JPH07138187A - Oral immunotolerance, oral rheumatoid arthritis therapeutic agent and functional food - Google Patents

Abstract

PURPOSE:To obtain an oral tolerogen useful for treating and preventing rheumatoid arthritis (RA) and an oral therapeutic agent for the RA and a functional food containing the tolerogen. CONSTITUTION:This oral tolerogen comprises a peptide having a partial sequence in II type collagen. The mechanism of onset of RA is considered as an autoimmune disease using a self-component containing the II type collagen as an antigen. Since the immune response to the II type collagen is suppressed in vivo by administering the oral tolerogen, the RA can be treated and prevented to manifest effects on the practicable treatment and prevention of the RA by the oral administration in which the administration thereof is especially simple.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an oral immunotolerogen, an oral rheumatoid arthritis therapeutic agent and a functional food. More specifically, the present invention relates to an oral immunotolerogen that is composed of a peptide having a specific amino acid sequence and is useful for the treatment / prevention of rheumatoid arthritis, an oral rheumatoid arthritis therapeutic agent and a functional food containing the same.

[0002]

2. Description of the Related Art Rheumatoid Arthrit
is, hereinafter referred to as RA) is one of the chronic diseases with many affected patients and is a systemic disease that causes inflammation in connective tissue. The disease mainly causes nonspecific inflammation in the synovium of joints, presents with the pathology of polyarthritis throughout the body, and causes cartilage and bone damage. Although the pathogenic mechanism of RA has not been sufficiently analyzed, it is considered to be an autoimmune disease involving lymphocyte antigen (HLA) -DR4 and involving activated T cells (Lancet 341 , 283, 1993). . Since type II collagen is a major structural protein in cartilage and administration of type II collagen to experimental animals causes morphologically similar symptoms to rheumatoid arthritis, type II collagen is one of the autoantigens of this disease. (J. Exp. Med. 146 , 857,
1977; Lab. Invest. 54 , 26, 1986). Anti-rheumatic agents (gold salt preparations, D-penicillamine, etc.), non-steroidal agents, immunosuppressants, etc. are commonly used as therapeutic agents for RA, but it seems that sufficient effects cannot be obtained by administration of these agents. In that case, a steroid drug having a strong anti-inflammatory action and immunosuppressive action is used.

[0003]

RA by the above-mentioned agents
The treatment of is a symptomatic treatment and cannot be said to be a curative treatment. It has been pointed out that steroids may cause serious side effects, and therefore it is necessary to exercise caution when using them and always consider dose reduction or discontinuation. The most desirable treatment for RA is to reduce joint inflammation based on a disease-specific mechanism and it is desirable that the drug used be non-toxic.
From this point of view, a therapeutic method for RA based on immunological tolerance is drawing attention. Immune tolerance is a phenomenon in which when an animal is treated with an antigen under certain conditions, an immune response such as antibody production does not occur even if an appropriate immunization with this antigen is performed next, leading to immunological tolerance. The substance is called the tolerogen. As a method for treating RA by immunological tolerance, a peptide having a partial sequence of type II collagen is used as an immunotolerant, and this peptide can be intravenously or intraperitoneally administered to neonatal rats to suppress the onset of RA. Reported (J. Exp. Med. 170 , 1999, 1989; J. Immunology
151 , 500, 1993).

In the above-mentioned method, the immunotolerogen is intravenously or intraperitoneally administered, but the administration of the immunotolerogen by intravenous or intraperitoneal administration is not only complicated, but the peptide of the immunotolerogen is administered to RA patients. In the case of continuous intravenous or intraperitoneal administration to RA, it is expected that a severe allergic reaction or shock may be caused in RA patients. Therefore, a more convenient and safer method for treating RA is desired. Further, it is more preferable not only as a medicine but also for treating / preventing RA through daily intake of food, and functional foods for treating / preventing RA are also demanded. In order to solve the above problems, the present inventors have made earnest studies on a method for treating RA by a simple administration method, and have found that RA can be treated / prevented by oral tolerance. Oral immunological tolerance is a phenomenon in which the systemic immune response to an antigen is lost when the antigen enters orally through the intestinal tract. In oral tolerance, the antigen is orally absorbed through the intestinal tract. When
Since it is affected by various organs and functions such as Peyer's patches, intestinal epithelial cells and adjacent lymphocytes, portal vein, liver, etc., it does not cause allergic reactions and shocks, and immunosuppressive therapy for allergy and organ transplantation. Has been tried as. The present inventors have focused their attention on this oral tolerance, and as a result of repeated research, when the peptide consisting of the partial sequence in type II collagen was orally administered, RA
The present invention has been completed by finding that the onset of the disease can be suppressed and the peptide is useful as an oral immunotolerant. The present invention was made on the basis of such findings, and prevents RA
It is an object of the present invention to provide an oral immunotolerogen used for therapeutic purposes, an oral RA therapeutic agent and a functional food containing the same.

[0005]

Means for Solving the Problems The oral immunotolerogen of the present invention made to solve the above problems comprises the peptide represented by SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof. Further, the therapeutic agent for oral RA of the present invention contains the above-mentioned oral immunotolerance as an active ingredient, and the functional food of the present invention further contains the above-mentioned oral immunotolerance.

The oral immunotolerogen of the present invention comprises the peptide represented by SEQ ID NO: 1 or a pharmacologically acceptable salt thereof. The pharmacologically acceptable salts include inorganic metal salts such as alkali metal salts (eg sodium salt, potassium salt etc.) and alkaline earth metal salts (magnesium salt, calcium salt etc.), ammonium salts, organic bases. Salt (eg,
Trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, etc.), organic acid salt (for example, formate salt, acetate salt, maleate salt, tartrate salt, methanesulfonate salt, benzenesulfonate salt, toluenesulfonate salt, etc.), Examples thereof include inorganic acid salts (for example, hydrochloride, hydrobromide, sulfate, phosphate, etc.).

The peptide represented by SEQ ID NO: 1 (hereinafter referred to as the peptide of the present invention for convenience) can be usually prepared by a stepwise introduction of amino acids by an organic chemical synthesis method. , Type II collagen or type II collagen-derived gelatin and / or the content thereof can also be prepared by a hydrolysis treatment using an enzyme, an acid or an alkali.

Solid-phase peptide synthesis or liquid-phase peptide synthesis is known as an organic chemical synthesis method, and is described in detail, for example, in Nobuo Izumiya et al., "Basics and Experiments of Peptide Synthesis (published by Maruzen)". ing. More specifically, in the liquid phase peptide synthesis method, the carboxyl group of the amino acid to be located at the C-terminus is replaced with an appropriate protecting group [eg, benzyl group (Bzl), t-butyl group (t-Bu), etc.]. The amino group of the amino acid to be protected and located at the second position from the C-terminus is protected by a suitable protecting group [eg, t-butyloxycarbonyl group (Boc),
Benzyloxycarbonyl group (Z), etc.], and these amino acids are dissolved in an appropriate solvent [eg, dimethylformamide (DMF), tetrahydrofuran, etc.] and condensed with a condensing agent [eg, dicyclohexylcarbodiimide (DCC)]
And, if necessary, in the presence of a racemization inhibitor [eg, 1-hydroxybenzotriazole (HOBT), etc.] at low temperature (4
The reaction is performed at about 10 ° C for about 24 hours. Then, the amino protecting group of the product is removed by a conventional method using trifluoroacetic acid or the like to obtain a dipeptide. Then, the obtained dipeptide is reacted with a third amino acid (which also has an amino group protected) in the same manner as above to obtain a tripeptide. Further, the same procedure is repeated to sequentially bond the required amino acids to obtain the target peptide with the protecting group attached. When the amino acid to be reacted has a side chain functional group, it needs to be protected prior to the peptide synthesis reaction. For example, the amino group at the 6-position of lysine is protected by a tosyl group (Tos). After completion of the final reaction, these protective groups can be removed by catalytic reduction, hydrogen fluoride (HF) or the like to obtain the target peptide.

The solid phase peptide synthesis method can be carried out by using a conventional peptide synthesizer (for example, Model 430A manufactured by Applied Biosystems). In this method, an amino acid protected by a Boc group is sequentially bonded to the N side of a phenylacetamidomethyl (PAM) resin (ie, amino acid-OCH ₂ -PAM) to which the C-terminal amino acid of a target peptide is bonded by automatic control. Then, a sample in which the protective group and PAM resin are bound to the target peptide can be obtained. Next, a scavenger such as anisole is added to this sample, HF is introduced, and the target peptide can be released by reacting at -2 ° C for 1 hour. The liberated peptide is washed with anhydrous ether or the like, extracted with water containing acetic acid, and lyophilized to give the desired peptide.

The peptide of the present invention can also be obtained by a genetic engineering method. For example, a DNA fragment encoding the amino acid sequence of SEQ ID NO: 1 is synthesized, and this DNA fragment is inserted into an appropriate expression vector by a conventional method and the expression thereof is expressed. The desired peptide can be prepared by transforming an appropriate host with the vector, culturing the obtained transformant, and isolating and purifying from the culture. In addition, when bacteria and / or yeast are expressed as hosts for the purpose of providing them to functional foods, in addition to isolation and purification of the peptide of the present invention from the culture, the bacteria and / or yeast are killed. The bacterium or its powder may be used as it is.

The peptide of the present invention can also be obtained by a method of hydrolyzing type II collagen or type II collagen-derived gelatin and / or the content thereof with an enzyme, an acid or an alkali. For example, type II Collagen or type II collagen-derived gelatin and / or the inclusion thereof is used as an enzyme (for example, chymotrypsin, trypsin,
V8 protease) and heat treatment to deactivate the enzyme, and then solid-liquid separation is performed by means such as filtration and centrifugation, and the obtained liquid is separated by means such as ultrafiltration and gel filtration. By purifying, the target peptide can be prepared. When an acid or an alkali is used as a catalyst, the same hydrolysis treatment may be performed using an acid or an alkali instead of the enzyme in the above method.

The thus obtained peptide of the present invention can be purified by subjecting it to conventional means such as high performance liquid chromatography and ion exchange column chromatography.
In the present invention, the peptide of the present invention may have one or more amino acids added to its N-terminal and / or C-terminal, as long as it is useful as an oral immunotolerant for treating / preventing RA. One or more amino acids in the amino acid sequence of the peptide may be deleted or replaced with another amino acid. The salt of the peptide of the present invention can be prepared by adding an acid or base to the peptide according to a conventional method.

[0013] The oral immunotolerogen of the present invention has an RA onset inhibitory action, and treats RA in mammals including humans,
It is useful for prevention and is used as medicines, functional foods, etc. In particular, since the peptide of the present invention is a peptide consisting of the partial sequence of type II collagen, it is presumed that it is highly safe.

The RA therapeutic agent of the present invention contains the above-mentioned oral immunotolerance as an active ingredient, and is orally administered for the purpose of treating / preventing RA. Upon administration, the active ingredient is mixed with a solid or liquid pharmaceutical non-toxic carrier suitable for oral administration, and is administered in the form of a conventional pharmaceutical preparation. Examples of such a preparation include tablets, granules, Examples include solid agents such as powders and capsules, solutions such as solutions, suspensions and emulsions, and freeze-dried preparations. These preparations can be prepared by conventional means for preparation. Examples of the nontoxic carrier for pharmaceutical use include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride,
Polyethylene glycol, hydroxyethyl starch,
Examples thereof include ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, albumin, water and physiological saline. If necessary, conventional additives such as stabilizers, lubricants, wetting agents, emulsifiers and binders can be added as appropriate. In the therapeutic agent for RA of the present invention, the dose of the active ingredient is appropriately selected depending on the patient's age, body weight, symptoms, degree of disease, administration schedule, formulation form, etc.
It is determined, for example, 0.05 to 500 μg / kg per day
The dose may be about the body weight, and the administration may be divided into several times a day.

Further, the functional food of the present invention comprises the above-mentioned oral immunotolerant, which can be used as it is or with various nutrients added or in foods or drinks to treat or prevent RA. It is eaten as a useful functional food (or food material). For example, after adding the appropriate auxiliaries described above, using conventional means, an edible form,
For example, it may be formed into granules, granules, tablets, capsules, pastes, etc. for use in food, and various foods (for example, processed meat products such as ham and sausages, processed fish products such as kamaboko, chikuwa, and bread). , Butter, milk powder, etc.) or added to drinks such as water, fruit juice, milk, and soft drinks. Ingestion amount of the peptide of the present invention in the form of such functional food, age, weight, symptoms,
It is appropriately selected and determined depending on the degree of disease, the form of food, etc., and is, for example, about 0.05 to 500 μg / kg body weight per day, but the peptide of the present invention has an advantage that it does not adversely affect the living body even if ingested in a large amount. Therefore, you may ingest more than that.

[0016]

As described above, the pathogenic mechanism of RA is
It is considered to be an autoimmune disease in which self-components including type II collagen are used as antigens, and by administering the oral immunotolerogen of the present invention, an immune response at the site of inflammation where type II collagen is present in vivo Is suppressed, RA
Can be treated and prevented. Therefore, it is possible to treat RA that has already developed illness and to prevent RA, and in particular, by administering to infants, children or adults before illness in a family with frequent RA, RA
Can be effectively prevented. Further, there is an effect that RA can be treated / prevented by oral administration which is easy to administer. Further, the therapeutic agent for RA and functional food of the present invention comprises an oral immunotolerogen having the above-mentioned effects, and is useful for treating / preventing RA.

[0017]

The present invention will be described in more detail based on the following examples and test examples, but the present invention is not limited to these examples. Production Example Synthesis of Peptide The peptide shown in SEQ ID NO: 1 was prepared by a solid phase method using a commercially available solid phase synthesizer (manufactured by Applied Biosystems) according to a conventional method. The obtained peptide was subjected to high performance liquid chromatography using a Shiseido column CAPCELL SG120 (C18 column) (mobile phase; 0.1% TFA: acetonitrile = 95: 5 to 45:55 linear gradient for 50 minutes). When the dissolution behavior was tested, it was eluted in about 24 minutes.

Test Example 1 Oral Tolerance Method and Immunization Method As a model of arthritis, collagen-induced arthritis (CI
A) was used. CIA is a system that causes polyarthritis by sensitizing mice and rats with type II collagen, and is often used as an animal experimental model of human RA. More specifically, the peptide of the present invention was orally administered to DBA / 1 mice 5 times at a dose of 5 μg / animal / day, and 100 μg of bovine type II collagen was immunized intradermally in the ridge with a complete adjuvant. The same amount of bovine type II collagen was boosted with incomplete adjuvant.
As a control, ovalbumin (OVA) was orally administered instead of the above peptide, and the same test was conducted. After that, the symptoms of CIA were observed and evaluated from 0 to 3 (evaluation 0 is a healthy state, evaluation 1 is a slightly reddish and swollen state, evaluation 2
Was completely red and swollen and developed, and evaluation 3 was a state in which swelling reached its peak) according to the intensity of arthritis. Using this evaluation, the average evaluation value (Index of Arthritis, IA) for each group was determined based on the following formula.

As shown in this equation, the IA sums the evaluation values of each mouse for each group and gives the most C
This is a calculation of what percentage of the total evaluation value in the state where the IA is deteriorated, and the larger the value is, the more severe the symptoms of arthritis are. The results of the above test are shown in FIG. ● in the figure
Indicates a group to which the peptide of the present invention was orally administered, and O indicates a group (control) to which OVA was orally administered. As is clear from FIG.
In the control group, IA was high and the symptoms of arthritis were observed, whereas in the group to which the peptide of the present invention was orally administered, the onset of arthritis was significantly suppressed.

Test Example 2 Examination of Dose Dependence In order to examine the difference in inhibitory effect depending on the dose, the amount of peptide administered per administration was 1, 5 and 25 μg / animal.
The test day was changed and tested in the same manner as in Test Example 1 above.
The result is shown in FIG. In the figure, Δ, ●, and ∇ indicate groups to which the peptide of the present invention was orally administered at 1, 5 or 25 μg / animal · day, respectively, and ∘ indicate groups to which OVA was orally administered (control). As shown in FIG. 2, there was a difference in the suppressive ability depending on the dose, and the effect was observed in the dose groups of 5 μg and 25 μg.

Example 1 Peptide of the present invention 0.5 mg Magnesium stearate 5 mg Corn starch 20 mg Lactose 174.5 mg According to a conventional method, the mixture having the above composition was tableted to give tablets.

Example 2 Peptide of the present invention 0.5 mg Magnesium stearate 5 mg Lactose 194.5 mg According to a conventional method, a mixture having the above composition was filled in a hard gelatin capsule to obtain a capsule.

Example 3 Natural juice (concentrated juice reduction) was mixed with the peptide of the present invention at a ratio of 0.5 mg per 200 ml of natural juice, followed by sterilization according to a conventional method and aseptic packaging to obtain a juice product. .

Example 4 To a ground meat for wiener sausage, the peptide of the present invention was mixed at a ratio of 30 μg per 15 g of the ground meat, and the sausage casing was filled, smoked and sterilized according to a conventional method.
After cooling, it was packaged to obtain a wiener sausage.

[0025]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 26 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ala Ser Gly Pro Leu Gly Pro Lys Gly Gln Thr Gly Glu Pro Gly Ile 1 5 10 15 Ala Gly Phe Lys Gly Glu Gln Gly Pro Lys 20 25

[Brief description of drawings]

FIG. 1 is a diagram showing IA (Index of Arthritis) in a mouse tolerated by oral administration of the peptide of the present invention.

FIG. 2 shows the IA (Index of Arthriti) in mice tolerated by oral administration of the peptide of the present invention at different doses.
It is a figure which shows s).

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Toshishi Yamura 1-1-1, Yayoi, Bunkyo-ku, Tokyo Tokyo University Faculty of Agriculture, Faculty of Agriculture, Department of Agricultural Chemistry, Animal Products Research Laboratory (72) Inventor, Tatsuhiro Hisatsune, Bunkyo-ku, Tokyo 1-1-1 Yayoi Laboratory, Department of Animal Science, Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo (72) Takayuki Matsumoto, 3-3 Midorigahara, Tsukuba, Ibaraki Pref.

Claims (3)

[Claims] 1. An oral immunotolerogen comprising a peptide having the amino acid sequence shown by SEQ ID NO: 1 or a pharmacologically acceptable salt thereof. 2. A therapeutic agent for rheumatoid arthritis, which comprises the oral immunotolerogen according to claim 1 as an active ingredient. 3. A functional food containing the oral immunotolerant according to claim 1.