WO1996023002A1 - Lipopolysaccharide de faible poids moleculaire - Google Patents

Lipopolysaccharide de faible poids moleculaire Download PDF

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Publication number
WO1996023002A1
WO1996023002A1 PCT/JP1996/000135 JP9600135W WO9623002A1 WO 1996023002 A1 WO1996023002 A1 WO 1996023002A1 JP 9600135 W JP9600135 W JP 9600135W WO 9623002 A1 WO9623002 A1 WO 9623002A1
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molecular weight
lps
low
weight
lipopolysaccharide
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PCT/JP1996/000135
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English (en)
Japanese (ja)
Inventor
Den-Ichi Mizuno
Gen-Ichiro Soma
Takashi Nishizawa
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Taiho Pharmaceutical Co., Ltd.
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Publication of WO1996023002A1 publication Critical patent/WO1996023002A1/fr

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    • 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
    • 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

Definitions

  • the present invention relates to a novel low molecular weight lipopolysaccharide having specific physicochemical and biological properties, extremely high safety (low toxicity), and high biological activity.
  • Lipopolysaccharide (hereinafter sometimes referred to as LPS) consists of lipids and sugars present in the outer membrane surrounding peptide glycans in the cell wall of Gram-negative bacteria such as Escherichia coli, Salmonella, and B. pertussis. It is a complex compound and is known as the active ingredient of the 0 antigen and endotoxin [edit by JM Ghuysen and R. Hakenbeck, edited by “New Comprehensive New Comprehensive Biochemistry j, Vol.
  • LPS The basic structure is composed of lipid 8, which has a unique lipid, an oligosaccharide called an R-core covalently bonded thereto, and 0-specific polysaccharide. Noroji latest term Dictionary ", the fourth 3 1 page, Nikkei McGraw-Hill, Inc., 1 9 8 5 years).
  • the basic structure of lipid A is common to many strains, and the basic skeleton is composed of ⁇ -1,6-linked glucosaminyl and glucosamine, with phosphoric acid bound to C-1 and C-4 ', respectively. Often. Each amino group binds a 3-hydroxy fatty acid, and the hydroxyl group binds several kinds of saturated fatty acids or hydroxy fatty acids to form a unique glycolipid. Although there are a few cases, the basic skeletal structure is completely different, and an example consisting of 2,3-diamino-2,3-dideoxy D-glucose alone has been reported (Edited by Nomichi Noma, “Vol. 49 of the Dictionary of Medical Science”) , Page 82, Kodansha, 1984).
  • the structure of the R-coa is common to most of the bacterial species associated with it, such as Salmonella JR, and there are cases where several partially different structures are known, such as Escherichia coli [JM Ghuysen and R. Hackenbeck, eds., “New Comprehensive Biochemistry”, Volume 27, Bacterial 'cells' Bacterial Cell Wall, pp. 283, Elsevea, 1989.
  • Escherichia coli JM Ghuysen and R. hackenbeck, eds., “New Comprehensive Biochemistry”, Volume 27, Bacterial 'cells' Bacterial Cell Wall, pp. 283, Elsevea, 1989.
  • KDO 2-keto-3-doxyctonate
  • the structure of the 0-specific polysaccharide is the most diverse of the constituents, is specific to the bacterial species, and exhibits activity as a so-called 0 antigen. Generally, it is characterized by a repeating structure of oligosaccharides composed of several types of monosaccharides, but those composed of the same monosaccharide or those not having a repeating structure are also known.
  • the biosynthesis of 0-specific polysaccharides is governed by a different gene from that of the R-core, and it is possible to replace 0-specific polysaccharides of different strains by conjugation or transduction, and to increase the virulence of bacteria and vaccines. [JM Ghuysen and R. Hackenbeck, edited by JM Ghuysen and R. hackenbeck, “New Comprehensive Biochemistry:”, Vol. 27, Bacterial Cell Wall, pages 265-267, Elsevea, 1991].
  • LPS has a wide variety of pharmacological effects, for example, simultaneous administration of an antigen and LPS enhances the immune response, so LPS is currently used as a type of adjuvant to enhance vaccine efficacy. (Honma et al., "Bacterial Endotoxins", p. 312, Kodansha, 1973).
  • Escherichia coli-derived SDS-PAGE molecular weight of 40,000 ⁇ 10,000 or 8,000 ⁇ 4,000, phosphorus number LPS with a molecular weight of 30,000, a hexosamine number of 45 ⁇ 6Z molecular weight of 30,000, a fatty acid number of 18 molecular weight of 30,000, and a KDO number of 5 ⁇ 1Z molecular weight of 30,000 (JP-A-6-40937), Serratia.
  • LPS having a phosphorus number of 4 molecular weight of 6,000, a hexosamine number of 12Z molecular weight of 6,000, and a KDO number of SilZ molecular weight of 6,000 (JP-A-5-155778, Japanese Patent Application Laid-Open No. 6-40993), SDS-PAGE of B. pertussis molecular weight 6,000 ⁇ 1,000 or 9,500 ⁇ 1,500, phosphorus number 5 / LPS having a molecular weight of 8,000, a hexosamine number of 16 ⁇ 2, a molecular weight of 8,000, a KDO number of 2 ⁇ 1, and a Z molecular weight of 8,000 (Japanese Unexamined Patent Publication No.
  • LPS with a molecular weight of around 5,000 has already been reported, but the main staining band in these SDS-PAGE is 5,000 or 6,000, There were also more than 30,000 stained bands. That is, The original LPS with a molecular weight of around 5,000 was a mixture with LPS with a molecular weight of 30,000 or more.
  • the inventors of the present invention have proposed anti-toxoplasma agents (JP-A-4-49245) and cholesterol-lowering agents (JP-A-4924943).
  • An anti-herbal agent Japanese Patent Application Laid-Open No. 4-42942
  • an anti-rheumatic agent Japanese Patent Application Laid-Open No. 419,241
  • an antidiabetic agent Japanese Patent Application Laid-Open No. No. 4
  • an anti-digestive hamper Japanese Patent Application Laid-Open No. 419/240
  • an immune function activator Japanese Patent Application Laid-Open No. 419/4981
  • the conventional LPS has been pointed out to be a problem in clinical application from the viewpoint of safety (Japanese Society for Tissue Culture, “Cell Growth Factor part II”, No. 1). 21 Page, Asakura Damage Store, 1987).
  • Hei 9-194481 discloses that The SDS-PAGE image is shown, but in addition to the stained band with a molecular weight of around 6,000, there is clearly a stained band with a molecular weight of 30,000 or more. Further, in Japanese Unexamined Patent Application Publication No. Hei 4-187640, Japanese Unexamined Patent Publication No. Hei 4-92440 and Japanese Unexamined Patent Publication No. Hei 5-155578, the molecular weight is 5, 0000 or 6, 000. Although low molecular weight LPS of 0 is disclosed, these are all purified products by the thermal phenol method and ion exchange, and have not been subjected to a step of completely eliminating high molecular weight LPS, High molecular weight LPS was mixed.
  • conventionally reported low-molecular-weight LPS is a mixture containing high-molecular-weight LPS.
  • a drug component such as an immune function activator
  • the present invention has been made in view of the above circumstances, and provides a novel LPS having higher safety (that is, lower toxicity) and superior biological activity than conventional LPS. It is intended to be. Disclosure of the invention
  • the inventors of the present invention have conducted intensive studies to solve the above-mentioned problems, and as a result, have discovered a novel low-molecular-weight LPS that is different from LPS that has been conventionally reported, and have been able to improve the strength.
  • This new low molecular weight LPS 'has extremely high safety compared to conventional LPS, and has been found to be superior in biological activity to conventional LPS, thus completing the present invention.
  • the present invention is obtained from microbial cells and has the following physicochemical properties a) to c)
  • the present invention provides the following physicochemical and biological properties obtained from microbial cells:
  • the nucleic acid content is 1% (weight) or less
  • the microorganism is a gram-negative microorganism
  • the gram-negative microorganism is a microorganism belonging to Pantoea (Pantoea) or a microorganism belonging to the genus Salmonella. Is also a desirable embodiment.
  • the low molecular weight LPS of the present invention is highly safe and has excellent biological activity, and has the above-mentioned anti-toxoplasma agent, cholesterol lowering agent, anti-herpes agent, anti-rheumatic agent, anti-diabetic agent, anti-digestion agent It is effective as a pharmacologic agent such as a sexually active cancer drug, an immunostimulant (immune function activator), an analgesic, a growth promoter, an anti-abstinent symptom, etc., as well as a wound treatment, a hemorrhoid agent, an anti-tumor pain agent, etc. It is.
  • the present invention provides a pharmaceutical composition comprising the low-molecular-weight LPS of the present invention and a pharmaceutical carrier.
  • the present invention provides a medicine containing the low-molecular-weight LPS of the present invention as an active ingredient, particularly an immunostimulant and a wound treatment.
  • This drug can also be used as a veterinary drug.
  • crude lipopolysaccharide obtained by extraction from microbial cells is subjected to anion-exchange chromatography, and then the treated product is subjected to the presence of a surfactant.
  • the present invention provides a method for producing a low-molecular-weight LPS according to the present invention, which is characterized in that gelation occurs in the presence.
  • the surfactant is preferably deoxycholic acid.
  • the low-molecular-weight LPS of the present invention is obtained by culturing a Gram-negative microorganism, for example, a microorganism belonging to the genus Pantoea or a microorganism belonging to the genus Salmonella, by a conventional method, collecting cells from the culture medium, and using a known method from the collected cells.
  • a Gram-negative microorganism for example, a microorganism belonging to the genus Pantoea or a microorganism belonging to the genus Salmonella
  • a conventional method collecting cells from the culture medium, and using a known method from the collected cells.
  • the thermal phenol method [O-I 'Westphal, Ed., Methods in Carbohydrate Chemistry, Vol. 5, p. 83, Ademick Press Academic Press), 1965]
  • microbial cells are suspended in distilled water, this suspension is added to a mixture of distilled water and an equal volume of hot phenol, and the mixture is stirred, and then centrifuged to collect the water brow.
  • the layer is dialyzed to remove phenol, concentrated by ultrafiltration to collect the crude LPS fraction, and this fraction is subjected to conventional anion exchange chromatography (eg, mono-Q-Sepharose or Q-phase). -Sepharose) and desalting by a conventional method.
  • the purified LPS thus obtained is disclosed in JP-A-4-18764, JP-A-4-49240, JP-A-4-949841, and JP-A-5-948. — Although it is said that this corresponds to LPS with a molecular weight of about 5,000 to about 6,000 disclosed in Japanese Patent Publication No. 1557778, the actual purification of this LPS has not been completed. And a mixture containing a high molecular weight fraction as described above. This has been clarified for the first time in the present invention, and it is considered that the purified low-molecular-weight LPS did not exist as a single product in the past.
  • the number of hexosamines of the low-molecular-weight LPS of the present invention described later is significantly different from that of the conventional unpurified LPS. That is, it is considered that the number of hexosamines was calculated to be larger than that of the present invention because the low molecular weight LPS was conventionally mixed with the low molecular weight LPS. Therefore, unpurified LPS containing such high molecular weight LPS is gel-filtered in the presence of a surfactant such as sodium deoxycholate to contain low molecular weight LPS. By recovering only the fractions which are present and removing the mixed low molecular weight LPS, a highly purified novel low molecular weight LPS of the present invention can be obtained.
  • the step of gel filtration in the presence of a surfactant is described in JP-A-4-187640, JP-A-4-149240 and JP-A-5-157778. This is for purifying the LPS having a molecular weight of about 5,000 to 6,000, which is disclosed in the gazette, to a higher degree. This step completely eliminates the high-molecular-weight LPS that is present in the mixture.
  • the hexosamine content measured by the Elson-Morgan method is 1 to 3 and the Z molecular weight is 5,000.
  • the term “pieces /” molecular weight of 5,000 ”in paragraphs b) and c) refers to the number of hexosamine or KDO per low-molecular-weight LPS molecule having a molecular weight of 5,000.
  • the hexosamine number and the KDO number when the molecular weight of the low molecular weight LPS is other than 5,000 can be converted as those proportional to the molecular weight of 5,000.
  • the respective numbers at the molecular weight of 5,000 are assumed to be proportional to the molecular weight of the LPS. The number of each can be calculated.
  • the immunostimulatory ability of the low-molecular-weight LPS of the present invention was confirmed by the effect of producing endogenous TNF through the activity of the macula phage, as shown in Test Example 4 described below, It turned out that there is.
  • the low molecular weight LPS of the present invention can be used not only for each compound but also for any combination of two or more of them, or further in combination with other pharmaceuticals, as long as they do not adversely affect the intended use. You can also.
  • the low molecular weight LPS of the present invention can be used as a pharmaceutical composition according to a usual method using an appropriate pharmaceutical carrier.
  • the carrier it is possible to use various ones commonly used for ordinary drugs, for example, excipients, binders, disintegrants, lubricants, coloring agents, flavoring agents, flavoring agents, surfactants, and the like. it can.
  • the dosage unit form when using the medicament or the pharmaceutical composition of the present invention is not particularly limited and can be appropriately selected depending on the purpose of treatment. Specifically, injections, suppositories, external preparations (descendants, patches) , Liniments, lotions, etc.), parenteral preparations such as aerosol preparations, tablets, coated tablets, powders, granules, capsules, pills, troches, and liquid preparations (suspension, emulsion, etc.). Can be In particular, skin * contains a large amount of macrophages, so that a higher effect can be obtained when administered as a skin application.
  • the carrier When molding into an injection, the carrier may be, for example, diluted with water, ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc.
  • Agents, pH adjusters such as sodium citrate, sodium acetate, sodium phosphate and the like, and biting agents, stabilizers such as sodium pyrosulfite, ethylenediaminetetracarboxylic acid, thioglycolic acid, thiolactic acid and the like can be used.
  • a sufficient amount of saline, glucose or glycerin to prepare an isotonic solution may be included in the pharmaceutical preparation, and a usual solubilizing agent, soothing agent, local anesthetic Etc. may be added.
  • a usual solubilizing agent, soothing agent, local anesthetic Etc. may be added.
  • suitable carriers include, for example, polyethylene glycol, cocoa butter, lanolin, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, witezbazole (registered trademark: Dynamite Nobel) and the like. Accelerators can be added and used.
  • ointments such as pastes, creams and gels, commonly used bases, stabilizers, wetting agents, preservatives, etc. are added as necessary, and mixed and formulated according to the usual methods. Is done.
  • As the base for example, white petrolatum, paraffin, glycerin, cellulose derivatives, polyethylene glycol, silicon, bentonite and the like can be used.
  • a preservative methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate and the like can be used.
  • the above-mentioned ointment, cream, gel, base or the like may be applied to a usual support in a usual manner.
  • a woven or nonwoven fabric made of cotton, cloth, or synthetic fiber, an ointment film of vinyl chloride, polyethylene, polyurethane, or the like, or a foam sheet is suitable.
  • carriers include, for example, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, and gay acid.
  • excipients such as simple syrup, dextrose, damp
  • the tablet can be a tablet coated with a usual coating, if necessary, for example, a sugar-coated tablet, a gelatin-coated tablet, an enteric-coated tablet, a film-coated tablet, a double tablet, a multilayer tablet and the like.
  • Capsules are prepared by mixing with various carriers exemplified above, and filling them in hard gelatin capsules, soft capsules, or the like.
  • carriers include, for example, excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, talc, and binders such as gum arabic, powdered tragacanth, gelatin, and ethanol.
  • disintegrating agents such as laminaran and agar.
  • Liquid preparations may be aqueous or oily suspensions, solutions, syrups and elixirs, which are prepared according to the usual methods using ordinary additives.
  • the amount of the compound of the present invention to be contained in the above-mentioned preparation varies depending on the dosage form, administration route, administration schedule and the like and cannot be specified unconditionally, and is appropriately selected from a wide range, but is usually 1 to 70 in the preparation. It is good to be about weight%.
  • the administration method of the above preparation is not particularly limited, and may be enteral administration, oral administration, rectal administration, oral administration depending on the form of the preparation, the age of the subject of administration such as a patient, gender and other conditions, the degree of symptoms, etc.
  • the administration method such as transdermal administration is appropriately determined. For example, tablets, pills, solutions, suspensions, emulsions, granules and capsules are administered orally, and suppositories are administered rectally.
  • a normal replenisher such as glucose or amino acid
  • Ointments are applied to the skin, oral mucosa and the like.
  • the dosage and administration interval of the low-molecular-weight LPS of the present invention are naturally determined individually under the strict control of the attending physician in consideration of the age, symptoms, weight, and administration effect of the administration subject. (60 kg), 1 / zg to 100 mg for oral administration, 100 ng to 10 mg for intravenous administration, 100 ng to lmg power for dermal administration, This is a rough guide.
  • These preparations can be administered once a day or divided into 2 to 4 times a day.
  • Test example 1 The low molecular weight LPS of the present invention will be described in more detail.
  • Test example 1 The low molecular weight LPS of the present invention will be described in more detail.
  • LPS Purified LPS containing high-molecular-weight LPS (hereinafter, also simply referred to as “LPS”) was prepared by the same method as in Reference Example 1, and low-molecular-weight LPS of the present invention was prepared by the same method as in Example 1.
  • Each of low molecular weight LPS and LPS was dissolved in distilled water to prepare a solution having a concentration of 2 mgZm1, and 10 g of the solution was transferred to a 1.5 ml plastic tube.
  • 1 of 0.5M Tris-HCl ( PHDS 6.8) and SDS-treated solution 10i1 prepared by adding 22.5 1 distilled water were added to each of the above sample solutions and mixed well, then immersed in a boiling water bath for 5 minutes, and immediately thereafter, ice water It was immersed in and quenched.
  • the content of hexosamine was determined by the Elson-Morgan method (edited by The Biochemical Society of Japan, “Biochemical Experiment Course”, Vol. 4, pp. 377-379, 1st edition, Tokyo Chemical Dojin Press, 1976 ) was determined as follows. LPS was dissolved in distilled water to prepare a solution having a concentration of 2 mg / ml. 100/1 was weighed into a Spitz with screw cap (manufactured by Iduki Glass Co., Ltd.). 1 was added, the mixture was heated at 110 and heated for 16 hours, and then the pH was adjusted to 7 by adding about 200 ⁇ 1 of 4N NaOH.
  • Reagent A A mixture of 751 acetylacetone and 2.5 ml of 1.25N sodium carbonate.
  • Reagent B l. A mixture of 6 g of p-dimethylpenzaldehyde, 30 ml of concentrated hydrochloric acid and 30 ml of 96% ethanol.
  • the KDO content was quantified by the diphenylamine method [Analytical Biochemistry, Vol. 58, No. 1, pp. 123-129, pp. 1974] as follows.
  • the absorbance at 0 nm was measured (the measured values are described as A420, A470, A630, and A650, respectively).
  • an aqueous solution of KDO ammonium salt manufactured by Sigma having a concentration of 0.5 mol (250 u ⁇ ) was used.
  • Limulus activity is a horseshoe crab blood cell extract created by Levin in 1968.
  • Limulus test a method for quantifying endotoxin using effluent and a chromogenic substrate (Ikuo Suzuki, “Development of Pharmaceuticals, Vol. 14, Pharmaceutical Quality Control and Test Methods”, 227-
  • the protein content was determined by the Lowry method [Journal of Biological Chemistry, Vol. 193, page 65, 1995].
  • FIG. 1 is an SDS-PAGE electrophoresis diagram, in which lane 1 is a protein and peptide molecular weight marker [94 kD, 67 kD, 43 kD, 30 kD, 20.lkD, 17.2 kD, 14.6 kD, 14.4 kD, 8.24 kD, 6.38 kD, 2.56 kD (Pharmacia)], lanes 2, 3 and 4 are LPS (20 g , 5 ug and 1.25 g), lanes 5, 6, 7 and 8 are low molecular weight LPS (20 g, 5 g, 1.25 2 and 0.3 1 g), and the vertical axis of the figure is And the molecular weight.
  • lane 1 is a protein and peptide molecular weight marker [94 kD, 67 kD, 43 kD, 30 kD, 20.lkD, 17.2 kD, 14.6 kD, 14.4 kD, 8.24 kD, 6.38 kD
  • the molecular weight of the low molecular weight LPS (calculated from lane 8) was calculated from the size marker in lane 1 and was 5 kD at the center value of the stained band, and the range of the stained band was 3 kD to 7 kD. Also, in Lane 5, no high-molecular-weight LPS was observed at all, unlike in Lane 2, although 20 g of low-molecular-weight LPS was electrophoresed.
  • the molecular weight of the low molecular weight LPS of the present invention was 5,000 ⁇ 2,000, indicating that the high molecular weight was completely removed.
  • the number of hexosamines in the low-molecular-weight LPS of the present invention was 2 / molecular weight of 5,000.
  • KDO contained in the low molecular weight LPS of the present invention was 2.4 Z molecular weight was 5,000
  • the low molecular weight LPS of the present invention had a limulus activity of 43 SEUZng, whereas the conventional LPS prepared by the same method as in Reference Example 1 had a limulus activity of 8.4 EUZng.
  • the protein inclusion of the low molecular weight LPS of the present invention was 0.68% or less.
  • the nucleic acid content of the low molecular weight LPS of the present invention was 0.50% or less.
  • the purity of the low molecular weight LPS of the present invention was 98% or more.
  • the toxicity of low-molecular-weight 3 ⁇ 4L PS prepared by the same method as in Example 1 and LPS prepared by the same method as in Reference Example 1 was evaluated for 7-week-old C 3 HZHe mice (purchased from Japan Rivers Japan). Tested using Each sample was dissolved in saline and administered intravenously at a ratio of 5.0, 10, 20, and 4 OmgZkg per mouse to a group of four mice per group (except for 4 OmgZkg). Administration is low molecular weight LPS only). The mice were observed for life and death for 72 hours after administration.
  • the same low molecular weight LPS as in Test Example 2 was administered intravenously at a rate of 40, 80, and 16 OmgZ kg per animal, and LPS was administered per animal 5.0, or The test was performed in the same manner as in Test Example 2 except that the dose was administered intravenously at a rate of 1 Omg / kg.
  • the TNF activity in each serum thus obtained was measured by a method based on toxicity to L929 cells. That is, L 929 cells were prepared in a MEM medium containing 5% fetal calf serum to a concentration of 8 ⁇ 10 4 cells 1001, and 1001 was added to each well of a 96-well flat bottom plate. , 37 for 2 hours, and cultured in 5% C0 2 presence. Then add actinomycin D to 11 / ml and add 50 1 serum sample or positive control human TNF- ⁇ (manufactured by Asahi Kasei Co., Ltd.) serially diluted in MEM medium. Cultured.
  • the cultured cells were inoculated into a 10-liter tabletop fermenter (manufactured by Marubishi Bio-enge) containing 7 liters of L broth medium, aerated under the same conditions, and then collected. About 70 g of wet cells were collected and stored frozen.
  • the obtained crude LPS lyophilized product is dissolved in distilled water, filter-sterilized, buffer is added, and subjected to anion exchange chromatography (Pharmacia Co., Ltd., Q-Sepharose 'fast' flow).
  • the sample solution was passed through the column with a buffer solution containing 1 to 1 (pH 7.5) and 1 OmM NaC1, and then 20 to 40 OmM NaC11 OmM Tris-HC1 (pH 7
  • the limulus active fraction was eluted with 5).
  • the eluate was subjected to ultrafiltration under the same conditions as described above, desalted and concentrated, and lyophilized to obtain about 30 mg of purified LPS from about 70 g of wet cells.
  • FIG. 1 and FIG. 2 are SDS-PAGE diagrams of each LPS sample. BEST MODE FOR CARRYING OUT THE INVENTION
  • Solubilization buffer [3% sodium deoxycholate (manufactured by Wako Pure Chemical Industries, Ltd.), 0.2 M sodium chloride, 5 mM EDTA—purified LPS 10 Omg obtained in the same manner as in Reference Example 1 at a concentration of 5 mg / m 1 It consists of 2Na and 2OmM Tris-HCl, dissolved in H8.3], purified and gently layered 2Oml of PS solution on top of Sephacryl S-200 HR column (Pharmacia), elution buffer [0.25% sodium deoxycholate (manufactured by Wako Pure Chemical Industries, Ltd.), 0.2 M sodium chloride, 5 mM EDTA and 1 OmM tris-hydrochloric acid, pH 8.3] at a flow rate of 16 ml Time) eluted.
  • the obtained eluate was fractionated by a fraction collector (Advantech, SF 2 120), and the first 24 Om 1 (24 fractions) was collected. ) was discarded, and then fractionated up to 80 fractions with a 10 ml Z fraction.
  • the eluted fractions were treated with the stock solution or diluent using the phenol Z sulfuric acid method (Sakuzo Fukui, “Quantitative method for Tetsugen sugar 'second edition”, pp. 50-52, Gakkai Shuppan Center, 1990). Was determined and the elution state was examined.
  • the cultured cells were inoculated into a 10-liter tabletop fermenter (manufactured by Marubishi Bio-engagement Co., Ltd.) containing 7 liters of magnesium-malachite togulin medium, aerated under the same conditions, and then collected. And collect about 50 g of wet cells and freeze them.
  • the crude LPS lyophilizate obtained by ultra-concentration under nitrogen gas at 2 atm with a membrane is dissolved in distilled water, filter-sterilized, and added to the mouth liquid.
  • Anion exchange chromatography (Pharmacia) The sample solution was passed through the column with a buffer containing 10 mM Tris-HC1 (pH 7.5) and 1 01111 ⁇ & ⁇ 1. Limulus active fraction with ⁇ 40 OmM NaClZl OmM Tris-HC1 (pH 7.5) was eluted.
  • the eluate was subjected to ultrafiltration under the same conditions as described above, desalted and concentrated, and lyophilized to obtain about 21 Omg of purified LPS from about 50 g of wet cells.
  • FIG. 2 shows an SDS-PAGE diagram of low-molecular-weight LPS purified from Salmonella's Minnesota strain.
  • lane 1 shows the protein and peptide markers [94 kD, 67 kD, 43 kD, 30 kD, 20.lkD, 17.2 kD, 14.6 kD, 14.4 kD D, 8.24 kD, 6.38 kD and 2.56 kD (Pharmacia)]
  • lanes 2, 3 and 4 were gels in the presence of sodium deoxycholate.
  • lanes 5, 6, 7 and 8 have low molecular weight LPS (20 g.5 g. 1.25 ⁇ 0.31 ⁇ g) ).
  • the following is a formulation example of a preparation containing the low molecular weight LPS of the present invention.
  • the low-molecular-weight LPS amount in Formulation Examples 1 to 4 is the low-molecular-weight LPS-converted amount by the Limulus test.
  • a liquid preparation was prepared at the above mixing ratio according to a conventional method.
  • a descendant was prepared according to a conventional method at the above mixing ratio.

Abstract

Nouveau lipopolysaccharide de faible poids moléculaire présentant une sécurité élevée qui lui permet d'être utilisé en tant que médicament, et une forte activité biologique. Il est obtenu à partir de cellules microbiennes et possède des propriétés physico-chimiques et biologiques telles que (a) il possède un poids moléculaire de 5.000 ± 2.000 tel que déterminé par électrophorèse sur gel SDS à l'aide de marqueurs protéiques et qu'il est pratiquement exempt d'autres zones tachées, (b) il possède une teneur en hexosamine de 1 à 3 par poids moléculaire de 5.000 telle que déterminée par le procédé Elson-Morgan, (c) il possède une teneur en 2-céto-3-désoxyoctonate de 1 à 3 par poids moléculaire de 5.000 telle que déterminée par le procédé à la diphénylamine et (d) il possède une activité limulus d'au moins 10 EU/ng.
PCT/JP1996/000135 1995-01-27 1996-01-25 Lipopolysaccharide de faible poids moleculaire WO1996023002A1 (fr)

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Cited By (7)

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WO2015050179A1 (fr) * 2013-10-03 2015-04-09 日東電工株式会社 Composition de vaccin par voie muqueuse
WO2015050180A1 (fr) * 2013-10-03 2015-04-09 日東電工株式会社 Composition de vaccin par voie muqueuse
WO2015050181A1 (fr) * 2013-10-03 2015-04-09 日東電工株式会社 Composition de vaccin injectable
WO2015050178A1 (fr) * 2013-10-03 2015-04-09 日東電工株式会社 Composition de vaccin muqueux nasal
WO2021060127A1 (fr) * 2019-09-24 2021-04-01 有限会社バイオメディカルリサーチグループ Procédé de production de lipopolysaccharides
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JP6539400B1 (ja) * 2018-10-24 2019-07-03 株式会社アンチエイジング・プロ Ｌｐｓ高含有組成物の製造方法
JP7365007B2 (ja) * 2020-04-27 2023-10-19 株式会社島津製作所 血清型判別方法
JP2024042229A (ja) * 2022-09-15 2024-03-28 Ｅｎｅｏｓ株式会社 リポ多糖を含む組成物

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WO2000057719A1 (fr) * 1999-03-26 2000-10-05 Taiho Pharmaceutical Company, Ltd. Additifs pour aliments de crustacees ou de poissons et aliments
CN105555308A (zh) * 2013-10-03 2016-05-04 日东电工株式会社 粘膜疫苗组合物
WO2015050181A1 (fr) * 2013-10-03 2015-04-09 日東電工株式会社 Composition de vaccin injectable
US10071155B2 (en) 2013-10-03 2018-09-11 Nitto Denko Corporation Nasal mucosal vaccine composition
US10092642B2 (en) 2013-10-03 2018-10-09 Nitto Denko Corporation Mucosal vaccine composition
JP2015091794A (ja) * 2013-10-03 2015-05-14 日東電工株式会社 注射ワクチン組成物
CN105530954A (zh) * 2013-10-03 2016-04-27 日东电工株式会社 鼻粘膜疫苗组合物
CN105530958A (zh) * 2013-10-03 2016-04-27 日东电工株式会社 粘膜疫苗组合物
WO2015050179A1 (fr) * 2013-10-03 2015-04-09 日東電工株式会社 Composition de vaccin par voie muqueuse
US9962439B2 (en) 2013-10-03 2018-05-08 Nitto Denko Corporation Injectable vaccine composition
WO2015050180A1 (fr) * 2013-10-03 2015-04-09 日東電工株式会社 Composition de vaccin par voie muqueuse
WO2015050178A1 (fr) * 2013-10-03 2015-04-09 日東電工株式会社 Composition de vaccin muqueux nasal
US10391167B2 (en) 2013-10-03 2019-08-27 Nitto Denko Corporation Mucosal vaccine composition
WO2021060127A1 (fr) * 2019-09-24 2021-04-01 有限会社バイオメディカルリサーチグループ Procédé de production de lipopolysaccharides
JPWO2021060127A1 (fr) * 2019-09-24 2021-04-01
CN114450312A (zh) * 2019-09-24 2022-05-06 生物医学研究集团有限公司 脂多糖的制造方法
EP4036245A4 (fr) * 2019-09-24 2024-01-24 Biomedical Res Group Inc Procédé de production de lipopolysaccharides
CN114450312B (zh) * 2019-09-24 2024-02-23 生物医学研究集团有限公司 脂多糖的制造方法
US20210353732A1 (en) * 2020-05-14 2021-11-18 Zivo Bioscience, Inc. Use of tlr4 modulator in the treatment of coccidiosis

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