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WO2023147555A2 - Méthodes de traitement de la douleur - Google Patents

Méthodes de traitement de la douleur Download PDF

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Publication number
WO2023147555A2
WO2023147555A2 PCT/US2023/061588 US2023061588W WO2023147555A2 WO 2023147555 A2 WO2023147555 A2 WO 2023147555A2 US 2023061588 W US2023061588 W US 2023061588W WO 2023147555 A2 WO2023147555 A2 WO 2023147555A2
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WIPO (PCT)
Prior art keywords
par2
agent
endocytosis
subject
dynamin
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Ceased
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PCT/US2023/061588
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WO2023147555A3 (fr
Inventor
Nigel \W. BUNNETT
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Endosome Therapeutics Inc
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Endosome Therapeutics Inc
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Publication of WO2023147555A3 publication Critical patent/WO2023147555A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors

Definitions

  • This disclosure features chemical entities and compositions containing the same that are useful for treating a condition associated with gastrointestinal (“GI”) inflammation and/or pain.
  • GI gastrointestinal
  • G protein-coupled receptors are the largest family of transmembrane receptors, control most physiological and pathological processes, and are the target of >30% approved drugs (1). The effects of disease on the function and subcellular distribution of GPCRs continues to be investigated. GPCRs at the plasma membrane interact with extracellular ligands and couple to intracellular heterotrimeric G proteins. However, GPCR signaling at the plasma membrane is often transient. GPCR kinases phosphorylate activated receptors, increasing their affinity for P-arrestins (PARRs) (2). PARRs uncouple GPCRs from G proteins, which mediates desensitization, and couple GPCRs to clathrin and adaptor protein- 2, which mediate endocytosis.
  • PARRs P-arrestins
  • GPCRs can also continue to signal from other intracellular compartments, including the Golgi apparatus (10, 11).
  • Protease- activated receptor-2 is a mediator of protease-evoked inflammation and pain.
  • PAR2 is expressed by primary' sensory neurons that control neurogenic inflammation and pain transmission. Multiple proteases that are generated during injury' and inflammation can activate PAR? on sensory? nerves, including epithelial derived trypsin IV, mast cell tryptase, macrophage cathepsin S and neutrophil elastase. These proteases cleave and activate PAR2 on primary sensory nerves, leading to sensitization of transient receptor potential ion channels and the release of neuropeptides (substance P and calcitonin gene related peptide). These peptides cause peripheral inflammation and central transmission of pain. Trypsin and tryptase activate PAR2 by canonical mechanisms, leading to recruitment of P-arrestins and PAR2 endocytosis. See, e.g., WO 2017/197463.
  • WO 2017/197463 discloses compounds that inhibit endosomal protease-activated receptor-2 (PAR2) signalling and their use in the treatment of pain.
  • PAR2 endosomal protease-activated receptor-2
  • IBD Inflammatory Bowel Disease
  • IBD generally occurs as the result of inappropriate immune responses in individuals, which are mediated by a variety of interactions between environmental stimuli, microbial factors, and the intestinal immune system.
  • IBD is represented either by excessive immune responses that mediate gastrointestinal tissue damage, directly or through the release of soluble, pro-inflammatory mediators.
  • IBD typically results in frequent and bloody bowel movements accompanied with histopathological damage to the gastrointestinal mucosa (see, e.g., Zhang et al., 2017, Front Immunol, 8:942).
  • This disclosure features chemical entities and compositions containing the same that are useful for treating a condition associated with gastrointestinal (“GI”) inflammation and/or pain.
  • GI gastrointestinal
  • this disclosure features methods for treating a condition associated with gastrointestinal (“GI”) inflammation and/or pain in a subject (e.g., a subject in need thereof), the methods include administering to the subject an effective amount of an agent that that inhibits endosomal protease-activated receptor-2 (“PAR2”) signaling.
  • GI gastrointestinal
  • PAR2 endosomal protease-activated receptor-2
  • this disclosure features methods for treating a condition associated with gastrointestinal (“GI”) inflammation and/or pain in a subject (e.g., a subject in need thereof), the methods include contacting a colonocyte in the subject with an effective amount of an agent that inhibits endosomal PAR2 signalling in the colonocyte.
  • GI gastrointestinal
  • the condition is colitis.
  • the condition is autoimmune colitis.
  • the autoimmune colitis is an inflammatory bowel disease.
  • inflammatory bowel disease can be Crohn’s disease or ulcerative colitis.
  • the condition is selected from the group consisting of inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis (“UC”)), collagenous colitis, ileocolitis (e.g., granulomatous ileocolitis), granulomatous gastritis, idiopathic inflammatory bowel disease, ileitis, functional bowel disorders (e.g., such as non-ulcer dyspepsia and irritable bowel syndrome), lymphocytic colitis, regional enteritis, spastic colon, celiac disease, microscopic colitis, radiation enteritis, and iatrogenic autoimmune colitis.
  • inflammatory bowel disease e.g., Crohn's disease and ulcerative colitis (“UC”)
  • ileocolitis e.g., granulomatous ileocolitis
  • granulomatous gastritis e.g., granulomatous gastritis
  • idiopathic inflammatory bowel disease ileit
  • the methods described herein include treating pain associated with any one or more of the foregoing disorders.
  • the methods described herein include treating inflammation associated with any one or more of the foregoing disorders.
  • the methods described herein include treating pain and inflammation associated with any one or more of the foregoing disorders.
  • the agent is an agent that inhibits endocytosis of the PAR2.
  • the agent is an inhibitor of a process selected from the group consisting of clathrin-mediated endocytosis; clathrin-independent endocytosis; caveolae- mediated endocytosis; micropinocytosis; dynamin-mediated endocytosis; dynamin- independent endocytosis; endosome maturation; and P-arrestin-mediated endocytosis.
  • the agent is an inhibitor of a process selected from the group consisting of clathrin-mediated endocytosis; dynamin-mediated endocytosis; and P-arrestin- mediated endocytosis.
  • the agent is an inhibitor of a process selected from the group consisting of dynamin 1 -dependent endocytosis, dynamin 2-dependent endocytosis, and dynamin 3-dependent endocytosis.
  • the agent is an inhibitor of dynamin 2-mediated endocytosis.
  • dynamin 2 inhibitors include Dynasore, long chain amines and ammonium salts (MiTMABs, OcTMAB), dynoles, (DD-6), Phthaladyn-23, and dihydroxyl and trihydroxyl dynasore.
  • the agent includes an anti-sense molecule targeting Dynamin 2 resulting in decreased mRNA expression by at least about 25% to about 75%.
  • the agent is an oligonucleotide targeting Dynamin-2 resulting in decreased mRNA expression by at least about 25% to about 75% (e.g., from about 25% to about 50% or from about 50% to about 75%).
  • the agent is an siRNA targeting Dynamin-2 resulting in decreased mRNA expression by at least about 25% to about 75% (e.g., from about 25% to about 50% or from about 50% to about 75%).
  • the agent achieves about 10%, or about 20% , or about 30%, or about 40%, or about 50%, about 60% or about 70% or about 80% or about 90% or about 95% or about 99%, or about 10% to about 50%, or about 50% to about 90% inhibition of endocytosis of the PAR2.
  • the agent can include an nucleic acid molecule targeting Dynamin 2, such as an antisense oligonucleotide targeting Dynamin-2 resulting in decreased mRNA or a siRNA targeting Dynamin-2.
  • an nucleic acid molecule targeting Dynamin 2 such as an antisense oligonucleotide targeting Dynamin-2 resulting in decreased mRNA or a siRNA targeting Dynamin-2.
  • the agent is a PAR2 antagonist.
  • Non-limiting examples of PAR2 antagonists include AZ8838, AZ3451, ENMD-1068, SAM11, K-14585, K-12940, C391, P2pal-18S, PZ-235, OA-2351, OA-235c, GB83, GB88, AZ7188, 1-191, B5, MAB3949, FAB3949, MEDI0618, and PAR2 mAb, P2pal-18S, R284S, PZ-235, GB83, GB110, and GB88 (see, e.g., Biochem Soc Trans. 2020 Dec 18; 4S(6): 2525- 2537).
  • the PAR2 antagonist is encapsulated in a nanoparticle that is structurally predisposed to release the PAR2 antagonist in the endosome.
  • suitable nanoparticles can be found, e.g., in WO 2020/084471, which is incorporated herein by reference in its entirety.
  • the PAR2 antagonist includes a lipid anchor that promotes insertion of the antagonist into a plasma membrane (optionally the antagonist and lipid anchor are connect by a linker, thereby forming a tripartite compound).
  • lipid anchors and linkers as well as methods of using the same to form a tripartite compound can be found, e.g., in WO 2017/112792 and WO 2017/197463, each of which is incorporated herein by reference in its entirety.
  • the agent comprises a targeting moiety having an affinity for binding to a plasma membrane-expressed moiety on a colonocyte.
  • the targeting moiety selectively binds to plasma membrane- expressed protein (receptor, channel, enzyme) on the colonocyte.
  • the targeting moiety selectively binds to plasma membrane- expressed protein (receptor, channel, enzyme) on the colonocyte.
  • the targeting moiety is an antibody.
  • the agent is an antibody- nucleic acid molecule conjugate.
  • targeting moieties are described herein (e.g., lectins and neoglycoconjugates; see, e.g., Advanced Drug Delivery Reviews, Volume 56, Issue 4, 3 March 2004, Pages 491-509).
  • the targeting moiety is or includes an antibody (e.g., monoclonal antibody.
  • the level of stimulated proinflammatory cytokine release in the GI tract in the presence of the agent is less than pre-administration levels.
  • the level of stimulated proinflammatory cytokine release in the colon in the presence of the agent is less than pre-administration levels.
  • the severity and/or frequency of at least one of the symptoms is decreased after the first or subsequent administration of the agent.
  • the subject upon administration of the agent, the subject experiences a reduced number of symptom flare-ups and/or increased symptom remission period compared to pre- administration levels.
  • the agent is administered before onset of symptoms.
  • the agent is administered concurrently with, or after, the onset of symptoms.
  • the agent is administered to the GI tract of the subject.
  • the agent is administered locally to the GI tract of the subject.
  • the agent can be administered by enema.
  • the condition is refractory or resistant to other traditional or conventional therapies.
  • the agent is administered in combination with one or more additional therapies.
  • the one or more additional therapies are selected from the group consisting of sphingosine 1 -phosphate (SIP) receptor modulators (e.g., etrasimod or ozanimod); steroidal anti-inflammatory agents (e.g, beclomethasone 17 or budesonide); non-steroidal anti-inflammatory agents (e.g., 5-ASA); receptor-interacting protein kinase 1 (RIPK1) inhibitors (e.g., GSK2982772); EP4 modulators (e.g., KAG-308); toll-like receptor (e.g., TLR4, TLR9) modulators (e.g., JKB-122, cobitolimod); Janus kinase (JAK) inhibitors (e.g., TD-1473, tofacitinib, upadacitinib, filgotinib, PF-06651600, and PF- 06
  • SIP sphingo
  • the method further includes identifying the subject in need of such treatment.
  • this disclosure features methods of identifying a candidate agent for treating associated with GI inflammation and/or pain, the method includes:
  • this disclosure features chemical entities that includes (i) a targeting moiety that selectively targets a colonocyte; and (ii) a moiety that inhibits endosomal PAR2 receptor signaling in the colonocyte.
  • This disclosure contemplates both in vitro and in vivo methods.
  • mammals are a mammal, more preferably a human. Mammals also include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats.
  • calcitonin gene-related peptide and "CGRP” refers to any form of calcitonin gene-related peptide and variants thereof that retain at least part of the activity of CGRP.
  • CGRP may be .alpha. -CGRP or .beta. -CGRP.
  • CGRP includes all mammalian species of native sequence CGRP, e.g., human, canine, feline, equine, and bovine.
  • an "effective dosage” or “effective amount” of drug, compound, or pharmaceutical composition is an amount sufficient to effect beneficial or desired results.
  • beneficial or desired results include results such as eliminating or reducing the risk, lessening the severity, or delaying the onset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease.
  • beneficial or desired results include clinical results such as reducing pain intensity, duration, or frequency associated with any of the indications described herein, and decreasing one or more symptoms associated with any of the indications described herein (biochemical, histological and/or behavioral), including its complications and intermediate pathological phenotypes presenting during development of the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication, and/or delaying the progression of the disease of patients.
  • An effective dosage can be administered in one or more administrations.
  • an effective dosage of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly.
  • an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition.
  • FIGs. 1A-1E show generation and characterization of Par2-mugfp mice.
  • FIG. 1A shows a Par2-mugfp targeting construct comprising a downstream phosphoglycerine kinase neomycin cassette flanked by loxP sites and a downstream Kpnl site.
  • FIG. IB show Southern blot images confirming correct targeting.
  • FIG. 1C shows Southern blot and
  • FIG. ID shows RT- PCR blot confirming Par2-mugfp expression in mice. WT, wild-type.
  • FIGs. 4A-4G show colitis-evoked endocytosis of PAR2-muGFP.
  • FIGs. 4A-4C show DSS colitis in Par2-mugfp mice.
  • FIG. 4A shows body weight and disease activity index (DAI) after DSS or water (control).
  • FIG. 4C shows redistribution of PAR2-muGFP from the plasma membrane (arrowheads) to endosomes (arrows) after DSS.
  • FIG. 4D-4F show TNBS colitis in Par2-mugfp mice.
  • FIG. 4D shows grossly inflamed colon.
  • FIG. 4E shows histological sections showing infiltration of neutrophils in the submucosa (yellow arrowheads).
  • FIG. 4F shows redistribution of PAR2-muGFP from the plasma membrane to endosomes after TNBS.
  • FIGs. 5A-5U show PAR2 trafficking to endosomes.
  • FIG. 5A shows BRET assays of the proximity between PAR2-Rluc8 and Venus- or YFP-tagged proteins resident to plasma membrane (PM, CAAX or KRas), early endosomes (EE, Rab5a), late endosomes (LE, Rab7a), recycling endosomes (RE, Rabi la), cis-Golgi (CG, giantin) or trans-Golgi (TG, TGN38).
  • PM plasma membrane
  • CAAX or KRas early endosomes
  • EE early endosomes
  • LE late endosomes
  • RE Rabi la
  • cis-Golgi CG, giantin
  • trans-Golgi TG, TGN38
  • FIGs. 5B-5G show effects of 2F (100 pM) on translocation of PAR2-Rluc8 from the plasma membrane (RGFP-CAAX) to early endosomes (tdRGFP-Rab5a) of T84 cells treated with vehicle, hypertonic sucrose, DnmK44A or Dnm2 siRNA.
  • FIGs. 5H-5S show effects of 2F (10 pM) on translocation of PAR2-Rluc8 from the plasma membrane (KRas-Venus, FIGs. 5H, 51) to early endosomes (Rab5a-Venus, FIGs. 5J, 5K), late endosomes (Rab7a-Venus, FIGs.
  • FIGs. 6E shows NbBRET uses luciferase split into two fragments to detect BRET between receptor and effector (PAR2 and mGa or PARR) for proteins resident to the PM (CAAX) or EE (FYVE).
  • FIGs. 6F-6H show effects of 2F (100 pM) on recruitment of mGa S q, mGa Si (FIG. 6F) or PARR1 (FIG. 6G) to the PM (LgBiT- CAAX) or EM (LgBiT-FYVE) of T84 cells.
  • FIGs. 6I-6K show effects of 2F (10 pM) on recruitment of mGa S q, mGa Si (FIG.
  • FIG. 6J PARR1 or mGa s (FIG. 6J) to the PM (LgBiT- CAAX) or EE (LgBiT-FYVE) of HEK293T cells.
  • FIGs. 6D, 6K show 1-way ANOVA with Dunnett’s (FIG. 6D) or Holm-Sidak’s (FIG. 6K) test compared to vehicle.
  • FIG. 6H shows unpaired t-test.
  • FIG. 6H shows unpaired t-test.
  • 6L shows localization of immunoreactive HA-PAR2, Ga q and EEA1 plus YFP-PARRl and YFP-PARR2 in HEK293T cells.
  • Cells were unstimulated or incubated with 2F (10 pM, 30 min).
  • Arrowheads plasma membrane.
  • FIGs. 7A-7E show PAR2 endosomal signaling and colonic inflammation and pain.
  • FIG. 7C shows effects of trypsin (100 nM), 2F (10 pM) or vehicle (saline, control) on TEER of T84 cells after 6 h. Cells were preincubated with GB88 (PAR2 antagonist) or vehicle (DMSO, control).
  • 7D shows effects of trypsin (100 nM), 2F (10 pM) or vehicle (saline, control) on TEER of T84 cells after 5 h.
  • Cells were preincubated with Dnm2 or control siRNA.
  • FIG. 7E shows an exemplary proposed mechanism by which proteases (e.g., tryptase) from mucosal inflammatory cells (e.g., mast cells) activate PAR2 at the basolateral membrane of colonocytes to evoke PAR2 endocytosis and assembly of a PAR2, Goc, PARR signalosome in endosomes.
  • Endosomal signaling causes disassembly of tight junctions (TJ) and release of proinflammatory cytokines (e.g., IL-8) and possibly proteases (e.g., trypsins) from colonocytes.
  • proteases e.g., tryptase
  • mucosal inflammatory cells e.g., mast cells
  • Endosomal signaling causes disassembly of tight junctions (TJ) and release of proinflammatory cytokines (e.g., IL-8) and possibly proteases (e.g., trypsins) from colonocytes.
  • FIGs. 8A-8K show contribution of Dnm2 to PAR2-evoked inflammation and pain in the colon.
  • FIGs. 8A-8H show Dnm2 or control (Ctrl) shRNA was administered to Par2-mugfp mice by intracolonic injection. Mice were studied after 48 h.
  • FIG. 8A show localization of Dnm2 by RNAScope®. Representative images, scale bar, 100 pm.
  • FIG. 8B show quantification of Dnm2 in the colon.
  • FIG. 8C shows localization of GFP immunoreactivity in the colon at 5 h after intracolonic injection of 2F. Arrows denote endosomes; arrowheads denote cell surface. Scale bar, 10 pm.
  • FIGs. 8A-8C. N 5 mice.
  • FIGs. 8D-8F show levels of TNFa (FIG. 8D), CXCL1 (FIG. 8E) and IL-10 (FIG. 8F) in the colon 5 h after intracolonic 2F.
  • FIGs. 8G, 8H show abdominal von Frey filament withdrawal responses 1-5 h after intracolonic 2F.
  • FIG. 8G shows time course.
  • FIG. 8H shows area under curve (AUC) from 0- 5 h.
  • N 6 mice.
  • FIGs. 81, 8J show abdominal von Frey filament withdrawal responses of wild-type, Par2-mugfp and Pa.r2N0.vl.8 mice 1-6 h after intracolonic 2F.
  • FIG. 81 shows time course.
  • FIG. 8J shows area under curve from 0-6 h.
  • FIG. 8B **P ⁇ 0.01. Student’s t-test.
  • FIGs. 8D-8J *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001. 1- and 2-way ANOVA, Tukey’s test.
  • FIG. 8K **P ⁇ 0.01. Student’s t-test.
  • This disclosure features chemical entities and compositions containing the same that are useful for treating a condition associated with gastrointestinal (“GI”) inflammation and/or pain.
  • GI gastrointestinal
  • This disclosure features methods for treating, preventing, ameliorating, controlling, reducing incidence of, or delaying the progression of a condition associated with gastrointestinal (“GI”) inflammation and/or pain.
  • GI gastrointestinal
  • the condition is colitis, e.g., autoimmune colitis.
  • the autoimmune colitis can be an inflammatory bowel disease.
  • the inflammatory bowel disease can be Crohn’s disease.
  • the inflammatory bowel disease can be ulcerative colitis.
  • the colitis e.g., autoimmune colitis
  • the iatrogenic autoimmune colitis can result from Clostridium difficile infection, which is amond the leading cause of nosocomial diarrhea and colitis in the industrialized world and typically occurs in subjects taking broad spectrum antibiotics.
  • the colitis can be collagenous colitis, lymphocytic colitis, or microscopic colitis.
  • the condition is an autoimmune disease.
  • the condition is autoimmune colitis, e.g., an inflammatory bowel disease (e.g., Crohn’s disease or ulcerative colitis).
  • the condition is Crohn’s disease, autoimmune colitis, iatrogenic autoimmune colitis, ulcerative colitis, colitis induced by one or more chemotherapeutic agents, colitis induced by treatment with adoptive cell therapy, colitis associated by one or more alloimmune diseases (such as graft-vs-host disease, e.g., acute graft vs. host disease and chronic graft vs. host disease), and radiation enteritis.
  • alloimmune diseases such as graft-vs-host disease, e.g., acute graft vs. host disease and chronic graft vs. host disease
  • the condition is selected from the group consisting of inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis (“UC”)), collagenous colitis, ileocolitis (e.g., granulomatous ileocolitis), granulomatous gastritis, idiopathic inflammatory bowel disease, ileitis, functional bowel disorders (e.g., such as non-ulcer dyspepsia and irritable bowel syndrome), lymphocytic colitis, regional enteritis, spastic colon, celiac disease, microscopic colitis, radiation enteritis, and iatrogenic autoimmune colitis.
  • inflammatory bowel disease e.g., Crohn's disease and ulcerative colitis (“UC”)
  • ileocolitis e.g., granulomatous ileocolitis
  • granulomatous gastritis e.g., granulomatous gastritis
  • idiopathic inflammatory bowel disease ileit
  • the methods described herein include treating pain associated with any one or more of the foregoing disorders.
  • the methods described herein include treating inflammation associated with any one or more of the foregoing disorders.
  • the methods described herein include treating pain and inflammation associated with any one or more of the foregoing disorders.
  • the agent increases the barrier function of an epithelial cell layer (e.g., in an in vitro assay), wherein the increase is relative to the barrier function in the assay in the absence of the protein.
  • the in vitro assay is a transepithelial electrical resistance (TEER) assay.
  • the increase in barrier function is an increase in electrical resistance of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% greater than the electrical resistance in the assay in the absence of the agent.
  • the epithelial cell layer is an intestinal epithelial cell layer.
  • the intestinal epithelial cell layer is a cell layer which comprises enterocytes and goblet cells.
  • the agent decreases the secretion of a pro-inflammatory cytokine from a cell (e.g., in an in vitro assay).
  • the in vitro assay comprises incubation of monocytic cells with heat killed E. coli in the presence and absence of the protein.
  • the at least one pro-inflammatory cytokine is selected from the group consisting of TNF-. alpha., IL-17, IL-10, IL-2, IEN-. gamma., IL-6, IL-12, IL- 25, IL-33, IL-8, MCP-1, MIP-3. alpha., CXCL1, and IL-23.
  • the agent increases secretion of an anti-inflammatory cytokine from a cell (e.g., in an in vitro assay).
  • the in vitro assay comprises incubation of a monocyte with heat killed E. coli in the presence and absence of the agent.
  • the at least one anti-inflammatory cytokine is selected from the group consisting of IL-4, IL-10, IL-13, IFN-. alpha., and TGF-.beta.
  • the agent reduces intestinal tissue pathology in a subject administered the agent.
  • the subject was induced to have intestinal tissue damage by treatment with a chemical.
  • the subject was treated with the chemical dextran sodium sulfate (DSS) to induce intestinal tissue damage.
  • the subject is a mammal.
  • the animal is a rodent.
  • the subject is a non human primate.
  • the agent reduces gastrointestinal inflammation in a subject administered the agent. In other embodiments, the agent reduces intestinal mucosa inflammation in the subject. In still other embodiments, the agent improves intestinal epithelial cell barrier function or integrity in the subject.
  • the agent increases the amount of mucin in intestinal tissue in a subject administered said agent.
  • the agent increases intestinal epithelial cell wound healing in a subject administered the agent. In other embodiments, the agent increases intestinal epithelial cell wound healing in an in vitro assay.
  • the agent prevents or reduces colon shortening in a subject administered the agent.
  • the agent modulates (i.e. increases or decreases) a cytokine in the blood, plasma, serum, tissue and/or mucosa of a subject administered the agent.
  • the agent decreases the levels of at least one pro-inflammatory cytokine in the blood, plasma, serum, tissue and/or mucosa of the subject.
  • the at least one pro-inflammatory cytokine is selected from the group consisting of TNF-. alpha., IL-17, IL-l.beta., IL-2, IFN-.gamma., IL-6, IL-12, IL-25, IL-33, IL-8, MCP-1, MIP-3. alpha., CXCL1, and IL-23.
  • the agent increases the levels of at least one anti-inflammatory cytokine in the blood, plasma, serum, tissue and/or mucosa of the subject.
  • the at least one anti-inflammatory cytokine is selected from the group consisting of IL-4, IL-10, IL-13, IFN-. alpha., and TGF-.beta..
  • the agent decreases the level of at least one anti-inflammatory cytokine in the blood, plasma, serum, tissue and/or mucosa of the subject.
  • the at least one anti-inflammatory cytokine is selected from the group consisting of IL-4, IL-10, IL-13, IFN-. alpha., and TGF-.beta.
  • Any procedure that allows an assessment of the mucosa can be used. Examples include barium enemas, x-rays, and endoscopy.
  • An endoscopy may be of the esophagus, stomach and duodenum (esophagogastroduodenoscopy), small intestine (enteroscopy), or large intestine/colon (colonoscopy, sigmoidoscopy). These techniques are used to identify areas of inflammation, ulcers and abnormal growths such as polyps.
  • Scoring systems based on this visual examination of the GI tract exist to determine the status and severity of IBD, and these scoring systems are intended to ensure that uniform assessment of different patients occurs, despite the fact that patients may be assessed by different medical professionals, in diagnosis and monitoring of these diseases as well as in clinical research evaluations. Examples of evaluations based on visual examination of UC are discussed and compared in Dapemo M et al (J Crohns Colitis. 2011 5:484-98).
  • Clinical scoring systems also exist, with the same purpose.
  • the findings on endoscopy or other examination of the mucosa can be incorporated into these clinical scoring systems, but these scoring systems also incorporate data based on symptoms such as stool frequency, rectal bleeding and physician's global assessment.
  • IBD has a variety of symptoms that affect quality of life, so certain of these scoring systems also take into account a quantitative assessment of the effect on quality of life as well as the quantification of symptoms.
  • UCIS Ulcerative Colitis Endoscopic Index of Severity
  • Baron Score Baron et al., 1964, BMJ, 1 :89
  • Ulcerative Colitis Colonoscopic Index of Severity UCCIS
  • Rachmilewitz Endoscopic Index Rachmilewitz, 1989, BMJ, 298:82-86
  • Sutherland Index also known as the UC Disease Activity Index (UCDAI) scoring system; Sutherland et al., 1987, Gastroenterology, 92:1994-1998), Matts Score (Matts, 1961, QJM, 30:393-407), and Blackstone Index
  • CD Al Crohn's Disease Activity Index
  • Calculation of the CDAI score includes scoring of the number of liquid stools over 7 days, instances and severity of abdominal pain over 7 days, general well-being over 7 days, extraintestinal complications (e.g., arthritis/arthralgia, ulceris/uveitis, erythema nodosum, pyoderma gangrenosum, aphtous stomatitis, anal fissure/fistula/abscess, and/or fever >37.8. degree.
  • the CD is classified as either asymptomatic remission (0 to 149 points), mildly to moderately active CD (150 to 220 points), moderately to severely active CD (221 to 450 points), or severely active fulminant disease (451 to 1000 points).
  • the method of treatment comprising administering to a patient diagnosed with CD a therapeutically effective amount of SG-11 protein or variant or fragment thereof results in a decrease in a diagnostic score of CD.
  • the score may change the diagnosis from severely active to mildly or moderately active or to asymptomatic remission.
  • the Harvey-Bradshaw index is a simpler version of the CD Al which consists of only clinical parameters (Harvey et al., 1980, Lancet 1(8178): 1134-1135). The impact on quality of life is also addressed by the Inflammatory Bowel Disease Questionnaire (IBDQ) (Irvine et al., 1994, Gastroenterology 106: 287-296). Alternative methods further include CDEIS and SES CD (see, e.g., Levesque, et al. (2015) Gastroentrol. 148:37 57).
  • IBDQ Inflammatory Bowel Disease Questionnaire
  • the methods and compositions described herein are suitable for use in combination therapy with one or more additional therapeutic agents.
  • therapeutic agents useful for treating or preventing inflammatory bowel disease (IBD) e.g., Crohn's disease, ulcerative colitis.
  • Non-limiting examples of the additional therapeutic agents include: sphingosine 1 -phosphate (SIP) receptor modulators (e.g., etrasimod or ozanimod); steroidal anti-inflammatory agents (e.g, beclomethasone 17 or budesonide); non-steroidal antiinflammatory agents (e.g., 5-ASA); receptor-interacting protein kinase 1 (RIPK1) inhibitors (e.g., GSK2982772); EP4 modulators (e.g., KAG-308); toll-like receptor (e.g., TLR4, TLR9) modulators (e.g., JKB-122, cobitolimod); Janus kinase (JAK) inhibitors (e.g., TD-1473, tofacitinib, upadacitinib, filgotinib, PF-06651600, and PF-06700841); lanthionine synthetase C-like 2 (LANCL2) modulators (e
  • the one or more therapeutic agents can be: budenoside; epidermal growth factor; corticosteroids; cyclosporine; sulfasalazine; aminosalicylates; 6- mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide; antioxidants; thromboxane inhibitors; IL-1 receptor antagonists; anti- IL-1 monoclonal antibodies; anti-IL-6 monoclonal antibodies (e.g., anti-IL-6 receptor antibodies and anti-IL-6 antibodies); growth factors; elastase inhibitors; pyridinyl-imidazole compounds; TNF antagonists as described herein; IL-4, IL-10, IL-13 and/or TGF.beta.
  • cytokines or agonists thereof e.g., agonist antibodies
  • IL-11 glucuronide- or dextran- conjugated prodrugs of prednisolone, dexamethasone or budesonide
  • ICAM-1 antisense phosphorothioate oligodeoxynucleotides ISIS 2302; Isis Pharmaceuticals, Inc.
  • soluble complement receptor 1 TP10; T Cell Sciences, Inc.
  • slow-release mesalazine methotrexate
  • antagonists of platelet activating factor (PAF) ciprofloxacin
  • lignocaine e.g., agonist antibodies
  • the one or more additional therapeutic agents can be therapeutic agents and/or regimens for treating autoimmune colitis.
  • corticosteroids e.g., budesonide, prednisone, prednisolone, Beclometasone dipropionate
  • diphenoxy late/atropine infliximab
  • loperamide mesalamine
  • TIP60 inhibitors see, e.g., U.S. Patent Application Publication No. 2012/0202848
  • vedolizumab e.g., vedolizumab.
  • the chemical entities can be administered in combination with one or more conventional pharmaceutical excipients.
  • Pharmaceutically acceptable excipients include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, selfemulsifying drug delivery systems (SEDDS) such as d-a-tocopherol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens, poloxamers or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, tris, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose- based substances, polyethylene glycol, sodium carb
  • the chemical entities escribed herein or a pharmaceutical composition thereof can be administered to subject in need thereof by any accepted route of administration.
  • Acceptable routes of administration include, but are not limited to, buccal, cutaneous, endocervical, endosinusial, endotracheal, enteral, epidural, interstitial, intraabdominal, intra-arterial, intrabronchial, intrabursal, intracerebral, intracisternal, intracoronary, intradermal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraovarian, intraperitoneal, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratesticular, intrathecal, intratubular, intratumoral, intrauterine, intravascular, intravenous, nasal, intranasal,
  • the chemical entities described herein or a pharmaceutical composition thereof are suitable for local, topical administration to the digestive or GI tract, e.g., rectal administration.
  • Rectal compositions include, without limitation, enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly suppositories, and enemas (e.g., retention enemas).
  • PAR2- muGFP immunoreactivity was prominently localized to the basolateral plasma membrane of epithelial cells lining the small and large intestine.
  • RNAScope using probes to F2rll and Gfp confirmed expression by enterocytes and colonocytes.
  • the lack of GFP immunoreactivity and mRNA in wild-type mice verified selectivity.
  • the detection of PAR2 in intestinal epithelial cells confirms reports using receptor antibodies and functional assays (27, 43).
  • proteases that are activated in the colon of mice with colitis and patients with IBD (20-25). These include proteases from immune cells (e.g., neutrophil elastase, cathepsin S, (20-22)), proteases from colonocytes (e.g., trypsins, (45)) and proteases from the microbiome.
  • An understanding of whether PAR2 is principally expressed at the apical or basolateral membrane of intestinal epithelial cells has implications for mechanisms of activation.
  • Luminal proteases from epithelial cells, exocrine glands or colonic bacteria could activate apical PAR2, whereas proteases from mucosal immune or epithelial cells could activate PAR2 at the basolateral membrane.
  • the finding of predominant basolateral expression is consistent with reports that basolateral but not apical application of PAR2 agonists increases paracellular permeability (43), although PAR2 has also been detected at the apical membrane (27).
  • the endosomal complex was stable during the period of measurement (1200 s), which might give rise to sustained signals in subcellular compartments that control critical functions of colonocytes, including tight junction assembly and release or proinflammatory and pronociceptive factors.
  • the results do not exclude the possibility that PAR2 signals from other organelles, such as the Golgi apparatus, as is the case for thyroid stimulating hormone receptors and opioid receptors (10, 11).
  • Proteases of late endosomes, including cathepsin S and legumain can activate PAR2 even in acidic conditions, which could facilitate receptor activation in other subcellular compartments (17, 22).
  • signalosomes comprising PAR2 and Ga q , Gai or 0ARR1/2 assemble predominantly in early endosomes.
  • Dnm2 knockdown also abrogated 2F-evoked colonic hyperalgesia, a finding that could be secondary to reduced release from colonocytes of cytokines or proteases that sensitize nociceptors. Since deletion of PAR2 from Na v 1.8-positive neurons blunted 2F-evoked colonic hyperalgesia, the latter is likely. The identity of colonocyte-derived proteases remains to be determined, although colonocytes express isoforms of trypsin that activate PAR2 (45). Further studies are required to ascertain whether endocytosis is necessary for other actions of PAR2 in the intestine, such as inflammation associated with bacterial proteolysis and food allergies (46).
  • Pan-mugfp mice A segment of genomic DNA containing exon 2 of mouse Pan (P2rll) was sub-cloned from a genomic BAC.
  • the Pan-mugfp gene was synthesized by fusing muGFP to the 3’ end of exon 2.
  • the targeting construct comprised Pan-mugfp, a phosphoglycerine kinase neomycin cassette flanked by loxP sites.
  • the targeting vector was electroporated into C57BL/6J ES cells. Clones were screened by Southern blotting, sequenced and injected into mouse blastocysts. Chimeras were bred with wild-type female mice to generate black Fl progeny. Expression of Pan-mugfp was confirmed by Southern blotting and PCR.
  • Immunofluorescence - PAEG-muGFP was localized using a GFP antibody. Endosomes were detected using EEA1 and Rab7a antibodies and the plasma membrane was identified using E- cadherin antibodies. Neurons were identified using antibodies to NeuN or PGP9.5. RNAScope® in situ hybridization - Probes to F2rll, Gfp, Dnm isoforms were used for in situ hybridization.
  • PAR2-muGFP signaling - KNRK cells expressing PAR2-muGFP or PAR2-HAI I were stimulated with PAR2 agonists and IP-1 formation was measured.
  • HEK293T cells expressing PAR2-muGFP and CellLight organelle markers were challenged with PAR2 agonists and examined by confocal microscopy.
  • mice expressing mouse PAR2 fused at the intracellular C- terminus to muGFP were generated.
  • muGFP harbors stabilizing mutations (Q69L, N164Y), which improve hydrophobic packing in the core and facilitate hydrogen bonding, and a mutation at the dimer interface (F223D), which prevents dimerization (31).
  • muGFP retains fluorescence after harsh fixation and resists undesirable dimerization.
  • a targeting construct comprising Pan-mugfp, a floxed PGK neomycin cassette, and a downstream Kpnl site for Southern screening (Fig. 1A).
  • Five ES clones expressing the construct were identified by Southern blotting (1.25% incorporation); blotting at 3’ and 5’ arm of homology confirmed correct targeting (Fig. IB).
  • Screening with an internal probe to the NEO cassette excluded random integration or insertion of a concatemer. The presence and the integrity of LoxP sites and insertion of Pan-mugfp were verified by sequencing. Two clones were injected into BalbC mouse blastocysts.
  • Example 2 Characterization of Pa -mugfp mice
  • F2rll the gene encoding for PAR2, in Pan-mugfp and wild-type mice was compared using qRT-PCR.
  • F2rll expression in the stomach, duodenum, ileum, colon and dorsal root ganglia (DRG) was not different in Pan-mugfp and wild-type mice (Fig. IE).
  • F2rll was expressed at higher levels in the jejunum of Pan-mugfp mice, for unknown reasons. Thus, in most tissues F2rll is expressed at similar levels in Pan-mugfp and wild-type mice.
  • RNAScope® in situ hybridization was used with probes to F2rll and Gfp to confirm PAR2-muGFP localization in the colon.
  • F2rll and Gfp were prominently detected in colonocytes (Fig. 2C).
  • F2rll but not Gfp was detected in colonocytes.
  • RNAScope® was used to detect PAR2 expression with high sensitivity in DRG neurons.
  • F2rll and Gfp were coexpressed in a subpopulation of DRG neurons (Fig. 2D). F2rll but not Gfp was expressed in DRG neurons of wild-type mice (Fig. 2E).
  • GPCRs Upon activation, many GPCRs, including PAR2, undergo endocytosis (3, 7). GPCR trafficking is usually studied in cell lines overexpressing receptors. Little is known about agonist-evoked trafficking of endogenous GPCRs in intact tissues or organisms, largely due to the inadequate specificity or sensitivity of GPCR antibodies.
  • trypsin 140 nM
  • 2F 100 pM
  • vehicle 60 min, 37°C
  • PAR2-muGFP was principally localized to the basolateral plasma membrane of colonocytes (Fig. 3A, B, arrowheads).
  • Fig. 3A, B, arrows Vesicles containing PAR2-muGFP were co-stained with an antibody to early endosomal antigen- 1 (EEA1), and are thus early endosomes (Fig. 3C, D, arrows). Some vesicles were costained with an antibody to Rab7a, and are late endosomes. Quantification of endocytosis, assessed by the cytosol: plasma membrane pixel intensity, confirmed extensive endocytosis of PAR2-muGFP (Fig. 3E, F). Thus, agonists evoke robust endocytosis of endogenous PAR2 in the intact colon.
  • ESA1 early endosomal antigen- 1
  • Pan-mugfp mice were treated with 3% dextran sulphate sodium (DSS) in drinking water for 7 days to induce mucosal inflammation of the colon. Controls received plain water. DSS induced signs of colitis, including fecal blood, diarrhea, weight loss and colon shortening (Fig. 4A, B) PAR2-muGFP was localized at the basolateral plasma membrane of colonocytes in control mice (Fig. 4C arrowheads). At 24 h post-DSS, PAR2-muGFP was depleted from the plasma membrane and prominently detected in intracellular vesicles of colonocytes (Fig. 4C, arrows).
  • DSS dextran sulphate sodium
  • Pan-mugfp mice were also treated with trinitrobenzene sulphonic acid (TNBS, 4 mg/ml in 0.9% NaCl, 50% ethanol, 150 pl, enema) to induce transmural inflammation of the colon.
  • Control mice received vehicle (0.9%NaCl, 50% ethanol).
  • TNBS-treated mice had extensive transmural inflammation, with mucosal and submucosal infiltration of neutrophils (Fig. 4D, E).
  • PAR2-muGFP was prominently detected in intracellular vesicles (Fig. 4F, arrows) and also present at the plasma membrane (Fig. 4F, arrowheads) of colonocytes.
  • PAFb-muGFP was detected in immune cells of the mucosa (Fig. 4G, arrowheads) and in EEA1 -positive early endosomes of colonocytes (Fig. 4G, arrows).
  • PAFb-muGFP redistributes from the plasma membrane to endosomes of colonocytes. This redistribution is likely attributable to the activation of proteases in the inflamed colon.
  • PAR2 Activated PAR2 undergoes clathrin-mediated endocytosis and traffics to early endosomes and lysosomes (13). PAR2 ubiquitination is necessary for lysosomal targeting (33). Recovery of cellular responsiveness requires GPy-dependent activation of protein kinase D, which liberates newly synthesized PAR2 from the Golgi apparatus and stimulates translocation to the plasma membrane (34, 35).
  • Enhanced bystander bioluminescence resonance energy transfer was used, which capitalizes on the affinity of Renilla (R)-tagged proteins, to study the intracellular trafficking of PAR2 in T84 human colon carcinoma cells (26).
  • ebBRET Enhanced bystander bioluminescence resonance energy transfer
  • BRET1 sensors for PAR2 and subcellular compartments in HEK293T cells were expressed (Fig. 5 A), where BRET1 responses were larger.
  • 2F (10 pM) and trypsin (100 nM) decreased BRET between PARa-Rluc8 and Venus- KRas (plasma membrane) and increased BRET between PAR2-R1UC8 and Venus-Rab5a (early endosome) (Fig. 5H-K).
  • BRET between PAR2-R1UC8 and Venus-Rab7a (late endosome) was unaffected (Fig. 5L, M).
  • 2F increased BRET between PAR2-R1UC8 and Venus-Giantin (cisGolgi) and caused a transient decrease and then a sustained increase in BRET between PAR2- Rluc8 and YFP-TGN-38 (trans-Golgi) (Fig. 5N-Q). Trypsin did not affect BRET between PAR2-R1UC8 and Venus-Giantin but caused a transient decrease in BRET between PAR2- Rluc8 and YFP-TGN-38. 2F and trypsin stimulated BRET between PAR2-R1UC8 and Venus- Rabi la (recycling endosomes) (Fig. 5R, S). DnmK44A inhibited the recruitment of PAR2 to most compartments.
  • PAEG-muGFP was expressed in HEK293T cells expressing markers of early and late endosomes and the Golgi apparatus tagged with red fluorescent protein (RFP), and localized PAR2 and markers by confocal microscopy.
  • 2F (10 pM, 30 min) stimulated trafficking of PAR2-muGFP from the plasma membrane to early endosomes (Fig. 5T).
  • PAR2-muGFP was not detected in late endosomes or the Golgi apparatus by microscopy (Fig. 5U).
  • GPCRs can assemble Ga and PARR signaling complexes in endosomes, which enable sustained signaling of internalized receptors (3, 4, 36, 37).
  • Ga subunits can transduce signals and PARRs serve as scaffolds that organize GPCRs and signaling enzymes. Since agonists stimulated trafficking of PAR2 to early endosomes of T84 and HEK293T cells, the assembly of signaling complexes was examined in this compartment. The activation of Ga q , Gat and Ga s was studied using ebBRET to detect recruitment of mini (m) Ga coupled to Rluc8 to RGFP-CAAX or tdRGFP-Rab5a (Fig. 6A).
  • mGa proteins are N- terminally truncated Ga proteins that freely diffuse within the cytosol and bind to active conformations of GPCRs, which reflects Ga activation.
  • mGa sq and mGa Si were developed by mutating mGa s residues to equivalent Ga q and Gat residues.
  • Recruitment of PARR was assessed by measuring ebBRET between Rluc2-PARR2 and RGFP-CAAX or tdRGFP-Rab5a (Fig. 6A).
  • Luminescence occurs from a complex between PAR2-NP and LgBiT-CAAX or LgBiT-FYVE, which serves as an energy donor for fluorophore-tagged mGa or PARR.
  • 2F 100 pM
  • 2F and trypsin also stimulated nbBRET between PAR2, mGa S i, mGa S q or PARR1 and CAAX or FYVE (Fig. 6I-K).
  • nbBRET signals were maintained for at least 1200 s, suggesting assembly of stable PAR2/mGa or PAR2/PARR complexes.
  • Hypertonic sucrose inhibited agonist-stimulated assembly of PAR2, mGa S i, mGa S q or PARR1 complexes in early endosomes.
  • DynK44A had similar inhibitory effects on signalosome assembly but to a lesser extent.
  • the PAR2 antagonist GB88 prevented 2F and trypsin-stimulated signalosome assembly in endosomes of HEK293T cells. Immunofluorescence and confocal microscopy confirmed localization of HA-LgBiT-CAAX at the plasma membrane and HA-LgBiT-FYVE in endosomes.
  • Example 8 PAR2 endocytosis and the colonic epithelial barrier
  • Tight junctions between colonocytes form a barrier that prevents the ingress of proinflammatory macromolecules and microbes from the lumen. Alterations in tight junction structure contribute to impaired barrier function and inflammation in IBD. PAR2 agonists promote paracellular permeability of the colon (39) by a mechanism that involves PARR- dependent activation of ERK1/2 and redistribution of ZO1 and occludin from tight junctions (3, 26), suggesting a role for endosomal PAR2 signaling.
  • Dnm2 or control shRNA was administered into the colon of Par2-mugfp mice. After 48 h, RNAScope® in situ hybridization revealed ⁇ 50% knockdown of Dnm2 mRNA in colonocytes of mice treated with Dnm shRNA compared to control shRNA (Fig. 8A, B).
  • 2F 100 pM
  • vehicle was administered into the colon by enema (150 pl). Endocytosis of PARa-muGFP, colonic levels of proinflammatory factors, and colonic nociception were assessed.
  • mice receiving control shRNA stimulated endocytosis of PAR2-muGFP in colonocytes after 6 h; Dnm2 shRNA inhibited endocytosis (Fig. 8C).
  • 2F increased mRNA levels of tumor necrosis factor- a (TNF-a,), C-X-C Motif Chemokine Ligand 1 (CXCL1) and interleukin- 10 (IL- 10) in the colon (Fig. 8D-F).
  • Intracolonic 2F decreased withdrawal responses of the abdomen to calibrated von Frey filaments from 1-5 h, which indicates hyperalgesia (Fig. 8G, H).
  • the pronociceptive response to intracolonic 2F was similar in Par2-mugfp and wild-type mice, but was attenuated in PanNci v 1.8 mice that lack PAR2 in Na v 1.8-positive nociceptors (7) (Fig. 81, J). These results implicate PAR2 on Na v 1.8-positive neurons in colonic pain and are in line with reports that proteases induce somatic nociception by activating PAR2 on Na v 1.8 neurons (7).
  • IL-8 is proinflammatory cytokine that is released by colonocytes (40).
  • 2F 10 pM, 4 h
  • IL-8 release into culture medium.
  • 2F stimulated IL-8 release (Fig. 8K).
  • Cathepsin S is activated during colitis and causes visceral hyperalgesia by a PAR2-dependent mechanism in mice. Gastroenterology 141, 1864-1874 el861-1863 (2011).
  • Luminal trypsin may regulate enterocytes through proteinase-activated receptor 2. Proc Natl Acad Sci U S A 94, 8884-8889 (1997).

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Abstract

La présente divulgation concerne des entités chimiques et des compositions les contenant qui sont utiles pour traiter une affection associée à une inflammation et/ou à une douleur gastro-intestinale ("GI").
PCT/US2023/061588 2022-01-28 2023-01-30 Méthodes de traitement de la douleur Ceased WO2023147555A2 (fr)

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