JP2013087108A - Preventive or therapeutic agent for inflammatory bowel disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a preventive or therapeutic agent for an inflammatory bowel disease such as ulcerative colitis, Crohn's disease, and the like, which is highly safe and efficient, and which can be supplied at a low cost.SOLUTION: A composition includes at least one type of fullerene selected from hydroxylated fullerene, polyvinylpyrrolidone inclusion fullerene, fullerene, and pharmacologically acceptable salt thereof, as an active constituent.

Description

  The present invention relates to a preventive or therapeutic agent for inflammatory bowel diseases such as ulcerative colitis and Crohn's disease.

  Inflammatory Bowel Disease (IBD) is a general term for diseases of unknown cause that cause chronic inflammation or ulcers in the mucosa of the large and small intestine, such as Ulcerative Colitis (UC), Crohn's disease (Crohn's Disease: CD) is known. These are designated as specific intractable diseases by the Ministry of Health, Labor and Welfare.

  Ulcerative colitis is an inflammatory disease of the large intestine that causes erosions and ulcers in the mucous membrane of the large intestine. Occasionally, diarrhea with melena and frequent abdominal pain appear as characteristic symptoms. The lesion has the property of spreading continuously from the rectum and ascending (oral), and at most it extends from the rectum to the entire colon.

  In recent years, the number of patients with ulcerative colitis in Japan has increased by approximately 5,000 each year, and according to the number of patients receiving medical treatment for specific diseases, it is currently reported that it exceeds 100,000, and is said to be 1,000,000 in the United States. Yes.

  The peak age of onset is 20-24 years for males and 25-29 years for females.

  Possible causes include the involvement of enteric bacteria, the abnormal immune system that normally prevents the immune system that protects against external enemies, or the involvement of changes in dietary habits. Not.

  For patients with ulcerative colitis, surgery is necessary in severe cases or when drug therapy does not work, but in principle, medical treatment with drugs is performed. Currently, there is no medical treatment that completely cures ulcerative colitis, but 5-aminosalicylic acid (5-ASA) preparations, corticosteroids, immunomodulators, etc. as effective treatment methods to suppress intestinal inflammation Drug treatment and blood cell component removal therapy are known (see Patent Documents 1 and 2).

  Crohn's disease is a disease in which inflammation or ulceration can occur in any part of the digestive tract from the oral cavity to the anus, but the terminal part of the small intestine is a common site, causing abdominal pain, diarrhea, bloody stool, weight loss, etc. .

  In recent years, the number of patients with Crohn's disease in Japan has been increasing by about 1,500 each year, and according to the number of patients receiving medical treatment for specific diseases, it is now reported to exceed 30,000.

  It is common among young people in their 10s and 20s, with the most common onset being 20-24 years for men and 15-19 years for women.

  The cause is not yet clarified and there is no fundamental treatment, but in order to suppress inflammation in the intestinal tract and relieve symptoms and improve nutritional status, In the exacerbation period, medical treatment combining nutrition therapy and drug therapy is mainly performed.

  As therapeutic drugs, 5-aminosalicylic acid preparations, corticosteroids, and immunomodulators such as 6-MP and azathioprine are mainly used. In addition, 5-aminosalicylic acid preparations and immunomodulators are used to maintain remission. Anti-TNFα receptor antagonists have been used at relatively early stages in patients with intractable fistulas. In addition, blood cell component removal therapy may be performed.

JP 2005-509619A Special table 2009-543873

  However, it is desired to provide a therapeutic agent that is noninvasive, highly safe, and effective regardless of the patient, and it is desired to provide a therapeutic drug with a low drug price. Expected.

  The present invention has been made in view of the circumstances as described above, and an object of the present invention is to provide a preventive or therapeutic agent for inflammatory bowel disease that is highly safe and effective and can be supplied at a low price.

  In order to solve the above problems, the preventive or therapeutic agent for inflammatory bowel disease of the present invention is at least selected from hydroxylated fullerene, polyvinylpyrrolidone inclusion fullerene, fullerene, and pharmacologically acceptable salts thereof. It is characterized by containing one kind of fullerene as an active ingredient.

  According to the preventive or therapeutic agent for inflammatory bowel disease of the present invention, it can be supplied at a low price with high safety and effectiveness.

FIG. 1 is a graph showing the measurement results of changes in body weight of mice in the DSS administration group and the non-administration group in Example 1. FIG. 2 is a graph showing the measurement results of the length of the large intestine and blood stool score extracted from mice in the DSS administration group and the non-administration group in Example 1. FIG. 3 is a graph showing the measurement results of MPO activity in the large intestine extracted from mice in the DSS administration group and the non-administration group in Example 1. 4 is a graph showing the measurement results of leukocytes, lymphocytes and granulocytes in the large intestine excised from the mice of the DSS administration group and the non-administration group in Example 1. FIG. FIG. 5 is a graph showing the measurement results of cytotoxicity of hydroxylated fullerenes by measuring LDH activity in Example 1. FIG. 6 is a graph showing the measurement results of ELISA for the amount of IL-8 in the supernatant after stimulation with TNF-α in Example 1. FIG. 7 is a graph showing the measurement results by ELISA of the amount of IL-8 in the supernatant after IL-1β stimulation in Example 1. FIG. 8 is a graph showing measurement results of changes in body weight of mice in the DSS administration group and the non-administration group in the safety evaluation of Example 2. FIG. 9 is a graph showing measurement results of the weights of kidney, liver, and spleen extracted from mice in the DSS administration group and the non-administration group in the safety evaluation of Example 2. FIG. 10 is a graph showing the results of measuring the length of the large intestine and the weight of the large intestine excised from the mice of the DSS administration group and the non-administration group in the safety evaluation of Example 2. FIG. 11 is a graph showing the measurement results of body weight changes of mice in the DSS administration group and the non-administration group in the anti-inflammatory effect evaluation of Example 2. FIG. 12 is a graph showing the results of measuring the length of the large intestine and the weight of the large intestine excised from the mice of the DSS administration group and the non-administration group in the anti-inflammatory action evaluation of Example 2. FIG. 13 is a graph showing the measurement results of blood stool scores of mice in the DSS administration group and the non-administration group in the anti-inflammatory action evaluation of Example 2. FIG. 14 is a graph showing the measurement results by ELISA of the amount of IL-8 in the supernatant after IL-1β stimulation in Example 1. FIG. 15 is a graph showing measurement results of changes in body weight of mice in the DSS administration group and the non-administration group in the safety evaluation of Example 3. FIG. 16 is a graph showing measurement results of blood tests of mice in the DSS administration group and the non-administration group in the safety evaluation of Example 3. FIG. 17 is a graph showing measurement results of changes in body weight of mice in the DSS administration group and the non-administration group in the anti-inflammatory effect evaluation of Example 3. FIG. 18 is a graph showing measurement results of blood stool scores of mice in the DSS administration group and the non-administration group in the anti-inflammatory effect evaluation of Example 3.

  The present invention is described in detail below.

  The preventive or therapeutic agent for inflammatory bowel disease of the present invention comprises at least one fullerene selected from hydroxylated fullerene, polyvinylpyrrolidone inclusion fullerene, fullerene, and pharmacologically acceptable salts thereof as an active ingredient. Contains.

Examples of the fullerene in these fullerenes include C ₆₀ , C ₇₀ , and a mixture of C ₆₀ and C ₇₀ . Examples of the pharmacologically acceptable salt include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, salts with organic bases such as amines, and the like.

As the fullerene hydroxide, for example, one represented by C ₆₀ (OH) _n (n represents an integer of 30 to 48) can be used. Such fullerene hydroxide has infrared absorption spectrum analysis (having hydroxyl groups and no carbon-hydrogen single bond and no carbon-oxygen double bond), elemental analysis (having close values of the number of hydrogen and oxygen elements) , Water measurement (estimated water content), UV-visible spectrum analysis (decrease in the number of conjugated double bonds), water solubility measurement, or particle size distribution measurement in water, or a combination thereof.

  Polyvinylpyrrolidone inclusion fullerene (hereinafter referred to as “PVP fullerene”) is a composite of polyvinylpyrrolidone and fullerene. A commercially available PVP fullerene can be used, and can be produced by a known method. For example, an organic solvent solution of fullerene can be added to an organic solvent solution of polyvinylpyrrolidone at room temperature and mixed well, and then the organic solvent can be removed under reduced pressure or the like to obtain an aqueous solution or powder.

  The weight average molecular weight (Mw) of polyvinylpyrrolidone is not particularly limited, but for example, 3,000 to 3,000,000, especially 6,000 to 1,500,000 can be used according to a viscosity measurement method.

  The prophylactic or therapeutic agent for inflammatory bowel disease of the present invention can be in various pharmaceutical dosage forms according to the therapeutic purpose. Specifically, tablets, coated tablets, pills, powders, granules, capsules Oral preparations such as liquids, suspensions and emulsions, and parenteral preparations such as injections and suppositories. These administration agents can be formulated by a conventional formulation method generally known in this field using a pharmaceutically acceptable carrier or the like.

  In forming into a tablet form, as a carrier, for example, excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid; water, ethanol, propanol, corn starch, Simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, potassium phosphate, polyvinylpyrrolidone, etc .; dried starch, sodium alginate, agar powder, laminaran powder, Disintegrating agents such as sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, lactose; sucrose, stearic acid, kaka Disintegration inhibitors such as butter and hydrogenated oils; Absorption accelerators such as quaternary ammonium salts and sodium lauryl sulfate; Moisturizers such as glycerin and starch; Adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid A lubricant such as purified talc, stearate, boric acid powder, and polyethylene glycol can be used. Furthermore, the tablet can be made into a tablet coated with a normal coating as necessary, for example, sugar-coated tablet, gelatin-encapsulated tablet, enteric-coated tablet, film-coated tablet, double tablet, multilayer tablet and the like.

   In forming into a pill form, as a carrier, for example, excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, talc; binders such as gum arabic powder, tragacanth powder, gelatin, ethanol; Disintegrants such as laminaran powder and kanteng powder can be used.

  Capsules are prepared by mixing with various carriers exemplified above and filling them into hard gelatin capsules, soft capsules and the like according to a conventional method.

  When an oral liquid preparation is used, a liquid preparation, a syrup, an elixir or the like can be produced by a conventional method using a corrigent, a buffer, a stabilizer, a corrigent and the like. In this case, examples of the flavoring agent include sucrose, orange peel, citric acid, and tartaric acid, examples of the buffering agent include sodium citrate, and examples of the stabilizer include tragacanth, gum arabic, and gelatin.

  In forming into a suppository, for example, polyethylene glycol, cacao butter, higher alcohol, higher alcohol esters, gelatin, semi-synthetic glyceride and the like can be used as a carrier.

  In the case of injections, solutions, emulsions and suspensions are preferably sterilized and isotonic with blood. When forming into these forms, diluents such as water, aqueous lactic acid, ethyl alcohol, Propylene glycol, macrogol, ethoxylated isostearyl alcohol, polyoxyethylenated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters and the like can be used. In this case, a sufficient amount of sodium chloride, glucose, or glycerin to prepare an isotonic solution may be contained in the pharmaceutical preparation, and a normal solubilizing agent, buffer, soothing agent, etc. It may be added.

  Furthermore, you may mix | blend a coloring agent, a preservative, a fragrance | flavor, a flavor agent, a sweetening agent, etc. with said each formulation as needed. The content of the active ingredient, fullerene hydroxide, polyvinylpyrrolidone inclusion fullerene, fullerene, and pharmacologically acceptable salts thereof in the preventive or therapeutic agent for inflammatory bowel disease of the present invention is not particularly limited and is appropriately selected. However, in general, it is preferable that the content is usually about 1 to 80% by mass in the preparation.

  The method for administering the prophylactic or therapeutic agent for inflammatory bowel disease of the present invention is not particularly limited, and is appropriately determined according to various preparation forms, patient age, sex and other conditions, patient symptom severity, and the like. For example, tablets, pills, powders, granules, capsules, solutions, suspensions and emulsions are orally administered. The injection is administered intravenously alone or mixed with a normal fluid such as glucose or amino acid, and further administered alone, if necessary, intraarterially, intramuscularly, intradermally, subcutaneously, or intraperitoneally. Suppositories are administered rectally.

  The dosage of the active ingredient of the preventive or therapeutic agent for inflammatory bowel disease of the present invention can be appropriately selected depending on the usage, the age of the patient, sex and other conditions, the degree of the disease, and the like. The active ingredient of the present invention, fullerene hydroxide, polyvinylpyrrolidone inclusion fullerene, fullerene, and pharmacologically acceptable salts thereof have low toxicity and can be used safely. The dosage of active ingredients (fullerenes) is usually about 10 to 4000 mg / day, preferably about 100 to 1000 mg / day. These preparations of the present invention can be administered once a day or divided into 2 to 4 times a day.

  EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

  In the following Examples, the in vivo drug efficacy evaluation test for inflammatory bowel disease was conducted mainly by the DSS-induced mouse colitis model. A wide variety of models have been devised for the study of inflammatory bowel diseases, including spontaneous colitis models, colitis models based on chemical and immunological mechanisms, and genetically modified colitis models. The mouse colitis model is frequently used in studies for evaluating the efficacy of inflammatory bowel disease, and is a method for inducing induction of colitis by allowing a mouse to drink a dextran sulfate sodium (DSS) solution.

  C57BL / 6J male mice (SPF) purchased at 7 weeks of age were preliminarily raised for 1 week and randomly divided into 2 groups, one of which was used as a control group and the other as a DSS administration group. Thereafter, DSS (molecular weight: 36,000 to 40,000, MP Biomedicals) was dissolved in ultrapure water at a concentration of 2.5% and allowed to drink freely for 7 days as drinking water.

  The weights of mice in both groups were measured before and after DSS drinking water administration. Meanwhile, the body weight was measured once a day. On the final day, the mice were euthanized, and after removing the large intestine tissue, its full length and weight were measured. In addition to the normal autopsy of stool, the state of blood stool was observed using a fecal occult blood measurement kit (Shionogi), and scored in 5 stages from 5 strong positive to 1 negative.

For the body weight, bloody stool score, colon length, etc., the mean value ± standard error for each group of values obtained from 5 to 7 animals per group was calculated. About the data of body weight and large intestine length, the comparison between groups was performed by Student-t test or multiple comparison. In any test, p <0.05 was considered statistically significant.
<Example 1>
Mice C57BL / 6 were allowed to drink DSS (2.5%) freely, and mice in the DSS administration group had C ₆₀ (OH) ₃₆ 205 μg / mouse (fullerene alone concentration: 100 μg / mouse), C ₆₀ (OH) ₄₄ 224 μg / mouse (Concentration of fullerene alone: 100 μg / mouse), 5-aminosalicylic acid (5-ASA, manufactured by SIGMA) 5 mg / mouse, or N-acetylcysteine (NAC, manufactured by SIGMA) 1 mg / mouse was orally administered for 7 days.

  Thereafter, the body weight was measured, dissected 7 days after the start of DSS administration, an autopsy was performed, and the length of the large intestine and blood stool score were measured and a blood cell test was performed.

The measurement results of body weight are shown in FIG. Both C ₆₀ (OH) ₃₆ and C ₆₀ (OH) ₄₄ suppressed weight loss, and C ₆₀ (OH) ₃₆ suppressed weight loss more effectively than the existing drugs 5-ASA and NAC.

The measurement results of the length of the large intestine and blood stool score are shown in FIG. Both C ₆₀ (OH) ₃₆ and C ₆₀ (OH) ₄₄ suppressed the shortening of the length of the large intestine and the increase in blood stool score. C ₆₀ (OH) ₃₆ was confirmed to be more effective than the existing drugs 5-ASA and NAC.

The MPO activity in the large intestine mucosa was dissected and the large intestine was collected, homogenized, centrifuged, and the supernatant was measured by ELISA. MPO (Myeloperoxidase) is present only in neutrophils and monocytes and catalyzes the reaction in which hypochlorous acid is produced from hydrogen peroxide and chloride ions. The result is shown in FIG. It was suggested that both C ₆₀ (OH) ₃₆ and C ₆₀ (OH) ₄₄ suppressed infiltration of neutrophils and the like.

FIG. 4 shows the measurement results of leukocytes, lymphocytes, and granulocytes in the blood cell test in blood collected at the time of dissection. DSS administration increased white blood cells, lymphocytes, and granulocytes. There was a tendency to suppress the increase in blood cells by administering fullerene hydroxide (C ₆₀ (OH) ₃₆ , C ₆₀ (OH) ₄₄ ) or NAC.

Next, cytotoxicity of fullerene hydroxide was evaluated by LDH assay. Human intestinal epithelial cell line (Caco2) was seeded in a 96-well microtiter plate, hydroxylated fullerene (C ₆₀ (OH) ₃₆ , C ₆₀ (OH) ₄₄ ) was added and incubated for 24 hours, and then the supernatant was collected . The LDH activity due to the reduction of tetrazolium salt INT to formazan in the presence of NADH and hydrogen ions by a series of enzymatic reactions was quantified by measuring the absorbance of formazan to evaluate cytotoxicity.

  The result is shown in FIG. Hydroxyl fullerene showed little cytotoxicity against Caco2 cells.

Caco2 cells were seeded in a 96-well microtiter plate, and each hydroxylated fullerene (C ₆₀ (OH) ₃₆ , C ₆₀ (OH) ₄₄ ) was added. After 30 minutes, stimulation with inflammatory cytokine TNF-α was performed. did. After 24 hours, the supernatant was collected, and the amount of interleukin-8 (IL-8) in the supernatant was evaluated by ELISA. The result is shown in FIG. In addition, the stimulating substance is changed to IL-1β and suspended in each hydroxylated fullerene (C ₆₀ (OH) ₁₂ , C ₆₀ (OH) ₂₄ , C ₆₀ (OH) ₃₆ , C ₆₀ (OH) ₄₄ ) medium. The results of measuring the amount of IL-8 released after the addition are shown in FIG. By adding hydroxylated fullerene, the production of IL-8 by stimulation of TNF-α and IL-1β, which are various inflammatory cytokines, was suppressed in a concentration-dependent manner. This suggests the anti-inflammatory effect of fullerene hydroxide.
<Example 2>
The safety and anti-inflammatory effect of PVP-fullerene were evaluated.

  Safety evaluation was performed according to the following procedure. Mouse C57BL / 6 was orally administered with PVP-fullerene 51 mg / 0.5 ml / mouse (fullerene 150 μg / 0.5 ml / mouse), PVP 51 mg / 0.5 ml / mouse, or physiological saline for 7 days.

  Thereafter, body weight was measured and dissected, and whole blood and feces were collected, organs were measured, and colon length and colon weight were measured.

  The measurement results of body weight are shown in FIG. 8, the measurement results of kidney, liver and spleen weights are shown in FIG. 9, and the measurement results of colon length and colon weight are shown in FIG. PVP-fullerene was suggested to be extremely safe with no significant side effects. Although not shown in the figure, no abnormality was observed in blood cell examination and biochemical test.

  The usefulness of inflammatory bowel disease was evaluated by the following procedure. Mouse C57BL / 6 was allowed to drink DSS (2.5%) freely, and mice in the DSS administration group were given PVP-fullerene 51 mg / 0.5 ml / mouse (fullerene 150 μg / 0.5 ml / mouse), PVP 51 mg / 0.5 ml / mouse, 5- ASA 5 mg / mouse, Vitamin E 2.5 mg / 0.5 ml / mouse, and physiological saline were orally administered for 7 days.

  Meanwhile, body weight was measured and dissected 7 days after the start of DSS administration, and the colon length, colon weight, and blood stool score were measured.

  The results of body weight measurement are shown in FIG. 11, the results of colon length and colon weight are shown in FIG. 12, and the results of blood stool score are shown in FIG.

  Compared with existing drugs, PVP-fullerene was confirmed to have a more effective tendency to suppress weight loss, shorten colon length and colon weight, and increase blood stool score. Moreover, the tendency which is effective in these suppression was confirmed compared with the case of PVP single administration.

Caco2 cells were seeded in a 96-well microtiter plate, PVP-fullerene was added, and after 30 minutes, stimulation with IL-1β, an inflammatory cytokine, was performed. After 24 hours, the supernatant was collected, and the amount of interleukin-8 (IL-8) in the supernatant was evaluated by ELISA. The result is shown in FIG. By adding PVP-fullerene, IL-8 production by IL-1β stimulation is suppressed in a concentration-dependent manner, suggesting an anti-inflammatory effect by PVP-fullerene.
<Example 3>
Fullerene (C ₆₀ ) was evaluated for safety and anti-inflammatory activity.

  Safety evaluation was performed according to the following procedure. Fullerene 100 μg / 0.5 ml / mouse or ultrapure water was orally administered to mouse C57BL / 6 for 7 days.

  Thereafter, body weight measurement and blood test were performed.

  The measurement results of body weight are shown in FIG. 15, and the measurement results of blood tests (monocytes, platelet count, leukocytes, lymphocytes) are shown in FIG. Fullerenes were suggested to be extremely safe with no significant side effects.

  The anti-inflammatory effect was evaluated by the following procedure. Mouse C57BL / 6 was allowed to drink DSS (2.5%) freely, and mice in the DSS administration group were orally administered fullerene 100 μg / 0.5 ml / mouse, 5-ASA 5 mg / mouse, and Milli-Q water for 7 days.

  Thereafter, body weight was measured, dissected, and blood stool score was measured.

  The measurement result of the body weight is shown in FIG. 17, and the measurement result of the blood stool score is shown in FIG. Fullerene suppressed weight loss and increased blood stool score, confirming the same trend as 5-ASA, an existing drug.

Claims (4)

  A prophylactic or therapeutic agent for inflammatory bowel disease comprising, as an active ingredient, at least one fullerene selected from hydroxylated fullerene, polyvinylpyrrolidone inclusion fullerene, fullerene, and pharmacologically acceptable salts thereof.   The prophylactic or therapeutic agent for inflammatory bowel disease according to claim 1, comprising hydroxylated fullerene or a pharmacologically acceptable salt thereof as an active ingredient.   The preventive or therapeutic agent for inflammatory bowel disease according to claim 1, comprising fullerene or a pharmacologically acceptable salt thereof as an active ingredient.   The preventive or therapeutic agent for inflammatory bowel disease according to any one of claims 1 to 3, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.