+

WO2007000046A1 - Methodes de diagnostic de maladie intestinale fonctionnelle - Google Patents

Methodes de diagnostic de maladie intestinale fonctionnelle Download PDF

Info

Publication number
WO2007000046A1
WO2007000046A1 PCT/CA2006/001060 CA2006001060W WO2007000046A1 WO 2007000046 A1 WO2007000046 A1 WO 2007000046A1 CA 2006001060 W CA2006001060 W CA 2006001060W WO 2007000046 A1 WO2007000046 A1 WO 2007000046A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
cells
receptor
medicament
peripheral blood
Prior art date
Application number
PCT/CA2006/001060
Other languages
English (en)
Inventor
Elizabeth C. Colley
Ronald H. Stead
Original Assignee
Holburn Biomedical Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Holburn Biomedical Corporation filed Critical Holburn Biomedical Corporation
Publication of WO2007000046A1 publication Critical patent/WO2007000046A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9406—Neurotransmitters
    • G01N33/942—Serotonin, i.e. 5-hydroxy-tryptamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS

Definitions

  • the present invention relates to methods for the diagnosis and treatment of functional bowel diseases, in particular, irritable bowel syndrome, .and for methods to select the appropriate medicament for such treatment.
  • mast cells are involved in inflammatory and allergic responses. Thus, mast cells have been considered to be immune response elements, participating in a wide range of defense mechanisms against infectious diseases. Through the expression of immunoglobulin receptors, mast cells can participate in adaptive immune responses (GaIIi et al., 2005); and they can also regulate innate immune responses (Marshall & Jawdat, 2004). Over the last decade, it has become apparent that mast cells are also active participants in homeostatic processes, in part through their interaction with peripheral nerves (Gottwald et al., 1998). In fact, mast cells might act as sentinels, responding to a variety of stimuli by selective release of numerous mediators which they are known to synthesize and release (Stead et al.
  • mast cells activate mast cells.
  • Activation of mast cells causes the recruitment of neutrophils and macrophages, as well as eosinophils at a later stage.
  • stimulation of mast cells initiates a cascade of events known as inflammation (Marshall, 2004).
  • Mast cells are bone marrow derived and migrate through the peripheral blood (generally as precursors) before homing to various tissues and organs (Weller et al., 2005).
  • mast cells express receptors for mediators that they release, and such receptors act in inhibitory processes, down-regulating the ongoing release of the mediator (Lippert et ai, 2004).
  • Mast cells express three subtypes of histamine receptor (H 1 , H2 and H4; there is some suggestion that H3 might also be expressed).
  • H1 , H2 and H4 histamine receptor
  • mast cells express auto-receptors for the biogenic amine that they release upon stimulation.
  • serotonin can also modulate mast cell activity through different subtypes of serotonin receptors. However, these data are restricted to non-human species, and there is to this point, no published evidence for the expression of serotonin receptors on human mast cells.
  • gastrointestinal mast cells express immunoreactivity for 5-HT 4 receptor in mouse, rat and human tissues. It has also been found that in cultured mast cells this receptor is functional, in that after treatment with a serotonin agonist or antagonist the response to a challenge with a mast cell stimulator (e.g., a calcium ionophore such as ionomycin) causes the inhibition of the mediator release.
  • a mast cell stimulator e.g., a calcium ionophore such as ionomycin
  • Functional bowel diseases also called "functional bowel disorders” are, by definition, disorders with no known underlying pathology (Thompson et ai, 1999). They are generally considered to be collections of symptoms attributable to the gastrointestinal tract. Irritable bowel syndrome (IBS) is one of the functional bowel diseases.
  • Other functional bowel diseases include, by way of non-limiting example, functional abdominal bloating, functional constipation, functional diarrhea, functional abdominal pain, functional dyspepsia, non-cardiac chest pain (NCCP), and chronic abdominal pain.
  • Ethiopathogenic features of functional bowel diseases often include psychosocial disturbances, dysmotility and/or heightened sensitivity.
  • IBS afflicts up to 20% of the adult population and accounts for up to 50% of visits to gastroenterologists. In addition to suffering from abdominal pain and discomfort, IBS sufferers also have altered bowel habits. Some patients suffer from chronic constipation, while others have diarrhea.
  • Patients with diarrhea-predominant IBS benefit from treatment with 5-HT 3 receptor antagonists (De Ponti & Tonini, 2001), such as, e.g., alosetron (Lotronex produced by GSK).
  • 5-HT 3 receptor antagonists include ondansetron, granisetron, dolasetron, tropisetron, renzapride and zacopride.
  • Constipation-predominant IBS sufferers benefit from treatment with 5- HT 4 receptor agonists (De Ponti & Tonini, 2001 ) such as, e.g., tegaserod (Zelnorm produced by Novartis), renzapride and prucalopride.
  • 5-HT 4 receptor agonists include 5 methoxytryptamine, and substituted benzamide prokinetics such as, e.g., metoclopramide and cisapride.
  • Various mechanisms of action for these serotonin receptor acting drugs have been proposed, including actions on gastrointestinal nerves and muscle. However, the possibility that at least some of the responses to serotonin receptor-specific agents might be through modulation of other gastrointestinal cell activity and, in particular, mast cell activity has not been considered.
  • 5HT 3 receptor antagonists and 5HT 4 agonists are used here as examples only.
  • Theragnostics refers to diagnostic tests that point to a specific form of therapy. For example, the expression of estrogen receptors by breast cancer cells indicates that the tumor is likely to respond to tamoxifen therapy.
  • IBS is diagnosed by symptoms using standardized questionnaires. Patients with IBS are generally biopsied during the colonoscopy which is part of the diagnostic process. The biopsies are reviewed in pathology laboratories primarily to exclude other disorders which would indicate that the patients do not have IBS.
  • the choice of drug used to treat these patients is determined by symptoms and is not based upon the likelihood that any given patient will respond because of the relevant ligand or receptor in their gastrointestinal tissues.
  • receptors for serotonin, as well as serotonin itself can be localized and detected, respectively, and the extent of expression of the different molecules can be quantified.
  • the presence and level of expression of serotonin and serotonin receptors such as, e.g., 5-HT 3 receptors and 5-HT 4 receptors can be used as theragnostic markers for the treatment of functional bowel diseases such as, e.g., IBS. Accordingly, the methods of the present invention are expected to be of significant utility in the diagnosis, management and treatment of functional bowel disorders.
  • the present invention provides a method for diagnosing a functional bowel disease, in a mammal and, in particular, a human patient.
  • the method comprises the testing of gastrointestinal cells or cells circulating in peripheral blood of the patient for the presence of serotonin and/or at least one serotonin receptor.
  • Non-limiting examples of serotonin receptors include 5-HTi receptor, 5- HT 2 receptor, 5-HT 3 receptor, 5-HT 4 receptor, 5-HT 5 receptor, 5-HT 6 receptor, 5- HT 7 receptor and the various subtypes of these receptors such as, e.g., 5-HT ' i A receptor, 5-HT 18 receptor, 5-HT 10 receptor, 5-HTi E receptor, 5-HT 1 F receptor, 5- HT 2A receptor, 5-HT 2B receptor, 5-HT 20 receptor, 5-HT 3A receptor, 5-HT 3B receptor, 5-HT 4A receptor, 5-HT 4 B receptor, 5-HT 5 A receptor, 5-HT 5B receptor, 5- HT 6 receptor and 5-HT 7 receptor.
  • the gastrointestinal cells and/or circulating cells are tested for the presence of serotonin and/or a serotonin receptor such as, e.g., 5-HT 3 receptor and/or 5-HT 4 receptor.
  • the gastrointestinal cells may at least be tested for the presence of serotonin, and/or they may at least be tested for the presence of a serotonin receptor such as, e.g., 5-HT 3 receptor and/or 5-HT 4 receptor. In another aspect, they may be tested for the presence of at least 5-HT 3 receptor and 5-HT 4 receptor.
  • they may be tested for the presence of at least two species, e.g., serotonin and at least one serotonin receptor. In another aspect, they may be tested for three species, e.g., serotonin, 5-HT 3 receptor and 5-HT 4 receptor.
  • the method may comprise a quantitative determination of serotonin and/or at least one serotonin receptor such as, e.g., 5-HT 3 receptor and 5-HT 4 receptor, for example, a quantitative determination of serotonin and/or a quantitative determination of 5- HT 3 receptor and/or a quantitative determination of 5-HT 4 receptor.
  • serotonin and/or at least one serotonin receptor such as, e.g., 5-HT 3 receptor and 5-HT 4 receptor, for example, a quantitative determination of serotonin and/or a quantitative determination of 5- HT 3 receptor and/or a quantitative determination of 5-HT 4 receptor.
  • the testing is preferably carried out ex vivo, for example, on a tissue section and/or on cells isolated from tissue samples and/or on cells circulating in peripheral blood.
  • the gastrointestinal cells may comprise one or more of the following types of cells: mast cells, muscle cells, neurons, epithelial cells, lymphocytes, macrophages, granulocytes, dendritic cells, enteroglial cells and enteroendocrine cells.
  • the circulating blood cells may comprise one or more of the following types of cells: mast cells, lymphocytes, macrophages, granulocytes, dendritic cells and other circulating cells, particularly leucocytes..
  • the functional bowel disease may comprise irritable bowel syndrome.
  • the method may comprise the incubation of the gastrointestinal cells with one or more antisera for at least one of serotonin and a serotonin receptor such as, e.g., 5-HT 3 receptor and 5-HT 4 receptor, and/or the method may comprise the in situ hybridization of mRNA that is associated with at least one serotonin receptor, e.g., the in situ hybridization of mRNA that is associated with one or both of the 5-HT 3 and 5-HT 4 receptors.
  • the in situ hybridization may be carried out with at least one biotinylated probe and/or with at least one probe which comprises at least one of the following sequences:
  • ACACAGCCACCATCACCAGCAGGTTCCCCA (SEQ ID NO: 6)
  • AGCACCACCTTCTCCACTGACCCGAAACCC (SEQ ID NO: 21 ) GCCGAGCCAGAGGAAAGCAGTCCACACCTG (SEQ ID NO: 22) TGGCCCAGAATGGAAGGTCTTCGGTAGCGC (SEQ ID NO: 23) ACACTGACTCTCCCACTGGCCACCACACTC (SEQ ID NO: 24) GAGCAGGTGATGGCGTAGGGCTTGTTGACC (SEQ ID NO: 25)
  • CTCCTTAGCTGTGACATAGATGCGGTAATA SEQ ID NO: 28
  • CACAGCCACCATCACCAGCAGGTTCCCCAA SEQ ID NO: 29
  • Said probe may be labeled with any suitable label, e.g. with a fluorescent label such as fluorescein (FITC), a radiolabel, an enzyme label, a biotin label, digoxygenin (DIG) label, etc, as it is well known in the art.
  • FITC fluorescein
  • DIG digoxygenin
  • the gastrointestinal cells may comprise mast cells.
  • the method may comprise contacting
  • mast cell stimulators include a calcium ionophore (such as, e.g., ionomycin), IgE and antigen, a neuropeptide (such as, e.g., substance P), a neurotransmitter (such as, e.g., nitric oxide) and other mast cell secretagogues
  • the present invention also provides a method for diagnosing irritable bowel syndrome in a human patient.
  • the method comprises testing gastrointestinal mast cells of the patient for the presence of at least one of serotonin, 5-HT 3 receptor and 5-HT 4 receptor.
  • the mast cells may at least be tested for the presence of serotonin and/or they may at least be tested for the presence of 5-HT 3 receptor and/or they may at least be tested for the presence of 5-HT 4 receptor. In another aspect, they may at least be tested for the presence of 5-HT 3 receptor and 5-HT 4 receptor. In another aspect, they may be tested for the presence of serotonin, 5-HT 3 receptor and 5-HT 4 receptor.
  • the method may comprise a quantitative determination of one or more of serotonin, 5-HT 3 receptor and 5-HT 4 receptor, i.e., a quantitative determination of serotonin and/or a quantitative determination of 5-HT 3 receptor and/or a quantitative determination of 5-HT 4 receptor.
  • the testing is preferably carried out ex vivo, optionally on a tissue section or on cells isolated from a tissue or on a peripheral blood sample.
  • the method may comprise the incubation of the mast cells with one or more antisera for at least one of serotonin, 5-HT 3 receptor and 5-HT 4 receptor and/or the method may comprise the in situ hybridization of mRNA that is associated with at least one of the 5-HT 3 and 5-HT 4 receptors.
  • the in situ hybridization may be carried out with at least one biotinylated probe and/or with at least one probe which comprises at least one of the following sequences:
  • ACACAGCCACCATCACCAGCAGGTTCCCCA (SEQ ID NO: 6)
  • ACACTGACTCTCCCACTGGCCACCACACTC SEQ ID NO: 24
  • GAGCAGGTGATGGCGTAGGGCTTGTTGACC SEQ ID NO: 25
  • Said probe may be labeled with any suitable label, e.g. with a fluorescent label such as fluorescein (FITC(, a radiolabel, an enzyme label, a biotin label, a digoxygenin (DIG) label, etc, as it is well known in the art.
  • FITC fluorescein
  • DIG digoxygenin
  • the method may comprise contacting (challenging) the mast cells with a stimulator such as, e.g., a calcium ionophore, for example ionomycin.
  • a stimulator such as, e.g., a calcium ionophore, for example ionomycin.
  • the present invention also provides a method for diagnosing a serotonin-related gastrointestinal disorder in a human patient that may be a functional bowel disease, e.g. irritable bowel syndrome.
  • the method comprises testing gastrointestinal cells or cells circulating in peripheral blood of the patient for the presence of serotonin and/or a serotonin receptor such as, e.g., 5-HT 3 receptor and/or 5-HT 4 receptor.
  • aspects of this method include those which have been set forth above in connection with the method for diagnosing a functional bowel disease in a mammal.
  • the present invention also provides a method for the treatment of a functional bowel disease in a mammal, preferably a human patient, whose gastrointestinal cells and/or cells circulating in peripheral blood have been determined to comprise (express) one or more serotonin receptors.
  • the method comprises the administration to the mammal of at least one serotonin receptor antagonist and/or at least one serotonin receptor agonist in an effective amount for alleviating the symptoms of the functional bowel disease.
  • the functional bowel disease may comprise irritable bowel syndrome.
  • the gastrointestinal cells may comprise mast cells.
  • the present invention also provides a method for the treatment of a functional bowel disease in a mammal, preferably a human patient, whose gastrointestinal cells have been determined to comprise (express) 5-HT 3 receptor.
  • the method comprises the administration to the mammal of a 5-HT 3 receptor antagonist in an effective amount for alleviating the symptoms of the functional bowel disease.
  • the functional bowel disease may comprise irritable bowel syndrome.
  • the gastrointestinal cells may comprise mast cells.
  • the 5-HT 3 receptor antagonist may comprise alosetron.
  • the present invention also provides a method for the treatment of a functional bowel disease in a mammal, preferably a human patient, whose gastrointestinal cells and/or cells circulating in peripheral blood have been determined to comprise (express) 5-HT 4 receptor.
  • the method comprises the administration to the mammal of a 5-HT 4 receptor agonist in an effective amount for alleviating the symptoms of the functional bowel disease.
  • the present invention provides a method for determining which medicament out of the marketed medicaments for treating functional bowel disease is suitable for a particular individual.
  • the functional bowel disease may comprise irritable bowel syndrome.
  • the gastrointestinal cells may comprise mast cells.
  • the 5-HT 4 receptor agonist may comprise tegaserod.
  • the present invention further provides a medicament that comprises a serotonin receptor antagonist and/or a serotonin receptor agonist and is associated with instructions to administer the medicament to a patient whose gastrointestinal cells and/or cells circulating in peripheral blood comprise (express) the serotonin receptor.
  • the present invention also provides a label or leaflet on or in a packing of a medicament for treating functional bowel disease indicating the use thereof according to the type of HT receptor found on gastrointestinal cells and/or cells circulating in peripheral blood of the patient.
  • the gastrointestinal cells and/or cells circulating in peripheral blood may comprise mast cells.
  • the medicament may be indicated for treating a functional bowel disease such as, e.g., irritable bowel syndrome.
  • a functional bowel disease such as, e.g., irritable bowel syndrome.
  • the present invention further provides a medicament that comprises a 5-HT 3 receptor antagonist and is associated with instructions to administer the medicament to a patient whose gastrointestinal cells and/or cells circulating in peripheral blood comprise (express) 5-HT 3 receptor.
  • the gastrointestinal cells and/or cells circulating in peripheral blood may comprise mast cells.
  • the medicament may be indicated for treating a functional bowel disease such as, e.g., irritable bowel syndrome.
  • the present invention also provides a medicament that comprises a 5- HT 4 receptor agonist and is associated with instructions to administer the medicament to a patient whose gastrointestinal cells and/or cells circulating in peripheral blood comprise (express) 5-HT 4 receptor.
  • the gastrointestinal cells and/or cells circulating in peripheral blood may comprise mast cells.
  • the medicament may be indicated for treating a functional bowel disease such as, e.g., irritable bowel syndrome.
  • the present invention also provides a probe for the hybridization of mRNA that is associated with at least one 5-HT receptor as well as a diagnostic kit comprising the probe and the use of the diagnostic kit.
  • the probe comprises at least one of the following sequences:
  • TGATATAGCCGAGCCAGAGGAAAGCAGTCC (SEQ ID NO: 19) CAATTATGCCAATGTTATTCCAGCCTTGCA (SEQ ID NO: 20) AGCACCACCTTCTCCACTGACCCGAAACCC (SEQ ID NO: 21 )
  • ACACTGACTCTCCCACTGGCCACCACACTC (SEQ ID NO: 24) GAGCAGGTGATGGCGTAGGGCTTGTTGACC (SEQ ID NO: 25) GATGAGTGCTATGCTGGTCTGCCGACTGAG (SEQ ID NO: 26) TGCCGTTGTGAGCAGGACGTCCAGAGATGT (SEQ ID NO: 27) CTCCTTAGCTGTGACATAGATGCGGTAATA (SEQ ID NO: 28) CACAGCCACCATCACCAGCAGGTTCCCCAA (SEQ ID NO: 29)
  • Said probe may be labeled with any suitable label, e.g. with a fluorescent label such as fluorescein (FITC), a radiolabel, an enzyme label, a biotin label, a digoxygenin (DIG) label, etc, as it is well known in the art.
  • FITC fluorescein
  • DIG digoxygenin
  • Figure 1 Gel Electrophoresis confirmed the size of the plasmids for 5- HT 2B and 5-HT 4 .
  • Figure 2 Serotonin immunoreactivity in surgical resections of human bowel (A - F), rat jejunum (G) and mouse jejunum (H).
  • Figure 3 5-HT 3 immunoreactivity in surgical resections of human bowel.
  • Figure 4 5-HT4-immunoreactivity in human gut.
  • Figure 5 5-HT4- immunoreactivity in rat gut.
  • Figure 6 Localization of mRNA for 5-HT4 in human gut.
  • Figure 7 5HT4-immunoreactivity in human, rat and mouse cell lines.
  • Figure 8 5HT4-immunoreactivity in 5HT4b-transfected CHO cells.
  • FIG. 9 The 5-HT 4 receptor partial agonist, RS 67506, augments ionomycin-induced calcium mobilization in 5-HT 4B -transfected CHO cells.
  • Figure 10 The 5-HT 4 receptor partial agonist, RS 67506, attenuates ionomycin-induced calcium mobilization in BMMC.
  • Figure 1 1 The 5-HT 4 receptor partial agonist, RS 67506, attenuates ionomycin-induced calcium mobilization in RBL (A) and KU812 (B) cells.
  • FIG. 12 The 5-HT 4 receptor partial agonist, RS 67506, attenuates ionomycin-induced TNF ⁇ release from RBL cells (A) and BMMC (B).
  • FIG. 13 The 5-HT 4 receptor partial agonist, RS 67506, attenuates ionomycin-induced ⁇ -hexosaminidase release from KU812 cells.
  • Figure 14 Variable expression of 5-HT 4 in the lamina intestinal of terminal ileum endoscopic biopsies from six different patients (A - 007, B - 068, C - 075, D - 077, E - 095, F - 102).
  • Figure 15 Variable expression of 5-HT 4 in the lamina intestinal of rectal endoscopic biopsies from six different patients (A - 007, B - 068, C - 075, D - 077, E - 095, F - 102).
  • Figure 16 5-HT 4 immunoreactive cell counts in the lamina intestinal of terminal ileum and rectal endoscopic biopsies from patients illustrated in Figures 9 and 10.
  • Figure 17 5-HT 4 -immunoreactivity in the lamina intestinal of IBS patients in dependence of clinical symptoms.
  • BMMC Bone Marrow Mast Cells
  • Bone marrow mast cells were obtained from BALB/c mice and cultured in DMEM medium containing 10% FCS, penicillin/streptomycin with mouse rlL-3 and mouse rSCF (both at concentration of 10 ng/ml) in a 25 cm 3 flask at a cell density of 2 x 10 5 /ml. Prior to testing the cultures were maintained for a minimum of 3 weeks at 37 0 C and 5% CO 2 and the medium was changed weekly.
  • BMMC were washed and re-suspended in DMEM medium at a concentration of 10 6 AnI and placed in 3 ml sterile tubes.
  • Cells were pre-incubated with tested compounds for 15 minutes, followed by activation with the calcium ionophore ionomycin (1 ⁇ M). After a 4 hour incubation cells were spun down, the supernatant was removed and frozen at -2O 0 C for further analysis (released mediators). Tyrode's buffer was added to cell pellets, followed by 2 cycles of freezing and boiling after which supernatants were collected (total mediators) and frozen for further analysis.
  • Rat basophilic cells RBL-2H3 and human KU812 cells were cultured in RPMI medium containing 10% FCS, penicillin/streptomycin in 75 cm 3 flasks at a cell density of 2 x 10 6 /ml. Cultures were maintained at 37 0 C and 5% CO 2 and the medium was changed as necessary.
  • CHO cells Chinese Hamster ovary (CHO) cells were transfected with plasmid using Lipofectamine (Invitrogen) and successfully transfected cells were selected using Geneticin (G418). Cell cultures, cell smears and NBF-fixed cell blocks were prepared, as required.
  • FIG. 1 Gel Electrophoresis confirmed the size of the plasmids for 5-HT 2B and 5-HT 4 .
  • 5-HT 2 B located in lane 6 is 6651 basepairs (bp).
  • 5-HT 4 located in lane 8 had two bands of 2262bp and 4668 bp. Lanes 5 and 7 are supercoiled DNA.
  • the TNF- ⁇ assay may be used to assess the response to ionomycin.
  • BD OptEIA Set Mouse TNF- ⁇ ELISA or BD OptEIA Rat TNF- ⁇ ELISA kit was obtained from BD Biosciences (# 558874 and 558870 respectively). Briefly, plates were coated with 100 ⁇ l of capture antibody and incubated overnight at 4 0 C. Wells were washed (3x) with washing buffer and blocked with 300 ⁇ l of 2% BSA in PBS. After washing again (3x), standard or 100 ⁇ l of sample (cell supernatant) was loaded and incubated for 2 hr at room temperature. After washing detection antibody was added and incubation continued for 1 hr.
  • detection antibody was added followed up by wash (7x) and enzyme conjugate (Avidin-HRP). After a 30-minute incubation in darkness, the reaction was stopped by adding 50 ⁇ l of 1 N HCI. Absorbance was read at 450 nm using an ELISA plate reader and the concentration was determined using the KCJunior program (Bio-Tech Instrument).
  • ⁇ -Hexosaminidase assay is used to measure mast cell degranulation. Briefly, cells at density ⁇ 10 6 AnI are pre-incubated with tested substances for 15 minutes at 37 0 C after which ionomycin is added and incubation continued for additional
  • BMMC cells (1 *10 5 per ml), RBL-2H3 cells (1 ⁇ 10 5 per ml), KU812 cells (1 ⁇ 10 5 per ml) or CHP (wild type or 5-HT 4 transfected) (1 *10 5 per ml) were grown overnight on poly-P-lysine coated glass-bottom culture dishes (Mattek Corp., MA, USA). The cells were loaded with fura-2/AM (final concentration 1-5 ⁇ M, Invitrogen, PN, Canada) for 30 minutes at 37°C.
  • fura-2/AM final concentration 1-5 ⁇ M, Invitrogen, PN, Canada
  • Pishes were then placed on the stage of a Leica PRIRB inverted fluorescence microscope (Leica, PN, Canada) and perfused with Krebs solution (composition in mM: NaCI 118.0, KCI 4.7, NaH 2 PP 4 1.0, NaHCP 3 25.0, MgSP 4 1.2, CaCI 2 2.5, P-Glucose 11.1 , with pH adjusted to 7.3 by using NaPH) for 10-30 minutes (4.5 ml/min) to wash away background fura-2 fluorescence and any unattached cells.
  • Fura-2 signals were monitored using an lonoptix imaging system (lonoptix, MA, USA) under a 4Ox Leica FLUPSTAR lens.
  • Fura-2 excitation was achieved using a mercury arc lamp shuttered between 340 nm and 380 nm excitation filters and emission signals were passed through a 510 nm cutoff emission filter and collected by a Myocam CCP video camera (lonoptix, MA, USA). Data was acquired at 5 or 33 Hz and analyzed using IonWizard software (V4.4.13).
  • Specimens were promptly removed and routinely fixed in 10% neutral buffered formalin for 48 hours and processed to paraffin. Samples from rats and mice were handled similarly. Cultured cells were pelleted in 2% agarose and processed in parallel.
  • Sections were then deparaffinized with xylene, hydrated in alcohol and then immersed in 3% H 2 O 2 to remove endogenous peroxidase activity.
  • Sections from each block were cut at 2 ⁇ m on a Leica microtome.
  • Tissue sections were placed on microslides and left to dry overnight on a hotplate set to 60 0 C.
  • Sections were deparaffinized with xylene, three changes of 100% alcohol were used to clear the xylene. Sections were then hydrated in 70, 50 and
  • Sections were incubated at room temperature in a humidity chamber for 30 - 45 minutes. Sections were checked microscopically to ensure proper color development. [0073] Following appropriate color development, slides were washed in three changes of distilled water. The slides were mounted with an aqueous medium following standard protocols. [0074] The probes used were 30mer oligonucleotides synthesized commercially. These were 3'-biotinylated using Biotin-11-dUTP, dATP and TdT, using standard methodology before purification using spin-columns.
  • ACACAGCCACCATCACCAGCAGGTTCCCCA (SEQ ID NO: 6)
  • ACACTGACTCTCCCACTGGCCACCACACTC SEQ ID NO: 24
  • GAGCAGGTGATGGCGTAGGGCTTGTTGACC SEQ ID NO: 25
  • Probe SEQ ID NOS. 1-30 are particularly suitable for 5-HT 4 receptor.
  • Probe SEQ ID NOS. 31-40 are particularly suitable for 5-HT 3 receptor.
  • Probe SEQ ID NOS. 41-47 are particularly suitable for 5-HT 2 B receptor.
  • FIG. 2 Serotonin immunoreactivity in surgical resections of human bowel (A - F), rat jejunum (G) and mouse jejunum (H). Prominent serotonin immunoreactivity is apparent in enterochromaffin cells (in A and ⁇ in B, C, D, G and H). Mast cells are clearly serotonin immunoreactive in all rodent gut samples (* in G and H) and in approximately 1 in 5 human samples (* in C, D, E and F).
  • Figure 3 5-HT 3 immunoreactivity in surgical resections of human bowel. All samples are non-involved tissues from cancer resections. Note clear enteroendocrine cell staining (A - C) and minimal labelling in the lamina limbal (B, C and D).
  • FIG. 4 5-HT 4 -immunoreactivity in human gut.
  • 5-HT 4 receptors are localized to mast cells in the lamina propria (A), enteroendocrine cells (B) muscle (muscularis intestinal; C) and in a punctuate pattern in the lamina propria (D), at all levels of the human gastrointestinal tract.
  • FIG. 5 5-HT 4 - immunoreactivity in rat gut.
  • Mast cells in the submucosa (A) and mucosa in normal rat gut are 5-HT 4 -immunoreactive.
  • B 5-HT4-immunoreactivity
  • FIG. 6 Localization of mRNA for 5-HT 4 in human gut.
  • a submucosal mast cell expressing message for 5-HT 4 (antisense probe 36LA) is evident in (B) but mast cells do not bind the message sense probe (A).
  • muscle cells express 5-HT 4 mRNA (D) but do not label with message sense probes (C).
  • 5-HT 4 receptor immunohistochemistry was performed on human and rodent mast cell lines in order to attempt to verify this observation.
  • 5-HT 4 receptor immunoreactivity was observed on the human mast cell line KU812, primary mouse cultured bone marrow mast cells and rat basophil leukaemia cells.
  • Figure 7 5HT 4 -immunoreactivity in human, rat and mouse cell lines KU812 (human mast cell/basophil) cells express strong 5HT 4 -immunoreactivity (A); but staining is not observed using an equivalent concentration of normal rat immunoglobulins (negative control; B). Both primary mouse cultured bone marrow mast cells (C) and rat basophil leukaemia cells (mucosal mast cell like; RBL; D) also stain intensely for 5HT 4 .
  • C primary mouse cultured bone marrow mast cells
  • RBL rat basophil leukaemia cells
  • Figure 8 5HT4-immunoreactivity in 5HT 4b -transfected CHO cells. Smears of wild type (A & E) and 5HT 4 -transfected (B & F) CHO cells and paraffin processed cell pellets of wild type (C & G) and transfected (D & H) cells are illustrated, lmmunostaining for the V5 tag labels a small proportion of only the transfected cells (B & D) but not the wild type cells (A & C). Anti-5HT 4 antibodies detect a similar population of transfected CHO cells (F & H) but not wild type cells (E & G).
  • FIG. 9 The 5-HT 4 receptor partial agonist, RS 67506, augments ionomycin-induced calcium mobilization in 5-HT 4B -transfected CHO cells.
  • ionomycin (2 ⁇ M) resulted in a reproducible elevation of intracellular calcium as measured with Fura-2.
  • Stimulation of the 5-HT 4 transfected CHO cells with ionomycin resulted in an elevation of intracellular calcium, similar to the levels observed in wild type cells.
  • pre-incubation with RS resulted in a significant increase in the Fura-2 ratio, indicating an enhanced elevation of intracellular calcium.
  • FIG. 10 The 5-HT 4 receptor partial agonist, RS 67506, attenuates ionomycin-induced calcium mobilization in BMMC.
  • RS 67506 attenuates ionomycin-induced calcium mobilization in BMMC.
  • ionomycin resulted in a reproducible elevation of intracellular calcium as measured with Fura-2.
  • pre-incubation with RS resulted in a significant decrease in the Fura-2 ratio, suggesting that stimulation of the with the 5-HT 4 receptor partial agonist resulted in a decrease in the stimulated elevation of intracellular calcium.
  • FIG. 11 The 5-HT 4 receptor partial agonist, RS 67506, attenuates ionomycin-induced calcium mobilization in RBL (A) and KU812 (B) cells.
  • ionomycin resulted in a reproducible elevation of intracellular calcium as measured with Fura-2.
  • stimulation of the KU812 cells with ionomycin caused a reproducible elevation of intracellular calcium.
  • Preincubation of the RBL cells with 20 ⁇ M RS resulted in a significant decrease in the Fura-2 ratio.
  • pre-incubation of the KU812 cells with RS (2 - 20 ⁇ M) resulted in a dose-dependent decrease in the Fura-2 ratio.
  • FIG. 12 The 5-HT 4 receptor partial agonist, RS 67506, attenuates ionomycin-induced TNF ⁇ release from RBL cells (A) and BMMC (B). Stimulating either cell type resulted in a significant increase in the amount of TNF ⁇ released into the culture medium. Stimulation with 5-HT had no significant impact on the amount of TNF release in either RBL or BMMC cell types. Similarly, stimulation with the 5-HT 4 receptor antagonist caused no significant changes in the amount of TNF release. In contrast, stimulation of the RBL cells with RS resulted in a significant 72% decrease in TNF release. A similar trend was observed in the BMMC; however, this failed to reach statistical significance. TNF levels were measured using species specific ELISA.
  • 5-HT 4 receptors appear to be expressed in human intestine, especially on mast cells, and mast cell numbers and relationships with nerves are reported to change in irritable bowel syndrome (Barabara et al. 2006), this raises the possiblity that 5-HT 4 receptors could be used as a theragnostic tool to determine the most appropriate treatment for patients suffering from functional bowel disorders such as irritable bowel syndrome.
  • the levels of 5-HT 4 receptor expression was determined in patients suffering from irritable bowel syndrome and controls.
  • Quantitation of the 5HT4 immunoreactivity in the lamina limbalosies using image analysis software revealed numerical differences in the amount of staining in different patients.
  • Figure 14 Variable expression of 5-HT 4 in the lamina intestinal of terminal ileum endoscopic biopsies from six different patients (A - 007, B - 068, C - 075, D - 077, E - 095, F - 102). Compare with Figure 10.
  • Figure 15 Variable expression of 5-HT 4 in the lamina intestinal of rectal endoscopic biopsies from six different patients (A - 007, B - 068, C - 075, D - 077, E - 095, F - 102). Compare with Figure 9.
  • Figure 16 5-HT 4 immunoreactive cell counts in the lamina limbal ileum and rectal endoscopic biopsies from patients illustrated in Figures 9 and 10.
  • Figure 17 Quantitation of the 5-HT 4 -immunoreactivity in the lamina intestinal of rectal biopsies . Using imaging software numerical differences in the amount of staining in different patients was revealed. The classification according to patient group show a trend that in patients suffering from IBS with constipation, the level of 5-HT 4 receptor expression is close to healthy controls, whereas the level of 5-HT 4 receptor expression in patients suffering from IBS with diarrhea or alternating constipation and diarrhea is lower. [0100] It is noted that the foregoing examples have been provided merely for the purpose of explanation and are in no way to be construed as limiting of the present invention.
  • Irritable bowel syndrome new agents targeting serotonin receptor subtypes. Drugs 61 , 317-332.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne une méthode de diagnostic d'une maladie intestinale fonctionnelle chez un patient humain, la méthode consistant à tester des cellules gastro-intestinales et/ou des cellules circulant dans le sang périphérique du patient afin de déterminer la présence d'une sérotonine et/ou d'un récepteur de sérotonine.
PCT/CA2006/001060 2005-06-27 2006-06-27 Methodes de diagnostic de maladie intestinale fonctionnelle WO2007000046A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US69378405P 2005-06-27 2005-06-27
US60/693,784 2005-06-27

Publications (1)

Publication Number Publication Date
WO2007000046A1 true WO2007000046A1 (fr) 2007-01-04

Family

ID=37595023

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CA2006/001060 WO2007000046A1 (fr) 2005-06-27 2006-06-27 Methodes de diagnostic de maladie intestinale fonctionnelle

Country Status (1)

Country Link
WO (1) WO2007000046A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016520199A (ja) * 2013-05-24 2016-07-11 ネステク ソシエテ アノニム 過敏性腸症候群を診断するための経路特異的マーカー

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4910193A (en) * 1985-12-16 1990-03-20 Sandoz Ltd. Treatment of gastrointestinal disorders
WO2002036114A1 (fr) * 2000-11-01 2002-05-10 Respiratorius Ab Composition comprenant les antagonistes des recepteurs de serotonine 5 ht-2 et 5 ht-3
US6444477B1 (en) * 2000-11-28 2002-09-03 Pharmagene Laboratories Limited Assay method for detecting 5-HT2B antagonists
US20030036549A1 (en) * 1997-10-07 2003-02-20 Mangel Allen Wayne Methods for treating irritable bowel syndrome
WO2004009633A1 (fr) * 2002-07-19 2004-01-29 Inpharmatica Limited Recepteur de serotonine
WO2004104577A2 (fr) * 2003-05-21 2004-12-02 Bayer Healthcare Ag Approches diagnostiques et therapeutiques des maladies associees au recepteur 5-hydroxytryptamine 4 (5-ht4)(serotonine)
US20050032780A1 (en) * 2003-01-13 2005-02-10 Dynogen, Inc. Method of treating functional bowel disorders

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4910193A (en) * 1985-12-16 1990-03-20 Sandoz Ltd. Treatment of gastrointestinal disorders
US20030036549A1 (en) * 1997-10-07 2003-02-20 Mangel Allen Wayne Methods for treating irritable bowel syndrome
WO2002036114A1 (fr) * 2000-11-01 2002-05-10 Respiratorius Ab Composition comprenant les antagonistes des recepteurs de serotonine 5 ht-2 et 5 ht-3
US6444477B1 (en) * 2000-11-28 2002-09-03 Pharmagene Laboratories Limited Assay method for detecting 5-HT2B antagonists
WO2004009633A1 (fr) * 2002-07-19 2004-01-29 Inpharmatica Limited Recepteur de serotonine
US20050032780A1 (en) * 2003-01-13 2005-02-10 Dynogen, Inc. Method of treating functional bowel disorders
WO2004104577A2 (fr) * 2003-05-21 2004-12-02 Bayer Healthcare Ag Approches diagnostiques et therapeutiques des maladies associees au recepteur 5-hydroxytryptamine 4 (5-ht4)(serotonine)

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CROWELL M.D. ET AL.: "Enterochromaffin cells and 5-HT signaling in the pathophysiology of disorders of gastrointestinal function", CURR. OPIN. INVESTIG. DRUGS, vol. 5, no. 1, January 2004 (2004-01-01), pages 55 - 60, XP008074603 *
NAKAI A. ET AL.: "The effects of the 5-HT3 antagonist, alosetron, on brain serotonin synthesis in patients with irratable bowel syndrome", NEUROGASTROENTEROL MOTIL., vol. 17, no. 2, April 2005 (2005-04-01), pages 212 - 221, XP003006302 *
SMITH D.G. ET AL.: "Tegaserod treatment for IBS: a model of indirect costs", AM. J. MANAG. CARE, vol. 11, no. 1, SUPPL. 1, April 2005 (2005-04-01), pages S43 - S50, XP003006301 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016520199A (ja) * 2013-05-24 2016-07-11 ネステク ソシエテ アノニム 過敏性腸症候群を診断するための経路特異的マーカー
JP2019194600A (ja) * 2013-05-24 2019-11-07 ソシエテ・デ・プロデュイ・ネスレ・エス・アー 過敏性腸症候群を診断するための経路特異的マーカー

Similar Documents

Publication Publication Date Title
Kafaja et al. pDCs in lung and skin fibrosis in a bleomycin-induced model and patients with systemic sclerosis
Beroukas et al. Up-regulation of M3-muscarinic receptors in labial salivary gland acini in primary Sjögren's syndrome
US20200323948A1 (en) Treating Renal and Liver Dysfunction with TLR4 Antagonists
TR201821294T4 (tr) Böbrek Hastalığının Teşhisi Ve Tedavisine Yönelik Slit-robo Sinyal İletimi
WO2023179013A1 (fr) Application d'un réactif pour la détection d'un complexe immun iga
Thomsen et al. Immunohistochemical detection of C5b-9 (m) in myocardium: an aid in distinguishing infarction-induced ischemic heart muscle necrosis from other forms of lethal myocardial injury
Zhang et al. Effect of glomerular mannose-binding lectin deposition on the prognosis of idiopathic membranous nephropathy
RU2009106718A (ru) Применение белка с меланома-ингибирующей активностью (миа) в качестве раннего индикатора терапевтического ответа при меланоме
Zou et al. C/EBPβ isoform‐specific regulation of podocyte pyroptosis in lupus nephritis‐induced renal injury
Lv et al. Microbial imidazole propionate affects glomerular filtration rate in patients with diabetic nephropathy through association with HSP90α
WO2007000046A1 (fr) Methodes de diagnostic de maladie intestinale fonctionnelle
WO2013150375A2 (fr) Biomarqueurs d'activation des cellules endothéliales caractérisant le rejet médié par anticorps et utilisations associées
KR102314642B1 (ko) 파브리 병 진단용 바이오 마커 및 이의 용도
Akasaka et al. Normal saline remodels the omentum and stimulates its receptivity for transcoelomic metastasis
RU2358265C1 (ru) Способ диагностики активности воспалительного процесса при хроническом пиелонефрите
US8895511B2 (en) Use of sarcoplasmic CA2+-ATPase type 2 protein for diagnosing and treating learning or mental disorders
GOANŢĂ et al. Clinical and therapeutical approach to protein-losing nephropathy in dogs-a review.
WO2019077143A1 (fr) Procédés et compositions pour prédire et diagnostiquer précocement une forme active chronique d'abmr avec une glomérulopathie de greffe et une perte de greffe rénale
CN112557664A (zh) Cryab在急性肾损伤检测中的应用及检测试剂盒
Hattori et al. Intrarenal renin–angiotensin system activation and macrophage infiltrations in pediatric chronic glomerulonephritis
EP1137946B1 (fr) Utilisation du facteur de croissance endothelial vasculaire
RU2712225C2 (ru) Способ постановки прогноза и наборы, применимые в указанном способе
CN115951047A (zh) 自身免疫性水疱病PBMCs直接免疫荧光诊断试剂盒
Ren et al. TIGAR attenuates intervertebral disc degeneration via autophagy-mediated Keap1 degradation and Nrf2 nuclear translocation to suppress nucleus pulposus pyroptosis
RU2447440C1 (ru) Способ оценки эффективности лечения язвенного колита

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

WWW Wipo information: withdrawn in national office

Country of ref document: DE

122 Ep: pct application non-entry in european phase

Ref document number: 06775040

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载