JP6804859B2 - Rheumatoid arthritis preventive and therapeutic compositions - Google Patents

Description

The present invention relates to a composition for preventing and treating rheumatoid arthritis, which comprises an antibody and prebiotics as active ingredients.

In general, rheumatoid arthritis is a general term for diseases in which motor organs that support and move the body, such as bones, joints, and muscles, are affected with systemic inflammation. Of these, inflammatory joint diseases that begin with redness, pain, and swelling of the joints of the hands and feet, progress to joint destruction and deformation, and lead to functional paralysis of the joints with pain are called rheumatoid arthritis.

In drug therapy for rheumatoid arthritis, a combination of corticosteroids, immunosuppressants, acidic anti-inflammatory agents, organic gold compounds, anti-cytokine antibodies and other drugs is used to stop inflammation. All of these drug therapies are so-called symptomatic treatments.

Although the etiology of rheumatoid arthritis is generally unknown today, studies are being conducted 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 ingestion usually does not result in antibody production against these antigens. This phenomenon is called oral immune tolerance.

Antibody production is regulated by lymphocytes (helper T cells, Th) that promote antibody production and lymphocytes (suppressor T cells, Ts) that act suppressively. 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 the production of autoantibodies against type II collagen. Attempts have been made to administer a modified version of the derived peptide, type II collagen. Examples of this include Patent Documents 1 to 3, and all of these are patents intended for oral immune tolerance.

The present inventors have elucidated the etiology in order to contribute to the prevention and treatment of rheumatoid arthritis. The etiology of rheumatoid arthritis is that the patient's constitutional and genetic factors include disruption of oral immunity in patients with rheumatoid arthritis, and external factors include type II collagen derived from heterologous animals in food. , It is believed that the cause is the absorption of bacterial endotoxin from the digestive tract.

Bacterial endotoxin, also known as endotoxin, lipopolysaccharide, LPS, etc., is a component of the cell wall of Gram-negative bacteria, but it is released from the bacterial cells by decomposition of bacterial cells and exhibits various toxic effects on the living body. .. Gram-negative bacteria such as Escherichia coli are resident in the intestines of humans and animals, and the LPS concentration in 1 g of feces reaches several tens of micrograms.

The breakdown of oral immunity is the following conditions. Oral immune tolerance is a normal immune response to orally ingested antigens, but due to intrinsic, constitutional, and environmental factors, normal immune tolerance cannot be induced, leading to the production of autoantibodies. The idea is. To test this hypothesis, we examined the arthritis-inducing activity of similar antigens and bacterial endotoxins in mice for collagen arthritis. DBA / 1 strain mice are mice having a genetic trait that easily develops type II collagen arthritis. When these mice were orally fed with chicken-derived type II collagen or heat-denatured ones, antibodies against mouse type II collagen were produced in the blood of the mice, and arthritis developed. This arthritis was exacerbated by the combined use of bacterial endotoxin (also known as lipopolysaccharide or LPS). In addition, long-term administration of only bacterial endotoxin, excluding chicken-derived type II collagen, caused arthritis (Non-Patent Document 1).

These results, along with antibodies to type II collagen, indicate that non-specific activation or exhaustion of the innate immune system by bacterial endotoxin is involved in the development of rheumatoid arthritis, and that oral immune tolerance may not be established. There is. In this experiment, it was shown that even the heat-denatured type II collagen prepared with the intention of immune tolerance, which was shown in Patent Documents 1 to 3, may not be able to induce immune tolerance.

In addition, the present inventors obtained by immunizing livestock such as cows, chickens, sheep and goats with type II collagen, formalin-treated gram-negative bacteria or heat-killed bacteria as a vaccine, and separating them from milk, serum or eggs. We have proposed a composition for oral administration for preventing rheumatoid arthritis, which comprises the above-mentioned anti-type II collagen antibody and / or anti-bacterial toxin antibody as an active ingredient (Patent Document 4).

Japanese Unexamined Patent Publication No. 07-138187 Japanese Unexamined Patent Publication No. 09-059176 Japanese Unexamined Patent Publication No. 09-255581 Japanese Unexamined Patent Publication No. 2006-151914

K. Terato et al. Br. J. Rheum. 35,828-838, 1996

An object of the present invention is to provide a rheumatoid arthritis preventive and therapeutic composition having a higher effect than a rheumatoid arthritis preventive and therapeutic composition containing an antibody as an active ingredient.

While conducting various studies on the ingestion effect of the antibody, the present inventors happened to find that a composition containing the antibody and prebiotics is effective in the prevention and treatment of rheumatoid arthritis. Then, it was confirmed that a synergistic effect was observed between the antibody and the prebiotics, and the present invention was completed.

The present invention has been made based on such findings, and provides a composition for preventing and treating rheumatoid arthritis, which comprises an antibody and prebiotics as active ingredients.

According to the present invention, the combined use of an antibody and prebiotics can prevent and treat rheumatoid arthritis more effectively than the antibody alone or the prebiotics alone.

It is a figure which shows the result of having examined the enhancement effect by prebiotics of the anti-rheumatoid arthritis effect of a milk antibody.

The composition for preventing and treating rheumatoid arthritis according to the present invention will be described. The rheumatoid arthritis preventive and therapeutic composition according to the present embodiment contains an antibody and prebiotics as active ingredients, and is used for preventing and / or treating rheumatoid arthritis.

The antibody used in the present embodiment is a product of an immune system that binds to pathogens and their constituents that are foreign substances to the living body, eliminates the foreign substances from the body, and detoxifies them, and is chemically immunoglobulin. Is.

For antibody selection, plasma from domestic animals such as cows, buffaloes, horses, pigs, goats, rabbits and rabbits, and eggs such as first milk, raw milk and chickens are the starting materials for the preparation of compositions for the prevention and treatment of rheumatoid arthritis. Various human pathogenic bacteria can also be vaccinated to increase the antibody concentration. The starting materials are further freeze-dried and spray-dried after defiber treatment in the case of plasma, and after removing milk fat, lactose and casein in the case of primary milk and raw milk, water removal and concentration by ultrafiltration, sterile filtration. Further, it is pulverized by freeze-drying and spray-drying. These technologies have already been established and implemented.

Since the activity of an antibody is rapidly lost by heating in a liquid state, it is necessary to manage the sterilization by heating to satisfy both sterilization and prevention of antibody thermal denaturation. Practical forms of rheumatoid arthritis preventive and therapeutic compositions are aqueous solutions or dry powders.

Quality assessment is done by quantification of antibodies. For the measurement of the total antibody amount, for example, a dual antibody enzyme immunoassay method, an immunodiffusion method, a protein A or protein G column method, etc. are recommended according to the text. In addition, the enzyme immunoassay is used to measure antibodies against specific bacteria and bacterial toxins.

It is important to check the antigen specificity of the antibody in order to ensure the function of the composition having the function of lowering endotoxin in blood and feces. That is, it is an essential requirement to include an antibody against endotoxin-producing bacteria, a human-derived pathogenic bacterium, an antibody against endotoxin, and an antibody against exotoxin.

Antibodies to endotoxin-producing bacteria promote removal by excreting endotoxin-producing bacteria from the intestinal tract, and antibodies to endotoxin bind to endotoxin and act to prevent endotoxin translocation into the body.

On the other hand, Gram-positive human pathogens that do not produce endotoxin (Clostridium perfringens, Staphylococcus, etc.) produce exotoxin, destroy the barrier of the gastrointestinal tract, and enhance the transfer of endotoxin into the body. It binds to toxins and blocks endotoxin translocation into the body.

The endotoxin-producing bacterium is a bacterium corresponding to the following genera. For example, Fusobacterium, Veillonella, Megasphaera, Neisseria, Moraxella, Branhamella, Branhamella, Acinetobacter (Acinetobacter), Citrobacter (Acinetobacter) (Eschericia), Hafnia, Klebsiella, Morganella, Proteus, Providencia, Salmonella, Salmonella, Serratia, Serratia, Serratia, Serratia (Vibrio), Eromonas, Plesiomonas, Haemofilus, Pasteurella, Pseudomonas, Legionella and the like.

Formalin-treated or heat-killed bacteria of endotoxin-producing bacteria are selected as an antigen for antibody detection and quantification, if necessary. Formalin treatment is required for the detection and quantification of antibodies against finbrier and flagella. As the endotoxin, each of the above bacterial cells can be used as it is, or endotoxin (LPS) can be extracted from these bacterial cells by the trichloroacetic acid method or the phenol method according to the textbook and used.

The activity of anti-endotoxin antibody can be evaluated by measuring the amount of antibody by the ELISA method using endotoxin extracted from each bacterial cell or heated or formalin-treated bacterial cells as an antigen, and the endotoxin activity can be evaluated by the Limulus method on a commercially available kit. Can be used and measured.

The activity of the anti-endotoxin antibody is such that the anti-endotoxin antibody neutralizes the endotoxin and causes the activity of the endotoxin to be lost. By measuring with, the total endotoxin neutralizing (= detoxifying) activity of various antibody mixtures having different antigen specificities can be quantitatively measured.

In the present embodiment, it is preferable to use whey protein (WPC) containing an innate immune antibody derived from raw milk as a material for the antibody. "Innate immune antibody" refers to an antibody obtained as a result of natural exposure of an animal's immune system to a foreign antigen. WPC is a collection of proteins contained in whey, which is a by-product of cheese production, and innate immune antibodies are concentrated in this WPC by using a production method with a small heat history. As the WPC containing the innate immune antibody, a commercially available WPC can be used. As a commercially available WPC, for example, "Asama Whey Protein" (manufactured by Asama Kasei Co., Ltd.) can be used.

"Asama milk clear protein" is, among other natural immune antibodies, a) endotoxin derived from 33 pathogenic bacteria selected from human pathogenic bacteria, b) pathogenic Escherichia coli O-26 strain, O-55 strain, O An antibody selected using two types of endotoxins, endotoxin derived from the −111 strain, four types of endotoxin derived from Salmonella minesota, c) enterotoxin B derived from Staphylococcus aureus, and enterotoxin derived from Clostridium perfringens as indicators. (Yoshiko Kijima et al., Journal of Japan Society for Food Chemistry, 2009, 56: 475-482). Therefore, "Asama whey protein" contains a large amount of innate immune antibodies against human pathogenic bacteria and bacterial toxins. This was obtained as a result of the newly developed ELISA method, which is a highly sensitive and accurate measurement method superior to the ELISA method, which enables the selection of whey protein products containing antibodies against human pathogenic bacteria. It is a thing.

The 33 types of spontaneous immune antibodies against human pathogenic bacteria in "Asama milk clear protein" are Escherichia coli O-111, intestinal hemorrhagic Escherichia coli O-157, and Streptococcus salmonella (Eschericia coli O-157). Streptococcus, Shiga, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus Minnesota R595), Streptococcus epidermidis, Streptococcus aureus, Streptococcus cerevius, Streptococcus profringens, Campylobacter, Campylobacter, Campylobacter Streptococcus sanguis, Streptococcus salivarius, Streptococcus mutans, Aerogenes bacterium Enterobacter aerogenes, Alkaline bacter aerogenes, Alkaline streptococcus aeruginosa), Proteus, Helicobacter streptococcus, Group A Streptococcus type 1 (Group A Streptococci type-1), Group A Streptococcus type 12 (Group Acpic12) Group A Streptococcus type 12 Streptococcus pyogenic type 22 (Group A Streptococcus type-22), Streptococcus viridans, Streptococcus pneumoniae, Pneumoniae pneumoniae, Kleb It is an antibody against Haemophilus influenzae, and the innate immune antibody may include other anti-human pathogenic bacterial antibodies.

The natural immune antibodies against bacterial toxins in "Asama milk clear protein" are enterotoxins derived from pathogenic Escherichia coli O-26, O-55 and O-111 strains, Lipid A derived from Salmonella Minnesota, and Staphylococcus aureus. It is an antibody against enterotoxin B derived from S. aureus and enterotoxin derived from Clostridium perfringens, and the natural immune antibody may contain other antibacterial toxin antibodies.

The selection of innate immune antibodies is based on the fact that the antibody titer is 8 μg or more in 1 g of whey protein for a) above, and the antibody titer is 1 μg or more in 1 g of whey protein for b) and c) above. did.

In the rheumatoid arthritis preventive and therapeutic compositions according to the present embodiment, the antibody content required for exerting the effect is preferably 0.001% by weight or more, more preferably 0.01% by weight or more. Is preferable. The intake of the antibody is preferably 10 mg or more, more preferably 100 mg or more per day for an adult.

As used in this embodiment, prebiotics means indigestible foods that promote the growth of intestinal probiotics or health-promoting microorganisms. Prebiotics are degraded in the stomach and / or upper intestine of the person who ingested the prebiotics, or are not absorbed in the gastrointestinal tract, but are fermented by the gastrointestinal microbiota and / or prebiotics. Prebiotics include, for example, Glenn R. et al. Gibson and Marcel B. Robertfrod, Dietary Modulation of the Human Colonic Microbiota: Introducing the Concept of Prebiotics, J. Mol. Nutr. It is defined by 1995 125: 1401-1412.

The prebiotics that can be used in this embodiment are not particularly limited, and include all foods that promote the growth of probiotics or health-promoting microorganisms in the intestine. Preferably, the prebiotics are skim milk, oligosaccharides (galactooligosaccharides, fructo-oligosaccharides, soybean oligosaccharides, milk fruit oligosaccharides, xylooligosaccharides, isomal oligosaccharides, raffinose, lactulose, coffee bean manno-oligosaccharides, gluconic acid, etc.) and dietary fiber. It can be selected from the group consisting of (such as polydextrose), inulin, or mixtures thereof. Preferred prebiotics are skim milk, fructooligosaccharide (FOS), galactooligosaccharide (GOS), isomaltooligosaccharide (IOS), xylooligosaccharide (XOS), soybean oligosaccharide, glycosyl sucrose (GS), lactooligosaccharide (LS), lactulose. (LA), palatinose oligosaccharide (PAO), maltooligosaccharide (MOS), gum and / or its hydrolyzate, pectin and / or its hydrolyzate. Although the function of skim milk as prebiotics has not been known so far, the present inventors have clarified that skim milk can function as prebiotics.

In the rheumatoid arthritis preventive and therapeutic compositions according to the present embodiment, the prebiotic content required for exerting the effect is preferably 10% by weight or more, more preferably 20% by weight or more. Is preferable. The content ratio with the antibody is preferably 1: 1 to 1: 100, more preferably 1: 5 to 1:20.

In order to further make the preventive / therapeutic effect of rheumatoid arthritis more effective by using the rheumatoid arthritis preventive and therapeutic compositions according to the present embodiment, ingredients other than the above-mentioned active ingredients can be blended. Examples of active ingredients other than the above-mentioned active ingredients include gliadin-treated SOD of antioxidant food materials, probiotics, and the like.

Gliadin-treated SOD is a product in which gliadin, which is a wheat-insoluble protein, is stably bound to active oxygen scavenging enzyme (SOD) extracted from melon, and gliadin (trademark, Nutrition Act) is known as a commercially available product. ing. By adding to the rheumatoid arthritis preventive and therapeutic compositions according to the present embodiment, the symptoms of rheumatoid arthritis can be more effectively improved. The amount added is preferably 0.1 to 10% by weight based on the composition for preventing and treating rheumatoid arthritis.

Probiotics are viable additives that benefit the host by improving the balance of intestinal microbes, including Lactobacillus, Enterococcus, Streptococcus, and Bifidobacterium. Examples include lactic acid bacteria (including bacteria that produce lactic acid as a metabolite) such as the genus (Bifidobacterium) and butyric acid bacteria. By adding to the rheumatoid arthritis preventive and therapeutic compositions according to the present embodiment, the symptoms of rheumatoid arthritis can be more effectively improved. The amount added is preferably 0.1 to 10% by weight based on the composition for preventing and treating rheumatoid arthritis.

Examples of ingestion forms of the rheumatoid arthritis preventive and therapeutic compositions according to the present embodiment include powders, tablets, capsules, granules, cookies, ice cream, and beverages. However, it is premised that the active ingredient antibody is ingested under the condition that it is not inactivated.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.

1. 1. Preparation of composition for treating rheumatoid arthritis Asama whey protein (milk antibody manufactured by Asama Kasei Co., Ltd., antibody amount 30 mg / g) was used as an antibody, and skim milk (manufactured by Yotsuba Dairy Co., Ltd., lactose 53%, protein 34%) was used as prebiotics. Ashes (8%) was used. 30 kg of each of these was mixed at a ratio of 1: 1 and divided into 20 g per packet to prepare a powdery rheumatoid arthritis preventive and therapeutic composition (Example 1).

As a comparative example, 60 kg each of milk antibody alone (Comparative Example 1) and prebiotics alone (Comparative Example 2) were packaged in 20 g per packet to prepare a clinical trial preparation, which was tested in a test described later.

2. 2. Test method (1) Subject Patients are patients who do not show improvement by using normal rheumatoid arthritis chemotherapeutic agents (DMARDs), and have a disease activity DAS28-ESR score of 3.2, which is an index of the degree of disease of rheumatoid arthritis. The above patients were targeted.

(2) Administration test of composition for treating rheumatoid arthritis One packet of each of the three preparations prepared as described above, one group consisting of 32 patients with rheumatoid arthritis, was added to water for 12 weeks. It was administered by oral ingestion. In order to examine the prebiotics effect of skim milk, the feces of the subjects were collected before ingestion and at the end of 12 weeks of ingestion, and microbial analysis of the feces was performed by the PCR method.

The preventive / therapeutic effect of rheumatoid arthritis was evaluated by measuring the DAS28-ESR score, which is used as an international index showing the disease activity of rheumatoid arthritis, over time.

The DAS28-ESR score was calculated by the following procedure. Rheumatoid arthritis is characterized by pain and swelling in the joints of the limbs, and the diagnosis is for 20 joints of both hands, 6 joints of both arms, elbows, shoulders, and 2 joints of the knees, for a total of 28 joints. The doctor diagnoses the degree of swelling (swelling joint, SJ) and the degree of pain (joint that hurts when the joint is pressed or moved (tension joint, TJ), and determines the number of swollen joints (SJC) and the number of tender joints (TJC), respectively. Furthermore, blood was collected, placed in a glass tube and leaned against it, and the rate of erythrocyte precipitation in the glass tube (ESR = erythrocyte sedimentation rate) was measured (mm / hour h). Further, the patient jointed. Rheumatoid arthritis was self-evaluated, and the condition at the time of diagnosis was shown in the length from 0 mm (best) to 100 mm (worst) (patient VAS mm). Based on the above values, the DAS28-ESR score was calculated by the following formula. Calculated.

The DAS28-ESR score is used as an index of the therapeutic effect of rheumatoid arthritis, and the lower the score, the higher the therapeutic effect of rheumatoid arthritis.

3. 3. Results (1) Prebiotics of skim milk Table 1 shows the changes in fecal bifidobacteria over 12 weeks. As shown in Table 1, in the skim milk group of Comparative Example 2, a significant increase of 0.31 in logarithm, that is, twice the value at week 0 in real number conversion was observed. Bifidobacterium is a typical bacterium that acts to improve the intestinal flora, and the number of bifidobacteria after 12 weeks in Comparative Example 2 increased, indicating that skim milk has a prebiotic effect. It was.

(2) Therapeutic effect results of rheumatoid arthritis are shown in FIG. Example 1 was found to reduce the DAS28-ESR score by as much as 0.5. On the other hand, in Comparative Example 1 and Comparative Example 2, the DAS28-ESR score did not decrease so much even after 12 weeks had passed, and Comparative Example 1 had about 0.13 and Comparative Example 2 had about 0.23. It only decreased. As a result, Example 1 had a DAS28-ESR score reduction rate of 3.8 times that of Comparative Example 1 and 2.2 times that of Comparative Example 2, and the antibody had a synergistic effect when mixed with prebiotheotics. It became clear that Further, since the amount contained in the composition of Example 1 is half the amount of the mixture of Comparative Example 1 and Comparative Example 2, its usefulness in terms of cost was recognized. From this result, it was found that the antibody and the prebiotics synergistically enhance the preventive / therapeutic effect of rheumatoid arthritis as compared with the case where they are used alone.

Claims (6)

A composition for preventing and treating rheumatoid arthritis, which comprises an antibody and prebiotics as active ingredients.
The antibody is an antibody against human pathogenic bacteria and bacterial toxins, and is Escherichia coli O-111, intestinal hemorrhagic Escherichia coli O-157, Salmonella, Streptococcus serrata, Seleus, Listeria, Ersina, Seratia, and Nezumitifus. . , Green pus, Proteus, Pyrroli, Group A streptococcus type 1, Group A pyogenic streptococcus type 12, Group A pyogenic streptococcus type 22, Green lentococcus, Pneumonia streptococcus, Pneumococcus and Influenza Including
The prebiotics are skim milk,
Rheumatoid arthritis preventive and therapeutic compositions. The composition for preventing and treating rheumatoid arthritis according to claim 1, wherein the antibody is an innate immune antibody. The antibody is derived from an antibody produced by at least one selected from the group consisting of cows, goats, sheep, horses and chickens, and consists of milk, first milk, blood and chicken eggs of these animals. The composition for preventing and treating rheumatoid arthritis according to claim 1 or 2 , which is produced from at least one selected from the group as a raw material. The composition for preventing and treating rheumatoid arthritis according to any one of claims 1 to 3 , wherein the antibody has an antibody titer against human pathogenic bacteria and bacterial toxins of 8 μg or more in 1 g of whey protein . The rheumatoid arthritis prevention according to any one of claims 1 to 4 , further comprising at least one of the prebiotics that can be selected from the group consisting of oligosaccharides, dietary fiber, inulin or a mixture thereof. And therapeutic compositions. By adding Purebaiote I box to the antibody, a method for increasing the prevention or therapeutic effect of rheumatoid arthritis,
The antibody is an antibody against human pathogenic bacteria and bacterial toxins, and is Escherichia coli O-111, intestinal hemorrhagic Escherichia coli O-157, Salmonella, Streptococcus serrata, Seleus, Listeria, Ersina, Serratia, and Mouse typhi. . , Green pus, Proteus, Pyrroli, Group A streptococcus type 1, Group A pyogenic streptococcus type 12, Group A pyogenic streptococcus type 22, Green lentococcus, Pneumonia streptococcus, Pneumococcus and Influenza Including
The prebiotics are skim milk.
A method of enhancing the preventive or therapeutic effect of rheumatoid arthritis .