Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
  • A61K35/745—Bifidobacteria
• • 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
  • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
• • 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
  • A61P37/00—Drugs for immunological or allergic disorders
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to a process for the preparation of a macromolecule of bacterial origin, to the macromolecule thus obtained, and to the macromolecule used for the preparation of a macromolecule of bacterial origin, to the macromolecule and to the macromolecule. obtained and the use of said macromolecule for the prophylaxis and treatment of inflammatory rheumatism.
• rheumatoid arthritis many inflammatory rheumatisms such as rheumatoid arthritis, ankylosing spondylitis or post-infectious rheumatism have the common denominator presence of bacteria or bacterial signals (DNA, RNA, peptidoglycan-polysaccharides) in the synovial fluid
• RT-PCR analysis of the bacterial rRNA for detection and characterization of bacterial species in arthritis synovial tissue "- Kempsell KE, Cox CJ., Hurle M., Wong A., Wiiké S., Zanders ED, Gaston IS., Crowe IS., In Infect Immun.
• Antibiotic therapy applied to patients with a rheumatic condition reduces the number of certain bacteria in the intestinal flora and contributes to the reduction of inflammation; however, this method of treatment can not be used in the long term, given the risk of emergence of resistant bacteria.
• the present invention makes it possible to overcome the disadvantages presented by the existing methods of treating inflammatory rheumatism.
• the aim of the present invention is to propose another approach to the treatment of inflammatory rheumatism, based on the use of a macromolecule of bacterial origin, capable of regulating the intestinal flora and the bacterial translocation and, consequently, of decrease the reaction inflammation that accompanies inflammatory rheumatism.
• the invention relates to a process for preparing a macromolecule produced by bacteria belonging to the strain Bifidobacterium brief 1-2219 which has been deposited according to the Treaty of
• Said method is characterized in that it comprises the following steps: i) the inoculation and incubation, under aerobic or anaerobic conditions and at a temperature of between 30 ° C. and 40 ° C., of a strain of Bifidobacterium Brief 1-2219, in a culture medium having a regulated or unregulated pH and comprising as ingredients, a whey protein fraction enriched in native lactalbumin (undenatured) and lactose; ii) separating said bacteria from said culture medium after a incubation period between 16 and 48h; iii) ultrafiltration on filtration membranes with a cutoff threshold of 2 KDa at 50 KDa, leading to the production of a concentrated retentate; iv) macromolecule enrichment by washing with a volume of 5 to 25 times the volume of concentrated retentate; v) the dehydration of said enriched concentrated retentate; vi) purification of the macromolecule by passage over anion exchange resin; vii) recovering the fraction
• the process for the preparation of said macromolecule of bacterial origin has the following characteristics, where appropriate combined: the seeding of the bifidobacteria in said culture medium is made from a frozen concentrate or a meadow. -culture of 16-24h, which allow the proliferation of bacteria;
• the bacteria are inoculated in said culture medium at 5 to 10 October 10 cfu per ml of medium; the ingredients of the culture medium are present in the following quantities:
• fraction of whey proteins enriched in undenatured native lactalbumin 0.01 to 1 g / l of medium;
• lactose 30-70 g / liter of medium; the pH of said culture medium is not regulated;
• the pH of said culture medium is maintained between 4 and 6 units approximately.
• the invention relates to a macromolecule of bacterial origin produced by bacteria belonging to the strain Bifidobacterium brief 1-2219 and prepared by the setting artwork
• the present invention relates to the use of said macromolecule of bacterial origin for the preparation of medicaments for the prophylaxis or treatment of inflammatory rheumatism.
• Drugs comprising said macromolecule can be used as an adjunct to therapies currently used for the treatment of inflammatory rheumatism (leflunomide, monoclonal antibodies, anti-inflammatory drugs) or to replace these drugs, the side effects of which are well known.
• Drugs comprising said macromolecule can be administered orally or by subcutaneous injection; they have the effect of regulating the intestinal flora and bacterial translocation, and consequently reduce the inflammatory reaction that accompanies inflammatory rheumatism. These effects recommend them both for the prophylaxis of rheumatic diseases (in the case of patients from families at risk), and for the treatment of various forms of inflammatory rheumatism, including rheumatoid arthritis and ankylosing spondylitis.
• the medicaments comprising said macromolecule of bacterial origin prepared according to the invention may be stored at a temperature of -35 ° C. to + 4 ° C. for the liquid forms and up to +40 ° C. for dry forms for 3 years. Said drugs do not give a side reaction.
• a culture medium containing the following ingredients is prepared:
• lactalbumin enriched preparation corresponding to 0.01-0.05 g of lactalbumin / liter of prepared medium
• the lactalbumin-enriched preparation is prepared by precipitating a solution of whey proteins (for example Vitalarmor alpha 607 distributed by ARMOR PROTEINS) at pH 4.1 for 15 minutes in a water bath at 60 ° C. The supernatant is then recovered after centrifugation.
• whey proteins for example Vitalarmor alpha 607 distributed by ARMOR PROTEINS
• a first solution is reconstituted with lactose alone.
• a second solution is reconstituted with the rest of the ingredients. Both solutions are autoclaved for 30 minutes at 115 ° C. Discontinuous fermentation at regulated pH
• the pH of the medium is adjusted to a value of 5.8 once the medium is poured into the fermenter.
• the culture medium is inoculated with 6.67% (v / v) (100 ml of inoculum per 1500 ml of medium) of a 24-hour inoculum, containing between IxIO 6 and 2x10 8 colony forming units (CFU) of bifidobacteria per ml of culture medium.
• the bifidobacteria are cultured with agitation, without aeration of the medium, at a temperature of 37 ° C.
• the pH is maintained at 5.8 by addition of 3N sodium hydroxide.
• the fermentation lasts about 20 hours and the population of brief Bifidobacterium end of culture is between IxIO 9 and IxIO 10 CFU per ml of culture medium.
• the Bacterial growth corresponds to an average increase of a factor of 10 of the population.
• the pH of the medium is not adjusted when the medium is poured into the fermenter.
• the culture medium is inoculated with 6.67% (v / v) (100 ml of inoculum per 1500 ml of medium) of a 24-hour inoculum, containing between 1 ⁇ 10 6 and 2 ⁇ 10 8 CFU of bifidobacteria per ml. of culture medium.
• Bifidobacteria are grown without agitation, without aeration of the medium, at a temperature of 37 ° C. The fermentation lasts about 20 hours.
• the seeding of the bifidobacteria in said culture medium is made from a frozen concentrate containing 10 8 to 10 12 CFU of bifidobacteria per g of concentrate.
• the product yield recovered after ultrafiltration is 700-1000 mg of dry powder per liter of fermented medium.
• Lowry is 275 mg / g dry powder.
• the amount of sugar determined by the Dubois method is 220 mg / g of dry powder.
• the product yield recovered after ultrafiltration is of the order of 70-170 mg of dry powder per liter of fermented medium.
• the amount of protein, determined by the method of Lowry, is of the order of 71 mg / g of dry powder.
• the quantity of sugars, determined by the Dubois method, is 615 mg / g of dry powder.
• the bacteria are removed by centrifugation for 30 minutes at 8000 g.
• the supernatant is ultrafiltered at room temperature on an AMICON Proflux M12 device equipped with an AMICON spiral cartridge type S10Y10, with a cutoff threshold of 10 kDa.
• the supernatant is concentrated at most 10 times, then washed continuously with 15 liters of water RO.
• the retentate is concentrated to a volume of 500 ml. It is then preserved in frozen or freeze-dried form.
• the macromolecule contained in the retentate is then purified by passage of the concentrated solution of retentate or reconstituted retentate solution (DEAE-Sepharose fast flow resin distributed by Amersham Biosciences, equilibrated in a Tris-HCl buffer) on anion exchange resin. 50mM, pH 8.0 elution is by the same buffer to which is added a NaCl concentration ranging from 0 to 1M).
• DEAE-Sepharose fast flow resin distributed by Amersham Biosciences, equilibrated in a Tris-HCl buffer
• anion exchange resin 50mM, pH 8.0 elution is by the same buffer to which is added a NaCl concentration ranging from 0 to 1M).
• the bacterial macromolecule is recovered in the fraction eluted with 0.15-0.6M NaCl (preferentially 0.3M). This fraction is desalted and then freeze-dried.
• the macromolecule thus produced is composed of oligosaccharides with a molecular mass of between 5 and 30 kDa and a protein of bacterial origin with a molecular mass of between 50 and 70 kDa.
• EXAMPLE 2 Determination of the anti-rheumatic properties of the macromolecule derived from the Bifidobacterium strain Brief 1-2219
• the anti-rheumatic effects of the macromolecule of bacterial origin prepared according to the invention were evaluated in an experimental model of rheumatoid arthritis, namely induced arthritis. collagen II in male DBA mice 1.
• mice used were divided into two groups: a control group: 15 mice (immunized with collagen II); an experimental group: 5 mice (immunized with collagen II).
• Two collagen II injections are made at the base of the male DBA 1 male tail. The animals are 4-5 weeks old at the first injection. The recall is done three weeks later. In the following weeks, joint swellings appear.
• a score Arthritis is established by animal and reading is performed several times a week.
• control in place of drinking water, the animals in the control group receive the unfermented culture medium and treated by ultrafiltration at
• animals receiving either the culture medium (10 male DBA1 mice) or the macromolecule (6 male DBA1 mice) are included in the study. They are not immunized by collagen II, but are distributed in the cages of the immunized animals. They do not develop arthritis.
• facultative anaerobic and aero-anaerobic flora are counted in the cecum, Peyer's patches, heart blood, kidney, spleen , liver and lung. The animals are sacrificed at least two hours after the last feeding.
• Table 1 shows the results for total intestinal flora, expressed as an average and log deviation of the number of CFU / g stool, where n is the number of mice for each product tested (two-way ANOVA comparison).
• mice that developed arthritis an immunized control group
• the caecal flora was larger than in non-immunized animals (non-immune control group) (p ⁇ 0.0469).
• This bacterial proliferation is not found in immunized animals receiving the macromolecule (immunized test group).
• the macromolecule therefore regulates the intestinal flora.
• the macromolecule reduces the passage of bacteria in non-immune animals.
• log CFU / PP log colony forming units / Peyer's patches
• Bacteria that have passed the intestinal barrier are eliminated by macrophages and polynuclear cells in the blood and by macrophages and dendritic cells in the deep organs (lung, liver, spleen, kidney).
• the macromolecule therefore allows better control of bacterial contamination in the liver.

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• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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• 2004
  • 2004-10-14 FR FR0410886A patent/FR2876702B1/fr not_active Expired - Fee Related
• 2005
  • 2005-10-14 JP JP2007536225A patent/JP2008516933A/ja active Pending
  • 2005-10-14 US US11/577,307 patent/US20080038776A1/en not_active Abandoned
  • 2005-10-14 CA CA002583269A patent/CA2583269A1/fr not_active Abandoned
  • 2005-10-14 EP EP05809190A patent/EP1869195A2/de not_active Withdrawn
  • 2005-10-14 WO PCT/FR2005/002554 patent/WO2006040485A2/fr active Application Filing

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