WO2016172219A1 - Procédés et compositions visant à inhiber l'inflammation à médiation par les récepteurs de type toll (tlr) - Google Patents

Procédés et compositions visant à inhiber l'inflammation à médiation par les récepteurs de type toll (tlr) Download PDF

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Publication number
WO2016172219A1
WO2016172219A1 PCT/US2016/028451 US2016028451W WO2016172219A1 WO 2016172219 A1 WO2016172219 A1 WO 2016172219A1 US 2016028451 W US2016028451 W US 2016028451W WO 2016172219 A1 WO2016172219 A1 WO 2016172219A1
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tlr
peptide
mucl
composition
cell
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PCT/US2016/028451
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English (en)
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Kwang Chul Kim
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The Arizona Board Of Regents On Behalf Of The University Of Arizona
Temple University - Of The Commonwealth System Of Higher Education
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Publication of WO2016172219A1 publication Critical patent/WO2016172219A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4727—Mucins, e.g. human intestinal mucin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention is directed to a compound that binds to a Toll-like Receptor (TLR) and prevents TLR-mediated inflammation.
  • TLR Toll-like Receptor
  • the present invention is further directed to methods for blocking an interaction between TLR and MyD88 and/or TRIF, methods for inhibiting TLR-mediated inflammation in a mammalian cell, methods for preventing and/or treating a mammal suffering or at risk for suffering from TLR-mediated inflammation, and methods for preventing and/or treating a mammal suffering or at risk for suffering from a disorder related to TLR-mediated inflammation (e.g., chronic inflammatory disorders).
  • a disorder related to TLR-mediated inflammation e.g., chronic inflammatory disorders.
  • TLRs Toll-like receptors
  • PAMPs pathogen-associated molecular patterns
  • TLRs participate in the first line of defense against invading pathogens and play a significant role in inflammation, immune cell regulation, survival, and proliferation.
  • TLRl 11 members of the TLR family have been identified, of which TLRl, TLR2, TLR4, TLR5, TLR6, and TLRl 1 are located on the cell surface and TLR3, TLR7, TLR8, and TLR9 are localized to the endosomal/lysosomal compartment.
  • TLRl, TLR2, TLR4, TLR5, TLR6, and TLRl 1 are located on the cell surface and TLR3, TLR7, TLR8, and TLR9 are localized to the endosomal/lysosomal compartment.
  • TLR cytoplasmic Toll/IL-1 receptor
  • IL-1 receptor-associated kinase-4 IL-1 receptor-associated kinase-4
  • MyD88 recruits IL-1 receptor-associated kinase-4 (IRAK-4) to TLRs through interaction of the death domains of both molecules.
  • IRAK-1 is activated by phosphorylation and associates with TRAF6, thereby activating the IKK complex and leading to activation of MAP kinases (JNK, p38, ERK) and NF- ⁇ .
  • Tollip and IRAK-M interact with IRAK-1 and negatively regulate the TLR-mediated signaling pathways. Additional modes of regulation for these pathways include TRIF-dependent induction of TRAF6 signaling by RIP1 and negative regulation of TIRAP-mediated downstream signaling by ST2L, TRIAD3A, and SOCS1.
  • TRAF3 Activation of MyD88- independent pathways occurs via TRIF and TRAF3, leading to recruitment of ⁇ / ⁇ , phosphorylation of IRF3, and expression of interferon- ⁇ .
  • TIR domain containing adaptors such as TIRAP, TRIF, and TRAM regulate TLR-mediated signaling pathways by providing specificity for individual TLR signaling cascades.
  • TRAF3 plays a critical role in the regulation of both MyD88-dependent and TRIF-dependent signaling via TRAF3 degradation, which activates MyD88-dependent signaling and suppresses TRIF-dependent signaling (and vice versa).
  • Mucin 1(MUC1) is a membrane-tethered glycoprotein expressed in various mucosal epithelial cells as well as hematopoietic cells and plays an anti-inflammatory role during the resolution phase of airway bacterial infection. Recently, it was shown that the antiinflammatory effect of MUC1 is attributable to its cytoplasmic tail, specifically the presence of the EGFR phosphorylation site (YEKV (SEQ ID NO: 1)).
  • this MUCl- like synthetic compound may provide an alternative therapy for chronic inflammatory disorders such as COPD.
  • the invention is directed to a compound that binds to a Toll-like
  • TLR TLR Receptor
  • the present invention is further directed to methods for blocking an interaction between TLR and MyD88 and/or TRIF (see, e.g., Kato, et al, Am J Respir Cell Mol Biol 2014, Vol. 51(3):446-454; Ueno, et al, Am J Respir Cell Mol Biol 2008 Vol. 38:263-268), methods for inhibiting TLR-mediated inflammation in a mammalian cell, methods for preventing and/or treating a mammal suffering or at risk for suffering from TLR-mediated inflammation, and methods for preventing and/or treating a mammal suffering or at risk for suffering from a disorder related to TLR-mediated inflammation (e.g., chronic inflammatory disorders).
  • a disorder related to TLR-mediated inflammation e.g., chronic inflammatory disorders.
  • the present invention provides a composition comprising a compound that binds to a Toll-like Receptor (TLR) polypeptide and inhibits TLR-mediated inflammation, wherein the compound comprises a MUCl peptide or MUCl mimetic.
  • TLR Toll-like Receptor
  • the MUCl peptide or MUCl mimetic comprises at least a portion of the wild type amino acid sequence of MUCl. In some embodiments, the MUC-1 peptide or MUCl mimetic comprises at least a portion of the wild type MUCl amino acid sequence which permits the resulting compound to inhibit interaction between a TLR polypeptide (e.g., TLR5) and MyD88 and/or TRIF.
  • TLR polypeptide e.g., TLR5
  • the MUCl peptide or MUCl mimetic comprises at least a portion of the wild type MUCl amino acid sequence which permits the resulting compound to inhibit TLR-mediated inflammation (e.g., TLR- mediated inflammation resulting from interaction between a TLR polypeptide (e.g., TLR5) and MyD88 and/or TRIF).
  • TLR-mediated inflammation e.g., TLR- mediated inflammation resulting from interaction between a TLR polypeptide (e.g., TLR5) and MyD88 and/or TRIF.
  • the MUCl peptide or MUCl mimetic comprises at least a portion of the wild type MUCl amino acid sequence which permits the resulting compound to inhibit NF-kB activation resulting from interaction between a TLR polypeptide (e.g., TLR5) and MyD88).
  • the MUCl peptide or MUCl mimetic comprises at least a portion of the wild type MUCl amino acid sequence which permits the resulting compound to inhibit IL-8 release resulting from NF-kB activation (e.g., resulting from interaction between a TLR polypeptide (e.g., TLR5) and MyD88 and/or TRIF).
  • a MUCl peptide or MUCl mimetic comprises the following amino acid sequence: YEKV (SEQ ID NO: l).
  • such a MUCl peptide or MUCl mimetic comprises the following amino acid sequence:
  • TDRSPYEKVSA (SEQ ID NO:2).
  • the wild type MUCl nucleic acid sequence and amino acid sequence is provided at Figs. 9A (NM_013605.2) and 9B.
  • the MUCl peptide or MUCl mimetic further comprises a cell- penetrating peptide.
  • a cell- penetrating peptide is selected from the group consisting of HIV-derived TAT peptide, penetratins, transportans, SS peptides, and hCT derived cell-penetrating peptides.
  • the cell- penetrating peptide is a Trans-Activator of Transcription (TAT) sequence.
  • TAT sequence has the amino acid sequence RRRQRRKKRGY (SEQ ID NO: 3).
  • the MUCl peptide or MUCl mimetic further comprises at least one biocompatible carrier.
  • the biocompatible carrier is selected from the group consisting of poly-lactic acid, poly-gly colic acid, and copolymers of poly- lactic acid and poly-gly colic acid.
  • the biocompatible carrier comprises at least one biodegradable fatty acid or a metal salt thereof.
  • the biodegradable fatty acid is selected from the group consisting of palmitic acid, stearic acid, oleic acid, myristic acid, and metal salts thereof.
  • the biocompatible carrier comprises a salt selected from the group consisting of porous or non-porous calcium phosphates, porous or non-porous hydroxyapatites, porous or non-porous tricalcium phosphates, porous or non-porous tetracalcium phosphates, porous or non-porous calcium sulfates, and combinations thereof.
  • the MUCl peptide or MUCl mimetic is directly linked to the cell-penetrating peptide.
  • the cell-penetrating peptide is directly linked to the MUCl peptide or MUCl mimetic, and the MUCl peptide or MUCl mimetic is directly linked to the biocompatible carrier.
  • the cell-penetrating peptide is directly linked to the biocompatible carrier, and the cell-penetrating peptide is directly linked to the MUCl peptide or MUCl mimetic.
  • the compound is within a composition comprising a pharmaceutically acceptable excipient.
  • the compound inhibits TLR-mediated inflammation through inhibiting an interaction between MyD88 and/or TRIF and a TLR polypeptide.
  • the TLR polypeptide is selected from TLRl , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11 , TLRl 2, and TLRl 3.
  • the present invention provides methods for inhibiting interaction between MyD88 and/or TRIF and a TLR polypeptide in a mammalian cell, the method comprising contacting the TLR polypeptide with a compound as described herein, wherein the mammalian cell expresses MyD88 and/or TRIF and a TLR polypeptide.
  • the mammalian cell is experiencing or is at risk for experiencing TLR- mediated inflammation.
  • the mammalian cell is a mucosal epithelial cell.
  • the mammalian cell is in a living human.
  • the TLR polypeptide is selected from TLRl , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR1 1, TLR12, and TLRl 3.
  • the present invention provides methods of inhibiting TLR- inflammation in a mammalian cell, the method comprising treating the cell with a compound as described herein, wherein the mammalian cell is experiencing or is at risk for experiencing TLR-mediated inflammation.
  • the mammalian cell is a mucosal epithelial cell.
  • the mammalian cell is in a living human.
  • the compound inhibits TLR-mediated inflammation through inhibiting an interaction between MyD88 and/or TRIF and a TLR polypeptide.
  • the TLR polypeptide is selected from TLRl , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLRl 1, TLRl 2, and TLR13.
  • the present invention provides methods for preventing and/or treating a mammal experiencing or at risk for TLR-mediated inflammation, the method comprising administering the composition of claim 1 to the mammal.
  • the mammal is suffering from or is at risk for developing a disorder related to TLR-mediated inflammation.
  • the disorder is selected from pulmonary fibrosis,
  • the mammal is a human.
  • the compound inhibits TLR- mediated inflammation through inhibiting an interaction between MyD88 and/or TRIF and a TLR polypeptide.
  • the TLR polypeptide is selected from TLRl, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR1 1, TLR12, and TLRl 3.
  • the present invention provides an isolated polypeptide comprising the amino acid sequence TDRS P YEKVS ARRRQRRKKRGYK (SEQ ID NO:4).
  • the present invention provides a peptide or mimetic comprising the amino acid sequence NH2-TDRSPYEKVSARRRQRRKKRGYK-Palmitic Acid (SEQ ID NO: 4).
  • FIG. 1 A-B Structure of Peptide 1
  • A The sequence of 72 amino acids consisting of the MUC1 cytoplasmic tail (CT).
  • B The structure of Peptide 1 which consists of three components - 11 mer, TATp, and CI 6 palmitic acid, which are covalently linked through an extra lysine (K) moiety.
  • FIG. 3 Effect of Peptide 1 mutants on PAK-induced IL-8 release 293-hTLR5-HA cells were pretreated with 10 ⁇ of Peptide 1 or its derivatives for 1 h prior to stimulation with PAK (10 6 CFU/ml) for 2 h. While Peptide 1 has YEKV (SEQ ID NO: 1) sequence, Peptide 2 and 3 have FEKV (SEQ ID NO: 10) and YKEV (SEQ ID NO: 11) sequence, respectively (see Figure 1 A and Methods).
  • TATp-C16 refers to the carrier molecule used for delivering the peptides into the cell.
  • FIG 4A-B Effect of Peptide 1 on PAK-induced NF- ⁇ activation
  • A 293-hTLR5- HA cells were transiently transfected with both aNF- ⁇ dependent luciferase plasmid (reporter gene) and a Renilla luciferase plasmid (reference gene) as described in Methods. After transfection, cells were incubated for 1 h with Peptide 1 or PBS prior to stimulation with PAK (10 6 CFU/ml) for 2 h. Relative luciferase activities of cell lysates were measured by a luminometer using the Dual-luciferase reporter system.
  • B 293-hTLR5 NF- ⁇ /SEAPorter cells were treated with Peptide 1 and PAK for 4 h under the same condition as above. Aliquots of spent media were subjected to the SEAP assay as described in Methods.
  • FIG. 5 Effect of Peptide 1 on PAK-induced ⁇ - ⁇ degradation and p65
  • phosphorylation 293-hTLR5-HA cells were pretreated with Peptide 1 (10 ⁇ ) for 1 h prior to treatment with PAK (10 6 CFU/ml) for the indicated time periods. Equal protein amounts of cell ly sates were subjected to Western blotting with indicated antibodies. The results are representative of three independent experiments.
  • FIG. 6 Effect of Peptide 1 on PAK-induced nuclear translocation of p65 293-hTLR5- HA cells were pretreated with Peptide 1 (10 ⁇ ) for 1 h prior to treatment with PAK (10 6
  • FIG. 7 Effect of Peptide 1 mutants on PAK-induced NF- ⁇ activation All the experimental conditions were identical with those in Figure 4B except for using various
  • FIG. 8A-B Effect of Peptide 1 on PAK-induced TLR5/MyD88 interaction 293- hTLR5-HA cells were pretreated with Peptide 1 (10 ⁇ ) for 1 h followed by treatment with PAK (10 6 CFU/ml) for 30 min. Equal protein amounts of cell lysates were used for immunoprecipitation with anti-MyD88 Ab or isotype-matched normal rabbit IgG, and the immunoprecipitated proteins were subjected to Western blotting with the indicated Abs (A).
  • FIG. 9A and 9B show the wild type MUC1 nucleic acid sequence and amino acid sequence.
  • COPD chronic obstructive pulmonary disease
  • MUC1 (MUC in human and Muc in animals) is a membrane-tethered mucin-like glycoprotein expressed in mucosal epithelial cells as well as some hematopoietic cell types (13, 14).
  • WT wild type
  • MUCl/Mucl binds to TLRs and thus preventing the interaction between TLRs and MyD88 (10) or TRIF (9), the two adaptor proteins required for TLR signaling following activation of TLRs by their agonists (1).
  • TLR Toll-like receptor
  • TAT peptide is derived from the transactivator of transcription
  • TAT human immunodeficiency virus (3) and has been successfully used as a CPP (18).
  • a TAT sequence (YGRKKRRQRRR; amino acids 47 to 57 of TAT)(4) and palmitic acid was used to introduce an 11-mer MUC1 CT domain containing Y46 in the center in order to determine whether this peptide can mimic the anti -inflammatory activity of MUC1 without cytotoxicity.
  • YGRKKRRQRRR amino acids 47 to 57 of TAT
  • palmitic acid was used to introduce an 11-mer MUC1 CT domain containing Y46 in the center in order to determine whether this peptide can mimic the anti -inflammatory activity of MUC1 without cytotoxicity.
  • this MUC1- like synthetic compound may provide an alternative therapy for chronic inflammatory disorders such as COPD.
  • the present invention is based on the discovery that peptides having a portion of the sequence of MUC1 inhibits interaction between TLR and MyD88, which thereby inhibits TLR-mediated inflammation.
  • the invention is directed to compounds that bind to a TLR polypeptide and prevent binding between the TLR polypeptide and MyD88 and/or TRIF.
  • the compounds are not limited to binding a particular TLR polypeptide.
  • the TLR polypeptide is selected from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLRl l, TLR12, and TLR13.
  • the invention is directed to compounds that bind to TLR5 and prevent binding between the TLR5 polypeptide and MyD88.
  • the invention is not limited to a particular type or kind of compound that binds to a
  • the compound is a peptide or mimetic of MUC1.
  • such peptides or mimetics comprise a portion of the amino acid sequence of MUC1 which permits the resulting peptide or mimetic to bind to a TLR polypeptide and thereby prevent TLR-mediated inflammation.
  • the MUC1 peptide or MUC1 mimetic comprises the amino acid sequence YEKV (SEQ ID NO: l).
  • the peptides or mimetics comprise the amino acid sequence TDRSPYEKVSA (SEQ ID NO:2).
  • the MUC1 peptide or MUC1 mimetic can also comprise one or more functional groups, such as a moiety that facilitates purification, e.g., a (His)6 moiety or an antibody - binding epitope.
  • a moiety that facilitates purification e.g., a (His)6 moiety or an antibody - binding epitope.
  • the MUC1 peptide or MUC1 mimetic further comprises at least one biocompatible carrier (e.g., poly-lactic acid, poly-gly colic acid, and copolymers of poly-lactic acid and poly-gly colic acid).
  • the MUC1 peptide or MUC1 mimetic further comprises at least one biocompatible carrier selected from the group consisting of porous or non-porous calcium phosphates, porous or non-porous hydroxyapatites, porous or non-porous tricalcium phosphates, porous or non-porous tetracalcium phosphates, and porous or non-porous calcium sulfates, or a combination thereof.
  • the biocompatible carrier is palmitic acid.
  • the MUC1 peptide or MUC1 mimetic further comprises a cell- penetrating moiety that facilitates delivery of the peptides to an intracellular space, e.g., HIV- derived TAT peptide, penetratins, transportans, SS peptides (alternating aromatic residues and basic amino acids (aromatic-cationic peptides)), SA, SM, or SNL peptides, or hCT derived cell-penetrating peptides (see, e.g., Caron et al, (2001) Mol. Ther. 3(3):310-8; Langel, Cell- Penetrating Peptides: Processes and Applications (CRC Press, Boca Raton Fla. 2002); El- Andaloussi et al, (2005) Curr Pharm Des. 11(28):3597-611; Lindgren et al, Trends
  • the cell- penetrating moiety is linked to the MUC1 peptide or MUC1 mimetic, e.g., as a single fusion protein; thus, the invention includes fusion proteins comprising a MUC1 peptide as described herein and a cell-penetrating peptide, e.g., TAT, penetratins, transportans, or hCT derived cell-penetrating peptides.
  • a cell-penetrating peptide e.g., TAT, penetratins, transportans, or hCT derived cell-penetrating peptides.
  • the cell-penetrating peptide is attached to the N-terminus of the MUC1 peptide; in some embodiments, the cell-penetrating peptide is attached to the C-terminus of the MUC1 peptide.
  • the fusion protein further comprises a cleavable moiety as known in the art between the cell-penetrating peptide and the MUC1, that cleaves off the cell-penetrating peptide, leaving the MUC1 peptide intact.
  • An additional useful functional group here is a moiety that facilitates detection of the MUC1 peptide or MUC1 mimetic, such as a fluorescent moiety, a radioactive moiety, or an antigen.
  • the compound comprises a TAT sequence and a palmitic acid sequence.
  • the MUC1 peptide or MUC1 mimetic may be linked directly to the TAT sequence, and the TAT sequence is directly linked to the palmitic acid sequence.
  • the TAT sequence is directly linked to the MUC1 peptide or MUC1 mimetic, and the MUC1 peptide or MUC1 mimetic is directly linked to the palmitic acid sequence.
  • the TAT sequence is directly linked to the palmitic acid sequence, and the palmitic acid sequence is directly linked to the MUC1 peptide or MUC1 mimetic.
  • the MUC1 peptide or MUC1 mimetic is preferably in a pharmaceutically acceptable excipient.
  • Such compositions can be formulated without undue experimentation for administration to a mammal, including humans, as appropriate for the particular application. Additionally, proper dosages of the compositions can be determined without undue experimentation using standard dose-response protocols.
  • the MUC1 peptide or MUC1 mimetic compositions designed for oral, lingual, sublingual, buccal and intrabuccal administration can be made without undue experimentation by means well known in the art, for example with an inert diluent or with an edible carrier.
  • the compositions may be enclosed in gelatin capsules or compressed into tablets.
  • the pharmaceutical compositions of the present invention may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like.
  • Tablets, pills, capsules, troches and the like may also contain binders, recipients, disintegrating agent, lubricants, sweetening agents, and flavoring agents.
  • binders include microcrystalline cellulose, gum tragacanth or gelatin.
  • excipients include starch or lactose.
  • disintegrating agents include alginic acid, com starch and the like.
  • lubricants include magnesium stearate or potassium stearate.
  • An example of a glidant is colloidal silicon dioxide.
  • sweetening agents include sucrose, saccharin and the like.
  • flavoring agents include peppermint, methyl salicylate, orange flavoring and the like. Materials used in preparing these various compositions should be pharmaceutically pure and nontoxic in the amounts used.
  • the MUC1 peptide or MUC1 mimetic compositions of the present invention can easily be administered parenterally such as for example, by intravenous, intramuscular, intrathecal or subcutaneous injection.
  • Parenteral administration can be accomplished by incorporating the compositions of the present invention into a solution or suspension.
  • solutions or suspensions may also include sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents.
  • Parenteral formulations may also include antibacterial agents such as for example, benzyl alcohol or methyl parabens, antioxidants such as for example, ascorbic acid or sodium bisulfite and chelating agents such as EDTA.
  • Buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be added.
  • the parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.
  • Rectal administration includes administering the pharmaceutical peptide or mimetic compositions into the rectum or large intestine. This can be accomplished using suppositories or enemas.
  • Suppository formulations can easily be made by methods known in the art. For example, suppository formulations can be prepared by heating glycerin to about 120° C, dissolving the composition in the glycerin, mixing the heated glycerin after which purified water may be added, and pouring the hot mixture into a suppository mold.
  • Transdermal administration includes percutaneous absorption of the composition through the skin.
  • Transdermal formulations include patches (such as the well-known nicotine patch), ointments, creams, gels, salves and the like.
  • the present invention includes nasally administering to the mammal a therapeutically effective amount of the composition.
  • nasally administering or nasal administration includes administering the composition to the mucous membranes of the nasal passage or nasal cavity of the patient.
  • pharmaceutical compositions for nasal administration of a composition include therapeutically effective amounts of the composition prepared by well-known methods to be administered, for example, as a nasal spray, nasal drop, suspension, gel, ointment, cream or powder. Administration of the MUC 1 peptide or MUC1 mimetic composition may also take place using a nasal tampon or nasal sponge.
  • the invention is directed to methods of inhibiting interaction between a TLR polypeptide (e.g., TLR5) and MyD88 and/or TRIF.
  • TLR polypeptide e.g., TLR5
  • MyD88 and/or TRIF any of the compounds described above.
  • the TLR polypeptide (e.g., TLR5) and MyD88 and/or TRIF are is in a mammalian cell. Addition of the peptides and mimetics as described herein inhibits such binding between the TLR polypeptide (e.g., TLR5) and MyD88 and/or TRIF.
  • such mammalian cells are expressed in a living mammal. The invention methods would be expected to work in any mammal, however, in the most preferred embodiments, the mammal is a human.
  • the invention is directed to methods of inhibiting TLR- mediated inflammation in a mammal.
  • methods of inhibiting TLR- mediated inflammation in a mammal is accomplished through inhibiting interaction between a TLR polypeptide (e.g., TLR5) and MyD88 and/or TRIF.
  • TLR polypeptide e.g., TLR5
  • MyD88 and/or TRIF e.g., TLR5
  • Such methods comprise contacting the cells expressing a TLR polypeptide (e.g., TLR5) and MyD88 and/or TRIF with any of the compounds described above.
  • such mammalian cells are expressed in a living mammal.
  • the invention methods would be expected to work in any mammal, however, in the most preferred embodiments, the mammal is a human.
  • the invention is directed to methods of preventing and/or treating an individual suffering from or at risk for suffering from TLR-mediated
  • methods of inhibiting TLR-mediated inflammation in a mammal is accomplished through inhibiting interaction between a TLR polypeptide (e.g., TLR5) and MyD88 and/or TRIF.
  • TLR polypeptide e.g., TLR5
  • MyD88 and/or TRIF e.g., TARF5
  • Such methods comprise administering to the individual a composition comprising any of the compounds described above.
  • the individual is a living mammal (e.g., a living human).
  • the invention is directed to methods of treating an individual suffering from or at risk for suffering from a disorder related to TLR-mediated inflammation.
  • methods of treating a disorder related to TLR-mediated inflammation is accomplished through inhibiting interaction between a TLR polypeptide (e.g., TLR5) and MyD88 and/or TRIF.
  • TLR polypeptide e.g., TLR5
  • MyD88 and/or TRIF e.g., a TLR polypeptide
  • Such methods comprise administering to the individual a composition comprising any of the compounds described above.
  • the individual is a living mammal (e.g., a living human). Examples of disorders related to TLR-mediated inflammation include, but are not limited to, pulmonary fibrosis, COPD, asthma,
  • cardiovascular disorder diabetes, obesity, metabolic syndrome, autoimmune disorders, neuroinflammatory disorders, schizophrenia, bipolar disorder, autism, clinical depression, chronic fatigue syndrome, alcohol abuse, and toluene inhalation.
  • the compound comprises a peptide or mimetic that comprises the sequence of SEQ ID NO: l or SEQ ID NO:2.
  • the present invention provides a polypeptide comprising SEQ ID NO: 1
  • the invention is further directed to polypeptides comprising SEQ ID NO: l or SEQ ID NO:2.
  • polypeptides comprising SEQ ID NO: l or SEQ ID NO:2.
  • polynucleotides encoding this polypeptide, and vectors comprising this polynucleotide.
  • MUC1 CT 11-mer has an ability to suppress PAK- induced IL-8 release without cytotoxicity. It has been shown that the anti-inflammatory effect of MUC1 resides in its CT and requires tyrosine phosphorylation of the CT by EGFR on Y46 (10, 17, 20). To determine whether a small peptide containing Y46 can mimic the antiinflammatory effect of MUC1, we chose an 11-mer from MUC1 CT that contains Y46 in its center (Fig. 1A). To introduce the 11-mer into the cell, it was covalently linked to both TATp sequence (4) and palmitic acid (Fig. IB). The resulting peptide is referred to as Peptide 1 throughout this study. Confluent 293-hTLR5-HA cells were treated with various
  • Fig. 2A shows that treatment with PAK drastically increased IL-8 release from the cells, which was suppressed by pretreatment with Peptide 1 in a dose-dependent manner except for 100 ⁇ which showed an increase, suspecting a possible cytotoxicity.
  • the spent media were analyzed for LDH activity.
  • Fig. 2B shows that treatment with Peptide 1 didn't cause a significant release of LDH until the concentration reached 100 ⁇ - suggesting that a significant increase in IL-8 release by 100 ⁇ of Peptide 1 was most likely due to the plasma membrane damage at this concentration.
  • TLR5 regulates IL-8 gene expression mainly through the NF- KB signaling pathway (12, 20).
  • NF- ⁇ activation we measured the activation of PAK-induced NF- ⁇ following pretreatment with Peptide 1 using two different methods - the NF- ⁇ dependent ELAM-1 promoter assay and the SEAP assay.
  • NF- ⁇ activation involves the phosphorylation and degradation of ⁇ - ⁇ with a concomitant phosphorylation and nuclear translocation of p65, a subunit of NF-KB (2, 19).
  • pretreatment with Peptide 1 inhibits PAK- induced degradation of ⁇ - ⁇ with a concomitant increase in the levels of p65 compared with its control (PBS treatment) (Fig. 5) and nuclear translocation of p65 (Fig. 6).
  • Peptide 1 TATp-C16-l lmer [NH2-TDRSPYEKVSA(RRRQRRKKRGY)K-Pal] (SEQ ID NO: 4)
  • Peptide 2 TATp-C 16-11 mer with a mutation (YEKV (SEQ ID NO. : l) to FEKV (SEQ ID NO: 10))
  • Peptide 3 TATp-C16-l l-mer with a mutation (YEKV (SEQ ID NO.: l) to YKEV (SEQ ID NO: 11))
  • the resins were treated with Fmoc-amino acid (4.4 equiv) in the presence of HBTU (4 equiv) and DIPEA (4 equiv) in DMF (1 ml) at room temperature. After shaking for 2 h, the reaction mixture was drained and the resins were washed with DMF (3 x), CH2C12 (2x), MeOH (2x), and DMF (3 x). This process was repeated respectively with the amino acid residues.
  • the products were cleaved from the resins using a cleavage cocktail (95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane, and 2.5% water) for 2 h at room temperature. The mixture was purified by Prep-HPLC.
  • 293-hTLR5-HA cells were obtained by stable transfection of HEK293 cells with the pUNO-hTLR5-HA plasmid, which expresses the human TLR5 gene fused at the 3'end to the influenza hemagglutinine (HA) (InvivoGen, San Diego, CA).
  • 293-hTLR5 NF-kB/SEAPorter cells were obtained by co-transfection of HEK293 cells with the human TLR5 gene and an inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene.
  • SEAP embryonic alkaline phosphatase
  • 293/hTLR5-HA cells and 293-hTLR5 NF-kB/SEAPorter cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 50 U/ml penicillin, 50 ⁇ g/ml streptomycin and 10% FBS.
  • DMEM Dulbecco's modified Eagle's medium
  • Cytotoxicity was monitored by measuring LDH release using a LDH assay kit as previously described (15).
  • HEK 293-hTLR5 NF-KB/SEAPorter cells (InvivoGen), a stably co-transfected cell line which expresses full-length human TLR5 and the SEAP reporter gene under the transcriptional control of an NF- ⁇ response element, were seeded in 48-well plates. When cells reached 90% confluence, the cells were pretreated with peptides (10 ⁇ ) for 1 h and then treated with PAK for 4 h, and cell culture media were assayed using SEAP Assay Kit (Novus Biologicals, Littleton, CO) following the manufacture's protocol.
  • SEAP Assay Kit Novus Biologicals, Littleton, CO
  • the cells were incubated overnight with anti-NF- ⁇ p65 rabbit monoclonal Ab followed by incubation for 1 h with Alexa-Fluor-488 -conjugated goat anti -rabbit IgG Ab (Invitrogen) at RT. Nuclei were counterstained with 1 ⁇ g/ml of 4', 6-diamidino-2-phenylindole (DAPI).
  • DAPI 6-diamidino-2-phenylindole
  • TNF-alpha is a key regulator of MUC1, an anti -inflammatory molecule, during airway Pseudomonas aeruginosa infection.
  • MUC1 regulates epithelial inflammation and apoptosis by PolyLC through inhibition of Toll/IL-1 receptor-domain-containing adapter- inducing IFN-beta (TRIF) recruitment to Toll-like receptor 3.
  • TNF IFN-beta
  • Neutrophil elastase stimulates MUC1 gene expression through increased Spl binding to the MUC1 promoter.
  • American journal of physiology Lung cellular and molecular physiology 289: L355-362, 2005.
  • Neutrophil elastase induces IL-8 gene transcription and protein release through p38/NF- ⁇ kappa ⁇ B activation via EGFR transactivation in a lung epithelial cell line.
  • American journal of physiology Lung cellular and molecular physiology 291 : L407-416, 2006.
  • PDCdelta Pulmonary endothelial protein kinase C-delta
  • MUC1 mucin is a negative regulator of Toll-like receptor signaling.

Abstract

L'invention concerne un composé qui se lie à un récepteur de type Toll (TLR) et empêche l'inflammation à médiation par les TLR. La présente invention concerne en outre des procédés de blocage de l'interaction entre les TLR et MyD88 et/ou TRIF, des procédés d'inhibition de l'inflammation à médiation par les TLR dans une cellule de mammifère, des méthodes de prévention et/ou de traitement d'un mammifère souffrant ou risquant de souffrir d'une inflammation à médiation par les TLR, et des méthodes de prévention et/ou de traitement d'un mammifère souffrant ou risquant de souffrir d'un trouble lié à une inflammation à médiation par les TLR (de type affections inflammatoires chroniques).
PCT/US2016/028451 2015-04-24 2016-04-20 Procédés et compositions visant à inhiber l'inflammation à médiation par les récepteurs de type toll (tlr) WO2016172219A1 (fr)

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CN108084269A (zh) * 2017-12-28 2018-05-29 桂林医学院 一种多肽自组装纳米载体及其制备方法
WO2020243787A1 (fr) * 2019-06-06 2020-12-10 The University Of Sydney Agents anti-inflammatoires
EP3737703A4 (fr) * 2018-01-11 2021-12-08 UTI Limited Partnership Traitement du syndrome de l'x fragile

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EP3737703A4 (fr) * 2018-01-11 2021-12-08 UTI Limited Partnership Traitement du syndrome de l'x fragile
WO2020243787A1 (fr) * 2019-06-06 2020-12-10 The University Of Sydney Agents anti-inflammatoires
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EP3980440A4 (fr) * 2019-06-06 2023-07-05 The University Of Sydney Agents anti-inflammatoires

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