WO2025057155A1 - Modulateurs peptidiques de l'axe 4-neurexine 1-bêta de la neuroligine pour le traitement d'affections impliquant une réponse inflammatoire anormale - Google Patents

Modulateurs peptidiques de l'axe 4-neurexine 1-bêta de la neuroligine pour le traitement d'affections impliquant une réponse inflammatoire anormale Download PDF

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WO2025057155A1
WO2025057155A1 PCT/IL2024/050910 IL2024050910W WO2025057155A1 WO 2025057155 A1 WO2025057155 A1 WO 2025057155A1 IL 2024050910 W IL2024050910 W IL 2024050910W WO 2025057155 A1 WO2025057155 A1 WO 2025057155A1
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composition
seq
peptide
subject
use according
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Rifaat Safadi
Johnny AMER
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Hadasit Medical Research Services and Development Co
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Hadasit Medical Research Services and Development Co
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • the terms “essentially” and “substantially” are synonymous and when referring to a stated material such as a composition, a substance, and the like, is meant to encompass variations of in some embodiments, ⁇ 0.1%, or in some embodiments, ⁇ 1%, or in some embodiments, ⁇ 2%, or in some embodiments, ⁇ 5% or in some embodiments, ⁇ 10% from a stated amount, as such variations/deviations are appropriate to perform the disclosed methods. Each possibility is a separate embodiment.
  • compositions comprising isolated inhibitory peptides, capable of effectively and safely affecting NLGn4X-Nrxip interactions, for treating colon/intestine-related conditions, namely IBD.
  • the isolated peptide includes or consists of an amino acid sequence as denoted by any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and/or SEQ ID NO: 6, or any combination thereof, wherein the isolated peptide is capable of affecting interactions between neuroligin 4 (NLGn4X) and Neurexin ip (Nrxip).
  • NLGn4X neuroligin 4
  • Nrxip Neurexin ip
  • the isolated peptide includes or consists of an amino acid sequence as denoted by any one of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 or any combination thereof. Each possibility is a separate embodiment.
  • the isolated peptide is an isolated modulator peptide.
  • the terms “modulate”, “affect”, “alter” and “interfere”, may be used interchangeably.
  • the phrase “capable of affecting/modulating/interfering” as used herein refers to the capability of the isolated peptide(s) of the invention to inhibit or prevent interaction or binding between NLGn4 and Nrxip or to interact with a target of NLGn4 or Nrxip, such that the target becomes less accessible, preferably inaccessible, to binding by its corresponding binding partner (for example, Nrxip or NLGn4), for example the receptor's natural antigen.
  • the isolated peptide is capable of inhibiting binding between NLGn4 and Nrxip.
  • binding site refers to the region of NLGn4 and/or Nrxip that specifically reacts with a particular modulator peptide.
  • peptide and polypeptide as used herein are intended to encompass any amino acid sequence including modified sequences.
  • the terms “peptide” and “polypeptide” are specifically intended to cover naturally occurring proteins, as well as those, which are recombinantly or synthetically produced.
  • the inhibitory peptides of the invention also include modified peptides (with amino acid substitutions, both conservative and non-conservative as described below) that have the same or improved activity as a wild-type or unmodified peptide. “Salts” of the peptides of the invention contemplated by the invention are physiologically and pharmaceutically acceptable organic and inorganic salts. The invention also provides conservative amino acid variants of the peptides according to the invention.
  • Variants according to the invention also may be made that conserve the overall molecular structure of the encoded peptides. Given the properties of the individual amino acids comprising the disclosed peptide products, some rational substitutions will be recognized by the skilled worker. Amino acid substitutions, i.e., “conservative substitutions,” may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • the amino acids used in this invention are those, which are available commercially or are available by routine synthetic methods. Certain residues may require special methods for incorporation into the peptide, and either sequential, divergent or convergent synthetic approaches to the peptide sequence are useful in this invention. Natural coded amino acids and their derivatives are represented by three- letter codes according to IUPAC conventions. When there is no indication, the L isomer was used. The D isomers are indicated by “D” before the residue abbreviation.
  • Conservative substitutions of amino acids as known to those skilled in the art are within the scope of the present invention.
  • Conservative amino acid substitutions include replacement of one amino acid with another having the same type of functional group or side chain, e.g., aliphatic, aromatic, positively charged, negatively charged. These substitutions may enhance oral bioavailability, penetration into the islets, targeting to specific beta cell populations, immunogenicity, and the like.
  • One of skill will recognize that individual substitutions, deletions or additions to a peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid.
  • Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • the peptides of the present invention may be produced by any method known in the art, including recombinant and synthetic methods. Synthetic methods include exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, or classical solution synthesis. Solid phase peptide synthesis procedures are well known to one skilled in the art and described, for example by John Morrow Stewart and Janis Dillaha Young, Solid Phase Polypeptide Syntheses (2nd Ed., Pierce Chemical Company, 1984). In some embodiments, synthetic peptides are purified by preparative high-performance liquid chromatography (Creighton T. (1983) Proteins, structures and molecular principles. WH Freeman and Co. N.Y.) The peptide sequence may be confirmed by amino acid sequencing using methods known to one skilled in the art.
  • isolated means either: 1) separated from at least some of the components with which it is usually associated in nature with respect of the source protein; 2) prepared or purified by a process that involves the hand of man; 3) not occurring in nature.
  • isolated peptide refers to the peptides of the present invention produced by any method known in the art, including recombinant and synthetic methods and thereafter purified using any method known in the art so as to become at least a major component of the solution, mixture, or extract and dry forms thereof (i.e., powder), which may also include smaller amounts of other molecules; or so as to become essentially the only component of the solution, mixture, or extract and dry forms thereof (i.e., powder) (i.e. other components are present in residual amounts only).
  • the isolated peptide refers to solution, mixture, or extract and dry forms thereof (i.e., powder) containing at least 60% (w/w or w/v), at least 70% at least 80%, or at least 90%, or at least about 95%, or at least about 96%, or at least about 97% or at least about 98%, or at least about 99% of the isolated peptide.
  • solution, mixture, or extract and dry forms thereof i.e., powder
  • the isolated peptide refers to solution, mixture, or extract and dry forms thereof (i.e., powder) containing at least 60% (w/w or w/v), at least 70% at least 80%, or at least 90%, or at least about 95%, or at least about 96%, or at least about 97% or at least about 98%, or at least about 99% of the isolated peptide.
  • the solution, mixture, or extract and dry forms thereof (i.e., powder) containing essentially the isolated peptide contains less than about 10% (w/w or w/v) or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1%, or less than 0.1% of other molecules than the isolated peptide.
  • concentration w/w or w/v
  • 5% 5%
  • 4% 5%
  • 4% or less than 3%
  • 2% or less than 1%, or less than 0.1% of other molecules than the isolated peptide.
  • recombinant protein techniques are used to generate the peptide of the present invention.
  • recombinant protein techniques are used for generation of polypeptides or nucleic acid sequences or viral or bacterial vectors for vaccine formulation. Recombinant techniques are described for example by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 3:17-311, Coruzzi et al. (1984) EMBO J.
  • Isolated polynucleotide sequences comprising at least one sequence encoding a peptide, peptide analog, peptide homolog conjugate or fusion protein of a modulator peptide are also included in the scope of the present invention.
  • the polynucleotide sequence encoding the peptide or peptide analog, peptide homolog is translationally linked to another polynucleotide sequence such as an RNA or DNA molecule and is recombinantly expressed within target cells.
  • the polynucleotide sequence is part of a recombinant viral or bacterial vector.
  • analog refers to a molecule, which has the amino acid sequence according to the invention except for one or more amino acid changes.
  • Analogs according to the present invention may include peptidomimetics.
  • “Peptidomimetic” refers to a peptide modified in such a way that it includes at least one non-coded residue or non-peptidic bond. Such modifications include, e.g., alkylation and more specific methylation of one or more residues, insertion of or replacement of natural amino acid by non-natural amino acids, replacement of an amide bond with another covalent bond.
  • a peptidomimetic according to the present invention may optionally comprise at least one bond, which is an amide -replacement bond such as urea bond, carbamate bond, sulfonamide bond, hydrazine bond, or any other covalent bond.
  • an amide -replacement bond such as urea bond, carbamate bond, sulfonamide bond, hydrazine bond, or any other covalent bond.
  • the design of appropriate “analogs” may be computer assisted. Analogs are included in the invention as long as they remain pharmaceutically acceptable.
  • the polypeptide comprises one or more amino acid substitutions, additions, or deletions. In some embodiment, the polypeptide comprises one or more substitutions corresponding to a conservative variant of the amino acid.
  • a composition comprising an isolated peptide comprising an amino acid sequence as denoted by any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and/or SEQ ID NO: 6, or any combination thereof, for use in treating, attenuating, and/or preventing progression of an Inflammatory Bowel Disorder (IBD) in a subject in need thereof.
  • IBD Inflammatory Bowel Disorder
  • the isolated peptide comprises an amino acid sequence as denoted by SEQ ID NO: 1 and/or SEQ ID NO: 6. Each possibility is a separate embodiment.
  • the isolated peptide comprises an amino acid sequence as denoted by SEQ ID NO: 1. In some embodiments, the isolated peptide comprises an amino acid sequence as denoted by SEQ ID NO: 6.
  • the isolated peptide consists of an amino acid sequence as denoted by any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and/or SEQ ID NO: 6, or any combination thereof. Each possibility is a separate embodiment.
  • the isolated peptide consists of an amino acid sequence as denoted by SEQ ID NO: 1 and/or SEQ ID NO: 6. Each possibility is a separate embodiment.
  • the amino acid sequence of the peptide has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or at least 99.9% sequence homology/identity/similarity to the amino acid sequence as set forth in any one of SEQ ID NO: 1, SEQ ID NO: 2 SEQ ID NO: 3 and/or SEQ ID NO: 6. Each possibility is a separate embodiment.
  • sequence similarity may be interchangeably used and refer hereinafter to the level of identities between two homologous sequences when they are compared by aligning them using an alignment tool.
  • sequence similarity is with respect to any one of the amino acid sequences disclosed herein and denoted by any one of SEQ ID NO: 1, SEQ ID NO: 2 SEQ ID NO: 3 and/or SEQ ID NO: 6.
  • the level of similarity or homology is determined by the number of identities in the amino acid sequence when they are aligned.
  • the composition includes a plurality of isolated peptides.
  • plural or “plurality of isolated peptides” refer to a combination/mixture including two or more of the isolated peptides of the invention as set forth in any one of SEQ ID NO: 1, SEQ ID NO: 2 SEQ ID NO: 3 and/or SEQ ID NO: 6 and/or variants or homologs thereof including peptides having conservative amino acid substitutions and/or peptide analog/modified peptide thereof.
  • the composition includes a combination of two or more isolated peptides selected from SEQ ID NO: 1, SEQ ID NO: 2 SEQ ID NO: 3 and SEQ ID NO: 6, or any combination thereof.
  • a method of treating, attenuating and/or preventing progression of Inflammatory Bowel Disorder (IBD) in a subject in need thereof comprising administering a therapeutically effective amount of a composition comprising an isolated peptide comprising an amino acid sequence as denoted by any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and/or SEQ ID NO: 6.
  • the administration includes systemic and/or localized administration. Each possibility is a separate embodiment.
  • composition comprising the isolated peptide is administered in combination with at least one additional therapeutic agent.
  • subject and “patient” are interchangeable and as used herein refer to any individual suffering from IBD.
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures.
  • those in need of treatment include those already having a disorder.
  • those in need of treatment include those in which the progression of the disorder is to be prevented.
  • those in need of treatment include those in which the disorder is to be prevented.
  • the terms “prevent”, “reduce”, “attenuate”, “ameliorate”, “inhibit”, “mitigate”, “alleviate” may be used interchangeably.
  • administration refers to providing or giving a subject a therapeutic agent (e.g. an isolated peptide or composition comprising the same), by any effective route.
  • routes of administration include, but are not limited to, injection or infusion (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intrathecal, intravenous, intracerebroventricular, intrastriatal, intracranial and into the spinal cord), oral, intraductal, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes. Each possibility is a separate embodiment.
  • any suitable route of administration to a subj ect may be used for the compositions comprising the isolated peptides of the present invention, including but not limited to, local and systemic routes.
  • exemplary suitable routes of administration include, but are not limited to: orally, intra-nasally, parenterally, intravenously, topically, enema or by inhalation.
  • systemic administration of the composition is via an injection.
  • the composition may be formulated in an aqueous solution, for example in a physiologically compatible buffer including, but not limited, to Hank’s solution, Ringer’s solution, or physiological salt buffer.
  • Formulations for injection may be presented in unit dosage forms, for example, in ampoules, or in multi -dose containers with, optionally, an added preservative.
  • parenteral administration is administration intravenously, intra-arterially, intramuscularly, intraperitoneally, intradermally, intravitreally, or subcutaneously.
  • parenteral administration is performed by bolus injection.
  • parenteral administration is performed by continuous infusion.
  • preparations of the composition of the invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions, each representing a separate embodiment of the present invention.
  • the administration may include any suitable administration regime, depending, inter aha, on the medical condition, patient characteristics, administration route, and the like.
  • administration may include administration twice daily, every day, every other day, every third day, every fourth day, every fifth day, once a week, once every second week, once every third week, once every month, and the like.
  • IBD indicaciones
  • colon/intestinal-related disorders may be used interchangeably, and refer to pathologies that cause the bowel/colon/intestine to function improperly as generally known in the art.
  • IBD pathologies include at least one of any one of: (i) body weight loss; (ii) morphological changes related to colon size; (iii) chronic inflammation; (iv) fibrosis and/or fibrostenotic strictures; (v) increased paracellular permeability or colon susceptibility; and (vi) changes in lipid homeostasis, or any combination thereof.
  • body weight loss e.g., body weight loss
  • morphological changes related to colon size e.g., chronic inflammation, fibrosis and/or fibrostenotic strictures; (v) increased paracellular permeability or colon susceptibility; and (vi) changes in lipid homeostasis, or any combination thereof.
  • IBD pathologies may also include loss of body weight.
  • the colon/intestinal-related disorders/IBD includes but is not limited to one or more of: ulcerative colitis (UC), Crohn's disease (CD) Collagenous colitis, Lymphocytic colitis, Ischemic colitis, Diversion colitis, Behcet' s disease, and Indeterminate colitis, or any combination thereof.
  • UC ulcerative colitis
  • CD Crohn's disease
  • Collagenous colitis Lymphocytic colitis
  • Ischemic colitis Ischemic colitis
  • Diversion colitis Diversion colitis
  • Behcet' s disease Diversion colitis
  • Indeterminate colitis or any combination thereof.
  • the IBD is selected from any one or more of: ulcerative colitis (UC), Crohn's disease (CD) Collagenous colitis, Lymphocytic colitis, Ischemic colitis, Diversion colitis, Behcet's disease, and Indeterminate colitis, or any combination thereof.
  • UC ulcerative colitis
  • CD Crohn's disease
  • Collagenous colitis Lymphocytic colitis
  • Ischemic colitis Diversion colitis
  • Behcet's disease Diversion colitis
  • Indeterminate colitis or any combination thereof.
  • composition and “pharmaceutical composition” are used interchangeably and as used herein refers to any composition comprising at least one isolated peptide of the invention., or a composition which comprises a mixture/plurality of isolated peptides.
  • the composition can include a plurality of peptides.
  • the plurality of peptides includes a combination of several peptides.
  • the composition may include a combination of peptide 1, peptide 2 and/or peptide 3, and in other embodiments, the composition may include a mixture of peptide 1, peptide 3 and/or peptide 6.
  • the composition includes the modulator peptide and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to any carrier conventional used in the production of pharmaceutical compositions. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition, 1975, describes compositions and formulations suitable for pharmaceutical delivery of the compositions disclosed herein.
  • the present invention provides compositions including novel isolated peptides, and uses thereof for treating IBD by modulating, interfering with, inhibiting and/or preventing NLGn4-Nrxip interaction, in particular, by preventing protein-protein interaction ("PPI") thereof.
  • PPI protein-protein interaction
  • the isolated peptides may be derived from NLGN4X.
  • the peptides may include different amino acid sequences that can vary in length, and may be derived from the same binding site as that of p-neurexin (i.e., the ligand of the NLG4NX receptor).
  • the active binding site is in a domain containing amino acids 359-364 in NLGN4X protein.
  • epitopes involved in binding may include, E361, L363, H267, Y463 and E270.
  • the isolated peptides are derived from E361 site epitope.
  • the isolated peptide is used in a method of treating, attenuating and/or preventing progression of a bowel disorder, the method includes administering a therapeutically effective amount of peptide (or a composition including the same), the peptide having an amino acid sequence as denoted by any one of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and/or SEQ ID NO: 6, wherein said peptide is capable of interfering with, inhibiting and/or preventing neuroligin 4 (NLGn4X) - Neurexin ip (Nrxip) interaction.
  • a therapeutically effective amount of peptide or a composition including the same
  • the peptide having an amino acid sequence as denoted by any one of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and/or SEQ ID NO: 6, wherein said peptide is capable of interfering with, inhibiting and/or preventing neuroligin 4 (NLGn4X) - Neurexin
  • the peptide may be used in combination with at least one additional therapeutic agent, such as, but not limited to PD1 and PDL1 inhibitors.
  • the additional therapeutic agent comprises a classical therapeutic agent.
  • the isolated peptide may be comprised in a single composition with the additional therapeutic agent.
  • the peptide may be comprised in a separate composition than the additional therapeutic agent.
  • the isolated peptide may be a synthetic peptide or a recombinant peptide. Each possibility is a separate embodiment.
  • isolated peptides for affecting the NLGn4-Nrxip interaction have an amino acid sequence as set forth in SEQ ID NO: 6: EQGEFLNY.
  • the isolated peptide having amino acid sequence as denoted by SEQ ID NO: 6 is designated "peptide 6".
  • the isolated peptide may consist or comprise of amino acid sequence as denoted by SEQ ID NO: 6.
  • the isolated peptide sequence of NLGn4- Nrxip PPI may have an amino acid sequence as denoted by SEQ ID NO: 1: MEQGEFLNYD.
  • the isolated peptide having amino acid sequence as denoted by SEQ ID NO: 1 is designated "peptide 1".
  • peptide 1 may comprise or consist of amino acid sequence as denoted by SEQ ID NO: 1.
  • the isolated peptide sequence may have an amino acid sequence as denoted by SEQ ID NO: 2: QILMEQGEFLNYDIM.
  • the isolated peptide having amino acid sequence as denoted by SEQ ID NO: 2 is designated "peptide 2".
  • peptide 2 may comprise or consist of amino acid sequence as denoted by SEQ ID NO: 2.
  • the isolated peptide sequence may have an amino acid sequence as denoted by SEQ ID NO: 3: DPQILMEQGEFLNYDIMLGV.
  • the isolated peptide having amino acid sequence as denoted by SEQ ID NO: 3 is designated "peptide 3".
  • peptide 3 may comprise or consist of amino acid sequence as denoted by SEQ ID NO: 3.
  • composition comprising an isolated peptide capable of affecting interactions between neuroligin 4 (NLGn4X) and Neurexin ip (Nrxip), and wherein the isolated peptide comprises or consist of an amino acid sequence as denoted by SEQ ID NO: 6.
  • the isolated peptide comprises or consist an amino acid sequence as denoted by any one of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. Each possibility is a separate embodiment.
  • the isolated peptide comprises or consist of an amino acid sequence as denoted by any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and/or SEQ ID NO: 6. Each possibility is a separate embodiment.
  • the isolated peptide comprises an amino acid sequence as denoted by SEQ ID NO: 1. In some embodiments, the isolated peptide comprises an amino acid sequence as denoted by SEQ ID NO: 6.
  • the present invention provides methods and compositions for treating, attenuating, and/or preventing progression of IBD.
  • the present invention provides methods and compositions for treating, attenuating, and/or preventing progression of autoimmune disease.
  • the inhibitory peptide can modulate/disrupt/block the NLGn4X/p-neurexin axis.
  • the isolated peptide can ameliorate or reduce the severity of one or more pathological characteristics associated with IBD.
  • the isolated peptide can ameliorate or reduce the severity of one or more pathological characteristics associated with autoimmune disease.
  • pathological characteristics associated with IBD may be evident through examination of serum/blood, and/or colon/inte stine samples.
  • pathological characteristics associated with IBD may be evident through examination of mRNA and/or protein levels biomarkers.
  • the pathological characteristics associated with IBD includes, but are not limited to: weight loss, morphological variations related to colon size, persistent inflammation, fibrotic tissue/stricture, gut barrier permeability, and changes in lipid homeostasis, or any combination thereof.
  • the isolated peptide does not affect diet/food uptake and water intake.
  • a isolated peptide has no adverse effects and does not negatively affect the well-being of a treated subject, estimated by food intake and/or water intake.
  • treatment of IBD patients using the isolated peptide or composition including the same includes prevention of or protection from morphological variations related to colon size.
  • morphological variations related to colon size includes prevention of or protection from morphological variations related to colon size.
  • morphological variations related to colon size include, one or more of colon weight, colon length and/or ratio thereof, or any combination thereof. Each possibility is a separate embodiment.
  • prevention of or protection from morphological variations related to colon size includes prevention of an increase in colon weight and/or prevention of reduction in colon length. Each possibility is a separate embodiment.
  • prevention of or protection from morphological variations related to colon size includes prevention of an increase in the ratio of colon weight/colon length. Each possibility is a separate embodiment.
  • treatment of IBD patients using the isolated peptide or composition including the same includes prevention of or protection from increased paracellular permeability and/or susceptibility of the intestine thereby reducing leakiness through the gut barrier.
  • Each possibility is a separate embodiment.
  • prevention of or protection from paracellular permeability and/or susceptibility of the intestine includes prevention of increased expression of one or more of: Occludin, ZO-1, Claudin and/or Mucin, or any combination thereof. Each possibility is a separate embodiment.
  • treatment of IBD patients using the isolated peptide or composition including the same includes prevention of or protection from inflammation.
  • Each possibility is a separate embodiment.
  • prevention of or protection from inflammation includes prevention of increased expression of one or more of: IL-ip, TNF-a, IL-6 and/or IL- 4, or any combination thereof. Each possibility is a separate embodiment.
  • treatment of IBD patients using the isolated peptide or composition including the same includes prevention of or protection from fibrosis/fibrostenotic strictures.
  • fibrosis/fibrostenotic strictures include prevention of or protection from fibrosis/fibrostenotic strictures.
  • prevention of or protection from fibrosis/ fibrostenotic strictures includes prevention of increased expression of one or more of: aSMA, collagen I, and collagen III expression, or any combination thereof. Each possibility is a separate embodiment.
  • prevention of or protection from fibrosis/ fibrostenotic strictures includes prevention of upregulation of one or more of: TGF-P expression.
  • TGF-P expression is a separate embodiment.
  • the isolated peptide can prevent or reduce P- neurexin expression and/or activity in fibrotic colon/intestine.
  • the isolated peptide of the invention can inhibit fibrostenotic strictures in vivo.
  • the NLGn4-Nrxip inhibitory peptides can advantageously promote anti-fibrotic effects, by modulating NLGN4X/p-neurexin axis.
  • a method of preventing or protecting from fibrostenotic strictures in the colon/intestine including administering to a subject in need thereof the isolated peptide of the invention, or compositions including the same; in some embodiments the subject in need thereof suffers from IBD.
  • a method of inhibiting or ameliorating fibrosis in a subject in the need thereof includes administrating to a subject in need thereof an inhibitory peptide of the invention, or a composition including the same; in some embodiments the subject in need thereof suffers from IBD.
  • treatment of IBD patients using the isolated peptide or composition including the same includes prevention of or protection from changes in lipid homeostasis.
  • each possibility is a separate embodiment.
  • prevention of or protection from changes in lipid homeostasis includes prevention of increased expression and/or variability in expression of one or more of: Cholesterol (Choi), Triglycerides (TRG), SREBP2, HMGCR, and HMGCS, or any combination thereof. Each possibility is a separate embodiment.
  • treatment of IBD patients using the isolated peptide or composition including the same includes prevention of or protection from increase in P-Nrxn expression.
  • the present invention provides methods and compositions for treating, attenuating, and/or preventing progression of acute lung injury (ALI).
  • ALI acute lung injury
  • the inhibitory peptide can modulate/disrupt/block the NLGn4X/p-neurexin axis.
  • the isolated peptide can ameliorate or reduce the severity of one or more pathological characteristics associated with ALI.
  • pathological characteristics associated with ALI may be evident through examination of serum/blood, and/or lung samples.
  • pathological characteristics associated with ALI may be evident through examination of mRNA and/or protein levels biomarkers.
  • the pathological characteristics associated with ALI includes, but are not limited to: increased lung weight, acute/chronic inflammation in the lung, fibrosis, increment in the number of trNK cells in the lungs, increment in the levels of soluble RAGE, increment in the levels of surfactant protein D (SP-D), or any combination thereof.
  • SP-D surfactant protein D
  • treatment of ALI patients using the isolated peptide or composition including the same includes prevention of or protection from increased lung weight.
  • Each possibility is a separate embodiment.
  • the isolated peptide does not affect diet/food uptake and water intake.
  • a isolated peptide has no adverse effects and does not negatively affect the well-being of a treated subject, estimated by food intake and/or water intake.
  • treatment of ALI patients using the isolated peptide or composition including the same includes prevention of or protection from inflammation.
  • Each possibility is a separate embodiment.
  • prevention of or protection from inflammation includes prevention of increased expression of one or more of: IFN-y, IL-6 and IL-4, or any combination thereof. Each possibility is a separate embodiment.
  • treatment of ALI patients using the isolated peptide or composition including the same includes prevention of or protection from fibrosis/fibrostenotic strictures.
  • fibrosis/fibrostenotic strictures include prevention of or protection from fibrosis/fibrostenotic strictures.
  • prevention of or protection from fibrosis/fibrostenotic strictures includes prevention of increased expression of: aSMA and/or GFAP. Each possibility is a separate embodiment.
  • the isolated peptide can prevent or reduce - neurexin expression and/or activity in fibrotic lung.
  • the isolated peptide of the invention can inhibit fibrostenotic strictures in vivo.
  • treatment comprises prevention or reduction of number of trNK cells in the lungs.
  • the NLGn4-Nrxip inhibitory peptides can advantageously promote anti-fibrotic effects, by modulating NLGN4X/p-neurexin axis.
  • a method of preventing or protecting from fibrosis/fibrostenotic strictures in the lung including administering to a subject in need thereof the isolated peptide of the invention, or compositions including the same; in some embodiments the subject in need thereof suffers from IBD.
  • a method of inhibiting or ameliorating fibrosis in a subject in the need thereof includes administrating to a subject in need thereof an inhibitory peptide of the invention, or a composition including the same; in some embodiments the subject in need thereof suffers from ALL
  • treatment of ALI patients using the isolated peptide or composition including the same includes prevention of or protection from increase in P-Nrxn expression.
  • composition comprising an isolated peptide including an amino acid sequence as denoted by any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and/or SEQ ID NO: 6, or any combination thereof, for use in treating, attenuating, and/or preventing progression of one or more conditions that involve an abnormal inflammatory response in a subject in need thereof.
  • SEQ ID NO: 1 SEQ ID NO: 2, SEQ ID NO: 3 and/or SEQ ID NO: 6, or any combination thereof.
  • the isolated peptide comprises an amino acid sequence as denoted by SEQ ID NO: 1.
  • the isolated modulator peptide comprises an amino acid sequence as denoted by SEQ ID NO: 6.
  • the abnormal inflammatory response is acute and/or chronic.
  • the condition that involves an abnormal inflammatory response is localized and/or systemic.
  • the inflammatory response is localized and/or systemic.
  • administration of the composition is localized and/or systemic.
  • the one or more conditions that involve an abnormal inflammatory response are selected from one or more of an autoimmune disease, Acute lung injury (ALI), and cancer, or any combination thereof. Each possibility is a separate embodiment.
  • the conditions at involve an abnormal inflammatory response include an autoimmune disease.
  • the autoimmune disease is selected from one or more of: inflammatory bowel disorder (IBD), Thyroid diseases including Graves' disease and Hashimoto’s thyroiditis, Adrenal diseases including Addisson disease, Thymus disease including Myasthenia gravis, Multiple sclerosis (MS), Rheumatoid arthritis, Psoriasis and Psoriatic arthritis, Type 1 diabetes, Coeliac disease, Systemic lupus erythematosus, Scleroderma, Polymyositis, Dermatomyositis, and Pernicious anemia, or any combination thereof. Each possibility is a separate embodiment.
  • IBD inflammatory bowel disorder
  • Thyroid diseases including Graves' disease and Hashimoto’s thyroiditis
  • Adrenal diseases including Addisson disease
  • Thymus disease including Myasthenia gravis
  • MS Multiple sclerosis
  • Rheumatoid arthritis Psoriasis and Psoriatic arthritis
  • Type 1 diabetes Coeliac disease
  • the autoimmune disease includes inflammatory bowel disorder (IBD).
  • IBD inflammatory bowel disorder
  • the IBD is selected from any one or more of: ulcerative colitis (UC), Crohn's disease (CD) Collagenous colitis, Lymphocytic colitis, Ischemic colitis, Diversion colitis, Behcet's disease, and Indeterminate colitis, or any combination thereof.
  • UC ulcerative colitis
  • CD Crohn's disease
  • Collagenous colitis Lymphocytic colitis
  • Ischemic colitis Diversion colitis
  • Behcet's disease Diversion colitis
  • Indeterminate colitis or any combination thereof.
  • the conditions that involve an abnormal inflammatory response include acute lung injury (ALI).
  • the Acute lung injury (ALI) includes acute respiratory distress syndrome (ARDS).
  • the conditions that involve an abnormal inflammatory response include cancer.
  • the cancer includes lung cancer.
  • the cancer is not liver cancer, colorectal cancer, breast cancer, and prostate cancer, or any combination thereof. Each possibility is a separate embodiment.
  • composition comprising an isolated peptide including an amino acid sequence as denoted by any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and/or SEQ ID NO: 6, or any combination thereof, for use in treating, attenuating, and/or preventing progression of an autoimmune disease in a subject in need thereof.
  • SEQ ID NO: 1 SEQ ID NO: 2, SEQ ID NO: 3 and/or SEQ ID NO: 6, or any combination thereof.
  • the autoimmune disease includes abnormal inflammatory response.
  • the abnormal inflammatory response is acute and/or chronic.
  • the autoimmune disease is associated with cancer.
  • the cancer includes lung cancer.
  • the association of autoimmunity with cancer comprises a higher risk of developing the cancer.
  • the autoimmune disease is associated with Acute lung injury (ALI). In some embodiments, the autoimmune disease is associated with acute respiratory distress syndrome (ARDS).
  • ALI Acute lung injury
  • ARDS acute respiratory distress syndrome
  • the association of autoimmunity with ALI or ARDS comprises a higher risk of developing ALI or ARDS.
  • the Acute lung injury includes acute respiratory distress syndrome (ARDS).
  • ARDS acute respiratory distress syndrome
  • composition comprising an isolated peptide including an amino acid sequence as denoted by any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and/or SEQ ID NO: 6, or any combination thereof, for use in treating, attenuating, and/or preventing progression of Acute lung injury (ALI) in a subject in need thereof.
  • ALI Acute lung injury
  • the Acute lung injury includes abnormal inflammatory response.
  • the abnormal inflammatory response is acute and/or chronic.
  • the Acute lung injury includes acute respiratory distress syndrome (ARDS).
  • ARDS acute respiratory distress syndrome
  • a composition comprising an isolated peptide including an amino acid sequence as denoted by any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and/or SEQ ID NO: 6, or any combination thereof, for use in treating, attenuating, and/or preventing progression of cancer in a subject in need thereof.
  • SEQ ID NO: 1 amino acid sequence as denoted by any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and/or SEQ ID NO: 6, or any combination thereof.
  • the cancer includes abnormal inflammatory response.
  • the isolated peptide of the invention inhibits/attenuates proliferation and/or oncogenicity of cancer cells.
  • the cancer includes lung cancer.
  • a method of promoting anti- cancerous effects in the lung including administering to a subject in need thereof a composition comprising isolated peptide of the invention, or compositions including the same.
  • composition comprising an isolated peptide including an amino acid sequence as denoted by any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and/or SEQ ID NO: 6, or any combination thereof, for use in treating, attenuating, and/or preventing progression of one or more immune associated disease/condition in a subject in need thereof.
  • SEQ ID NO: 1 SEQ ID NO: 2, SEQ ID NO: 3 and/or SEQ ID NO: 6, or any combination thereof.
  • the inflammatory response is acute and/or chronic.
  • the one or more immune associated disease/conditions are selected from one or more of an autoimmune disease, Acute lung injury (ALI), and cancer, or any combination thereof. Each possibility is a separate embodiment.
  • the immune associated disease/condition includes an abnormal inflammatory response.
  • the immune associated disease/condition that involves an abnormal inflammatory response include an autoimmune disease.
  • the autoimmune disease is selected from one or more of: inflammatory bowel disorder (IBD), Thyroid diseases including Graves' disease and Hashimoto’s thyroiditis, Adrenal diseases including Addisson disease, Thymus disease including Myasthenia gravis, Multiple sclerosis (MS), Rheumatoid arthritis, Psoriasis and Psoriatic arthritis, Type 1 diabetes, Coeliac disease, Systemic lupus erythematosus, Scleroderma, Polymyositis, Dermatomyositis, and Pernicious anemia, or any combination thereof. Each possibility is a separate embodiment.
  • the autoimmune disease includes inflammatory bowel disorder (IBD).
  • IBD inflammatory bowel disorder
  • the IBD is selected from any one or more of: ulcerative colitis (UC), Crohn's disease (CD) Collagenous colitis, Lymphocytic colitis, Ischemic colitis, Diversion colitis, Behcet's disease, and Indeterminate colitis, or any combination thereof.
  • UC ulcerative colitis
  • CD Crohn's disease
  • Collagenous colitis Lymphocytic colitis
  • Ischemic colitis Diversion colitis
  • Behcet's disease Diversion colitis
  • Indeterminate colitis or any combination thereof.
  • the immune associated disease/condition that involves an abnormal inflammatory response includes acute lung injury (ALI).
  • the Acute lung injury (ALI) includes acute respiratory distress syndrome (ARDS).
  • the immune associated disease/condition that involves an abnormal inflammatory response includes cancer.
  • the cancer includes lung cancer.
  • the cancer is not liver cancer, colorectal cancer, breast cancer, and prostate cancer, or any combination thereof. Each possibility is a separate embodiment.
  • the abnormal inflammatory response related to IBD is associated with one or more of (i) morphological changes related to colon size; (ii) acute/chronic inflammation in the bowel; (iii) fibrosis/fibrostenotic strictures in the bowel; and their association with (iv) increased paracellular permeability; and (v) changes in lipid homeostasis, or any combination thereof.
  • morphological changes related to colon size e.g., chronic fibronic inflammation in the bowel, or chronic fibrosis, or chronic fibrosis, or any combination thereof.
  • the abnormal inflammatory response related to ALI is associated with one or more of (i) acute/chronic inflammation in the lung; (ii) fibrosis of the lung; (iii) increment in the number of trNK cells in the lungs; (iv) increment in the levels of soluble RAGE; (v) increment in the levels of surfactant protein D (SP-D), or any combination thereof.
  • SP-D surfactant protein D
  • the condition involving an abnormal inflammatory response in the subject involves one or more of: inflammation in the subject, fibrosis and/or fibrostenotic strictures in the subject, increased levels of trNK cells in the subject, morphological variations related to colon size or lung weight in the subject, increased paracellular permeability and/or susceptibility of an intestine or lung in the subject, and changes lipid homeostasis in the subject, or any combination thereof.
  • inflammation in the subject fibrosis and/or fibrostenotic strictures in the subject
  • increased levels of trNK cells in the subject morphological variations related to colon size or lung weight in the subject
  • increased paracellular permeability and/or susceptibility of an intestine or lung in the subject increases lipid homeostasis in the subject, or any combination thereof.
  • the fibrosis and/or fibrostenotic strictures are not liver fibrosis and/or liver fibrostenotic strictures. Each possibility is a separate embodiment.
  • a nucleic acid encoding for the isolated peptides of the invention there is provided a DNA construct/vector (such as, an expression vector) harboring or comprising a nucleic acid encoding for the peptides (optionally in addition to one or more regulatory sequences, non-coding sequences, and the like).
  • a DNA construct/vector such as, an expression vector
  • suitable vectors are known to those skilled in art, and the choice of which depends on the function desired. Such vectors include, for example, plasmids, cosmids, viruses, bacteriophages and other vectors.
  • the polynucleotides and/or vectors harboring the same can be reconstituted into vehicles, such as, for example, liposomes for delivery to target cells.
  • vehicles such as, for example, liposomes for delivery to target cells.
  • Any cloning vector and/or expression vector known in the art may be used, depending on the purpose, the host cell, and the like. Such vectors may be used for in- vitro and/or in-vivo introduction/expression.
  • the encoding nucleic acid molecules and/or the vectors disclosed herein may be designed for direct introduction or for introduction via carrier, such as, liposomes, viral vectors (adenoviral, retroviral) into target cells/tissue.
  • carrier such as, liposomes, viral vectors (adenoviral, retroviral) into target cells/tissue.
  • kits comprising the isolated peptides or composition comprising the same, as disclosed herein.
  • such kits/compositions can be used, for example, in the treatment of IBD or ALL Each possibility is a separate embodiment.
  • the kit may further include one or more reagents, buffers and/or instructions for using the same.
  • the composition may further include a pharmaceutically acceptable carrier.
  • mice used in the Examples described hereinbelow were housed in a barrier facility and received care according to the Hebrew University ethical regulations and NIH guidelines.
  • the institutional animal care ethics committee of the Hebrew University approved all animal protocols under the ethics number of MD-18- 15494-3.
  • IBD Inflammatory Bowel Disease
  • Groupl wild-type mice drinking regular water without DSS with scrambled peptide (8pM), i.p. injection daily for five days.
  • mice drinking regular water without DSS with NLGn4-Nrxip inhibitor peptide 1 (8pM), i.p. injection daily for five days.
  • Group3 wild-type mice drinking regular water with DSS 3% with scrambled peptide (8pM), i.p. injection daily for five days.
  • Group4 wild-type mice drinking regular water with DSS 3% with NLGn4-Nrxip inhibitor peptide 1 (8pM), i.p. injection daily for five days.
  • mice were weighed, and water intake and diet uptake were measured daily. Prior to sacrifice, animals were anesthetized by 5% isoflurane inhalation for 10 seconds before cervical dislocation. Colon protein extract were assessed for pro-inflammatory cytokine profile assessments by ELISA, and mRNA was obtained for RT-PCR as described below.
  • RNA isolation, cDNA preparation, and real-time PCR - Total cellular of 2mg/ml RNA (purity 98%) determined using a NanoDrop ND- 1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) was isolated from all mice groups (n 5) using 2 ml of TRI reagent (Bio Lab; Cat# 90102331). The samples were centrifuged (1,400 rpm) for 15 minutes at 4°C to collected RNA-supematant. For RNA precipitation, the supernatant of each sample was transferred to a new microcentrifuge tube, and 0.5 ml of isopropanol (Bio Lab; Cat# 16260521) was added and incubated at 25°C for 10 minutes.
  • cDNA was prepared with a High-Capacity cDNA Isolation Kit (R&D; Cat# 1406197).
  • Real-time PCR was performed with TaqMan Fast Advanced Master Mix (Cat# 4371130, Applied Biosystems) to quantify b-Nrxn, aSMA, Col I, Col III, SREBP2, HMGCR, HMGCS gene expression with normalization to the expression of the housekeeping gene GAPDH.
  • Cycling condition for Thermo Fisher One Step RT-PCR kit involved RT step for 30 min at 50 °C and denaturation for 15 min at 95 °C. Further the reaction mixture was incubated for 40 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min followed by 72 °C for 10 min. Data analysis were analyzed using QuantStudio TM 5 Real-Time PCR System (Cat# A34322, Applied Biosystem).
  • a multiplexed sandwich enzyme-linked immunosorbent assay -based technology (Cat# MHSTCMAG-70K; R&D Systems) was used to simultaneously determine the concentration of multiple cytokines of IL- lb, IL-6, IL-4, TNF-a, TGF-b.
  • cholesterol (Abeam; ab285242), triglyceride (Abeam; ab65336), were performed using ELISA kits according to the manufacture protocols. Briefly, all reagents and samples were brought to room temperature (18 - 25 °C) before use. A volume of 100 pL of each standard and protein sample were added into appropriate wells and incubate for 2.5 hours at room temperature with gentle shaking.
  • IBD Inflammatory Bowel Disease
  • DSS Dextran sulfate sodium salt
  • the experiments were carried out by comparing between four groups of mice, according to the experimental procedure of IBD-animal model described in detail in the material and methods section and summarized herein, as follows: group 1 - wt mice administered with 8pM of scrambled peptide (SEQ ID NO: 7); group 2 - wt mice administered with 8pM of peptide 1 (SEQ ID NO: 1); group 3 - IBD-model mice (3% DSS) administered with 8pM of scrambled peptide (SEQ ID NO: 7); and group 4 - IBD-model mice (3% DSS) administered with 8pM of peptide 1 (SEQ ID NO: 1).
  • mice Initially, the general well-being of the mice participating in these experiments was assessed. Assessment was performed by daily measurements of food and water consumed by the mice, as well as their body weight (FIGs. 1A-1C).
  • mice's food and water consumption were meticulously recorded throughout the study, with daily measurements taken to capture precise intake levels. At the culmination of the study, mice were sacrificed, and their body weights were measured.
  • FIG. 1A Analysis of the data revealed no significant disparities in either food (FIG. 1A) consumption or water intake (FIG. IB) among experimental groups 1-4, indicating consistent consumption patterns emphasizing the meticulous nature of the research design and indicate that factors beyond food and water availability might be influential in any observed effects.
  • One such observed effect is related to a reduction of about 2g in the measured body weight exhibited by mice induced with IBD using 3% DSS and receiving treatment with 8pM scrambled peptide relative to wt mice (FIG. 1C; compare group 3 with groups 1-2).
  • mice induced with an IBD model with 8pM peptide 1 improved this effect, by preventing about 50% (about 1g) of the loss of weight (compare group 3 with group 4).
  • Example 2 NLGn4-NrxlB inhibitory peptide protected IBD model mice from morphological variations related to colon size
  • mice belonging to same experimental groups 1-4 presented in the previous example were assessed in mice belonging to same experimental groups 1-4 presented in the previous example.
  • Changes in colon length may provide additional insights into other potential morphological variations, and its proportion with respect to the weight of the colon constitutes a predictive measure/index indicative of colon health.
  • Example 3 NLGn4-NrxlB inhibitory peptide protected IBP model mice from changes in gene expression related to paracellular permeability and colon susceptibility
  • mice induced with 3% DSS - such as changes in paracellular permeability
  • NLGn4-Nrxip inhibitory peptide 1 - changes in gene expression of specific genes related to leakiness in the gut barrier, namely, Occludin, ZO-1, and Claudin was evaluated by quantifying mRNA levels of these genes in mice belonging to all experimental groups 1-4, as in previous examples (FIGs. 3A-3C).
  • Mucin mRNA levels which relates to colon susceptibility was also evaluated using RT-PCR (FIG. 3D).
  • Results of the quantification of the expression of Occludin, ZO-1, Claudin, and Mucin show the levels of the mRNAs are increased by 2-fold to 4-fold in the IBD- induced mice model using 3% DSS and treated with 8pM scrambled peptide relative to wt mice (FIG. 3A, FIG.3B FIG.3C and FIG.3D; compare group 3 with groups 1-2).
  • treatment of IBD-induced mice with peptide 1 improved this effect, by preventing the potential increase in mRNA expression, keeping it at the range of about 1-fold to about 3 -fold for Occludin, ZO-1 and Mucin (FIG. 3A, FIG. 3B, and FIG. 3D, respectively; compare groups 4 with groups 3, while completely diminishing the potential increase in Claudin keeping it close to wt levels (FIG. 3C, respectively; compare group 4 with groups 1-2)
  • NLGN4X peptide administration to IBD- induced mice led to substantial enhancements in intestinal and colonic permeability through inhibitions in down expression of Occludin, Zo-1 and claudin protein levels indicating reduced leakiness in the gut barrier, which is often compromised in IBD.
  • ELISA sandwich enzyme-linked immunosorbent assay
  • mice exposed to 3% DSS and treated with 8pM scrambled peptide exhibit a strong and highly significant increment in the levels of all four cytokines tested, compared to wt mice (compare group 3 with groups 1-2).
  • ECM extracellular matrix
  • NLGN4X peptide may prevent fibrostenotic strictures associated with IBD.
  • Example 6 NLGn4-NrxlB inhibitory peptide protected IBD-induced mice by preserving lipid homeostasis
  • lipid homeostasis was performed in mice belonging to all experimental groups 1-4, as in any of the previous examples, by measuring levels of cholesterol (Choi), triglyceride (TGR), and related genes SREBP2, HMGCR2 and HMGCS III.
  • the analysis of the data is indicative of increased levels and/or variability in each of the above-mentioned molecules and protein when measured in colon extract of mice induced for IBD and treated with 8pM scrambled peptide relative to wt mice (FIGs. 6A-6C; compare group 3 with groups 1-2).
  • mice Similarly, treatment of IBD-induced mice with peptide 1 protected mice from potential increment in levels and/or variability of expression of SREBP2, HMGCR2 or HMGCS III (FIG. 6C; compare group 4 with group 3) also keeping the level of expression of these genes at about the same levels and/or variability of wt.
  • peptide 1 protected IBD- induced mice from changes in lipid homeostasis, preserving the level of homoeostasis comparable to that of wt.
  • NLGN4X peptide show a marked reduction in lipidemia reproduced by cholesterol and triglycerides levels in intestinal and colon tissues, and is indicative of its beneficial impact on lipid metabolism in the context of IBD.
  • Example 7 NLGn4-NrxlB inhibitor peptide modulates B-neurexin expression in-vivo
  • P-Nrxn expression was evaluated in mice belonging to all experimental groups 1-4, as in any of the previous examples, by qPCR (FIG. 7).
  • mice induced with 3% DSS and treated with 8pM scramble peptide were induced with 3% DSS and treated with 8pM scramble peptide, relative to wt mice (compare group 3 with groups 1-2).
  • NLGn4-Nrxip inhibitor peptide 1 prevented any increase in the levels of P-Nrxn mRNA expression, keeping it at about same or even below wt levels (compare group 4 with groups 1-2). This is in accordance with its demonstrated ability to prevent any of the established effects of the IBD model, exemplified throughout any one of previous examples 1-6, including for example, preventing potential establishment of fibrostenotic strictures.
  • NLGN4X peptides holds a potential as a valuable therapeutic option for mitigating the pathological effects of IBD, in particular fibrostenotic strictures.
  • ALI Acute Lung Injury mice - Twelve-week-old male weight 25 ⁇ 1.5 g wild-type C57BL/6J obtained from Envigo (Harlan, Israel).
  • ALI administration/induction was performed by i.p injection of the mice with Oleic acid (OA; Merck, O1008-1G >99% purity, as determined by LC/MS analyses) in the concentration of 100 mg/kg twice a week for two weeks.
  • OA Oleic acid
  • NLGn4-Nrxip inhibitor peptide 1 was prepared fresh daily in phosphate buffered saline (PBS) at a concentration of 8pM at the volume of lOOpl day following OA administration.
  • PBS phosphate buffered saline
  • Acute lung injury and its most severe manifestation, the acute respiratory distress syndrome (ARDS) is a clinical syndrome defined by acute hypoxemic respiratory failure, bilateral pulmonary infiltrates consistent with edema, and normal cardiac filling pressures.
  • An ALI-induced animal model was generated by injection of oleic acid to the animal, reproducing known risk factors for ARDS.
  • Groupl wild-type mice without OA with scrambled peptide (8pM), i.p. injection twice a week for two weeks.
  • mice wild-type mice without OA with NLGn4-Nrxip inhibitor peptide 1 (8pM), i.p injection twice a week for two weeks.
  • mice and diet were weighed, and water intake was measured daily. Prior to sacrifice, animals were anesthetized by 5% isoflurane inhalation for 10 seconds before cervical dislocation. On the sacrificing day whole blood was withdrawn through cardiac puncture collected in heparinized tubes for studying immune cells. Lung BALF extract were assessed for pro-inflammatory cytokine profile assessments by the ELISA and white blood cells (WBCs) count, mRNA was obtained from lung for gene expressions assessment by RT-PCR as described below.
  • WBCs white blood cells
  • cDNA was prepared with a High-Capacity cDNA Isolation Kit (R&D; Cat# 1406197).
  • Real-time PCR was performed with TaqMan Fast Advanced Master Mix (Cat# 4371130, Applied Biosystems) to quantify P-Nrxn, aSMA, GFAP and SP-D gene expressions with normalization to the expression of the housekeeping gene GAPDH.
  • Cycling condition for Thermofisher one step RT-PCRkit involved RT step for 30 min at 50 °C and denaturation for 15 min at 95 °C. Further the reaction mixture was incubated for 40 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min followed by 72 °C for 10 min. Data analysis were analyzed using QuantStudioTM 5 Real-Time PCR System (Cat# A34322, Applied biosystem).
  • Cytokines assessment in BALF - A multiplexed sandwich enzyme-linked immunosorbent assay -based technology (Cat# MHSTCMAG-70K; R&D Systems) was used to simultaneously determine the concentration of multiple cytokines of IL-6, IL- 4, IFN-y and sRAGE concentrations. Briefly, all reagents and samples were brought to room temperature (18 - 25 °C) before use. A volume of 100 pL of each standard and BALF sample were added into appropriate wells and incubate for 2.5 hours at room temperature with gentle shaking. The solution was discarded, wells were wash 4 times with IX Wash Solution, of note; washing was done by filling each well with Wash Buffer (300 pL) using a multi-channel Pipette or auto washer.
  • a volume of 100 pL of lx prepared detection antibody were added to each well for 1 hour at room temperature with gentle shaking.
  • a volume of 100 pL of TMB One-Step Substrate Reagent (Item H) were added to each well for 30 minutes at room temperature in the dark with gentle shaking.
  • 50 pL of Stop Solution (Item I) were added to each well. Absorbance was read at 450 nm immediately using ELISA reader (Tecan Ml 00 Plate Reader).
  • trNK cells isolation from the lung - Isolation of lung trNK cells were prepared by digesting the lung tissue samples at 37°C for 40 minutes with 20 ml of GBSS solution containing 0.1% (wt/vol) pronase, 0.1% (wt/vol) collagenase, and 0.01% (wt/vol) deoxyribonuclease I (DNase I) (all enzymes from Roche Diagnostics GmbH, Mannheim, Germany).
  • DNase I deoxyribonuclease I
  • the cell suspension was fdtered through a 150-mm steel mesh and centrifuged on a FICOLL (Sigma, St. Louis, USA) at 1,400 x g for 20 minutes at 25 °C to produce a WBCs-enriched fraction in the middle opaque layer.
  • mice induced with Acute Lung Injury were carried out by comparing between four groups of mice, according to the experimental procedure of ALI-induced animal model described hereinabove and summarized herein below, as follows: group 1 - wt mice administered with 8pM of scrambled peptide (SEQ ID NO: 7); group 2 - wt mice administered with 8pM of peptide 1 (SEQ ID NO: 1); group 3 - ALI-induced mice model (Oleic acid-treated) administered with 8pM of scrambled peptide (SEQ ID NO: 7); and group 4 - ALI-induced mice model (Oleic acid-treated) administered with 8pM of peptide 1 (SEQ ID NO: 1).
  • mice were meticulously recorded throughout the study, with daily measurements taken to capture precise intake levels. Analysis of the data revealed no significant disparities in either food consumption, or water intake, among experimental groups 1-4, indicating consistent consumption patterns emphasize the meticulous nature of the research design and indicate that factors beyond food and water availability might be influential in any observed effects (Data not shown).
  • mice were sacrificed, and their weights were measured in grams. No significant disparities were observed in mice body weight among experimental groups 1-4 (Data not shown).
  • NLGn4-NrxlB inhibitory peptide protected ALI-induced mice from increased lung weight - Next, variations related to the weight of the lung were assessed in mice belonging to same experimental groups 1-4.
  • lungs from each subject were carefully harvested, the weight of the lungs was measured, and the ratio between lungs weight and mice weight (lungs weight: mice weight ratio) was also calculated to ascertain any proportional changes.
  • NLGn4-Nrxip inhibitory peptide 1 protected the lungs of ALI mice from increased weight (FIG. 8A; compare group 4 with groups 3 relative to groups 1-2), and from increment in the calculated ration of lungs weight : mice weight (FIG. 8B; compare group 4 with groups 3 relative to groups 1-2).
  • NLGn4-NrxlB inhibitory peptide protected ALI-induced mice from increased B- Nrxn expression in the lungs - Quantification of B-Nrxn mRNA expression was performed in lung tissues obtained from mice belonging to experimental groups 1-4. The analysis of the data suggests that B-Nrxn is important for mediating lung injury in ALI mice of the current model (FIG. 9; compare group 4 with groups 3 relative to groups 1-2).
  • NLGn4-NrxlB inhibitory peptide protected ALI-induced mice from fibrosis in the lungs - Lung fibrosis was assessed by measuring mRNA expression levels of aSMA and GFAP in mice belonging to all experimental groups 1-4. The analysis of the data suggests that NLGn4-NrxlB inhibitory peptide 1 protected the lungs of ALI mice from increased expression of fibrosis markers aSMA and GFAP (FIG. 10; compare group 4 with groups 3 relative to groups 1-2).
  • NLGn4-NrxlB inhibitory peptide protected ALI-induced mice from increment in inflammation in the lungs - lung inflammation was assessed by measuring the levels of pro-inflammatory cytokines, including IFN-y, IL-6 and IL-4, in fluid recovered by bronchoalveolar lavage (BAL fluid or BALF) from mice belonging to all experimental groups 1-4.
  • BAL fluid or BALF bronchoalveolar lavage
  • NLGn4-NrxlB inhibitory peptide protected ALI-induced mice from increment in the number of trNK cells in the lungs - the number of trNK cells purified/isolated from the lungs was assessed in mice belonging to all experimental groups 1-4, and was calculated as (cells ⁇ mg lung tissue weight). The analysis of the data suggests that NLGn4-NrxlB inhibitory peptide 1 protected the lungs of ALI mice from increased amount of trNK cells (FIG. 12; compare group 4 with groups 3 relative to groups 1-2).
  • NLGn4-NrxlB inhibitory peptide protected ALI-induced mice model from increment in the levels of soluble RAGE associated with lung injury - the soluble form of the receptor for advanced glycation end products (sRAGE) was assessed by measuring its levels in fluid recovered by bronchoalveolar lavage (BAL fluid or BALF) from mice belonging to all experimental groups 1-4.
  • BAL fluid or BALF bronchoalveolar lavage
  • NLGn4-NrxlB inhibitory peptide protected ALI-induced mice from increment in the levels of surfactant protein D (SP-D) - the level of SP-D was assessed in mice belonging to all experimental groups 1 -4.
  • SP-D surfactant protein D
  • the analysis of the data suggests that NLGn4- Nrxi inhibitory peptide 1 protected the lungs of ALI mice from increment in levels of SP-D (FIG. 14; compare group 4 with groups 3 relative to groups 1-2).
  • NLGN4X peptides hold a potential as a valuable therapeutic option for mitigating the pathological effects of ALI, in particular those effects related to fibrosis and inflammation of lung tissue.

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Abstract

L'invention concerne des méthodes et des compositions comprenant des peptides isolés pouvant interférer avec l'interaction protéine-protéine (PPI) de la neuroligine-4 (NLGn4)-neurexine 1β (Nrx1β) et leurs utilisations pour traiter une affection qui implique une réponse inflammatoire anormale.
PCT/IL2024/050910 2023-09-13 2024-09-11 Modulateurs peptidiques de l'axe 4-neurexine 1-bêta de la neuroligine pour le traitement d'affections impliquant une réponse inflammatoire anormale Pending WO2025057155A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016157195A1 (fr) * 2015-04-01 2016-10-06 Hadasit Medical Research Services And Development Ltd. Inhibiteurs de l'interaction protéine-protéine neuroligine 4-neurexine 1-bêta pour le traitement de troubles hépatiques
WO2023170678A1 (fr) * 2022-03-07 2023-09-14 Hadasit Medical Research Services And Development Ltd. Modulateurs peptidiques de l'axe 1-bêta de la neuroligine 4-neurexine pour le traitement de troubles hépatiques

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016157195A1 (fr) * 2015-04-01 2016-10-06 Hadasit Medical Research Services And Development Ltd. Inhibiteurs de l'interaction protéine-protéine neuroligine 4-neurexine 1-bêta pour le traitement de troubles hépatiques
WO2023170678A1 (fr) * 2022-03-07 2023-09-14 Hadasit Medical Research Services And Development Ltd. Modulateurs peptidiques de l'axe 1-bêta de la neuroligine 4-neurexine pour le traitement de troubles hépatiques

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