JPH10152497A - Helicobacter pylori lipid a - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new Helicobacter pylori lipid A having a specific chemical structure, low in toxicity, low in pyrogenicity, and useful for immunostimulators, infectious disease medicines, antitumor medicines, antiviral medicines, etc., as a new immunologically active substance. SOLUTION: A new Helicobacter pylori lipid A having a chemical structure of the formula. The compound has an adjuvant activity, a B-cell activation action, a natural killer(NK) cell activity, an antitumor activity, an antiviral activity, and specific and nonspecific infection-preventing actions, and is low in toxicity, very low in pyrogenicity and useful for agents for producing various kinds of lymphokines, immunostimulators, medicines for treating and preventing various kinds of infections diseases, septicemia, rheumatoid arthritis, etc., antitumor medicines, antiviral medicines, etc. The compound is obtained by culturing Helicobacter pylori, centrifugally collecting the bacteria from the culture solution, lyophilizing the obtained bacterium pellets, defatting the product, subjecting the defatted product to an extraction treatment by a warm phenol/water method, hydrolyzing the extract, and subsequently fractionating the hydrolyzate by silica gel chromatrography.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel lipid A, and more particularly, to a lipid A isolated from Helicobacter pylori and a pharmaceutical composition containing the same as an active ingredient.

[0002]

2. Description of the Related Art Lipopolysaccharide (LPS; lipopolysaccharide) contained in the cell wall outer membrane of Gram-negative bacteria
e) is a glycolipid called lipid A in which various saccharides are bound, and has long been known to be a main component of endotoxin (Raetz, CRH, Bio).
synthesis of lipid A, Vol.
I. (Morrison, DC and Rya
n, J. et al. L. eds. ), CRC Press I
nc. , BocaRaton, 1992, 67-
80).

The above LPS exerts various immunobiological activities on the host, and it has been revealed that the site of its activity is the above-mentioned lipid A, and Escheric ( Escheric)
Hiacoli) Various lipid A derived from or Salmonella (Salmonella) and the like are various isolated, its chemical structure is also solved, for lipid A of this like, various immunological agents such as immunomodulatory effect and anti-tumor effect pharmacological Effects have been reported (Zahr
inger, U. et al., Adv. Carbohydr. Chem. Biochem.,
1994, 50 , 211-276; Takada, H. and Kotani, S., Bacte
rial endotoxic lipopolysaccharides (Morrison, D.
C. and Ryan, JL eds), CRC Press, New York, 1992,
pp. 107-134; Rietschel, ET et al., Prog. Clin.
Biol. Res., 1987, 231 , 25-53; Homma, JY, et a
l., DrugsFuture, 1989, 4 , 645-665).

[0004] The present inventor has previously reported that L of Porphyromonas gingivalis, a gram-negative anaerobic bacillus which is considered to be one of the causative bacteria of periodontal disease.
Focusing on the fact that PS has a very weak endotoxin effect such as pyrogenicity and lethal toxicity, PS was isolated and prepared, and its structure was successfully determined by focusing on the fact that it exerts a strong immunopotentiating effect (Ogawa,
T., FEBS Lett., 1993, 332 , 197-201;
No. 6893).

On the other hand, Helicoba pylori ( Helicoba)
cter pylori ) is frequently isolated from patients with chronic active gastritis and is associated with human duodenal and gastric ulcers (Wa
rren, JR and Marshall, B., Lancet, 1983, i , 127
3-1275; Buck, GE, et al., J. Infect. Dis., 198
6, 153 , 664-669; O'Connor, HJ, Scand. J. Gastro
enterol., 1994, 201 , 11-15; Goodwin, CS, Med.M
icrobiol. Lett., 1995, 4 , 155-164).

The bacterium is a gram-negative, microaerobic bacillus, having LPS on the cell surface, and the LPS molecule has characteristics chemically and biologically different from those of the enteric bacteria LPS. (Geis, G., et al., J. Clin. Microbiol.,
1990, 28 , 930-932; Conrad, RS, et al., Curr. Mi
crobiol., 1992, 24 , 165-169; Moran, A., et al., J.
Bacteriol., 1992, 174 , 1370-1377).

If the pylori-derived LPS lipid A can be provided, it is considered to be useful in the pharmaceutical field as a new immunoactive substance.

[0008]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide the above-mentioned lipid A of Helicobacter pylori.

[0009]

According to the present invention, there is provided Helicobacter pylori lipid A having a chemical structure represented by the following formula (1) (hereinafter referred to as "lipid A of the present invention").

[0010]

Embedded image

Further, according to the present invention, there is provided a pharmaceutical composition containing the lipid A of the present invention as an active ingredient.

The lipid A according to the present invention has a (R) -3-hydroxyoctadecanoyl group and a (R) -3-octadecanoyloxyoctadecayl group at the 2-position and the 2′-position, respectively, as represented by the above chemical structural formula. Glucosaminyl β (1-6) glucosamine-α-aminoethyl phosphate having a noyl group, unlike a conventional lipid A derived from Escherichia coli or Salmonella, having no phosphate group at the 4′-position, That the hydroxyl groups at the 3 and 3 'positions remain free,
And the amino acid group at the 1-position phosphate group.

The lipid A of the present invention has various biological activities, for example, an activity of producing IL-6, an activity of producing IL-8, a human natural killer (NK) cell activity and the like, and has low (no) toxicity. Also, it has a characteristic that the exothermicity is very low.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the method for isolating and preparing lipid A of the present invention will be described in detail. It can be produced from a microorganism of its origin according to a general method. Here, the source microorganisms include Helicobacter pylor isolated from the gastric mucosa and gastric juice of various higher animals.
i ) can be used. The standard strain is, for example, AT
Deposited as CC43504.

In the separation and purification of LPS from the above-mentioned source microorganism, cells are collected from a culture of the source microorganism by a conventional method such as filtration and centrifugation, and the cells are based on the physicochemical properties of the LPS. Various means, for example, extraction means such as hot phenol water extraction, etc., various enzyme treatment means, centrifugation, solvent fractionation means, column chromatography means using various resins, etc. Can be implemented.

The production of the lipid A of the present invention from the LPS thus obtained can be carried out by a general hydrolysis means, preferably a hydrolysis reaction using a weak acid such as acetic acid of about 0.05 to 0.2 N, for example. Possible and intended lipid A
Can be purified by ordinary purification means, for example, solvent extraction, various types of chromatography and the like.

The chemical structure of lipid A of the present invention can be determined by known chemical manipulation, gas-liquid chromatography, nuclear magnetic resonance spectroscopy (N
MR), mass spectrometry (MS) and the like.

The lipid A of the present invention can also be chemically synthesized based on the chemical structure analyzed as described above. This is because, for example, an N-glucosamine derivative corresponding to the above-mentioned chemical structure protected by an ordinary protecting group is converted into a disaccharide derivative according to a glycoside bond formation reaction, and an appropriate fatty acid is subjected to the N-acylation reaction. Alternatively, an N-glucosamine derivative corresponding to the above-mentioned chemical structure, which is N-acylated by a suitable fatty acid and protected with a usual protecting group, is subjected to a glycoside bond-forming reaction to give a disaccharide derivative. The resulting derivative is phosphorylated at position 1 of the reducing end by reacting with a phosphoramidite having an N-protected aminoethyl group, and then oxidized to a phosphate ester, and finally catalytically reduced to remove all the protecting groups. It can be carried out by desorption.

The lipid A of the present invention thus obtained can form a pharmaceutically acceptable salt at the phosphate group. Examples of the salt include salts of alkali metals such as sodium and potassium, salts of alkaline earth metals such as calcium and magnesium, salts of ammonium such as tetramethylammonium, methylamine, triethylamine,
Examples thereof include salts of organic amines such as benzylamine, pyridine, piperidine, diethanolamine, lysine, and arginine.

The lipid A of the present invention and a pharmaceutically acceptable salt thereof are used as an active ingredient, and in the form of a general pharmaceutical preparation composition using an appropriate pharmaceutical carrier together with the active ingredient, and are used in pharmaceutical applications. Practical for Therefore, the present invention also provides such a pharmaceutical composition.

The pharmaceutical composition of the present invention has a biological activity possessed by the active ingredient, for example, an adjuvant activity, a B cell activating effect, an NK cell activity, an antitumor activity, an antiviral activity,
Based on specific / non-specific protective action against infection, etc., various lymphokine producing agents, immunostimulants, various infectious disease treatment and prevention agents, sepsis treatment and prevention agents, treatment and prevention agents for rheumatoid arthritis, antitumor agents, It is useful as an antiviral agent and the like.

In the pharmaceutical composition of the present invention, the pharmaceutical carrier to be used includes a filler, a bulking agent, a binder, a humectant, a disintegrant, a surfactant, a lubricant, which are generally used depending on the use form of the pharmaceutical. Diluents or excipients such as powders can be exemplified, and these can be appropriately selected and used depending on the dosage unit form of the obtained preparation.

As the dosage unit form of the pharmaceutical composition of the present invention, various forms can be selected according to the purpose of treatment, and typical examples are tablets, pills, powders, solutions, suspensions, emulsions and granules. Preparations, capsules, suppositories, injections (solutions, suspensions, etc.), ointments and the like.

In the case of molding into a tablet form, the above-mentioned preparation carriers include excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, potassium phosphate and the like.
Water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, methylcellulose, binders such as polyvinylpyrrolidone, sodium carboxymethylcellulose, calcium carboxymethylcellulose, low-substituted hydroxypropylcellulose, dried Disintegrants such as starch, sodium alginate, agar powder, laminaran powder, sodium bicarbonate, calcium carbonate, surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, and monoglyceride stearate; sucrose, stearin, cocoa butter; Disintegration inhibitors such as hydrogenated oil, quaternary ammonium bases, absorption enhancers such as sodium lauryl sulfate, glycerin, starch and the like Moisturizers, starch, lactose, kaolin, bentonite, adsorbent such as colloidal silicic acid, purified talc, stearates, boric acid powder, a lubricant such as polyethylene glycol can be used.

Further, the tablets can be made into tablets coated with a usual coating, if necessary, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, double tablets and multilayer tablets.

In the case of molding into the form of pills, for example, glucose, lactose, starch, cocoa butter,
Excipients such as hardened vegetable oil, kaolin and talc, gum arabic powder, tragacanth powder, binders such as gelatin and ethanol, and disintegrants such as laminaran and agar can be used.

For shaping in the form of suppositories, for example, polyethylene glycol, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides and the like can be used as pharmaceutical carriers. Capsules are usually prepared by mixing the active ingredient of the present invention with the various pharmaceutical carriers exemplified above and filling the mixture into hard gelatin capsules, soft capsules and the like according to a conventional method.

When the medicament of the present invention is prepared as an injection, such as a solution, an emulsion or a suspension, it is preferable that the medicament is sterilized and isotonic with blood. As the diluent, for example, water, ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxyisostearyl alcohol, polyoxyethylene sorbitan fatty acid esters and the like can be used. In this case, a sufficient amount of salt, glucose or glycerin to adjust the isotonic solution may be contained in the drug of the present invention, and ordinary solubilizers, buffers, soothing agents and the like are added. May be.

Further, the drug of the present invention may contain a coloring agent, a preservative, a fragrance, a flavoring agent, a sweetening agent and the like, and other pharmaceuticals, if necessary.

When shaping into the form of an ointment such as a paste, cream, gel or the like, for example, white vaseline, paraffin, glycerin, a cellulose derivative, polyethylene glycol, silicone, bentonite and the like can be used as a diluent.

The amount of the active ingredient to be contained in the medicament of the present invention is not particularly limited and may be appropriately selected from a wide range, but it is generally assumed that the pharmaceutical preparation contains about 0.1 to 70%. Is good.

The administration method of the above pharmaceutical preparation is not particularly limited, and is determined according to various preparation forms, age, sex and other conditions of the patient, degree of disease, and the like. For example, tablets, pills, solutions, suspensions, emulsions, granules, and capsules are orally administered, and injections are administered intravenously, alone or as a mixture with ordinary replenishers such as glucose and amino acids. Intramuscularly, intradermally, subcutaneously or intraperitoneally, and suppositories may be administered rectally alone.

The dose of the above-mentioned pharmaceutical preparation is appropriately selected depending on its usage, the age of the patient, gender and other conditions, the degree of the disease, etc. Usually, the amount of the active ingredient is about 0.01 per adult per day. The dosage is preferably about 100 mg.
It can be administered in 1 to 4 times a day.

[0034]

EXAMPLES Hereinafter, in order to explain the present invention in more detail, a production example of lipid A of the present invention will be described as an example, and then a biological activity test example conducted on lipid A of the present invention will be described.

Incidentally, C57BL /
Six mice (male, 8 weeks old) were obtained from SLC Japan (Hamamatsu), and domestic Japanese white rabbits were purchased from Oriental Yeast (Osaka). In addition, natural products and synthetic products as reference samples in the test examples were prepared as follows.

That is, lipid A of P. gingivalis 381 is
It was prepared according to the method described in JP-A-6-206893. This is hereinafter referred to as " P. gingivalis lipid A". E. coli type synthetic lipid A (Imoto, M., et al., T
etrahedron Lett., 1985, 26 , 1545-1548) was purchased from Daiichi Pure Chemicals (Tokyo). Hereinafter, this is referred to as “Compound 50”.
6 ". Both were dissolved in a 0.1% aqueous solution of triethylamine at a concentration of 2 mg / ml, and the stock solution was heated at 4 ° C.
Prior to the assay and used after appropriately diluting with pyrogen-free physiological saline (PBS; Micro Lab, Osaka) or cell culture medium.

From the results in each test example, a statistically significant difference between the test group and the control group was examined by Student's t-test. Each test was repeated at least three times, and it was confirmed that similar results were obtained by this repetition.

[0038]

Example 1 Preparation and Purification of Lipid A of the Present Invention H. pylori (clinical isolate, obtained from Hyogo College of Medicine)
% O _2, in the 15% CO ₂ and 80% N ₂ atmosphere, 7% horse blood, Skirrow Supplement (OXOID, Hampshire, UK)
And 0.4% Fungizone (Life Technology, Inc., New
York, USA) with Brucella medium (Becton Dickinson and
(Company, Cockeysville, USA) plates at 37 ° C. for 72 hours. The bacterial cells were collected in pyrogen-free water, and collected by centrifugation at 5000 g and 4 ° C. for 30 minutes. After washing this twice, the bacterial pellet was freeze-dried.
The freeze-dried cells were defatted by treatment with chloroform / methanol (1/2).

From the freeze-dried defatted cells, a warm phenol / water method (Westphal, O. and Jann, K., Meth. Carbohyd
r., 1965, 5 , 83-91).

The obtained LPS (300 mg) was heated at 105 ° C.
For 2.5 hours in 0.6% acetic acid, and then cooled and neutralized. The hydrolyzate is made up to 100 ml with water and then
CHCl ₃ / methanol / water / triethylamine (30
/12/2/0.1), and the lower layer was evaporated under reduced pressure to give lipid A fraction.

The obtained lipid A fraction was subjected to silica gel column chromatography (Merck No. 9385, Darmstadt, Ger.
many) and separated by CHCl ₃ / methanol / water / triethylamine (30/15 / 0.5 / 0.1). The eluate was monitored by thin layer chromatography (TLC) on Merck No. 5715 plates. The plate was washed with CHCl ₃ / methanol / water / triethylamine (3
0/8 / 0.7 / 0.1), and anisaldehyde-sulfuric acid (organic chemistry experiment guide 1, Chemical Doujinshi, 198)
8, page 120) and ninhydrin reagent.

The compound (Rf = 0.4) was observed as a single spot, and was found to be the main component.

The following results were obtained by conducting various instrumental analyzes on this lipid A. These were comprehensively analyzed and compared with the analysis results of other lipid A, and the chemical structure was identified as the above formula (1).

(1) NMR spectrum: As a result of measuring COSY (two-dimensional shift correlation ¹ H-NMR) and TOCSY (two-dimensional total correlation ¹ H-NMR), FIG. 1 and FIG. D1 (ppm) and the horizontal axis is D2 (p
pm)). From these results, the structure of the lipid A of the present invention, such as the position of the substituent, was supported.

FIGS. 3 and 4 are charts showing the results of PH—HMBC (two-dimensional heteronuclear multiple bond correlation NMR).
(Partial enlarged view of FIG. 3) (In each figure, the vertical axis is F1 (p
pm) is shown in F2 (ppm) on the horizontal axis).

From these figures, coupling between the phosphorus atom and hydrogen at the 1-position and hydrogen at the aminoethyl group was confirmed.
Each of the above NMRs was 1: 1 methanol-d4:
The reaction was performed in a solvent obtained by adding a small amount of heavy pyridine to heavy chloroform.

(2) Mass spectrum: FAB-MS-MS in m-nitrobenzyl alcohol matrix
(High-speed electron impact tandem MS) and ESI-MS-M
S (ion spray tandem MS) was performed. The results are shown in FIG. 5 (FAB-MS-MS, normal scan), FIG. 6 (FAB-MS-MS, linked scan) and FIG.
(ESI-MS-MS).

From each figure, 1293 [(M−H) ⁻ ]
, And 1009, 745, 727, 584, 56
6, 463, 140 and 79 major peaks were observed,
The dissociation pattern of MS, including these, supported the type of each substituent such as a (2-aminoethyl) phosphate group, the mother nucleus structure, and the like.

The result of FAB high-resolution MS analysis is as shown in FIG.
Is confirmed, and this value is determined by the molecular formula C ₆₈
H ₁₃₁ N ₃ O ₁₇ P ^- values calculated on the basis of 1292.92
152 well matched.

Further, the results of GC and GC-MS confirmed the presence of an octadecanoyl group and a 3-hydroxyoctadecanoyl group.

(3) Phosphorus quantification: Phosphorus quantification is described in the literature (FE
MS Immunol. Med. Microbiol., 1995, 12 , 97-112), it was confirmed that 1 mol of phosphorus atom was present in one molecule.

[0052]

[Bioactivity test example 1] Lethal toxicity in galactosamine-administered mice C57BL / 6 mice (5 mice per group) were treated with PBS 0.5
D-galactosamine hydrochloride in ml (Wako Pure Chemical, Osaka) 1
6 mg was intraperitoneally administered, and the test sample in 0.2 ml of PBS was prepared according to the method of Galanos et al. (Galanos, C., et. Al.).
al., Proc. Natl. Acad. Sci., USA, 1979, 76 , 59
39-5943), and injected immediately via the tail vein in stepwise doses. The death of mice due to poisoning was observed for 24 hours, and the 50% lethal dose (LD ₅₀ ) of each group was determined by the method of Karber (Karber, G., Arch. Exp. Pathol. Pharmakol., 193).
1, 162 , 480-483).

The results of the above test are shown in the following table.

[0054]

[Table 1]

From Table 1, it was found that lipid A of the present invention had no lethal toxicity at a maximum dose of 10 μg per mouse, and the 50% lethal dose (LD ₅₀ ) was 31.6 μg / mouse. As shown in Table 1, the lethal toxicity of P. gingivalis lipid A was low, and its LD ₅₀ was 12.6 μm per mouse.
g, but the toxicity (LD ₅₀ ) of compound 506 was 0.1 g.
0079 μg / mouse was found to be quite strong.

[0056]

[Bioactivity test example 2] pyrogenicity 3 Japanese domestic white rabbits in each group (male; 2.2-2.5
kg), a test sample in 10 ml of pyrogen-free PBS was injected at a prescribed dose through the ear vein (Ogawa, T.,
Eur. J. Biochem., 1994, 219 , 737-742).

The rectal temperature of each rabbit was measured and continuously recorded with an automatic measuring instrument (EP670, 12; Iio Denki, Tokyo). In this test, the measurement was simplified, and the temperatures of the first phase and the second phase were measured at 1-1.
The rectal temperature of each rabbit was examined every 30 minutes until 5 hours (Peak 1) and 2.5-4 hours (Peak 2), and an increase in rectal temperature exceeding 0.6 ° C was regarded as a positive exothermic reaction.

The results of the above test are shown in the following table.

[0059]

[Table 2]

As shown in Table 2, the lipid A of the present invention did not induce any pyrogenic reaction in rabbits. On the other hand, the pyrogenicity of P. gingivalis lipid A is low, and peak 2
, A very low pyrogenicity was observed at the dose of 10 μg / kg, but not at peak 1. In addition, rabbits to which compound 506 was administered at 0.01 μg / kg showed a remarkable exothermic reaction at both peaks 1 and 2.

[0061]

[Bioactivity test example 3] Limulus test Pregel (Pre Ge), which is an amoebocyte lysate prepared from Tachypleus tridentatus , according to the usage instructions attached to the reagent
This test was performed using l). The dose that produced a stronger response than the stated increase in viscosity was taken as the minimum effect.

The results of the above test are shown in the following table.

[0063]

[Table 3]

From Table 3 it can be seen that Compound 506 was tested for T.
strongly activates the agglutinating enzyme cascade of tridentatus amoeba cell lysates, in contrast to the lipid A of the present invention.
In addition, P. gingivalis lipid A was much weaker than compound 506, and the activity of lipid A of the present invention was found to be 1/1000 that of compound 506.

[0065]

[Biological activity test example 4] Cytokine assay Heparin-added venous blood collected from a healthy donor was Histopaq
ue (Sigma Chemical Co., St. Louis, MO, USA) to obtain peripheral blood mononuclear cells (PBMC). The above P
The BMC was washed with PBS, and RPMI1640 medium supplemented with 10% (v / v) fetal bovine serum (FBS, Hyclone) (Micro Lab.
(Osaka) The cells were suspended at a density of 1.5 × 10 ⁶ cells per ml. PBMCs (1.5 × 10 ⁵ / well) were placed in 96-well culture plates (Becton Dickinson) with or without different concentrations of test samples at 37 ° C. for 72 hours.
After incubation in humid air containing% (v / v) CO ₂ , α-type tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) assays were performed.

The normal human gingival fibroblast cell line (Gin-
1, purchased from Dainippon Pharmaceutical Co., Ltd.)
Improved Du with 0% FBS (Hyclone Laboratories, Inc.)
5% CO in lbecco's Eagle's medium (DMEM; micro-lab)
_It was kept at 37 ° C. in _two atmospheres and passages 10 and 12 were used for the assay.

Gin-1 cells (2.0 × 10 ⁴ /0.1
ml / well) in a 96-well culture plate (Becton Dickinson) at 37 ° C. for 24 hours in the presence or absence of the prescribed amount of the test sample, and the IL-
8 were assayed. After incubation, supernatants were collected and used for cytokine assays.

The cytokines (TNF-α, IL-6 and IL-8, Amersham International plc., Amersham, U.S.A.)
The production amount of K) was measured in the culture supernatant using a commercially available ELISA kit. Assays were performed according to the manufacturer's instructions and data for each assay was determined by a standard curve.

The results of the above test are shown in FIGS.

The figures show the TNF-α production (pg / ml), IL-6 production (pg / ml) and IL-8 production (pg / ml) for each dose of each test sample.
6 is a bar graph showing.

From FIG. 9 (the results of assaying the lipid A of the present invention and the lipid A of P. gingivalis and the compound 506 for the TNF-α production activity), the lipid A of the present invention and the P. gi
It is evident that ngivalis lipid A shows lower TNF-α producing activity than compound 506 in human PBMC culture supernatant.

From FIG. 10 (similarly showing the activity of each lipid A for producing IL-6), it can be seen that the lipid A of the present invention shows the compound 506 in contrast to the TNF-α producing activity shown in FIG.
And P. gingivalis was found to induce IL-6 production comparable to lipid A.

As shown in FIG. 11, the lipid A of the present invention stimulated the human gingival fibroblast Gin-1 strain, and IL-
8 was found to be produced in a dose-dependent manner. Its activity is
It fell short of that of compound 506.

[0074]

[Bioactivity test example 5] Cell lysis assay The cell lysis assay was performed by radioactive release assay (Anders
son, LC, et al., Int. J. Cancer, 1979, 23 , 143-1
47). That is, human PBMC (1.0 × 10 ⁶
/ Well) with 15% FBS (Hyclone Laboratories, In
c.) 5% (v / v) CO in supplemented RPMI medium (Micro Lab)
Incubation was carried out at 37 ° C. for 72 hours with a prescribed amount of test sample in a humid air atmosphere containing ₂ . RPMI16
After washing with 40, the NK cell sensitive red and white blood cell line ⁵¹
Incubation with 2 × 10 ⁴ Cr-labeled K562 cells at an effect / target cell ratio of 50: 1 at 37 ° C. for 4 hours. After the incubation, the culture supernatant was collected and counted using a gamma counter.

The% cell activity was calculated as follows:

Natural killer (NK) cell activity (%)
= 100 x (experimental release value-spontaneous release value) / (total release-spontaneous release) where the experimental release value is the average cpm released with the effector cells, and the spontaneous release value is the average released from the target cells alone cpm, the total release is 0.1N
⁵¹ C incorporated into target cells dissolved in aOH
The total amount of r is shown.

FIG. 12 shows the results of the above test.

From the figure, it can be seen that lipid A of the present invention, P. gingivalis
In human PBMC incubated with each of lipid A and compound 506, almost equivalent induction of NK cell activity was observed.

From the above test results and the results of other biological activity tests, the following became clear.

That is, lipid A of the present invention has no or very low toxin in terms of endotoxin, such as lethal toxicity in mice administered galactosamine, pyrogenicity to rabbits, and limulus test activity, etc. (Tables 1 to 3). This low toxicity
As in P. gingivalis lipid A, C-2 'position and C-
It is significantly weaker than that of compound 506, which has two acyloxyacyl groups at the 3'-position.

The lipid A of the present invention was observed to have IL-6 production and human NK cell activity comparable to those of compound 506 and P. gingivalis lipid A.

The structural characteristics of the lipid A of the present invention are summarized as follows. That is, it has a disaccharide skeleton of glucosamine β (1-6) glucosamine, and at the first position of the reducing end, α-linked phosphoric acid in which ethanolamine is bonded by a phosphate bond. Furthermore, there is no phosphate group at the 4'-position of glucosamine on the non-reducing terminal side, and there is no ester-linked fatty acid at the 3- and 3'-hydroxyl groups. The fatty acid is a (R) -β hydroxy fatty acid having 18 carbon atoms in the form of an amide bond to the 2-position amino group of the reducing terminal glucosamine and a 3-acyloxyacyl-type fatty acid at the 2′-amino group of the non-reducing terminal glucosamine. Carbon number 18
(R) -β hydroxy fatty acid having 18 carbon atoms in which the above fatty acid is ester-bonded. From the above points, it is clear that the intestinal bacteria are clearly distinguished from lipids A.

The lipid A of the present invention can also be obtained from B. fragilis (We
intraub, A., et al., Eur. J. Biochem., 1989, 183 ,
425-431), P. gingivalis , Prevotella intermedia (Jo
hne, B., et al., Oral Microbiol. Immunol., 1988, 3 ,
Like lipid A of 22-27), it does not have a phosphate group at the 4'-position, which can be considered to be a very weak causative factor of toxicity.

The lipid A of the present invention has only one 3-acyloxyacyl group, thereby having no or very low endotoxinity (weak TNF-α production), but producing other cytokines and NK. Cell activation is as strong as compound 506 and such low endotoxin and strong immune bioactivity
It is thought to be due to the position of the 3-acyloxyacyl group bonded to the skeletal structure.

The lipid A of the present invention, unlike lipid A of the intestinal bacteria, has a chemical structure similar to that of P. gingivalis lipid A, has immunostimulatory properties, but has extremely low endotoxinity.
Therefore, it is thought to play an important role in controlling inflammation, immune biological characteristics, and the like.

[Brief description of the drawings]

FIG. 1 is a graph showing the results of two-dimensional shift correlation NMR (COSY) spectrum analysis of lipid A of the present invention obtained in Example 1.

FIG. 2 is a graph showing the results of two-dimensional total correlation NMR (TOCSY) spectrum analysis of lipid A of the present invention obtained in Example 1.

FIG. 3 is a graph showing the results of two-dimensional heteronuclear multiple bond correlation NMR (P-H.HMBC) spectrum analysis of lipid A of the present invention obtained in Example 1.

FIG. 4 is a graph showing the results of two-dimensional heteronuclear multiple bond correlation NMR (P-H-HMBC) spectrum analysis (partially enlarged in FIG. 3) of lipid A of the present invention obtained in Example 1.

FIG. 5 is a graph showing the mass spectrum (FAB-MS-MS, normal scan) analysis result of lipid A of the present invention obtained in Example 1.

FIG. 6 is a graph showing a mass spectrum (FAB-MS-MS, linked scan) analysis result of lipid A of the present invention obtained in Example 1. FIG. 7 (ESI-MS-MS)

FIG. 7 is a graph showing the results of mass spectrometry (ESI-MS-MS) analysis of lipid A of the present invention obtained in Example 1.

FIG. 8 is a graph showing the result of FAB high-resolution mass spectrum analysis of lipid A of the present invention obtained in Example 1.

FIG. 9 is a graph showing the TNF-α producing activity of lipid A of the present invention determined in accordance with Test 4 of Biological Activity.

FIG. 10 is a graph showing the IL-6 producing activity of lipid A of the present invention determined in accordance with Test 4 of Biological Activity.

FIG. 11 is a graph showing the IL-8 producing activity of lipid A of the present invention determined in accordance with Test 4 of Biological Activity.

FIG. 12 is a graph showing the NK cell activity of lipid A of the present invention determined according to Test Example 5 for biological activity.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 31/70 ADY A61K 31/70 ADY ADZ ADZ (72) Inventor Toshihide Tamura 2-8-5 Aoyamadai, Suita-shi, Osaka (72) Inventor Takashi Shimoyama 6-12-34-301 Okamoto, Higashinada-ku, Kobe City, Hyogo Prefecture

Claims (2)

[Claims] (1) Formula (1) Helicobacter pylori lipid A having a chemical structure represented by: 2. A pharmaceutical composition comprising the lipid A of Helicobacter pylori according to claim 1 as an active ingredient.