Patent Application
20090028840
1. Field of the Invention
The present invention relates to a composition for preventing or treating arthritis.
2. Description of the Related Art
Arthritis is one of the chronic diseases in which five percentages of worldwide human population are suffering. Its etiology involves inflammation at synovial membrane of articular capsule, which ultimately causes edema and pain in systemic joints. Arthritis is progressive degenerative disease and induces deformation and difficulties in bending of joints, and is very likely to produce serious effects if left untreated.
Little is known about the direct cause of arthritis. Various therapeutic drugs have been used for arthritis, including steroidal anti-inflammatory agents such as cortisone and other adrenocortical hormones, non-steroidal anti-inflammatory agents such as aspirin, piroxicam and indomethacin, anti-rheumatic agents such as chloroquinones and D-penicillamine, anti-gout agents such as colchicine, and immune suppressive agents such as cyclophosphamide, azathioprine, methotrexate and levamisol. However, these drugs are not considered to be fundamental therapeutics and the administration of steroidal drugs is somewhat restricted due to their side effects. Aspirin-based drugs used to relieve pain and remove inflammation associated with arthritis exhibit adverse effects against stomach; therefore, their continuous administration for treating arthritis is nearly impossible.
Since the conventional chemotherapeutic drugs described above have serious shortcomings including adverse effects, and limited anti-inflammation efficacy on developed arthritis, non-steroidal anti-inflammatory drug such as indomethacin is currently administered as an alternative therapeutic realm.
Accordingly, there is a long-felt need to develop novel therapeutics for arthritis showing better anti-inflammatory and pain relief efficacies together with overcoming drawbacks of conventional drugs described above. Since the prolonged administration of drugs is typically required for arthritis, the development of therapeutics with much less adverse effects is a significant consideration factor. Drugs administered via intravenous and intraperitoneal route are tedious to be administered and are likely to be accompanied with side effects such as allergy and shock. Therefore, drugs showing safety and convenience in administration are in demand.
Most of health foods for treating arthritis comprise cartilage constituents as a main ingredient. Korean Pat. Appln. No. 10-1997-0035934 discloses a therapeutic for arthritis comprising peptides derived from type II collagen, and Korean Pat. Appln. No. 10-2003-00433119 suggests a health food composition for treating degenerative arthritis comprising 40-45% of gluosamine, 30% of mucopolysaccharide protein, vitamin C, Achyranthes japonica, Chaenomelis sinensis, Eucommia ulmoides and shark cartilage extract.
As examples of drugs containing lactic acid bacteria, U.S. Pat. Appln. Publication No. 2005/007441 discloses that irritable bowel syndrome and inflammation could be alleviated by ingesting Bifidobacteria. In addition, Ehud Baharav et al. reported that the oral administration of live Lactobacillus GG or heat-inactivated Lactobacillus could prevent the development of arthritis (Ehud Baharav et al., Nutritional Immunology, 134:1964-1969 (2004)). B. Sheil et al. revealed that Lactobacillus salivarus 118 administered via subcutaneous or oral route would alleviate irritable bowel syndrome and arthritis in arthritis model of mice. Given that the immunomodulatory action of Lactobacillus is exhibited in arthritis and bowel syndrome, it can be demonstrated that Lactobacillus modulates immune hypersensitivity and autoimmune response through regulating TGF-β level in T-lymphocytes (B Sheil et al., Gut, 53:694-700 (2004)).
Yang et al. reported that peptides derived from type II collagen carrying T cell antigenic determinants are orally administered to collagen-induced arthritis of mouse models to prevent or treat collagen-induced arthritis (H. I. Yang, et al., J. of The Korean Rheumatism Association, 12:7(04)(2000)).
Throughout this application, various publications and patents are referred and citations are provided in parentheses. The disclosures of these publications and patents in their entities are hereby incorporated by references into this application in order to fully describe this invention and the state of the art to which this invention pertains.
The present inventors have made intensive studies to develop novel compositions for preventing or treating arthritis, having enhanced therapeutic efficacies and dramatically reduced adverse effects. As results, we have discovered that lactic acid bacteria administered to subjects together with collagen serving as self antigens for arthritis, greatly enhanced immunetolerance and suppressed joint-specific inflammation, and finally helped the successful prevention and treatment of arthritis.
Accordingly, it is an object of this invention to provide a composition for preventing or treating arthritis.
It is another object of this invention to provide a method for preventing or treating arthritis.
It is still another object of this invention to provide a use of a composition comprising a lactic acid bacterium and collagen for manufacturing a medicament for preventing or treating arthritis.
Other objects and advantages of the present invention will become apparent from the following detailed description together with the appended claims and drawings.
In one aspect of this invention, there is provided a composition for preventing or treating arthritis, which comprises a lactic acid bacterium and collagen as active ingredients.
In another aspect of this invention, there is provided a method for preventing or treating arthritis, which comprises administering to a subject a pharmaceutical composition comprising (a) a pharmaceutically effective amount of a lactic acid bacterium and collagen, and (b) a pharmaceutically acceptable carrier.
In still another aspect of this invention, there is provided a use of a composition comprising a lactic acid bacterium and collagen for manufacturing a medicament for preventing or treating arthritis.
The present inventors have made intensive studies to develop novel compositions for preventing or treating arthritis, having enhanced therapeutic efficacies and dramatically reduced adverse effects. As results, we have discovered that lactic acid bacteria administered to subjects together with collagen serving as self antigens for arthritis, highly enhanced immunetolerance and suppressed joint-specific inflammation, and finally helped the successful prevention and treatment of arthritis.
In the composition of this invention, the active ingredient comprises lactic acid bacteria and collagen.
The lactic acid bacteria as an active ingredient have been reported to have therapeutic efficacies on intestinal disorders, e.g., alleviation of irritable bowel syndrome and improvement in intestinal function. The present invention is based on the novel findings of the present inventors in which lactic acid bacteria allows for the synergic increase in immunetolerance induced by collagen. In addition, the lactic acid bacteria in the present composition play a role in up-regulating the production of anti-inflammatory cytokines such as IL-10 and TGF-β.
The term used herein “lactic acid bacteria” refers to bacteria capable of producing lactic acid as main metabolites of carbohydrates, e.g., Lactococcus, Lactobacillus, Leuconostoc, Propionibacterium, Enterococcus, Bifidobacterium, Streptococcus and Pediococcus. Preferably, the lactic acid bacterium suitable in this invention is at least one lactic acid bacterium selected from the group consisting of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus gasseri, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus bulgaricus, Lactobacillus helveticus, Streptococcus thermophilus, Streptococcus lactis, Enterococcus faecium, Enterococcus faecalis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium brave and Bifidobacterium longum. Most preferably, the lactic acid bacterium suitable in this invention is Lactobacillus casei.
The preferable amount of lactic acid bacteria in the present composition is 105 cfu-1011 cfu per unit weight (g) of compositions.
Another active ingredient, collagen serves as self antigens to induce immunetolerance. Arthritis-specific drugs have not been successfully developed because self antigens involved in immune reactions are extremely diverse. The principle underlying the present invention is to employ immunetolerance elicited in the gut associated lymphoid tissue (GALT). Immunetolerance refers to immune reactions in which the immune system (GALT) present under epithelial cells of small intestine elicits specific immuno-suppressive reactions against materials or substances orally delivered. Collagen as an active ingredient suppresses selectively arthritis-specific inflammation through immunetolerance responses in the GALT.
Collagen used in this invention is type I or type II collagen, preferably, type II collagen. The preferable amount of collagen in the present composition is 0.1-2.0 wt %, more preferably, 0.2-1.5 wt %, most preferably 0.2-1.1 wt %.
According to a preferred embodiment, the composition of this invention further comprises glucosamine, chondroitin and their combination. All of collagen, glucosamine and chondroitin are cartilage constituents. Therefore, cartilage constituents (collagen) acting as self antigens permit to induce enhanced immunetolerance reactions. In addition, glucosamine allows promoting the formation and regeneration of cartilage. Chondroitin inhibits enzyme destructing cartilage thereby protect cartilage as well as induces immunetolerance responses.
In the present composition, glucosamine is preferably present in the form of D-(+)-glucosamine hydrochloride. In the present composition, chondroitin is preferably present in the form of chondroitin 6-sulfate salt. The preferable amount of glucosamine in the present composition is 1.0-4.0 wt %, more preferably 1.3-3.0 wt %, most preferably 2.0-3.0 wt %. The preferable amount of chondroitin in the present composition is 0.5-4.0 wt %, more preferably 0.7-3.0 wt %, most preferably 1.4-2.3 wt %.
According to a preferred embodiment, the composition of this invention further comprises a malt extract, Vitamin D and their combination.
Preferably, the malt extract is a fermented malt extract, more preferably, a malt extract containing about 2 wt % of chloride and 60-70 wt % of reducing sugars (e.g., maltose, sucrose and dextrose). The malt extract in the present composition allows suppression of inflammatory responses. The amount of malt extract in the present composition is preferably 10-70 wt %, more preferably 20-60 wt % and most preferably 35-55 wt %.
Vitamin D used in this invention is a lipophilic Vitamin involved in bone metabolism and homeostasis of calcium phosphate. Preferably, Vitamin D used in this invention is Vitamin D3. Vitamin D in the present composition promotes the productions of anti-inflammatory cytokine IL-10 and regulatory T cell (Tr1).
According to a preferred embodiment, the composition of this invention further comprises at least one extract selected from the group consisting of extracts of Actinidia polygama, cactus leaves, Eucommia ulmoides, seed of Carthamus tinctorius, Coix lachrymal and Chaenomelis sinensis. The oriental medicines have been known to relieve pain and reduce inflammation. The amount of the oriental medicine in the present composition is preferably 5-50 wt %, more preferably 10-35 wt %, most preferably 20-35 wt %.
The present composition for preventing or treating arthritis may be prepared to provide a pharmaceutical composition by use of active ingredients and a pharmaceutically acceptable carrier. In the pharmaceutical compositions of this invention, the pharmaceutically acceptable carrier may be conventional one for formulation, including lactose, dextrose, sucrose, sorbitol, mannitol, starch, rubber arable, potassium phosphate, arginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oils, but not limited to. The pharmaceutical composition according to the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative. Details of suitable pharmaceutically acceptable carriers and formulations can be found in Remington's Pharmaceutical Sciences (19th ed., 1995), which is incorporated herein by reference.
Since the pharmaceutical composition of this invention exerts its efficacies through immunetolerance, it should be administered via the oral route.
A suitable dose of the pharmaceutical composition of the present invention may vary depending on pharmaceutical formulation methods, administration methods, the patient's age, body weight, sex, severity of diseases, diet, administration time, administration route, an excretion rate and sensitivity for a used pharmaceutical composition. Physicians of ordinary skill in the art can determine an effective amount of the pharmaceutical composition for desired treatment. Preferably, the pharmaceutical composition of the present invention is administered with a daily dose of 0.01-2000 mg/kg (body weight).
According to the conventional techniques known to those skilled in the art, the pharmaceutical composition may be formulated with pharmaceutically acceptable carrier and/or vehicle as described above, finally providing several forms including a unit dose form and a multi-dose form. Non-limiting examples of the formulations include, but not limited to, a solution, a suspension or an emulsion in oil or aqueous medium, an extract, an elixir, a powder, a granule, a tablet and a capsule, and may further comprise a dispersion agent or a stabilizer.
The present composition for preventing or treating arthritis may be prepared to provide a food composition, in particular a health food composition. The food composition may comprise conventional additives for preparing food compositions, e.g., proteins, carbohydrates, lipids, nutritive substances and flavors. For example, where the food composition of this invention is provided as a drink, it may further comprise flavors and natural carbohydrates as well as lactic acid bacteria and collagen as active ingredients. Non-limiting examples of natural carbohydrates include, but not limited to, monosaccharide (e.g., glucose and fructose), disaccharide (e.g., maltose and sucrose), oligosaccharide, polysaccharide (e.g., dextrin and cyclodextrin) and sugar alcohol (e.g., xylitol, sorbitol and erythritol). Non-limiting examples of flavors include, but not limited to, natural flavors (e.g., thaumatin and extract of Stevia) and synthetic flavors (e.g., saccharin and aspartame). Considering availability to foods, the food composition of this invention is very useful in preventing or treating arthritis.
As demonstrated and illustrated in Examples, the composition of this invention induces immunetolerance by down-regulating the expression of proinflammatory cytokines such as TNF-α, IL-1β and IL-12, and up-regulating the expression of anti-inflammatory cytokines such as IL-10, Foxp3 and TGF-β, thereby enhancing Th2 type immune responses and reducing Th1 type immune responses. As results, the present invention suppresses dramatically arthritis-specific autoimmune responses and inflammation, leading to the successful prevention or treatment of arthritis.
The composition of this invention exerts excellent prevention or treatment efficacies on arthritis (osteoarthritis and rheumatoid arthritis), specifically rheumatoid arthritis which is one of inflammatory autoimmune diseases. In addition, since the active ingredients used in the composition of this invention have been recognized to be safe to human, the present composition is also considerably safe to human. Surprisingly, the present composition comprising active ingredients approved as raw materials of foods exhibits much better prevention or treatment efficacies than conventional chemical drugs (e.g., methotrexate).
According to conventional therapies for arthritis, there are limitations in the senses that self antigens involved in immune responses are extremely diverse and no arthritis-specific drugs can be developed. In addition, the conventional drugs for arthritis intend mostly to suppress only inflammation and their prolonged administration is very likely to induce adverse effects. Meanwhile, most of conventional health foods for arthritis comprise glucosamine, one of cartilage constituents, and extract of shark cartilage as main ingredients, thus possessing restricted efficacies. In contrast, the composition of this invention induces immunetolerance in the gut associated lymphoid tissue to suppress autoimmune responses and inflammation elicited specifically in joints, finally successfully preventing and treating arthritis.
The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.
The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.
a)-(d) show the results of ELISA to analyze the antibody concentrations altered by the present composition.
Six oriental medicines (Gyeondong Market, Korea), 200 g of Actinidia polygama, 120 g of Eucommia ulmoides, 120 g of Chaenomelis sinensis, 120 g of Coix lachryma, 120 g of seed of Carthamus tinctorius and 120 g of cactus leaves were dried for 2 days at 75° C. and pulverized to form mixed powder. To this mixed powder, 60 g of glucosamine (Sigma), 50 g of chondroitin (Sigma), 25 g of type I collagen (Sigma), 1200 g of malt extract (Bacto), 36 mg of Vitamin D, 60 g of Lactobacillus casei in the form of a powder (Cell Biotech) were added and mixed, producing the exemplified immunomodulatory composition of this invention.
Daily dose for animal test in Lewis rats was determined to be 25 g (1.8 g of the exemplified composition of this invention and 23 g of feed powder). Feeds for rats were prepared by mixing 2160 g of the exemplified composition of this invention and 27600 g of feed powder.
Details of the exemplified composition of this invention are summarized in Table 1.
Lactobacillus casei
Actinidia polygama
Chaenomelis sinensis
Coix lachryma
Eucommia ulmoides
The comparative compositions were prepared as Table 2.
Lactobacillus casei
Actinidia polygama
Chaenomelis sinensis
Coix lachryma
tinctorius
One animal model for rheumatoid arthritis is collagen-induced arthritis (CIA) induced by one of joint constituents, type II collagen, showing similarities to human rheumatoid arthritis in pathological clinical and immunological views. The animal model shows autoimmune diseases elicited by collagen-specific T cells and antibodies.
Another animal model for rheumatoid arthritis is for adjuvant-induced arthritis (AIA) induced by Mycobacterium tuberculosis germs rather than self antigens, exhibiting pathological symptoms and phenotypes of chronic arthritis. Even though molecules serving as self antigens have not yet reported, the animal model shows autoimmune diseases elicited by T cell-induced inflammation and tissue disruption in joint. This model shows similar symptoms to human spontaneous arthritis.
These two types of animal models were employed to accurately verify and confirm the efficacies of the present invention in the experimental examples, because causes and factors involved in the development of rheumatoid arthritis have not yet unraveled.
Animal models for collagen-induced arthritis (CIA) were generated by injecting collagen involved in the development of rheumatoid arthritis as follows:
Four mg of chicken type II collagen (Sigma) were dissolved overnight at 4° C. in 1 ml of 100 mM acetic acid. The dissolved collagen was emulsified in 1 ml of Freund's complete adjuvant (Difco) containing 4 mg/ml of Mycobacterium tuberculosis. Lewis rat aged 6-8 weeks was immunized by the intradermal injection of 150 μl (300 μg) of the emulsion into the base of tail of rat. Separately, the dissolved collagen was emulsified in 1 ml of Freund's incomplete adjuvant (Difco). After 7 days of the primary immune response, 150 μl (300 μg) of the emulsion was intradermally injected into the base of tail of rat to induce the secondary immune (boosting) response. The symptoms of arthritis such as erythema and swelling could be observed 1-2 weeks after the secondary immune response.
Animal models for adjuvant-induced arthritis (AIA) also exhibit pathological characteristics similar to human rheumatoid arthritis and are predominantly used in the art for animal tests along with collagen-induced arthritis animal models.
Ten mg of Mycobacterium tuberculosis were pulverized to give powder and mixed with 1 ml of Freund's incomplete adjuvant (Difco). Lewis rat aged 6-8 weeks was immunized by the intradermal injection of 100 μl (1 mg) of the mixture into the base of tail of rat. After 1 week of immunization, symptoms associated with arthritis appeared.
From the next day of the immunization, the compositions to be tested were orally administered everyday to the rats with a gastric feeding tube. Ten Lewis rats per group were selected. One group of adjuvant-induced arthritis animals was administered with a daily dose (25 g; 11.5 g/kg of the present composition) of mixtures containing animal feed powder and the present composition; the other group as a negative control consisted of adjuvant-induced arthritis animals administered with only animal feed powder. Following the administration, the severity of arthritis was evaluated by observing joint swelling, paw thickness and erythema, and the body weight of rats was measured.
For verifying the prevention efficacy on arthritis, AIA-inducing immunization was carried out 2 weeks after oral administration of the composition in Example 1. The experiment was carried out for total of 9 weeks. In addition, to reveal the treatment efficacy on arthritis, AIA-inducing primary immunization was initially performed and in turn the composition in Example 1 was orally administered. The experiment was also carried out for total of 9 weeks. Ten rats per group were employed. The appearance of each paw of rats such as paw thickness and erythema was observed and the clinical severity of arthritis was calculated according to scoring index of Tables 3 and 4.
As shown in the clinical score of
As shown in the clinical score of
The composition of Example 1 revealed to have considerable efficacies on arthritis, hence more accurate and reliable tests were carried out to confirm the prevention and treatment efficacies on arthritis.
The treatment efficacy of compositions of this invention (in particular, composition of Example 1) was compared with that of other compositions as indicated in Comparative Examples of Table 2. The experimental procedures are similar to Experimental Example 1-3. Each group consists of 10 rats.
The control group is the non-administered group and MTX group is administered twice a week with 60 μg unit dosage of a conventional drug for arthritis, methotrexate. Oriental medicine group is administered with a mixed extract of Actinidia polygama, Eucommia ulmoides, cactus leaves, seed of Carthamus tinctorius, Coix lachryma and Chaenomelis sinensis. Joint constituent group is administered with a mixture of glucosamine, collagen and chondroitin. Present composition group is administered with the present composition of Example 1. Each composition was orally administered for 2 week and the primary immunization was induced using collagen. One week later, the secondary immunization was induced and the clinical score and paw thickness were measured.
As shown in
Accordingly, these results clearly show that the compositions of this invention give similar or improved treatment efficacy on arthritis compared with MTX. Moreover, the enhance treatment efficacy of the present composition is suggested to be ascribed to the synergic contribution of lactic acid bacteria to immunetolerance responses induced by collagen, leading to the synergic increase in immunetolerance responses.
The experiment for treatment efficacy on arthritis was carried out as Experimental Example 2. After 35 days of arthritis-inducing immunization, rats having the mean clinical score were sacrificed and their swelled ankle joint were histologically analyzed. Joints dissected were washed with PBS and fixed for 24 hr in 4% paraformaldehyde, followed by decalcification for 2-3 days in decalcification solution (Merck). The decalcified joint tissues were embedded in paraffin, sectioned to 0.6-1 μm, deparaffinized, stained with hematoxylin and eosin, and observed under microscope. A basic dye, hematoxylin binds to nucleus containing negative-charged DNA and RNA molecules and an acidic dye, eosin binds to cytoplasmic proteins and collagen with positive charge.
As shown in
Accordingly, it would be recognized that the composition of this invention inhibits effectively lymphocyte-induced immune inflammation.
The experiment for treatment efficacy on arthritis was carried out as Experimental Example 2. After 35 days of arthritis-inducing immunization, rats having the mean clinical score were sacrificed and their spleen and draining lymph nodes were dissected, which were ground to give mixed lymphocytes. The mixed lymphocytes (5×106 cells/well) were aliquoted into 24-well plate and stimulated for 6 hr with 20 μg/well of type II collagen. Then, cells were harvested and treated with Trizol to yield the total RNAs. From 1 μg RNA of each group, cDNA was synthesized using oligo dT primer and reverse transcriptase (Promega). The quantitative real-time PCR was carried out using the synthesized cDNA as template, 10 pmol primers for cytokines and SYBR premix to analyze and compare the levels of cytokine expression (
The results were normalized to the expression of the β-actin gene as a housekeeping gene. The data indicated in
Accordingly, our invention shows that the composition of this invention inhibits the expression of proinflammatory cytokines to suppress inflammation reactions, leading to the treatment of arthritis.
Spleen from normal rats were dissected, ground and treated for 30 min with 25 μg/ml mitomycin C (Sigma) to obtain splenocytes as APC (antigen presenting cell). 105 cells/well of the splenocytes were aliquoted into 24-well plates and stimulated with 20 μg/well of type II collagen for 30 min.
The experiment for treatment efficacy on arthritis was carried out as Experimental Example 2. After 70 days of arthritis-inducing immunization, rats having the mean clinical score were sacrificed and their draining lymph nodes were dissected, which were ground to give mixed lymphocytes. From the mixed lymphocytes, CD4+ T cells were separated using CD4+ magnetic beads (Miltenyi Biotec) and their aliquots (5×106 cells/well) were transferred into the 24-well plates containing APC and collagen to stimulate for 24 hr. Then, cells were harvested and treated with Trizol to yield a total RNA. From 1 μg RNA of each group, cDNA was synthesized using oligo dT primer and reverse transcriptase (Promega). The quantitative real-time PCR was carried out using the synthesized cDNA as template, 10 pmol primers for cytokines and SYBR premix to analyze and compare the levels of cytokine expression (
The results were normalized to the expression of the β-actin gene as a housekeeping gene. The data indicated in
On the basis of results of cytokine expressions in CD4+ T cells, which play a pivotal role in inflammation reactions, it can be concluded that the composition of the present invention enhances the expressions of anti-inflammatory cytokines as well as inhibits the expressions of proinflammatory cytokines.
The experiment for treatment efficacy on arthritis was carried out as Experimental Example 2. After 75 days of arthritis-inducing immunization, rats having the mean clinical score were sacrificed and their popliteal lymph nodes and inguinal lymph nodes surrounding joints were dissected, which were ground to give mixed lymphocytes.
The mixed lymphocytes (5×106 cells/well) were aliquoted into 24-well plate and stimulated for 24 hr with 40 μg/well of type II collagen. Then, cells were harvested and treated with Trizol to yield the total RNAs. From 1 μg RNA of each group, cDNA was synthesized using oligo dT primer and reverse transcriptase (Promega). The quantitative real-time PCR was carried out using the synthesized cDNA as template, 10 pmol primers for cytokines and SYBR premix to analyze and compare the levels of cytokine expression (
The results were normalized to the expression of the β-actin gene as a housekeeping gene. The data indicated in
Accordingly, these results lead us to reason that the present composition induces oral immunetolerance in lymphocytes surrounding joints. Interestingly, the present invention up-regulated the expression of the regulatory T cell marker Foxp3 as well as the expression of TGF-β, which is capable of suppressing inflammation.
Taken together, our works suggest that the composition of the present invention induces oral immunotolerance in lymph nodes surrounding joints directly involving the development of arthritis, which results in the up-regulation of expressions of anti-inflammatory cytokines such as Foxp3, IL-10 and TGF-β as well as the down-regulation of expressions of proinflammatory cytokines such as IL-12, TNF-α and IL-1β.
Spleen from normal rats were dissected, ground and treated for 30 min with 25 μg/ml mitomycin C (Sigma) to obtain splenocytes as APC (antigen presenting cell). 105 cells/well of the splenocytes were aliquoted into 96-well plates and stimulated with 20 μg/well of type II collagen for 30 min.
The experiment for treatment efficacy on arthritis was carried out as Experimental Example 2. After 70 days of arthritis-inducing immunization, rats having the mean clinical score were sacrificed and their draining lymph nodes were dissected, which were ground to give mixed lymphocytes.
From the mixed lymphocytes, CD4+ T cells were separated using CD4+ magnetic beads (Miltenyi Biotec) and their aliquots (105 cells/well) were transferred into the 94-well plates containing APC and collagen to stimulate for 56 hr. Then, cells were pulsed with 0.5 μCi [3H] thymidine (Perkin Elmer) for 16 hr and the thymindine incorporation was measured (
In the case that arthritis is actively developed, the levels of the thymindine incorporation are measured to be higher because CD4+ T cells responding to arthritis antigens proliferate rapidly. In other words, the thymindine incorporation becomes higher as the severity of arthritis become larger. As represented in
Accordingly, it could be concluded that CD4+ T cells in the group administered with the present composition lowered immune responses to collagen, demonstrating that the composition of the present invention confers immunetolerance to CD4+ T cells.
The experiment for treatment efficacy on arthritis was carried out as Experimental Example 2. After 75 days of arthritis-inducing immunization, rats having the mean clinical score were sacrificed and their popliteal lymph nodes and inguinal lymph nodes surrounding joints were dissected, which were ground to give mixed lymphocytes. The mixed lymphocytes (2×105 cells/well) were aliquoted into 96-well plate and stimulated for 56 hr with 40 μg/well of type II collagen.
Then, cells were pulsed with 0.5 μCi [3H] thymidine (Perkin Elmer) for 16 hr and the thymindine incorporation was measured (
As represented in
The experiment for treatment efficacy on arthritis was carried out as Experimental Example 2. After 35 days and 70 days of arthritis-inducing immunization, whole blood samples were prepared from rats having the mean clinical score and their serum components were separated for ELISA.
First, we measured the concentration of anti-collagen Ab present in serum samples. 100 μl/well of chicken type II collagen (1 μg/ml) aliquoted into 96-well plates were incubated for 12 hr at 4° C., and incubated with 100 μl of 1/10000 diluted serum of each group for 1 hr at 37° C., followed by incubating with 100 μl of anti-rat secondary Ab for 1 hr at 37° C. The color development was carried out using OPD and its absorbance was measured at 495 nm. As shown in
The IgG isotyping was conducted using anti-Ab against each IgG isotype (Immunology Consultant Laboratory). Various isotypes of IgGs such as IgG1, IgG2a, IgG2b and IgG2c have been reported and their analysis permits to reveal whether disease conditions are induced by Th1 or Th2 immune reactions. IgG1 is related to Th2 type immune reactions and IgG2 to Th1 type immune reactions. 100 μl/well of anti-Ab (2 μg/ml) to each IgG isotype aliquoted into 96-well plates were incubated for 12 hr at 4° C., and incubated with 100 μl of diluted serum of each group for 1 hr at 37° C., followed by incubating with 100 μl of anti-rat secondary Ab for 1 hr at 37° C. The color development was carried out using OPD and its absorbance was measured at 495 nm. As shown in
In contrast, the group administered with the present composition exhibited the lowest levels of IgG2a and IgG2b on both 35 days and 70 days of immunization. These results correspond to the down-regulation of proinflammatory cytokines such as TNF-α, IFN-γ and IL-1β by the present composition, addressing that the composition of this invention suppresses Th1 type immune reactions.
According to conventional therapies for arthritis, there are limitations in the senses that self antigens involved in immune reactions are extremely diverse and no arthritis-specific drugs can be developed. In addition, the convention drugs for arthritis intend mostly to suppress only inflammation and their prolonged administration is very likely to induce adverse effects. Meanwhile, most of conventional health foods for arthritis comprise glucosamine, one of cartilage constituents, and extract of shark cartilage as main ingredients, thereby possessing restricted efficacies. In contrast, the composition of this invention induces immunotolerance in the gut associated lymphoid tissue to suppress autoimmune reactions and inflammation generated specifically in joints, finally successfully preventing and treating arthritis.
Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2005-0088465 | Sep 2005 | KR | national |
| 10-2006-0071796 | Jul 2006 | KR | national |
This is a National Stage Application under 35 U.S.C. § 371 of PCT/KR2006/003774 filed Sep. 22, 2006, which claims priority from Korean Patent Application 10-2005-0088465 filed on Sep. 23, 2005, and Korean Patent Application 10-2006-0071796 filed on Jul. 29, 2006, all of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/KR2006/003774 | 9/22/2006 | WO | 00 | 4/23/2008 |
