WO1996023002A1

WO1996023002A1 - Low-molecular-weight lipopolysaccharide

Info

Publication number: WO1996023002A1
Application number: PCT/JP1996/000135
Authority: WO
Inventors: Den-Ichi Mizuno; Gen-Ichiro Soma; Takashi Nishizawa
Original assignee: Taiho Pharmaceutical Co., Ltd.
Priority date: 1995-01-27
Filing date: 1996-01-25
Publication date: 1996-08-01
Also published as: JPH08198902A; JP4043533B2

Abstract

A nobel low-molecular-weight lipopolysaccharide having such a high safety as to permit the use thereof as a medicine and a high biological activity. It is obtained from microbial cells and has physicochemical and biological properties such that (a) it has a molecular weight of 5,000 ± 2,000 as determined by SDS-PAGE using protein markers and is substantially free from other stained zones, (b) it has a hexosamine content of 1 to 3 per molecular weight of 5,000 as determined by the Elson-Morgan method, (c) it has a 2-keto-3-deoxyoctonate content of 1 to 3 per molecular weight of 5,000 as determined by the diphenylamine method, and (d) it has a limulus activity of at least 10 EUng.

Description

明

Low molecular S lipopolysaccharide

Technical field

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. Background art

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. 27, Bacterial Cell Wall, page 18, Elsevea, 1989] 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 β-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. In general, heptose and 2-keto-3-doxyctonate (KDO) Is a common component of many R cores, and it is also known that LPS lacks one or both depending on the bacterial species that binds to lipid A via KDO. JM Ghuysen and R. Hackenbeck, "New Comprehensive Biocheraistry", Volume 27, Bacterial "Cell" All B acterial Cell Wall), pp. 294-295, Elsevea, 1989].

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].

Although 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).

Conventionally, a wide variety of LPS have been reported, even LPS extracted generally by any method, 1 0 ⁶ have a very large molecular weight of ~ 1 0 ⁷ known (Honma Herikuda Other editions, "Bacterial endotoxin", page 211, Kodansha, 1973 ). Later, LPS with a relatively small molecular weight was also reported, and the molecular weight was 8,00 0 ± 1,000 or 5, 000 ± 2, determined by wheat-derived SDS-PAGE (SDS-polyacrylamide gel electrophoresis). , 0000, phosphorus number 1-4 Z molecular weight 8,000, hexosamine number 6 ± 2 molecular weight 8,000, fatty acid number 6 ± 2Z molecular weight 8,000, KDO number 5 ± 1 / LPS with a molecular weight of 8,000 (Japanese Patent Application Laid-Open Nos. Hei 4-149,245, Hei 4-149,243, Hei 4-149,242, Hei 4 Japanese Patent Application Laid-Open No. 49224/1994, Japanese Patent Application Laid-Open No. 4-49224, Japanese Patent Application Laid-Open No. 4-49240, Japanese Patent Application Laid-Open No. 5-15557778, Japanese Patent Application Laid-Open No. No. 4,093,737), Chlorella-derived SDS-PAGE molecular weight 40,000 to 90,000, phosphorus number 4 ± 1 molecular weight 10,000, hexosamine number 7 ± 1Z molecular weight 10,000, fatty acid LPS with number 6 ± 1Z molecular weight 10,000, KDO number 2 ± 1 molecular weight 10,000 (JP-A-4-149254, JP-A-4-149) JP-A-243, JP-A-4-49242, JP-A-4-14921, JP-A-4-1492, JP-A-4-149240 Japanese Patent Application Laid-Open No. H05-157579, Japanese Patent Application Laid-Open No. 6-409937), a molecular weight of 30,000 ± 5,000, obtained by E. coli-derived SDS-PAGE. LPS with phosphorus number 1 2 molecular weight 30,000, hexosamine number 4 5 ± 6Z molecular weight 30,000, fatty acid number 18 / molecular weight 30,000, KDO number 5 ± 1 molecular weight 30,000 (Maihei Hei 4-1-49245) Japanese Unexamined Patent Publication Nos. Hei 4-449243, Japanese Patent Laid-Open No. Hei 4-92424, Japanese Patent Laid-open No. Hei 4-49241, Japanese Laid-open Patent No. Heisei 4-92424, Japanese Unexamined Patent Publication No. No. 4,924,040), SDS derived from B. pertussis—molecular weight by PAGE 6, 000 ± 1, 0000 or 9,000 ± 1, 0000, phosphorus number 5 MW LPS with 8,000, hexosamine number 1 6 ± 2 molecular weight 8,000, fatty acid number 5Z molecular weight 8,000, KDO number 2 ± 1 / molecular weight 8,000 (Japanese 4 9 2 No. 45, Japanese Unexamined Patent Application Publication No. 4-49243, Japanese Unexamined Patent Application Publication No. 4-49224, Japanese Unexamined Patent Application Publication No. 4-49241, Japanese Unexamined Patent Application Publication No. , Japanese Patent Application Laid-Open No. 419/240), 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. Bacterial SDS-Molecular weight by PAGE 5,000 ± 1,000, Phosphorus 2 ± 1 Molecular weight 5,000 LPS with 0, hexosamine number 9 ± 1 molecular weight 5, 000, KDO number 2 ± 1 molecular weight 5, 000 (JP-A-6-40993, JP-A-5-157) No. 78, Japanese Patent Application Laid-Open No. 6-65092, Japanese Patent Application Laid-Open No. 419948/1994, Japanese Patent Application Laid-Open No. 6-97545), by SDS-PAGE derived from Enterobacter bacteria. Molecular weight 6,500 ± 2,500, phosphorus number 1-2Z molecular weight 5,500, hexosamine number 7 ± 1, no molecular weight 5,000, KDO number 1-2 molecular weight 5,000 LPS (Japanese Unexamined Patent Publication No. Hei 6-40997, Japanese Unexamined Patent Publication No. Hei 5-155778, Japanese Unexamined Patent Publication No. Hei 6-65092, Japanese Unexamined Patent Publication No. Hei 4-949948) Gazette, Japanese Patent Application Laid-Open No. 6-97045), SDS-PAGE derived from Pantoea genus bacteria, molecular weight of 6,500 ± 2,500, phosphorus number 2 ± 1, molecular weight of 5,000, LPS having a hexosamine number of 5 ± 1 Z molecular weight of 5,000 and a KDO number of 2 ± 1Z molecular weight of 5,000 (JP-A-6-40993, JP-A-6-650) No. 92, Japanese Patent Application Laid-Open No. 4-949641, Japanese Patent Application Laid-Open No. 6-90745), B. pertussis-derived SDS-PAGE with a molecular weight of 6,000 ± 1,000. 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. ), SDS-PAGE molecular weight of Aeromonas' Hydrophyca sp. 5,000 ± 1,500, Phosphorus number 2 ± 1 Molecular weight 5,000, Hexosamine number 9 ± 1 Molecular weight 5, 0 0 0, 00 number 0.

LPS of 8 ± 0.5 molecular weight of 5,000 (Japanese Patent Laid-Open No. 6-141489), SDS-PAGE derived from Pantoea bacteria, molecular weight of 5,000, phosphorus number 2 / Molecular weight 5, 000, hexosamine number 2, molecular weight 5, 000, KDO number 5, molecular weight 5, 000 [Biotherapy (B10THERAPY), Volume 6, Issue 3, page 357 , 1 9

9 2 years].

As described above, 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.

Regarding the use of LPS, 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), Japanese Patent Application Laid-Open No. 6-14. No. 1,849, JP), oral and percutaneous immune function promoter (JP-A-4-187640), analgesic (JP-A-6-40937), growth promoter ( Japanese Unexamined Patent Application Publication No. Hei. 5-155778, an anti-withdrawal agent (Japanese Unexamined Patent Publication No. Hei 6-65092) and the like have been proposed.

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).

On the other hand, a method for purifying LPS from the cell wall of bacteria has been described by a conventional phenol-water extraction method [O. Westpal, edited by Methods in Carbohydrate Chemistry] (Methods in Carbohydrate Chemistry). , Vol. 5, pp. 83, Academic 'Breath (Academic Press :), 196 5], Tric oleic acid extraction method [AM Staub, Ed., Methodology in Imno Logi-Ant * Methods in Immunology and Immunochemistry, Volume 1, Page 28, Academic Press, 1967, EDTA extraction method [Journal 'ob' bio] Logical 'Chemistry (Journal of Biological Chemistry), No. 243, pp. 6384, pp. 1968] is known, and LPS obtained in this way is sodium dexoxylate. Such as It has been reported that in the presence of a surfactant, it is further dissociated into subunits having a molecular weight of about 200,000 (Honma et al., "Bacterial endotoxin", pp. 229, Kodansha , 197 3). On the other hand, there has been no report on a method for obtaining only LPS of extremely low molecular weight of about S500, which does not contain LPS with a molecular weight of 200,000 or more. For example, Japanese Patent Application Laid-Open No. 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.

As described above, conventionally reported low-molecular-weight LPS is a mixture containing high-molecular-weight LPS.For example, when used clinically as a drug component such as an immune function activator, from the viewpoint of safety, Or they were not always satisfactory in terms of medicinal properties.

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.

That is, the present invention is obtained from microbial cells and has the following physicochemical properties a) to c)

a) The molecular weight measured by SDS-PAGE using a protein marker is 5,000 ± 2,000, and there is virtually no other staining band.

b) The hexosamine content measured by the Elson-Morgan method is 1-3, and the molecular weight is 5,000.

c) The content of 2-keto 3-dexoxyctonate measured by the diphenylamine method is 1-3, and the molecular weight is 5,000. A low molecular weight lipopolysaccharide having the following formula:

Further, the present invention provides the following physicochemical and biological properties obtained from microbial cells:

b) The content of hexosamine measured by the Elson-Morgan method is 1 to 3 and the Z molecular weight is 5,000.

c) The content of 2-keto 3-dexoxytonate measured by the diphenylamine method is 1 to 3 and the molecular weight is 5,000.

d) Limulus activity of at least 10 EUZng

e) Protein inclusion is less than 1% (weight)

f) The nucleic acid content is 1% (weight) or less

And a low molecular weight ribopolysaccharide having the formula:

Also, in the present invention, the microorganism is a gram-negative microorganism, and 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.

Therefore, the present invention provides a pharmaceutical composition comprising the low-molecular-weight LPS of the present invention and a pharmaceutical carrier.

Further, 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.

According to the present invention, 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. In this case, the surfactant is preferably deoxycholic acid.

Next, the present invention will be described in detail.

In the following description, percentages are by weight unless otherwise specified.

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. For example, the thermal phenol method [O-I 'Westphal, Ed., Methods in Carbohydrate Chemistry, Vol. 5, p. 83, Ademick Press Academic Press), 1965], and purified by anion exchange resin. That is, 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. One of the reasons is that 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 novel low-molecular-weight LPS of the present invention produced by the above-described method is, as shown in Test Example 1 described below,

b) The hexosamine content measured by the Elson-Morgan method is 1 to 3 and the Z molecular weight is 5,000.

c) The content of 2-keto 3-dexoxyctonate measured by the diphenylamine method is 1 to 3 and the Z molecular weight is 5,000.

d) Limulus activity of at least 10 EU / ng

e) Protein content is 1% or less

f) Nucleic acid content is 1% or less

Physicochemical and biological properties, and has a purity of at least 98%. However, depending on the intended use, the degree of purification can be reduced (eg, 90%). 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. Also, when the number of hexosamines and the number of KDOs are known when the molecular weight of the low molecular weight LPS is other than 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. As 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.

Each of the above compositions is formulated by a formulation method generally known in the art. 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. In this case, 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. By adding these carriers, injections for subcutaneous, intramuscular, and intravenous injections can be produced by a conventional method.

When molded into suppositories, 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. When preparing 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. As a preservative, methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate and the like can be used.

When a patch is produced, the above-mentioned ointment, cream, gel, base or the like may be applied to a usual support in a usual manner. As the support, 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.

When transforming into the form of solid oral preparations such as tablets, powders, and granules, carriers include, for example, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, and gay acid. , Methyl cellulose, glycerin, sodium alginate, gum arabic, etc., excipients such as simple syrup, dextrose, dampening solution, gelatin solution, polyvinyl alcohol, polyvinyl ether, borobinyl pyrrolidone, carboxymethyl cellulose, shellac, methyl cellulose, ethyl cellulose Binders such as water, ethanol, potassium phosphate, etc., dried starch, sodium alginate, powdered agar, powdered laminaran, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, Disintegrators such as sodium rill sulfate, monoglyceride stearate, starch, lactose, decay inhibitors such as sucrose, stearic acid, cocoa butter, hydrogenated oil, quaternary ammonium salt, absorption of sodium lauryl sulfate, etc. Use accelerators, humectants such as glycerin and starch, adsorbents such as starch, lactose, kaolin, bentonite, colloidal gay acid, lubricants such as purified talc, stearate, borate powder, polyethylene glycol, etc. it can. Further, 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. When formed into pill form, 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. And 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. In the case of an injection, it is intravenously administered alone or mixed with a normal replenisher such as glucose or amino acid, and further administered alone as needed, intraarterially, intramuscularly, intradermally, subcutaneously or intraperitoneally. 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. For animals, cattle, horses, and other large animals use 60/100 of the above amount as a guideline per kg of body weight, and for medium-sized and small animals such as pigs, dogs, and cats, twice the amount Can be administered as a guideline for the amount per kg of body weight, and for birds such as chickens, twice the amount can be used as a guideline for the amount per kg of body weight. These preparations can be administered once a day or divided into 2 to 4 times a day.

Next, test examples will be shown, and the low molecular weight LPS of the present invention will be described in more detail. Test example 1

This test was conducted to determine the physicochemical and biological properties of the low molecular weight LPS of the present invention. " 1) Sample preparation

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.

2) Test method

① Measurement of molecular weight

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. Separately, 180% of 10% (wZv) SDS, 5% of 451; 3-mercaptoethanol, 901 of CBB dye solution, 12.5. 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.

Dissolve 10 ml of 10% (w / v) SDS, 17.9 g of Tricine and 3.03 g of Tris in 1 liter of distilled water, and transfer the electrophoresis buffer to a slab gel electrophoresis tank. (Made by Marisol). Fix the 20% polyacrylamide gel in the electrophoresis tank, put the sample in the sample groove, set the voltage to 50 V for 1 hour, then fix the voltage to 150 V, and continue electrophoresis until the dye elutes from the gel. I did. After the electrophoresis, silver staining was performed at room temperature with a silver staining kit 161-1024 (manufactured by Bio-Rad) to confirm the behavior.

②Quantification of hexosamine content

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. Weigh out 10 Ο χί 1 and put it into another screw cap with screw cap, add 200 1 reagent 、, heat at 105 for 1.5 hours, and run And cooled. Then, 1001 was fractionated, added with 670% of 96% ethanol, further added with 671 of reagent B, left at room temperature for 1 hour, and measured for the absorbance at 5335 nm. It was measured. As a standard sample for preparing a calibration curve, N-acetyldarcosamine (manufactured by Wako Pure Chemical Industries, Ltd.) of 0 to 800 g / m1 was used.

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.

③ Quantification of KDO content

The KDO content was quantified by the diphenylamine method [Analytical Biochemistry, Vol. 58, No. 1, pp. 123-129, pp. 1974] as follows.

50 Omg diphenylamine (Wako Pure Chemical), 5 ml ethanol (Wako Pure Chemical), 45 ml glacial acetic acid (Wako Pure Chemical), 50 ml concentrated hydrochloric acid (Wako Pure) (Produced by Yakuhin) was mixed to prepare a KDO detection reagent. The 500〃1 was mixed with an aqueous solution of 250 // 1 containing each sample at a concentration of 0.5 Omg / m1 and heated in a boiling water bath at 100 ° C for 30 minutes, After cooling in constant temperature water (24 to 25 ° C) for 30 minutes, the spectrophotometer (Hitachi Ltd. model U21010) was used to cool the sample to 420, 470, 63, and 65. The absorbance at 0 nm was measured (the measured values are described as A420, A470, A630, and A650, respectively). As a reference sample, an aqueous solution of KDO ammonium salt (manufactured by Sigma) having a concentration of 0.5 mol (250 u \) was used.

From four measurements of the test sample and the standard sample, determine the S value by equation (1), S value of the test body sample and standard sample were as S, and S _s, respectively. Next, the number of moles X of KD0 was calculated by equation (2).

S = A 4 2 0—A 4 7 0 + A 6 3 0—A 6 5 0 (1)

X = (0. 5 x Ss x LPS 1 mole of molecular weight) / (0. 5 x S, x 1 0 6) - ... (2)

④Limulus activity measurement

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-

243 page, Hirokawa Damage Store, 1990), and this limulus test is known as an LPS detection method. The measurement was carried out using a toxic color system (manufactured by Seikagaku Corporation) using 345 pg / EU of E. coli 0111: B4 as a standard.

⑤Protein content

The protein content was determined by the Lowry method [Journal of Biological Chemistry, Vol. 193, page 65, 1995].

⑥Nucleic acid content

The nucleic acid content was measured at the OD (260 nm-300 nm) (1 OD = 40 // g).

⑦Purity

Purity (%) was calculated by the following equation.

Purity-[(dry yield-(protein content + nucleic acid content)} dry yield] X 100

3) Test results

① Molecular weight

The results of the molecular weight measurement are as shown in FIG. 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.

In general, when a substance having a sugar chain is electrophoresed, when the amount of sample per lane is excessive, the staining band becomes wide, and the apparent molecular weight range becomes wide. In the SDS-PAGE of Figure 1, lanes 5 to 8 show changes in the amount of low molecular weight LPS in the same sample. The width of the stained band increased as the amount of electrophoresis of the sample increased. Therefore, for the purpose of examining the accurate molecular weight, an amount of about 1 "g is appropriate, and Lane 8 is equivalent. Lanes 2 and 5 are used to confirm the presence of high molecular weight LPS. A large amount of sample was electrophoresed.

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.

From the above results, 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.

②Hexosamine content

The number of hexosamines in the low-molecular-weight LPS of the present invention was 2 / molecular weight of 5,000.

③ KDO content

KDO contained in the low molecular weight LPS of the present invention was 2.4 Z molecular weight was 5,000

④ Limulus activity

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.

⑤Protein content

The protein inclusion of the low molecular weight LPS of the present invention was 0.68% or less.

⑥Nucleic acid content

The nucleic acid content of the low molecular weight LPS of the present invention was 0.50% or less.

⑦Purity

The purity of the low molecular weight LPS of the present invention was 98% or more.

Although tested by changing the microorganism and the production method, _c test example 2 is almost the same results were obtained

This test was performed to determine the acute toxicity of the low molecular weight LPS of the present invention. (1) Sample preparation and test method

The toxicity of low-molecular-weight ¾L 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.

(2) Test results

The results of this test are shown in Table 1. As is evident from Table 1, the case of intravenous administration, deaths in mice was not observed even at a low molecular weight any dose in LPS of the invention, LD _5. Was more than 4 OmgZkg, but all died at 10 and 2 OmgZkg doses in LPS and LDs. Was 7.l mgZkg. The test was performed by changing the type of microorganism and the method for producing low-molecular-weight LPS, and almost the same results were obtained.

table 1

Test example 3

This test was performed to examine acute toxicity when the low molecular weight LPS of the present invention was administered in a larger amount than in Test Example 2.

(1) Sample preparation and test method

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.

(2) Test results

The results of this test are shown in Table 2. As is evident from Table 2, 25% died at the LPS 5. Omg / kg dose and 75% at the 10 mg / kg dose. In contrast, no mortality at a dose of 4 Omg Roh kg in the low molecular weight LPS, 8 0 and 1 6 In a dose of OmgZk g, 1 0 0% test example died _c before the test example 2 Toko From the results of 3, LD ₅ . Table 3 shows the calculated values. As is evident from Table 3, LD _{5 of} low molecular weight LPS. The value of iv was approximately 8 times higher than that of LPS by intravenous administration.

These results Ri your show that differences in the molecular weight of LPS affects the toxicity, low molecular weight LPS is _c Table 2 that extremely low toxicity as compared with the conventional LPS was found

Table 3

Test example 4

This test was performed to confirm the TNF-producing effect of the low-molecular-weight LPS of the present invention. In the tail vein of 7-week-old male C 3HZHe mice (purchased from Charles River Japan, Inc.) of 3 mice in each group, 0.1, 1.0, or 10 g per animal as in Example 1 0.2 ml of physiological saline containing the low-molecular-weight LPS produced by the method described in 1) or the LPS obtained by the same method as in Reference Example 1 was injected, and one hour later, blood was collected and serum was separated by a conventional method.

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-α (manufactured by Asahi Kasei Co., Ltd.) serially diluted in MEM medium. Cultured. After removing the medium with an aspirator, the cells were washed with 37 PBS to completely remove dead cells, and a 1% methyl alcohol solution containing 0.1% crystal violet was added to stain live cells. The degree of staining was measured using the absorbance at OD (590 nm) as an index, and the TNF activity was calculated based on the relationship between the dilution ratio of TNF-α used as a positive control and the absorbance. The results are as shown in Table 4. In Table 4, the TNF activity is the average of three animals in each group. From these results, it has been clarified that the TNF production effect of the low-molecular-weight LPS of the present invention exceeds that of the conventional LPS obtained by the method of Reference Example 1. Table 4

Reference example 1

Trypton (Difco) 10 g, Yeast extract (Difco) 5 g, Na Add 10 g of C1 (manufactured by Wako Pure Chemical Industries, Ltd., special grade) to 1 liter of distilled water, adjust the pH to 7.5 with NaOH, sterilize with autoclave, and separately sterilize glucose (Wako Pure Chemical Industries, Ltd.). Medium (supplemented by Kogyo Co., Ltd.) at a rate of 0.1% (hereinafter referred to as L-grass medium). Isolate and inoculate a single colony from the Pantoea agglomerans stock strain stored at, and incubate overnight at 35 ° C with shaking. A 3 liter Sakaguchi flask containing a broth medium was inoculated and cultured in the same manner.

Furthermore, 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.

About 70 g of the cryopreserved cells were suspended in 500 ml of distilled water, 500 ml of 90% hot phenol was added, and the mixture was stirred for 20 to 65 to 70 minutes, cooled, and cooled. After centrifugation at 100,000 G and 4 ° C for 20 minutes, the aqueous layer was collected. The same operation as above was repeated once more for the phenol layer, and the collected two eyebrows were combined. The phenol was removed by dialysis overnight, and the dialysis solution was subjected to an ultrafiltration apparatus (Adventech Toyo). It was ultra-concentrated using a UK-200) ultra-thin layer under nitrogen gas at 2 atm with a molecular weight of 200,000 cut-off membrane.

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. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 and FIG. 2 are SDS-PAGE diagrams of each LPS sample. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in more detail and specifically with reference to Examples, but the present invention is not limited to the following Examples.

Example 1

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.

While controlling the flow rate using a peristaltic pump PI (Pharmacia), 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. From the results of the elution states obtained, of the fractions (fractions 30 to 60) in which LPS is expected to be present, SDS-PAGE was performed using 0.5 ml of each fraction of fractions 37 to 55, and LPS _c As a result of examining the fraction pattern, fraction 45-55, a low molecular weight only (molecular weight of about 5 k D) LPS was observed, fractions 37- 44 were observed LPS of both high and low molecular weight Therefore, the low molecular weight LPS fraction of fractions 45-55 was further purified as follows.

Each fraction was mixed, freeze-dried, suspended in ethanol, centrifuged to remove ethanol-soluble dexocholic acid, and the low-molecular-weight LPS was recovered as an insoluble fraction.c Low-molecular-weight LPS fraction ethanol The treatment was repeated two more times to remove dexcholate, then resuspended in 70% ethanol, and centrifuged to remove loose mouth liquid components. This operation was repeated three more times, and the low-molecular-weight LPS was recovered in the insoluble fraction, lyophilized, and purified to obtain about 2 Omg of low-molecular-weight LPS.

Example 2

5 g of tryptone (manufactured by Difco), 1.6 g of potassium dihydrogen phosphate and 8 g of sodium chloride were dissolved in 1,000 mL of purified water, and sterilized at 121 ° C for 15 minutes (hereinafter referred to as “this”). Is called a basal medium). 10 ml of a 40% magnesium chloride solution and 3 ml of a 0.4% malachite togulin solution were aseptically added to 100 ml of the basal medium, and this was used as a magnesium-malachite toglin medium.

Separate and inoculate a single colony of Salmonella minnesota preserved bacteria into a 500 ml Sakaguchi flask containing 100 ml of magnesium-malachite togulin medium. The culture was shake-cultured at ° C overnight, and the whole amount was inoculated into a 3-liter Saka lofrasco containing 1,000 ml of a magnesium-malachite togulin medium, and cultured under the same conditions.

Furthermore, 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.

About 50 g of the cryopreserved cells were suspended in 500 ml of distilled water, 500 ml of 90% hot phenol was added, and the mixture was stirred at 65 to 70 for 20 minutes and cooled. Then, the mixture was centrifuged at 10, 00 G, 4 for 20 minutes, and the eyebrows were collected. The phenol layer was treated once more in the same manner as above. The two aqueous layers are combined, dialyzed overnight to remove the phenol, and the dialysis solution is subjected to an ultrafiltration apparatus (Advantok Toyo Co., Ltd., UK—200,000). 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 ^ & 〇1. 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.

80 mg of this purified LPS was dissolved in a solubilization buffer containing 3% sodium deoxycholate in the same manner as in Example 1, and developed on a Cefacryl S_20 OHR column (Pharmacia), and only low molecular weight LPS was used. Was collected, lyophilized, suspended in ethanol, the buffer components such as deoxycholic acid were removed by centrifugation, and lyophilized to obtain about 5 mg of low molecular weight LPS.

The molecular weight, KDO number and hexosamine number of this low molecular weight LPS were measured in the same manner as in Test Example 1 above. It was 6,000.

For reference, FIG. 2 shows an SDS-PAGE diagram of low-molecular-weight LPS purified from Salmonella's Minnesota strain. In the figure, 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. / g, 5 ^ 8 ぉ and 1.25 g), 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.

Formulation example 1 (tablet)

Low molecular weight LPS obtained in Example 1 0.04 g

6% HPC lactose 1 78 g

8 g of talc stearate

No, '14 g of starch

The above were mixed and tableted according to a conventional method to prepare a 0.5 g tablet containing low molecular weight LPS.

Formulation Example 2 (Liquid for internal use) Low molecular weight LPS obtained in Example 1 1 mg

Purified water 100 ml

A liquid preparation was prepared at the above mixing ratio according to a conventional method.

Formulation Example 3 (Paster)

Low molecular weight LPS obtained in Example 1 0.1

80 g of purified lanolin

Yellow Serine qs

1 0 0 0 g

A descendant was prepared according to a conventional method at the above mixing ratio.

Prescription example 4 (injection)

0.5 mg of low molecular weight LPS obtained in Example 2

Appropriate amount of distilled water for injection

1 0 0 0m l

Injections were prepared at the above mixing ratios according to a conventional method. Industrial applicability

As described in detail above, according to the present invention, highly safe and biologically active high L and low molecular weight LPS that can be used as pharmaceuticals and the like are provided.

Claims

The scope of the claims

1. Obtained from microbial cells and have the following physicochemical properties a) to c)

b) The content of hexosamine measured by the Elson-Morgan method is 1-3 per molecule / molecular weight of 5,000.

c) The content of 2-keto-3-dexoxytonate measured by the diphenylamine method is 1 to 3 / molecular weight of 50,000.

A low molecular weight lipopolysaccharide having:

2. The following physicochemical and biological properties of a) to f) obtained from microbial cells: 0, with virtually no other staining bands

c) The content of 1-3 keto-3-doxyctonates measured by the diphenylamine method is 1-3, and the molecular weight is 5,000.

d) Limulus activity of at least 1 O EUZng

e) Protein content is less than 1% (weight)

f) The nucleic acid content is 1% (weight) or less

A low molecular weight lipopolysaccharide having:

3. The low-molecular-weight lipopolysaccharide according to claim 1 or 2, wherein the microorganism is a gram-negative microorganism.

4. The low molecular weight lipopolysaccharide according to claim 3, wherein the gram-negative microorganism is a microorganism belonging to Pantoea or a microorganism belonging to Salmonella.

5. A pharmaceutical composition comprising the low-molecular-weight lipopolysaccharide according to any one of claims 1 to 4 and a pharmaceutical carrier.

6. The low molecular weight lipopolysaccharide according to any one of claims 1 to 4. A medicinal product containing as an active ingredient.

7. The medicament according to claim 6, which is an immunostimulant.

8. Use of the low-molecular-weight lipopolysaccharide according to any one of claims 1 to 4 as a medicament.

9. The crude lipopolysaccharide obtained by extraction from microbial cells is treated by anion exchange chromatography, and then the treated gel is subjected to gel filtration in the presence of a surfactant. 3. The method for producing a lipopolysaccharide according to claim 1 or 2.

10. The method for producing lipopolysaccharide according to claim 9, wherein the surfactant is deoxycholic acid.