WO1999048510A1 - Inhibiteur de la production d'anticorps d'immunoglobuline m et medicament contre la polyarthrite rhumatoide - Google Patents

Inhibiteur de la production d'anticorps d'immunoglobuline m et medicament contre la polyarthrite rhumatoide Download PDF

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Publication number
WO1999048510A1
WO1999048510A1 PCT/JP1998/004891 JP9804891W WO9948510A1 WO 1999048510 A1 WO1999048510 A1 WO 1999048510A1 JP 9804891 W JP9804891 W JP 9804891W WO 9948510 A1 WO9948510 A1 WO 9948510A1
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Prior art keywords
klebsiella
rha
component
capsular
solution
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PCT/JP1998/004891
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English (en)
Japanese (ja)
Inventor
Hitoshi Oomori
Ryosuke Sugihara
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Tayca Corporation
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Publication of WO1999048510A1 publication Critical patent/WO1999048510A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof

Definitions

  • the present invention relates to an anti-chronic joint which can be obtained from a capsule of a bacterium belonging to the genus Klebsiella, in particular, Klebsielella oxytoca and Klebsiel 1 apneumoniae.
  • rheumatoid arthritis has been treated with steroids such as corticosteroid, an anti-inflammatory agent.
  • steroids such as corticosteroid, an anti-inflammatory agent.
  • long-term use of the above-mentioned steroids can damage skin tissues and adrenocortical. This can result in reduced functioning and sometimes undesirable side effects.
  • the rheumatoid factor detected in the serum of patients with rheumatoid arthritis is IgM antibody, and it is considered that if the production of IgM antibody can be suppressed, it may have an anti-rheumatic rheumatoid arthritis effect. I have.
  • the present inventors have found that a component obtained from the capsule of a bacterium belonging to the genus Klebsiella has an inhibitory effect on IgM antibody production and exhibits an anti-rheumatic rheumatoid arthritis effect.
  • the present invention provides a capsule component of Klebsiella oxalate or Klebsiella 'newmonium, or a fragment obtained by treating it with an acid, a base or a reducing agent. Characterized by containing 8/04891
  • Klebsiella Axitol is particularly preferred as the Klebsiella Axitol TNM 3 strain (Accession No. FERMBP — 46969: Institute of Biotechnology, Industrial Technology Institute, Ministry of International Trade and Industry, Ministry of International Trade and Industry) ) Or a mutant thereof.
  • Klebsiella pneumoniae are Klebsiella 'Pneumonia K19 strain (Michel Beurret et al., Structual investigation of the capsular polysaccharide from Klebsiella K19 by chemical and NMR analyses ", Carbohydrate Research, 157 : 13-25 (1986).) Or mutants thereof.
  • These mutants include three strains of Klebsiella oxytoca TNM or K19 strain of Klebsiella pneumoniae, such as ultraviolet rays, X-rays, etc. Exposure to radiation or chemical mutagens such as ethyl methane sulfonic acid (EMS), N-methyl-N'12-tro-N-2-torosogazidin (MNNG) These capsular components of these bacteria, or by treating them with an acid, a base or a reducing agent, can be produced by contacting with known mutagenesis means. Yo Anti rheumatoid rheumatism effects or I g M presence and intensity of antibody production suppressing action of the resulting off La Gume down bets can and this is readily determined by methods described below.
  • EMS ethyl methane sulfonic acid
  • MNNG N-methyl-N'12-tro-N-2-torosogazidin
  • the capsular component has the formula
  • a-then-Rt 1 [Where L-Rha is L-rhamnose residue, D-Ga1 is D-galactose residue, D-Glc is D-glucose residue, and D-G 1 c UA represents D-gluconic acid residue, and the number in parentheses indicates the position of the glycosidic bond. ] It has a repeating unit of this polysaccharide.
  • Each of the three strains of Klebsiella oxalate TNM and Klebsiella pneumoniae K19 has a polysaccharide composed of the following molar ratios as the constituent saccharides, and a polysaccharide composed of the following molar ratios.
  • Rha (l ⁇ :- ⁇ 2) Rha (l ⁇ : ⁇ 2) Glc (l ⁇ : ⁇ 3) Gal (l ⁇ ⁇ : — 3) GlcUA (l
  • R ha represents a rhamnose residue
  • G 1c represents a glucose residue
  • G a1 represents a galactose residue
  • G 1 c UA represents a glucuronic acid residue.
  • the numbers adjacent to the parentheses indicate the positions of glycoside bonds at each residue.
  • the present invention provides a capsular polysaccharide of Klebsiella oxytoca or Klebsiella newmonia, or a fragment obtained by treating this with an acid, a base or a reducing agent.
  • the present invention also provides an anti-rheumatic rheumatoid arthritis agent and an IgM antibody production inhibitor characterized by containing.
  • the capsular polysaccharide is released from the cells by, for example, wet heat sterilization or ultrasonic treatment, and the culture supernatant from which the cells have been removed, for example, by centrifugation is subjected to ultrafiltration (eg, Millipore).
  • Pore's ultrafiltration membrane can be obtained as a component with a molecular weight cut-off of 1,000 and remaining on the high molecular weight side .
  • the product resulting from the fragmentation of the capsular component can be obtained by treating the capsular component with an acid, a base or a reducing agent. Further, the treatment with the reducing agent can be performed while culturing by adding, for example, iron sulfate and EDTA to the medium.
  • these bacteria particularly preferred in the present invention is a Klebsiella oxytoxin TNM3 strain bacterium.
  • Culturing of the genus Klebsiella to obtain the capsular component of the present invention can generally be performed according to the following method. That is, as a culture medium, carbon and nitrogen sources, inorganic salts, and micronutrients necessary for the growth of the genus Klebsiella and the production of the desired capsular component can be obtained. Any medium may be used as long as the medium is contained, and there is no particular limitation.
  • carbon sources examples include glucose, lactose, maltose, xylose, mannitol, saccharose, ramnoose, arabinose, trehalose, Raffinose can be used.
  • Nitrogen sources include, for example, synthetic compounds such as nitrates, ammonium salts, and urea, polypeptone, corn steep liquor, yeast extract, meat extract, defatted soy extract, peptides, Natural organic substances such as MIN can be used.
  • inorganic salts for example, phosphates, potassium salts, sulfates, magnesium salts and the like can be used.
  • micronutrients for example, yeast extract and various vitamins can be used.
  • the medium may be supplemented with calcium salts, manganese salts, or the like.
  • the medium used may be a solid medium or a liquid medium.
  • Use liquid medium When used, static culture may be used, but shaking culture or aeration-agitation culture is more preferable in order to obtain the desired capsular component at a high yield.
  • the medium pH at the time of culture is not particularly limited as long as it is suitable for the growth of the microorganism and does not hinder the production of the desired capsular component, but the appropriate pH is usually 4 to 8.
  • the culture temperature is not particularly limited, but is usually preferably from 20 to 35 ° C.
  • the culture time is appropriately set so that the production of the desired capsular component is maximized, but usually 1 to 7 days is preferable.
  • the culture obtained by the above culture can be used as an anti-rheumatic rheumatoid arthritis agent or an IgM antibody production inhibitor without purification after sterilization.
  • the cells can be removed by a conventional method using centrifugation or filtration after the capsule is released from the cells by, for example, wet heat sterilization.
  • a water-miscible organic solvent such as methanol, ethanol, isopronol, and acetate is added to the culture solution after the removal of the cells to form a precipitate, and then the precipitate is formed.
  • Is dissolved in water then dialyzed against water, and then purified by drying the dialysis solution by methods such as through-air drying, hot-air drying, spray drying, drum drying, reduced-pressure drying, and freeze-drying. Processing may be performed.
  • ultrafiltration for example, a fraction having a fractionation amount of 1,000 from Millipore, which can be used for concentration
  • the method of subjecting the liquid to a drying step, and if necessary, precipitation or salting out with various types of column chromatography such as ion exchange, gel filtration, and affinity, quaternary ammonium salts, and organic Precipitation with a solvent is used.
  • the culture itself or the capsular component purified above may be subjected to various treatments such as addition of a reducing agent, acid or base hydrolysis, heating under pressure, or ultrasonic treatment, if desired. good.
  • a reducing agent such as sulfuric acid or hydrochloric acid or an aqueous solution such as sodium hydroxide aqueous solution or ammonia water is added to the culture after completion of the culturing operation or the capsular component purified above. It is preferred to add and adjust the conditions.
  • a reducing agent may be added to the medium to such an extent that the production of the capsular component in the culture does not decrease.
  • a reducing agent to be used in this case for example, a combination of ferrous sulfate and Z or ferrous chloride and ethylenamine tetracarboxylic acid is preferable.
  • Polysaccharides which are capsular components of Klebsiella bacteria obtained in this way, or fragments obtained by treating these with an acid, base or reducing agent, are anti-fragments. It has the effect of rheumatoid arthritis and the effect of suppressing IgM antibody production.
  • a 50 OmL volume Sakaguchi flask is charged with 10 OmL of the medium having the composition shown in Table 1, and sterilized by wet heat at 121 ° C for 20 minutes, followed by Klebsiella box.
  • One platinum loop was inoculated from three test strains (Klebsiel 1 aoxytoca) TNM 3 (FERMBP-46669) and shaken at 1 10 strokes per minute at 28 ° C. Cultivation was carried out two days before with reciprocating shaking for three days.
  • a 50 O mL volume Sakaguchi flask was sterilized with 100 O mL of the medium shown in Table 2 in the same manner as above, and then inoculated with lm L of the pre-preculture, and shaken. Preculture was performed by reciprocating for 1 day at 28 ° C. with 110 strokes per minute.
  • the rotation speed during the main culture was 200 rpm until the 36th hour of culture, and 400 rpm until the 96th hour thereafter.
  • the ventilation rate was 1.5 L / min up to 48 hours, and 4 L / min until the 96th hour.
  • Vitamin B 1 0. 0 0 0 5%
  • Vitamin B 1 0.0 0 0 5%
  • Beta tin 0.00 0 0 0 6% 0 Calcium titanate 0.01 0 1% Nicotinamide 0.00 0 0 5%
  • the culture obtained by the main culture of PH 7 was sterilized by wet heat at 121 ° C. for 20 minutes, and then the cells were removed by centrifugation to precipitate acetate.
  • the precipitate was washed with a 70% aqueous acetone solution, and dried with warm air to obtain 19 g of a component mainly composed of a fragment from a capsular polysaccharide per 1 L of the culture medium.
  • 20 g of a fragment-main component from the obtained capsular polysaccharide was brought to 500 mL with 0.1 M citrate buffer (pH 4.5).
  • the solution After passing through an exchange resin, the solution is neutralized with a 1 M aqueous sodium hydroxide solution, and the solution that has passed through a 0.2 m membrane filter is freeze-dried to obtain a capsular polysaccharide. As a result, 8 g of a purified product mainly composed of a fragment from the product was obtained.
  • Example 1 Anti-rheumatic rheumatoid arthritis effect evaluation (effect on collagen arthritis)
  • a purified product of a component mainly composed of a fragment from the capsular polysaccharide obtained in Production Example was dissolved in physiological saline, and the dose was 100 mg / kg for 1 mouse of DBA.
  • the thus prepared solution (150 L) was subcutaneously administered from the 4th day before the primary sensitization to the day before the primary sensitization and from 4 days before the secondary sensitization to the day before the secondary sensitization a total of 8 times.
  • the control group received only saline.
  • TNP — KLH prepared using 8-week-old male BDF1 mice (8 mice / group) by reacting key honorelinpet hemosinin with trinitrobenzenesulfonate as antigen 0.2 ml of physiological saline containing a mixture of 7 // g of the above and 1 mg of aluminum hydroxide as an adjuvant was injected intraperitoneally to sensitize.
  • a purified product of a component mainly composed of a fragment from the capsular polysaccharide obtained in Production Example was dissolved in physiological saline, and the dose was increased to 100 mg / kg per mouse of BDF.
  • the thus adjusted solution 150 ⁇ L was subcutaneously administered to the back four times from the day before sensitization to 2 days after sensitization.
  • the control group received saline only.
  • blood was collected from the fundus, and the amount of anti-III-IgM antibody produced was measured.
  • the production of the anti-TNP-IgM antibody was measured according to the ELISA method reported in Ohomori, H., Immunol. Lett., 23: 251-256 (1990). The sample measurement method is described below.
  • anti-mouse IgM antibody ( CAPTEL: GOATF (ab ') 2 FRAGMENTTOM 0 USEI g M) is diluted to a concentration of 20 g / mL, and 50 L is added to a 96-well plate at 50 ° C. And left for 12 hours for coating. Remove the supernatant and add 250 ⁇ L of BSA solution (bovine serum albumin) diluted with A1 buffer to a concentration of 0.2% by weight. Block for 12 hours. The supernatant was removed and used for the test. Table 5
  • Each sample can be measured in advance for fluorescence using the BSA solution diluted to a concentration of 0.1% by weight with the A2 buffer solution of the composition shown in Table 6. It was diluted to a concentration within an appropriate range, and added to the above-described plate in 50 ⁇ L portions, and then left at 4 ° C. for 12 hours to react. After removing the supernatant of each sample after the reaction and washing twice with the A2 buffer-diluted BSA solution described above, adjust to the appropriate concentration (approximately several gmL) using the A2 buffer-diluted BSA solution. Add 50 L of TNP--3—galactosidase solution and leave at 4 ° C for 4 hours. I responded. The supernatant was removed and washed three times with Tween 20 solution diluted with A2 buffer to a concentration of 0.02% by volume. Table 6
  • Ig M antibody production inhibition rate [C-S] / C] 100 (where, C: anti-TNP in control—Ig M antibody production, S: anti-TNP in test sample administration group TNP-Ig M antibody production) Table 7 Average value of antibody TNP-IgM antibody production . ⁇ g / mL) Test sample Control Suppression rate (%
  • the component mainly composed of a fragment from the capsular polysaccharide has an inhibitory effect on IgM antibody production.
  • Fragment-based product from capsular polysaccharide obtained in Production Example at a concentration of 5 mg / 400 / L with respect to the above DBA1 mouse As described above, a solution dissolved in a 0.05M aqueous acetic acid solution was orally administered in a dose of 400 L once a day from 10 days before the primary immunization, a total of 10 times. The same operation was performed using a 0.05 M aqueous acetic acid solution as a control group.
  • the degree of arthritis swelling was visually observed, and one point was calculated when one hand or one foot was out of normal condition, and the sum was divided by the number of mice in each group.
  • Table 8 shows the results.
  • the incidence of arthritis was determined by visual observation, when one hand or one foot was detached compared to the normal state, and the number was divided by the number of each group. Multiplied by 100.
  • Table 9 shows the results.
  • bovine cartilage-derived type I collagen was dissolved in a 0.05 M aqueous acetic acid solution at a concentration of 4 mg Z mL, and IFA as an adjuvant (Wako Pure Chemical Industries, Ltd.)
  • the second sensitization was carried out by injecting 100 ⁇ L of an emulsion consisting of an equal mixture with the above mixture into the skin of ⁇ ai1Base.
  • the concentration of a product mainly comprising a fragment from the capsular polysaccharide prepared in the production example was adjusted to 0.05 mg / 400 ⁇ L to give a concentration of 0.05 M.
  • the dissolved solution was orally administered in an aqueous solution of acetic acid at a dose of 400 ⁇ L once a day from the day after the primary immunization, a total of 10 times, and then orally once every three days. The same operation was performed using a 0.05 M aqueous acetic acid solution as a control group.
  • test sample 0.1 0.1 0.1 0.3 0.3 0.6 0.6 0.6 0.6 0.6
  • the oral administration of the capsular component of the bacterium of the genus Klebsiella or the fragmented product thereof after the sensitization with collagen resulted in a higher collagen content than the control group.
  • Arthritis can be suppressed with a high suppression rate.

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Abstract

Ce polysaccharide, obtenu à partir de la capsule de Klebsiella oxytoca ou de Klebsiella pneumoniae, ou l'un de ses fragments, inhibe la production in vivo d'anticorps IgM et est donc utile pour soigner la polyarthrite rhumatoïde.
PCT/JP1998/004891 1998-03-24 1998-10-28 Inhibiteur de la production d'anticorps d'immunoglobuline m et medicament contre la polyarthrite rhumatoide WO1999048510A1 (fr)

Applications Claiming Priority (2)

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JP10/96861 1998-03-24
JP9686198 1998-03-24

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH04211017A (ja) * 1990-02-16 1992-08-03 Lab Om Sa 皮膚炎治療薬
JPH06313001A (ja) * 1993-04-28 1994-11-08 Kureha Chem Ind Co Ltd Ｂｓ−５物質およびその製造方法
JPH07188035A (ja) * 1993-12-24 1995-07-25 Kureha Chem Ind Co Ltd 軟骨保護剤

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH04211017A (ja) * 1990-02-16 1992-08-03 Lab Om Sa 皮膚炎治療薬
JPH06313001A (ja) * 1993-04-28 1994-11-08 Kureha Chem Ind Co Ltd Ｂｓ−５物質およびその製造方法
JPH07188035A (ja) * 1993-12-24 1995-07-25 Kureha Chem Ind Co Ltd 軟骨保護剤

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