FIG. 8C), leukocyte composition (FIG. 8D), epithelial mucin expression (FIG. 8E), or airway T2 cytokines (FIGs 8F-8H) in BALF.

The costs of severe asthma are projected to exert a substantial economic and societal burden in the coming decade. FAO presents an intractable problem for the treatment of patients with acute severe symptoms as it is frequently associated with reduced MCh responsiveness (Damera et al., PLoS One 7, e28504 (2012); Varricchi et al., Allergy 77, 3538-3552 (2022); Shimizu et al., J Allergy Clin Immunol 149, 934-942 e938 (2022)). The efficacy of P2 agonist bronchodilators to relax ASM is further adversely impacted by P2 adrenergic receptor de sensitization and a complex signaling pathway that may be antagonized by type 2 inflammatory pathways (Camoretti-Mercado et al., J Allergy Clin Immunol 147, 1983-1995 (2021)).

N-cadherin may represent an attractive target for the treatment for both acute and chronic airway obstruction in asthma. N-cadherin expression in ASM is increased in patients with asthma, which may result from chronic inflammation. TGFp, which was previously implicated in increased N-cadherin expression in bronchial epithelial cells (associated with reduced E-cadherin expression), has a similar effect on ASM cells (Maneechotesuwan et al., J Asthma Allergy 16, 343-354 (2023)). Chronic airway remodeling is associated with increased airway TGFp in patients with established asthma, and TGFp levels are higher in sputum from patients with FAO than in those without FAO (Ojiaku et al., Am J Respir Cell Mol Biol 58, 575-584 (2018); Li et al., Inflammation, (2024)). A non-toxic, reversible antagonist of N-cadherin-mediated intercellular interactions (ADH-1) reduced AHR in allergen challenged mice and prevented bronchoconstriction ex vivo. Since ADH-1 also elicited bronchodilation in pre-constricted airways, rapidly acting inhaled N-cadherin antagonists might be developed for the treatment of acute airway obstruction even in patients with FAO. ADH-1 is well tolerated by patients with few side effects in clinical trials for solid tumors (Yarom et al., Curr Clin Pharmacol 8, 81-88 (2013)). Since ADH-l’s bronchoprotective benefits are independent of inflammation, it may also have benefits for other obstructive lung diseases (COPD, RSV bronchiolitis). To constrict the bronchial lumen, airway smooth muscle (ASM) cells must contract as a collective. Although it is recognized that multicellular migration within tissues requires long distance, cooperative transmission of intercellular forces (De Pascalis et al., Mol Biol Cell 28, 1833-1846 (2017)), the mechanisms underlying ASM cell-cell contraction are as not well understood. The above Examples demonstrated that intercellular forces were coordinated amongst -50% of cells in the untreated ASM monolayer but reduced to -30% cells within 24 hours of N-cadherin inhibition by ADH- 1. Subsequent histamine stimulation did not alter the overall force landscape but increased contraction in pre-existing hotpots in control HASM monolayers. The Examples also indicated that these biophysical changes were independent of canonical pathways leading to shortening in isolated ASM cells (Ca²⁺ flux, MLC phosphorylation, RhoA activation).

Rather, ADIT-1 prevented bronchoconstrictor (histamine)-evoked F-actin stress fiber formation and co-alignment. In contrast to endothelial cells, which form adherens junction- mediated single layer contacts on either side, ASM cells form intercalated contacts with multiple cells at once, in all directions (Gupta et al., Fac Rev 10, 56 (2021)). Because of this unique organizational structure, contraction of the ASM bundle requires complex spatio-temporal remodeling, which is shown in the traction force microscopy experiments. F-actin was found to be primarily cortical (at the periphery) in ADH- 1 pretreated cells exposed to histamine, suggesting that by promoting a more static/less coordinated force terrain, N-cadherin blockade effectively impaired the formation of actin planar F-actin stress fibers within individual cells. Without intending to be bound by theory, this remodeling was expected to impede cell contraction and synchronized intercellular force transmission in response to a spasmogen.

Without intending to be bound by theory, the intracellular pathway by which N-cadherin- dependent pathway transmits force within the ASM bundle likely depends on p-catenin, a cytosolic protein with multiple links to the actin cytoskeleton. Knockdown of P-catenin by siRNA or inhibition of the N-cadherin-P-catenin interaction with a small molecule antagonist impairs contraction of ASM cells or tracheal rings ex vivo (Jansen et al., Am J Physiol Lung Cell Mol Physiol 299, L204-214 (2010); Wang et al., J Biol Chem 290, 8913-8924 (2015)). N- cadherin-mediated mechanotransduction might also impact other pathways needed for efficient contraction. Bronchi/bronchioles perceive contractile agonist concentrations in concert by generating synchronized Ca²⁺ oscillations propagated around their perimeter to constrict the airway. Agonist-induced Ca²⁺ oscillation frequency increases in proportion to the degree of bronchoconstriction and depends strictly on ECM stiffness and force transmission between adjacent ASM cells rather than intercellular Ca²⁺ diffusion or gap junction mediated transport (Stasiak et al., Sci Adv 6, eabal 149 (2020)). Although it was found that ADH-1 did not reduce the immediate intracellular Ca²⁺ flux elicited by agonist, it might nonetheless regulate Ca²⁺ oscillations over longer times.

METHODS OF THE EXAMPLES

The following methods were employed in the above examples.

Experimental design

The main objective of this study was to elucidate the functions of N-cadherin on ASM contraction at homeostasis and in asthma models. A genetic model (selective Cdh2 haploinsufficiency in ASM) or effects of a pharmacological antagonist (ADH-1) on HASM monolayer or PCLS contraction ex vivo and AHR induced by fungal allergen challenge was studied. Sample size justification was derived from previous animal studies that were sufficiently powered to detect differences in lung resistance (Wong et al., J Allergy Clin Immunol 146, 1152- 1164 el l l3 (2020)). Animals were randomly assigned to treatment with vehicle or ADH-1 prior to the start of each study.

Cell lines

HASM cells were extracted post-mortem from tracheas of de-identified donors [International Institute for the Advancement of Medicine (Edison, NJ) or the National Disease Research Interchange (Philadelphia, PA)] as described previously (Deeney et al., Am J Physiol Lung Cell Mol Physiol 323, L142-L151 (2022)). Demographic information on these subjects is presented in Table 1. Cells were cultured in Ham’s F-12 medium supplemented with L- glutamine and 10% FBS at 37 °C in 5% CO2.

Table 1 Donor Demographics

* Mean ± SEM Reagents

TGFp, EGF, ADH-1, formoterol, MCh, CCh and histamine were from Sigma. Recombinant human IL-4, IL- 13, TSLP, IL-33, PDGFp were purchased from R&D Systems.

Mice

Cdh2ⁿ mice were obtained from Jackson Laboratories and backcrossed with Balb/cJ mice for 4 generations. SMAA-Cre mice were provided by Dr. Fred Finkelman (University of Cincinnati School of Medicine). All mice were bred and maintained under pathogen-free conditions at an American Association for the Accreditation of Laboratory Animal Care accredited animal facility at the NIAID and housed in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals under an animal study proposal (LAD3E) approved by the NIAID Animal Care and Use Committee.

RNA isolation and quantitative RT-PCR

RNA was extracted using the RNeasy kit (Qiagen) according to the manufacturer's instructions. Total RNA (500 ng) was reverse transcribed into cDNA using SuperScript™ IV VILO™ Master Mix (Thermo Fisher). TaqMan probes (ThermoFisher) were as follows: Cdh2. Mm01162497_ml; Actb, Mm026I9580_gI.

Immunoblotting and immunofluorescence

Lysates were prepared from HASM cells using IX radioimmunoprecipitation (RIP A) lysis buffer (Millipore/Sigma) containing protease (cOmplete) and phosphatase inhibitor (PhosSTOP) inhibitor cocktails (Roche) and clarified by centrifugation at 15,000 rpm for 10 minutes at 4°C. Samples were boiled with SDS sample buffer at 95 °C, electrophoresed on 4- 12% Tris-glycine gels, and transferred to nitrocellulose membranes. Primary antibody staining was detected using near-infrared conjugated secondary antibodies and quantified with the LiCor Odyssey Imaging System and Image Studio software (LiCor Biosciences). For immunofluore scent staining, cells were plated in Chamberwell slides (Nunc), fixed in 4% paraformaldehyde and permeabilized with PBS containing 0.2% vol/vol Triton X-100. Cells were then blocked in 2% (wt/vol) BSA in PBS containing 5% goat serum. Cells were then incubated primary antibodies or phalloidin and then with AlexaFluor-conjugated goat anti-mouse or goat anti -rabbit secondary antibodies. Slides were mounted on coverglass with ProLong Gold antifade reagent with DAPI. Images were obtained at 63x original magnification using a Leica SP8 confocal microscope and analyzed using ImageJ and anisotropy using the FibrilTool plugin.

Cell viability assay

Viability, cytotoxicity, and apoptosis of HASM cells were evaluated using the ApoTox Gio assay (Promega) according to the manufacturer’s instructions.

Ca²⁺ measurements

HASM cells were plated in 96-well black-walled plates (1 x 10⁵ cells/well). Ca²⁺ Fluo-6 indicator and (FLIPR Calcium 6 assay kit, Molecular Devices) and probenecid (1 mM) was added to each well containing serum-free medium and analyzed using a FlexStation III instrument (Molecular Devices) after addition of agonist as described previously (Wong et al., J Allergy Clin Immunol 146, 1152-1164 el l 13 (2020)).

Traction force microscopy

HASM cells were plated upon deformable substrates (Y oung’s Modulus, 3kPa) prepared in a standard 6-well plate and treated with either vehicle or ADH-1 for 4 hours. Cells were then stimulated with histamine (3 pM) for 30 minutes. Contraction (% increase) was determined by normalizing histamine induced ASM contraction to its pre-treatment value. All force measurements were performed using the method of traction microscopy adapted for ASM cell monolayers (Y oshie et al., Biophys J 114, 2194-2199 (2018)).

Allergic airway inflammation model

8-12-week-old female mice were sensitized with a mixture of one part alum and one part PBS containing Aspergillus fumigatus (Af) extract (25 mg protein, Hollister Stier Allergy), by i.p. injection on days 0 and 7. 1-2 weeks later mice were then challenged intranasally with either PBS or A/(20 mg) daily for three consecutive days. 24 hours after the final challenge, mice were euthanized following analysis of lung resistance. BALF was collected injection and collection of PBS/1 mM EDTA (1 ml) through a tracheal cannula. Red blood cells were lysed with ACK lysis buffer and clarified BALF supernatants frozen and stored at -80°C. Cell pellets were resuspended in PBS/EDTA counted by hemocytometry and dispersed on glass microscope slides by cytospin. Diff-Quick stained slides were used to determine cell composition by microscopy (n = 300 cells/slide). Left lungs were fixed in 10% neutral buffered formalin for generation of paraffm- embedded sections. Sections were stained with hematoxylin and eosin, and PAS. Cytokines in BALF supernatants were analyzed using a customized Multiplex bead array (BioRad) as previously described (Wong et al., J Allergy Clin Immunol 146, 1152-1164 el 113 (2020)).

Lung resistance measurements

Mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg) i.p. The trachea was dissected and cannulated with a 20 gauge cathether. Mice were then paralyzed with vecuronium bromide (200 mg via i.p. injection) and mechanically ventilated using the FlexiVent FX1 respirator (Scireq). Lung resistance was measured by the pulse oscillometry technique at baseline and after inhalation of increasing doses of MCh as described previously (Wong et al., J Allergy Clin Immunol 146, 1152-1164 el 113 (2020)).

Analysis of airway contraction in PCLS

PCLS were generated as described previously (Wong et al., J Allergy Clin Immunol 146, 1152-1164 e 1113 (2020)) and incubated overnight at 37°C in a tissue culture incubator (5% CO2). Intact slices were cryopreserved (Rosner et al., Am J Respir Cell Mol Biol 50, 876-881 (2014)). On the day of the experiment, the slices were rapidly thawed, placed in 12- or 24-well plates, and stimulated with MCh (10 pM). Images of the airways in the lung slices were compared before and at t=20 minutes after MCh stimulation using ImageJ to calculate luminal area. Airway constriction was expressed as the percentage of the pre-stimulation luminal area.

Statistical analysis

Data were analyzed using GraphPad Prism 9.0 and expressed as means ± SEM of n=3 or more biological replicates. Unpaired, two-tailed Student’s t test was used for comparison of 2 groups and 1- or 2-way analysis of variance (ANOVA) for multiple groups. Non-parametric analyses were used for non-normally distributed data. P values < 0.05 was considered statistically significant.

Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the disclosure described herein to adapt it to various usages and conditions. Such embodiments are also within the scope of the following claims. The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or sub-combination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

What is claimed is:

1. A method of inhibiting N-cadherin activity in an airway smooth muscle cell, the method comprising contacting an airway smooth muscle cell with an effective amount of an agent having N-cadherin antagonist activity, thereby inhibiting N-cadherin activity in the airway smooth muscle cell.

2. A method of reducing contractile agent-induced airway smooth muscle cell contraction, the method comprising contacting an airway smooth muscle cell with an agent having N- cadherin antagonist activity, wherein the airway smooth muscle cell will be contacted with a contractile agent, was concurrently contacted with a contractile agent, or has been contacted with a contractile agent.

3. A method of inducing bronchodilation in an airway smooth muscle cell, the method comprising contacting an airway smooth muscle cell with an agent having N-cadherin antagonist activity.

4. The method of any one of claims 1-3, wherein the N-cadherin antagonist is a linear peptide or a cyclic peptide comprising a His-Ala-Val amino acid sequence that binds N-cadherin competitively and reversibly inhibits homotypic intercellular interactions.

5. The method of any one of claims 1-4, wherein the airway smooth muscle cell is in vitro or in vivo.

6. The method of claim 5, wherein the airway smooth muscle cell is in vivo, and the N- cadherein antagonist is administered via inhalation.

7. A method of inhibiting N-cadherin activity in an airway smooth muscle cell, the method comprising contacting an airway smooth muscle cell with an N-cadherin antagonist that is a small molecule compound, linear peptide, a cyclic peptide, or an antibody or antigen binding portion thereof that binds N-cadherin competitively and reversibly inhibits homotypic intercellular interactions.

8. The method of claim 7, wherein the method reduces contractile agent-induced airway smooth muscle cell contraction.

9. The method of claim 2 or 8, wherein the contractile agent is selected from the group consisting of cholinergic agonists, histamine, prostaglandins, leukotrienes, adenosine, NSAIDs, tachykinins, bradykinin, and endotoxins.

10. The method of claim 9, wherein the contractile agent is acetylcholine, methacholine, carbachol, histamine, or bradykinin.

11. The method of claim 7, wherein the method induces bronchodilation in the airway smooth muscle cell.

12. The method of any one of claims 1-11, wherein the agent is a linear or cyclic peptide comprising a His-Ala-Val amino acid sequence.

13. The method of claim 12, where the peptide is a cyclic peptide comprising the following sequence:

, wherein XI and X2 are optional, and if present, are independently selected from the group consisting of amino acid residues linked by peptide bonds, and wherein Xi and X₂ independently range in size from 0 to 10 residues, such that the sum of residues contained within Xi and X₂ ranges from 1 to 12; wherein Y i and Y₂ are amino acid residues, and wherein a covalent bond is formed between residues Y i and Y₂; and wherein Zi and Z₂ are optional, and if present, are amino acid residues linked by peptide bonds.

14. The method of any one of claims 1-13, wherein the peptide comprises the sequence AHAVSE or LRAHA VDVNG.

15. The method of any one of claims 1-14, wherein the peptide is cyclized via a disulfide bond; an amide bond between terminal functional groups, and/or a thioether bond between terminal function groups, between residue side-chains, or between one terminal functional group and one residual side chain.

16. The method of any of claims 1-11, wherein the N-cadherin antagonist is ADH-1 or LCRF006.

17. A method of treating a lung disorder in a subject, the method comprising administering to the subject an agent having N-cadherin antagonist activity, wherein the subject has a lung disorder selected from the group consisting of asthma, bronchiolitis, bronchiectasis, bronchitis, and chronic obstructive pulmonary disease (COPD), and wherein the agent is a linear peptide, a cyclic peptide, or an antibody or antigen binding portion thereof, wherein the agent binds N- cadherin competitively and reversibly inhibits homotypic intercellular interactions.

18. The method of claim 17, wherein the subject has a fixed airway obstruction associated with the lung disorder.

19. A method of reducing or preventing airway hyperresponsiveness (AHR) in a subject, the method comprising administering to the subject an N-cadherin antagonist, thereby reducing or preventing AHR in the subject.

20. The method of any one of claims 17-19, wherein the method reduces actin remodeling, reduces histamine-induced airway smooth muscle cell contraction, and/or induces bronchodilation in the airway smooth muscle cell.

21. The method of any one of claims 17-19, wherein the agent is a linear or cyclic peptide comprising a His-Ala-Val amino acid sequence.

22. The method of claim 21, where the peptide is a cyclic peptide comprising the following sequence: wherein XI and X2 are optional, and if present, are independently selected from the group consisting of amino acid residues linked by peptide bonds, and wherein Xi and X2 independently range in size from 0 to 10 residues, such that the sum of residues contained within Xi and X2 ranges from 1 to 12; wherein Y 1 and Y2 are amino acid residues, and wherein a covalent bond is formed between residues Y 1 and Y2; and wherein Zi and Z2 are optional, and if present, are amino acid residues linked by peptide bonds.

23. The method of claim 21 or 22, wherein the peptide comprises the sequence AHAVSE or LRAHAVDVNG.

24. The method of claim 22, wherein the peptide is cyclized via a disulfide bond; an amide bond between terminal functional groups, and/or a thioether bond between terminal function groups, between residue side-chains, or between one terminal functional group and one residual side chain.

25. The method of any one of claims 17-19, wherein the N-cadherin antagonist is ADH-1 or LCRF006.

26. The method of any one of claims 17-25, further comprising administering to the subject a second agent selected from the group consisting of: leukotriene receptor antagonists; corticosteroids; theophylline; beta agonists; anti-cholinergic agents; and anti-inflammatory agents.

27. The method of claim 26, wherein the second agent is monte lukast, zafirlukast, zileuton, prednisone, methylprednisone, fluticasone propionate, budesonide, ciclesonide, beclomethasone, mometasone, fluticasone furonate, theophylline, albuterol, levalbuterol, ipratropium, or tiotropium.

28. A pharmaceutical composition comprising an N-cadherin antagonist and a pharmaceutically acceptable excipient, wherein the composition is formulated for inhalation.

29. The pharmaceutical composition of claim 28, wherein the N-cadherin antagonist is ADH- 1 or LCRF006.

30. A kit comprising a N-cadherin antagonist and instructions for using the N-cadherin antagonist or pharmaceutical composition in the method of any of claims 1-27.

31. The kit of claim 30, wherein the agent is a linear or cyclic peptide comprising a His-Ala- Val amino acid sequence.

32. The kit of claim 31, wherein the peptide comprises the sequence AHAVSE or LRAHAVDVNG.

33. The kit of claim 30, wherein the N-cadherin antagonist is ADH-1 or LCRF006.

34. A method of inducing bronchodilation in an airway smooth muscle cell, the method comprising contacting an airway smooth muscle cell with an agent having N-cadherin antagonist activity and an agent having beta agonist activity.

35. A method of inducing bronchodilation in an airway smooth muscle cell, the method comprising contacting an airway smooth muscle cell with an agent having N-cadherin antagonist activity and an agent having corticosteroid activity.

36. A pharmaceutical composition comprising an N-cadherin antagonist, a beta agonist, and a pharmaceutically acceptable excipient.

37. A pharmaceutical composition comprising an N-cadherin antagonist, a corticosteroid, and a pharmaceutically acceptable excipient.

38. The pharmaceutical composition of claim 36 or claim 37, wherein the pharmaceutical composition is formulated for inhalation.