JP2003513995A - Inhibitors of gastrointestinal diseases induced by Helicobacter pylori - Google Patents

Abstract

  (57) [Summary] The present invention relates to a method for producing a medicament for treating or preventing a Helicobacter mediated disease in a mammal, and a method for treating or preventing a Helicobacter mediated disease.

Description

Detailed Description of the Invention

Description [0001] The present invention relates to a method for producing a drug for treating or preventing a Helicobacter-mediated disease in a mammal, and a method for treating or preventing a Helicobacter-mediated disease.

[0002] Helicobacter pylori is a pathogen known to cause gastrointestinal disorders. Recent treatments against Helicobacter pylori include the application of two antibiotics: tetracycline, amoxicillin, clarithromycin or metronidazole in combination with inhibitors of proton pump-like ranitidine or bismuth salts such as bismuth citrate. Combination therapy is included. However, a newly emerged resistance to metronidazole among Helicobacter strains has already been found worldwide and is associated with treatment failure (Alarcon et al., Int. J. Antimicrob.
Agents 1 (1999), 19-26).

Helicobacter pylori combats the acidic gastric juices of the stomach by releasing a urease that converts stomach urea to bicarbonate and ammonia. As a result, the gastrin receptor, a G protein-coupled receptor (GPCR), is used to initiate peripheral regulation of gastric acid secretion by the release of gastrin from G cells. Furthermore, Helicobacter pylori is critically involved in the upregulation of gastric epithelial interleukin 8 (IL-8) expression. This up-regulated secretion of epithelial IL-8 and the concomitant activation of chemotactic and immunocompetent cells have been implicated in tissue damage, ulceration and gastric carcinogenesis (Crabtree and Cradtree & Lindley, Eur. J. Gastrointer
ol. Hepatol. 6 Suppl 1 (1994), 33-38; Crabtree et al., J. Am. Clin. Pathol. 48 (1995
), 41-45; Crabtree, Aliment. Pha
rmacol. Ther. 10 Suppl. 1 (1996), 29-37).

Helicobacter pylori strains are divided into two main groups: groups with loci encoding immunodominant proteins of indeterminate function (cagA island).
); CagA + strain) and a group without this gene region (Crabtree (
Crabtree) et al. (1995), same as above; cagA-strain). Since the Helicobacter pylori strain that does not have this gene region is still infectious, this cagA pathogenic island is presumed to be the cause of the Helicobacter pylori-induced disease, but the patient remains asymptomatic ( Acoustics (A
Kopyants) et al., Mol. Microbiol. 28 (1998), 37
-53).

Helicobacter pylori cagA- (TX30a, ATCC 51932) / c
By hybridizing the cDNA sequence to a human gastric cancer cell line infected or not with the agA + (60190, ATCC 49503) strain,
The following results were obtained: upregulation of cytokines, epidermal growth factor (EGF) family-like heparin-binding EGF (HB-EGF), amphiregulin and transforming growth factor (TGF) -α members as well as A
Upregulation of genes of the DAM20, ADAM family (proteins containing disintegrin and metalloprotease domains) occurred. In stark contrast to the results of Akopyants, et al., The cagA + strain of Helicobacter pylori is not the only cause of disease, and the cagA- strain is also described by Prenzel et al., Nature 402 (1999).
), 884-888, and the triple transmembrane signaling cascade (t
triple membrane passing signaling cas
Cade) shows the same molecular effect as the cagA + strain,
This is verified by the results of the present inventors.

There are about 500,000 cases of gastric cancer each year in Japan and about 200,000 in Europe (SCRIP No 2467, p. 18), many of which are probably caused by chronic Helicobacter pylori infection. Therefore, cagA- / c
The inventors reasoned that by blocking the cascade of events caused by the agA + Helicobacter pylori strain, the deleterious chronic effects of the pathogen should be suppressed.

Thus, the present invention provides a method for producing a drug for treating or preventing a Helicobacter-mediated disease in a mammal, wherein the drug comprises (a) inhibition of gastrin / cholecystokinin (CCK) -B receptor. An agent, (b) an inhibitor of protein kinase C, (c) an inhibitor of a membrane-bound metalloproteinase, (d) an inhibitor of growth factor receptor activation, (e) an inhibitor of growth factor receptor kinase activity, The present invention relates to a method comprising as an active ingredient at least one compound selected from (f) mitogen-activated protein kinase cascade inhibitor, and (g) transcription inhibitor.

The Helicobacter-induced disease to be treated or prevented may be selected from gastric cancer and inflammatory diseases such as non-erosive chronic gastritis, peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma and intestinal adenocarcinoma. preferable.

The active ingredient is selected from compounds (a) to (g). The drug can contain only one active ingredient or several active ingredients, which can be selected from compounds targeted to the same target or compounds directed to two or more different targets.

Inhibitors of the gastrin / cholecystokinin (CCK) -B receptor (a) are as described by Level (Revel) and Makovec (Drugs of the Future 23 (1998), 751). −
766), nonapeptide CCK-B antagonist, amino acid derivative, benzodiazepine, dipeptoid, pyrazolidinone, quinazolinone, ureidoacetamide, bicyclic compound such as dibenzobicyclo [2.2.2] octane, aromatic compound such as binaphthalene derivative, ureidobenz It is preferably selected from azepine and ureidomethylcarbamoylphenylketone. Further suitable compounds are Crawley (J. Neuroscience 12 (19)
92), 3380-3391) and Hughes (Proc. N.
atl. Acad. Sci. U. S. A. 87 (1990), 6728-673.
2). The disclosures of these documents are incorporated herein by reference.

Detailed examples of amino acid derivatives include glutamic acid derivatives such as spiroglumide, for example CR-2622 (Makovec et al., J. Med. Chem.
． 39 (1996), 135-142). A detailed example of a benzodiazepine is given in L-365260 (Bock et al., J. Med. Chem. 32 (1).
989), 13-16), YM-022 (Sato et al., Chem. Pharm. B.
all. (Tokyo) 43 (1995), 2159-2167) and GR-
199114X (Bailey et al., Bioorg. Med. Che.
m. Lett. 7 (1997), 281-286). Detailed examples of pyrazolidinones are LY-288513 and LY-262691 (Helton.
on) et al., Pharmacol. Biochem. Behav. 53 (1996
), 493-502). A detailed example of quinazolinone is LY-247348.
(Yu et al., J. Med. Chem. 34 (1991), 1505-15.
08). Detailed examples of 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)
Is. Detailed examples of ureidoacetamide are RP-69758 and RP-7.
3870 (Pendley et al., J. Pharmacol. Exp.
． Ther. 273 (1995), 1015-1022). Detailed examples of ureidobenzazepines are described in CP-212454 (Lowe III et al., J.
． Med. Chem. 37 (1994), 3789-3811) and the like, 5-phenyl-3-ureidobenzazepin-2-one. Detailed examples of ureidomethylcarbamoylphenylketones are described in S-0509 (Hagishita et al., Bioorg.
Med. Chem. 8 (1997), 1695-1714). The disclosures of these documents are incorporated herein by reference.

Inhibitors of protein kinase C (b) can be ATP competitive inhibitors, antisense oligonucleotides, peptides and lipids. Particularly preferred are indole carbazole, bis indolyl maleimide, valanol analogs, antisense oligonucleotides and alkyl-lysophospholipids (gauge ((
Goekjian) and Jirousek, Curr. Me
d. Chem. 6 (1999), 877-903). The disclosure of this document is incorporated herein by reference.

Detailed examples of the indolecarbazole compound are, for example, EP-A-032800.
0, EP-A-0370236, EP-A-0410389 and EP-A-0.
The compounds described in 434057 or the compounds described in EP 99116426.0. Detailed examples of suitable indolecarbazoles include Go7612 and C
GP41251 (Ikegami et al., Jpn. J. Pharmacol. 70 (199
6), 65-72). A detailed example of bis-indolyl maleimide is LY33
3531, GF109203x, Ro32-0432 and Ro31-8220.
(Jirousek et al., J. Med. Chem. 39 (199
6), 2664-267). A detailed example of a valanol analog is SPC1
00840 (Goekjian and Jirouk
sk), the same as above). A detailed example of an antisense oligonucleotide is CGP64128A (Goekjian and zirousec (
Jirousek), the same as above). Detailed examples of alkyl-lysophospholipids are ET-18-OCH3 (Civoli and Daniel.
el), Cancer Chemother. Pharmacol. 42 (19
98), 319-326). The disclosures of these documents are incorporated herein by reference.

Inhibitors of membrane-bound metalloproteases (c), especially ADAM family members, or TACE (Moss et al., J. Neuroimmunol. 7).
2 (1997), 127-129) can be selected from inhibitors of proteinase activity or compounds that block the access of metalloproteinases to their substrate, for example by masking the substrate. These inhibitors can be selected, for example, from compounds containing hydroxamic acid groups. Detailed examples of hydroxamic acid derivatives can be found in GW 9471 and GW 9277 (Moss et al.,
J. Neuroimmunol. 72 (1997), 127-129), BB-
94 (Batimastat), BB-2516 (Marimastat) and BB-1101 (Wojotwicz-Praga).
) Et al., Invest. New Drugs 15 (1997), 61-75),
CT1418 (Epps et al., J. Protein Chem. 17 (
1998), 699-712), N- (D, L- [2- (hydroxyaminocarbonyl) methyl] -4-methylpentanoyl) -L-3- (2'naphthyl) -alanyl-L-alanine, 2 -Aminoethylamide (Mohler et al., Nature 370 (1994), 218-220), GI129471 (
McGeehan et al., Nature 370 (1994), 5
58-561), CGS27023A and RO31-9790 (Lombard (
Lombard) et al., Cancer Res 58 (1998), 4001-4.
007). Other examples are TIMP (tissue inhibitors of metalloprotease) family members such as TIMP-1 and TIMP-2 (Lombard (Lom).
same as above)). The disclosure of this document is incorporated herein by reference.

Growth factor receptor inhibitors (d) are epidermal growth factor receptor family members,
It can be, for example, an inhibitor of the EGF receptor, HER2, HER3, HER4, or another growth factor receptor, such as the TNF-α receptor. Inhibitors of growth factor receptors are released from low molecular weight compounds or from the binding of receptor ligands to their receptors, especially from membrane bound precursors (fe HB-EGF),
It can be selected from antibodies that inhibit the binding of receptor ligands of the EGF-like ligand family. Detailed examples include anti-receptor antibodies or fragments thereof, such as the antibodies Herceptin or Trastuzumab (Goldenberg, Clin. Ther. 21 (1999).
), 309-318). The disclosure of this document is incorporated herein by reference.

An inhibitor of growth factor receptor kinase activity (e) is phenylaminoquinazoline,
Substituted pyridimines including pyrido-, pyrrolo-, pyrazolo-, pyrimido- and phenylaminopyrimidines, tyrphostins, lavendustin and dianilino-phthalimides (Traxler and Lydon), Drugs of Drags of.
Future 20 (1995), 1261-1274), the disclosure of which is incorporated herein by reference.

A detailed example of phenylaminoquinazoline is PD153030 (Fry (Fry)
) Et al., Science 265 (1994), 1093-1095), ZD18.
39 (Woodburn et al., Proc. Am. Assoc. C.
ancer Res. 38 (1997), 633) and CP-358, 774.
(Proc. Am. Assoc. Cancer Res. 38 (1997), 6
33). Detailed examples of substituted pyrimidines include PD158780 (Rewcastle et al., J. Med. Chem. 39 (1996), 18).
23-1835), PD166285 (Panek et al., J. Pha.
rmacol. Exp. Ther. 283 (1997), 1433-1444).
, CGP59326, CGP60261 and CGP62706 (Traxler et al., J. Med. Chem. 40 (1997), 3601-.
3616). Detailed examples of tyrphostins are AG1478, RG1.
3022 and AG825 (Levitski and Gagit (
Gazit), Science 267 (1995), 1782-1788). A detailed example of lavendustin is lavendustin A (Onoda et al., J. Nat. Prod. 52 (1998), 1252-1257). A detailed example of dianilino-phthalimide is CGP54698 (Buchdanger (
Buchdunger) et al., Clin. Cancer Res. 8 (1995)
, 813-821). The disclosures of these documents are incorporated herein by reference.

The inhibitor (f) of the mitogen-activated protein cascade is MAPKKK (
It is preferably selected from inhibitors of Raf), MAPKK (Mek) and MAPK (Erk). A detailed example can be found in PD098059 (Dudley et al., Proc. Nat. Acad. Sci. U.S.A. 9).
2 (1995), 7686-7689; He et al., Cell Growth.
h Differ. 10 (1999), 307-315), U0126 (Favata et al., J. Biol. Chem. 273 (1998), 186.
23-18632) and SB203580, L167307, RWI6835.
4, SK & F86002, SC102, SB226882, VK19911 and RWI67657 (Bhagwat et al., DDT 4 (1999
), 472-479). The disclosures of these documents are incorporated herein by reference.

The transcription inhibitor (g) can be a general transcription inhibitor or a transcription inhibitor having selectivity to suppress transcription of c-fos gene, eg, antisense oligonucleotide.

Administration of compounds (a)-(g) may be part of a combination therapy with several compounds belonging to the same class of inhibitors and / or different classes of inhibitors Is. In addition, the treatment can include the administration of other active ingredients that are effective against Helicobacter infections. Other active ingredients are antibiotics such as tetracycline, amoxicillin, pantoprazole, clarithromycin or metronidazole, ranitidine, lansoprazole, omeprazole,
It can be selected from proton pump inhibitors such as rabeprazole, benzimidazole, or bismuth salts and Helicobacter pylori vaccines such as recombinant urease or urease subunit vaccines. This other active ingredient may be an immunogenic adjuvant, such as a compound that provides a general or non-specific immunostimulatory action, such as aluminum hydroxide or especially a CpG motif-containing adjuvant (WO96 / 02555; WO98 / 40100; WO99 / 51259). See also) or an immunoadjuvant such as a cytokine. It should be noted that each active ingredient of the combined drugs may be administered together or separately.

The dose and type of administration depend on the extent of the disease and the drugs administered respectively, or the combination of the drugs administered respectively. In this context, reference is made to the documents referenced above. Generally, the drug is administered in effective amounts, respectively, over a period of days and weeks, and the treatment intervals are repeated several times if necessary.

Further provided are methods of identifying novel drugs that treat or prevent Helicobacter-mediated diseases in mammals. This method uses the classes (a) to ((
g) Compound screening assay. This screening assay consists of CCK-B receptor, protein kinase C, membrane-bound metalloproteinase,
Based on identifying modulators of target molecules selected from growth factor receptor and mitogen activated protein kinase cascade members and c-fos. This screening method determines the effect of the test compound on the target molecule in a cell assay, for example, an assay that previously determines the effect of the test compound on cells that overexpress the designated target molecule, or And can be a molecular assay in which the target molecule can be present in a substantially purified and isolated state.

[0023]   The invention is further described by the following examples.

Example 1 1. Materials and Methods: 1.1 Preparation of RNA for hybridizing cDNA sequences: In principle, after removing the medium, wash the gastric cancer cell line with 15 ml of ice-cold phosphate buffered saline (PBS). , Trypsinized, in a 15m vial tube at 4 ° C and 2
Pelletized at 000 rpm for 5 minutes. After centrifugation, the supernatant was removed, and the cells were lysed with 1 ml of TRIZOL reagent for 1.5 × 10 ⁶ cells. TRIZOL
Transfer the cell lysate to an eppendorf tube at 4 ° C and 13000r
Centrifuge at pm for 15 minutes. Transfer the supernatant to a new Eppendorf tube and use BCP (1-bromo-3-chloropropane) for each 1 ml of TRIZOL.
0.1 ml was added. The sample was vortexed for 15 seconds and incubated at room temperature for 5 minutes. The sample was then centrifuged at 4 ° C. and 13000 rpm for 15 minutes. Transfer the obtained upper aqueous phase to a new Eppendorf tube and use TR
0.5 ml isopropanol was added to each 1 ml IZOL; vortexed; incubated at room temperature for an additional 8 minutes. 1 at 4 ° C and 13000 rpm
After centrifugation for 0 minutes, the supernatant was completely removed, the precipitated RNA was washed twice with ice cold 75% ethanol and air dried. Tris RNA pellets
Resuspended in 50 μl of HCl (pH 7.5). The amount of RNA was monitored by UV spectroscopy and its quality was measured by gel electrophoresis on a 1.2% agarose gel containing formaldehyde. When preparing RNA to be used for Northern blotting, the protocol and reaction buffer attached to “RNeasy (registered trademark) mini kit” (Qiagen) were used.

1.2 cDNA preparation: 10 μg of total RNA was reverse transcribed into first strand cDNA by adding 1 μg of oligo-dT primer. The mixture was incubated at 60 ° C. for 5 minutes in a final reaction volume of 12 μl, chilled rapidly on ice for 2 minutes, followed by centrifugation at 13000 rpm for 30 seconds. 5 x single-stranded buffer (250 mMT
ris / HCl (pH 8.3), 375 mM KCl, 15 mM MgCl ₂
, GibcoBRL, Superscript) 4 μl, 0.1 M DTT 2 μ
1, 10 mM dNTP 1 μl and Superscript II (= 200 U
) 1 μl of reverse transcriptase was added and first incubated for 10 minutes at room temperature and then for another 60 minutes at 38 ° C. The reaction was stopped by adding 5 μl 0.5 M EDTA and 25 μl 0.6 N NaOH.

1.3 Purification of cDNA using a ProbeQuant Sephadex G-50 column (registered trademark) (Amersham): After vortexing the column, the bottom closure was removed and the column was placed in a 2 ml tube, 735 xg.
It was centrifuged for 1 minute. It was then placed in a new 1.5 ml Eppendorf tube without a cap and the sample was carefully pipetted into the center of the ready-made resin. After centrifugation at 735 xg for 2 minutes, the resulting flow-through containing the cDNA was added to 5M NaCl (final concentration 0.2M).
Precipitated by adding 1/25 volume, 1 μl of polyacrylic carrier, 2.5 volumes of 100% ethanol, vortexed, incubated on ice for 10 minutes and pelleted by centrifugation at 4 ° C. and 13000 rpm for 10 minutes. . Finally, the supernatant was removed, the cDNA pellet was washed with 80% ethanol, air dried and resuspended in 30 μl Tris / EDTA (10 mM / 0.1 mM).

1.4 Random Prime Labeling of cDNA Using Random Primer Labeling System (GibcoBRL): 50 ng of cDNA template was denatured at 95 ° C. for 5 minutes and rapidly cooled on ice for 5 minutes. After centrifuging the sample, the following components: 0.5 mM dCTP 2 μl, 0.5 mM dGTP 2 μl, 0.5 mM dTTP 2 μl
, P ³² -α-dATP (50 μCi) and Klenow enzyme (3 U / μl
) 1 μl was added to a final volume of 50 μl. The sample was incubated at 25 ° C. for 60 minutes and the reaction was stopped by adding 5 μl of 0.5 M EDTA (pH 8.0).

1.5 Removal of unincorporated P ³² -α-dATP: After vortexing the column, remove the bottom closure and place the column in a 2 ml tube,
Centrifuged at 735 xg for 1 minute. It was then placed in a new 1.5 ml Eppendorf tube without a cap and the radioactive sample was carefully pipetted into the center of the ready-made resin. Unincorporated P ³² -α-dATP nucleotides were removed by centrifugation at 735 xg for 2 minutes.

1.6 Pre-hybridization, pre-association, hybridization and filter wash: filter in TE buffer 25m for 5 minutes at room temperature in a rotating bottle.
Incubated with 1. Following the incubation, the TE solution was added to the pre-warmed hybridization solution (5xSSPE, 10xDenhar).
dts solution, 50% formamide, 1% SDS, denatured and fragmented salmon sperm-
DNA 100 μg / ml) was replaced with 8 ml. Before hybridization, radiolabeled probe was added to yeast RNA (final concentration 0.7 μg / μl), poly A (0.7 μg / μl).
μg / μl), CotDNA (0.07 μg / μl), SDS (1%) and 5
Pre-associated in a solution containing xSSPE. Denaturation was allowed to occur at 95 ° C for 5 minutes, followed by a pre-association step at 65 ° C for 60 minutes. The pre-associated probe was then added to the filter and the hybridization reaction was carried out at 42 ° C for 2 days. After hybridization, the filters were washed with 2 × SSC for 10 minutes at room temperature, twice with pre-warmed 2 × SSC / 0.5% SDS for 30 minutes at 65 ° C. and finally pre-warmed. 30% at 65 ℃ in 0.5 × SSC / 0,5% SDS
Washed twice per minute. Filter on fluorescent imager-screen (Fuji) 3
Exposed for days.

1.7 Northern Blot Analysis: 5 μg of total RNA was size fractionated (0.8 mA / cm ² ) on an agarose gel containing 1.2% formamide. After visualization of bands corresponding to 28S RNA and 18S RNA, ethidium bromide (1 μg /
gel) was equilibrated for 30 minutes in 2 × SSC and exposed to capillary transfer of RNA to nitrocellulose membrane using 2 × SSC as transfer buffer. After transfer was completed, RNA was immobilized on the filter using UV light to crosslink. Subsequently, the filter was treated with the following genes: heparin-binding epidermal growth factor (HB-EGF, gene bank accession number M60278), amphiregulin (AR, gene bank accession number M30704),
Tumor necrosis factor α converting enzyme (TACE / ADAM17, Gene Bank Accession No. U6
9611), ADAM20 (gene bank accession number AF029899) and glyceraldehyde phosphate dehydrogenase (GAPDH, gene bank accession number AF261085) or β-actin (gene bank accession number NM_0011).
Hybridized with a specific cDNA probe for 01). 25 ng of each cDNA template was denatured at 95 ° C. for 5 minutes and rapidly cooled on ice for 5 minutes. After centrifugation of the sample, the following components: 0.5 mM dCTP 1 μl, 0.5 mM dGT
P 1 μl, 0.5 mM dTTP 1 μl, P ³² -α-dATP (50 μCi) 5
μl, random primer solution (125 mM Tris / HCl (pH 6.8),
12.5 mM MgCl ₂ , 25 mM 2-mercaptoethanol, 50 μg /
20 ml of random octamer primer) and Klenow enzyme (40 U / μl)
) 1 μl was added to a final volume of 50 μl. The sample was incubated at 37 ° C. for 10 minutes and the reaction was stopped by adding 2 μl of 0.5 M EDTA (pH 8.0). Unincorporated P ³² -α-dATP nucleotides were removed as follows: ProbeQuant Sephadex G-50 column (
After vortexing (registered trademark) (Amersham), the bottom closure was removed, the column was placed in a 2 ml tube and centrifuged at 735 xg for 1 minute.
The column was then placed in a new 1.5 ml Eppendorf tube without a cap and the radioactive probe was carefully pipetted into the center of the ready-made resin. 73
Unincorporated P ³² -α-dATP nucleotides were removed by centrifugation at 5 × g for 2 minutes. Prior to initiating hybridization, the nitrocellulose filter was washed with the following solution: 50% formamide, 5xSSC, 5xDenhard.
Prehybridization was performed in t's solution, 0.1% SDS at 42 ° C for 4 hours. After prehybridization and denaturation at 100 ° C. for 10 minutes, each radiolabeled probe was added to the filter together with 20 μg / ml of salmon sperm DNA. Hybridization was carried out at 42 ° C. for 16 hours. The filter is then placed at 42 ° C
2 × SSC / 0.1% SDS, washed twice for 10 minutes, 0.2 ° C. at 42 ° C.
Washed twice in SSC / 0.1% SDS for 10 minutes. Hybridization signals were detected using Kodak BioMax MS film.

1.8 Western blot analysis of tyrosine phosphorylated content of EGFR: cag
To investigate EGF receptor tyrosine phosphorylation induced by A- and cagA + Helicobacter pylori strains, gastric cancer cells (eg, MKN-1, MKN-28) were treated with fetal calf serum (FCS) 24 hours before the incubation experiment. ) -Free PRMI1640 medium (starvation stage). cagA- and cagA
+ 0 min, 30 min, 90 min, 150 min and 2 with Helicobacter pylori strain
Incubated for 10 minutes each. Then, on ice, a lysis buffer (20 mM Hepes (pH 7.5), 150 mM NaCl, 1% Triton X-100,
10% glycerol, 1 mM PMSF, 10 μg / ml aprotinin, 1 mM
The cells were lysed with 420 μl of orthovanadate. Centrifuge the lysed cells (15 minutes, 13000 rpm, 4 ° C)
The necrotic tissue pieces were removed by. Immunoprecipitation of the clear lysate at 4 ° C. (40 μl protein A / G sepharose per lysate, anti-human EGF antibody 108
． 4 μl) for 3 hours. HNTG (20 mM Hepes, pH 7.5; 15
0 mM NaCl, 10% glycerin; 0.1% Triton X-100) 7
After three consecutive washes in 50 μl, the immunoprecipitate was dissolved in 40 μl of 3 × Laemmli buffer, denatured at 100 ° C. for 5 minutes and subjected to SDS-PAGE (gradient gel 7).
% To 12%). 4 ℃ on nitrocellulose filter (Amersham)
The gel was blotted for 3 hours (0.8 mA / cm ² ) with EGF receptor-specific phosphorylation using the ECL kit (Amersham) and 4G10 (UBI No. 05-32).
1) Detected with antibody.

1.9 Quantification of Interleukin 8 (IL-8) Concentration in Gastric Cancer Cell Line Culture Supernatants by Enzyme-Linked Immunosorbent Assay (ELISA) Human gastric cancer cell lines (KatoIII, MKN-1, MKN-28) Was cultured in RPMI 1640 medium supplemented with 10% FCS at 37 ° C. and 7% CO ₂ .
24 hours before the incubation experiment with cagA- or cagA + Helicobacter pylori strains, the medium was changed and FCS-free RPMI1640 was added.
Cells were cultured in medium (starvation stage). In the IL-8 induction experiment, the following compounds: PD 134,308 (final concentration 1 μM; gastrin / cholecystokinin (C
CK) -B receptor inhibitor); Batimastat (BB94; final concentration 1 μM, inhibitor of substrate metalloproteinase enzyme); [Glu ⁵² ] -diphtheria toxin (CRM197, strong mitogenic activity of HB-EGF) Non-toxic mutant of diphtheria that suppresses S. cerevisiae; Mitamura et al., J. Bi
ol. Chem. 270, 1015-1019 (1995); final concentration 5 μg /
Tyrphostin AG1478 (final concentration 250 nM; inhibitor of EGF receptor phosphorylation); PD 098,059 (final concentration 10 μM; inhibitor of MEK1);

Cells were pre-incubated with these inhibitors for 30 minutes at 37 ° C. and 7% CO ₂ , followed by Helicobacter pylori concentration of 10 ⁷ / ml in the presence or absence of specific inhibitors. And incubated for 24 hours. After the incubation period, cell supernatants were harvested and removed from bacterial necrotic debris by low speed centrifugation (5 min, 3000 rpm) and IL-8 according to the manufacturer's recommended (R & D) system as follows. ELISA detection was performed on: 96-well polystyrene microtiter plates were coated with a mouse monoclonal antibody against human IL-8. 100 μL of assay diluent buffer containing blue dye
Was added to each well. 50 μL of cell culture supernatant was then added to the plate, followed by 100 μL per well of polyclonal antibody against IL-8 conjugated to horseradish peroxidase. Test sample at room temperature 2
． Incubated for 5 hours. After the incubation period, aspirate the wells,
Washed. Then 200 μl of substrate solution containing equal amounts of hydrogen peroxide and tetramethylbenzidine were added to each well and incubated for 30 minutes at room temperature. Finally, 50 μL of 2N sulfuric acid was added to each well to stop the reaction. The optical density of each well was measured using a microplate reader set at 450 nm.

2. Results From the expression data of the present inventors' studies, the human gastric cancer cell lines MKN-1 or MKN
Helicobacter pylori infection of −28 has been shown to interfere with mRNA expression levels of the “Triple Transmembrane Signal” (TMPS) cascade members described by Prenzel et al. (1999).

Briefly, this signaling pathway is linked to the G protein-coupled receptor (GPCR).
), Is involved in an as yet unknown signaling element that leads to activation of ADAM gene family members of proteases. Protease activation involves the processing of membrane-bound ligands of the EFG-like family of EGFR ligands. After processing, they are able to stimulate EGFR activity in an autocrine or paracrine fashion, reflected by the increased tyrosine phosphorylation of this receptor.

First, we confirmed expression data from the cDNA sequence by Northern blot analysis. MKN-1, ST42 and MKN-2 in time course experiments
8 cells were treated with cagA- (TX30a, ATCC 51932) and cagA +.
(60190, ATCC 49503) Incubated with Helicobacter pylori strain for 0, 45 and 180 minutes. As shown in FIG. 1, within 45 minutes, a significant increase in the expression levels of HB-EGF- and ADAM20-mRNA species was observed. In addition, examination of later time points clearly shows a more significant increase in appropriate mRNA expression levels.

From this observation, incubation of a human gastric cancer cell line with the cagA− / cagA + Helicobacter pylori strain is essential for the establishment of diseases caused by Helicobacter pylori, such as peptic ulcers involving EGFR activation or gastric cancer. It has been shown that the expression levels of cascade members are upregulated.

To test whether EGFR tyrosine phosphorylation was induced upon incubation of human gastric cancer cell lines with TX30a and 60190 Helicobacter pylori strains, MKN-1 and MKN-28 cells were treated with a bacterial count of 1. × 10 ⁷ /
Incubated with the above Helicobacter pylori strain at a concentration of ml at different time points (0, 45, 90, 150 minutes). Both Helicobacter pylori strains induced increased tyrosine phosphorylation of EGFR (Fig. 2). This observation is similar to the increase in HB-EGF and ADAM20-mRNA expression levels.

If EGFR tyrosine phosphorylation is mediated by the TMPS cascade, inhibition of one of the involved signaling members should abrogate this activation. To investigate this possibility, MKN-1 and ST42 cells were treated with C
Preincubated with RM197, a specific inhibitor of HB-EGF for 30 minutes. Then, in the presence or absence of CRM197, the number of cells was 1 × 10 ^7.
Helicobacter pylori strain 60190 at a concentration of / ml was used to
I was stimulated for 0 minutes. As can be seen from FIG. 3, Helicobacter pylori-induced tyrosine phosphorylation of EGFR is completely abrogated in the presence of CRM197 as compared to untreated cells. This experiment shows that the Helicobacter pylori-induced EGFR activation involves, at least in part, the TMPS cascade.

To investigate the involvement of the TMPS cascade in Helicobacter pylori-induced IL-8 production, a well characterized cellular response to this pathogen, in the presence of specific inhibitors of TMPS cascade members or In the absence of M
KN-1 and MKN-28 gastric cancer cell lines were incubated with Helicobacter pylori 60190 and TX30a strains. IL-8 immunoassay (EL
IL-8 release was measured from cell supernatants using ISA). The following compounds: Inhibitors of gastrin / cholecystokinin (CCK) -B receptor (PD1343
08); an inhibitor of the substrate metalloproteinase enzyme (batimastat = BB-94)
Interfering with TMPS signaling); Inhibitor of proHB-EGF (CRM197, a non-toxic mutant of diphtheria toxin that strongly and specifically suppresses the mitogenic activity of HB-EGF; Mitamura et al., J. Chem.
Biol. Chem. 270, 1015-1019 (1995); interferes with TMPS signaling); Inhibitor of epidermal growth factor receptor (Tyrphostin AG1478; TMP).
Block S signaling); an inhibitor of MEK1 (PD098059);

Each inhibitor was added prior to incubation with Helicobacter pylori cagA- and cagA + strains. The presence of PD134308 had a minor effect on Helicobacter pylori-induced IL-8 production, while batimastat and C
The effect of RM197 was more pronounced (mean inhibition of about 25%). In the case of AG1478, the observed suppression was 30-70%, and PD098059 dramatically reduced the Helicobacter pylori-induced IL-8 response in human gastric cancer cells by about 60-75% (see Figure 4). In repeated experiments, we found that 1 μM P
D134308, 5 μM BB-94, 5 μg / ml CRM197, 250n
The inhibitory effects of M1AG1478 and 1 μM PD098059 were confirmed.

Therefore, the present inventors have made the following compounds: BB-94, BB-2516, B
B-1101, BB-94, GI129471, 2-aminoethylamide, CT
1418, RO31-9790, CGS27023A, PD134, 308, C
AM-1028, L-365, 260, Spiroglumide CR2622, YM-0
22, 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 183, AG 1478, PD 153035, P
D 158780, ZD1839, CP-358,774, CGP 59326.
, CGP 60261, CGP 62706 interfere with Helicobacter pylori-induced gastrointestinal disease.

In a further experimental set-up, we have HB- in the gastric cancer cell lines MKN-1 and MKN-28, which play a major role in TMPS signaling.
The effect of the above inhibitors on the expression of EGF mRNA was investigated. As can be seen in FIG. 5, Helicobacter pylori strain 60190 was used to test HB in the cell lines tested.
-Caused an increase in EGF and mRNA expression. BB-94, CRM197
, BB-94 in combination with CRM197 or AG1478 was present at the time of infection, the pathogen-induced increase in HB-EGF mRNA accumulation was significantly reduced. When PD098059 was used as an inhibitor, it disappeared almost completely. In other experiments, we found that not only was HB-EGF mRNA expression sensitive to inhibitors of TMPS signaling, but also ADAM.
It could be demonstrated that the expression of 20-mRNA was also sensitive (Fig. 6). Both mRNA expression levels were BB-94 and CR when compared to untreated cells.
Significantly reduced in the presence of M197. These results clearly demonstrate that the major players of TMPS signaling are induced by Helicobacter pylori infection of the gastric cancer cell line responsible for disease development, establishing an autocrine signaling cascade.

Without intending to be bound by theory, our results establish the following model (FIG. 7): via stress-induced signaling pathways, Helicobacter pylori infection leads to EGFR-mediated downstream. TMP causing signal transduction phenomenon
Guide a specific cellular defense program that is involved in the expression of S cascade members. Depending on the severity of Helicobacter pylori infection prior to eradication, an auto-paracrine signaling cascade emerges that is essentially responsible for the development of Helicobacter pylori-induced disease. In this model, Helicobacter pylori-induced pH changes would cause activation of GPCR-signalling or even chronic stimulation to be triggered in response to pH changes. This uncontrolled signaling phenomenon, involved in the GPCR and TMPS cascades, may also contribute to the establishment of the pathogenic phenotype.

[Brief description of drawings]

FIG. 1 Confirmation result of expression data from cDNA sequence by Northern blot analysis.

FIG. 2: Test results whether EGFR tyrosine phosphorylation is induced when human gastric cancer cell lines are incubated with TX30a and 60190 Helicobacter pylori strains.

FIG. 3: Test results that if EGFR tyrosine phosphorylation is mediated by the TMPS cascade, inhibition of one of the involved signaling members abrogates this activation.

FIG. 4 shows the test results of the effect of each inhibitor on IL-8 production induced by Helicobacter pylori.

FIG. 5. HB-EGF mR in gastric cancer cell lines MKN-1 and MKN-28.
Results of investigation of the influence of inhibitors on the expression of NA.

FIG. 6: Results demonstrating that ADAM20-mRNA expression is also sensitive.

FIG. 7: Expression model of a signal transduction cascade that causes disease development.

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Claims (15)

[Claims] 1. A method for producing a drug for treating or preventing a Helicobacter-mediated disease in a mammal, wherein the drug comprises (a) an inhibitor of gastrin / cholecystokinin (CCK) -B receptor, and (b) a protein. Kinase C inhibitor, (c) Membrane-bound metalloproteinase inhibitor, (d) Growth factor receptor activation inhibitor, (e) Growth factor receptor kinase activity inhibitor, (f) Mitogenic factor A method comprising as an active ingredient at least one inhibitor of a triple transmembrane signaling cascade selected from an inhibitor of an activated protein kinase cascade and (g) a transcription inhibitor. 2. The method of claim 1, wherein the Helicobacter-mediated disease is selected from gastrointestinal cancer and inflammatory disease. 3. The gastrin / cholecystokinin (CCK) -B receptor inhibitor is a peptide, amino acid derivative, benzodiazepine, dipeptoid, pyrazolidinone, quinazolidinone, bicyclic heteroaromatic compound, ureidoacetamide, ureidobenzazepine. The method according to claim 1 or 2, which is selected from and ureidomethylcarbamoylphenylketone. 4. The method according to claim 1 or 2, wherein the inhibitor of protein kinase C is selected from indolecarbazole, bisindolylmaleimide, valanol analogues, alkyl-lysophospholipids, and antisense oligonucleotides. . 5. The method according to claim 1 or 2, wherein the inhibitor of the membrane-bound metalloproteinase is selected from compounds containing a hydroxamic acid group and a TIMP family member. 6. The method according to claim 1 or 2, wherein the inhibitor of growth factor receptor activation is selected from anti-receptor antibodies or fragments thereof. 7. The inhibitor of growth factor receptor kinase activity is phenylamino-quinazoline, substituted pyridimine, tyrphostin,
3. The method according to claim 1 or 2 selected from lavendustin and dianilinophthalimide. 8. The inhibitor of a mitogen-activated protein kinase cascade inhibitor is selected from an inhibitor of MAPKKK (Raf), an inhibitor of MAPKK (Mek) and an inhibitor of MAPK (Erk). The method according to Item 1 or 2. 9. The inhibitor of the transcription inhibitor is a general transcription inhibitor or c-
The method according to claim 1 or 2, which is selected from fos-specific transcription inhibitors. 10. The method according to claim 1 or 2, wherein the drug contains at least two active ingredients selected from compounds of classes (a) to (g). 11. The drug according to claim 1, wherein the drug comprises a combination of at least one active ingredient selected from compounds (a) to (g) and another active ingredient effective against Helicobacter infection. The method according to 2 or 9. 12. The method of claim 11, wherein said other active ingredient is selected from antibiotics and proton pump inhibitors. 13. The method of claim 11, wherein said other active ingredient is selected from Helicobacter pylori vaccine. 14. The drug according to claim 1, which comprises a combination of at least one active ingredient selected from compounds (a) to (g) and an immunogenic adjuvant.
The method of 2 or 9. 15. The Helicobacter is Helicobacter pylori cagA +.
The method according to any one of claims 1 to 14, which is a strain.