JP4911894B2 - Composition for oral administration for preventing rheumatoid arthritis - Google Patents

Description

  The present invention relates to a therapeutic composition for rheumatoid arthritis, and more particularly to a composition having an effect of treating or preventing rheumatoid arthritis, comprising an antibody having an anti-rheumatic action.

  Rheumatoid arthritis is an inflammatory joint disease that begins with redness, pain and swelling of the joints of the hands and feet, progresses to destruction and deformation of the joints, and leads to functional paralysis of the joints with pain. In the drug therapy, in order to stop inflammation, treatment with a combination of corticosteroids, immunosuppressants, acidic anti-inflammatory agents, organic gold compounds, anti-cytokine antibodies, and other drugs is performed. These drug therapies are so-called symptomatic treatments.

  The etiology of rheumatoid arthritis is generally unknown today, but studies based on the theory that autoimmunity to type II collagen, the main component of articular cartilage, is involved.

  There are many antigenic substances in the diet, but antibody production against these antigens usually does not occur when taken orally. This phenomenon is called oral tolerance.

  Antibody production is regulated by lymphocytes (helper T cells, Th) that work to promote antibody production and suppressive lymphocytes (suppressor T cells, Ts). Since antibody production is suppressed when suppressor T cells are dominant, animal-derived type II collagen is intended to activate suppressor T cells against type II collagen in order to suppress autoantibody production against type II collagen. Attempts have been made to administer a modified peptide derived from type II collagen. Examples of this include Patent Documents 1 to 3, which are all patents intended for oral tolerance.

  In order to contribute to the prevention and treatment of rheumatoid arthritis, the present inventors have elucidated the etiology. The etiology of rheumatoid arthritis is an internal factor, a constitutional and genetic factor of the patient, a breakdown of oral immunity in patients with rheumatoid arthritis, an external factor such as type II collagen derived from different animals in food, and The research started under the hypothesis that bacterial endotoxin absorption is caused by the digestive tract.

  The breakdown of oral immunity is as follows. Oral tolerance is a normal immune response to antigens taken orally, but due to intrinsic, constitutional, and environmental factors, normal tolerance cannot be induced and autoantibodies are produced. This is the idea. For the verification of this hypothesis, the arthritis-inducing activity of similar antigens and bacterial endotoxins was examined using mice for collagen arthritis in mice. A DBA / 1 mouse is a mouse having a genetic trait that easily develops type II collagen arthritis. When these mice were orally given chicken-derived type II collagen or heat-denatured, antibodies against mouse type II collagen were produced in the blood of the mice and arthritis developed. This arthritis was exacerbated by the combination of bacterial endotoxin (also known as lipopolysaccharide or LPS). Arthritis also developed after long-term administration of bacterial endotoxin alone, excluding chicken-derived type II collagen (Non-patent Document 1).

These results, together with antibodies to type II collagen, indicate that nonspecific activation or exhaustion of the immune system by bacterial endotoxins is involved in the development of rheumatoid arthritis, and that oral tolerance may not be upright. Yes. In this experiment, it was shown that heat tolerance could not be induced even for heat-denatured type II collagen prepared for the purpose of immune tolerance shown in Patent Documents 1 to 3 described above.
JP 07-138187 A JP 09-059176 A JP 09-255551 A K. Terato et al. Br. J. Rheum. 35,828-838, 1996

  An object of the present invention is to provide a composition having a function of preventing and treating rheumatoid arthritis and preventing recurrence.

  As a result of intensive studies to solve the above problems, the present inventors immunized cattle, goats, sheep, and chickens with vaccines each of type II collagen, gram-negative enterobacterial bacterial cells, or a mixture thereof, In the case of cows, goats and sheep, the antibody is produced and secreted as milk, and the chicken is secreted as chicken egg yolk antibody. These antibodies are used as food or pharmaceutical compositions to achieve the task. We have found out that we can do it and have reached the present invention. In other words, by ingesting foods or medicines containing these antibodies, it is inactive by reacting food-derived type II collagen, endotoxins produced by bacteria in the oral cavity or intestine with the corresponding antibodies. It has been found that it is possible to prevent absorption from the gastrointestinal tract or gastrointestinal tract and, as a result, prevent rheumatoid arthritis, or improve the pathological condition and be useful for treatment.

That is, the present invention is as follows.
1) a composition for treating rheumatoid arthritis comprising an antibody having an anti-rheumatic activity as an active ingredient,
2) The rheumatoid arthritis therapeutic composition according to 1) above, wherein the antibody having an anti-rheumatic action is an anti-type II collagen antibody,
3) The composition for treating rheumatoid arthritis according to claim 2, wherein the anti-type II collagen antibody is at least one of an anti-chicken type II collagen antibody, an anti-bovine type II collagen antibody, and an anti-pig type II collagen antibody,
4) The composition for treating rheumatoid arthritis according to claim 1, wherein the antibody having antirheumatic activity is an antibacterial endotoxin antibody,
5) The composition for treating rheumatoid arthritis according to claim 1, wherein the antibody having antirheumatic activity is a mixture of an anti-type II collagen antibody and an antibacterial endotoxin antibody at an arbitrary ratio, and 6) anti-type II The therapeutic agent composition for rheumatoid arthritis according to claim 1, comprising at least one of a collagen antibody and an antibacterial endotoxin antibody, and a total antibody amount of 0.1% by mass or more.

  According to the present invention, arthritis is caused by a reaction between a type II collagen derived from food or a food-derived pharmaceutical containing it, a toxin produced by bacteria in the oral cavity or intestine, and an antibody in the composition. It is possible to provide a therapeutic composition for rheumatoid arthritis that can be used for the prevention or treatment of rheumatoid arthritis.

  In the present invention, two kinds of antigens that have been found to be one of the factors of rheumatoid arthritis are inoculated and immunized to livestock animals. The antibody obtained after separation from blood is used as a composition for food or medicine. Each item will be described below.

1. Antigen used for immunization In the present invention, animal type II collagen and Gram-negative bacteria or endotoxin (LPS) thereof are preferably used as antigens.
1) Commercially available products (Chondrex) can be used for type II collagen of bovine, porcine, and chicken. Antigens may be used alone or in combination, but a mixed vaccine prepared by mixing chicken, bovine, porcine type II collagen in any combination is efficient and economical.

2) Antigens used for immunization to produce antibodies against bacterial endotoxins are human parasitic Gram-negative bacteria that fall into the genus shown below. That is, Fusobacterium, Veillonella, Megasphaera, Neisseria, Moraxella, Branhamella, Acinetobacter, Citrobacter, enterobacter Enterobacter (Escherichia), Hafnia, Klebsiella, Morganella, Proteus, Providencia, Salmonella, Serratia, Shigella, Yia Formalin-treated or heat-killed bacteria such as Vibrio, Aeromonas, Prelesiomonas, Haemophillus, Pasteurella, Pseudomonas, Legionella, etc. To do. The vaccine may be a single vaccine or a mixed vaccine, but a mixed vaccine is efficient. The culturing of the fungi may be performed by the usual method described in the book.

3) Animals live in symbiosis with microorganisms, and in the digestive tract of each animal species, microorganisms common to humans and microorganisms common to humans survive, and these animals become antigens and animals produce and possess antibodies. . These are called natural antibodies. Since antibodies against gram-negative bacterial endotoxins common with humans also exist as natural antibodies, these can also be used.

2. Selection of animals It is generally known that an animal does not produce an antibody against an autologous or cognate antigen when injected. Furthermore, the amount of antibody produced tends to increase as the heterogeneous distance between the animal species immunized with the antigen increases. Therefore, for example, for the production of antibodies against bovine type II collagen, it is desirable to immunize chickens with bovine type II collagen and recover them as chicken egg yolk antibodies. In addition, an antibody against chicken type II collagen can be obtained by immunizing a cow with chicken type II collagen and obtaining the antibody in milk or serum. Thus, animals immunized with type II collagen are inoculated with heterologous type II collagen. If it is derived from different species, it is efficient and economical to mix a plurality of collagens and inject them as a mixed vaccine.
Since bacterial components are antigenic for all animals, they can be mixed and injected as a vaccine.

3. Antigen inoculation method Animals, such as cows and dairy goats, sheep, and chickens, can be in the form of an aqueous solution or suspension. However, proteinaceous antigens such as collagen can be mixed with various immune adjuvants. Is desirable. For a single immunization, dissolve 10-1000 μg of protein dry weight in 0.1-10 ml and suspend in 0.1-10 ml and use an equal volume of Freund's complete and incomplete adjuvants such as TiterMax Gold or Ribi Adjuvant R-730. As a mold emulsion, inject into 0.1 to 10 ml at one site, divided into 1 to 10 sites, intradermal, subcutaneous and intramuscular. Do this once a week or once in February.

  For the production of antibodies against endotoxin, 0.1 to 10 ml of a mixture of one type of killed bacteria or a mixture of several killed bacteria of 0.01% to 10% wet weight is given subcutaneously, intramuscularly, subcutaneously or intravenously. Administer once every 4 weeks. It is also possible to immunize with an adjuvant to produce antibodies against endotoxins. The dead bacteria solution is injected as a water-in-oil emulsion using Freund's complete and incomplete adjuvants, such as TiterMax Gold or Ribi Adjuvant R-730, divided into 1 to 10 sites intradermally, subcutaneously, or intramuscularly. Do this once a week or once in February.

4). Antibody collection 1) Milk antibody collection Milking is carried out by hand or using a milking machine in the usual manner.
Since antibodies lose activity upon heating above 72 ° C., avoid excessive heating to the final product. For sterilization of raw milk, treatment at 72 ° C. or lower, ultraviolet irradiation sterilization or radiation irradiation sterilization are suitable. Milk is centrifuged to remove fat, spray dried to skim milk, fat is removed, hydrochloric acid is added to pH 4.6, precipitated casein is removed by filtration, and low molecular weight substances such as lactose and minerals are added. Components obtained by removing the components by ultrafiltration to make whey, and fractions mainly consisting of antibody proteins excluding lactalbumin and lactoglobulin by ultrafiltration are cryospray-dried or freeze-dried, and the antibody content is 0.1%. It can be set as the powdery antibody composition containing about 90% from a stand. Usually, 1 to 100 g of total antibody is obtained from 1 ton of raw cow milk excluding colostrum, and an average of 30 g. Commercially available whey, whey protein concentrate (WPC), and whey protein isolate (WPI) containing antibodies can also be used.

2) Collection of serum antibodies The blood of immunized animals has a high antibody content and is excellent as a raw material.
The collected blood is centrifuged to obtain serum, and then the gamma globulin fraction that precipitates at 1/3 saturation of ammonium sulfate is collected by filtration, dissolved in water, desalted, and low-temperature spray-dried or freeze-dried to obtain a powder. Usually, the antibody content is 70 to 90%, and about 15 g of total antibody is obtained from 1 kg of blood.

3) Collection of chicken egg antibody From the egg yolk of immunized chicken egg, 0.9 to 1.5 g of 90% pure IgY antibody is generally obtained from 1 kg of chicken egg using GIT kit [Cosmo Bio Co., Ltd.]. The antibody solution is powdered by low temperature spray drying or freeze drying.

5. Antibody quantification method The amount of antibody is independent of the antigen specificity of the antibody, and the total antibody amount is measured, and the ELISA method (Fujii K, et al. J Immunol Both measurement methods according to Methods 1989; 124: 63-70) are used.

1) Measurement of total antibody amount Protein G columns are commercially available as antibody purification affinity columns for efficiently purifying antibodies (Amersham Biosciences, etc.). When a solution containing an antibody is passed at a pH near neutral, the antibody can be adsorbed by nearly 100%. After washing the column, when an acidic solution having a pH of 2.5 to 2.8 is passed, the antibody is released from the column and collected. Since the absorbance at an ultraviolet wavelength of 280 nm of the antibody 1% solution is about 14 in common with the species difference, the amount of antibody is determined from the absorbance.

2) Measurement of total IgY A commercially available egg yolk antibody adsorption column (HiTrapIgYPurificationHP, Amersham Bioscience) can be easily quantified. The antibody-containing solution is adsorbed with 0.5 M potassium sulfate-containing 20 mM phosphate buffer solution pH 7.5, washed with the same solution, eluted with 20 mM phosphate buffer solution pH 7.5, and the collected IgY antibody is calculated from the absorbance.

3) Measurement of affinity-purified specific antibody by ELISA method Collagen and bacterial endotoxin (LPS) are covalently bound to Actigel (Steligen) or CNBr activation (Sepharose Amersham Biosciencen) and used as an antigen affinity column for antibody purification. The antibody solution to be purified was allowed to flow through the column, only the target antibody was adsorbed to the column, and immunoglobulins that were not adsorbed on the column were washed out with a neutral buffer. The antibody bound to the column was eluted with 0.1 M glycine buffer at pH 2.8. The eluted antibody was further applied to a protein G column, a calibration curve was prepared by ELISA (enzyme immunoassay) using the purified antibody as a standard, and quantified. Conditions for sensitization of wells with each antigen and blocking solutions are described below.

・ ELISA measurement of anti-collagen antibody:
The collagen was diluted to 0.15M potassium phosphate buffer pH 7.2 to 10 μg / ml, and 50 μl thereof was placed in an ELISA plate well and coated at 4 ° C. overnight. For blocking, a solution obtained by adding 0.05 M Tris and 0.15 M salt to rabbit serum heated at 56 ° C. for 30 minutes and adjusting the pH to 7.8 is used as a blocking solution. This blocking solution is also used for dilution of the antibody measurement specimen.

・ ELISA measurement of antibody against bacterial endotoxin:
The ELISA plate well is coated with a solution containing LPS extracted from gram-negative bacterial cells with trichloroacetic acid in 0.05 M carbonate buffer at pH 9.5 at a concentration of 5 μg / ml. Blocking is performed with phosphate buffered saline containing 0.1% bovine serum albumin.

  The amount of antibody was determined by reacting peroxidase-labeled anti-bovine antibody, anti-goat antibody, anti-sheep antibody, and anti-IgY antibody, developing OPD as a substrate, measuring the absorbance, and using a standard antibody. The total antibody amount or specific antibody amount is determined from the prepared calibration curve.

6). Preparation of Antibody-Containing Composition An antibody-containing composition can be prepared by mixing a preparation containing an antibody against Gram-negative bacterial endotoxin and an antibody-containing preparation immunized with type II collagen, or a mixture of both. If necessary, food materials and excipients other than antibody-containing preparations can be added to produce foods and pharmaceuticals. The total antibody content of the final product is preferably 1% or more.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. In Examples,% is% by mass unless otherwise specified.

[Example 1]
a) Immune chicken type II collagen (chondrox) 2 mg with bovine collagen was dissolved in 1 ml of 0.05M acetic acid to give a 2 mg / ml collagen acetate solution. 2 ml of phosphate buffered saline (PBS) of the same amount as this was mixed to make 2 ml, and 1 ml of this was added to 1 ml of TiterMax gold, and stirred well to prepare an emulsion. Two ml of emulsion was subcutaneously divided into four necks of 4 pregnant cows, divided into two 1 ml portions, and immunized by four injections 60 days before, 30 days before, 30 days after delivery, and 60 days after delivery.

b) Bovine immunization with Escherichia coli O-111: B4 strain E. coli O-111: B4 strain is cultured and grown on a normal agar plate, scraped with 1% formalin-added physiological saline, and sterilized by heating at 80 ° C for 1 hour. did. After washing the heated bacterial solution, the turbidity was adjusted to an absorbance of 1.0 at a wavelength of 660 nm. 1 ml of the bacterial solution was added to 1 ml of TiterMax gold and stirred well to prepare an emulsion. Each 1 ml was injected subcutaneously into the neck skin of 3 pregnant cows twice, 60 days before and 30 days before the scheduled delivery.

c) Colostrum of chicken type II collagen immunized cow, Escherichia coli O-111: Adjusting whey from colostrum of cow immunized with B4 strain Pool raw milk for 3 days after delivery of each cow immunized in a) and b) Collected. A portion of 8 liters was centrifuged at 20000 g for 30 minutes to remove the fat layer and obtain skim milk. The skim milk is sterilized by heating at 62.5 degrees for 30 minutes, then hydrochloric acid is added to pH 4.6, casein is removed by centrifugation at 20000 g for 30 minutes, sodium hydroxide solution is added to pH 7.0, freeze-dried, and dried. 707 g and 780 g of product were obtained.

[Example 2]
Specific purification of anti-LPS bovine antibody The E. coli immunized bovine whey prepared in Example 1c) contained 0.25% of an antibody against E. coli endotoxin O-111. This is a solution containing 1% in 50 l of phosphate buffered saline, applied to a 500 ml O-111 solid phase affinity column, washed, pH 2.8 eluted, neutralized, and further concentrated and purified on a protein G column. 580 mg of antigen-specific antibody was obtained.

[Example 3]
Specific Purification of Anti-Chicken Type II Collagen Bovine Antibody The whey prepared from the colostrum of chicken type II collagen immunized cow prepared in Example 1c) contained 0.6% of antibody against chicken type II collagen. This is made into a solution containing 1% in 30 liters of phosphate buffered saline, applied to a 500 ml O-111 solid phase affinity column, washed sequentially, pH 2.8 eluted, neutralized, and further concentrated on a protein G column. Purification was performed to obtain 930 mg of antigen-specific antibody.

[Example 4]
Anti-LPS Bovine Antibody Inhibition of Arthritis Development in an Arthritis Development Model Using a Mouse Anti-Collagen Monoclonal Antibody Mixture When 4 mg of a monoclonal antibody against articular cartilage collagen (arthritis cocktail, Chondrex) is administered by injection, mice develop arthritis. In 2 days, joint swelling is maximized. However, reducing the amount of arthritic cocktail, 2 mg does not cause arthritis at all. However, when E. coli LPS (E. coli O-111: B4 Sigma) was orally administered to this mouse three times at a dose of 3 mg / mouse / day (0, 1, and 2 days with the arthritis cocktail as 0 days) Arthritis developed and the swelling reached its maximum in 6 days. Three groups of six 6-week-old BALB / c male mice per group, with three groups: arthritis cocktail group, arthritis cocktail and LPS oral combination group, arthritis cocktail and LPS oral administration, and anti-LPS antibody oral administration The swelling of arthritis was evaluated by measuring both foot volumes. As the anti-LPS bovine antibody, the purified specific antibody shown in Example 2 was orally ingested to 3 mg / mouse / day, on the day of LPS administration. The results are shown in Table 1.

  As shown in Table 1, the arthritis development promoting effect of oral LPS was blocked by oral administration of a specifically purified anti-LPS antibody.

[Example 5]
Inhibitory effect of anti-collagen antibody and anti-LPS antibody against the onset of arthritis in mice by long-term oral administration of chicken type II collagen and LPS When LPS is added to chicken type II collagen and orally administered to mice, antibody production is produced compared to the group administered with type II collagen alone And the onset of arthritis is enhanced (K. Terato et al. Br. J. Rheum. 35,828-838, 1996). Thus, the arthritis inhibitory action of each antibody was examined using DBA / 1 mice. Arthritis-prone DBA / 1 male 6-week-old mice consisted of 6 mice per group, collagen (chicken II collagen chondrox) alone group, and LPS (E.coli O-111: B4 Sigma) added to collagen A group and a group to which an anti-collagen antibody (Example 3) and an anti-LPS antibody (Example 2) are further added and administered are set, and 4 courses of oral immunization with 2 weeks as 1 course over 14 weeks Went. In oral administration during the oral immunization period, in order to promote oral absorption of collagen and LPS, 40 μg of indomethacin was added to all groups including Control Group 1, and a total volume of 0.4 ml was orally administered. On the first day of 16 weeks, blood was collected from the fundus and serum was obtained. Then, LPS was orally administered for the purpose of developing arthritis for one week. On the first day of each immunization course and on the first day of the 17th week, the swelling of the joint was scored (documents described in Table 2) and scored. Table 2 shows the composition of the animal group, treatment conditions, and footpad volume. All doses are orally administered per mouse.

  Table 2 shows that arthritis develops by oral sensitization by long-term administration of collagen and LPS alone, and arthritis develops strongly when both are administered. On the other hand, it was shown that anti-collagen antibody and anti-bacterial endotoxin antibody (anti-LPS antibody) can be blocked together.

[Example 6]
Anti-rheumatoid arthritis therapeutic composition Chicken obtained by immunization with commercially available bovine type II collagen and porcine type II collagen, 50% antibody content powder containing chicken egg antibody, chicken type II collagen and commercially available E. coli vaccine Prepare whey powder with a total antibody content of 5% from raw milk obtained from cows immunized with (imocoliv), mix them at a ratio of 1: 9, add excipients, Granules having a total antibody content of 0.1% were prepared at a temperature according to a conventional method.

Claims (4)

A oral group forming compounds to prevent the onset of rheumatoid arthritis induced by the body shifts from the gastrointestinal tract of type II collagen or bacterial endotoxins from animals in food,
A step of inoculating and immunizing a livestock animal with an antigen containing type II collagen derived from a heterologous animal species, and / or separating the produced anti-type II collagen antibody from the domestic animal, and / or the bacterial endotoxin Inoculating a livestock animal with an antigen containing the antibacterial endotoxin antibody produced from the livestock animal,
An anti-type II collagen antibody and / or anti-bacterial endotoxin antibody obtained by a process comprising
For oral administration sets composition as to prevent rheumatoid arthritis. 2. The oral administration for preventing rheumatoid arthritis according to claim 1, wherein the anti-type II collagen antibody is at least one of anti-chicken type II collagen antibody, anti-bovine type II collagen antibody, and anti-pig type II collagen antibody . set Narubutsu. Wherein said anti-type II collagen antibody with the oral administration group composition as to prevent rheumatoid arthritis according antibacterial endotoxin antibodies to claim 1 or 2 were mixed at an arbitrary ratio. The rheumatoid arthritis according to any one of claims 1 to 3, which contains at least one of the anti-type II collagen antibody and the anti-bacterial endotoxin antibody, and contains 0.1% by mass or more as a total antibody amount. for oral administration sets composition as to prevent.