EP1229925A2 - Inhibiteurs de maladies gastro-intestinales induites par helicobacter pilori - Google Patents

Inhibiteurs de maladies gastro-intestinales induites par helicobacter pilori

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Publication number
EP1229925A2
EP1229925A2 EP00990605A EP00990605A EP1229925A2 EP 1229925 A2 EP1229925 A2 EP 1229925A2 EP 00990605 A EP00990605 A EP 00990605A EP 00990605 A EP00990605 A EP 00990605A EP 1229925 A2 EP1229925 A2 EP 1229925A2
Authority
EP
European Patent Office
Prior art keywords
inhibitors
compounds
pylori
helicobacter
medicament
Prior art date
Legal status (The legal status 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 status listed.)
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Application number
EP00990605A
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German (de)
English (en)
Inventor
Christian Wallasch
Dorian Bevec
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Axxima Pharmaceuticals AG
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Axxima Pharmaceuticals AG
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Publication date
Application filed by Axxima Pharmaceuticals AG filed Critical Axxima Pharmaceuticals AG
Priority to EP00990605A priority Critical patent/EP1229925A2/fr
Publication of EP1229925A2 publication Critical patent/EP1229925A2/fr
Withdrawn legal-status Critical Current

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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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • 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
    • A61K31/275—Nitriles; Isonitriles
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/4152—1,2-Diazoles having oxo groups directly attached to the heterocyclic ring, e.g. antipyrine, phenylbutazone, sulfinpyrazone
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • 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
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A61K31/5513—1,4-Benzodiazepines, e.g. diazepam or clozapine
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the invention refers to a method for the manufacture of a medicament for treating or preventing Helicobacter mediated diseases in a mammal and a method for treating or preventing Helicobacter mediated diseases.
  • H. pylori is a pathogen which is known to induce gastrointestinal diseases.
  • Recent therapy against H. pylori includes a combination therapy comprising the application of two of the following antibiotics: tetracycline, amoxicillin, clarithromycin or metronidazole combined with an inhibitor of the proton pump like ranitidine or a bismuth salt, e.g. bismuth citrate.
  • tetracycline tetracycline
  • amoxicillin amoxicillin
  • clarithromycin or metronidazole metronidazole
  • an inhibitor of the proton pump like ranitidine or a bismuth salt e.g. bismuth citrate.
  • emerging resistance against metronidazole among Helicobacter strains is already found worldwide and correlates with treatment failure (Alarcon et al., Int. J. Antimicrob. Agents 1 ( 1 999), 1 9-26).
  • H. pylori fights the acidic gastric juice of the stomach by releasing urease which converts gastric urea into bicarbonate and ammonia.
  • peripheral regulation of gastric acid secretion is initiated by the release of gastrin from G-cells, employing the gastrin receptor, a G- protein coupled receptor (GPCR).
  • GPCR G- protein coupled receptor
  • H. pylori is crucially involved in the upregulation of gastric epithelial interleukin 8 (IL-8) expression. This upregulated epithelial IL-8 secretion and concommitant chemotaxis and activation of immunocompetent cells are discussed to be involved in tissue damage, ulceration and gastric carcinogenesis (Crabtree & Lindley, Eur. J.
  • H. pylori strains there is division into two major groups: those having genetic loci coding for immunodominant proteins of uncertain functions (cagA islands; cagA - strains) and those lacking this genetic area (Crabtree et al. (1 995), Supra; cagA- strains). This cagA pathogenicity island is supposed to be responsible for the disease induction by H.
  • cagA + strains of H. pylori are responsible for disease development, but also cagA- strains show the same molecular effects as cagA + strains by employing the triple membrane passing signaling cascade published by Prenzel et al., Nature 402 (1 999), 884-888.
  • the present invention refers to a method for the manufacture of a medicament for treating or preventing Helicobacter mediated diseases in a mammal, wherein said medicament comprises as an active ingredient at least one compound selected from
  • the active ingredient is preferably a compound capable of inhibiting the"triple membrane passing signal" cascade.
  • a pathogenic stimulation of this cascade caused by H. pylori infection resulting in a pathogenic phenotype will be at least partially suppressed. This leads to the improvement or the disappearance of clinical symptoms associated with H. pylori infections.
  • the Helicobacter induced diseases to be treated or prevented are preferably selected from gastrointestinal cancer and inflammatory conditions, e.g. chronic non-erosive gastritis, peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma and intestinal-type adenocarcinoma.
  • chronic non-erosive gastritis e.g. chronic non-erosive gastritis, peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma and intestinal-type adenocarcinoma.
  • the active ingredient is selected from compounds (a) to (g).
  • the medicament may comprise only one active ingredient or several active ingredients which may be selected from compounds which are directed to the same target or from compounds which are directed to two or more different targets.
  • the inhibitors of gastrin/cholecystokinin (CCK)-B receptor (a) are preferably selected from nonapeptide CCK-B antagonists, amino acid derivatives, benzodiazepines, dipeptoids, pyrazolidinones, quinazolinones, ureidoacetamides, bicyclic compounds such as dibenzobicyclo [2.2.2]octane, aromatic compounds such as binaphthalene derivatives, ureidobenzazepines and ureido methylcarbamoyl phenylketones as described by Revel and Makovec (Drugs of the Future 23 (1998), 751- 766). Further suitable compounds are described by Crawley (J. Neuroscience 12 (1992), 3380-3391) and Hughes et al. (Proc. Natl. Acad. Sci. U.S.A.87 (1990), 6728-6732). The disclosure of these documents is herein incorporated by reference.
  • amino acid derivatives are glutamic acid derivatives such as spiroglumides, e.g. CR-2622 (Makovec et al., J. Med. Chem. 39 (1996), 135-142).
  • benzodiazepines are L-365260 (Bock et al., J. Med. Chem. 32 (1989), 13-16), YM-022 (Satoh et al., Chem. Pharm. Bull. (Tokyo) 43 (1995), 2159-2167) and GR-199114X (Bailey et al., Bioorg. Med. Chem. Lett. 7 (1997), 281-286).
  • pyrazolidinones are LY-288513 and LY-262691 (Helton et al., Pharmacol. Biochem. Behav. 53 (1996), 493-502).
  • a specific example of quinazolinones is LY-247348 (Yu et al., J. Med. Chem.34 (1991), 1505- 1508).
  • dipeptoids are PD-136450, CI-988 and PD- 134308 (Boden et al., J. Med. Chem.36 (1993), 552-565; Maldonado et al., Br. J. Pharmacol. 114 (1995), 1031-1059).
  • ureidoacetamides are RP-69758 and RP-73870 (Pendley et al., J. Pharmacol. Exp. Ther. 273 (1995), 1015-1022).
  • ureidobenzazepines are 5-phenyl-3-ureidobenzazepin-2-ones such as CP- 212454 (Lowe III et al., J. Med. Chem. 37 (1994), 3789-3811).
  • a specific example of ureidomethylcarbamoylphenylketones is S-0509 (Hagishita et al., Bioorg. Med. Chem. 8 (1997), 1695-1714). The disclosure of these documents is herein incorporated by reference.
  • Inhibitors of protein kinase C may be ATP-competitive inhibitors, antisense oligonucleotides, peptides and lipids. Particularly preferred are indolocarbazoles, bisindolylmaleimides, balanol analogs, antisense oligonucleotides and alkyl-lysophospholipids (Goekjian and Jirousek, Curr. Med. Chem.6 (1999), 877-903). The disclosure of this document is herein incorporated by reference.
  • indolocarbazole compounds are e.g. the compounds dislosed in EP-A-0328000, EP-A-0370236, EP-A-0410389 and EP-A- 5 0434057 or the compounds disclosed in EP 991 1 6426.0.
  • suitable indolocarbazoles are G5761 2 and CGP41 251 (Ikegami et al., Jpn. J. Pharmacol. 70 (1 996), 65-72) .
  • Specific examples of bisindolyimaleimides are LY333531 , GF1 09203x, Ro32-0432 and Ro31 -8220 (Jirousek et al., J. Med. Chem. 39 ( 1 996), 2664-2671 ) .
  • balanol analogs is SPC100840 (Goekjian and Jirousek, Supra) .
  • a specific example of an antisense oligonucleotide is CGP641 28A (Goekjian and Jirousek, Supra) .
  • a specific example of alkyl- lysophospholipids is ET-1 8-OCH3 (Civoli and Daniel, Cancer Chemother. Pharmacol. 42 (1 998), 31 9-326). The disclosure of these documents is s herein incorporated by reference.
  • Inhibitors of membrane-associated metalloproteinases may be selected from inhibitors of proteinase activity or 0 compounds which block the metalloproteinase's access to its substrate, e.g. by masking the substrate. These inhibitors may be selected from e.g. compounds containing a hydroxamic acid group. Specific examples of hydroxamic acid derivatives are GW9471 and GW9277 (Moss et al., J. Neuroimmunol.
  • TACE tumor necrosis factor-alpha converting enzyme
  • thiol and hydroxamic acid derivatives have been used as TACE inhibitors: acetylated ortho- sulfonamido or phosphinic acid amido bicyclic heteroaryl hydroxamic acids o (WO0044749), heteroaryl acetylenic suifonamide or phosphinic acid amide hydroxamic acids (WO0044740), acetylenic suifonamide thiols (WO004471 6), alkynyl containing hydroxamic acids (WO004471 3), acetylenic ( ⁇ )-sulfonamido or phosphinic acid amide hydroxamic acids (WO004471 1 ), acetylenic aryl suifonamide
  • MMP matrix metalloproteinase
  • lactams e.g. lactams, alkenyldiarylmethanes, amides and sulfonamides, azepans or carboxylic 5 acids and derivatives thereof.
  • lactams As members of the lactam family have been cited N-carboxymethyl substituted benzolactams (AU 1 836900) or 3-thio substituted amidolactams (AU 1 926700) .
  • Alkenyldiarylmethanes are mentioned in WO9936384, WO9740072 and GB748400.
  • Suitable amides, sulfonamides or succinamides for MMP inhibiton are N- 0 hydroxyformamide (WO004471 2), amidomalonamides (WO0040552), N- hydroxy-2-(alkyl, aryl or heteroaryl sulfanyl, sulfinyl or sulfonyl)-3- substituted alky, aryl or heteroarylamides (CN 1 25271 3T), CN 1 252790T), thiadiazole amides (WO9748688), amino acid amides of 5-amino-1 ,3,4- thiadiazones (WO9640745); sterically hindered sulfonamides (US61 1 4568), cyclic suifonamide derivatives (WO9941 246), biphenylsulfonamides (CN 1 21 91 66); heteroaryl succinamides (EP0937042), N-(amino acid) substituted succinic acid amides
  • azepan derivatives like 1 -caboxymethyl-2-oxoazepan derivatives or 3- mercaptoacetylamino-1 ,5-substituted-2-oxoazepans have been shown to exhibit MMP inhibitor activity.
  • carboxylic acids, carboxylic acid derivatives and carboxylic acid group containing substances have been cited as metalloproteinase inhibitors.
  • Cited members of the carboxylic acid family are carboxylic acid derivatives (US61 1 8001 ), carboxylic acid substituted heterocycles (EP1 042297, WO9932452), aminomalonic acid derivatives (WO0002904), butyric acid (US6020366), malonic acid (WO991 1 608), bile acid (US564631 6).
  • MMP inhibitors sulfonated amino acids (AU3022200, EP0757037), phosphono derivatives of amino acids (WO931 4096), natural amino acid derivatives (US5643964), N- sulfonylamino derivatives of dipeptides (US55301 28), peptidyl compounds (US5981490), US5300501 ), arylpiperazines (WO001 2478), N- hydroxyacylamino compounds (WO001 2467), hydroxylamine derivatives (HU99041 65), sulfonylaminophosphinic and phosphonic acids (DE 1 9831 980), C-terminal ketones (US598591 1 ) macrocyclic compounds (US5952320), aporphinoid (WO991 6441 ), dioxolane (EP075688), mercaptoketones and mercaptoalcohols (EP081 8443), mercaptosulfides (US54
  • TIMP tissue inhibitor of metalloproteinase
  • TIMP-1 and TIMP-2 tissue inhibitor of metalloproteinase-2
  • Inhibitors of growth-factor receptors may be inhibitors of members of the epidermal growth factor receptor family, e.g. EGF receptor, HER2, HER3, HER4 or inhibitors of other growth-factor receptors, e.g. TNF-alpha receptor.
  • the inhibitors of growth-factor receptors may be selected from low-molecular weight compounds or from antibodies which inhibit the binding of receptor ligands to their receptors, particularly of receptor ligands of the EGF-like ligand family, which are released from membrane- bound ligand precursors (f.e. HB-EGF) .
  • Specific examples are anti-receptor antibodies or fragments thereof, e.g. the antibody Herceptin or Trastuzumab (Goldenberg, Clin. Ther. 21 (1 999), 309-31 8) .
  • the disclosure of this document is herein incorporated by reference.
  • Inhibitors of growth-factor receptor kinase activity may be selected from phenylaminoquinazolines, substituted pyrimidines comprising pyrido-, pyrrolo-, pyrazolo-, pyrimido- and phenylaminopyrimidines, tyrphostins, lavendustins and dianilino-phthalimides (Traxler and Lydon, Drugs of the Future 20 (1 995), 1 261 -1 274), the disclosure of which is herein incorporated by reference.
  • phenylaminoquinazolines are PD1 53035 (Fry et al., Science 265 (1 994), 1 093-1 095), ZD1 839 (Woodburn et al., Proc. Am. Assoc. Cancer Res. 38 (1 997), 633) and CP-358,774 (Proc. Am. Assoc. Cancer Res. 38 ( 1 997), 633) .
  • Specific examples of substituted pyrimidines are PD 1 58780 (Rewcastle et al., J. Med. Chem. 39 (1 996), 1 823-1 835), PD 1 66285 (Panek et al., J. Pharmacol. Exp. Ther.
  • tyrphostins AG 1 478, RG 1 3022 and AG825 (Levitzki and Gazit, Science 267 (1 995), 1 782-1 788) .
  • lavendustins A specific example of lavendustins is Lavendustin A (Onoda et al., J. Nat. Prod. 52 ( 1 998), 1 252-1 257) .
  • dianilino-phthalimides is CGP54698 (Buchdunger et al., Clin. Cancer Res. 8 ( 1 995), 81 3-821 ) . The disclosure of these documents is incorporated herein by reference.
  • Inhibitors of the mitogen-activated protein cascade are preferably selected from inhibitors of MAPKKK (Raf), inhibitors of MAPKK (Mek) and inhibitors of MAPK (Erk).
  • Specific examples are PD098059 (Dudley et al., Proc. Nat. Acad. Sci. U.S.A. 92 (1 995), 7686-7689; He et al., Cell Growth Differ. 1 0 ( 1 999), 307-31 5), U01 26 (Favata et al., J. Biol. Chem.
  • Transcription inhibitors (g) may be general transcription inhibitors or transcription inhibitors which have a selectivity for inhibiting the transcription of the c-fos gene, e.g. antisense oligonucleotides.
  • the administration of compounds (a) to (g) may be part of a combination therapy wherein several compounds belonging to the same inhibitor class and/or belonging to different inhibitor classes are employed.
  • the therapy may comprise the administration of a further active ingredient which is effective against Helicobacter infections.
  • This further active ingredient may be selected from antibiotics, e.g. tetracycline, amoxicillin, pantoprazole, clarithromycin or metronidazole, proton pump inhibitors such as ranitidine, lansoprazole, omeprazole, rabeprazole, benzimidazoles or bismuth salts and H. pylori vaccines, e.g. recombinant urease or urease subunit vaccines.
  • the further active ingredient may also be an immunogenic adjuvant, e.g. a compound which provides a general or unspecific immune stimulation, e.g. an immune adjuvant such as aluminum hydroxide or particularly a CpG-motif containing adjuvant (cf.
  • an immunogenic adjuvant e.g. a compound which provides a general or unspecific immune stimulation, e.g. an immune adjuvant such as aluminum hydroxide or particularly a CpG-motif containing adjuvant (cf.
  • WO96/02555 WO98/401 00; WO99/51 259 or a cytokine. It should be noted that the respective active ingredients of a combination medicament may be administered together or separately.
  • the dosage and type of administration depend on the degree of the disease and on the medicament administered each and or combination of medicaments administered each. In this context it is referred to the documents referenced above.
  • the medicaments are administered over a period of several days and several weeks, respectively, in an effective amount, with the treatment intervals being repeated several times, if necessary.
  • a method for identifying new medicaments for treating or preventing Helicobacter mediated diseases in a mammal comprises a screening assay for compounds of the classes (a) to (g) as defined above.
  • This screening assay is based on an identification of modulators of targets molecules selected from CCK-B receptor, protein kinase C, membrane-associated metalloproteinases, growth-factor receptors and members of the mitogen-activated protein kinase cascade and c-fos.
  • the screening method may be a cellular assay, e.g.
  • an assay wherein the effect of test compounds on cells overexpressing a target molecule as specified above is determined, or a molecular assay, wherein the effect of test compounds on the target molecule is determined in a cell- free system, and wherein the target molecule may be present in a substantially purified and isolated form.
  • Example 1 The invention shall be further illustrated by the following Examples.
  • Example 1 The invention shall be further illustrated by the following Examples.
  • Example 1 The invention shall be further illustrated by the following Examples.
  • RNA Preparation for hybridizing cDNA arrays In principle, gastric cancer cell lines were washed after removal of medium with 1 5 ml ice-cold phosphate-buffered saline (PBS), trypsinized and pelleted in a 1 5 ml flacon tube for 5 min at 4°C and 2000 rpm. After centrifugation the supernatant was removed and the cells were lysed in 1 ml of TRIZOL reagent per 1 .5 x 1 0 s cells. The TRIZOL/cell lysate was transferred to an eppendorff tube and centrifuged for 1 5 min at 4°C and 1 3000 rpm.
  • PBS ice-cold phosphate-buffered saline
  • the supernatant was transferred into a new eppendorff tube and 0.1 ml of BCP (1 -bromo-3-chlorpropane) for each ml of TRIZOL was added.
  • the samples were vortexed for 1 5 seconds and incubated for 5 min at room temperature. Then the samples were centrifuged for 1 5 min at 4°C and 1 3000 rpm.
  • the resulting upper aqueous phase was transferred into a new eppendorff tube, 0.5 ml of isopropanol for each ml of TRIZOL was added; vortexed; and incubated for additional 8 min at room temperature.
  • RNA for Northern Blotting was prepared, the protocol and reaction buffers included with the "RNeasy ® Mini Kit" (QIAGEN) were used.
  • RNA was reverse transcribed into first strand cDNA by adding 1 ⁇ g of oligo-dT primer. In a final reaction volume of 1 2 ⁇ , the mixture was incubated for 5 min at 60°C, and quickly cooled for 2 min on ice, followed by a 30 second centrifugation at 1 3000 rpm.
  • the reaction was stopped by addition of 5 ⁇ 0.5 M EDTA and 25 ⁇ 0.6 N NaOH.
  • the resulting flow-through containing the cDNA was precipitated by adding 1 /25 volume 5 M NaCI (final concentration 0.2 M), 1 ⁇ l polyacryl carrier, 2.5 volumes 1 00% ethanol, vortexed, incubated for 10 min on ice and pelleted by centrifugation for 1 0 min at 4°C with 1 3000 rpm. Finally the supernatant was removed, the cDNA-pellet washed with 80% ethanol, air-dried and resuspended in 30 ⁇ l Tris/EDTA (1 0 mM/0.1 mM) .
  • Random primed labeling of cDNA using Random Primers Labeling System 50 ng of cDNA template were denatured for 5 min at 95°C and quickly chilled on ice for 5 min. After spinning down the sample the following ingredients were added to a final volume of 50 ⁇ l: 2 ⁇ l 0.5 mM dCTP, 2 ⁇ l 0.5 mM dGTP, 2 ⁇ l 0.5 mM dTTP, 5 ⁇ l P 32 - ⁇ -dATP (50 ⁇ Ci) and 1 ⁇ l Klenow-enzyme (3 U/ ⁇ l) . The sample was incubated for 60 min at 25°C and the reaction was stopped by adding 5 ⁇ l 0.5 M EDTA, pH 8.0.
  • Prehybridization, preassociation, hybridization and filterwash Filters were incubated for 5 min at room temperature with 25 ml TE buffer in roller bottles. Following incubation TE solution was replaced by 8 ml prewarmed hybridization-solution (5 x SSPE, 1 0 x Denhardt's solution, 50% formamide, 1 % SDS, 1 00 ⁇ g/ml denatured and fragmented salmon sperm-DNA). Before hybridization, the radioactively labelled probe was pre-associated in a solution containing yeast RNA (final concentration 0.7 ⁇ g/ ⁇ l), polyA (0.7 ⁇ g/ ⁇ l), Cot DNA (0.07 ⁇ g/ ⁇ l), SDS (1 %) and 5 x SSPE. Denaturation occured for 5 min at 95°C and was followed by a pre-association step for 60 min at 65°C. Then the preassociated probe was added to the filters and hybridization reaction was performed for 2 days at
  • HB-EGF heparin-binding epidermal growth factor
  • AR Amphiregulin
  • AR Genebank accession no. M30704
  • TACE/ADAM 1 tumor necrosis factor ⁇ converting enzyme
  • ADAM20 Genebank accession no. AF029899
  • GPDH Genebank accession no. AF261 085
  • ⁇ -Actin Genebank accession no. NM_001 1 01
  • 25 ng of the respective cDNA template were denatured for 5 min at 95°C and quickly chilled on ice for 5 min. After spinning down the sample, the following ingredients were added to a final volume of 50 ⁇ l: 1 ⁇ l 0.5 mM dCTP, 1 ⁇ l 0.5 mM dGTP, 1 ⁇ l 0.5 mM dTTP, 5 ⁇ l
  • P 32 - ⁇ -dATP (50 ⁇ Ci) 20 ⁇ l random primer solution (1 25 mM Tris/HCI pH 6.8, 1 2.5 mM MgCI 2 , 25 mM 2-mercaptoethanol, 50 ⁇ g/ml random octamer primer) and 1 ⁇ l Klenow-enzyme (40 U/ ⁇ l) .
  • the sample was incubated for 1 0 min at 37°C and the reaction was stopped by adding 2 ⁇ l 0.5M EDTA, pH 8.0.
  • Unincorporated P 32 - ⁇ -dATP nucleotides were removed as follows: After vortexing a ProbeQuant Sephadex G-50 column TM (Amersham), the bottom closure was snapped off, the column placed in a 2 ml tube and centrifuged for 1 minute at 735 x g. Then the column was placed in a new 1 .5 ml eppendorff tube without cap and the radioactive probe was pipetted carefully on the center of the preformed resin. Centrifugation for 2 min at 735 x g removed the unincorporated P 32 - -dATP nucleotides.
  • the nitrocellulose filter was prehybridized for 4 hours at 42°C in the following solution: 50% formamide, 5 x SSC, 5 x Denhardt's solution, 0, 1 % SDS.
  • the respective radioactively labeled probe was added after denaturation at 1 00°C for 1 0 min together with 20 ⁇ g/ml salmon sperm DNA to the filters.
  • Hybridization was performed for 1 6 hours at 42°C. Then the filters were washed twice for 1 0 min with 2 x SSC/0.1 % SDS at 42°C and twice for 10 min in 0.2 x SSC/0.1 % SDS at 42°C.
  • Hybridization signals were detected using Kodak BioMax MS films.
  • gastric cancer cells e.g. MKN-1 , MKN-28
  • RPMI 1 640 medium without fetai calf serum (FCS) starvation phase.
  • FCS fetai calf serum
  • Incubation with the cagA- and cagA + H. pylori strains occured for 0, 30, 90, 1 50 and 21 0 min, respectively.
  • Cells were then lysed with 420 ⁇ l of lysis buffer (20 mM Hepes pH 7.5, 1 50 mM NaCI, 1 % Triton X-1 00, 1 0% glycerol, 1 mM PMSF, 1 0 ⁇ g/ml Aprotinin, 1 mM orthovanadate) on ice.
  • Lysed cells were cleared from debris by centrifugation (1 5 min, 1 3000 rpm, 4°C) . Cleared lysates were subjected to immunoprecipitation (40 ⁇ l Protein A/G sepharose, 4 ⁇ l anti-human EGF antibodies 1 08.1 per lysate) for 3 hours, 4°C.
  • IL-8 interleukin 8
  • Human gastric cancer cell lines (Katolll, MKN-1 , MKN-28) were cultured in RPMI 1 640 medium supplemented with 1 0% FCS at 37°C and 7% CO 2 . 24 hours prior to the incubation experiments with cagA- or cagA -t- H. pylori strains, medium was changed and cells were cultivated inRPMI 1 640 medium without FCS (starvation phase) .
  • PD 1 34,308 endconcentration 1 ⁇ M; inhibitor of gastrin/chole-cystokinin (CCK)-B receptor
  • Batimastat (BB94; endconcentration 1 ⁇ M, inhibitor of matrix metallo-proteinase enzymes)
  • Tyrphostin AG 1478 endconcentration 250 nM; inhibitor of EGF-receptor phosphorylation
  • PD 098,059 endconcentration 1 0 ⁇ M; inhibitor of MEK1
  • this signal transduction pathway involves a G-protein coupled receptor (GPCR), a still unknown signaling element(s) leading to the activation of members of the ADAM gene-family of proteases.
  • GPCR G-protein coupled receptor
  • Activation of the protease is followed by the processing of membrane-bound ligands of the EGF-like family of ligands of the EGFR. After being processed they can stimulate in an autocrine or paracrine manner EGFR-activity, reflected by increased tyrosine phosphorylation of this receptor.
  • H. pylori strains MKN-1 and MKN-28 cells were incubated for distinct time points (0, 45, 90, 1 50 min) with the above mentioned H. pylori strains at a concentration of 1 x10 7 bacteria per ml. Both H. pylori strains induced increased tyrosine phosphorylation of the EGFR (Fig.2) . This observation parallels the increase in HB-EGF and ADAM20 mRNA expression levels.
  • EGFR tyrosine phosphorylation is mediated via TMPS cascade
  • inhibition of one of the involved signaling members should abrogate this activation.
  • MKN-1 and ST42 cells were preincubated for 30 min with CRM 1 97, the specific inhibitor of HB-EGF. Both cells lines were then stimulated for 1 50 min with H. pylori strain 601 90 at a cone, of 1 x 1 0 7 cells per ml in presence or absence of CRM 1 97.
  • H. pylori-induced tyrosine phosphorylation of the EGFR is completely abrogated in the presence of CRM 1 97 in comparison to untreated cells.
  • the experiment clearly demonstrates that H. pylori-induced EGFR activation involves, at least by part, TMPS cascade.
  • TMPS cascade H. pylori-induced IL-8 production (a well characterized cellular response for this pathogen) MKN-1 and MKN-28 gastric cancer cell lines were incubated with 601 90 and TX30a strains of H. pylori in the presence or absence of specific inhibitors of members of TMPS cascade. IL-8 release was measured from the supernatant of cells using the IL-8 Immunoassay (ELISA). The following compounds were used:
  • 25 pylori induced gastrointestinal diseases BB-2516, BB-1101, BB-94, GI129471, 2-Aminoethyl-amide, CT1418, RO31-9790, CGS27023A, PD134,308, CAM-1028, L-365,260, spiroglumide CR2622, YM-022, RB210, RB213, LY-288,513, LY-262,691, DA-3934, RP-73870, S-0509, CP-212,454, CI-1015, YF-476, L-740,093, Lavendustin A, AG 112, AG
  • H. pylori infection leads via stress-induced signaling pathways to a specific cellular defense program, which involves the expression of members of the TMPS cascade leading to EGFR-mediated downstream signaling events.
  • a specific cellular defense program which involves the expression of members of the TMPS cascade leading to EGFR-mediated downstream signaling events.
  • an auto-paracrine signaling cascade is manifested which essentially contributes to H. pylori- induced disease development.
  • H. pylori-induced pH changes will also lead to the activation or even chronic stimulation of GPCR- signaling as response to pH change. This disregulated signaling event involving GPCRs and TMPS cascade, will contribute as well to the establishment of the pathogenic phenotype.

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Abstract

L'invention concerne une méthode de préparation d'un médicament permettant de traiter ou de prévenir des maladies induites par Helicobacter chez un mammifère, et une méthode permettant de traiter ou de prévenir des maladies induites par Helicobacter.
EP00990605A 1999-11-19 2000-11-17 Inhibiteurs de maladies gastro-intestinales induites par helicobacter pilori Withdrawn EP1229925A2 (fr)

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EP1174129A1 (fr) * 2000-07-17 2002-01-23 Zenner, Hans Peter, Prof. Dr. med. Utilisation d'un inhibiteur de la métalloprotéase matricielle pour le traitement du cancer
ES2359136T3 (es) * 2002-03-08 2011-05-18 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Uso de inhibidores de la transactivación de egfr en cáncer humano.
EP3750551A1 (fr) 2008-02-29 2020-12-16 Acorda Therapeutics, Inc. Compositions permettant de fournir les niveaux de plasma désirés du facteur 2 de croissance gliale
BR112016011727A2 (pt) 2013-11-22 2017-08-08 CL BioSciences LLC Antagonistas de gastrina para o tratamento e prevenção de osteoporose
WO2018151285A1 (fr) * 2017-02-20 2018-08-23 学校法人順天堂 Médicament pour la prévention ou le traitement des maladies de peau s'accompagnant de prurit

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EP0979100A1 (fr) * 1997-04-30 2000-02-16 Merieux OraVax SNC COMPOSITION VACCINALE ANTI-$i(HELICOBACTER) COMPRENANT UN ADJUVANT DE TYPE TH1
JPH11124368A (ja) * 1997-10-22 1999-05-11 Takeda Chem Ind Ltd 生理活性物質、その製造法及び剤
CA2315715C (fr) * 1997-12-22 2010-06-22 Bayer Corporation Inhibition de la kinase p38 par des diphenyl-urees symetriques et dissymetriques
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EP1073435B1 (fr) * 1998-04-30 2004-07-07 Abbott GmbH & Co. KG Derives substitues de pyrazole tricyclique exer ant une activite de proteine kinase
ATE263572T1 (de) * 1998-06-18 2004-04-15 Vecta Ltd Verwendung von gastrin fragmenten oder sythetischen analogen von gastrin zur herstellung eines arzneimittels zur behandlung der helicobacter pylori assozierten störungen

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JP2003513995A (ja) 2003-04-15

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