JPH07267856A - Oral immunodepressant - Google Patents

Abstract

PURPOSE:To provide an oral immunodepressant which contains docosa-hexaenoic acid and its derivative, and can be applied to autoimmune diseases such as collagen disease without side-effects such as nephrotic and enterogastric disorders. CONSTITUTION:At least one selected from docosahexaenoic acid(DHA) and its derivatives such as free DHA, DHA alkyl esters, DHA glycerides is used as an active ingredient to give the objective immunodepressant where the DHA preparation is in the form of a fatty acid mixture in which the content of DHA or its derivative exceeds 95%. Thus, DHA or its derivative are efficiently absorbed to manifest their effect promptly, while other fatty acids depress the side-effects. Further, the dose form per one application can be minimized to facilitate the administration. It is useful for treating Wegener's granulomatosis, systemic lupus erythematosus, collagen diseases such as Behcet's disease, nephroses, a variety of nephritis, ulcerative colitis, psoriasis, multiple sclerosis, Basedow disease, pernicious anemia, autoimmune disease such as Addison disease and for inhibition of rejection in organ transplantation.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an oral immunosuppressant.

[0002]

2. Description of the Related Art Immunosuppressive agents such as cyclosporine are used for autoimmune diseases such as collagen disease. However, most of the immunosuppressive agents currently in use are drugs that are also used as anticancer agents, and are accompanied by side effects such as renal damage, gastrointestinal damage, bone marrow suppression, and hair loss.

[0003]

DISCLOSURE OF THE INVENTION Based on the above findings, the present inventors have conducted earnest research to develop a novel immunosuppressive drug which is safe and has no side effects. As a result, docosahexaenoic acid (DHA), which is a polyunsaturated fatty acid contained in fish oil and has been attracting attention as a health food that is expected to have a heart disease preventive action and a brain function improving action, has an immunosuppressive action by oral administration. The present invention has been completed by finding out that and further researching.

[0004]

That is, the present invention provides DH
It is an oral immunosuppressive agent containing a class A compound. In the present invention, DHAs include DHA, DHA alkyl ester, DHA glyceride, DHA phospholipid, DHA choline body, DHA nicotinic acid body, and DHA amino acid body.

DHA alkyl ester has 1 to 4 carbon atoms.
Is a DHA monoester having a straight chain or branched chain alkyl group, specifically methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl. DHA glyceride is DHA mono, di or triglyceride. The DHA phospholipid refers to a mono- or di-DHA ester with a phospholipid, and examples thereof include DHA phosphatidylcholine, DHA phosphatidylethanolamine, DHA phosphatidylserine, DHA phosphatidylinositol and DHA phosphatidic acid. What is DHA amino acid body?
DHA in which DHA and various amino acids are peptide-bonded
Derivatives, such as DHA glycine and DHA taurine. Particularly preferred as DHAs are DHA, DHA ethyl ester and DHA triglyceride.

In the oral immunosuppressant of the present invention, DH
It is desirable that the type A is blended as a mixture of fatty acids containing 95% or more, preferably 97% or more, and more preferably 99% or more of DHAs. The DHA content is 95
This is because when it is at least%, DHAs, which are the active ingredients, are efficiently absorbed, the drug effect is rapidly exhibited, and the problem of side effects due to other fatty acid components can be avoided.

As fatty acids, in addition to the above DHAs, eicosapentaenoic acid, docosapentaenoic acid, arachidonic acid, oleic acid, α-linolenic acid, γ-linolenic acid, myristic acid, palmitic acid, vaccenic acid, etc. Other fatty acids and their alkyl esters, glycerides, esters with phospholipids, choline bodies, nicotinic acid bodies, amino acid bodies are included.

DHAs can be obtained in various ways. As a typical method, a method in which DHA extracted, separated, and purified from fish oil or the like is esterified by a known method, and DH
Examples include a method of separating and purifying DHAs from a mixture containing As. For example, as described in JP-A-4-95048, page 2, lower left column, line 4 to page 4, lower left column, line 9, a method of adsorbing to a clay mineral is disclosed.
No. 159398, page 2, upper right column, line 6 to same page, lower right column, line 12 and JP-A-4-243849, page 2, column 2, line 33 to page 3, column 4, column 1, line 1. As described above, by a method of forming a complex with a silver compound,
D containing a small amount of other fatty acids such as myristic acid, palmitic acid, and vaccenic acid from a mixture containing DHAs
HAs are obtained.

DHA triglyceride can also be prepared by a known method. For example, it can be prepared according to the method described in JP-A No. 62-129216, from page 2, upper right column, lower line, to page 2, lower left column, lower column, third line. That is, DHA and N, N '
-High-purity DHA triglyceride can be obtained by reacting with carbonyldiimidazole to prepare an acylimidazole of DHA and then reacting this with glycerol.

The indications of the oral immunosuppressive agent of the present invention include Wegener's disease, polyarteritis nodosa, systemic lupus erythematosus,
Collagen diseases such as Behcet's disease, nephrosis, various nephritis,
Ulcerative colitis, localized ileitis, chronic active hepatitis, psoriasis,
Myasthenia gravis, multiple sclerosis, pulmonary fibrosis, Hashimoto thyroiditis, primary myxedema, Basedow's disease, pernicious anemia, Addison's disease, premature menopause, male infertility, good pasture syndrome, vulgaris vulgaris Psoriasis, pemphigoid, sympathetic ophthalmitis, lens uveitis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leukopenia, primary biliary cirrhosis, cryptogenic cirrhosis, ulcer Colitis, Sjogren's syndrome, dermatomyositis (polymyositis), scleroderma, rheumatoid arthritis, discoid lupus erythematosus, mixed connective tissue disease,
It is an organ-specific or non-specific autoimmune disease such as contact dermatitis, atopic dermatitis, bronchial asthma, and is also indicated for suppressing rejection in organ transplantation.

The oral immunosuppressive agent of the present invention is prepared in the form of tablets, capsules, granules, powders, liquids and the like. Orally administered carriers include commonly used emulsifiers, excipients,
Binders, lubricants, coloring agents, disintegrating agents and the like can be used. As the excipient, for example, lactose, sucrose, starch, talc, magnesium stearate, crystalline cellulose, methyl cellulose, carboxymethyl cellulose,
Glycerin, sodium alginate, gum arabic, etc., as the binder, polyvinyl alcohol, polyvinyl ether, ethyl cellulose, gum arabic, shellac, sucrose, etc. can be mentioned, and other known coloring agents, disintegrating agents, etc. should be used. You can The tablets may be coated by a known method. In addition, liquid agents are aqueous or oily suspensions, emulsifiers, solutions, syrups,
It may be an elixir or the like and is prepared by a commonly used method.

The preferred dosage forms of the oral immunosuppressant of the present invention are capsules and granules. For example, a soft capsule in which DHAs are encapsulated with a capsule base having increased plasticity by adding glycerin or sorbitol to gelatin is easy to take and has a DHA blending ratio of 95.
It is particularly preferable in that it can be set to at least%. Also,
Granules are also particularly preferable in that they are easy to take and can be made into an enteric preparation or a sustained preparation by a known coating agent. Granules are prepared by wet or dry granulation using the above-mentioned excipients, binders, disintegrants and the like.

The oral immunosuppressant of the present invention contains DHA 9
It is desirable to contain 5% or more. 95% DHA
In the case of containing the above, DHA which is an active ingredient is efficiently absorbed, the onset of drug effect is promptly performed, the side effect problem due to other contained ingredients can be avoided, and further, the preparation (soft capsule) (Forms such as agents) can be made smaller and the administration becomes easier.

The oral immunosuppressive agent of the present invention is orally administered to mammals such as humans, cows, horses, dogs, cats, mice and rats alone or in combination with known agents.
The dose of the oral immunosuppressant of the present invention is not particularly limited, but for humans, it is preferably 0.1 to 10 as a DHA.
It is 10 g / day, more preferably 0.5 to 5 g / day.

The preferred embodiments of the present invention are listed below. 1. In the oral immunosuppressant of the present invention, DHAs are D
An embodiment which is at least one compound selected from HA, DHA ethyl ester and DHA triglyceride. 2. In the oral immunosuppressant of the present invention, DHAs are D
An embodiment in which it is blended as a mixture of fatty acids containing 95% or more of HAs. 3. The above 2. Of oral immunosuppressants
An embodiment containing 5% or more. 4. An embodiment in which the oral immunosuppressive agent of the present invention is a capsule or granule.

[0016]

EXAMPLES Reference examples, examples and experimental examples are given below for illustrating the present invention in more detail, but the present invention is not limited to these.

Reference Example 1 An aqueous solution prepared by dissolving 11.7 g of silver acetate in 10 ml of distilled water was added to 20.24 g of a fatty acid mixture containing 70% DHA and erucic acid as an impurity, and the mixture was shielded from light under a nitrogen atmosphere at -5 ° C. It was stirred for 1 hour. After stirring, the reaction solution was extracted twice with 200 ml of hexane, and the aqueous phase was recovered. Toluene in the recovered aqueous phase
200 ml was added, and the mixture was refluxed for 1 hour while shielding from light under a nitrogen atmosphere.

The toluene phase was recovered and 400 ml of distilled water was collected.
It was washed twice with 200 ml of saturated saline and then dehydrated with magnesium sulfate. Concentration under reduced pressure gave 1.4 g of fatty acid. As a result of examining the composition of fatty acids, the purity of DHA was
It was 98.2%.

Reference Example 2 30 g of a fatty acid ethyl ester mixture containing 42.3% DHA ethyl ester and palmitic acid as an impurity
An aqueous solution of 25 g of silver nitrate dissolved in 15 ml of distilled water is added to
The mixture was stirred for 2 hours under a nitrogen atmosphere in the dark. After the reaction, the aqueous phase was separated and obtained from the fatty acid ethyl ester mixture.

The obtained aqueous phase was extracted twice with 150 ml of benzene, the obtained benzene phase was washed with distilled water and saturated saline, and dehydrated with magnesium sulfate. Concentration under reduced pressure gave 6.3 g of fatty acid ethyl ester. As a result of examining the composition of the fatty acid ethyl ester, the purity of the DHA ethyl ester was 96.4%.

Reference Example 3 12.85 g of N, N'-carbonyldiimidazole and a fatty acid (purity of DHA obtained in the same manner as in Reference Example 1)
98.5%) 21.9 g and dissolved in 30 ml of anhydrous acetonitrile,
The mixture was stirred under a nitrogen atmosphere at room temperature for 1 hour. To this reaction solution was added 20 ml of anhydrous dimethyl sulfoxide solution containing 1.84 g of glycerol, and 20 ml of dimethyl sulfoxide solution containing sodium imidazole [5 g of imidazole and 1.44 g of sodium hydride (50% in oil) in anhydrous dimethyl sulfoxide (20 ml). Prepared by reacting for 1 hour with 5 ml of pyridine, and stirred at room temperature for 3 hours under a nitrogen atmosphere.

A hexane / 1N hydrochloric acid / methanol mixture (300 ml: 100 ml: 100 ml) was added to the obtained reaction liquid, and the mixture was separated with a separating funnel. The aqueous layer was further extracted 3 times with 200 ml of hexane and combined with the previous hexane layer. The hexane layer was mixed with 100 ml of saturated aqueous sodium hydrogen carbonate solution and 200 ml of saturated saline solution.
Washed with ml. The hexane layer was concentrated, and the concentrate was dissolved in a hexane / ether mixture (96: 4) and passed through a column filled with 400 g of silica gel treated with the same solvent system, and the eluate eluted with 8 l of the same solvent was added. A thin-layer chromatography was performed to obtain 18.53 g of DHA glyceride containing DHA triglyceride. The purity of DHA triglyceride was 97.3%.

Example 1 A gelatin soft capsule was filled with an oily substance obtained by adding 0.3% by weight of α-tocopherol to 99.7% by weight of the fatty acid obtained in Reference Example 1. The content of DHA in the capsule was about 96%.

Example 2 A gelatin soft capsule was filled with an oily substance obtained by adding 0.3% by weight of α-tocopherol to 99.7% by weight of the fatty acid ethyl ester obtained in Reference Example 2. The content of DHA ethyl ester in the capsule was about 95%.

Example 3 18 g of DHA glyceride obtained in Reference Example 2 Lactose 31
5 g, 120 g corn starch and 25 crystalline cellulose
g were uniformly mixed and granulated by an extrusion granulator using a screen with a diameter of 0.5 mm. The granules were coated with 1.9 kg of a film coating solution having the following composition using a fluidized granulator to obtain enteric coated granules.

Coating liquid composition: Hydroxypropyl methylcellulose phthalate 5.0 (w / w)% Stearic acid 0.25 (w / w)% Methylene chloride 50.0 (w / w)% Ethanol 44.75 (w / w)%

Example 4 20 g of the fatty acid obtained in Reference Example 1, 100 g of lactose, 36 g of corn starch, 30 g of crystalline cellulose, 10 g of carboxymethyl cellulose calcium and 4 g of magnesium stearate were uniformly mixed, and then mixed in a single tableting machine. Diameter 7.
One tablet of 200 mg was made with a 5 mm punch.

The immunosuppressive effect exhibited by the oral immunosuppressant of the present invention was confirmed by the following experimental examples. Experimental Example 1 (DH for mouse oxazolone-induced ear edema
Effect of A) The effect of DHA on the delayed hypersensitivity reaction [Arch. Int. Pharmacodyn. 269, 153-166 (1984)] induced by oxazolone in mice was examined. ICR of 60 animals
Male male mice (around 20 g) were divided into a control group, a vehicle administration group, a 97% DHA administration group and a 99% DHA administration group, and each drug was orally administered for 10 consecutive days. The vehicle group was administered with a solution of 5% gum arabic at 10 ml / kg / day, and the 97% DHA administration group and the 99% DHA administration group each received D
A suspension of 5% gum arabic containing 300 mg / ml of an HA-containing oily substance was administered at 10 ml / kg / day.

On the 4th day from the start of administration, the abdomen of each group of mice was shaved, and then 50% of a 2% oxazolone-ethanol solution was added.
l was applied twice for sensitization. 6 days after sensitization (start of administration 10
On the first day, 10 μl of 2% oxazolone-acetone solution was applied to the left ear once. The thickness of the ears before and 24 hours after the application was measured with a dial gauge, and the increase in the thickness of the ears after the application was determined and summarized in Table 1 and FIG.

In addition, the hypersensitivity inhibitory rates for the control group and the vehicle administration group were calculated, respectively, and the significant difference test (Bonf
erroni's method). The results are also summarized in Table 1 and FIG.

[0031]

[Table 1]

Experimental Example 2 (Effect of DHA on rat carrageenin paw edema) For the purpose of investigating the action of DHA on rat carrageenin paw edema which is an acute inflammation model, a vehicle administration group and 97
Divided into% DHA administration groups (similar to Experimental Example 1), 3 g / kg of each drug was continuously orally administered for 10 days from 9 days before carrageenin administration. The left paw volume (a) of the rat was measured using a paw volume measuring device (PLETHYSMO METER). Next, 1% Seakem carrageenin 402 saline solution as a inflammatory substance
0.1 ml was subcutaneously injected into the footpad, and the paw volume (b) was measured 3 hours after the injection.

The difference between the foot volumes (b) and (a) is the edema degree (Sw
2 is shown in FIG. As shown in Figure 2, 97% D
It was revealed that the HA-administered group showed a significant foot edema inhibitory action as compared with the vehicle-administered group, and DHA had an anti-inflammatory action.

Experimental Example 3 (Effect of DHA on Type II Collagen-Induced Mouse Arthritis) A 1: 1 emulsion of bovine type II collagen (3 mg / ml) and FCA (complete Freund's adjuvant) was applied to DBA / 1J male mice. 100 μl / body in the left footpad
(Collagen 150 μg / body) was administered (primary immunization). twenty one
After a day, bovine type II collagen (3 mg / ml) and FI
A 1: 1 emulsion with A (Incomplete Freund's adjuvant) was intradermally administered to the ridge at 100 μl / body (secondary immunization).

For the purpose of comparing the efficacy of DHA for type II collagen-induced mouse arthritis with EPA, a vehicle administration group, a 97% DHA administration group (same as in Experimental Example 1) and an EPA administration group (97% EPA-containing oily substance) 300 mg / ml
Suspension of 5% arabic gum containing), and oral administration of the test drug was started simultaneously with the primary immunization for 28 days, that is, 2
The drug was administered until 7 days after the next immunization (Day 7 in FIGS. 3 and 4).

After the second immunization, 2,4,7,8,9,10,11,12,13,14,1
The joints of the extremities were visually observed on days 6, 18, 21, 24 and 29, and each extremity of the mouse was classified according to the following five-step criteria.

Score Symptom 0: No symptom 1: Reddened swelling with only one finger 2: Reddened swelling of two or more fingers or reddened swelling of wrist or ankle 3: Reddened swelling of hand or entire foot 4: Hand reaching maximum Alternatively, the red swelling of the whole foot The obtained results are shown in FIG. 3 and FIG. 4, respectively, as the incidence (Incidence) and the average score.

In the 97% DHA administration group, a clear inhibitory effect was observed in the incidence of arthritis and the average score, as compared with the control vehicle administration group. In the 97% DHA-administered group, the onset was suppressed during the administration period as well as the onset over 5 days after the end of drug administration. Even in the mean arthritis score, inhibition of the rise in the mean score for 5 days after the end of drug administration was observed, and a tendency for exacerbation suppression was also observed during the subsequent observation period.

On the other hand, in the comparative EPA-administered group, the suppressive effect on the incidence and score of arthritis was not observed at all as in the 97% DHA-administered group.

Experimental Example 4 The change in body weight during the period of Experimental Example 1 was traced, and the change was almost the same as that of the control group. Therefore, it was confirmed that there was no problem of side effects due to oral administration of DHA.

[0041]

As described above, the oral immunosuppressive agent of the present invention contains fatty acids containing DHAs as an active ingredient, and therefore, renal disorders against autoimmune diseases such as collagen disease,
It can be used without side effects such as gastrointestinal disorders. In particular, 95% or more of fatty acids are DHAs, and more preferably 95% or more of DHAs are contained in the preparation, the DHAs as an active ingredient are efficiently absorbed, and the onset of drug effect is rapid. In addition, the problem of side effects due to other contained components can be avoided, and the dosage form of a preparation can be reduced, which facilitates administration.

[Brief description of drawings]

FIG. 1 is a diagram showing the effect of DHA on ear edema in Experimental Example 1.

FIG. 2 is a diagram showing the effect of DHA on foot edema in Experimental Example 2.

[Fig. 3] DH for the incidence of arthritis in Experimental Example 3.
It is a figure which shows the influence of A.

FIG. 4 is a diagram showing the influence of DHA on the average score of arthritis in Experimental Example 3.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yoshihito Nawa 2-25-1 Otani Otani, Hirakata-shi, Osaka Prefecture Midori Cross Central Research Institute Co., Ltd. (72) Junko Wada Invited 2-25 Otani, Hirakata-shi, Osaka No. 1 Incorporated Midori Cross Central Research Institute (72) Inventor Fujio Kobayashi Invited by Hirakata City, Osaka Prefecture 2-25 Otani Incorporated Midori Cross Central Research Institute (72) Inventor Akio Umemura Invited by Hirakata City, Osaka Prefecture 2-25-1 Otani Inside the Midori Cross Central Research Institute (72) Inventor Masanori Sugiura Invited to Hirakata City, Osaka Prefecture 2-25-1 Otani Inside the Midori Cross Central Research Institute (72) Inventor Kazuyoshi Yazawa 1-28-10 Unomori, Sagamihara City, Kanagawa Prefecture

Claims (5)

[Claims] 1. An oral immunosuppressive agent containing docosahexaenoic acids. 2. The oral immunosuppressive agent according to claim 1, wherein the docosahexaenoic acids are at least one compound selected from docosahexaenoic acid, docosahexaenoic acid ethyl ester, and docosahexaenoic acid triglyceride. 3. The oral immunosuppressant according to claim 1, wherein the docosahexaenoic acids are blended as a mixture of fatty acids containing 95% or more of docosahexaenoic acids. 4. The oral immunosuppressant according to claim 3, which comprises 95% or more of docosahexaenoic acids. 5. A capsule or a granule as claimed in claim 1.
The oral immunosuppressive agent according to any one of 4 to 4.