KR20060025607A - Preventive and/or therapeutic agent for inflammatory bowel disease - Google Patents

Abstract

A medical agent that is available for bowel diseases, especially inflammatory bowel disease. In particular, a preventive and/or therapeutic agent for bowel diseases, comprising as an active ingredient a heat shock protein inducer or its salt, or a hydrate thereof. Specifically, there is provided a preventive and/or therapeutic agent for bowel diseases, comprising a prenyl ketone compound, 6,10,14,18- tetramethyl-5,9,13,17-nanodecatetraen-2-one, as an active ingredient.

Description

Prophylactic and / or therapeutic agents for inflammatory bowel disease {PREVENTIVE AND / OR THERAPEUTIC AGENT FOR INFLAMMATORY BOWEL DISEASE}

The present invention relates to a prophylactic and / or therapeutic agent for inflammatory bowel disease, and more particularly, to a prophylactic and therapeutic agent for inflammatory bowel disease such as ulcerative colitis or Crohn's disease.

Human Inflammatory Bowel Disease (hereinafter referred to simply as IBD) is an inflammatory disease that causes chronic inflammation or ulceration in the large intestine and small intestine mucosa and undergoes a chronic course, and the pathogenesis of the disease is unknown (for example, , See Non-Patent Document 1). This IBD is widely used, including ulcerative colitis or fibrosis or ulcer, an unknown non-specific inflammation that causes erosion or ulceration to invade the large intestine mucosa, and Crohn's disease, an inflammatory disease involving nonspecific granulomas. This includes intestinal Behcet's disease and ischemic enteritis. Currently, the number of patients with these diseases in Japan is on the rise, according to the report of the Ministry of Health, Labor and Welfare of the Intestinal Disorders Study Group, the number of ulcerative colitis and Crohn's disease totaled 100,000, especially among young people in their 10s to 20s. It is one of the incurable diseases that have to suffer and fight these diseases for the rest of their lives.

The mechanism of action of inflammation and the immune response in IBD have not been elucidated, but it has been found that various inflammatory mediators, such as tumor necrosis factor (hereinafter referred to as "TNF") α and macrophage endosperm inhibitors, are involved in lesions or exacerbations. (See, for example, Non Patent Literatures 2 to 5).

Modern treatments for IBD mainly use 5-aminosalicylic acid (simply referred to as "5-ASA"), glucocorticoids, immunosuppressants or immunomodulators. However, there are also patients who are resistant to this treatment. Recently, anti-TNF-α monoclonal antibodies have been developed as novel therapeutic agents (see, for example, Non-Patent Documents 6 and 7). This antibody inhibits the activity of TNF-α and rapidly improves enteritis in patients with Crohn's disease. However, side effects such as infections and malignant tumors frequently occur (see, eg, Non-Patent Documents 8 and 9). These side effects make it difficult to continue treatment with anti-TNF-α monoclonal antibodies.

Heat shock proteins (hereinafter referred to simply as "HSP") are stress-inducing proteins that exhibit protective properties and control the immune response (see, for example, Non-Patent Document 10). The primary function of HSP is to act as an intracellular chaperone for abnormally folded or mutated proteins, conferring cellular protection to cells under stress conditions. One of many HSPs is named HSP70 because its molecular weight is about 70 kDa. Some of its subfamilies have a strong cellular protective function against stress to the stomach, liver and heart (see, for example, Non Patent Literatures 11 to 14). HSP70-transgenic mice are resistant to heart injury models (see, eg, Non-Patent Documents 15 and 16). In the human intestine, expression of HSP70 is increased in ulcerative colitis compared with nonspecific colitis in the colonic mucosa (see Non-Patent Document 17). However, the role of HSP in the condition of IBD is not fully examined.

By the way, geranyl geranyl acetone (hereinafter, simply referred to as "GGA") is an acyclic polyisoprenoid which protects the stomach from obstacles such as ulcers. This compound is known to be effective in protecting the gastric mucosa against various stresses without affecting gastric acid secretion (see, for example, Non-Patent Documents 18 to 20). In addition, GGA enhances the synthesis and secretion of glycoproteins and surface-active phospholipid components (for example, see Non-Patent Document 22) as well as the synthesis and secretion of gastric mucin. Reported. Several years ago, GGA was reported to induce the expression of HSP70 in gastric mucosal cells (see, eg, Non-Patent Document 23). In addition, oral administration of GGA induces the expression of HSP70 in many local tissues in vivo to protect tissues against destruction and inflammation (see, eg, Non-Patent Literatures 24-26).

Further, although a patent application has been filed stating that GGA is effective for inflammatory bowel disease (see Patent Document 1), it is merely described in the claims, and in the detailed description, geranylgeranylacetone is used for inflammatory bowel disease. Since no experimental examples have been described that are effective, it is unclear whether they are effective against inflammatory bowel disease.

Many experimental animal models have been used to investigate factors that can induce intestinal inflammation or immune responses. Due to the reliability and convenience of the models themselves, dextran sodium sulfate (hereinafter simply referred to as "DSS") and 2,4,6-trinitrobenzenesulfonic acid (hereinafter referred to as "TNBS") induced enteritis models are widely used. (For example, see nonpatent documents 27-29).

On the other hand, ekabetosodium is known as the same gastric mucosa protective agent, and is known as a prophylactic / treatment agent for inflammatory bowel disease as in the present invention (see Patent Document 2, for example), but ekabetosodium has a chemical structure of GGA. Since it is not similar, and oral administration makes it difficult to reach the lower digestive tract such as the large intestine, for use in the treatment of inflammatory bowel disease, at least by intestinal infusion, as shown in many experimental examples of Patent Document 1 mentioned above. Administration is necessary. In the case of such intestinal infusion, since the drug is directly administered around the inflammatory site, in addition to placing a large burden on the patient, only a portion of the descending colon or the transverse colon can be reached, thereby limiting the treatment range.

Non Patent Literature 1: Podolsky DK. Inflammatory bowel disease. N Engl J Med 2002; 347: 417-29.

Non Patent Literature 2: Sartor RB. Pathogenesis and immune mechanisms of chronic inflammatory bowel diseases. Am J Gastroenterol 1997; 92 (12 Suppl.): 5S-11S.

[Non-Patent Document 3] Papadakis KA, Targan SR. Role of cytokines in the pathogenesis of inflammatory bowel disease. Annu Rev Med 2000; 51: 289-98.

[Non-Patent Document 4] de Jong YP, Abadia-Molina AC, Satoskar AR, et al. Development of chronic colitis is dependent on the cytokine MIF. 2001; 2: 1061-66.

[Non-Patent Document 5] Ohkawara T, Nishihira J, Takeda H, et al. Amelioration of dextran sulfate sodium-induced colitis by anti-macrophage migration inhibitory factor antibody in mice. Gastroenterology, 2002; 123: 256-70.

[Non-Patent Document 6] van Dullemen HM, van Deventer SJ, Hommes DW, et al Treatment of Crohn's disease with anti-tumor necrosis factor chimeric monoclonal antibody (cA2). Gastroenterology 1995; 109: 129-35.

[Non-Patent Document 7] Targan SR, Hanauer SB, van Deventer SJ, et al. A short-term study of chimeric monoclonal antibody cA2 to tumor necrosis factor alpha for Crohn's disease. Crohn's Disease cA2 Study Group. N Eng J Med 1997; 337: 1029-35.

Non-Patent Document 8: Bickston SJ, Lichtenstein GR, Arseneau KO, et al. The relationship between infliximab treatment and lymphoma in Crohn's disease. Gastroenterology 1999; 117: 1433-37.

[Non-Patent Document 9] Keane J, Gershon S, Wise RP, et al. Tuberculosis associated with infliximab, a tumor necrosis factor alpha-neutralizing agent. N Engl J Med 2001; 345: 1098-104.

Non Patent Literature 10: Pockley AG. Heat shock protein as regulators of the immune response. Lancet 2003; 9; 362 (9382): 469-76

[Non-Patent Document 11] Nakamura K, Rokutan K, Marui N, et al. Induction of heat shock proteins and their implication in protection against ethanol-induced damage in cultured guinea pig gastric mucosal cells. Gastroenterology 1991; 101: 161-166

Non Patent Literature 12: Mestril R, Chi SH, Sayen MR, et al. Expression of inducible stress protein 70 in rat heart myogenic cells confers protection against stimulated ishemia-induced injury. J Chn Invest 1994; 93: 759-767.

[Non-Patent Document 13] Urayama S, Musch MW, Retskv J, et al. Dexamethasone protection of rat IEC against oxidant injury mediated by induction of heat shock protein 72. J Clin Invest 1998; 102: 1860-1865.

Non Patent Literature 14: Fujimori S, Otaka M, Otani S, et al Induction of a 72-kDa heat shock protein and cytoprotection against thioacetamide-induced liver injury in rats. Dig Dis Sci 1997; 42: 1987-1994.

Non-Patent Document 15: Marber MS, Mestril R, Chi SH, et al. Overexpression of the rat inducible 70-kD heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury. J Clin Invest 1995; 95: 1446-56.

[Non-Patent Document 16] Hutter JJ, Mestril R, Tam EK, et al. Overexpression of heat shock protein 72 in transgenic mice decreases infarct size in vivo. Circulation 1996; 94: 1408-11.

Non-Patent Document 17: Ludwig D, Stahl M, Ibrahim ET, et al. Enhanced intestinal expression of heat shock protein 70 in patients with inflammatory bowel disease. Dig Dis Sci 1999; 44: 1440-47.

Non Patent Literature 18: Terano A, Shiga J, Hiraishi H, et al. Protective action of tetraprenylacetone against ethanol-induced damage in rat gastric mucosa. Digestion 1986; 35: 182-88.

Non-Patent Document 19: Pappas TN, Mulvihil 1 SJ, Goto Y, et al. Advances in drug therapy for peptic ulcer disease. Arch Surg 1987; 122: 447-50.

Non Patent Literature 20: Bilski J, Sarosiek J, Murty VL, et al. Enhancement of the lipid content and physical properties of gastric mucus by geranylgeranylacetone. Biochem Pharmaco 1 1987; 36: 4059-65.

Non-Patent Document 21: Fujimoto M, Yamanaka T, Bessho M, et al. Effects of geranylgeranylacetone on gastrointestinal secresion. Eur J Pharmacol 1982; 77: 113-18

Non-Patent Document 22: Ito M, Tanaka T, Suzuki Y. Increasing action of teprenone, a new antiulcer agent, on high-molecular-weight glycoprotein in gastric mucus during the healing process of acetic acid-induced ulcer in rats. Jpn J Pharmacol 1986; 41: 117-25.

Non-Patent Document 23: Hirakawa T, Rokutan K, Nikawa T, et al. Geranylgeranylacetone induces heat shock proteins in cultured guinea pig gastric mucosal cells and rat gastric mucosa. Gastroenterology 1996; 111: 345-57.

Non-Patent Document 24: Ooie T, Takahashi N, Saikawa T, et al. Single oral dose of Geranylgeranylacetone induces Heat-shock protein 72 and renders protection against ischemia / reperfusion injury in rat heart. Circulation 2001; 104: 1837-43.

Non-Patent Document 25: Yamagami K, Yamamoto Y, Ishikawa Y, et al. Effects of geranyl-geranyl-acetone administration before heat shock preconditioning for conferring tolerance against ischemia-reperfusion injury in rat livers. J Lab Clin Med 2000; 135: 465-75.

Non-Patent Document 26: Unoshima M, Iwasaka H, Eto J, et al. Antiviral effects of geranylgeranylacetone: enhancement of MxA expression and phosphorylation of PKR during influenza virus infection. Antimicrob Agents Chemother 2003; 47: 2914-21.

Non-Patent Document 27: Elson CO, Sastor RB, Tennyson GS, et al Experimental models of inflammatory bowel disease. Gastroenterology. 1995; 109: 1344-67.

Non-Patent Document 28: Okayasu I, Hatakeyama S, Yamada M, et al. A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice. Gastroenterology 1990; 98: 694-702.

Non-Patent Document 29: Morris GP, Beck PL, Herridge MS, et al. Hapten-induced model of chronic inflammation and ulceration in the rat colon. Gastroenterology 1989; 96: 795-803.

Patent Document 1: WO02 / 098398

Patent Document 2: Japanese Patent Laid-Open No. 2002-104962

<Start of invention>

<Technical problem to be solved>

It is an object of the present invention to provide a novel drug which is safe and effective in the prevention and / or treatment of inflammatory bowel disease throughout the intestinal tract without imposing great burden on the patient.

Means for solving the problem

As a result of earnest examination, the present inventors have found that a specific acyclic polyisoprenoid compound induces HSP in the intestinal tract, and is effective for the prevention and / or treatment of inflammatory bowel disease.

That is, according to the first aspect of the present invention, there is provided an agent for the prevention and / or treatment of intestinal diseases containing a heat shock protein inducing agent or a salt thereof or a hydrate thereof as an active ingredient.

In a preferred embodiment of the prophylactic and / or therapeutic agent for enteric diseases according to the present invention, the heat shock protein inducing agent is a prenyl ketone compound.

In a preferred embodiment of the prophylactic and / or therapeutic agent for enteric diseases according to the present invention, the prenyl ketone compound is represented by the formula (I) 6,10,14,18-tetramethyl-5,9,13,17- Nanodecatetraen-2-one.

In a preferred embodiment of the prophylactic and / or therapeutic agent for bowel disease according to the invention, the bowel disease is an inflammatory bowel disease.

In a preferred embodiment of the prophylactic and / or therapeutic agent for intestinal diseases according to the present invention, the inflammatory bowel disease is a disease selected from the group consisting of Crohn's disease, ulcerative colitis, enteric Behcet's disease and ischemic enteritis.

In a preferred embodiment of the prophylactic and / or therapeutic agent for enteric diseases according to the invention, the heat shock protein is heat shock protein 70.

The present invention also provides a prophylactic agent for inflammatory bowel disease comprising (6,10,14,18-tetramethyl-5,9,13,17-nanodecatetraen-2-one represented by the formula (I) as an active ingredient and / or ) To provide treatment.

<Formula I>

Inflammatory bowel disease in the prophylactic and / or therapeutic agent according to the present invention is selected from the group consisting of Crohn's disease, ulcerative colitis, intestinal Behcet's disease and ischemic enteritis.

According to a second aspect of the present invention, there is also provided a method for preventing and / or treating intestinal diseases, comprising the step of administering a heat shock protein inducing agent or salt thereof or a hydrate thereof.

In a preferred embodiment of the method of preventing and / or treating intestinal diseases according to the present invention, the heat shock protein inducing agent is a prenyl ketone compound.

In a preferred embodiment of the method for preventing and / or treating intestinal diseases according to the present invention, the prenyl ketone compound is represented by the formula (I) 6,10,14,18-tetramethyl-5,9,13,17 -Nanodecatetraen-2-one.

<Formula I>

In a preferred embodiment of the method of preventing and / or treating bowel disease according to the invention, said bowel disease is an inflammatory bowel disease.

In a preferred embodiment of the method of preventing and / or treating intestinal diseases according to the present invention, the inflammatory bowel disease is a disease selected from the group consisting of Crohn's disease, ulcerative colitis, enteric Behcet's disease and ischemic enteritis.

In a preferred embodiment of the method of preventing and / or treating intestinal diseases according to the invention, the heat shock protein is heat shock protein 70.

The present invention also provides a method for preventing inflammatory bowel disease comprising 6,10,14,18-tetramethyl-5,9,13,17-nanodecatetraen-2-one represented by the formula (I) as an active ingredient, and ( Or) provide a method of treatment.

<Formula I>

Inflammatory bowel disease in the prophylactic and / or therapeutic method according to the present invention is selected from the group consisting of Crohn's disease, ulcerative colitis, intestinal Behcet's disease and ischemic enteritis.

Further, according to the third aspect of the present invention, a heat shock protein inducing agent or salt thereof for preparing a prophylactic and / or therapeutic agent for enteric diseases containing a heat shock protein inducing agent or salt thereof or a hydrate thereof as an active ingredient The use of these hydrates is provided.

In a preferred embodiment of the use according to the invention, the heat shock protein inducer is a prenylketone compound.

In a preferred embodiment of the use according to the present invention, the prenyl ketone compound is 6,10,14,18-tetramethyl-5,9,13,17-nanodecatetraene-2- represented by the formula (I). It's on.

<Formula I>

In a preferred embodiment of the use according to the invention, said intestinal disease is an inflammatory bowel disease.

In a preferred embodiment of the use according to the present invention, the inflammatory bowel disease is a disease selected from the group consisting of Crohn's disease, ulcerative colitis, intestinal Behcet's disease and ischemic enteritis.

In a preferred embodiment of the use according to the invention, the heat shock protein is heat shock protein 70.

The present invention also provides a prophylactic agent for inflammatory bowel disease containing 6,10,14,18-tetramethyl-5,9,13,17- or nodecatetraen-2-one represented by the formula (I) as an active ingredient or The use of heat shock protein inducers or salts thereof or hydrates thereof for the preparation of a therapeutic is provided.

<Formula I>

Inflammatory diseases in the use according to the invention are selected from the group consisting of Crohn's disease, ulcerative colitis, intestinal Behcet's disease and ischemic enteritis.

Effect of the Invention

According to the invention, the compound according to the invention 6,10,14,18-tetramethyl-5,9,13,17-nanodecatetraen-2-one induces a heat shock protein 70 in the intestine, Effective for preventing and / or treating diseases, more specifically inflammatory bowel disease. In addition, since the compounds according to the present invention can be administered orally, rather than by intestinal infusion, it is possible to prevent and / or treat intestinal diseases without undue strain on the patient.

Best Mode for Carrying Out the Invention

The following embodiment is an illustration for demonstrating this invention, and is not intended to limit this invention only to this embodiment. The present invention can be implemented in various forms without departing from the gist of the invention.

The present invention provides non-toxic prophylactic and / or therapeutic agents that are effective against the treatment of bowel disease, particularly inflammatory bowel disease. Hereinafter, the present invention will be described in detail.

In the present invention, the compound which is an active ingredient is a prenyl ketone compound represented by the formula (I).

<Formula I>

The compound name of the compound preferably used in the present invention is 6,10,14,18-tetramethyl-5,9,13,17-nanodecatetraen-2-one (alias: geranylgeranylacetone, generic name: Tefrenone, hereinafter also referred to simply as "GGA." The GGA used in the present invention has a double bond in four parts of the structure, and a total of eight types of geometric isomers exist, but the present invention is not particularly limited, and any one isomer may be used, or a mixture of two or more kinds thereof. It may be. Among these, preferred compounds include (5E, 9E, 3E) -6,10,14,18-tetramethyl-5,9,13,17-nonadecatetraen-2-one and (5Z, 9E, 13E)- And 6,10,14,18-tetramethyl-5,9,13,17-nonadecatetraen-2-one.

GGA used in the present invention is a known compound and can be obtained as a reagent or an industrial raw material. As a synthesis method of GGA, it can synthesize | combine according to the method disclosed by Unexamined-Japanese-Patent No. 53-145922, for example.

GGA, which is an active ingredient used in the present invention, may include a polymorphic form, but is not limited thereto. Any one crystal form may be single, or a crystalline mixture may be used. In addition, metabolites resulting from degradation of GGA used in the present invention are also included in the claims of the present invention.

The prophylactic and / or therapeutic agent according to the present invention can use GGA as it is, or known pharmaceutically acceptable carriers such as excipients (specifically lactose, white sugar, starch, mannitol, etc.), binders (specifically Examples include α-ized starch, gum arabic, carboxycellulose, polyvinylpyrrolidone, etc.), lubricants (specifically, magnesium stearate, talc, etc.), disintegrants, colorants, copulation agents, stabilizers, emulsifiers, absorption accelerators, interfaces It can be formulated by the method generally used by mix | blending the components generally used as a raw material of pharmaceutical preparations, such as an active agent, a pH adjuster, a preservative, antioxidant. If necessary, components such as blood flow promoters, bactericides, anti-inflammatory agents, cell activators, vitamins, amino acids, humectants, and keratin solubilizers can be further blended.

Formulations of the formulation of the prophylactic and / or therapeutic agents according to the invention include tablets, powders, granules, granules, coated tablets, capsules, syrups, troches, inhalants, suppositories, injections, lyophilizers, ointments, eye ointments, Eye drops, eye drops, ear drops, cataplasms, lotions, and the like.

In addition, although the dosage form of GGA is not specifically limited in this invention, It is preferable to administer orally. GGA can be obtained as a brand name "Servex" (made by Ezai Kabushiki Kaisha).

GGA-containing prophylactic and / or therapeutic agents according to the invention are useful for the prevention and / or treatment of intestinal diseases in mammals (e.g. humans, mice, rats, malmots, rabbits, dogs, horses, monkeys, etc.), in particular humans Useful for the treatment and / or prevention of bowel disease.

The dosage of the GGA-containing prophylactic and / or therapeutic agent according to the present invention is appropriately selected depending on conditions such as symptoms, age, weight, etc., but is 20 to 2000 mg, preferably 50 to 1000 mg, more preferably per adult day. Is 100 to 500 mg.

The diseases in which the GGA compound according to the present invention is effective are intestinal diseases, Crohn's disease, ulcerative colitis, enteric Behcet's disease, simple ulcers, radiation enteritis and ischemic enteritis, and are particularly effective against Crohn's disease or ulcerative colitis. .

Although this invention is demonstrated further in detail by the following example, the scope of the present invention is not limited to this. Various changes and modifications are possible to those skilled in the art based on the description of the present invention, and these changes and modifications are also included in the present invention.

Experimental Example 1

(Experimental animal)

Mice were used SPF (specific pathogen free) BALB / c male mice (manufactured by Charles River Japan Co., Ltd.) at 8 to 10 weeks of age and weighing 22 to 25 g. All mice were fed free negatively by providing standard experimental feed. The present invention has been made in compliance with the Helsinki Declaration and with the approval of the Animal Experimentation Ethics Regulations Committee of the Hokkaido University School of Medicine.

Statistical interpretation

Unless otherwise noted, all data described below are expressed as mean ± standard error. Results were statistically interpreted using the F test and the Student's t test (StatView, SAS Institute, Cray, North Carolina). P <0.05 was considered statistically significant.

(Preparation and Oral Administration of GGA)

GGA (trade name "Servex", manufactured by Ezai Kabushiki Kaisha) used a mixture of 5% Arabian rubber solution and 0.0008% tocopherol. Mice were orally administered 5 ml / kg / volume of GGA at 300 mg / kg / dose via a metal tube attached to a 1 ml syringe. Mice in the control group received an equivalent amount of tocopherol-containing gum arabic (hereinafter referred to as control reagent). Mice in each group received GCA or control reagents (four times in total) two hours before DSS administration and every other day for seven days after the initial administration of DSS. Dose dependent experiments were evaluated with different doses of GGA (50, 100, 300, 500 mg / kg) in clinical evaluation of DSS colitis. To examine the effect of GGA on the induction of HSP70 by Western blot, mice were treated with a single oral dose of 300 mg / kg GGA. Samples of colon tissue were collected from each mouse before and after GGA or control reagent administration.

Sodium dextran sulfate: induction and evaluation of DSS colitis

Mice were induced by enteric membranes by free negative aqueous solution of 3.0% DSS (molecular weight: 40,000; ICN Biochemicals Inc, California, USA) for 7 days. The body weights of the mice were observed daily from the start of administration, and rectal bleeding and diarrhea were observed. The weight of each mouse was measured. Colonic tissues were taken at various time intervals for histological evaluation, cytokine, HSP and myeloperoxidase (hereinafter referred to as "MPO") activity at each stage of the experiment. The colon was stored at -80 ° C until just before use. In addition, the severity of colitis was evaluated by the length of the colon and histological examination 7 days after the start of DSS administration.

1 is a diagram showing the frequency of diarrhea and rectal bleeding at the time of DSS colitis induction in the control reagent administration group and the GGA treatment group to the mouse in the embodiment of the present invention. As can be seen from FIG. 1, the 300 mg / kg / time and 500 mg / kg / time GGA treated groups significantly reduced the frequency of diarrhea and rectal bleeding compared to the control reagent dosed group. In the control reagent-administered group, when the body weight before DSS administration was 100%, the body weight of the mouse decreased to 86.3 ± 2.4% after 7 days of DSS administration, but in the GGA-treated group, the change in body weight was compared with the treatment group. Significantly reduced (93.8 ± 4.1% in the 300 mg / kg / hour group and 94.7 ± 5.0% in the 500 mg / kg / hour group). In clinical evaluation of the dose dependent effect of GGA, 300 mg / kg / dose of GGA was found to be sufficient to inhibit the disease.

Colonization and tissue changes are known to be well correlated, and colon length is routinely used as a morphological parameter of the degree of inflammation. In order to evaluate the length of the large intestine, mice in each group were killed at a predetermined time. Figure 2 shows the colon length after 7 days of DSS administration. 2 shows that the colon length (6.8 ± 0.3 cm) in the GGA treated group was significantly longer than the colon length (5.1 ± 0.1 cm) in the control reagent treated group.

Histological Interpretation of DSS Colitis

Colon tissue was opened in the longitudinal direction, immobilized with 10% neutral formalin buffer, and immersed in paraffin. After removing the paraffin from the tissue portion on a glass slide, the samples were stained with hematoxylin eosin (hereinafter referred to as "HE"). As a histological assessment of tissue damage, the area of inflammatory lesions is observed under a microscope, and Cooper et al. (Benjamin IJ, McMIllan DR. Stress (heat shock) proteins molecular chaperones. Circ Res. 1998 27; 83 : 117-32.]) And quantified by two pathologists. Colon damage was classified into six stages. Ie 0: normal mucosa, 1: infiltration of inflammatory cells, 2: less than half of crypts, 3: more than half of crypts, 4: loss of crypts, 5: epithelial cell loss (ulcer) Formation and erosion). In addition, the extent of inflammatory lesions was also evaluated. The extent of lesions throughout the large intestine was divided into six stages. That is, 0: 0%, 1: 1 to 20%, 2: 21 to 40%, 3: 41 to 61%, 4: 61 to 80%, 5: 81 to 100%.

3 shows the results of histological analysis of DSS colitis in the present invention. Histology in the large intestine of DSS treated BALB / c mice was examined to evaluate the effect of GGA on tissue damage using HE staining. Invasive cell number in the colon was minimal before DSS treatment (see FIG. 3A), but infiltrated cell number increased with loss of crypts and destruction of epithelial cells 7 days after DSS treatment (see FIG. 3B). In the GGA treated group, tissue damage was significantly improved and the infiltrating inflammatory cell number was reduced compared to the control reagent treated group (see FIGS. 3C and D). For GGA treated mice, the results of pathological histological scores for both tissue damage and lesion range were significantly reduced compared to untreated and control reagent treated mice (see FIG. 4). 4 shows the results of the histological scores of the mouse colon at 7 days after 3% DSS administration according to the present invention.

(Western Blot Analysis)

Tissues were ground in a Polytron homogenizer (Kinematica, Lucerne, Switzerland). Equal amounts of homogenate are 20 μl Tris-HCl, 50 mM (pH) containing 2-mercaptoethanol (1%), SDS (2%), glycerin (20%) and bromophenol blue (0.04%). 6.8) and the sample was heated at 100 ° C. for 5 minutes. Next, the sample was separated by electrophoresis using SDS-polyacrylamide gel (SDS-PAGE), and electrophoretically transferred to nitrocellulose membrane. The membrane was blocked with 1% nonfat dry milk in PBS, and the anti-HSP25, 40, 70, 90 antibodies (Stressgen, Victoria, BC) and β-actin (Sigma, St. Louis, MO) Sigma)) and then reacted with goat anti rabbit IgG antibodies bound to horseradish peroxidase (hereinafter referred to as "HRP"). The remaining complexes were processed for detection systems based on manufacturer protocols. Protein concentrations of cell homogenates were quantified using the Micro BCA Protein Assay Reagent Kit.

(Immunohistochemistry)

Immunohistochemical analysis for HSP70 was done using the Vectastain ABC kit based on the manufacturer's protocol. Paraffin-immersed colon tissue was excised into small pieces 4 μm thick. This fragment was pretreated with 3% H ₂ O ₂ at 4 ° C for 10 minutes, then treated with 10% normal goat serum at room temperature for 30 minutes, and then at 4 ° C with anti-HSP70 antibody (diluted to 1: 200, BC Incubated overnight with Stressgen, Victoria, Canada). HSP70 positive staining was visualized with diaminobenzidine.

Fig. 5 shows the results of Western blot analysis of HSP25, 40, 70 and 90 in the large intestine of colitis model mice treated with 3% DSS. Expression of HSP was examined by Western blot analysis using antibodies against HSP25, 40, 70, 90. HSP70 was detected weakly before GGA addition (300 mg / kg), but after 12 hours of GGA treatment its expression level increased. On the other hand, the expression of HSP25, 40 and 90 did not change even GGA treatment (see Fig. 5).

Fig. 6 shows the results of immunohistochemical analysis of HSP70 in the large intestine of DSS-induced colitis according to the present invention. In order to confirm the expression portion of HSP70 in the large intestine of mice suffering from DSS-induced colitis, immunohistochemistry of HSP70 was performed using specific antibodies of HSP70 in colon tissue. The immune reactivity of HSP70 was weakly positive in the crypts of epithelial cells and normal mice (see FIG. 6B). In DSS induction and control reagent treated mice, few HSP70 positive staining cells were present in the large intestine. Interestingly, staining in colon tissue of GGA treated mice was much stronger than that in control reagent treated mice (see FIG. 6D). These findings support the hypothesis that GGA induces endogenous HSP in the large intestine.

(Measurement of myeloperoxidase activity in the large intestine)

Tissue MPO activity is described by Krawisz et al. (Krawisz JE, Sharon P, Stenson WF. Quantitative assay for acute intestinal inflammation based on myeloperoxidase activity.Evaluation of inflammation in rat and hamster models.Gastroenterology 1984; 87: 1344 -50.]) Was obtained by a slightly modified method. That is, a tissue sample (about 300 mg) was homogenized in each sample for 30 seconds, 3 times in buffer (pH 6.0, 0.5% hexadecyltrimethylammonium bromide in 50 nM potassium phosphate buffer) using a polytron homogenizer on ice. I was. The sample was centrifuged at 20,000 × g for 20 minutes at 4 ° C. and its supernatant was collected. The sample (100 [mu] l) was added to 2.9 ml of 50 mM phosphate buffer (pH 6.0) containing 0.167 mg / ml o- dianisidine hydrochloride and 0.0005% hydrogen peroxide, using a spectrometer at 25 ° C. Measured. Protein concentration in the supernatant was determined using a Bradford assay kit (Bio-rad laboratories, Hercules, Calif.) For calibration, and the value was determined using human leukocytes ( Standardized using MPO purified from Sigma, St. Louis, Missouri.

Another parameter of inflammation is the level of myeloperoxidase (MPO) activity in colon tissue. MPO is an enzyme produced primarily by polymorphonuclear leukocytes and is an enzyme associated with granulocytes in tissues. Fig. 7 shows the results of MPO activity of the colon after 7 days of 3% DSS-induced colitis treatment in mice according to the present invention. In vehicle treated mice administered DSS, the level of MPO activity was significantly increased (see FIG. 7). In contrast, the level of MPO activity in GGA treated mice was significantly reduced (1.4 ± 0.4 units / g, P <0.03) compared to the level of MPO activity in the vehicle treated group (2.8 ± 0.3 units / g). . These results demonstrated that GGA inhibited granulocyte infiltration.

(Enzyme Immunoassay for Cytokines)

TNF-α in colon tissue was measured by a TNF-α specific ELISA kit (GT product, Minneapolis, Minnesota). Anti-mouse TNF-α IgG monoclonal antibody dissolved in 50 ml of phosphate buffered saline (PBS) was added to each well of a 96-well microtiter plate and left at room temperature for 30 minutes. After washing the plate three times with distilled water, PBS containing 0.5% bovine serum albumin (BSA) for blocking all wells was filled and left at room temperature for 20 minutes. After removal of the blocking solution, plasma or tissue samples were added twice to individual wells and incubated for 1 hour at room temperature. Plasma was obtained by cardiac puncture using a heparin-added syringe. For the preparation of colon tissue samples, tissue in PBS containing a mixture of protease inhibitors (1 μl-20 mg of tissue by the manufacturer's protocol) is homogenized in a polytron homogenizer (Kinematica, Lucerne, Switzerland) , The supernatant was analyzed by centrifugation at 12,000 xg for 10 minutes. Mouse recombinant TNF-α protein was used to obtain a standard curve. After washing the plate three times with PBS containing 0.05% Tween-20 (Wash Buffer), 50 μl of biotin binding anti TNF-α antibody was added to each well. After incubation for 1 hour at room temperature, the plates were washed again three times with washing buffer. Next, an avidin-bound goat anti-rabbit IgG antibody was added to each well, and the microtiter plate was incubated for 15 minutes at room temperature. After three washes with wash buffer, substrate solution (50 μL) was added to each well. The substrate solution (10 µl) contained 8 µg o-phenylenediamine and 4 µl of 30% H ₂ O ₂ in citric acid phosphate buffer (pH 5.0). After incubation for 20 minutes at room temperature, the reaction was terminated with 25 μl 4 N sulfuric acid. Absorbance was measured at 492 nm using an ELISA plate reader (Bio-Rad, Hercules, Calif., Model 3550).

Similarly, mouse IFN-γ in tissues was also measured using an IFN-γ specific ELISA kit.

In order to explain the site of action from the viewpoint of inhibiting cytokine production in the large intestine by GGA treatment, production of TNF-α and IFN-γ in colon tissues was measured using ELISA. 8 shows the production results of TNF-α and IFN-γ in the large intestine 7 days after 3% DSS treatment. In colon tissues, the contents of TNF-α and IFN-γ increased after 7 days of DSS administration (7.9 ± 1.0 and 9.7 ± 1.2 pg / mg proteins, respectively). On the other hand, production of these cytokines in GGA treated mice was significantly lower than that in vehicle treated mice (4.2 ± 0.8 and 4.1 ± 0.8 pg / mg protein, respectively). These results suggest that GGA inhibits the production of proinflammatory cytokines in the large intestine.

(2,4,6-trinitrobenzenesulfonic acid: induction and evaluation of TNBS colitis)

NBS colitis was induced in the BALB / c mice described above. That is, after fasting overnight, mice were anesthetized by intraperitoneal injection of bentobarbital (70 mg / kg). In order to evaluate the survival rate in TNBS colitis, 100 μl of TNBS (250 mg / kg) in 50% ethanol was prepared by polyethylene cannula (intermedic PE-20; manufactured by Becton Dickinson); Injection into the intestinal cavity (about 3 cm around the anus) by a 1 ml syringe with San Jose, CA. To measure body weight change by comparison with the first day, mice were treated with a dose of 100 mg / kg TNBS in 50% ethanol.

To evaluate the effects of GGA in other colitis models, the effects of GGA on survival and weight loss in TNBS induced colitis were examined. In this model, many mice died 7 days after TNBS administration because the 270 mg / kg TNBS dose induces a severe inflammatory response.

Figure 9 shows the results of the GGA effect on the survival rate in TNBS-induced severe colitis according to the present invention. In control reagent treated and TNBS treated mice, survival was reduced by 23.3 ± 8.8% (see FIG. 9). In contrast, the survival in TNBS induced colitis significantly increased by 56.7 ± 3.3% by oral administration of GGA.

10 shows the results of the GGA preventive effect of weight loss in TNBS induced colitis. Similar to survival, body weight loss was also minimal (-5.6 ± 1.2%) in GGA treated mice than control reagent treated mice (22.1 ± 3.3%) (see FIG. 10).

Experimental Example 2

(Onset of colitis by the DSS model)

Enteritis was developed by freely dehydrating 3.0% aqueous solution of sodium dextran sulfate (hereinafter referred to as "DSS") in 8-week-old mice (Balb / c mice, Charles River, Shizuoka) (n = 10) for 7 days. Mice caused diarrhea, bloody stool, and weight loss from 5 or 6 days after DSS aqueous solution administration. 100 mg / kg of GGA (manufactured by Ezai Kabushiki Co., Ltd.) was administered before and after DSS administration and 3 days after DSS administration in a mixed state in saline using Sonde.

(Inflammatory inhibitory effect of colitis model by GGA administration)

Seven days after the start of DSS administration, the intestines of mice were collected and evaluated for gross and histological findings. The histopathological diagrams of the non-administered group and the administration group are shown in FIGS. 11 and 12, respectively. As a result, tissue destruction progressed in the control administration of FIG. 11, while tissue destruction was hardly confirmed in the GGA administration group. In addition, six levels of scores produced by the inventors were scored for the extent of tissue destruction by inflammation and the extent of inflammation. Score criteria are as follows.

Degree of tissue destruction by inflammation

O: no inflammation

1: mild infiltration of inflammatory cells

2: shortening of crypts is longitude

3: high simplification of crypts

4: frost is lost

5: epithelial cells are missing

Scope of inflammation

0: none

1: 1 to 20%

2: 21-40%

3: 41 to 60%

4: 61 to 80%

5: 81-100%

The scores were compared in the GGA administered group and the non-administered group, and the results are shown in Table 1. As also shown in Table 1, the extent of tissue destruction was suppressed by the administration of GGA, and the extent of inflammation was reduced. Similarly, the incidence of diarrhea, the incidence of bloody stools, the weight loss rate, and the length of the intestine were compared. The results are shown in Table 2. As shown in Table 2, GGA administration suppressed the onset of diarrhea and bloody stool derived from DSS enteritis, and also had an inhibitory effect on weight loss and intestinal contraction.

From the above, it was found that GGA is very effective against DSS enteritis, a model of inflammatory bowel disease.

Inhibitory Effect of DSS Enteritis by GGA Administration Histological score Tissue destruction Scope of inflammation Control 3.7 ± 1.5 2.7 ± 0.6 GGA 1.3 ± 0.6 1.3 ± 0.6

Inhibitory Effect of DSS Enteritis by GGA Administration Diarrhea onset (%) Blood stool development rate (%) Weight loss (%) Length of the sheet (cm) Control 100 100 86.4 ± 4.4 5.1 ± 0.1 GGA 33 67 93.8 ± 5.1 6.7 ± 0.4

From the above results, it has been demonstrated that oral administration of GGA inhibits clinical disease activities such as diarrhea and rectal bleeding and weight loss induced by DSS. In addition, oral administration of GGA induced the expression of HSP70 in the large intestine, inhibiting the level of MPO activity and the production of inflammatory cytokines. In addition, unlike the DSS enteritis model, the effect of GGA was also confirmed in TNBS induced colitis. It is known that acute TNBS induced colitis is more severe than DSS induced enteritis in mice. Survival in mice treated with high doses of TNBS was improved by oral administration of GGA, and weight loss was also significantly inhibited by GGA. From these results, GGA has been found to have anti-inflammatory action through upregulation of HSP70 and to be useful for the treatment of human IBD.

1 is a view showing the results of the effect of oral GGA administration on clinical finding 7 days after DSS administration in the present invention. Data for diarrhea and rectal bleeding were interpreted by the F test. Data on changes in weight loss were interpreted by Student's t test. All values were compared to mice in DSS and control reagent treatments. * Indicates P <0.05, ** indicates P <0.01, *** indicates P <0.001, respectively, and a indicates the ratio when the first day of administration was 100%.

FIG. 2 shows the results of geranylgeranyl acetone (GGA) effects on the inhibition of colonic mononuclearization 7 days after administration of 3% sodium dextran sulfate (DSS) in drinking water. The left bar represents the results of normal control mice, the middle bar represents the results of control reagent administered mice, and the right bar represents the results of mice treated with 300 mg / kg GGA. The difference between control reagent-administered mice (n = 10) and GGA treated mice (n = 10) was statistically significant (P <0.05). The same result was confirmed in three independent experiments.

3 shows the results of histological examination of the GGA effect on DSS induced tissue damage in BALB / c mice. The colon tissue was opened in the longitudinal direction, fixed with 10% neutral buffered formalin buffer, and immersed in paraffin. After removing paraffin from the thin tissue fragments on the glass slide, the samples were stained with HE. (A) The micrograph of the mouse large intestine which provided only drink is shown. (B) Micrographs of the large intestine of mice treated with 3% DSS solution for 7 days are shown. Severe inflammatory cell infiltration and epithelial cell destruction were observed. (C) Micrographs of the mouse colon in which the control reagent was administered every other day in mice administered the 3% DSS solution are shown. It shows the same histological appearance as (B). (D) Micrographs of mouse colon treated with 300 mg / kg GGA every other day (four times in total) in mice administered 3% DSS solution are shown. GGA inhibited inflammatory cell infiltration in mucosal and submucosal tissues and showed protection against destruction of epithelial cells in the colonic mucosa. In addition, the magnification in this figure is 100 times.

4 shows the results of histological scores of the mouse colon at 7 days after 3% DSS administration. In histological evaluation of tissue damage, the extent of inflammatory lesions was evaluated under a microscope and quantified by the techniques described in the Examples herein. Bars with fine points represent the results of control reagent-administered mice (n = 10) and black bars represent the results of GGA treated mice (n = 10). Both tissue damage and the extent of lesions were significantly inhibited in the GGA treated mice (n = 10) when compared to the results of control reagent administered mice (n = 10).

5 shows the results of Western blot analysis of HSP25, 40, 70, and 90 in the large intestine of 3% DSS-induced colitis. Total colon homogenate was prepared from mice before and 12 hours after administration of GGA. Proteins were separated by SDS-PAGE and transferred to nitrocellulose and immunoblotting the expression of HSP25, 40, 70, 90 using HSP or β-actin specific antibodies. Expression of HSP70 was markedly increased in GGA treated mice compared to control reagent administered mice. The same results were confirmed in three independent experiments.

6 shows the results of immunohistochemical analysis of HSP70 in the large intestine of DSS-induced colitis. This shows a photograph of mouse colon treated with anti-HSP70 antibody or goat IgG (50-fold). (A) Photographs of small sections of colonic mucosa from normal mice treated with goat IgG are shown. (B) Photographs of colonic fragments from normal mice treated with anti-HSP70 antibodies are shown. (C) Photographs of small sections of vehicle-treated mouse colons treated with anti-HSP70 antibodies are shown. (D) Pictures of large intestine fragments from mice treated with DSS and GGA are shown. HSP70 expression in surface epithelial cells and infiltrating mononuclear cells was increased compared to untreated and control reagent administered mice. Photomicrographs show representative fragments obtained in the experiment. The same appearance was observed in all samples.

Fig. 7 shows the results of colonic myeloperoxidase (MPO) activity 7 days after 3% DSS-induced colitis treatment in mice. The left bar represents the results of normal control mice, the middle bar represents the results of vehicle treated mice, and the black bar represents the results of mice treated with 300 mg / kg of GGA. By oral administration of GGA, elevated MPO activity was inhibited by DSS administration. The difference between control reagent-administered mice (n = 10) and GGA treated mice (n = 10) was statistically significant.

FIG. 8 shows the results of production of TNF-α and IFN-γ in the large intestine 7 days after 3% DSS treatment. Cytokine content was analyzed by ELISA described in the Examples herein. The left bar represents the results of normal control mice, the middle bar represents the results of vehicle treated mice, and the black bar represents the results of mice treated with 300 mg / kg of GGA. Production of TNF-α and IFN-γ in the large intestine of GGA treated mice (n = 10) was significantly reduced compared to control reagent administered mice (n = 10).

9 shows the results of the GGA effect on survival in severe colitis of TNBS induction. The effect of GGA on survival of BALB / c mice treated with TNBS (270 mg / kg) was assessed after 7 days. The left bar represents the results of normal control mice, the middle bar represents the results of TNBS and vehicle treated mice, and the black bar represents the results of mice treated with TNBS and 300 mg / kg GGA. The results from three independent experiments are shown as mean ± SE.

10 shows the results of GGA prevention of weight loss in TNBS induced colitis. GGA showed a protective effect against weight loss by TNBS induction after 7 days. White bars represent the results of untreated mice (n = 10), slanted bars represent the results of TNBS and vehicle treated mice (n = 6), black bars represent mice treated with TNBS and 300 mg / kg GGA. The result of (n = 6) is shown. Results are shown as mean ± SE.

11 is a pathological histogram of the control group of DSS enteritis model mice.

12 is a pathological histogram of the GGA-administered group of DSS enteritis model mice.

According to the present invention, the administration of GGA induces a heat shock protein in the large intestine, which is effective for the prevention and / or treatment of intestinal diseases, especially inflammatory bowel diseases, and the prevention of intestinal diseases containing GGA as an active ingredient, and ( Or) a therapeutic agent is provided.

Claims (24)

A prophylactic and / or therapeutic agent for enteric diseases containing a heat shock protein inducing agent or a salt thereof or a hydrate thereof as an active ingredient. The agent for preventing and / or treating intestinal diseases according to claim 1, wherein the heat shock protein inducing agent is a prenyl ketone compound. The method of claim 2, wherein the prenyl ketone compound is 6,10,14,18-tetramethyl-5,9,13,17-nanodecatetraen-2-one represented by the formula (I) and (Or) therapeutic agent. <Formula I>

The preventive and / or therapeutic agent for intestinal diseases according to any one of claims 1 to 3, wherein the intestinal diseases are inflammatory bowel diseases. The agent for preventing and / or treating intestinal diseases according to claim 4, wherein the inflammatory bowel disease is selected from the group consisting of Crohn's disease, ulcerative colitis, intestinal Behcet's disease and ischemic enteritis. The preventive and / or therapeutic agent for intestinal diseases according to any one of claims 1 to 5, wherein the heat shock protein is heat shock protein 70. A prophylactic and / or therapeutic agent for inflammatory bowel disease, comprising 6,10,14,18-tetramethyl-5,9,13,17-nanodecatetraen-2-one represented by the formula (I) as an active ingredient. <Formula I>

The agent for preventing and / or treating inflammatory bowel disease according to claim 7, wherein the inflammatory bowel disease is selected from the group consisting of Crohn's disease, ulcerative colitis, intestinal Behcet's disease and ischemic enteritis. A method of preventing and / or treating intestinal disorders comprising the step of administering a heat shock protein inducing agent or salt thereof or a hydrate thereof. The method for preventing and / or treating intestinal diseases according to claim 9, wherein the heat shock protein inducing agent is a prenyl ketone compound. The method for preventing intestinal diseases according to claim 10, wherein the prenyl ketone compound is 6,10,14,18-tetramethyl-5,9,13,17-nanodecatetraen-2-one represented by Formula (I). And / or a method of treatment. <Formula I>

The method for preventing and / or treating intestinal diseases according to any one of claims 9 to 11, wherein the intestinal diseases are inflammatory bowel diseases. The method of claim 12, wherein the inflammatory bowel disease is selected from the group consisting of Crohn's disease, ulcerative colitis, intestinal Behcet's disease, and ischemic enteritis. The method for preventing and / or treating intestinal diseases according to any one of claims 9 to 13, wherein the heat shock protein is heat shock protein 70. A method for preventing and / or treating inflammatory bowel disease, comprising 6,10,14,18-tetramethyl-5,9,13,17-nanodecatetraen-2-one represented by the formula (I) as an active ingredient. <Formula I>

The method of claim 15, wherein the inflammatory bowel disease is selected from the group consisting of Crohn's disease, ulcerative colitis, intestinal Behcet's disease, and ischemic enteritis. Use of a heat shock protein inducer or a salt thereof or a hydrate thereof for the preparation of a prophylactic and / or therapeutic agent for enteric diseases containing a heat shock protein inducer or a salt thereof or a hydrate thereof as an active ingredient. 18. The use according to claim 17, wherein said heat shock protein inducer is a prenylketone compound. 19. The use according to claim 18, wherein the prenyl ketone compound is 6,10,14,18-tetramethyl-5,9,13,17-nanodecatetraen-2-one represented by the formula (I). <Formula I>

20. The use according to any one of claims 17 to 19, wherein said bowel disease is an inflammatory bowel disease. 21. The use according to claim 20, wherein said inflammatory disease is selected from the group consisting of Crohn's disease, ulcerative colitis, intestinal Behcet's disease and ischemic enteritis. 22. The use according to any one of claims 17 to 21, wherein the heat shock protein is heat shock protein 70. For preparing a prophylactic or therapeutic agent for inflammatory bowel disease containing 6,10,14,18-tetramethyl-5,9,13,17-nanodecatetraen-2-one represented by the formula (I) as an active ingredient, Use of a heat shock protein inducer or salt thereof or a hydrate thereof. <Formula I>

The use according to claim 23, wherein said inflammatory disease is selected from the group consisting of Crohn's disease, ulcerative colitis, intestinal Behcet's disease and ischemic enteritis.

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