EP0862458A1 - Urease destinee au traitement d'infections a helicobacter pylori - Google Patents

Urease destinee au traitement d'infections a helicobacter pylori

Info

Publication number
EP0862458A1
EP0862458A1 EP96932735A EP96932735A EP0862458A1 EP 0862458 A1 EP0862458 A1 EP 0862458A1 EP 96932735 A EP96932735 A EP 96932735A EP 96932735 A EP96932735 A EP 96932735A EP 0862458 A1 EP0862458 A1 EP 0862458A1
Authority
EP
European Patent Office
Prior art keywords
urease
pylori
mammal
coccoid
agent
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.)
Withdrawn
Application number
EP96932735A
Other languages
German (de)
English (en)
Inventor
Bow National University Of Singapore Ho
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.)
Cortecs International Ltd
Chapman Paul William
Original Assignee
Cortecs International Ltd
Chapman Paul William
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
Priority claimed from GBGB9520585.2A external-priority patent/GB9520585D0/en
Application filed by Cortecs International Ltd, Chapman Paul William filed Critical Cortecs International Ltd
Publication of EP0862458A1 publication Critical patent/EP0862458A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Definitions

  • the present invention relates to methods of treating H. pylori infection, pharmaceutical formulations for use in such methods, methods of rendering coccoid cultures of H. pylori viable, as well as methods of inducing the coccoid form to convert to the spiral form.
  • H. pylori is a Gram negative bacteria that has been strongly implicated in chronic active gastritis and peptic ulcer disease (Marshall et al, Medical Journal of Australia, 142:439-444 (1985); Buck, G.E. , Journal of clinical Microbiology, 3:1-12 (1990)) .
  • H. pylori exists in two distinct morphological forms, the culturable spiral form and the non-culturable coccoid form (Marshall et al , Microbios letters, 25:83-88 (1984); Rung, J.S.L., and HO, B., Workshop on Gastroduodenal Pathology and Campylobacter pylori (abstract P9) , edited by F.
  • the present invention provides a method of treating H. pylori infection, in a mammal, which comprises administering to the mammal an agent capable of inducing conversion of the coccoid form of H. pylori to the spiral form.
  • the agent is not in the form of a vaccine, i.e. it is not administered to elicit any immune response.
  • the method further comprises the step of administering to the mammal an effective amount of at least one antibiotic.
  • the administration of the one or more antibiotics will take place after administration of the agent. This will allow for the coccoid form to convert to the spiral form prior to the administration of the antibiotic(s) .
  • the present invention provides a method of treating H. pylori infection, in a mammal, which comprises at least two treatment cycles, each treatment cycle comprising:
  • the agent administered is an amount of urease sufficient to induce conversion of the coccoid form of H. pylori to the spiral form.
  • urease is intended to include all forms of urease, either bacterial (eg H. pylori or Protieus mirabi tis urease) or non-bacterial (eg Jackbean urease) , as well as one or more individual subunits of the urease enzyme, or indeed peptides derived from such subunits. In one embodiment, only the C and D subunits of the urease are administered.
  • the method can also comprise the administration of urea to the mammal.
  • the urea can be co-administered or administered separately.
  • the present invention provides a method of treating H. pylori infection, in a mammal, which comprises administering to the mammal an agent capable of preventing conversion of the spiral form of H. pylori to the coccoid form.
  • the agent will be urease, optionally together with urea.
  • the present invention provides a method of treating H. pylori infection, in a mammal, which comprises one or more treatment cycles, each treatment cycle comprising:
  • the agent capable of inducing conversion to the spiral form is urease and the agent capable of inducing conversion to the coccoid form is "anti-urease", eg a urease inhibitor or antibody specific for urease.
  • the present invention provides a method of treating H. pylori infection, in a mammal,which comprises administering to the mammal an agent capable of changing the pH in the stomach.
  • the lowering of the pH induces the spiral form of H. pylori present to increase urease production.
  • This increased urease level induces conversion of any coccoid form present to the spiral form, thus making antibiotic treatment more effective.
  • this method generally also includes the step of administering at least one antibiotic to the mammal.
  • Methods of changing the pH can include administration of edible acids or bases.
  • the mammal is preferably a human.
  • the methods of the present invention will generally employ the agent in the form of a pharmaceutical formulation.
  • the present invention provides a pharmaceutical formulation comprising an agent capable of inducing conversion of the coccoid form of H. pylori to the spiral form together with one or more pharmaceutically acceptable carriers and/or excipients.
  • the pharmaceutical formulation is not in the form of a vaccine.
  • the agent is urease and optionally the pharmaceutical formulation will also comprise urea.
  • the pharmaceutical formulations of the invention may be presented in unit dose forms containing a predetermined amount of the agent, eg urease (and optionally urea) per dose.
  • a predetermined amount of the agent eg urease (and optionally urea) per dose.
  • Such a unit may contain for example enough urease to convert 3 mg urea in 30 min at 37°C, depending on the age, weight and condition of the patient.
  • the pharmaceutical formulations of the invention will be adapted for oral administration and may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non ⁇ aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions, or any conventional or non-conventional pharmaceutical form.
  • the urease and urea if administered together, will be administered in a form which prevents mixing of the two components.
  • the present invention provides the use of an agent capable of inducing conversion of the coccoid form of H. pylori to the spiral form in the manufacture of a medicament for the treatment of H. pylori infection.
  • the agent is urease and the medicament will optionally also include urea.
  • the medicament is not a vaccine.
  • H. pylori in culture to the coccoid form which comprises adding to the spiral culture an agent capable of inducing the spiral form of H. pylori to convert to the coccoid form.
  • the agent can be provided in the form of culture medium in which the spiral form has been growing but from which the spiral form has been removed.
  • the spiral form has been growing in the medium for at least 3 days.
  • the agent is urease, optionally together with urea.
  • a preferred agent is "anti-urease", e.g. a urease inhibitor or an antibody specific for urease.
  • anti-urease e.g. a urease inhibitor or an antibody specific for urease.
  • a local H. pylori strain V 2 isolated from a patient with non-ulcer dyspepsia was used, although other wild type strains can be used equally well.
  • This strain was initially grown on chocolate blood agar (CBA) to check for purity. The plate culture was then used as inoculum for a 250ml Schott flat-bottomed round bottle containing 30ml BHIH (brain heart infusion supplemented with 10% horse serum and 0.4% yeast extract), and incubated at 37°C for 72h. This in turn serves as the inoculum for chemostat or batch cultures.
  • CBA chocolate blood agar
  • a 1.5L fermenter containing 540ml BHIH was set up as described in Ho and Vijayakumari ⁇ Microbios , 76:59-66
  • the medium was inoculated with 2x30ml of 3 day old H. pylori cul ture, giving a ratio of 1:10
  • the culture was maintained under these conditions for up to 3 months during which daily monitoring of the cells was continued.
  • the cells were harvested by centrifugation at 10,000g for 40min. and washed once.
  • the pellet was then used for preparing coccoid antigen by using the modified glycine method (Ho, B., and Jiang, B., European Journal of Gas tr center ology and Hepatology, 7:121-124 (1995) .
  • a IL Schott round-bottomed bottle or IL Erlenmeyer flask with a side-arm and fitted with a tight fitting rubber bung, containing 270ml BHIH was used.
  • a 7mm diameter hole was bored so as to accomodate the fitting of a disposable filter unit containing a 0.22 ⁇ m filter having a diameter of 50mm (e.g. Gelman) .
  • Each 270ml of BHIH was inoculated with 30ml of 3 day old H. pylori culture. Carbon dioxide was supplied twice daily via the 0.22 ⁇ m filter.
  • the culture was incubated in a 37°C shaker incubator (New Brunswick) maintained at 90rpm for up to nine weeks and the cells were subsequently harvested by centrifugation at 10,000g for 40min.
  • the coccoids thus obtained are stored at -80°C in glycercl-BHIH for up to two years. When required, the coccoids were collected by centrifugation at 10,000xg for 30min, and washed once with PBS (pH 7.2) .
  • H. pylori coccoids were inoculated into 30ml of supernatant (IB) and incubated at 37°C in a 5% carbon dioxide incubator to give a final concentration of 10 ⁇ coccoids per ml. Similar amounts of coccoids were inoculated into 30ml of fresh BHIH served as controls. Subsequently, fresh BHIH was added to the coccoid cultures over time (24hr) .
  • the cells were fixed in 2% glutaraldehyde in 0.1M cacodylate buffer (pH 7.0) for 2-3hrs or overnight at 4°C and then washed in two changes of 0.IM cacodylate buffer.
  • the cells were resuspended in distilled water and processed for negative staining as above. Drops of the cell suspension were placed on carbon coated 400 mesh copper grids for 1 minute. The excess fluid was blotted and the grids air dried to fix. the grids were then stained with a drop of 1% phosphotungstic acid for 1 minute before the excess stain was blotted. After air drying, the grids were examined using a Philips CM120 transmission electron microscope.
  • Urea was added at a concentration of 5mM to coccoids in IB or BHIH. Samples of the culture were withdrawn at time intervals for microscopic examination and were subcultured on CBA and in BHIH.
  • the coccoids appeared dense under phase contrast microscopy. It took 30min. for counter staining to take effect. After 24h induction in IB, the coccoids became loose, and began to extrude the foetal spiral cells. Some nascent spirals could be seen attached to the "maternal" coccoid shells. At 48h of induction, this "birthing" process became more pronounced. Upon introduction of BHIH, the new spirals changed into mature cells and became motile. The spirals were more active in 2x BHIH. Upon addition of urea to the IB and BHIH, the growth was more evident, with spirals appearing as early as 24h.
  • the "inducer” initiated the foetal outgrowth of the spiral form from within the thick polysaccharide layer of the coccoid form. Thus, clearly, nutrients/inducers can pass through this coat.
  • Data obtained previously has indicated that urease subunits C and D are lacking or are present at reduced levels within the coccoid form. The data given above indicates that urease is present in the IB. Thus, it can be concluded that subunits C and D of the urease enzyme are in fact the "inducer".

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Oncology (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Communicable Diseases (AREA)
  • Immunology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biotechnology (AREA)
  • Epidemiology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne des nouvelles thérapies destinées au traitement d'infections à H. pylori, ainsi que des formulations pharmaceutiques servant dans de telles thérapies.
EP96932735A 1995-10-09 1996-10-08 Urease destinee au traitement d'infections a helicobacter pylori Withdrawn EP0862458A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
GBGB9520585.2A GB9520585D0 (en) 1995-10-09 1995-10-09 Therapeutic method
GB9520585 1995-10-09
US1588296P 1996-04-19 1996-04-19
US15882P 1996-04-19
PCT/GB1996/002456 WO1997013527A1 (fr) 1995-10-09 1996-10-08 Traitements d'infections a helicobacter pylori

Publications (1)

Publication Number Publication Date
EP0862458A1 true EP0862458A1 (fr) 1998-09-09

Family

ID=26307915

Family Applications (1)

Application Number Title Priority Date Filing Date
EP96932735A Withdrawn EP0862458A1 (fr) 1995-10-09 1996-10-08 Urease destinee au traitement d'infections a helicobacter pylori

Country Status (9)

Country Link
EP (1) EP0862458A1 (fr)
JP (1) JPH11514998A (fr)
KR (1) KR19990064104A (fr)
CN (1) CN1201395A (fr)
AU (1) AU7141096A (fr)
BR (1) BR9611033A (fr)
MX (1) MX9802809A (fr)
NO (1) NO981594L (fr)
WO (1) WO1997013527A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8524250B2 (en) 2006-11-20 2013-09-03 Keio University Carrier

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7939079B2 (en) 2006-11-14 2011-05-10 Universiteit Gent Helicobacter species and cultivation thereof
EP2087095B1 (fr) * 2006-11-14 2014-07-16 Universiteit Gent Culture in vitro de l'especes d'helicobacter

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6290962B1 (en) * 1992-11-03 2001-09-18 Oravax, Inc. Urease-based vaccine and treatment for helicobacter infection

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9713527A1 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8524250B2 (en) 2006-11-20 2013-09-03 Keio University Carrier

Also Published As

Publication number Publication date
BR9611033A (pt) 1999-12-28
NO981594L (no) 1998-06-03
JPH11514998A (ja) 1999-12-21
MX9802809A (es) 1998-11-29
AU7141096A (en) 1997-04-30
WO1997013527A1 (fr) 1997-04-17
NO981594D0 (no) 1998-04-07
KR19990064104A (ko) 1999-07-26
CN1201395A (zh) 1998-12-09

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