WO2013111014A2 - Méthodes et compositions destinées au traitement de la perte neuronale dans des maladies intestinales inflammatoires - Google Patents

Méthodes et compositions destinées au traitement de la perte neuronale dans des maladies intestinales inflammatoires Download PDF

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WO2013111014A2
WO2013111014A2 PCT/IB2013/000463 IB2013000463W WO2013111014A2 WO 2013111014 A2 WO2013111014 A2 WO 2013111014A2 IB 2013000463 W IB2013000463 W IB 2013000463W WO 2013111014 A2 WO2013111014 A2 WO 2013111014A2
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pannexin
inhibitor
neurons
function
enteric
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WO2013111014A3 (fr
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Brian D. GULBRANSEN
Keith A. SHARKEY
Roger J. THOMPSON
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Uti Limited Partnership
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    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • 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
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the present invention relates generally to the fields of biology and medicine. More particularly, it concerns treatment of neuron loss in inflammatory bowel diseases (e.g., Crohn's disease, ulcerative colitis) with agents that inhibit pannexin-1.
  • inflammatory bowel diseases e.g., Crohn's disease, ulcerative colitis
  • GI gastrointestinal
  • Enteric neuropathies are associated with a wide array of disorders including the inflammatory bowel diseases (IBD) ulcerative colitis and Crohn's disease, achalasia, chronic intestinal pseudo-obstruction, chronic constipation/megacolon, infectious colitis and irritable bowel syndrome (IBS) (De Giorgio et ah, 2004).
  • IBD inflammatory bowel diseases
  • Crohn's disease achalasia
  • chronic intestinal pseudo-obstruction chronic constipation/megacolon
  • infectious colitis and irritable bowel syndrome IBS
  • neuroinflammation is a key process driving neuron death.
  • extracellular ATP levels increase dramatically and activate microglial P2X7 receptors (P2X7Rs), stimulating microglial release of proinflammatory, neurotoxic factors such as tumor-necrosis factor (TNF)-a, nitric oxide, and interleukin (IL)-l (Lazarowski et ah, 2000; Block et ah, 2007).
  • TNF tumor-necrosis factor
  • IL interleukin
  • P2X7Rs Pannexin-1 channels
  • inflammasome a multiprotein complex called the inflammasome
  • the inventors have addressed this issue by investigating the cause of neuron death in animal models of experimental colitis that replicate aspects of IBDs (Linden et al, 2005), increasingly prevalent and debilitating disorders associated with neuron loss in the enteric nervous system (ENS) (De Giorgio et al, 2004). Neurodegeneration and synaptic remodeling within the ENS are thought to underlie the long-term alterations in gastrointestinal motility and secretion that persist well beyond the initial inflammatory insult (Mawe et al, 2009), but the causal mechanisms initiating enteric neural death are unknown.
  • Altered purinergic signaling is increasingly recognized as a key contributing factor to pathophysiology in IBD in both humans and experimental models. Increased release (Wynn et al, 2004; Lomax et al, 2005) and decreased hydrolysis (Wynn et al, 2004; Friedman et al , 2009) elevates extracellular purine levels during inflammation and purine receptor genes and proteins are dysregulated in affected humans (Rybaczyk et al, 2009; Yiangou et al, 2001) and animals models (Guzman et al, 2006).
  • a method of inhibiting enteric neuron cell death comprising contacting an enteric neuron with an inhibitor of pannexin- 1 signaling.
  • the inhibitor of pannexin- 1 signaling may be an inhibitor of pannexin- 1 expression, such as a pannexin- 1 siRNA.
  • the inhibitor of pannexin- 1 signaling may be an inhibitor of pannexin- 1 function, such as an inhibitor that binds pannexin- 1 like an anti-pannexin- 1 antibody or fragment thereof.
  • the inhibitor of pannexin- 1 function may interfere with pannexin- 1 binding to a pannexin- 1 binding partner, such as a pannexin- 1 peptide or pannexin- 1 binding partner peptide, such as a P2X7 peptide.
  • the inhibitor of pannexin- 1 function may be an organopharmaceutical small molecule, such as carbenoxolone or probenecid or mefloquine.
  • a method of treating neuron loss associated with inflammatory bowel disease (IBD) in a subject comprising administering to said subject an inhibitor of pannexin- 1 signaling.
  • the inhibitor of pannexin- 1 signaling may be an inhibitor of pannexin- 1 expression, such as a pannexin- 1 siRNA.
  • the inhibitor of pannexin- 1 signaling may be an inhibitor of pannexin- 1 function, such as an inhibitor that binds pannexin- 1 like an anti-pannexin- 1 antibody or fragment thereof.
  • the inhibitor of pannexin- 1 function may interfere with pannexin- 1 binding to a pannexin- 1 binding partner, such as a pannexin- 1 peptide or pannexin- 1 binding partner peptide, such as a P2X7 peptide.
  • the inhibitor of pannexin- 1 function may be an organopharmaceutical small molecule, such as carbenoxolone or probenecid or mefloquine.
  • the IBD may be Crohn's disease, ulcerative colitis, achalasia, chronic intestinal pseudo-obstruction, chronic constipation/megacolon, infectious colitis or irritable bowel syndrome.
  • the inhibitor of pannexin-1 signaling may be administered to said subject a second time.
  • the inhibitor of pannexin-1 signaling may be administered orally, intravenously, by suppository or by inhalation.
  • the method may further comprise administering to said subject a second IBD therapy, such as an antibiotic, an antifungal, an anti-inflammatory agent, and/or surgery.
  • neuronal cell death may be inhibited or reduced
  • a post-inflammatory bowel functional deficit is inhibited or reversed, such as reduction or inhibition of contractile motility or inflammation- induced secretory defects
  • macroscopic bowel tissue damage may reduced or inhibited
  • weight loss may be reduced or inhibited
  • patient discomfort may be reduced or inhibited. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.
  • compositions and kits of the invention can be used to achieve methods of the invention.
  • FIG. 1 Myenteric neuron loss is a consistent feature of experimental colitis in animal models.
  • DNBS 2,4-dinitrobenzene sulfonic acid treatment
  • DSS
  • FIGS. 2(a)-(f) Activating neural P2X7Rs is necessary for neuron death during inflammation and stimulating neuronal P2X7Rs in situ replicates in vivo neural death in a process requiring caspase activation and pannexin-1 opening.
  • Inhibiting caspase activity with zVAD (80 ⁇ ) or pannexin-1 with lOPanx (100 ⁇ ) protects against P2X7R-mediated neural death.
  • enteric neurons express pannexin-1 channels (FIGS. 3a-a' ").
  • enteric glia GFAP, green, FIG. 3 a
  • pannexin-l-ir magenta, FIG. 3a'
  • Boxed area in FIG. 3 a" is enlarged in FIG. 3 a' " to demonstrate that while pannexin-1 expression is confined to neurons, pannexin-1 expressing neurons are intimately associated with enteric glia.
  • Scale bar 20 ⁇ .
  • Enteric neurons ubiquitously express ASC (FIGS.
  • FIG. 3b-b Enteric neurons immunoreactive for ASC (magenta, FIG. 3b') are surrounded by GFAP-ir glial cells (green, FIG. 3b). Overlay of FIG. 3b and FIG. 3b' demonstrates that ASC expression within the myenteric plexus is confined to neurons (FIG. 3b"). (FIG.
  • FIGS. 4a-a Representative images (pseudocolored) from a Ca 2+ imaging experiment where a whole-mount preparation of the myenteric plexus was challenged with the P2X7R agonist BzATP (100 ⁇ , 30 sec). At rest, baseline Ca 2+ levels in neurons and glia are low (FIGS. 4a, 4b), increasing rapidly following bath application of BzATP first in neurons (FIGS. 4a', 4b) and then recruiting surrounding glia (FIGS. 4a", 4b). (FIG.
  • FIG. 4i Summary data of mean ( ⁇ s.e.m.) peak Ca 2+ responses (normalized to initial BzATP response magnitude within the same ganglion) in neurons (red) or glia (blue) in response to sham block and BzATP alone or in combination with various inhibitors. Significance determined by ANOVA with *p ⁇ 0.05, **p ⁇ 0.001, ***p ⁇ 0.0001 versus sham block and BzATP alone.
  • FIG. 5b Representative photomicrographs showing nNOS-ir neurons in the normal (control) and inflamed (DNBS) colon myenteric plexus.
  • ANOVA; n 8 animals saline, 7 DNBS, 7 saline+PB, and 6 DNBS+PB).
  • FIG. 5e Impaired inhibitory nitrergic innervation enhances EFS-elicited contractions after DNBS colitis.
  • Pannexin- 1 inhibition is neuroprotective in a clinical model of colitis and changes in the P2X7-pannexin-l pathway expressed by human enteric neurons are involved in the pathophysiology of Crohn's disease.
  • FIG. 6a Experimental scheme to replicate the relapsing/remitting nature of colitis.
  • FIG. 6b Therapeutic intervention with probenecid before a second bout of colitis significantly improved myenteric neuron survival (28.2 ⁇ 3% reduction in neural packing density with saline treatment vs.
  • FIG. 6c Human enteric neurons express P2X7. Image of a representative myenteric ganglia from human colon show numerous P2X7-ir neurons (arrows).
  • FIGS. 6d-f Human enteric neurons express pannexin-1. Representative images of pannexin-l-ir in the human myenteric (FIG. 6d) and submucosal (FIG. 6e) plexuses of the colon. Dashed line in FIG.
  • FIGS. 6d outlines a myenteric ganglia filled with several pannexin-l-ir neurons (arrows) and surrounded by longitudinal (LM) and circular (CM) muscle layers.
  • FIG. 6g Normal pannexin-l-ir within the human colonic myenteric plexus (control, FIG. 6g) is maintained during ulcerative colitis (UC, FIG. 6g') but lost in Crohn's disease (CD, FIG. 6g").
  • FIG. 6h Model of enteric neuron death during colonic inflammation. Tissue damage and inflammation generate abnormally high levels of extracellular ATP that activates neural P2X7Rs, opening pannexin-1 channels. Pannexin-1 acts as a conduit for ATP release from stimulated neurons and activates neuronal caspases through Asc leading to cell death. Neuronally released ATP converted to ADP by ectonucleotidases activates surrounding enteric glial cells and ATP itself could propagate inflammatory responses by stimulating P2X7Rs on adjacent neurons.
  • FIG. 7 Representative whole-mount preparation used for an in vitro cell death assay stained with hematoxylin and eosin (H&E).
  • FIG. 8a-a Dogiel type I neurons identified by immunoreactivity to nNOS (magenta, FIG. 8a') are highly immunoreactive for pannexin-1 (green, FIG. 8a) and the majority of nNOS neurons co-express pannexin-1 (overlay in FIG. 8a"). Asterices denote three pannexin-1 -nNOS dual labeled neurons. 97 of 191 (50.8%) pannexin-1 immunoreactive neurons co-express nNOS. (FIGS. 8b-b”) Dogiel type II neurons (identified by immunoreactivity to calretinin, green, FIG. 8b') also express pannexin-1 (magenta, FIG.
  • pannexin-1 immunoreactive neurons 118 co-expressed calretinin (39.6%; overlay in FIG. 8b"; asterices identify two pannexin-1 -calretinin double-labeled neurons).
  • TTX tetrodotoxin
  • 1 ⁇ 21 ganglia BzATP glia, 14 BzATP- TTX glia, 20 BzATP neurons, and 15 BzATP-TTX neurons.
  • FIGS. 10 Acute sympathectomy with 6-hydroxydopamine (60HDA) does not affect enteric neuron or glial responses to BzATP.
  • FIGS. 10c-c' Images of Fluo-4 fluorescence in myenteric plexus- longitudinal muscle preparations maintained in culture for three days to sever all extrinsic connections at baseline (FIG. 10c) and when stimulated with BzATP (FIG. 10c'). Note that neurons and glia still respond to the P2X7 agonist in the absence of extrinsic innervation.
  • FIG. 12 8 animals saline, 3 saline-suramin, 5 saline-L- NAME, 7 saline+PB, 3 saline+PB-suramin, 4 saline+PB-L-NAME, 6 PB+DNBS, 3 PB+DNBS-sura
  • enteric neuronal death during colitis involves direct activation and participation of the P2X7R/Panxl/ASC/caspase inflammasome that is endogenous to nitrergic enteric neurons.
  • Their observations provide strong evidence that Panxl opening mediates neuron death in an in vivo model of neurodegeneration that is highly relevant to human IBDs and support a model whereby elevated levels of extracelluar ATP generated by tissue damage acts on neuronal P2X7Rs, consequently activating Panxl and caspases (FIG. 6g).
  • Panxl acts at a critical junction in the activation of neuron death during colitis making Panxl an attractive target for therapeutic interventions.
  • human enteric neurons express Panxl, inhibiting Panxl during, or between, bouts of colitis improves neuron survival and function and maintains colonic contractility.
  • Dysregulated gut immunity is generally thought to drive intestinal inflammation in IBD.
  • enteric neurons contribute to the severity of intestinal inflammation through an unknown mechanism (Margolis et al, 201 1).
  • the inventors also describe a prospective mechanism for this phenomenon by demonstrating activation of neuronal P2X7Rs and Panx-1 during intestinal inflammation.
  • P2X7R activation in macrophages has both autocrine and paracrine cytotoxic effects mediated at least in part by Panxl activation of the NLRP3 inflammasome and subsequent stimulation of caspase-1 and IL- ⁇ secretion (Pelegrin et al, 2008).
  • Panx-1 opening could exacerbate neuronal damage by activating P2X7Rs on adjacent neurons.
  • alterations to the intrinsic physiological properties of enteric neurons induce long-lasting changes in the enteric control of gut motility (Mawe et al, 2009).
  • Enhanced excitability of sensory neurons Liden et al, 2003
  • synaptic facilitation Limax et al, 2005
  • impaired inhibitory neuromuscular transmission Krauter et al, 2007
  • the inventors show that decreased inhibitory nitrergic neuromuscular transmission following inflammation is likely mediated by a loss of nitrergic neurons in the myenteric plexus; similar to previous findings in rats (Bossone et al, 2001 ; Mizuta et al, 2000) and rabbits (Depoortere et al, 2002) but unlike guinea pigs where inhibitory purinergic neuromuscular transmission is impaired (Krauter et al, 2007). Why nitrergic neurons are particularly susceptible to inflammatory insults is unknown, but the inventors speculate that the oxidative environment within nitrergic neurons could contribute.
  • IBD inflammatory bowel disease
  • IBD symptoms may include inflammation of the intestine and resulting in abdominal cramping and persistent diarrhea.
  • IBD diseases include ulcerative colitis (UC), Crohn's disease (CD), indeterminate colitis, chronic colitis, discontinuous or patchy disease, ileal inflammation, extracolonic inflammation, granulomatous inflammation in response to ruptured crypts, aphthous ulcers, transmural inflammation, microscopic colitis, diverticulitis and diversion colitis.
  • Ulcerative colitis is a disease that causes inflammation and sores, called ulcers, in the lining of the large intestine.
  • the inflammation usually occurs in the rectum and lower part of the colon, but it may affect the entire colon.
  • Ulcerative colitis rarely affects the small intestine except for the end section, called the terminal ileum. Ulcerative colitis may also be called colitis or proctitis.
  • the inflammation makes the colon empty frequently, causing diarrhea. Ulcers form in places where the inflammation has killed the cells lining the colon; the ulcers bleed and produce pus.
  • Ulcerative colitis may occur in people of any age, but most often it starts between ages 15 and 30, or less frequently between ages 50 and 70. Children and adolescents sometimes develop the disease. Ulcerative colitis affects men and women equally and appears to run in some families.
  • ulcerative colitis The most common symptoms of ulcerative colitis are abdominal pain and bloody diarrhea. Patients also may experience fatigue, weight loss, loss of appetite, rectal bleeding, and loss of body fluids and nutrients. About half of patients have mild symptoms. Others suffer frequent fever, bloody diarrhea, nausea, and severe abdominal cramps. Ulcerative colitis may also cause problems such as arthritis, inflammation of the eye, liver disease (hepatitis, cirrhosis, and primary sclerosing cholangitis), osteoporosis, skin rashes, and anemia. No one knows for sure why problems occur outside the colon. Peoples think these complications may occur when the immune system triggers inflammation in other parts of the body. Some of these problems go away when the colitis is treated.
  • a thorough physical exam and a series of tests may be required to diagnose ulcerative colitis.
  • Blood tests may be done to check for anemia, which could indicate bleeding in the colon or rectum. Blood tests may also uncover a high white blood cell count, which is a sign of inflammation somewhere in the body.
  • the doctor can detect bleeding or infection in the colon or rectum. The doctor may do a colonoscopy or sigmoidoscopy. For either test, the doctor inserts an endoscope - a long, flexible, lighted tube connected to a computer and TV monitor - into the anus to see the inside of the colon and rectum. The doctor will be able to see any inflammation, bleeding, or ulcers on the colon wall.
  • the doctor may do a biopsy, which involves taking a sample of tissue from the lining of the colon to view with a microscope.
  • a barium enema x-ray of the colon may also be required. This procedure involves filling the colon with barium, a chalky white solution. The barium shows up white on x-ray film, allowing the doctor a clear view of the colon, including any ulcers or other abnormalities that might be there.
  • ulcerative colitis Treatment for ulcerative colitis depends on the seriousness of the disease. Most people are treated with medication. In severe cases, a patient may need surgery to remove the diseased colon. Surgery is the only cure for ulcerative colitis. Some people whose symptoms are triggered by certain foods are able to control the symptoms by avoiding foods that upset their intestines, like highly seasoned foods, raw fruits and vegetables, or milk sugar (lactose). Each person may experience ulcerative colitis differently, so treatment is adjusted for each individual. Emotional and psychological support is important. Some people have remissions - periods when the symptoms go away - that last for months or even years. However, most patients' symptoms eventually return. This changing pattern of the disease means one cannot always tell when a treatment has helped. Some people with ulcerative colitis may need medical care for some time, with regular doctor visits to monitor the condition.
  • the goal of therapy is to induce and maintain remission, and to improve the quality of life for people with ulcerative colitis.
  • Several types of drugs are available:
  • Sulfasalazine is a combination of sulfapyridine and 5-ASA and is used to induce and maintain remission.
  • the sulfapyridine component carries the anti- inflammatory 5-ASA to the intestine.
  • sulfapyridine may lead to side effects such as include nausea, vomiting, heartburn, diarrhea, and headache.
  • Other 5-ASA agents such as olsalazine, mesalamine, and balsalazide, have a different carrier, offer fewer side effects, and may be used by people who cannot take sulfasalazine.
  • 5-ASAs are given orally, through an enema, or in a suppository, depending on the location of the inflammation in the colon. Most people with mild or moderate ulcerative colitis are treated with this group of drugs first.
  • Corticosteroids - such as prednisone and hydrocortisone also reduce inflammation.
  • Corticosteroids also known as steroids
  • These drugs can cause side effects such as weight gain, acne, facial hair, hypertension, mood swings, and an increased risk of infection. For this reason, they are not recommended for long-term use.
  • Immunomodulators such as azathioprine and 6-mercapto-purine (6-MP) reduce inflammation by affecting the immune system. They are used for patients who have not responded to 5-ASAs or corticosteroids or who are dependent on corticosteroids. However, immunomodulators are slow-acting and may take up to 6 months before the full benefit is seen. Patients taking these drugs are monitored for complications including pancreatitis and hepatitis, a reduced white blood cell count, and an increased risk of infection. Cyclosporine A may be used with 6-MP or azathioprine to treat active, severe ulcerative colitis in people who do not respond to intravenous corticosteroids.
  • Other drugs may be given to relax the patient or to relieve pain, diarrhea, or infection.
  • a person may have severe bleeding or severe diarrhea that causes dehydration.
  • the doctor will try to stop diarrhea and loss of blood, fluids, and mineral salts.
  • the patient may need a special diet, feeding through a vein, medications, or sometimes surgery.
  • proctocolectomy Surgery to remove the colon and rectum, known as proctocolectomy, is followed by one of the following:
  • Ileostomy in which the surgeon creates a small opening in the abdomen, called a stoma, and attaches the end of the small intestine, called the ileum, to it. Waste will travel through the small intestine and exit the body through the stoma.
  • the stoma is about the size of a quarter and is usually located in the lower right part of the abdomen near the beltline. A pouch is worn over the opening to collect waste, and the patient empties the pouch as needed.
  • Ileoanal anastomosis or pull-through operation, which allows the patient to have normal bowel movements because it preserves part of the anus.
  • the surgeon removes the diseased part of the colon and the inside of the rectum, leaving the outer muscles of the rectum.
  • the surgeon then attaches the ileum to the inside of the rectum and the anus, creating a pouch. Waste is stored in the pouch and passed through the anus in the usual manner. Bowel movements may be more frequent and watery than before the procedure. Inflammation of the pouch (pouchitis) is a possible complication.
  • Crohn's disease As with ulcerative colitis, O-glycans have been suggested as playing a role in Crohn's disease, another inflammatory disease of the gastro-intestinal tract. Crohn's disease is characterized by intestinal inflammation and the development of intestinal stenosis and fistulas; neuropathy often accompanies these symptoms.
  • a failure of the intestinal mucosal barrier possibly resulting from genetic susceptibilities and environmental factors (e.g., smoking), exposes the immune system to antigens from the intestinal lumen including bacterial and food antigens (e.g., Soderholm et al, 1999; Hollander et ah, 1986; Hollander, 1992).
  • Another hypothesis is that persistent intestinal infection by pathogens such as Mycobacterium paratuberculosis, Listeria monocytogenes, abnormal Escherichia coli, or paramyxovirus, stimulates the immune response; or alternatively, symptoms result from a dysregulated immune response to ubiquitous antigens, such as normal intestinal microflora and the metabolites and toxins they produce (Sartor, 1997).
  • pathogens such as Mycobacterium paratuberculosis, Listeria monocytogenes, abnormal Escherichia coli, or paramyxovirus
  • ASCA IgA and IgG anti-Sacccharomyces cerevisiae antibodies
  • Cytokines are small secreted proteins or factors (5 to 20 kD) that have specific effects on cell- to-cell interactions, intercellular communication, or the behavior of other cells. Cytokines are produced by lymphocytes, especially T H 1 and T H 2 lymphocytes, monocytes, intestinal macrophages, granulocytes, epithelial cells, and fibroblasts (reviewed in Rogler &. Andus, 1998; Galley & Webster, 1996).
  • cytokines are pro-inflammatory (e.g., TNF-a, IL-l(a and ⁇ ), IL-6, IL-8, IL-12, or leukemia inhibitory factor (LIF)); others are anti-inflammatory (e.g., IL-1 receptor antagonist, IL-4, IL-10, IL-1 1, and TGF- ⁇ ).
  • pro-inflammatory e.g., TNF-a, IL-l(a and ⁇ )
  • IL-6 IL-6
  • IL-8 IL-12
  • LIF leukemia inhibitory factor
  • anti-inflammatory e.g., IL-1 receptor antagonist, IL-4, IL-10, IL-1 1, and TGF- ⁇ .
  • TNF-a and IL-6 are secreted into the blood circulation, and TNF-a, IL-1, IL-6, and IL-8 are produced in excess locally by mucosal cells (id.; Funakoshi et al, 1998).
  • cytokines can have far-ranging effects on physiological systems including bone development, hematopoiesis, and liver, thyroid, and neuropsychiatric function.
  • Anti-inflammatory drugs such as 5-aminosalicylates (e.g., mesalamine) or corticosteroids
  • 5-aminosalicylates e.g., mesalamine
  • corticosteroids are typically prescribed, but are not always effective (reviewed in Botoman et al, 1998). Immunosuppression with cyclosporine is sometimes beneficial for patients resistant to or intolerant of corticosteroids (Brynskov et al, 1989). In Crohn's disease, a dysregulated immune response is skewed toward cell-mediated immunopathology (Murch, 1998).
  • immunosuppressive drugs such as cyclosporine, tacrolimus, and mesalamine have been used to treat corticosteroid-resistant cases of Crohn's disease with mixed success (Brynskov et al, 1989; Fellerman et al, 1998). Nevertheless, surgical correction is eventually required in 90% of patients; 50% undergo colonic resection (Leiper et al, 1998; Makowiec et al, 1998). The recurrence rate after surgery is high, with 50% requiring further surgery within 5 years (Leiper et al, 1998; Besnard et al, 1998).
  • cytokine antagonists e.g., IL-lra
  • inhibitors e.g., of IL- ⁇ ⁇ converting enzyme and antioxidants
  • anti-cytokine antibodies Rosman and Andus, 1998; van Hogezand & Verspaget, 1998; Reimund et ah, 1998; Lugering et ah, 1998; McAlindon et ah, 1998.
  • Monoclonal antibodies against TNF-a have been tried with some success in the treatment of Crohn's disease (Targan et ah, 1997; Stack et ah, 1997; van Dullemen et ah, 1995).
  • U.S. Patent 5,599,795 discloses a method for the prevention and treatment of Crohn's disease in human patients. Their method was directed to sterilizing the intestinal tract with at least one antibiotic and at least one anti-fungal agent to kill off the existing flora and replacing them with different, select, well-characterized bacteria taken from normal humans.
  • Pannexins are a family of vertebrate proteins (pannexin- 1, -2 and -3) identified by their homology to the invertebrate innexins. While innexins are responsible for forming gap junctions in invertebrates, the pannexins have been shown to predominantly exist as large transmembrane channels connecting the intracellular and extracellular space, allowing the passage of ions and small molecules between these compartments (such as ATP and sulforhodamine B).
  • Pannexins are involved in early stages of innate immunity through their interaction with the P2X7 purinergic receptor. Activation of the pannexin channel through binding of ATP to P2X7 receptor leads to the release of IL- ⁇ ⁇ . Hypothetical roles of pannexins in the nervous system include participating in sensory processing, synchronization between hippocampus and cortex, hippocampal plasticity, and propagation of the calcium waves. Calcium waves are supported by glial cells, which help maintain and modulate neuronal metabolism. According to one hypothesis, pannexins also may participate in pathological reactions, including the neural damage after ischemia and subsequent cell death. Pannexin channels are involved in the release of ATP from cells.
  • pannexins and connexins are very similar, consisting of 4 transmembrane domains, 2 extracellular and 1 intracellular loop, along with intracellular N- and C-terminal tails. Despite this shared topology, the protein families do not share enough sequence similarity to confidently infer common ancestry. Pannexins may also be involved in the process of tumorigenesis. Particularly, PANX2 expression levels predict post diagnosis survival for patients with glial tumors. III. THERAPIES
  • the present invention also permits therapeutic intervention, with respect to patients having various IBD's or pre-clinical conditions that could lead to IBD. Therapeutic approaches for neuronal loss in IBD are therefore discussed below.
  • Antisense methodology takes advantage of the fact that nucleic acids tend to pair with "complementary" sequences.
  • complementary it is meant that polynucleotides are those capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Inclusion of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.
  • Antisense polynucleotides when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability.
  • Antisense RNA constructs, or DNA encoding such antisense RNA's may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.
  • Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. It is contemplated that the most effective antisense constructs will include regions complementary to intron/exon splice junctions. Thus, it is proposed that a preferred embodiment includes an antisense construct with complementarity to regions within 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected.
  • complementary or “antisense” means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated. For example, an antisense construct which has limited regions of high homology, but also contains a non-homologous region (e.g., ribozyme; see below) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.
  • ribozyme e.g., ribozyme; see below
  • Ribozymes may be advantageous to combine portions of genomic DNA with cDNA or synthetic sequences to generate specific constructs. For example, where an intron is desired in the ultimate construct, a genomic clone will need to be used.
  • the cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.
  • Ribozymes are RNA- protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cook, 1987; Gerlach et ah, 1987; Forster and Symons, 1987).
  • ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cook et al, 1981 ; Michel and Westhof, 1990; Reinhold-Hurek and Shub, 1992).
  • This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.
  • IGS internal guide sequence
  • Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cook et ah, 1981).
  • U.S. Patent 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes.
  • sequence-specific ribozyme-mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et ah, 1991; Sarver et ah, 1990).
  • ribozymes elicited genetic changes in some cells lines to which they were applied; the altered genes included the oncogenes H-ras, c-fos and genes of HIV. Most of this work involved the modification of a target mRNA, based on a specific mutant codon that is cleaved by a specific ribozyme.
  • RNA interference also referred to as "RNA-mediated interference” or RNAi
  • RNAi Double-stranded RNA
  • dsRNA double-stranded RNA
  • dsRNA activates post-transcriptional gene expression surveillance mechanisms that appear to function to defend cells from virus infection and transposon activity (Fire et ah, 1998; Grishok et ah, 2000; Ketting et ah, 1999; Lin and Avery et ah, 1999; Montgomery et ah, 1998; Sharp and Zamore, 2000; Tabara et ah, 1999).
  • RNAi offers major experimental advantages for study of gene function. These advantages include a very high specificity, ease of movement across cell membranes, and prolonged down-regulation of the targeted gene (Fire et ah, 1998; Grishok et ah, 2000; Ketting et ah, 1999; Lin and Avery et ah, 1999; Montgomery et ah, 1998; Sharp et ah, 1999; Sharp and Zamore, 2000; Tabara et ah, 1999). Moreover, dsRNA has been shown to silence genes in a wide range of systems, including plants, protozoans, fungi, C.
  • RNAi acts post-transcriptionally, targeting RNA transcripts for degradation. It appears that both nuclear and cytoplasmic RNA can be targeted (Bosher and Labouesse, 2000).
  • siRNAs must be designed so that they are specific and effective in suppressing the expression of the genes of interest. Methods of selecting the target sequences, i.e., those sequences present in the gene or genes of interest to which the siRNAs will guide the degradative machinery, are directed to avoiding sequences that may interfere with the siR A's guide function while including sequences that are specific to the gene or genes. Typically, siR A target sequences of about 21 to 23 nucleotides in length are most effective. This length reflects the lengths of digestion products resulting from the processing of much longer RNAs as described above (Montgomery et ah, 1998).
  • siRNAs has been mainly through direct chemical synthesis; through processing of longer, double-stranded RNAs through exposure to Drosophila embryo lysates; or through an in vitro system derived from S2 cells. Use of cell lysates or in vitro processing may further involve the subsequent isolation of the short, 21-23 nucleotide siRNAs from the lysate, etc., making the process somewhat cumbersome and expensive.
  • Chemical synthesis proceeds by making two single stranded RNA-oligomers followed by the annealing of the two single-stranded oligomers into a double stranded RNA. Methods of chemical synthesis are diverse. Non-limiting examples are provided in U.S. Patents 5,889, 136, 4,415,723, and 4,458,066, expressly incorporated herein by reference, and in Wincott et ah (1995).
  • RNA sequences having di-nucleotide overhangs may provide the greatest level of suppression.
  • These protocols primarily use a sequence of two (2'-deoxy) thymidine nucleotides as the di- nucleotide overhangs. These dinucleotide overhangs are often written as dTdT to distinguish them from the typical nucleotides incorporated into RNA.
  • the literature has indicated that the use of dT overhangs is primarily motivated by the need to reduce the cost of the chemically synthesized RNAs. It is also suggested that the dTdT overhangs might be more stable than UU overhangs, though the data available shows only a slight ( ⁇ 20%) improvement of the dTdT overhang compared to an siRNA with a UU overhang.
  • siRNAs are found to work optimally when they are in cell culture at concentrations of 25-100 nM, but concentrations of about 100 nM have achieved effective suppression of expression in mammalian cells. siRNAs have been most effective in mammalian cell culture at about 100 nM. In several instances, however, lower concentrations of chemically synthesized siRNA have been used (Caplen, et ah, 2000; Elbashir et ah, 2001).
  • RNA for use in siRNA may be chemically or enzymatically synthesized. Both of these texts are incorporated herein in their entirety by reference.
  • the enzymatic synthesis contemplated in these references is by a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g., T3, T7, SP6) via the use and production of an expression construct as is known in the art. For example, see U.S. Patent 5,795,715.
  • the contemplated constructs provide templates that produce RNAs that contain nucleotide sequences identical to a portion of the target gene.
  • the length of identical sequences provided by these references is at least 25 bases, and may be as many as 400 or more bases in length.
  • RNA single-stranded RNA is enzymatically synthesized from the PCR products of a DNA template, preferably a cloned cDNA template and the RNA product is a complete transcript of the cDNA, which may comprise hundreds of nucleotides.
  • WO 01/36646 incorporated herein by reference, places no limitation upon the manner in which the siRNA is synthesized, providing that the RNA may be synthesized in vitro or in vivo, using manual and/or automated procedures.
  • RNA polymerase e.g., T3, T7, SP6
  • RNA interference no distinction in the desirable properties for use in RNA interference is made between chemically or enzymatically synthesized siRNA.
  • the templates used are preferably of between 40 and 100 base pairs, and which is equipped at each end with a promoter sequence.
  • the templates are preferably attached to a solid surface. After transcription with RNA polymerase, the resulting dsRNA fragments may be used for detecting and/or assaying nucleic acid target sequences. 4. Antibodies
  • Antibodies to pannexin-1 are another class of inhibitory molecules.
  • Antibodies may be produced by standard methods well known in the art (see, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; U.S. Patent 4, 196,265).
  • MAbs may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as FPLC or affinity chromatography.
  • Fragments of the monoclonal antibodies of the invention can be obtained from the purified monoclonal antibodies by methods including digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction.
  • monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer.
  • single-chain and chimeric antibodies the latter having specificity for pannexin-1 and another ligand on target cells.
  • RNA can be isolated from the hybridoma line and the antibody genes obtained by RT-PCR and cloned into an immunoglobulin expression vector.
  • combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the cell lines and phagemids expressing appropriate antibodies are selected by panning using viral antigens.
  • a monoclonal antibody that is of the Immunoglobulin M (IgM class).
  • IgM's are the primary antibodies against A and B antigens on red blood cells. IgM is by far the physically largest antibody in the human circulatory system. It is the first antibody to appear in response to initial exposure to antigen.
  • IgM forms polymers where multiple immunoglobulins are covalently linked together with disulfide bonds, mostly as a pentamer but also as a hexamer. IgM has a molecular mass of approximately 900 kDa (in its pentamer form). Because each monomer has two antigen binding sites, a pentameric IgM has 10 binding sites. Typically, however, IgM cannot bind 10 antigens at the same time because the large size of most antigens hinders binding to nearby sites.
  • the J chain is found in pentameric IgM but not in the hexameric form, perhaps due to space constraints in the hexameric complex. Pentameric IgM can also be made in the absence of J chain. At present, it is still uncertain what fraction of normal pentamer contains J chain, and to this extent it is also uncertain whether a J chain-containing pentamer contains one or more than one J chain.
  • IgM is a large molecule, it cannot diffuse well, and is found in the interstitium only in very low quantities. IgM is primarily found in serum; however, because of the J chain, it is also important as a secretory immunoglobulin. However, due to its polymeric nature, IgM possesses high avidity, and is particularly effective at complement activation. By itself, IgM is an ineffective opsonin; however it contributes greatly to opsonization by activating complement and causing C3b to bind to the antigen.
  • IgM is the first immunoglobulin expressed by mature B cells. It is also the first immunoglobulin expressed in the fetus (around 20 weeks) and also phylogenetically the earliest antibody to develop. IgM antibodies appear early in the course of an infection and usually reappear, to a lesser extent, after further exposure. IgM antibodies do not pass across the human placenta (only isotype IgG). These two biological properties of IgM make it useful in the diagnosis of infectious diseases. Demonstrating IgM antibodies in a patient's serum indicates recent infection, or in a neonate's serum indicates intrauterine infection (e.g., congenital rubella).
  • IgM in normal serum is often found to bind to specific antigens, even in the absence of prior immunization. For this reason IgM has sometimes been called a "natural antibody.” This phenomenon is probably due to the high avidity of IgM that allow it to bind detectably even to weakly cross-reacting antigens that are naturally occurring.
  • the IgM antibodies that bind to the red blood cell A and B antigens might be formed in early life as a result of exposure to A- and B-like substances that are present on bacteria or perhaps also on plant materials.
  • IgM antibodies are mainly responsible for the clumping (agglutination) of red blood cells if the recipient of a blood transfusion receives blood that is not compatible with their blood type.
  • IgM is more sensitive to denaturation by 2-mercaptoethanol than IgG. This technique was historically used to distinguish between these isotypes before specific anti-IgG and anti- IgM secondary antibodies for immunoassays became commercially available. Serum samples would be tested for reactivity with an antigen before or after 2-mercaptoethanol treatment to determine whether the activity was due to IgM or IgG.
  • reasons such as improved expression, improved cross-reactivity, diminished off-target binding or abrogation of one or more natural effector functions, such as activation of complement or recruitment of immune cells (e.g., T cells).
  • immune cells e.g., T cells
  • Hybridomas may cultured, then cells lysed, and total RNA extracted. Random hexamers may be used with RT to generate cDNA copies of RNA, and then PCR performed using a multiplex mixture of PCR primers expected to amplify all human variable gene sequences. PCR product can be cloned into pGEM-T Easy vector, then sequenced by automated DNA sequencing using standard vector primers. Assay of binding and neutralization may be performed using antibodies collected from hybridoma supernatants and purified by FPLC, using Protein G columns.
  • Lonza has developed a generic method using pooled transfectants grown in CDACF medium, for the rapid production of small quantities (up to 50 g) of antibodies in CHO cells. Although slightly slower than a true transient system, the advantages include a higher product concentration and use of the same host and process as the production cell line.
  • pCon VectorsTM are an easy way to re-express whole antibodies.
  • the constant region vectors are a set of vectors offering a range of immunoglobulin constant region vectors cloned into the pEE vectors. These vectors offer easy construction of full length antibodies with human constant regions and the convenience of the GS SystemTM.
  • Antibody molecules will comprise fragments (such as F(ab'), F(ab') 2 ) that are produced, for example, by the proteolytic cleavage of the mAbs, or single-chain immunoglobulins producible, for example, via recombinant means. Such antibody derivatives are monovalent. In one embodiment, such fragments can be combined with one another, or with other antibody fragments or receptor ligands to form "chimeric" binding molecules. Significantly, such chimeric molecules may contain substituents capable of binding to different epitopes of the same molecule.
  • Humanized antibodies produced in non-human hosts in order to attenuate any immune reaction when used in human therapy.
  • Such humanized antibodies may be studied in an in vitro or an in vivo context.
  • Humanized antibodies may be produced, for example by replacing an immunogenic portion of an antibody with a corresponding, but non- immunogenic portion (i.e., chimeric antibodies).
  • Humanized chimeric antibodies are provided by Morrison (1985); also incorporated herein by reference. “Humanized” antibodies can alternatively be produced by CDR or CEA substitution. Jones et al. (1986), Verhoeyen et al. (1988), Beidler et al. (1988), incorporated herein by reference.
  • the antibody is a derivative of the disclosed antibodies, e.g., an antibody comprising the CDR sequences identical to those in the disclosed antibodies (e.g., a chimeric, humanized or CDR-grafted antibody).
  • the antibody is a fully human recombinant antibody.
  • the present invention also contemplates isotype modification.
  • isotype modification By modifying the Fc region to have a different isotype, different functionalities can be achieved. For example, changing to IgG 4 can reduce immune effector functions associated with other isotypes.
  • Modified antibodies may be made by any technique known to those of skill in the art, including expression through standard molecular biological techniques, or the chemical synthesis of polypeptides. Methods for recombinant expression are addressed elsewhere in this document.
  • pannexin-1 peptides contemplates the use of various pannexin-1 peptides.
  • Peptides will have, at a minimum, these six consecutive residues of the pannexin-1. In general, the peptides will be 50 residues or less, and generally no more than 20 consecutive residues of pannexin.
  • the overall length may be 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 residues. Ranges of peptide length of 6-50 residues, 7-50 residues, 6-25 residues 7-25, residues, 6-20 residues, 7-20 residues, and 6-15 residues, and 6-15 residues are contemplated. The number of consecutive MUC1 residues may be 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19 or 20. Ranges of consecutive residues of 6-20 residues, 7-20 residues, 6-15 residues, and 7-15 residues are contemplated.
  • the present invention may utilize L-configuration amino acids, D-configuration amino acids, or a mixture thereof. While L-amino acids represent the vast majority of amino acids found in proteins, D-amino acids are found in some proteins produced by exotic sea- dwelling organisms, such as cone snails. They are also abundant components of the peptidoglycan cell walls of bacteria. D-serine may act as a neurotransmitter in the brain.
  • L and D convention for amino acid configuration refers not to the optical activity of the amino acid itself, but rather to the optical activity of the isomer of glyceraldehyde from which that amino acid can theoretically be synthesized (D-glyceraldehyde is dextrorotary; L- glyceraldehyde is levorotary).
  • Retro-inverso modification of naturally occurring polypeptides involves the synthetic assemblage of amino acids with a- carbon stereochemistry opposite to that of the corresponding L-amino acids, i.e., D-amino acids in reverse order with respect to the native peptide sequence.
  • a retro-inverso analogue thus has reversed termini and reversed direction of peptide bonds (NH-CO rather than CO- NH) while approximately maintaining the topology of the side chains as in the native peptide sequence. See U.S. Patent 6,261,569, incorporated herein by reference.
  • Peptides may be modified for in vivo use by the addition, at the amino- and/or carboxyl-terminal ends, of a blocking agent to facilitate survival of the peptide in vivo. This can be useful in those situations in which the peptide termini tend to be degraded by proteases prior to cellular uptake.
  • a blocking agent can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the peptide to be administered. These agents can be added either chemically during the synthesis of the peptide, or by recombinant DNA technology by methods familiar in the art. Alternatively, blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino and/or carboxyl terminal residues.
  • Probenecid is a uricosuric drug that increases uric acid excretion in the urine. It is primarily used in treating gout and hyperuricemia. Probenecid was developed as an alternative to caronamide. Their primary goal was to competitively inhibit renal excretion of some drugs, thereby increasing their plasma concentration and prolonging their effects.
  • probenecid was used to extend limited supplies of penicillin, and is still currently used to increase antibiotic concentrations in serious infections.
  • probenecid was shown to more than double blood concentrations of oseltamivir (trade name Tamiflu), an antiviral drug used to combat influenza, suggesting that this property applies to antivirals as well. It has also found use as a masking agent.
  • Probenecid Some of the important clinical interactions of Probenecid include those with Captopril, Indomethacin, Ketoprofen, Ketorolac, Naproxen, Cephalosporins, Quinolones, Penicillins, Methotrexate, Zidovudine, Gancyclovir, Aciclovir. In all these interactions the excretion of these drugs is reduced due to Probenecid. In the kidneys probenecid is filtered at the glomerulus, secreted in the proximal tubule and reabsorbed in the distal tubule.
  • Probenecid works by interfering with the kidney's organic anion transporter (OAT), which reclaims uric acid from the urine and returns it to the plasma. If probenecid (an organic acid) is present, the OAT binds preferentially to it (instead of to uric acid), preventing re- absorption of the uric acid. Hence, the urine retains more uric acid, lowering uric acid concentration in the plasma. Probenecid also inhibits gap junction (connexin) channels.
  • OAT organic anion transporter
  • Carbenoxolone a synthetic derivative of glycyrrhetinic acid, is a licensed drug (in the UK) for oesophageal ulceration and inflammation.
  • Other uses include treatment of oral and perioral lesions, and also as a blocker of the enzyme 11 ⁇ - hydroxysteroid dehydrogenase ( ⁇ ⁇ ⁇ -HSD), of pannexon membrane channels (comprising 6 subunits of pannexin) and the related innexon channels (consisting of invertebrate innexins), and at higher concentrations, as a blocker of connexon channels ("hemichannels" made up of 6 connexin subunits each) and of gap junctions (two connexons joined together).
  • hemichannels made up of 6 connexin subunits each
  • gap junctions two connexons joined together.
  • Carbenoxolone has also been investigated for nootropic effects. This research started from an observation that long-term exposure to glucocorticoids may have negative effects on cognition. Carbenoxolone may decrease the amount of active glucocortocoid in the brain, because the drug inhibits 1 1 ⁇ -hydroxysteroid dehydrogenase type 1, an enzyme which activates Cortisol from cortisone, a glucocorticoid. In the research trial investigating this use of carbenoloxone, it was shown that the drug improved verbal fluency in elderly healthy men (aged 55-75). In type 2 diabetics aged 52-70, the drug improved verbal memory.
  • Mefloquine hydrochloride also known as Lariam® or Mefaquin®
  • Mefloquine hydrochloride is an orally administered medication used in the prevention and treatment of malaria.
  • Mefloquine was developed in the 1970's at the United States Department of Defense's Walter Reed Army Institute of Research as a synthetic analogue of quinine.
  • the brand name drug, Lariam® is manufactured by the Swiss company Hoffmann-La Roche. In August 2009, Roche stopped marketing Lariam® in the United States. Generic mefloquine from other manufacturers, is still widely available. Rare but serious neuropsychiatric problems have been associated with its use. Mefloquine is used to both prevent and treat certain forms of malaria.
  • Mefloquine is useful for the prevention of malaria in all areas except for those where parasites may have resistance to multiple drugs. It is typically taken for one to two weeks before entering an area with malaria. Doxycycline and atovaquone/proguanil provide protection within one to two days and may be better tolerated. If a person becomes ill with malaria despite prophylaxis with mefloquine the use of halofantrine and quinine for treatment may be ineffective.
  • Mefloquine may cause abnormalities with heart rhythms that are visible on electrocardiogram. Combining mefloquine with other drugs that cause similar effects, such as quinine or quinidine, can increase these effects. Combining mefloquine with halofantrine can cause significant increases in QTc interval.
  • Mefloquine blocks the action of the chemical that the parasites produce to protect themselves once inside the red blood cells. There is increasing resistance to this drug, due to it being costly to produce, and to the fact that it may have serious adverse side effects in some recipients. Mefloquine is metabolized primarily through the liver. Elimination of mefloquine in anyone with impaired liver function may be prolonged, resulting in higher plasma levels and an increased risk of adverse reactions. The mean elimination plasma half-life of mefloquine is between 2 and 4 weeks. Total clearance is through the liver and the primary means of excretion is through the bile and feces as opposed to only 4% to 9% excreted through the urine. During long-term use, the plasma half-life remains unchanged. Liver function tests should be performed during long-term administration of mefloquine. Alcohol use should be avoided during treatment with mefloquine.
  • Mefloquine is a chiral molecule with two asymmetric carbon centres, which means it has four different stereoisomers.
  • the drug is currently manufactured and sold as a racemate of the (R,S)- and (S,R)-enantiomers by Hoffman-LaRoche, a Swiss pharmaceutical company. Essentially it is two drugs in one. Plasma concentrations of the (-)-enantiomer are significantly higher than those for the (+)-enantiomer and the pharmokinetics between the two enantiomers are significanly different.
  • the (+)-enantiomer has a shorter half-life than the (-)-enantiomer.
  • the (+)-enantiomer is more effective in treating malaria, and the (-)-enantiomer specifically binds to adenosine receptors in the central nervous system, which may explain some of its psychotropic effects. It is not known whether mefloquine goes through stereoisomeric switching in vivo.
  • any of the therapies discussed below may be combined with that of the present invention for the treatment of an inflammatory bowel disease.
  • any one treatment may precede, be co-current with and/or follow the other agent(s) by intervals ranging from minutes to weeks.
  • the various agent(s) are applied separately to a cell, tissue or organism, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that both agent(s) would still be able to exert an advantageously combined effect on the cell, tissue or organism.
  • one may contact the cell, tissue or organism with two, three, four or more modalities substantially simultaneously (i.e. within less than about a minute).
  • an agent may be administered within of from substantially simultaneously, about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours about 8 hours, about 9 hours, about 10 hours, about 1 1 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 5 days,
  • Aminosalicylates are anti-inflammatory drugs in the aspirin family. There are five aminosalicylate preparations available for use in the United States: sulfasalazine (Azulfidine), mesalamine (Asacol, Pentasa), olsalazine (Dipentum), and balsalazide (Colazal). These drugs can be given either orally or rectally (enema, suppository formulations).
  • Corticosteroids are powerful, fast-acting anti-inflammatory agents. Their use in IBD is for acute flare-ups only. Corticosteroids may be administered by a variety of routes, depending upon the location and severity of disease; they may be administered intravenously (methylprednisolone, hydrocortisone) in the hospital, orally (prednisone, prednisolone, budesonide, dexamethasone), or rectally (enema, suppository, foam preparations). Corticosteroids tend to provide rapid relief of symptoms as well as a significant decrease in inflammation, but their side effects limit their use (particularly longer-term use).
  • Immune modifiers include 6-mercaptopurine (6-MP, Purinethol) and azathioprine (Imuran).
  • Immune modifiers may work by causing a reduction in the lymphocyte count (a type of white blood cell). They are often used when aminosalicylates and corticosteroids are either ineffective or only partially effective. They are useful in reducing or eliminating some patient's dependence on corticosteroids. Immune modifiers may also prove helpful in maintaining remission in some persons with refractory ulcerative colitis.
  • Infliximab (Remicade) is an anti-TNF agent, acting by binding to TNF, thereby inhibiting its effects on the tissues. It is approved by the FDA for the treatment of persons with moderate-to-severe Crohn's Disease who have had an inadequate response to standard medications. In such persons, a response rate of 80% and a remission rate of 50% have been reported.
  • Metronidazole and ciprofloxacin are the most commonly used antibiotics in persons with IBD. Antibiotics are used sparingly in persons with ulcerative colitis because they have an increased risk of developing antibiotic-associated pseudomembranous colitis. In persons with Crohn's Disease, antibiotics are used for the treatment of complications (perianal disease, fistulae, inflammatory mass). 6. Symptomatic treatments
  • O-glycan compositions in one aspect, relate to mucins.
  • Mucins are high-molecular weight epithelial glycoproteins with a high content of clustered oligosaccharides O- glycosidically linked to tandem repeat peptides rich in threonine, serine, and proline.
  • mucins There are two structurally and functionally distinct classes of mucins: secreted gel-forming mucins (MUC2, MUC5AC, MUC5B, and MUC6) and transmembrane mucins (MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC 17), although the products of some MUC genes do not fit well into either class (MUC7, MUC8, MUC9, MUC13, MUC15, MUC 16).
  • secreted gel-forming mucins MUC2, MUC5AC, MUC5B, and MUC6
  • transmembrane mucins MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC 17
  • compositions of the present invention comprise an effective amount of an agent dissolved or dispersed in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.
  • the preparation of a pharmaceutical composition that contains at least one active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18 th Ed. Mack Printing Company, 1990, incorporated herein by reference.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18 th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.
  • the compounds of the invention may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection.
  • the present invention can be administered orally, or rectally, but may also be administered intratracheally, intranasally, subcutaneously, mucosally, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, or by other method or any combination of the foregoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18 th Ed. Mack Printing Company, 1990, incorporated herein by reference).
  • the actual dosage amount of a composition of the present invention administered to a patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration.
  • the practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
  • the composition may comprise various antioxidants to retard oxidation of one or more component.
  • the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
  • parabens e.g., methylparabens, propylparabens
  • chlorobutanol phenol
  • sorbic acid thimerosal or combinations thereof.
  • the compounds of the present invention may be formulated into a composition in a free base, neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.
  • a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods.
  • isotonic agents such as, for example, sugars, sodium chloride or combinations thereof.
  • the therapeutic compositions of the present invention are prepared for administration by such routes as oral ingestion.
  • the solid composition may comprise, for example, solutions, suspensions, emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatin capsules), delayed release capsules, sustained release formulations, buccal compositions, troches, elixirs, suspensions, syrups, wafers, or combinations thereof.
  • Oral compositions may be incorporated directly with the food of the diet.
  • Specific carriers for oral administration comprise inert diluents, assimilable edible carriers or combinations thereof.
  • the oral composition may be prepared as a syrup or elixir.
  • a syrup or elixir and may comprise, for example, at least one active agent, a sweetening agent, a preservative, a flavoring agent, a dye, a preservative, or combinations thereof.
  • an oral composition may comprise one or more binders, excipients, disintegration agents, lubricants, flavoring agents, and combinations thereof.
  • a composition may comprise one or more of the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.; or combinations thereof the
  • the dosage unit form When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both.
  • suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum. After insertion, suppositories soften, melt or dissolve in the cavity.
  • traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof.
  • suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients.
  • the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof.
  • the liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose.
  • the preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.
  • composition should be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.
  • mice All experimental protocols adhered to the guidelines of the Canadian Council on Animal Care and were approved by the University of Calgary Animal Care and Use Committee.
  • Male C57B1/6 mice (6-12 weeks old; Charles River; Montreal, QC) were used for all studies except where the use of transgenic strains is indicated.
  • Male Asc _/" mice and Nlrp3 _/ ⁇ mice (6-9 weeks old, C57B1/6 background) were provided by Drs. Vishva M. Dixit (Genetech, San Francisco, CA) and Jurg Tschopp (University of Lausanne, Switzerland), respectively.
  • IL10 _/" mice and wild-type littermates were generated as described previously (McCafferty et al, 2000).
  • Human tissue Full-thickness specimens of normal and diseased human colon were obtained after informed consent from a minimum of 3 patients with active Crohn's disease, 3 with ulcerative colitis, and 3 undergoing resection for colon cancer. Marginal safe tissue bordering tumorous bowel served as control tissue for inflamed tissue. Human tissue was fixed with Holland's fixative and embedded in paraffin before being sectioned. After de- paraffinization in a graded ethanol series, antigen retrieval with sodium citrate (pH 6.0, 80°C, 30 min) was preformed before proceeding with immunohistochemistry (see below).
  • mice were anesthetized with isoflurane, a gavage needle inserted 3 cm into the colon and 0.1 mL of a solution containing either 5 mg DNBS or oxazolone dissolved in 50% ethanol/saline, or saline alone for controls, was injected into the colon. Animals were closely monitored and weight loss recorded daily. Animals were sacrificed approximately 48 hrs following induction of colitis and scored for macroscopic damage (Storr et al, 2009).
  • DNBS dinitrobenzene sulphonic acid
  • ozazolone ozazolone
  • DSS dextran sodium sulfate
  • Whole-mount immunohistochemistry Whole-mount preparations of the myenteric plexus were prepared by removing the mucosa, submucosa and circular muscle of the colon by microdissection. The resulting longitudinal muscle myenteric plexus (LMMP) preparations were processed for immunohistochemistry as described by Gulbransen and Sharkey (2009).
  • Primary and secondary antibodies are listed in Table 1. Specificity of primary antibodies was confirmed by pre-absorption with control peptides or lack of labeling in respective knockout animals. Specificity of secondary antibodies was assessed by omission of primary antibodies.
  • LMMPS whole-mounts were prepared as for Ca 2+ imaging, pinned flat in Sylgar-dcoated 35 mm cell culture dishes, and maintained in a humid oxygenated chamber at near 34°C.
  • a total of 6 LMMPs were obtained from each animal and 2 were incubated with buffer alone, 2 with 300 ⁇ BzATP, and 2 with 300 ⁇ BzATP plus inhibitor for 2 hrs.
  • Tissues were then rinsed with buffer and maintained in either buffer alone or buffer with inhibitor for an additional 2 hrs before being fixed with Zamboni's fixative overnight.
  • Modified Krebs buffer contained (in mmol/L): 121 NaCl, 5.9 KC1, 2.5 CaCl 2 , 1.2 MgCl 2 , 1.2 NaH 2 P0 4 , 10 HEPES, 21.2 NaHC03, 1 pyruvic acid, 8 glucose (Sigma- Aldrich, St. Louis, MO) (pH adjusted to 7.4 with NaOH) with 3 ⁇ /L nicardipine (Sigma) and 1 ⁇ /L scopolamine (Sigma) to inhibit muscle contractions.
  • Low Ca 2+ /Mg 2+ buffer was prepared as above except CaCl 2 was lowered to 0.5 and MgCl 2 omitted.
  • Neuronal packing density per animal was determined by averaging counts from a minimum of 10 ganglia from that animal except for in situ experiments were averaged counts from a minimum of 20 ganglia from 2 LMMPS from each treatment group per animal were counted to obtain values for neural packing density.
  • Confocal images were obtained on through the 60X objective (PlanApo N, 1.42 n.a., oil) of an Olympus BX50 Fluoview laser scanning confocal microscope (Olympus Canada Inc., Markham, ON, Canada).
  • Ca 2+ imaging data was analyzed as described by Gulbransen et al. (2010). Regions of interest (ROIs) were drawn and relative fluorescence measured using Imaging Workbench 6 (INDEC BioSystems). Analysis and generation of traces was performed using Prism 5 software (Graphpad Software Inc., La Jolla, CA). Traces show the change in fluorescence (AF/F) over time representing the mean response of all glial or neuronal RIOs within a single ganglion. Responses (magnitude of peak AF/F over baseline) were determined as increases in intracellular Ca 2+ >3 standard deviations from baseline lasting more than 3 sec. Responses are reported as the percentage of an initial response to 100 ⁇ BzATP in the same ganglion.
  • EFS tetrodotoxin
  • 6-Hydroxydopamine (6-OHDA) chemical sympathectomy. 6-OHDA treatment was performed as described by Gulbransen et al. (2010) with minor modifications for mice. Briefly, mice received one subcutaneous injection of 100 mg/kg 6-OHDA (Sigma) dissolved in H20 with 0.1% sodium metabisulfite as a stabilizing agent. Tissue was taken two days after injection. Loss of tyrosine hydroxylase (TH)-immunoreactive nerve fibers in the myenteric plexus confirmed the effectiveness of the 6-OHDA treatment.
  • 6-OHDA 6-Hydroxydopamine
  • LMMP Longitudinal muscle - myenteric plexus
  • P308 is a small molecule peptide inhibitor of Src.
  • Whole-mount preparations of the myenteric plexus from the mouse colon were incubated for 2 hr at 37°C either buffer (control), 300 ⁇ BzATP or 300 ⁇ BzATP in the presence of 10 ⁇ P308. Preparations were rinsed in buffer after the 2 h incubation and then incubated an additional 2 hr in buffer alone before being preserved for immunohistochemistry in Zamboni's fixative.
  • Immunoreactivity for HuC/D was used to identify enteric neurons and the number of enteric neurons per ganglionic area was determined. Values are normalized to the packing density of control (buffer-treated) tissue.
  • DNBS 2,4-dinitrobenzene sulfonic acid
  • the inventors examined the effects of pharmacological or genetic disruption of P2X7Rs, Panxl and inflammasome components on neuron survival and colonic motor function. Further, the inventors evaluated immunoreactivity for key signaling components in murine and human enteric neurons, characterized the effects of stimulating enteric neuron P2X7Rs using Ca 2+ imaging, and used models of colon motor function to analyze the long-term benefit of neuroprotection during inflammation.
  • P2X7Rs inhibiting neuronal P2X7Rs in vivo protects against neural loss during inflammation.
  • Constitutively elevated extracellular ATP during tissue inflammation is proposed to contribute to cytotoxicity by chronically activating P2X7Rs (Lazarowski et ah, 2000; Sperlagh et ah, 2006; Wang et ah, 2004).
  • P2X7Rs are expressed by enteric neurons (Hu et ah, 2001) so the inventors hypothesized that P2X7R activation mediates inflammation-induced enteric neuron death. Because the distribution of P2X7Rs within the mouse enteric nervous system is unknown, the inventors first used immunohistochemistry to localize P2X7Rs within the mouse colonic myenteric plexus.
  • mice were injected (i.p.) with the irreversible P2X7R antagonist oxidized-ATP (o-ATP; 7.5 mg/kg) prior to induction of colitis.
  • o-ATP oxidized-ATP
  • myenteric ganglia are filled with enteric neurons (FIG. 2b; identified by immunoreactivity to the pan-neuronal protein HuC/D) at a density of 2913 ⁇ 89 neurons/mm 2 (mean ⁇ s.e.m.).
  • P2X7R driven neural loss depends on activation of pannexin-1 and caspases but not IL- ⁇ .
  • the inventors tested whether activation of neuronal P2X7Rs is sufficient to drive neuron death.
  • the inventors acutely dissected whole-mount preparations of the mouse colonic myenteric plexus, challenged neurons with the selective P2X7R agonist BzATP (300 ⁇ , 2 hrs) and assayed the effects on neuron survival.
  • Control ganglia from whole-mounts maintained in buffer alone had an average of 2446 ⁇ 48 neurons/mi ⁇ .
  • Treatment with BzATP reduced neural packing density by 34.4 ⁇ 2% (FIG. 2f), which closely resembled the in vivo situation.
  • P2X7R stimulation causes apoptotic cell death through the activation of multiple caspases (Ferrari et ah, 1999).
  • the inventors treated wholemounts with the cell-permeable pan-caspase inhibitor zVAD(OMe)-fmk (zVAD; 80 ⁇ ) prior to, and during BzATP treatment.
  • zVAD prevented neuronal loss, suggesting that P2X7R- mediated enteric neuron death depends on caspase activation (FIG. 2f).
  • P2X7R activation causes caspase- 1 cleavage and IL- ⁇ release from cultured macrophages (Ferrari et ah, 1997) and IL- ⁇ ⁇ can be a key mediator of neurodegeneration (Allan and Rothwell, 2001) zVAD, being a pan-caspase inhibitor, inhibits caspase- 1 and may protect enteric neurons by decreasing IL- ⁇ ⁇ processing.
  • Panxl pannexin-1 channels
  • Panxl-ir is distributed throughout the mouse colon myenteric plexus and localizes to the cell bodies and processes of most, if not all, enteric neurons (FIG. 3a).
  • Dual labeling with GFAP revealed an intimate association between enteric glia and Panxl -ir neurons with GFAP-ir glia often embracing Panxl-ir neuronal processes and cell bodies (FIG. 3a" ').
  • Panxl-ir was restricted to neurons and the inventors did not observe Panxl expression in GFAP-ir glia. Dual labeling with P2X7 indicates that Panx-1 -expressing myenteric neurons co-express P2X7 (FIGS. 8(a)-(c")). Double-labeling with nNOS to identify inhibitory motor neurons and interneurons (Dogiel Type I) or calretinin to identify intrinsic primary afferent neurons (Dogiel Type II) demonstrates that both major classes of neurons in the mouse myenteric plexus express Panxl (50.8% of Panxl-ir neurons are nNOS- ir and 39.6% are calretinin-ir; see FIGS. 8(a)-(c")).
  • Neural loss during colitis depends on pannexin-1 and ASC but not theNLRP3 inflammasome.
  • the inventors show above that enteric neural death during inflammation depends on P2X7R stimulation, and inhibition of neuronal Panxl protects against P2X7Rmediated neuron death in situ. Therefore, the inventors asked if inhibition of Panxl in vivo would protect against neural loss during colitis.
  • the inventors injected mice with the Panxl inhibitor probenecid (PB, 177.5 mg/kg) (Silverman et ah, 2009; Silverman et ah, 2008), a drug used clinically to treat gout.
  • PB Panxl inhibitor probenecid
  • enteric neurons were rapidly lost following DNBS-colitis and neuronal density within ganglia significantly decreased by 22 ⁇ 5% (FIG. 3c).
  • mice injected with probenecid prior to DNBS treatment did not exhibit a loss of neurons.
  • probenecid-treated mice had equal macroscopic damage and weight loss (FIG. 3c-e), suggesting that Panxl inhibition protects against neural death without modifying the underlying tissue inflammation.
  • Panxl opening may contribute to neuron death by activating a large protein complex termed the inflammasome (Silverman et ah, 2009).
  • the inventors tested if inflammasome activation is required for neuron death in vivo using mice deficient in either the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) or NLRP3 (nucleotide-binding domain and leucine-rich repeat containing gene family pyrin domain containing 3).
  • ASC apoptosis-associated speck-like protein containing a CARD
  • NLRP3 nucleotide-binding domain and leucine-rich repeat containing gene family pyrin domain containing 3
  • Asc _/" and Nlrp3 _/ ⁇ mice lose weight (FIG. 3e) and have similar macroscopic damage scores (FIG. 3d) compared to wild-type mice treated with DNBS.
  • Panxl acts as a conduit for ATP release from apoptotic cells to recruit phagocytes (Chekeni et ah, 2010).
  • phagocytes Several non-neural cell types and expression models directly release ATP through Panxl, but whether Panxl activation mediates ATP release from neurons endogenously expressing this channel remains unknown (MacVicar and Thompson, 2010). Therefore, the inventors tested if Panxl opening, activated by P2X7R, stimulates ATP release from enteric neurons. Since enteric glial surrounding enteric neurons are highly responsive to ATP (Gulbransen and Sharkey, 2009), the inventors utilized glia as endogenous "sniffer cells" and monitored purinergic receptor activation using Ca 2+ imaging.
  • Enteric glia respond to ATP through a phospholipase C (PLC) signaling cascade initiated by P2YR activation (Gulbransen and Sharkey, 2009). If glial responses to BzATP were due to neuronal release of ATP then inhibition of either PLC or P2YRs should preferentially block glial responses to BzATP.
  • PLC inhibitor U73122 (10 ⁇ ) had no effect on neural responses to BzATP, but glial responses were reduced by 70.4 ⁇ 6% (FIG. 4i).
  • Murine enteric glia respond to ATP through P2Y1 receptors (Gomes et al, 2009), so the inventors tested if blockade of P2YlRs with MRS2179 (10 ⁇ ) blocked glial responses to BzATP.
  • MRS2179 provided a nearly complete block of glial responses to BzATP, reducing glial peak responses by 90.7 ⁇ 3% while having no significant inhibitory effect on neurons (FIG. 4e, i).
  • ADP is the endogenous ligand for P2Y1 receptors and P2Y1 receptors display higher affinity for ADP than ATP.
  • glial P2YlRs likely occurs following extracellular degradation of neuronally -released ATP by ectonucleotidases.
  • POM1 50 ⁇
  • Neural Ca 2+ responses to BzATP were not significantly affected by POM1, but glial responses were significantly inhibited (by 81.7 ⁇ 6%; FIG. 4f, FIG. 4i).
  • These results indicate that ATP is released from enteric neurons by P2X7R stimulation. This ATP is quickly degraded to ADP by ectonucleotidases to act on glial P2Y1 receptors, stimulating Ca 2+ responses through a PLC-mediated signaling cascade.
  • P2X7R activation promotes the activation of Panxl (Pelegrin and Surprenant, 3006) and Panxl mediates ATP release in several non-neuronal expression systems (Iglesias et al, 2009; Locovei et al, 2006; Romanov et al, 2007).
  • the inventors asked if P2X7R-stimulated ATP release from enteric neurons depends on Panxl. First, the inventors inhibited Panxl with probenecid (PB; 2 mM) (Silverman et al, 2009; Silverman et al, 2008) and found that glial responses to BzATP were reduced by 50.3 ⁇ 12% (FIG. 4g, FIG. 4i).
  • the inventors then tested the more specific peptide inhibitor of Panxl, lOPanx (100 ⁇ ), and found that lOPanx significantly inhibited glial responses to BzATP by 46.4 ⁇ 1 1% (FIG. 4h, FIG. 4i). However, lOPanx had no effect on neural responses to BzATP (FIG. 4h, FIG. 4i). From these results, the inventors conclude that P2X7R stimulation of enteric neurons elicits a direct release of ATP through Panxl .
  • Pannexin-1 inhibition during colitis protects colonic motor function.
  • Inflammation-induced alterations to the enteric nervous system contribute to long-term alterations in gastrointestinal motility that often persist following resolution of inflammation (Collins, 1996; Vasina et ah, 2006; Krauter et ah, 2007).
  • the inventors' results indicate that inhibiting Panxl during colitis is neuroprotective, prompting us to test if the sparing of enteric neurons translates into long-term functional benefits and preserves normal colonic motor activity.
  • Pannexin-1 inhibition protects enteric neurons during recurrent bouts of colitis.
  • mice are relapsing-remitting conditions and therapeutic intervention would occur after an initial inflammatory bout.
  • Panxl inhibition represents a clinically relevant therapeutic strategy to improve neuron survival during recurrent bouts of inflammation
  • the inventors developed a model of recurrent colitis in mice (FIG. 6a).
  • mice treated with saline during the remission period had a 28.2 ⁇ 3% reduction in myenteric neuron packing density (FIG. 6b).
  • probenecid intervention during remission protected against further neuron loss during recurrent inflammatory bouts (FIG. 6b).
  • Panxl inhibition improves neuron survival in a clinically relevant model of colitis.
  • Human enteric neurons express pannexin-1 and P2X7Rs. Translation of these mouse models to humans depends on the expression of P2X7Rs and Panxl by human enteric neurons. Therefore, the inventors assayed the human enteric nervous system for P2X7Rs and Panxl. In full thickness sections from human colon, the inventors find robust P2X7R expression in myenteric (FIGS. 6c-c') and submucosal neurons. Similarly, neurons with Panxl immunoreactivity decorating their surface are abundant in the human myenteric and submucosal plexuses (FIGS. 6d-f ).
  • An interfering peptide that inhibits Src-dependent activation of pannexin improves neuron survival.
  • the inventors assessed mean packing density of HuC/D immunoreactive myenteric neurons in the mouse colon after in situ activation of P2X7Rs with BzATP (300 ⁇ ) alone or in combination with P308 (10 ⁇ ; Pannexin residues SOS- S I S; LKVYEILPTFDVLH, SEQ ID NO: 1), an interfering peptide that mimics the Src target consensus sequence of pannexin. Immunoreactivity for HuC/D was used to identify enteric neurons and the number of enteric neurons per ganglionic area was determined.
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Abstract

La présente invention concerne des méthodes de traitement de la perte neuronale dans des maladies intestinales inflammatoires (par exemple maladie de Crohn ou colite ulcéreuse), comprenant l'inhibition de la pannexine-1 chez un sujet.
PCT/IB2013/000463 2012-01-25 2013-01-25 Méthodes et compositions destinées au traitement de la perte neuronale dans des maladies intestinales inflammatoires WO2013111014A2 (fr)

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Publication number Priority date Publication date Assignee Title
US20170157038A1 (en) * 2008-11-13 2017-06-08 Gholam A. Peyman Ophthalmic drug delivery method
US11116737B1 (en) 2020-04-10 2021-09-14 University Of Georgia Research Foundation, Inc. Methods of using probenecid for treatment of coronavirus infections
WO2023143513A1 (fr) * 2022-01-26 2023-08-03 羽晓瑜 Composé polypeptidique et son utilisation

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CHEKENI, F. B. ET AL.: 'Pannexin 1 channels mediate `find-me' signal release and membrane permeability during apoptosis' NATURE vol. 467, October 2010, ISSN 1476-4687 pages 863 - 867 *
GULBRANSEN, B. D. ET AL.: 'Activation of neuronal P2X7 receptor-Pannexin-1 mediates death of enteric neurons during colitis' NATURE MED. vol. 18, October 2012, ISSN 1546-170X pages 600 - 604 *
SILVERMAN, W. R ET AL.: 'The pannexin 1 channel activates the inflammasome in neurons and astrocyrtes' J. BIOL. CHEM. vol. 284, July 2009, ISSN 1083-351X pages 18143 - 18151 *
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170157038A1 (en) * 2008-11-13 2017-06-08 Gholam A. Peyman Ophthalmic drug delivery method
US11116737B1 (en) 2020-04-10 2021-09-14 University Of Georgia Research Foundation, Inc. Methods of using probenecid for treatment of coronavirus infections
US11903916B2 (en) 2020-04-10 2024-02-20 University Of Georgia Research Foundation, Inc. Methods of using probenecid for treatment of coronavirus infections
WO2023143513A1 (fr) * 2022-01-26 2023-08-03 羽晓瑜 Composé polypeptidique et son utilisation

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