JPH0733656A - Immunopotentiator - Google Patents

Abstract

PURPOSE:To obtain an immunopotentiator for preventing and treating infectious diseases due to virus and bacteria, etc., by administering to patients lowered in immunity, containing an ornithine-containing lipid consisting of a straight- chain fatty acid as an active ingredient, having high immune-potentiating action and reduced in side effects. CONSTITUTION:Chloroform, etc., is added to a humid bacterial cell of Achromobacter xylosoxidans ATCC 17458, etc., to extract total lipid and a chloroform layer is collected and the solvent is evaporated and the resultant lipid is subjected to thin-layer chromatography and a band located immediately below phosphatidyl ethanol is scraped together and this band is subjected to extraction with a solvent and the extract is concentrated and the concentrate is treated with phospholipase A2 to provide an ornithine-containing lipid. Then, the lipid is made to react with a straight-chain fatty acid (e.g. myristic acid) to afford a compound of the formula (R<1> is 11-17C alkyl; R<2> is acyl residue of myristic acid, etc.) and this compound is used as an active ingredient and blended with vehicles, etc., and the blend is prepared to provide the immunopotentiator.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an immunostimulant containing an ornithine-containing lipid consisting of a straight-chain fatty acid as an active ingredient.

[0002]

2. Description of the Related Art The immune system in vivo is composed of bacteria, yeasts, molds,
It plays an important role in the defense function of the body against infection by viruses and tumors. That is, in vivo, macrophages play an important role in foreign substance elimination function and specific immunity, T cells of lymphocytes are responsible for immune response, and B cells of lymphocytes produce antibodies, so that pathogenic bacteria or tumor cells are Have been removed from.

[0003] Various immunostimulants have been proposed so far, but the immune mechanism is complicated, and few of them are satisfactory in effectiveness, and there is a demand for the development of drugs with particularly few side effects. ing.

[0004]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide an immunostimulant having a high immunostimulatory action and few side effects.

[0005]

Means for Solving the Problems The present inventors have investigated the physiological activity of ornithine-containing lipids, and the ornithine-containing lipids already composed of branched fatty acids have the effect of activating macrophages and promoting the proliferation of B lymphocytes. Have been found (Y. Kawai, K. Akagawa, Infec
t. Immun. 57, 2086, 1989). In general, it is known that the biological activity of fatty acids is not always found to be linear, even if branched is found to be active (for example, Y. Song, et al., The Journal of Antibiot
ics, XXXIV, No.8, 980 (1981)). However, this time, it was found that even an ornithine-containing lipid composed of a linear fatty acid has a strong immunostimulatory action, and the present invention has been completed.

That is, the present invention has the general formula

[Chemical 2]

(In the formula, R ¹ represents a linear alkyl group having 11 to 17 carbon atoms, and R ² represents myristic acid, pentadecylic acid, palmitic acid, heptadecyl acid, stearic acid, nonadecanoic acid, or arachidic acid. The present invention relates to an immunostimulant containing an ornithine-containing lipid consisting of a linear fatty acid represented by (expressing an acyl residue) as an active ingredient.

As the ornithine-containing lipid consisting of the straight chain fatty acid represented by the above general formula, for example, N (α)-(3-myristoyloxymyristoyl) ornithine (hereinafter, Orn-3-OH-14: 0-14: Abbreviated as 0), N (α)
-(3-palmitoyloxymyristoyl) ornithine (hereinafter abbreviated as Orn-3-OH-14: 0-16: 0), N (α)-
(3-myristoyloxypalmitoyl) ornithine (hereinafter abbreviated as Orn-3-OH-16: 0-14: 0), N (α)-
(3-palmitoyloxypalmitoyl) ornithine (hereinafter abbreviated as Orn-3-OH-16: 0-16: 0), N (α)-
(3-palmitoyloxystearoyl) ornithine (hereinafter abbreviated as Orn-3-OH-18: 0-16: 0), and the like.

These compounds are, for example, Achromobacte
r xylosoxidans ATCC 17458, Bordetella pertussis Fu
kasawa (phase I), Pseudomonas fluorescens ATCC 135
It is produced by bacteria such as 25. It can also be chemically synthesized (see the reference example below).

Unlike the lipopolysaccharide (LPS) and other immunostimulants, the ornithine-containing lipid consisting of the linear fatty acid of the present invention has low toxicity, and even if it is administered at 7 mg / mouse, no lethal toxicity is observed. I couldn't do it. Further, neither pyrogenicity nor tumor necrosis factor (TNF) inducing activity was observed.

The ornithine-containing lipid comprising a linear fatty acid of the present invention has an ornithine-containing lipid 5 comprising a linear fatty acid as an action on a mouse peritoneal exudate macrophage.
It has prostaglandin E ₂ generation-inducing activity at a concentration of 0 mg / ml, and also has mouse hypothermic activity and hemagglutination activity.

The immunostimulant of the present invention can be administered orally or parenterally for therapeutic purposes. As the orally-administered agent, solid preparations such as powder, granules, capsules and tablets, or liquid preparations such as syrups and elixirs can be used. Moreover, an injection can be prepared as a parenteral preparation. These preparations are manufactured according to a conventional method by adding pharmacologically and pharmaceutically acceptable manufacturing aids to the active ingredient. Further, it is also possible to prepare a sustained-release preparation by a known technique. When the manufacturing aid is used, the amount of ornithine-containing lipid in the immunostimulant of the present invention is usually 0.1 to 10% by weight, preferably 0.2.
~ 5% by weight.

[0013] The above-mentioned additives are used as oral preparations (oral preparations), injectable preparations (injection preparations), and mucosal administration preparations (buccal, trocar).
Appropriate components for formulation depending on the administration route such as H., suppositories) and external preparations (ointments, patches, etc.) are used. For example, in the case of oral preparations and mucous membrane preparations, excipients (eg starch,
Lactose, crystalline cellulose, calcium lactose, magnesium aluminometasilicate, silicic acid anhydride), disintegrants (eg: carboxymethyl cellulose, carboxymethyl cellulose calcium), lubricants (eg: magnesium stearate, talc), co- In the case of injectable preparations, such as tong agents (eg, hydroxyethyl cellulose), flavoring agents, and the like, solubilizing agents or solubilizing agents (eg, distilled water for injection, which can constitute an aqueous injectable agent). Formulation components such as physiological saline, propylene glycol), suspending agents (eg, surfactants such as polysorbate 80), pH adjusters (eg, organic acid or its metal salt), stabilizers, etc. For external preparations, aqueous or oily solubilizers or solubilizers (eg:
Pharmaceutical ingredients such as alcohols, fatty acid esters), adhesives (eg carboxyvinyl polymers, polysaccharides), emulsifiers (eg surfactants) are used.

The immunostimulant of the present invention having the above constitution is
It can be produced by a known production method, for example, the method described in the Japanese Pharmacopoeia 10th Edition General Rules for Preparation or a method with appropriate modification.

When the immunostimulant of the present invention is administered as an injection, the dose of the active ingredient (per adult per day)
It is selected from the range of 0.1 to 100 mg.

The immunostimulant of the present invention is used for preventing or treating infectious diseases caused by viruses or bacteria by administering it to infants, the elderly, post-operative patients, AIDS patients and other immunocompromised patients. Useful for.

[0017]

The present invention will be described in detail below with reference to Reference Examples, Preparation Examples and Test Examples. However, the present invention is not limited to these reference examples, examples and test examples.

Reference Examples 1-3. Achromobacter xylosoxi
dans ATCC 17458, Bordetella pertussis Fukasawa
(phase I) cells and Pseudomonas fluorescens ATCC
13525, Isolation of ornithine-containing lipids from Flavobacterium meningosepticum cells. Total lipid (free lipi) was isolated from each wet cell by Bligh and Dyer method.
d) was extracted. Lipids were obtained by evaporating the chloroform layer. The resulting lipid was subjected to thin layer chromatography (chloroform / methanol / water = 65/25 /
4), scrape the band immediately below phosphatidylethanol, and remove it with chloroform / methanol (2/1).
Extraction by adding a mixed solvent of, and sonicating,
It was then concentrated. The concentrate was treated with phospholipase A ₂ and then subjected to thin layer chromatography again in the same manner as above to obtain an ornithine-containing lipid derived from each bacterium.
The composition of the ornithine-containing lipid obtained from each bacterium is shown below. Ornithine-containing lipid from Achromobacter xylosoxidans ATCC 17458 Orn-3-OH-16: 0-16: 0 (76%) Orn-3-OH-14: 0-16: 0 (20%) Orn-3-OH-18 : 0-16: 0 Ordine-containing lipid from Bordetella pertussis Fukasawa (phase I) Orn-3-OH-16: 0-16: 0 (85%) Orn-3-OH-14: 0-16: 0 (11 %) Pseudomonas fluorescens ATCC 13525 derived ornithine-containing lipid Orn-3-OH-16: 0-16: 0 (65%) Orn-3-OH-18: 1-16: 0 Orn-3-OH-16: 0 -18: 1 Ornithine-containing lipid derived from Flavobacterium meningosepticum (Comparative example) Orn-3-OH-iso 17-iso 15 (80%)

Reference Example 4. Synthesis of Orn-3-OH-16: 0-16: 0 N (α)-(R) -3-hydroxypalmitoyl-N (δ) -benzyloxycarbonyl- (S) -ornithine benzyl ester 3-hydroxypalmitic acid (550mg, 2.20mmol), N (δ)-
Benzyloxycarbonyl- (S) -ornithine benzyl ester p-toluenesulfonate (1.10 g, 1.92 mmol), and 1-hydroxybenzotriazole (288 mg, 2.12 mmol)
Was dissolved in DMF (10 ml), and 1- (3-N-dimethylaminopropyl) -3-ethylcarbodiimide (423 μl, 2.30 mmol) was added. After stirring overnight at room temperature, the mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate, washed successively with 1N hydrochloric acid and saturated brine, dried over magnesium sulfate, and concentrated under reduced pressure. The two obtained diastereomers were separated by silica gel column chromatography (170 g, toluene: ethyl acetate = 1: 1) and then recrystallized from acetonitrile to give colorless crystals. Yield 565 mg (32%). Mp: 104.5-105.5 ° C. [Α] ²⁷ D -4.6 ° (c 1.0, CHCl ₃ ). Analytical value (%): C, 70.70; H, 8.94; N, 4.55. C ₃₆ Calculated value for H ₅₄ O ₆ N ₂ (%): C, 70.79; H, 8.91; N,
4.59.

N (α)-(R) -3-palmitoyloxypalmitoyl-N (δ) -benzyloxycarbonyl- (S) -ornithine benzyl ester N (α)-(R) -3-hydroxypalmitoyl-N ( δ) -Benzyloxycarbonyl- (S) -ornithine benzyl ester
(200mg, 0.327mmol) and hexadecanoic acid (125mg, 0.491mmo
l) was dissolved in dichloromethane (8 ml) and 1- (3-N-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (9
4mg, 0.491mmol) and dimethylaminopyridine (4.0mg, 0.0
(33 mmol) was added and the mixture was stirred at room temperature overnight. Hexadecanoic acid
(84mg, 0.33mmol) and 1- (3-N-dimethylaminopropyl) -3
-Ethylcarbodiimide hydrochloride (63 mg, 0.33 mmol) was added and the mixture was further stirred overnight, then the reaction solution was washed successively with 1N hydrochloric acid and saturated brine. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (50 g, toluene: ethyl acetate = 3: 1) and recrystallized from acetonitrile to give colorless crystals. Yield 242 mg (87%). Mp: 86.5-87 ° C. [Α] ²⁸ D + 2.8 ° (c 1.0, CHCl ₃ ). Analytical values (%): C, 73.44; H, 9.94; N, 3.34. C ₅₂ Calculated as H ₈₄ O ₇ N ₂ (%): C, 73.54; H, 9.97; N,
3.30.

N (α)-(R) -3-palmitoyloxypalmitoyl- (S) -ornithine [Orn-3-OH-16: 0-16: 0] N (α)-(R) -3-palmitoyl Oxypalmitoyl-N (δ)
-Benzyloxycarbonyl- (S) -ornithine benzyl ester (151 mg, 0.178 mmol) was dissolved in a mixed solvent of methanol (10 ml) and chloroform (5 ml), acetic acid (10 μl) and palladium black (10 mg) were added, and the mixture was stirred under hydrogen atmosphere at room temperature. Stir overnight. The palladium black was removed by filtration, the filtrate was concentrated under reduced pressure, and recrystallized from ethyl acetate and hexane. Yield 82.2 mg (75%). Mp: 155.5-156 ° C. [Α] ²⁵ D + 1.8 ° (c 0.28, AcOH). Analytical values (%): C, 65.27; H, 10.93; N, 4.15. C ₃₇ H ₇₂ O ₅ N ₂・ CH ₃ CO ₂ H ・ 1.8H ₂ O calculated (%): C, 65.
29; H, 11.18; N, 3.90. ¹ H-NMR (270MHz, CDCl ₃ ): δ 0.8
8 (6H, t, CH ₃ ), 1.26 (46H, m, CH ₂ ), 1.6 (4H, m), 1.8
(4H, m), 2.00 (3H, s, C H ₂ CO ₂ H), 2.29 (2H, t, CH ₂ C
O ₂ ), 2.6 (2H, d, C H ₂ CONH), 3.10 (2H, m, CH ₂ NH ₂ ), 4.4
7 (1H, m, NHC H ), 5.21 (1H, m, CH (OH) CH ₂ CO ₂ ).

Reference Example 5. Synthesis of Orn-3-OH-16: 0-14: 0 N (α)-(R) -3-myristoyloxypalmitoyl-N (δ)
-Benzyloxycarbonyl- (S) -ornithine benzyl ester N (α)-(R) -3-hydroxypalmitoyl-N (δ) -benzyloxycarbonyl- (S) -ornithine benzyl ester
(140 mg, 0.229 mmol) and pyridine (111 μl, 1.38 mmol) in dichloromethane (5 ml) under ice-cooling myristoyl chloride (170 m
g, 0.69 mmol) was added, and the mixture was stirred at room temperature overnight. After concentration under reduced pressure, the residue was dissolved in ethyl acetate and washed successively with 1N hydrochloric acid and saturated brine. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (20 g, toluene: ethyl acetate = 5: 1) and recrystallized from acetonitrile to give colorless crystals. Yield 184 mg (98%). Mp: 75-76 ° C. [Α] ³¹ D + 2.6 ° (c0.50, CHCl ₃ ). Analysis (%): C, 72.78; H, .974; N, 3.43. Calculated as C ₅₀ H ₈₀ O ₇ N ₂ (%): C, 73.13; H, 9.82; N,
3.41.

N (α)-(R) -3-Myristoyl Oxypalmitoyl- (S) -Ornithine [Orn-3-OH-16: 0-14: 0] N (α)-(R) -3-Myristoyl Oxypalmitoyl-N (δ)
-Benzyloxycarbonyl- (S) -ornithine benzyl ester (135 mg, 0.164 mmol) was dissolved in a mixed solvent of methanol (6 ml) and tetrahydrofuran (3 ml) to prepare palladium black (10 mg).
(mg) was added and the mixture was stirred under a hydrogen stream at room temperature for 4 hours. After chloroform was added to dissolve the precipitate, palladium black was removed by filtration. The filtrate was concentrated under reduced pressure and then recrystallized from ethyl acetate and hexane. Yield 76.0 mg (78%). Mp: 127-129 ° C. [Α] ²⁷ D + 4.2 ° (c 0.33, AcOH). Analytical values (%): C, 69.70; H, 11.39; N, 4.57. C ₃₅ Calculated as H ₆₈ O ₅ N ₂ 0.25H ₂ O (%): C, 69.90; H,
^{. 11.48; N, 4.66 1 H} -NMR (270MHz, CDCl 3): δ 0.88 (6H, t, CH 3), 1.26 (42
H, m, CH ₂ ), 1.6 (4H, m), 1.8 (4H, m), 2.28 (2H, m, CH
₂ CO ₂ ), 2.49 (2H, d, C H ₂ CONH), 3.01 (2H, m, C H ₂ NH ₂ ),
4.23 (1H, m, NHC H ), 5.17 (1H, m, C H (OH) CH ₂ CO ₂ ).

Reference Example 6. Synthesis of Orn-3-OH-14: 0-14: 0 N (α)-(R) -3-myristoyloxymyristoyl-N (δ)
-Benzyloxycarbonyl- (S) -ornithine benzyl ester (R) -3-myristoyloxymyristic acid (980mg, 2.75mm
ol) and N (δ) -benzyloxycarbonyl- (S) -ornithine benzyl ester (1.25 g, 2.75 mmol) in THF (15
ice-cooled dicyclohexylcarbodiimide (0.57 g,
2.75 mmol) was added. After stirring at room temperature for 16 hours, the insoluble material was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (40 g, toluene: ethyl acetate = 7: 1) and recrystallized from acetonitrile to give colorless crystals. Yield 1.29 g (59%). Mp: 82-84 ° C. [Α] ¹⁹ D + 3.3 ° (c 1.0, CHCl ₃ ). Analytical values (%): C, 72.62; H, 9.72; N, 3.55. C Calculated as ₄₈ H ₇₆ O ₇ N ₂ (%): C, 72.62; H, 9.66; N,
3.53.

N (α)-(R) -3-myristoyl Oxymyristoyl- (S) -ornithine [Orn-3-OH-14: 0-14: 0] N (α)-(R) -3-myristoyl Oxypalmitoyl-N (δ)
-Benzyloxycarbonyl- (S) -ornithi [Orn-3-OH-1
[6: 0-14: 0] as in the synthesis of palladium black
(α)-(R) -3-Myristoyloxymyristoyl-N (δ)-
Benzyloxycarbonyl- (S) -ornithine benzyl ester (620 mg, 0.78 mmol) was catalytically reduced. Yield 399 mg (9
0%). Mp: 131-132 ° C. [Α] ²⁷ D + 5.1 ° (c 1.0, AcOH). Analytical value (%): C, 69.13; H, 11.34; N, 4.89. C ₃₃ H ₆₄ O ₅ Calculated value as N ₂ (%): C, 69.13; H, 11.34;
N, 4.89. ¹ H-NMR (270MHz, CDCl ₃ ): δ 0.87 (6H, t, CH ₃ ), 1.26 (38
H, m, CH ₂ ), 1.60 (4H, m), 1.80 (4H, m), 2.30 (2H, t,
CH ₂ CO ₂ ), 2.58 (2H, d, C H ₂ CONH), 3.08 (2H, m, C H ₂ N
H ₂ ), 4.62 (1H, m, NHC H ), 5.18 (1H, m, C H (OH) CH ₂ CO ₂ ).

Preparation Example 1. The ornithine-containing lipid obtained as described above is used in the following Examples 3
0 minutes before, it was made into liposome or a sufficiently good suspension. That is, a thin layer of ornithine-containing lipid was formed in a 10 ml eggplant-shaped flask or a test tube, a culture solution or a phosphate buffer solution (PBS) was added thereto, and the mixture was heated to 33 ° C., and then 1-2. Minute sonication was performed. When the desired suspension was not obtained by this treatment, phosphatidylglycerol (PG) was added in an amount of about 20% to prepare a thin film, and the above operation was performed.

Test Example 1. Prostaglandin E ₂ production inducing activity test Protease-peptone-induced mouse peritoneal exudate macrophages were adhered to a plate and the concentration of ornithine-containing lipid or lipopolysaccharide (E. coli 011) shown in Table 1 was used.
1: B4) 1 ml was added and the mixture was cultured at 37 ° C. for 20 hours.
The supernatant was taken and a prostaglandin E ₂ [ ¹²⁵ I] RIA kit manufactured by Dupont-New England Nuclear was used to
The amount of prostaglandin E ₂ (PGE ₂ ) produced and released was measured. The results are shown in Table 1.

[0028]

[Table 1] Table 1. Results of PGE ₂ production inducing activity test ──────────────────────────────── Ornithine dosage PGE ₂ release Lipid content (μg ) (ng / 10 ⁶ cells) ──────────────────────────────── A. xylosoxidans origin 50 25 Synthetic Orn-3 -OH-16: 0-16: 0 100 22 Synthetic Orn-3-OH-16: 0-14: 0 100 3 Synthetic Orn-3-OH-14: 0-14: 0 100 2 ────── ────────────────────────── F. meningosepticum origin 50 23 Lipopolysaccharide 10 22 Vehicle only 0.5 ─────────── ───────────────────── Thus, the ornithine-containing lipid of the present invention activates macrophages involved in immunity to generate prostaglandin E ₂ . Was induced.

Test Example 2. Hemagglutination activity test First, on a plate, lipids containing ornithine (initial concentration 2
50 μg / ml) was diluted 2-fold. Then, human A
Red blood cells (0.5% in PBS) were added and mixed well, then incubated at 28 ° C. for 2 hours. The same test was performed by increasing the dilution sequentially, and the minimum concentration of red blood cell agglutination was measured. The results are shown in Table 2.

[0030]

[Table 2] Table 2. Hemagglutination activity test results ──────────────────────────────── Ornithine Minimum hemagglutination concentration containing lipid (μg / ml) ─ ─────────────────────────────── A. xylosoxidans derived 4-8 A. xylosoxidans derived (PG added) ^* 1-4 Derived from B. pertussis 4-8 Derived from P. fluorescens 4-8 Synthetic Orn-3-OH-16: 0-16: 0 (PG added) ^* 4-8 Synthetic Orn-3-OH-16: 0-14: 0 (PG added) ^* 8-16 Synthetic Orn-3-OH-14: 0-14: 0 (PG added) ^* 31 ─────────────────────── ────────── F. meningosepticum origin 4-8 ──────────────────────────────── ^* PG means phosphatidyl glycerol, and the added amount is 20%. Generally, in ornithine-containing lipids, the higher the hemagglutination activity, the higher the immunostimulatory activity. The above results indicate that the ornithine-containing lipid composed of the linear fatty acid of the present invention has almost the same activity as the ornithine-containing lipid composed of the branched fatty acid.

Test Example 3. Lethal Toxicity Test The above-mentioned ornithine-containing lipid 500 μg to 7 mg PBS suspension was injected into the abdominal cavity of ICR or C3H / He mice. Observation was continued for one week after the injection, but no dying mouse was observed.

Test Example 4. Fever test The rabbit suspension (body weight: about 2.5 kg) was intravenously injected with 1-2 mg of the above-mentioned lipid containing ornithine in PBS from the ear.
As a result of observation for 5 hours, no exotherm was observed.

Test Example 5. Tumor necrosis factor (TNF) production induction test Twenty-four thioglycollate-induced mouse peritoneal macrophages
Stick on a well plate (10 ⁶ cells / well) and place 2 on top
Lipids containing ornithine at a concentration of 00, 100, 50, 25 μg / ml were placed and incubated at 37 ° C. for 16 hours. The supernatant was applied to _L929 cells in the presence of actinomycin D to measure the cell necrosis activity. [The test method is described in R.
E. Drysdale et al. J. Immuno
l. 131, p. 2362 (1983)]. As a result, no cell necrosis activity was observed, and it was revealed that the ornithine-containing lipid of the present invention does not induce the production of TNF.

Claims (2)

[Claims] 1. The following general formula: (In the formula, R ¹ represents a linear alkyl group having 11 to 17 carbon atoms, R ² represents an acyl residue of myristic acid, pentadecylic acid, palmitic acid, heptadecyl acid, stearic acid, nonadecanoic acid, or arachidic acid. And an ornithine-containing lipid consisting of a straight-chain fatty acid as an active ingredient. 2. An immunostimulant comprising an ornithine-containing lipid consisting of a straight-chain fatty acid as an active ingredient according to claim 1, wherein R ¹ is a tetradecyl group and R ² is a palmitoyl group.