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WO1997011721A1 - Procede pour identifier des composes therapeutiquement actifs - Google Patents

Procede pour identifier des composes therapeutiquement actifs Download PDF

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Publication number
WO1997011721A1
WO1997011721A1 PCT/SE1996/001183 SE9601183W WO9711721A1 WO 1997011721 A1 WO1997011721 A1 WO 1997011721A1 SE 9601183 W SE9601183 W SE 9601183W WO 9711721 A1 WO9711721 A1 WO 9711721A1
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WO
WIPO (PCT)
Prior art keywords
pylori
animal
mice
strain
mouse
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.)
Ceased
Application number
PCT/SE1996/001183
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English (en)
Inventor
Wubshet Mamo
Björn MELLGÅRD
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.)
AstraZeneca AB
Original Assignee
Astra AB
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 Astra AB filed Critical Astra AB
Priority to AU71024/96A priority Critical patent/AU7102496A/en
Publication of WO1997011721A1 publication Critical patent/WO1997011721A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates

Definitions

  • the present invention relates to a method for identification of compounds and vaccine candidates suitable for the therapeutic treatment of gastric disorders associated with Helicobacter infections. More particularly, the present invention relates to providing in vivo animal models useful in the screening and evaluation of prophylactic and therapeutic agents and vaccines for the treatment of gastritis, ulcer and other gastroduodenal diseases associated with Helicobacter pylon infections. The animal models provided by the present invention may also be useful in the development of diagnostic tests for such infections.
  • H pylori The relationship between gastroduodenal disorders and infections with Helicobacter pylori (H pylori ) is well established today.
  • Helicobacter pylori was previously named Campylobacter pylori or Campylobacter pyloridis or just Campylobacter like organisms.
  • the names H pylori , C pylori and C pyloridis are used interchangable.
  • the above-mentioned relationship has been discussed by, for instance, Marchall et al, Microbios Lett. 25: 83 - 88 (1984). Marchall isolated C pyloridis from human gastric mucosa. Goodwin et al, J.Clin. Pathol. 39: 353 - 365 (1986) also suggested that gastroduodenal ulceration is associated with C pyloridis.
  • H pylori invades and colonizes the stomach, its mode of action, such as persistence and role in gastroduodenal ulceration are important for the studies concerning the development of new therapies for the treatment of H pylori infections and related gastroduodenal diseases. At present there is no single compound therapy or treatment regimen which consistently provides eradication of said infections. Experimental work for studying H pylori related infections cannot be successfully investigated on human patients, due to ethical regulations.
  • Immunologically compentent rodents and especially some specific mouse strains, have now surprisingly been found to be capable of developing infections by H pylori .
  • Fresh isolates obtained directly from human gastric mucosa are used to establish H pylori infection.
  • the new animal models can also be used in the development of vaccines against such infections and related diseases. Further, the animal models may be useful in the development of diagnostic tests for such infections.
  • the H pylori infection is detectable 8 - 10 days following inoculation.
  • Fresh isolates obtained from H pylori strains are used for inoculation. These fresh isolates are isolated from human patients. The isolates have not been passaged in animals, but they have in most instances been stored in a -70°C to minirnize laboratory passage (in vitro passage).
  • New Zealand Black inbred mouse strain is New Zealand Black inbred mouse strain, shortly named NZB mice.
  • Other inbred mouse strains which can be used in the method according to the present invention are the mouse strains named KK and DBA-1.
  • KK and DBA-1 mouse strains have been previously used in biomedical research concerned with autoimmune diseases, see for instance, the International Index of Laboratory Animals, 6th edition, Michael F.W. Festing (1993).
  • Another rodent of interest for the present invention is an immunocompetent rat, such as Sprague-Dawley rats.
  • the latter have been inoculated by bacteria reisolated from colonized mice, i.e. human isolates which have been passaged in mice.
  • H pylori In order to be useful as a screening model for H pylori, animals must be susceptible to infection with H pylori and especially with fresh isolates of H pylori obtained from humans. Moreover, to establish adequate H pylori infection in the animals, the immunological status of the specific animal and the virulence of the infecting organism are important factors.
  • NZB mice are developed by Dr. Bielschowsky in 1970 as black-coated inbred mouse strains (Bieleshowsky et al. Cancer Res. 30: 834 ). It has to be noted that the NZB mice used according to the present invention are not genetically or immunologically transformed. The mice can be either of male or female sex. NZB mice are not previously known to have been infected by H pylori. They are hithereto used as standard animal for studies of the ethiology and pathogenisis of autoimmune diseases and therapeutic effect of immunosuppressive agents.
  • the new animal models can be used in a method for identifying therapeutically active compounds for treatment of H pylori infections in mammals and man, wherein the method includes the following steps
  • Immunologically compentent rodent of interest are inbred mouse strains, such as New Zealand Black mouse, DBA-1 mouse and KK mouse as well as rats.
  • the invention discloses a method for identifying biological probes clinically useful for detecting H pylori infections in mammals including humans.
  • Such methods include for instance a biological indicator suspected of interacting selectively with H pylori or a substance interacting with a product released by the bacteria and identifying a selective positive interaction with such a released product for detecting H pylori infections in mammals.
  • Figure 1 shows the number of H pylori colony forming units, i e bacteria, recovered from the stomach biopsies of NZB mice 1 to 10 weeks post inoculation.
  • Figures 2 (a) and 2 (b) show the number of H pylori colony forming units, i e bacteria, recovered from the stomach biopsies of DBA-1 mice and KK mice respectively 1 to 5 weeks post inoculation.
  • Figure 3 shows that H pylori colonization occurs around the crypts in the lamina propia of infected mouse stomach.
  • Figure 4 shows that H pylori- specific antibodies bind to the H pylori colonizing the stomach, i e are present on the thin-sectioned samples of infected NZB mouse stomach, confirr ing the colonizing bacteria of H pylori, by fluorescence staining.
  • Figure 5 shows in 5(a) a scanning electromicroscope (SEM) picture showing the presence of H pylori in the mucus blanket of the gastric epithelium.
  • 5(b) shows H pylori cultivated in vitro and sedimented on a filter paper, used for comparison.
  • Figure 6 shows the results from therapeutic immunisation in H pylori infected mice. Mice infected were NZB and DBA, respectively.
  • mice of the inbred mouse strain NZB were used.
  • the mice were of male or female sex.
  • the mice were bred and maintained in specific pathogen-free conditions and were examined to ensure the absence of specific bacteria and common murine diseases. They were housed in conventional Mak III cages and kept in room temperature, 50 - 60% relative humidity and fresh air exchange in accordance with Swedish regulations on Laboratory Animal Care.
  • the mice were fed with autoclaved commercial rodent diet and sterile drinking water ad libitum. In some instances the animals did not receive any food, i e only water, 24 hours before the inoculation of bacteria.
  • mice Prior to inoculation with bacteria some of the mice were given antibiotics per os for 3 days. Such optional antibiotic treatment of the animals was made to eliminate interference of normal flora competing with the experimentally inoculated H pylori. Doses of antibiotics were 40mg/ml Ampicillin; 200 mg/ml Nalidixan; 40 mg/ml metronidazole; 160 mg/ml Vancomycin; 200 mg/ml Trimethoprin and each mouse received 0.3 ml of antibiotics twice a day up to 4 hours before adrninistration of bacteria. Some of the mice were not pretreated with antibiotics before the inoculation with H pylori.
  • mice were pretreated with an inhibitor of intragastric acid secretion to increase the gastric pH in the animal before inoculation with H pylori.
  • an inhibitor of intragastric acid secretion used in the experimental studies was omeprazole.
  • mice from the inbred mouse strains DBA-1 and KK were also tested as animals for the in vivo model useful in the screening method according to the present invention. These mice from the mouse strains DBA-1 and KK were treated in the same way as the NZB mice. Also immunocompetent Sprague-Dawley rats were inoculated with H pylori.
  • mice 9 male sex.
  • the mice were inoculated per os with 0.2 ml of H pylori (6 x 10 bact/ml) in peptone water.
  • H pylori 6 x 10 bact/ml
  • peptone water As a control mice of the different species were inoculated with 0.2 ml peptone water.
  • Bacterial colonization and infection were followed up to 5 weeks post inoculation for DBA-1 and KK mice, and up to 10 weeks post inoculation for NZB mice.
  • H pylori strain A-9 and another strain AH 69 isolated from the stomach of a human patient with duodenal ulcer were used for inoculation of NZB mice, female sex.
  • the strain AH 69 was also used for infection of NZB and DBA mice.
  • the therapeutic effect was evaluated in oral immunization experiment four weeks post infection.
  • mice were inoculated by bacteria reisolated from mice colonized with H pylori.
  • the colonized mice had been inoculated with the H pylori strain AH 69 isolated from a human patient with duodenal ulcer.
  • the rats were pretreated with omeprazole to increase the intragastric pH before inoculation with the bacteria.
  • H pylori strain A-9 was recovered from the pyloric- antrum region of the stomach of 8 out of 9 mice, two weeks post inoculation. Four weeks later H pylori could be recovered from all mice inoculated. An average number of 1 000 bacteria/ 25 mm 2 stomach biopsies could be recovered.
  • the biopsies were homogenized and were analysed for urease, catalase and oxidase production. All analysed biopsy homogenates showed a rapid positive urease, catalase and oxidase reaction. None of the biopsy homogenates from the control group showed positive reactions.
  • strain A-9 produced large proportions of spreading colonies and all were motile.
  • the motility was rapid and directional.
  • Bacteria isolated from the stomach of inoculated mice were assayed for DNA dependent discriminary ribotyping and quantitative PCR tests.
  • the DNA pattern as well as PCR confirmed that those bacteria isolated from the stomach of the infected mice were identical to the inoculum H pylori strain A-9 or strain A-l 8 originally isolated from human gastritis patients.
  • H pylori adhesion to epithelial tissue is partly mediated via Lewis b (Le ) receptors.
  • mice were infected with mouse passaged fresh isolates H pylon, strain AH69. Oral immunizations were started four weeks post infection and from the first immunization day (day 1), mice were subsequently immunized on day 15, 25 and 35. One group mice was dosed with vehicle including Cholera toxin (CT) 10 ⁇ g/ mouse/ dose, and one group with the combination of CT + Membrane proteins (Mp). There were ten mice in each group.
  • CT Cholera toxin
  • Mp Membrane proteins
  • CFU Colony forming units

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Environmental Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Zoology (AREA)
  • Rheumatology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Diabetes (AREA)
  • Endocrinology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Pathology (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Toxicology (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Husbandry (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé pour identifier des composés et vaccins thérapeutiques actifs contre les infections à Helicobacter pylori, faisant intervenir un modèle animal in vivo. Selon ce procédé, on inocule une souche de H. pylori associée à une gastrite chez les humains à un animal rongeur immunologiquement compétent, sélectionné dans un des groupes de souris NZB, DBA, KK ou à un rat. Ensuite, on administre à l'animal infecté un composé test ou un vaccin candidat afin de déterminer l'efficacité dudit composé ou vaccin contre l'infection en question. L'invention concerne également l'utilisation dudit modèle animal dans le développement de tests diagnostiques pour de telles infections ainsi qu'une souris Nouvelle-Zélande Black (NZB) infectée par des souches de Helicobacter pylori humaines.
PCT/SE1996/001183 1995-09-25 1996-09-23 Procede pour identifier des composes therapeutiquement actifs Ceased WO1997011721A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU71024/96A AU7102496A (en) 1995-09-25 1996-09-23 Method to identify therapeutically active compounds

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE9503309-8 1995-09-25
SE9503309A SE9503309D0 (sv) 1995-09-25 1995-09-25 Method to identify therapeutically active compounds

Publications (1)

Publication Number Publication Date
WO1997011721A1 true WO1997011721A1 (fr) 1997-04-03

Family

ID=20399582

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SE1996/001183 Ceased WO1997011721A1 (fr) 1995-09-25 1996-09-23 Procede pour identifier des composes therapeutiquement actifs

Country Status (3)

Country Link
AU (1) AU7102496A (fr)
SE (1) SE9503309D0 (fr)
WO (1) WO1997011721A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999035907A3 (fr) * 1998-01-16 1999-09-23 Chiron Spa Modele
RU2186394C2 (ru) * 2000-01-31 2002-07-27 Белая Юлия Александровна Способ получения диагностикума для выявления антигенов helicobacter pylori в реакции коагглютинации

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996009757A1 (fr) * 1994-09-28 1996-04-04 Biocine Spa Modele souris d'infection a helicobacter pylori
WO1996018291A1 (fr) * 1994-12-13 1996-06-20 Yoshitomi Pharmaceutical Industries, Ltd. Gerbille de mongolie colonisee par helicobacter pylori, procede pour realiser cette colonisation, milieu pour la separation de helicobacter pylori et procede d'evaluation preliminaire de substances actives contre helicobacter pylori

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996009757A1 (fr) * 1994-09-28 1996-04-04 Biocine Spa Modele souris d'infection a helicobacter pylori
WO1996018291A1 (fr) * 1994-12-13 1996-06-20 Yoshitomi Pharmaceutical Industries, Ltd. Gerbille de mongolie colonisee par helicobacter pylori, procede pour realiser cette colonisation, milieu pour la separation de helicobacter pylori et procede d'evaluation preliminaire de substances actives contre helicobacter pylori

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
EUROPEAN H PYLORI STUDY GROUP, A.A. McCOLM et al., "Screening of Anti-Helicobacter Therapies in Mice Colonised with H. Pylori"; VIIIth International Workshop on Gastro-Duodenal Pathology an Helicobacter Pylori 7-9th July 1995 Edinburgh, Scotland, page A92. *
THE AMERICAN JOURNAL OF GASTROENTEROLOGY, VOlume 89, No. 2, 1994, MIKIO KARITA et al., "Establishment of a Small Animal Model for Human Helicobacter Pylori Infection Using Germ-Free Mouse", page 208. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999035907A3 (fr) * 1998-01-16 1999-09-23 Chiron Spa Modele
RU2186394C2 (ru) * 2000-01-31 2002-07-27 Белая Юлия Александровна Способ получения диагностикума для выявления антигенов helicobacter pylori в реакции коагглютинации

Also Published As

Publication number Publication date
SE9503309D0 (sv) 1995-09-25
AU7102496A (en) 1997-04-17

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