WO2010011117A2 - Ndga-containing composition for the suppression of transglutaminase - Google Patents

Ndga-containing composition for the suppression of transglutaminase Download PDF

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WO2010011117A2
WO2010011117A2 PCT/KR2009/004175 KR2009004175W WO2010011117A2 WO 2010011117 A2 WO2010011117 A2 WO 2010011117A2 KR 2009004175 W KR2009004175 W KR 2009004175W WO 2010011117 A2 WO2010011117 A2 WO 2010011117A2
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cancer
disease
transglutaminase
inflammatory
ndga
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Korean (ko)
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WO2010011117A3 (en
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김수열
이창훈
정경채
이병일
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국립암센터
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a transglutaminase inhibitor comprising NDGA, and more particularly, a transglule comprising NDGA for inhibiting the action of transglutaminase involved in the pathological mechanism of disease in the case of aberrant expression Rutaminase inhibitors and their novel use.
  • Transglutaminase is an enzyme that acts as a protective agent in blood clotting and wound healing. However, if the expression is unregulated, it is known to play a major role in the pathological mechanisms of many diseases (see, Soo-Youl Kim: New Target against Inflammatory Diseases: Transglutaminase 2. Archivum Immunologiae & Therapiae Experimentalis 52, 332-). 337, 2004).
  • transglutaminase is increased in Alzheimer's, Parkinson's, Huntington's, encephalitis and Celiac disease.
  • increased expression of NF- ⁇ B and increased expression of transglutaminase were observed in metastasis, chemo-resistance and radio-resistance of cancer (Soo-Youl Kim. Transglutaminase). 2 in inflammation.Front Biosci. 11, 3026-3035, 2006).
  • NF- ⁇ B is known to be activated by kinases according to signal transduction systems.
  • the activation of NF- ⁇ B is known without the help of kinase, the effectiveness of kinase inhibitors has been reduced (Tergaonkar et al., IkappaB kinase-independent IkappaBalpha degradation pathway: functional NF-kappaB activity and implications for cancer therapy.Mol Cell Biol. 2003 Nov; 23 (22): 8070-83.).
  • transglutaminase activates NF- ⁇ B by binding I- ⁇ B ⁇ without the help of kinases (IKK, NAK) (Jongmin Lee, et al. Transglutaminase 2 induces). NF-kB activation via a novel pathway in BV-2 microglia.J . Biol. Chem. 279, 53725-53735, 2004). Since transglutaminase is a calcium-dependent enzyme, it is possible to activate NF- ⁇ B only by increasing intracellular calcium.
  • transglutaminase Since the expression of inflammatory factors including transglutaminase and its inhibitor I- ⁇ B ⁇ is increased by the activity of NF- ⁇ B transcription factor in inflammatory reactions, the normal activity of NF- ⁇ B is normally suppressed by I- ⁇ B ⁇ . Although not achieved, NF- ⁇ B continues to be activated in chronic inflammatory diseases.
  • An interesting phenomenon is that the expression of transglutaminase is induced by the activity of NF- ⁇ B by TNF- ⁇ or lipopolysaccharide (LPS). Therefore, abnormally activated transglutaminase is expected to play a role in sustaining inflammation by directly activating NF- ⁇ B in inflammatory cells or by further maintaining activated NF- ⁇ B.
  • these malignant circuits may be a major cause of cancer metastasis and drug resistance in cancer tissues (Jongmin Lee, et al. Transglutaminase 2 induces NF-kB activation via a novel pathway in BV-2 microglia . J. Biol. Chem. 279, 53725-53735, 2004).
  • the inhibitor of transglutaminase may be an important substance to break the continuous ring of NF- ⁇ B, and the effect of replacing the steroidal agent shown by the present inventors may be based on this (Sohn, J. , Kim, T.-I., Yoon, Y.-H., and Kim, SY: Transglutaminase Inhibitor: A New Anti-Inflammatory Approach in Allergic Conjunctivitis.J. Clin. Invest. 111, 121-8, 2003).
  • An amine compound is known as a substance that inhibits transglutaminase activity.
  • cystamine nature Genetics 18, 111-117, 1998; Nature Medicine 8, 143-149, 2002
  • Putrescine Putrescine.
  • monodansylcadaverine J. Med. Chem. 15, 674-675, 1972
  • w-dibenzylaminoalkylamine J. Med. Chem. 18, 278-284, 1975
  • 3-halo-4,5-dihydroisoxazole Mol. Pharmacol.
  • elafin Anti-flammin protein (PLA2 inhibitor) and protein called elafin (a very strong transglutaminase substrate, Nara, K., et al. 1994.
  • Elastase inhibitor elafin is a new type of proteinase Inhibitor which has a transglutaminase-mediated anchoring sequence termed "cementoin" .J Biochem (Tokyo. 115: 441-448) was used to synthesize a peptide that mimics the transglutaminase catalytic site.
  • the expression of transglutaminase is increased in diseases with inflammation. Examples of such diseases include degenerative arthritis, diabetes mellitus, autoimmune myositis, arteriosclerosis, stroke, liver cirrhosis, malignant breast cancer, meningitis, and inflammatory gastric ulcer. have.
  • Nordihydroguaiaretic acid is a non-toxic phenolic lignan isolated from an effusion containing a resin of North American small oak bushes such as Larrea divaricata Cav. , As an antioxidant in foods (Lundberg, WO et al., Oil & Soap, 21, pp 33-35, 1944).
  • NDGA as an inhibitor of transglutaminase has not been reported to date.
  • NDGA inhibits the polymerization of I- ⁇ B ⁇ , thereby inhibiting the activity of transglutaminase, thereby causing abnormal activity of transglutaminase. It is not known that it can be easily applied without side effects in a safe manner and effectively prevented and treated in diseases induced by, for example, inflammatory diseases, neurological diseases and cancer diseases.
  • the present inventors have screened substances useful as transglutaminase inhibitors among natural substances which have already been recognized for safety and commercialized. As a result, the present inventors have newly discovered the activity as transglutaminase inhibitors of NDGA and have now reached the present invention.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating a neurological disease, an inflammatory disease or a cancer disease, which is a disease caused by an increase in transglutaminase activity, comprising NDGA as an active ingredient.
  • Still another object of the present invention is to provide a method for inhibiting the activity of transglutaminase comprising the NDGA.
  • Another object of the present invention is to provide a method for treating or preventing a nervous system disease, an inflammatory disease or a cancer disease caused by an increase in the activity of transglutaminase, comprising administering the pharmaceutical composition.
  • the pharmaceutical composition comprising the NDGA of the present invention and a method for treating and preventing diseases using the same have no side effects in a subject having a disease induced by abnormal activity of transglutaminase, such as an inflammatory disease, a neurological disease and a cancer disease. It can be applied safely, which is useful for effectively preventing and treating these diseases.
  • Figure 1 shows the degree of inhibition of transglutaminase activity according to the concentration of NDGA, specifically measuring the binding of transglutaminase to [1,4, -14 C] putrescine to succinylated casein, A graph showing the results of in vitro experiments in which NDGA competes with putrescine to inhibit its response.
  • the present invention relates to a pharmaceutical composition for preventing or treating a nervous system disease, an inflammatory disease or a cancer disease which is a disease caused by an increase in transglutaminase activity, comprising NDGA as an active ingredient.
  • the molecular formula of NDGA is C 18 H 22 O 4 , molecular weight is 302.365, 4- [4- (3,4-dihydroxyphenyl) -2,3-dimethylbutyl] benzene-1,2 according to the IUPAC system -Diol (4- [4- (3,4-dihydroxyphenyl) -2,3-dimethylbutyl] benzene-1,2-diol).
  • NDGA The origin of the NDGA used in the present invention is not limited thereto, and any NDGA may be used in the present invention.
  • the structural formula of NDGA is as follows (formula 1).
  • NDGA can be used separately from exudates containing resins of North American small oak bushes, such as Larrea divaricata Cav., As described above, and synthesized according to conventional methods in the art. It may be.
  • There are various methods for preparing NDGA for example, by demethylation of dimethyl ether of hydrogenated (-)-guaiaretic acid (Schroter, G. et al., Ber). 51, p1587, 1918), and a more systematic synthesis is carried out using the reaction of 1-piperonyl-1-bromoethane and the Grignard reagent derived therefrom as a key reaction.
  • Remethylation of dimethyl ether of hydrogenated (-)-guaiaretic acid Schroter, G. et al., Ber). 51, p1587, 1918
  • a more systematic synthesis is carried out using the reaction of 1-piperonyl-1-bromoethane and the Grignard reagent derived therefrom as a key reaction
  • NDGA transglutaminase binding [1,4, -14 C] putrescine to succinylated casein
  • Nervous system disease of the present invention is a disease associated with death or damage of nerve cells, in particular Alzheimer's disease, multi-infarct dementia, a combination of Alzheimer's disease and multi-infarct dementia, Parkinson's disease (Parkinson's disease), hypothyroidism, alcoholic dementia, Huntingtun disease, such as central nervous system diseases due to damage and death of the central nervous system cells are typical.
  • the main symptoms of these diseases include cognitive dysfunction, language, judgment, abstract spatial and temporal history, and other new skill acquisition disorders. Symptoms of personality changes, emotional instability, and eventually death.
  • the pharmaceutical composition of the present invention can be used for diseases in which the activity is increased, such as overexpression of transglutaminase in neural tissues.
  • ⁇ synuclein can be used in Parkinson's disease (PNAS, February 18,2003, Vol. 100, no.4, pp2047-2052), which is aggregated by transglutaminase, but is not limited thereto.
  • PNAS Parkinson's disease
  • it can be used to treat any disease in which transglutaminase is overexpressed in nervous tissues.
  • 'Inflammatory disease' of the present invention is a concept that includes all of the diseases causing inflammation, inflammation occurs when immune competent cells are activated in response to an external individual or antigenic protein.
  • Inflammatory processes may be advantageous, such as in the case of causing invading individuals to be phagocytized or neutralized, but may also be harmful, such as in the case of arthritis, which causes destruction of bone and cartilage and consequent limitations of joint function.
  • Inflammatory responses are usually induced by trauma or antigens, such as viral, bacterial, protozoan, or fungal antigens.
  • Inflammatory diseases suitable for the prophylaxis or treatment according to the present invention include degenerative arthritis, diabetes mellitus, autoimmune myositis, arteriosclerosis, stroke, cirrhosis, meningitis, inflammatory gastric ulcer, gallbladder stones, kidney stones, sinusitis, rhinitis, conjunctivitis, asthma, dermatitis, inflammatory Intestinal disease, inflammatory collagen vascular disease, glomerulonephritis, inflammatory skin disease, and sarcoidosis, and the like.
  • the inflammatory disease of the present invention is a) rheumatoid inflammatory disease (rheumatoid arthritis), systemic lupus erythematosus, Ankylosing spondylitis, Behcet's disease, autoimmune myositis ( inflammatroy mysitis, b) ulcerative colitis (UC), Crohn disease (C), an intestinal inflammatory disease, c) psoriasis, atopic dermatitis, and contact dermatitis, a dermatitis.
  • rheumatoid arthritis rheumatoid arthritis
  • systemic lupus erythematosus Ankylosing spondylitis
  • Behcet's disease autoimmune myositis ( inflammatroy mysitis
  • UC ulcerative colitis
  • Crohn disease Crohn disease
  • an intestinal inflammatory disease c
  • psoriasis psoriasis, atopic dermatitis,
  • PID pelvic inflammatory disease
  • the term 'cancer disease' of the present invention refers to a phenomenon in which cells multiply unlimitedly in living tissues to cause malignant tumors, and has metastasis, chemical resistance and radiation resistance of cancer in relation to transglutaminase. Significant increases in the expression of transglutaminase have been identified in the present invention, the inhibition of transglutaminase is also important in the prevention and treatment of cancer.
  • Specific cancers that can be prevented or treated using the pharmaceutical composition comprising the NDGA of the present invention include cancers in which an increase in transglutaminase occurs, specifically colon cancer, small intestine cancer, rectal cancer, anal cancer, esophageal cancer and pancreatic cancer. , Stomach cancer, kidney cancer, uterine cancer, breast cancer, lung cancer, lymph cancer, thyroid cancer, prostate cancer, leukemia, skin cancer, colon cancer, brain tumors, bladder cancer, ovarian cancer, gallbladder cancer and the like.
  • prevention refers to any action that suppresses or delays the onset of all diseases resulting from increased transglutaminase activity due to the administration of a composition comprising NDGA.
  • administration of the pharmaceutical composition means any action that improves or beneficially alters all diseases caused by increased transglutaminase activity.
  • the present invention relates to a method for treating or preventing neurological diseases, inflammatory diseases and cancer diseases caused by increased transglutaminase activity comprising NDGA as an active ingredient.
  • Methods of treating or preventing using the pharmaceutical compositions of the present invention are cattle, horses, sheep, pigs, goats, camels, nutrition, dogs, which can develop diseases caused by increased transglutaminase activity in humans as well. It can also be applied to mammals such as cats.
  • composition comprising the NDGA of the present invention may be used as a single agent, or may be prepared and used as a complex including a recognized pharmaceutical composition.
  • compositions comprising NDGA can be prepared by filling the capsule comprising NDGA without excipients or uniformly and sufficiently in contact with the particulate solid carrier, liquid carrier or both. The product can then be molded into the desired formulation and used if necessary.
  • suitable carrier excipients include starch, water, saline, ethanol, glycerol, Ringer's solution and dextrose solution, and the like (Remington's Pharmaceutical Science, 19 th Ed., 1995, Mack Publishing Company, Easton PA) and the like. As disclosed, it may be formulated into a suitable formulation known in the art.
  • composition comprising the NDGA of the present invention can be applied in any formulation and can be prepared for oral or parenteral use.
  • the parenteral formulation may be in the form of a spray such as injectable, application or aerosol, preferably in the form of a spray such as injectable or aerosol, and oral formulations are also preferred.
  • Formulations for oral administration comprising the pharmaceutical composition of the present invention include, for example, tablets, troches, lozenges, water-soluble or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs, and the like. Can be formulated.
  • lactose For formulation into tablets and capsules, lactose, saccharose, sorbitol, mannitol, starch, amylopectin, binders such as cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, stearic acid masne It may include lubricating oils such as calcium, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax, and in the case of capsule formulations, it may further contain a liquid carrier such as fatty oil in addition to the above-mentioned materials.
  • lubricating oils such as calcium, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax
  • parenteral administration is for injection, such as subcutaneous injection, intravenous injection or intramuscular injection, suppository injection method, or aerosol for inhalation through the respiratory system. It can be formulated.
  • injectable formulations NDGA is mixed in water with stabilizers or buffers to prepare solutions or suspensions, which are formulated for unit doses of ampoules or vials.
  • solutions or suspensions which are formulated for unit doses of ampoules or vials.
  • a rectal composition such as suppositories or body enema including conventional suppository bases such as cocoa butter or other glycerides.
  • a propellant or the like may be combined with the additives to disperse the dispersed dispersion or wet powder.
  • compositions of the invention may be administered in a conventional manner via the rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, nasal, inhalation, intraocular or intradermal routes.
  • Parenteral administration means administration modes including intravenous, intramuscular, intraperitoneal, sternum, transdermal and intraarterial injection and infusion.
  • Parenteral administration of the pharmaceutical composition comprising the NDGA of the present invention may be carried out under desired purity in admixture with a pharmaceutically acceptable carrier, i.e., a carrier which is nontoxic to the receptor and compatible with other agent components at the concentrations and dosages employed. It is preferred to formulate in unit dosage form. In particular, the formulation preferably does not contain oxidizing agents and other compounds known to be harmful to the human body.
  • the NDGA of the invention can be administered in a pharmaceutical composition with one or more pharmaceutically acceptable excipients.
  • the specific therapeutically effective amount for a particular patient may be based on the specific composition, including the type and severity of the response to be achieved, whether other agents are used in some cases, the age, body weight, general health, sex and diet, time of administration, It is desirable to apply differently depending on various factors and similar factors well known in the medical field, including the route of administration and the rate of release of the composition, the duration of treatment, and the drugs used with or concurrent with the specific composition. Therefore, the effective amount of NDGA suitable for the purpose of the present invention is preferably determined in consideration of the above-mentioned matters.
  • Transglutaminase measured the binding of [1,4, -14 C] putrescine to succinated casein and observed that NDGA competes with putrescine to inhibit its response.
  • NDGA N-(Sigma-Aldrich, Cat No. C3878) and diluted with distilled water to a final concentration of 1 unit / ml.
  • a stock solution of NDGA was prepared by dissolving NDGA (Sigma-Aldrich, Cat No. C3878) in DMSO at a concentration of 10 mM and then diluting it with DMSO at each concentration.
  • Substrate solution was prepared by mixing the biotinylated succinyl casein solution and [1,4- 14 C] Fu Trail New dihydrochloride solution 50 ⁇ l of 450 ⁇ l. Each sample was prepared by mixing 96 ⁇ l of reaction buffer, 3 ⁇ l of NDGA stock solution, and 1 ⁇ l of transglutaminase stock solution. After incubating the samples at 37 ° C. for 10 minutes, 500 ⁇ l of substrate solution and 100 ⁇ l of sample solution were mixed well and these mixtures were incubated at 37 ° C. for 2 hours. 4.5 ml of cold (4 ° C.) 7.5% TCA was added to stop the reaction and the entire solution was stored at 4 ° C. for 1 hour.
  • the TCA-protein precipitate was filtered through a GF / glass fiber filter, washed with 25 ml of cold 5% TCA and dried. Radioactivity of the crosslinked protein was measured by beta ( ⁇ ) -Counter (Beckman Coulter) and corrected with the activity of DMSO-control as a standard. The measured value is shown as the activity of transglutaminase. This assay was repeated three times under the same conditions and the values are shown in Table 1 below.
  • IC 50 values were calculated by the general nonlinear regression method, which was determined to be 1.4915 ( ⁇ 0.1229) ⁇ M.
  • the relative transglutaminase inhibitory activity measured according to the concentration of NDGA is shown in FIG. 1. As shown in Figure 1, it can be seen that the activity of transglutaminase is inhibited in a concentration-dependent manner of NDGA.
  • the pharmaceutical composition comprising the NDGA of the present invention and a method for treating and preventing diseases using the same can effectively prevent and treat diseases induced by abnormal activity of transglutaminase, such as inflammatory diseases, neurological diseases and cancer diseases. .

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Abstract

The present invention relates to a novel use of nordihydroguaiaretic acid (NDGA) for suppressing transglutaminase activity. More specifically, the present invention relates to a pharmaceutical composition which contains NDGA as an active ingredient and which is used for the prevention or treatment of neurological diseases, inflammatory diseases and cancerous diseases which are medical conditions induced by increased transglutaminase activity. Further, the present invention relates to a method for preventing or treating neurological diseases, inflammatory diseases and cancerous diseases, which are induced by increased transglutaminase activity, by the administration of the pharmaceutical composition. The novel method of the present invention for suppressing transglutaminase by using NDGA is advantageous in that it can be applied safely and without side effects in subjects having diseases involving the abnormal expression of transglutaminase, and can effectively treat or prevent such diseases.

Description

ＮＤＧＡ를 포함하는 트란스글루타미나제 억제용 조성물Composition for inhibiting transglutaminase containing NDA
본 발명은 NDGA를 포함하는 트란스글루타미나제 억제제에 관한 것으로, 더욱 상세하게는 이상 발현의 경우 질병의 병리적 기전에 관여하는 트란스글루타미나제의 작용을 억제하기 위한 NDGA를 포함하는 트란스글루타미나제 억제제와 이들의 신규한 용도에 관한 것이다.The present invention relates to a transglutaminase inhibitor comprising NDGA, and more particularly, a transglule comprising NDGA for inhibiting the action of transglutaminase involved in the pathological mechanism of disease in the case of aberrant expression Rutaminase inhibitors and their novel use.
트란스글루타미나제(Transglutaminase)는 정상적인 상황에서는 혈액 응고나 상처 치유 등의 보호적 작용을 하는 효소이다. 그러나, 그 발현이 조절 받지 못하는 경우에는 많은 질병의 병리적 기전에서 주요한 역할을 하는 것으로 알려져 있다(종설. Soo-Youl Kim : New Target Against Inflammatory Diseases: Transglutaminase 2.　 Archivum Immunologiae & Therapiae Experimentalis 52, 332-337, 2004).Transglutaminase is an enzyme that acts as a protective agent in blood clotting and wound healing. However, if the expression is unregulated, it is known to play a major role in the pathological mechanisms of many diseases (see, Soo-Youl Kim: New Target Against Inflammatory Diseases: Transglutaminase 2. Archivum Immunologiae & Therapiae Experimentalis 52, 332-). 337, 2004).
특히 염증을 동반하는 질병, 즉 퇴행성 관절염 (rheumatoid arthritis), 당뇨병 (diabetes), 자가면역 근육염 (inflammatory myositis), 동맥경화 (atherosclerosis), 뇌졸증 (stroke), 간경화 (liver cirrhosis), 유방암 (breast cancer), 알츠하이머 (Alzheimer), 파킨슨 (Parkinson), 헌팅턴 (Huntington), 뇌막염 (encephalitis) 및 염증성 위궤양 (Celiac disease) 등에서 트란스글루타미나제의 발현이 많이 증가 된다. 또한, 암의 전이(metastasis), 화학적 내성(chemo-resistance), 방사선내성(radio-resistance)에서도 NF-κB의 발현 증가 및 트란스글루타미나제의 발현 증가가 관찰되었다(Soo-Youl Kim. Transglutaminase 2 in inflammation. Front Biosci. 11, 3026-3035, 2006).In particular, diseases associated with inflammation, such as degenerative arthritis, diabetes, diabetes, inflammatory myositis, atherosclerosis, stroke, liver cirrhosis, breast cancer The expression of transglutaminase is increased in Alzheimer's, Parkinson's, Huntington's, encephalitis and Celiac disease. In addition, increased expression of NF-κB and increased expression of transglutaminase were observed in metastasis, chemo-resistance and radio-resistance of cancer (Soo-Youl Kim. Transglutaminase). 2 in inflammation.Front Biosci. 11, 3026-3035, 2006).
아직까지 암의 화학적 내성과 트란스글루타미나제가 어떻게 연관되어 있는지는 밝혀지지 않고 있으나, 화학적 내성을 가지는 유방암 세포주에 대해 트란스글루타미나제 발현을 억제하는 경우 암세포가 항암제에 민감하게 반응하여 사멸되는 것으로 드러났다(Antonyak et al., Augmentation of tissue transglutaminase expression and activation by epidermal growth factor inhibit doxorubicin-induced apoptosis in human breast cancer cells. J Biol Chem. 2004 Oct 1;279(40):41461-7.; Dae-Seok Kim et al. Reversal of Drug Resistance in Breast Cancer Cells by Transglutaminase 2 Inhibition and Nuclear Factor-KB Inactivation. Cancer Res. 2006. in press).The relationship between the chemical resistance of cancer and transglutaminase has not been established yet.However, the inhibition of transglutaminase expression in breast cancer cell lines with chemical resistance indicates that cancer cells are sensitive to anticancer drugs and die. (Antonyak et al., Augmentation of tissue transglutaminase expression and activation by epidermal growth factor inhibit doxorubicin-induced apoptosis in human breast cancer cells.J Biol Chem. 2004 Oct 1; 279 (40): 41461-7 .; Dae-Seok Kim et al. Reversal of Drug Resistance in Breast Cancer Cells by Transglutaminase 2 Inhibition and Nuclear Factor-KB Inactivation. Cancer Res. 2006. in press).
또한, 최근 트란스글루타미나제가 활성화되는 분자수준의 병리기전이 밝혀지면서, 트란스글루타미나제 억제의 타당성이 한층 더 구체화되었다(Key Chung Park, Kyung Cheon Chung, Yoon-Seong Kim, Jongmin Lee, Tong H. Joh, and　 Soo-Youl Kim. Transglutaminase 2 induces nitric oxide synthesis in BV-2 microglia.　 Biochem. Biophys. Res. Commun. 323, 1055-1062, 2004; Jongmin Lee,Yoon-Seong Kim, Dong-Hee Choi, Moon S. Bang, Tay R. Han , Tong H. Joh, and Soo-Youl Kim. Transglutaminase 2 induces NF-κB activation via a novel pathway in BV-2 microglia.　 J. Biol. Chem. 279, 53725-53735, 2004; ; Dae-Seok Kim et al. Reversal of Drug Resistance in Breast Cancer Cells by Transglutaminase 2 Inhibition and Nuclear Factor-KB Inactivation. Cancer Res. 2006. in press).In addition, the recent pathological mechanism of transglutaminase activation has been revealed to further refine the feasibility of transglutaminase inhibition (Key Chung Park, Kyung Cheon Chung, Yoon-Seong Kim, Jongmin Lee, Tong). .... H. Joh, and Soo-Youl Kim Transglutaminase 2 induces nitric oxide synthesis in BV-2 microglia Biochem Biophys Res Commun 323, 1055-1062, 2004;.. Jongmin Lee, Yoon-Seong Kim, Dong-Hee Choi , Moon S. Bang, Tay R. Han, Tong H. Joh, and Soo-Youl Kim.Transglutaminase 2 induces NF-κB activation via a novel pathway in BV-2 microglia.J. Biol. Chem. 279, 53725-53735 Dae-Seok Kim et al. Reversal of Drug Resistance in Breast Cancer Cells by Transglutaminase 2 Inhibition and Nuclear Factor-KB Inactivation. Cancer Res. 2006. in press).
염증은 주로 NF-κB 전사 물질의 활성화에 기인한다. NF-κB는 신호 전달체계에 따른 카이네이즈(kinase)들에 의해 활성화되는 것으로 알려졌다. 하지만 카이네이즈의 도움없이도 NF-κB의 활성화가 이루어지는 것이 알려지면서, 카이네이즈 억제제(kinase inhibitor)들의 실효성이 감소하게 되었다(Tergaonkar et al., IkappaB kinase-independent IkappaBalpha degradation pathway: functional NF-kappaB activity and implications for cancer therapy. Mol Cell Biol. 2003 Nov;23(22):8070-83.).  Inflammation is mainly due to the activation of NF-κB transcription material. NF-κB is known to be activated by kinases according to signal transduction systems. However, as the activation of NF-κB is known without the help of kinase, the effectiveness of kinase inhibitors has been reduced (Tergaonkar et al., IkappaB kinase-independent IkappaBalpha degradation pathway: functional NF-kappaB activity and implications for cancer therapy.Mol Cell Biol. 2003 Nov; 23 (22): 8070-83.).
본 발명자는, 이전의 연구에서 트란스글루타미나제가 카이네이즈들(IKK, NAK) 도움없이, I-κBα를 교결합하여 NF-κB를 활성화 시킨다는 것을 밝힌바 있다(Jongmin Lee, et al. Transglutaminase 2 induces NF-kB activation via a novel pathway in BV-2 microglia.　 J. Biol. Chem. 279, 53725-53735, 2004). 트란스글루타미나제는 칼슘의존성 효소이므로 세포 내 칼슘의 증가만으로도 NF-κB를 활성화시킬 수 있는 것이다.In a previous study, the inventors found that transglutaminase activates NF-κB by binding I-κBα without the help of kinases (IKK, NAK) (Jongmin Lee, et al. Transglutaminase 2 induces). NF-kB activation via a novel pathway in BV-2 microglia.J . Biol. Chem. 279, 53725-53735, 2004). Since transglutaminase is a calcium-dependent enzyme, it is possible to activate NF-κB only by increasing intracellular calcium.
염증반응에서 NF-κB 전사인자의 활성으로 트란스글루타미나제를 포함하는 염증성 인자들과 그 억제제인 I-κBα의 발현이 증가되므로, 정상적인 경우에는 I-κBα에 의해 NF-κB의 연속활성이 이루어지지 않지만, 만성 염증질환에서는 계속해서 NF-κB가 활성화된다. 흥미로운 현상은 TNF-α 또는 LPS(lipopolysaccharide) 등에 의한 NF-κB의 활성으로 트란스글루타미나제의 발현이 유도된다는 것이다. 따라서 비정상적으로 활성화된 트란스글루타미나제가 염증 세포에서 NF-κB를 직접 활성화시키거나, 활성화된 NF-κB를 더욱 유지시킴으로써 염증을 지속시키는 역할을 할것으로 예상된다. 또한 이러한 악성 회로는 암 조직에서 암의 전이 및 약물저항성을 유발하는 주원인이 될 수 있다(Jongmin Lee, et al. Transglutaminase 2 induces NF-kB activation via a novel pathway in BV-2 microglia.　 J. Biol. Chem. 279, 53725-53735, 2004).Since the expression of inflammatory factors including transglutaminase and its inhibitor I-κBα is increased by the activity of NF-κB transcription factor in inflammatory reactions, the normal activity of NF-κB is normally suppressed by I-κBα. Although not achieved, NF-κB continues to be activated in chronic inflammatory diseases. An interesting phenomenon is that the expression of transglutaminase is induced by the activity of NF-κB by TNF-α or lipopolysaccharide (LPS). Therefore, abnormally activated transglutaminase is expected to play a role in sustaining inflammation by directly activating NF-κB in inflammatory cells or by further maintaining activated NF-κB. In addition, these malignant circuits may be a major cause of cancer metastasis and drug resistance in cancer tissues (Jongmin Lee, et al. Transglutaminase 2 induces NF-kB activation via a novel pathway in BV-2 microglia . J. Biol. Chem. 279, 53725-53735, 2004).
따라서, 트란스글루타미나제의 억제제는 NF-κB의 연속 고리를 끊는 중요한 물질이 될 수 있으며, 본 발명자가 보여 주었던 스테로이드제제를 대체할 만한 효과는 바로 여기에 근거한 것으로 사료 된다(Sohn, J., Kim, T.-I., Yoon, Y.-H., and Kim, S.Y.: Transglutaminase Inhibitor: A New Anti-Inflammatory Approach in Allergic Conjunctivitis. J. Clin. Invest. 111, 121-8, 2003).Therefore, the inhibitor of transglutaminase may be an important substance to break the continuous ring of NF-κB, and the effect of replacing the steroidal agent shown by the present inventors may be based on this (Sohn, J. , Kim, T.-I., Yoon, Y.-H., and Kim, SY: Transglutaminase Inhibitor: A New Anti-Inflammatory Approach in Allergic Conjunctivitis.J. Clin. Invest. 111, 121-8, 2003).
트란스글루타미나제 활성을 억제하는 물질로는 아민 화합물(amine compound)이 알려져 있으며, 대표적으로 시스타민(cystamine, nature Genetics 18, 111-117, 1998; Nature Medicine 8, 143-149, 2002)과 푸트레신(putrescine)이다. 또한, 모노단실카다베린(monodansylcadaverine) (J. Med. Chem. 15, 674-675, 1972), w-디벤질아미노알킬아민(w-dibenzylaminoalkylamine)(J. Med. Chem. 18, 278-284, 1975), 3-할로-4,5-디하이드로이소자졸(3-halo-4,5-dihydroisoxazole)(Mol. Pharmacol. 35, 701-706, 1989), 2-[(2-oxopropyl)thio]imidazolium derivatives (Blood, 75, 1455-1459, 1990)등의 화학적 억제제들이 개발되어 있으나, 모두 생체에서 비특이적으로 다른 효소의 억제를 유발하는 독성이 알려져 있다. An amine compound is known as a substance that inhibits transglutaminase activity. Representatively, cystamine (nature Genetics 18, 111-117, 1998; Nature Medicine 8, 143-149, 2002) and Putrescine. In addition, monodansylcadaverine (J. Med. Chem. 15, 674-675, 1972), w-dibenzylaminoalkylamine (J. Med. Chem. 18, 278-284, 1975), 3-halo-4,5-dihydroisoxazole (Mol. Pharmacol. 35, 701-706, 1989), 2-[(2-oxopropyl) thio] Chemical inhibitors such as imidazolium derivatives (Blood, 75, 1455-1459, 1990) have been developed, but all are known to be nonspecifically toxic to cause inhibition of other enzymes in vivo.
따라서, 안전하고 효과적인 트란스글루타미나제 특이적인 억제제의 개발이 요구되고 있다. 최근, 손 등은 기니픽에 꽃가루를 이용한 눈 알레르기 모델에서, 펩타이드로 만든 트란스글루타미나제 억제제를 사용해 스테로이드 수준의 효과를 거두는데 성공했다(Sohn, J., Kim, T.-I., Yoon, Y.-H., and Kim, S.-Y.: Transglutaminase Inhibitor: A New Anti-Inflammatory Approach in Allergic Conjunctivitis. J. Clin. Invest. 111, 121-8, 2003). 안티플라민(anti-flammin) 단백질(PLA2 억제제)과 엘라핀(elafin)이라는 단백질(아주 강력한 트란스글루타미나제 기질, Nara, K., et al. 1994. Elastase inhibitor elafin is a new type of proteinase inhibitor which has a transglutaminase-mediated anchoring sequence termed "cementoin". J Biochem (Tokyo). 115:441-448)에서 트란스글루타미나제 촉매 부위를 흉내내어 합성한 펩타이드를 사용하였다. 특히 염증을 동반하는 질병들에서 트란스글루타미나제의 발현이 많이 증가되며, 이러한 질병의 예로서 퇴행성 관절염, 당뇨병, 자가 면역 근육염, 동맥경화, 뇌졸중, 간경화, 악성유방암, 뇌막염, 염증성 위궤양 등이 있다.Accordingly, there is a need for the development of safe and effective transglutaminase specific inhibitors. Recently, Son et al. Have succeeded in achieving steroid-level effects using transglutaminase inhibitors made of peptides in an eye allergy model using pollen in Guinea Pigs (Sohn, J., Kim, T.-I., Yoon). , Y.-H., and Kim, S.-Y .: Transglutaminase Inhibitor: A New Anti-Inflammatory Approach in Allergic Conjunctivitis.J. Clin.Invest . 111, 121-8, 2003). Anti-flammin protein (PLA2 inhibitor) and protein called elafin (a very strong transglutaminase substrate, Nara, K., et al. 1994. Elastase inhibitor elafin is a new type of proteinase Inhibitor which has a transglutaminase-mediated anchoring sequence termed "cementoin" .J Biochem (Tokyo. 115: 441-448) was used to synthesize a peptide that mimics the transglutaminase catalytic site. In particular, the expression of transglutaminase is increased in diseases with inflammation. Examples of such diseases include degenerative arthritis, diabetes mellitus, autoimmune myositis, arteriosclerosis, stroke, liver cirrhosis, malignant breast cancer, meningitis, and inflammatory gastric ulcer. have.
상기한 화합물들 외에도 다른 화학적 억제제들이 개발되었지만 모두 생체적으로 이용되는 경우 비특이적인 다른 효소의 억제를 유발하는 독성이 알려지고 있는 실정이다. 본 발명자에 의해 이전에 제조된 펩티드 억제제가 트란스글루타미나제의 억제제로 유효하나(대한민국 특허출원 제10-2006-98921호), 비용과 안전성면에서 그 실용화에는 여전히 문제들이 남아 있다.In addition to the above compounds, other chemical inhibitors have been developed, but all are known to cause toxicity when they are used in vivo, causing inhibition of other non-specific enzymes. Peptide inhibitors previously prepared by the present inventors are effective as inhibitors of transglutaminase (Korean Patent Application No. 10-2006-98921), but problems remain in practical use in terms of cost and safety.
노르디하이드로갈리아레틱산(Nordihydroguaiaretic acid; NDGA)는 라레아 디베러케이트(Larrea divaricata Cav.)와 같은 북아메리카산 작은떡갈나무덤불의 수지를 함유한 삼출액으로부터 분리시킨 무독성 페놀성 리그난(phenolic lignan)이며, 식품의 산화방지제로써(Lundberg, W. O. et al., Oil & Soap, 21, pp 33-35, 1944) 사용된다.Nordihydroguaiaretic acid (NDGA) is a non-toxic phenolic lignan isolated from an effusion containing a resin of North American small oak bushes such as Larrea divaricata Cav. , As an antioxidant in foods (Lundberg, WO et al., Oil & Soap, 21, pp 33-35, 1944).
그러나 현재까지 트란스글루타미나제의 억제제로 NDGA의 사용이 보고된 바는 없으며, 특히 NDGA가 I-κBα의 중합을 저해하여 트란스글루타미나제의 활성을 억제함으로써 트란스글루타미나제의 비정상적 활성에 의해 유도된 질환, 예컨대 염증성 질환, 신경계 질환 및 암 질환에서 안전한 방법으로 부작용없이 용이하게 적용되어 효과적으로 예방 및 치료할 수 있다는 사실에 대해서는 알려진 바가 없었다.However, the use of NDGA as an inhibitor of transglutaminase has not been reported to date. In particular, NDGA inhibits the polymerization of I-κBα, thereby inhibiting the activity of transglutaminase, thereby causing abnormal activity of transglutaminase. It is not known that it can be easily applied without side effects in a safe manner and effectively prevented and treated in diseases induced by, for example, inflammatory diseases, neurological diseases and cancer diseases.
본 발명자는 이미 안전성이 인정되어 상용화되고 있는 천연물질들 중에서 트란스글루타미나제 억제제로서 유용한 물질들을 스크리닝 한 결과, NDGA의 트란스글루타미나제 억제제로서의 활성을 새로이 발견하여 본 발명에 이르게 되었다. The present inventors have screened substances useful as transglutaminase inhibitors among natural substances which have already been recognized for safety and commercialized. As a result, the present inventors have newly discovered the activity as transglutaminase inhibitors of NDGA and have now reached the present invention.
본 발명의 목적은 NDGA를 유효성분으로 포함하는 트란스글루타미나제 억제용 조성물을 제공하는 것이다.It is an object of the present invention to provide a composition for inhibiting transglutaminase comprising NDGA as an active ingredient.
본 발명의 다른 목적은 NDGA를 유효성분으로 포함하는, 트란스글루타미나제 활성 증가에 의해 초래되는 질병인 신경계 질환, 염증성 질환 또는 암 질환을 예방 또는 치료하기 위한 약제학적 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating a neurological disease, an inflammatory disease or a cancer disease, which is a disease caused by an increase in transglutaminase activity, comprising NDGA as an active ingredient.
본 발명의 또 다른 목적은 상기 NDGA를 포함하는 트란스글루타미나제의 활성 억제 방법을 제공하는 것이다.Still another object of the present invention is to provide a method for inhibiting the activity of transglutaminase comprising the NDGA.
본 발명의 또 다른 목적은 상기 약제학적 조성물을 투여하는 것을 포함하는, 트란스글루타미나제의 활성 증가에 의해 초래되는 신경계 질환, 염증성 질환 또는 암 질환의 치료 또는 예방 방법을 제공하는 것이다.Another object of the present invention is to provide a method for treating or preventing a nervous system disease, an inflammatory disease or a cancer disease caused by an increase in the activity of transglutaminase, comprising administering the pharmaceutical composition.
본 발명의 NDGA를 포함하는 약제학적 조성물 및 이를 이용한 질환 치료 및 예방 방법은, 트란스글루타미나제의 비정상적 활성에 의해 유도된 질환, 예컨대, 염증성 질환, 신경계 질환 및 암 질환을 가진 대상에게 부작용없이 안전하게 적용될 수 있어, 이들 질환을 효과적으로 예방 및 치료하는 데 유용하다.The pharmaceutical composition comprising the NDGA of the present invention and a method for treating and preventing diseases using the same have no side effects in a subject having a disease induced by abnormal activity of transglutaminase, such as an inflammatory disease, a neurological disease and a cancer disease. It can be applied safely, which is useful for effectively preventing and treating these diseases.
도 1은 NDGA 농도에 따른 트란스글루타미나제 활성 억제 정도를 나타낸 것으로서, 구체적으로 트란스글루타미나제가 [1,4,-14C] 푸트레신을 숙시닐레이티드 카제인에 결합시키는 것을 측정하여, NDGA가 푸트레신과 경쟁하여 그 반응을 억제하는 것을 관찰한 인 비트로(in vitro) 실험 결과를 나타낸 그래프이다.Figure 1 shows the degree of inhibition of transglutaminase activity according to the concentration of NDGA, specifically measuring the binding of transglutaminase to [1,4, -14 C] putrescine to succinylated casein, A graph showing the results of in vitro experiments in which NDGA competes with putrescine to inhibit its response.
도 2는 NDGA 투여에 따라 I-κBα 폴리머 형성이 억제되는지 여부를 확인한 웨스턴 블롯팅 결과이다.2 is a result of Western blotting confirming whether I-κBα polymer formation is inhibited by NDGA administration.
따라서, 하나의 양태로서, 본 발명은 NDGA를 유효성분으로 포함하는, 트란스글루타미나제 활성 증가에 의해 초래되는 질병인 신경계 질환, 염증성 질환 또는 암 질환을 예방 또는 치료하기 위한 약제학적 조성물에 관한 것이다.Accordingly, in one aspect, the present invention relates to a pharmaceutical composition for preventing or treating a nervous system disease, an inflammatory disease or a cancer disease which is a disease caused by an increase in transglutaminase activity, comprising NDGA as an active ingredient. will be.
NDGA의 분자식은 C18H22O4이고, 분자량은 302.365이며, IUPAC 체계에 따라 4-[4-(3,4-다이하이드록시페닐)-2,3-다이메틸부틸]벤젠-1,2-다이올 (4-[4-(3,4-dihydroxyphenyl)-2,3-dimethylbutyl]benzene-1,2-diol)로 명명된다. The molecular formula of NDGA is C 18 H 22 O 4 , molecular weight is 302.365, 4- [4- (3,4-dihydroxyphenyl) -2,3-dimethylbutyl] benzene-1,2 according to the IUPAC system -Diol (4- [4- (3,4-dihydroxyphenyl) -2,3-dimethylbutyl] benzene-1,2-diol).
본 발명에서 사용되는 NDGA의 기원은 이에 한정되지 않으며 어떠한 NDGA라도 본 발명에 이용될 수 있다. NDGA의 구조식은 다음과 같다(화학식 1).The origin of the NDGA used in the present invention is not limited thereto, and any NDGA may be used in the present invention. The structural formula of NDGA is as follows (formula 1).
화학식 1
Figure PCTKR2009004175-appb-C000001
 Formula 1
Figure PCTKR2009004175-appb-C000001
NDGA는 상기 서술한 바와 같이 라레아 디베러케이트(Larrea divaricata Cav.)와 같은 북아메리카산 작은떡갈나무덤불의 수지를 함유한 삼출액으로부터 분리하여 사용할 수 있고, 당업계의 통상적인 방법에 따라 합성하여 사용할 수도 있다. NDGA의 제조방법은 다양하며, 그 예로 수소화된 (-)-갈리아레틱 산(guaiaretic acid)의 디메틸에테르(dimethyl ether)의 탈메틸화(dimethylation)의 방법으로 수행(Schroter, G. et al., Ber. 51, p1587, 1918)될 수 있으며, 보다 체계적인 합성법으로 1-피페로닐-1-브로모에탄과 그것으로부터 유래한 그리그나드 시약(Grignard reagent)의 반응을 주반응(key reaction)으로 하여 합성할 수도 있다(Liebermann, S. V. et al., J. Am. Chem. Soc. 69, p 1540, 1947; Sakakibara, Y., Nippon Kagaku Zasshi, 73, p 235, 1952; Blears, J. G. et al., J. Chem. Soc., Abstracts 1958, 1985). 이 밖에도 베타-케토 에스테르의 산화적인 커플링 방법(Gu, W. X. et al., Chin. Chem. Letters, 11, p 15, 2000), 디에틸 숙시네이트(diethyl succinate)와 피페로날을 이중 축합한 후 두 단계의 환원단계를 거치는 방법(Xia, Y. M. et al.,Chin. Chem. Letters, 14, p 359, 2003), 금속-포스핀 복합체에 의하여 촉매되는 할로티오펜의 그리그나드 커플링 반응과 이에 따른 촉매환원 방법 (Minato, A. et al., Tetrahderon Letters, 21, p 4017, 1980), 또는 대한민국 등록특허 제10-0783626호의 제조 방법 등에 의해 합성하여 사용될 수 있다.NDGA can be used separately from exudates containing resins of North American small oak bushes, such as Larrea divaricata Cav., As described above, and synthesized according to conventional methods in the art. It may be. There are various methods for preparing NDGA, for example, by demethylation of dimethyl ether of hydrogenated (-)-guaiaretic acid (Schroter, G. et al., Ber). 51, p1587, 1918), and a more systematic synthesis is carried out using the reaction of 1-piperonyl-1-bromoethane and the Grignard reagent derived therefrom as a key reaction. (Liebermann, SV et al., J. Am. Chem. Soc. 69, p 1540, 1947; Sakakibara, Y., Nippon Kagaku Zasshi, 73, p 235, 1952; Blears, JG et al., J. Chem. Soc., Abstracts 1958, 1985). In addition, the method of oxidative coupling of beta-keto esters (Gu, WX et al., Chin. Chem. Letters, 11, p 15, 2000), double condensation of diethyl succinate and piperonal A two-stage reduction step (Xia, YM et al., Chin. Chem. Letters, 14, p 359, 2003), Grignard coupling reaction of halothiophenes catalyzed by metal-phosphine complexes And the catalytic reduction method (Minato, A. et al., Tetrahderon Letters, 21, p 4017, 1980), or the preparation method of Korean Patent No. 10-0783626.
본 발명의 구체적인 실시예에서는 트란스글루타미나제가 [1,4,-14C] 푸트레신을 숙시닐레이티드 카제인에 결합시키는 것을 측정하여, NDGA가 푸트레신과 경쟁하여 그 반응을 억제하는 것을 관찰하였다. 인 비트로(in vitro)에서 14C로 표지된 푸트레신을 숙시닐레이티드 카제인과 결합시키는 트란스글루타미나제의 반응에 NDGA를 첨가하였을 때, 트란스글루타미나제의 활성은 NDGA의 농도가 증가할수록 감소됨을 확인하였다(도 1). 따라서, NDGA가 트란스글루타미나제의 억제제임을 알 수 있으며, 트란스글루타미나제가 과발현될 때 NDGA를 사용하여 증가된 트란스글루타미나제의 활성을 줄일 수 있음을 알 수 있다.In a specific embodiment of the present invention, the measurement of transglutaminase binding [1,4, -14 C] putrescine to succinylated casein, observing that NDGA competes with putrescine to inhibit its response. It was. When NDGA was added to the reaction of transglutaminase which binds 14 C-labeled putrescine to succinylated casein in vitro, the activity of transglutaminase increased the concentration of NDGA. It was confirmed that the decrease as the (Fig. 1). Thus, it can be seen that NDGA is an inhibitor of transglutaminase, and it can be seen that NDGA can be used to reduce the activity of increased transglutaminase when transglutaminase is overexpressed.
뿐만 아니라, 본 발명의 NDGA 조성물을 이용하는 경우 I-κBα의 중합체 형성을 억제시킴으로써, I-κBα 중합체 형성에 따른 트란스글루타미나제 활성 증가를 저해할 수 있음을 확인하였고, 이 역시 NDGA 농도에 의존적임을 알 수 있었다(도 2).In addition, by using the NDGA composition of the present invention by inhibiting the formation of I-κBα polymer, it was confirmed that it can inhibit the increase of transglutaminase activity due to the formation of I-κBα polymer, which is also dependent on the NDGA concentration It was found that (Fig. 2).
본 발명의 '신경계 질환'은 신경 세포의 사망 또는 손상과 관련되는 질환으로, 특히 알츠하이머 질환(Alzheimer disease), 다경색 치매(multi-infarct dementia), 알츠하이머 질환과 다경색 치매의 혼합형, 파킨슨 씨 질환(Parkinson's disease), 저갑상선증, 알코올성 치매, 헌팅턴 병(Huntingtun disease)등 중추 신경계 세포의 손상 및 사망에 따른 중추신경계 질환이 대표적이다. 이러한 질병의 주요 증상은 인지 기능 장애와 언어, 판단, 추상력 공간시간적 증력 및 기타 새로운 기술 습득의 장애 등을 포함하며, 성격변화, 정서적 불안정 등의 증세가 나타나며 종국적으로는 사망에 이르게 된다. 이중 본 발명의 약제학적 조성물은 신경 조직에서 트란스글루타미나제가 과발현되는 등 그 활성이 증가된 질병에 쓰일 수 있는데, 구체적으로는 뇌에서 트란스글루타미나제가 과발현되는 헌팅턴 병(Nature Medicine, Vol 8. Number 2 , February 2002 pp143-149), 소뇌와 대뇌 피질에서 트란스글루타미나제가 과발현되는 알츠하이머병(The Journal of Biological Chemistry, Vol. 274. No. 43. Issue Of October 22, pp 30715-30721), α 시누클레인(α synuclein)이 트란스글루타미나제에 의해 응집되는 파킨슨씨 병(PNAS, February 18,2003, Vol. 100, no.4, pp2047-2052) 등에 사용될 수 있으나, 이에 제한되지 않으며, 신경조직에서 트란스글루타미나제가 과발현되는 모든 질병의 치료에 사용될 수 있다. Nervous system disease of the present invention is a disease associated with death or damage of nerve cells, in particular Alzheimer's disease, multi-infarct dementia, a combination of Alzheimer's disease and multi-infarct dementia, Parkinson's disease (Parkinson's disease), hypothyroidism, alcoholic dementia, Huntingtun disease, such as central nervous system diseases due to damage and death of the central nervous system cells are typical. The main symptoms of these diseases include cognitive dysfunction, language, judgment, abstract spatial and temporal history, and other new skill acquisition disorders. Symptoms of personality changes, emotional instability, and eventually death. Among them, the pharmaceutical composition of the present invention can be used for diseases in which the activity is increased, such as overexpression of transglutaminase in neural tissues. Specifically, Huntington's disease in which transglutaminase is overexpressed in the brain (Nature Medicine, Vol 8 Number 2, February 2002 pp143-149), Alzheimer's Disease Overexpressing Transglutaminase in the Cerebellum and Cerebral Cortex (The Journal of Biological Chemistry, Vol. 274. No. 43. Issue Of October 22, pp 30715-30721) , α synuclein (α synuclein) can be used in Parkinson's disease (PNAS, February 18,2003, Vol. 100, no.4, pp2047-2052), which is aggregated by transglutaminase, but is not limited thereto. In addition, it can be used to treat any disease in which transglutaminase is overexpressed in nervous tissues.
본 발명의 '염증성 질환'은 염증을 유발하는 질환을 모두 포함하는 개념이며, 염증은 면역 적격 세포가 외부 개체 또는 항원성 단백질에 대한 반응에서 활성화되는 경우 일어난다. 염증성 과정은 침입 개체가 식세포화 되거나 중화되도록 하는 경우에서와 같이 유리할 수 있지만, 또한 골 및 연골의 파괴 및 결과적인 관절 기능의 제한을 야기하는 때의 관절염의 경우에서와 같이 해로울 수도 있다. 염증성 반응은 통상 외상 또는 항원, 예컨대 바이러스성, 박테리아성, 원생동물성, 또는 진균성 항원에 의해 유도된다. 'Inflammatory disease' of the present invention is a concept that includes all of the diseases causing inflammation, inflammation occurs when immune competent cells are activated in response to an external individual or antigenic protein. Inflammatory processes may be advantageous, such as in the case of causing invading individuals to be phagocytized or neutralized, but may also be harmful, such as in the case of arthritis, which causes destruction of bone and cartilage and consequent limitations of joint function. Inflammatory responses are usually induced by trauma or antigens, such as viral, bacterial, protozoan, or fungal antigens.
본 발명에 의한 예방 또는 치료에 적합한 염증성 질환에는 퇴행성 관절염, 당뇨병, 자가면역 근육염, 동맥 경화, 뇌졸증, 간경화, 뇌막염, 염증성 위궤양, 담낭 결석, 신장 결석, 부비강염, 비염, 결막염, 천식, 피부염, 염증성 장질환, 염증성 콜라겐 혈관 질환, 사구체신염, 염증성 피부 질환, 및 유육종증 등이 포함되지만, 이에 제한되는 것은 아니다.Inflammatory diseases suitable for the prophylaxis or treatment according to the present invention include degenerative arthritis, diabetes mellitus, autoimmune myositis, arteriosclerosis, stroke, cirrhosis, meningitis, inflammatory gastric ulcer, gallbladder stones, kidney stones, sinusitis, rhinitis, conjunctivitis, asthma, dermatitis, inflammatory Intestinal disease, inflammatory collagen vascular disease, glomerulonephritis, inflammatory skin disease, and sarcoidosis, and the like.
보다 바람직하게 본 발명의 염증성 질환은 a) 류마티스 염증성 질환인 퇴행성 관절염(rheumatoid arthritis), 전신성 홍반성 루푸스(Systemic lupus erythematosus), 강직성 척추염(Ankylosing spondylitis), 베체트 병(Behcet’s disease), 자가면역 근육염(inflammatroy mysitis), b) 장 염증성 질환인 궤양성대장염(UC: ulcerative colitis), 크론병(Crohn disease), c) 피부염증성 질환인 건선(psoriasis), 아토피성 피부염(atopic dermatitis), 접촉성 피부염. 습진성 피부염, 지루성 피부염, 편평 태선(lichen planus), 만성 단순 태선(lichen simplex chronicus), 천포창(pemphigus), 불러스 천포창, 표피 수포증(Epidermolysis Bullosa), 담마진(Urticaria), 혈관 부종(angioedema), 맥관염(vasculitis), 홍반 또는 피부 호산구증다증(Eosinophilia), 화폐상 피부염(nummular dermatitis), 전신성 박탈 피부염, 정체 피부염, 모낭 및 여드름과 같은 피지샘의 질환, 입 주위 피부염, 수염 가성모낭염 및 약물 발진, 다형 홍반(erythema multiforme), 결절 홍반(Erythema nodosum), 및 환상 육아종(Granuloma annulare)과 같은 염증 반응, 및 d) 이외의 염증성 위궤양(celiac disease), 뇌막염(encephalitis), 골반염증성질환(PID: Pelvic Inflammatory Disease) 등일 수 있으나, 이에 한정되는 것은 아니다.More preferably, the inflammatory disease of the present invention is a) rheumatoid inflammatory disease (rheumatoid arthritis), systemic lupus erythematosus, Ankylosing spondylitis, Behcet's disease, autoimmune myositis ( inflammatroy mysitis, b) ulcerative colitis (UC), Crohn disease (C), an intestinal inflammatory disease, c) psoriasis, atopic dermatitis, and contact dermatitis, a dermatitis. Eczema Dermatitis, Seborrheic Dermatitis, lichen planus, lichen simplex chronicus, pemphigus, Bullus pemphigus, Epidermolysis Bullosa, Urticaria, Angioedema , Diseases of the sebaceous glands, such as vasculitis, erythema or cutaneous eosinophilia, nummular dermatitis, systemic deprivation dermatitis, stagnant dermatitis, hair follicles and acne, percutaneous dermatitis, whisker pseudofolliculitis and drugs Inflammatory reactions such as rash, erythema multiforme, erythema nodosum, and granuloma annulare, and d) inflammatory gastric ulcer disease, encephalitis, pelvic inflammatory disease (PID) : Pelvic Inflammatory Disease), but is not limited thereto.
본 발명의 용어 '암 질환'의 경우, 생체 조직 안에서 세포가 무제한으로 증식하여 악성 종양을 일으키는 현상을 일컫는 용어로서, 트란스글루타미나제와 관련하여 암의 전이, 화학적 내성, 방사성 내성을 가지는 암에서 트란스글루타미나제의 현저한 발현 증가가 확인되었는바, 트란스글루타미나제의 억제는 암 예방 및 치료 측면에서도 중요성이 부각되고 있다. 본 발명의 NDGA를 포함하는 약제학적 조성물을 사용하여 예방 또는 치료될 수 있는 구체적인 암은 트란스글루타미나제의 증가가 나타나는 암, 구체적으로는 대장암, 소장암, 직장암, 항문암, 식도암, 췌장암, 위암, 신장암, 자궁암, 유방암, 폐암, 임파선암, 갑상선암, 전립선암, 백혈병, 피부암, 결장암, 뇌종양, 방광암, 난소암, 담낭암 등을 포함하나 이에 제한되는 것은 아니다.The term 'cancer disease' of the present invention refers to a phenomenon in which cells multiply unlimitedly in living tissues to cause malignant tumors, and has metastasis, chemical resistance and radiation resistance of cancer in relation to transglutaminase. Significant increases in the expression of transglutaminase have been identified in the present invention, the inhibition of transglutaminase is also important in the prevention and treatment of cancer. Specific cancers that can be prevented or treated using the pharmaceutical composition comprising the NDGA of the present invention include cancers in which an increase in transglutaminase occurs, specifically colon cancer, small intestine cancer, rectal cancer, anal cancer, esophageal cancer and pancreatic cancer. , Stomach cancer, kidney cancer, uterine cancer, breast cancer, lung cancer, lymph cancer, thyroid cancer, prostate cancer, leukemia, skin cancer, colon cancer, brain tumors, bladder cancer, ovarian cancer, gallbladder cancer and the like.
본 발명의 '예방 또는 치료'에서 '예방'이란 NDGA를 포함하는 조성물의 투여로 인한 트란스글루타미나제 활성 증가에 따르는 모든 질환의 발병을 억제시키거나 발병을 지연하는 모든 행위를 말하며, '치료'란 상기 약제학적 조성물의 투여로 트란스글루타미나제 활성 증가로 인한 모든 질병을 호전시키거나 이롭게 변경하는 모든 행위를 의미한다.In the "prevention or treatment" of the present invention, "prevention" refers to any action that suppresses or delays the onset of all diseases resulting from increased transglutaminase activity due to the administration of a composition comprising NDGA. By 'administration of the pharmaceutical composition' means any action that improves or beneficially alters all diseases caused by increased transglutaminase activity.
또 하나의 양태로서, 본 발명은 NDGA를 유효성분으로 포함하는 트란스글루타미나제 활성 증가에 의해 초래되는 신경성 질환, 염증성 질환 및 암 질환을 치료 또는 예방하는 방법에 관한 것이다. As another aspect, the present invention relates to a method for treating or preventing neurological diseases, inflammatory diseases and cancer diseases caused by increased transglutaminase activity comprising NDGA as an active ingredient.
본 발명의 약제학적 조성물을 이용하여 치료 또는 예방하는 방법은 인간 뿐만 아니라 트란스글루타미나제 활성의 증가로 인한 질병이 발병할 수 있는, 소, 말, 양, 돼지, 염소, 낙타, 영양, 개, 고양이 등의 포유동물에게도 적용될 수 있다. Methods of treating or preventing using the pharmaceutical compositions of the present invention are cattle, horses, sheep, pigs, goats, camels, nutrition, dogs, which can develop diseases caused by increased transglutaminase activity in humans as well. It can also be applied to mammals such as cats.
본 발명의 NDGA를 포함하는 조성물은 단일제로도 사용할 수 있으며, 공인된 약학 조성물을 포함하여 복합제로 제조하여 사용할 수도 있다. The composition comprising the NDGA of the present invention may be used as a single agent, or may be prepared and used as a complex including a recognized pharmaceutical composition.
NDGA를 포함하는 조성물은 NDGA를 포함하는 캡슐에 부형제 없이 충전하거나 미립된 고체 담체, 액체 담체 또는 그 양자와 균일하게 충분히 접촉시켜 제조될 수 있다. 그 후에 필요한 경우 생성물을 바람직한 제제로 성형하여 사용할 수 있다. 적합한 담체 부형제의 예로서 전분, 물, 식염수, 에탄올, 글리세롤, 링거액 및 덱스트로스 용액 등을 들 수 있으며, 문헌(Remington`s Pharmaceutical Science, 19thEd., 1995, Mack Publishing Company, Easton PA)등에 개시되어 있는 바에 따라 당해 기술 분야에 알려진 적합한 제제로 제제화할 수 있다.Compositions comprising NDGA can be prepared by filling the capsule comprising NDGA without excipients or uniformly and sufficiently in contact with the particulate solid carrier, liquid carrier or both. The product can then be molded into the desired formulation and used if necessary. Examples of suitable carrier excipients include starch, water, saline, ethanol, glycerol, Ringer's solution and dextrose solution, and the like (Remington's Pharmaceutical Science, 19 th Ed., 1995, Mack Publishing Company, Easton PA) and the like. As disclosed, it may be formulated into a suitable formulation known in the art.
본 발명의 NDGA를 포함하는 조성물은 어떠한 제형으로도 적용 가능하며, 경구용 또는 비경구용으로도 제조할 수 있다. 비경구용 제형으로는 주사용, 도포용, 에어로졸 등의 스프레이 형일 수 있으며, 바람직하게는 주사용 또는 에어로졸 등의 스프레이 형태이고, 경구용 제형 또한 바람직하다. The composition comprising the NDGA of the present invention can be applied in any formulation and can be prepared for oral or parenteral use. The parenteral formulation may be in the form of a spray such as injectable, application or aerosol, preferably in the form of a spray such as injectable or aerosol, and oral formulations are also preferred.
본 발명의 약제학적 조성물을 포함하는 경구 투여용 제형으로는, 예를 들어 정제, 트로키제, 로렌지, 수용성 또는 유성현탁액, 조제분말 또는 과립, 에멀젼, 하드 또는 소프트 캡슐, 시럽 또는 엘릭시르제 등으로 제제화 될 수 있다. 정제 및 캡슐 등의 제형으로 제제하기 위하여, 락토오스, 사카로오스, 솔비톨, 만니톨, 전분, 아밀로펙틴, 셀룰로오스 또는 젤라틴과 같은 결합제, 디칼슘 포스페이트와 같은 부형제, 옥수수 전분 또는 고구마 전분과 같은 붕괴제, 스테아르산 마스네슘, 스테아르산 칼슘, 스테아릴푸마르산 나트륨 또는 폴리에틸렌글리콜 왁스와 같은 윤활유를 포함할 수 있으며, 캡슐 제형의 경우 상기 언급한 물질 외에도 지방유와 같은 액체 담체를 더 함유할 수 있다.Formulations for oral administration comprising the pharmaceutical composition of the present invention include, for example, tablets, troches, lozenges, water-soluble or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs, and the like. Can be formulated. For formulation into tablets and capsules, lactose, saccharose, sorbitol, mannitol, starch, amylopectin, binders such as cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, stearic acid masne It may include lubricating oils such as calcium, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax, and in the case of capsule formulations, it may further contain a liquid carrier such as fatty oil in addition to the above-mentioned materials.
본 발명의 약제학적 조성물을 비경구로 투여하는 경우, 비경구 투여는 피하주사, 정맥주사 또는 근육내 주사 등의 주사용 형태, 좌제 주입방식 또는 호흡기를 통하여 흡입이 가능하도록 하는 에어로졸제 등 스프레이용으로 제제화 할 수 있다. 주사용 제형으로 제제화하기 위해서는 NDGA를 안정제 또는 완충제와 함께 물에서 혼합하여 용액 또는 현탁액으로 제조하고, 이를 앰플 또는 바이알의 단위 투여용으로 제제한다. 좌제로 주입하기 위해서는, 코코아버터 또는 다른 글리세라이드 등 통상의 좌약 베이스를 포함하는 좌약 또는 체료 관장제와 같은 직장투여용 조성물로 제제화 할 수 있다. 에어로졸제 등의 스프레이용으로 제형화하는 경우, 수분산된 농축물 또는 습윤 분말이 분산되도록 추진제 등이 첨가제와 함께 배합될 수 있다. When the pharmaceutical composition of the present invention is administered parenterally, parenteral administration is for injection, such as subcutaneous injection, intravenous injection or intramuscular injection, suppository injection method, or aerosol for inhalation through the respiratory system. It can be formulated. To formulate injectable formulations, NDGA is mixed in water with stabilizers or buffers to prepare solutions or suspensions, which are formulated for unit doses of ampoules or vials. For infusion into suppositories, it may be formulated in a rectal composition such as suppositories or body enema including conventional suppository bases such as cocoa butter or other glycerides. When formulated for spraying such as aerosols, a propellant or the like may be combined with the additives to disperse the dispersed dispersion or wet powder.
본 발명의 약제학적 조성물은 직장, 국소, 정맥 내, 복강 내, 근육 내, 동맥 내, 경피, 비측 내, 흡입, 안구 내 또는 피 내 경로를 통해 통상적인 방식으로 투여할 수 있다. 비경구 투여는 정맥 내, 근육 내, 복강 내, 흉골 내, 경피 및 동맥 내 주사 및 주입을 포함하는 투여 방식을 의미한다. 본 발명의 NDGA를 포함하는 약제학적 조성물의 비경구 투여는 바람직한 순도 하에 약제학적으로 허용가능한 담체, 즉 사용되는 농도와 투여량에서 수용체에게 비독성이고 다른 제제 성분과 화합할 수 있는 담체와 혼합하여 단위 투여량의 제형으로 조제하는 것이 바람직하다. 특히 제제는 산화제 및 인체에 유해한 것으로 알려진 기타 다른 화합물을 포함하지 않는 것이 바람직하다. The pharmaceutical compositions of the invention may be administered in a conventional manner via the rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, nasal, inhalation, intraocular or intradermal routes. Parenteral administration means administration modes including intravenous, intramuscular, intraperitoneal, sternum, transdermal and intraarterial injection and infusion. Parenteral administration of the pharmaceutical composition comprising the NDGA of the present invention may be carried out under desired purity in admixture with a pharmaceutically acceptable carrier, i.e., a carrier which is nontoxic to the receptor and compatible with other agent components at the concentrations and dosages employed. It is preferred to formulate in unit dosage form. In particular, the formulation preferably does not contain oxidizing agents and other compounds known to be harmful to the human body.
본 발명의 NDGA를 1 종 이상의 약제학적으로 허용가능한 부형제와 함께 약제학적 조성물로 투여될 수 있다. 인간 환자에게 투여되는 경우, 본 발명의 약제학적 조성물의 총 1일 사용량은 올바른 의학적 판단범위 내에서 처치의에 의해 결정될 수 있다는 것은 당업자에게 자명한 일이다. 특정 환자에 대한 구체적인 치료적 유효량은 달성하고자 하는 반응의 종류와 정도, 경우에 따라 다른 제제가 사용되는지의 여부를 비롯한 구체적 조성물, 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료기간, 구체적 조성물과 함께 사용되거나 동시 사용되는 약물을 비롯한 다양한 인자와 의약 분야에 잘 알려진 유사 인자에 따라 다르게 적용하는 것이 바람직하다. 따라서 본 발명의 목적에 적합한 NDGA의 유효량은 전술한 사항을 고려하여 결정하는 것이 바람직하다The NDGA of the invention can be administered in a pharmaceutical composition with one or more pharmaceutically acceptable excipients. When administered to a human patient, it will be apparent to those skilled in the art that the total daily usage of the pharmaceutical compositions of the present invention can be determined by the practitioner within the correct medical judgment. The specific therapeutically effective amount for a particular patient may be based on the specific composition, including the type and severity of the response to be achieved, whether other agents are used in some cases, the age, body weight, general health, sex and diet, time of administration, It is desirable to apply differently depending on various factors and similar factors well known in the medical field, including the route of administration and the rate of release of the composition, the duration of treatment, and the drugs used with or concurrent with the specific composition. Therefore, the effective amount of NDGA suitable for the purpose of the present invention is preferably determined in consideration of the above-mentioned matters.
이하, 본 발명을 하기 실시예를 참조하여 구체적으로 설명한다. 다만, 하기 실시예는 본 발명의 이해를 돕기 위한 것으로, 본 발명의 범위가 하기 실시예에 한정되지는 않는다.Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are provided to help the understanding of the present invention, and the scope of the present invention is not limited to the following examples.
실시예 1: 트란스글루타미나제 활성도 억제에 관한 in vitro 시험Example 1 In vitro Test on Inhibition of Transglutaminase Activity
트란스글루타미나제가 [1,4,-14C] 푸트레신을 숙시닐레이티드 카제인에 결합시키는 것을 측정하여, NDGA가 푸트레신과 경쟁하여 그 반응을 억제하는 것을 관찰하였다. Transglutaminase measured the binding of [1,4, -14 C] putrescine to succinated casein and observed that NDGA competes with putrescine to inhibit its response.
숙시닐화된 카제인은 Calbiochem(Cat. No. 573464)로부터 구입하고, 상기 분말 1g을 5mM DTT를 포함하는 반응 완충용액 50ml(0.1M Tris-acetate(pH 8.0), 10mM CaCl2, 0.15M NaCl, 1.0mM EDTA)에 용해시켰다. 이 용액은 사용 전에 딥 프리져(Deep freezer)에 보관하였다. [1,4-14C] 푸트레신 디하이드로클로라이드는 GE Healthcare(Cat. No. CFA301)로부터 구입하였고, 그 스톡 용액(stock solution)을 방사선적 용량(radiological dosage)이 5μCi/ml에 달할 때까지 증류수로 희석하였다. 트란스글루타미나제 2는 시그마-앨드리츠(Sigma-Aldrich(Cat. No.T5398))로부터 구입하였고 최종 농도가 1 unit/ml가 되게 증류수로 희석하였다. NDGA의 스톡 용액은 NDGA(Sigma-Aldrich, Cat No. C3878)을 DMSO에 10mM 농도로 녹여서 제조한 후 이를 DMSO로 희석하여 각각의 농도로 제조하였다.Succinylated casein was purchased from Calbiochem (Cat. No. 573464), and 1 g of the powder was obtained from 50 ml of reaction buffer containing 5 mM DTT (0.1 M Tris-acetate (pH 8.0), 10 mM CaCl 2 , 0.15 M NaCl, 1.0). mM EDTA). This solution was stored in a deep freezer before use. [1,4- 14 C] putrescine dihydrochloride was purchased from GE Healthcare (Cat. No. CFA301) and the stock solution was obtained at a radiological dosage of 5 μCi / ml. Dilute with distilled water until. Transglutaminase 2 was purchased from Sigma-Aldrich (Cat. No. T5398) and diluted with distilled water to a final concentration of 1 unit / ml. A stock solution of NDGA was prepared by dissolving NDGA (Sigma-Aldrich, Cat No. C3878) in DMSO at a concentration of 10 mM and then diluting it with DMSO at each concentration.
기질 용액은 450㎕의 숙시닐화된 카제인 용액 및 [1,4-14C] 푸트레신 디하이드로 클로라이드 용액 50㎕를 혼합하여 제조하였다. 각 샘플을 96㎕의 반응 완충액, 3㎕의 NDGA의 스톡 용액 및 1㎕의 트란스글루타미나제 스톡 용액을 혼합하여 제조하였다. 10분 동안 37℃에서 샘플을 인큐베이션 한 후에, 기질 용액 500㎕ 및 샘플 용액 100㎕을 잘 혼합하였고, 이들 혼합물을 37℃에서 2시간 동안 인큐베이션 하였다. 4.5ml의 차가운(4℃) 7.5% TCA를 부가하여 반응을 정지시키고 전체 용액을 1시간 동안 4℃에서 보관하였다. TCA-단백질 침전물을 GF/유리섬유 필터로 여과하여 차가운 5% TCA 25ml로 세척하고 건조하였다. 가교된 단백질의 방사능을 베타(β)-카운터(Beckman Coulter)로 측정하고 표준으로서 DMSO-대조군의 활성을 가지고 보정하였다. 측정된 수치를 트란스글루타미나제의 활성으로 나타내었다. 동일한 조건 하에서 이러한 어세이를 세 번 반복하였으며 그 값은 하기 표 1에 나타낸 바와 같다.Substrate solution was prepared by mixing the biotinylated succinyl casein solution and [1,4- 14 C] Fu Trail New dihydrochloride solution 50㎕ of 450㎕. Each sample was prepared by mixing 96 μl of reaction buffer, 3 μl of NDGA stock solution, and 1 μl of transglutaminase stock solution. After incubating the samples at 37 ° C. for 10 minutes, 500 μl of substrate solution and 100 μl of sample solution were mixed well and these mixtures were incubated at 37 ° C. for 2 hours. 4.5 ml of cold (4 ° C.) 7.5% TCA was added to stop the reaction and the entire solution was stored at 4 ° C. for 1 hour. The TCA-protein precipitate was filtered through a GF / glass fiber filter, washed with 25 ml of cold 5% TCA and dried. Radioactivity of the crosslinked protein was measured by beta (β) -Counter (Beckman Coulter) and corrected with the activity of DMSO-control as a standard. The measured value is shown as the activity of transglutaminase. This assay was repeated three times under the same conditions and the values are shown in Table 1 below.
표 1
농도 평균 SD(표준편차)
0.0μM 1.00000 0.00000
0.5μM 0.66457 0.01275
1.0μM 0.01608 0.00024
3.0μM 0.00125 0.00085
5.0μM 0.00269 0.00064
10.0μM 0.00635 0.00102
Table 1
density Average SD (standard deviation)
0.0μM 1.00000 0.00000
0.5 μM 0.66457 0.01275
1.0 μM 0.01608 0.00024
3.0 μM 0.00125 0.00085
5.0 μM 0.00269 0.00064
10.0 μM 0.00635 0.00102
또한, 그 평균값을 NDGA의 농도에 대하여 도시하였다. IC50 수치는 일반 비선형 회귀법에 의해 계산되었으며 이는 1.4915(±0.1229)μM로 결정되었다.In addition, the average value is shown with respect to the density | concentration of NDGA. IC 50 values were calculated by the general nonlinear regression method, which was determined to be 1.4915 (± 0.1229) μM.
NDGA의 농도에 따라 측정된 상대적인 트란스글루타미나제 억제 활성을 도 1에 나타내었다. 도 1에서 나타난 바와 같이, 트란스글루타미나제의 활성은 NDGA의 농도 의존적으로 억제되는 것을 알 수 있다.The relative transglutaminase inhibitory activity measured according to the concentration of NDGA is shown in FIG. 1. As shown in Figure 1, it can be seen that the activity of transglutaminase is inhibited in a concentration-dependent manner of NDGA.
실시예 2. NDGA의 I-κBα 중합체 형성 억제능 분석Example 2 Analysis of Inhibition of I-κBα Polymer Formation of NDGA
본 발명의 화합물이 트란스글루타미나제 2에 의한 I-κBα의 중합체 형성을 억제하는지 여부 및 I-κBα 폴리머(polymer) 수준 감소에 따른 모노머(monomer) I-κBα 수준을 회복시키는지 여부를 분석하기 위하여, 기니아피그(guinea pig) 트란스글루타미나제2 0.2mU을 37℃에서 10 분간 반응 완충액(0.1M Tris, pH8.0, 0.15M NaCl, 10mM CaCl2, 1.0mM EDTA)으로 사전배양(preincubation)하였다. 사전배양 후, 정제된 재조합 인간 I-κBα 20ng을 첨가하여, 대조군의 모노머의 양을 확인하는 한편, NDGA를 농도별로 처리한 후 37℃에서 1시간 동안 배양함으로써, NDGA에 의한 I-κBα 모노머 회복 여부를 관찰하였다. 반응 종결은 10mM EDTA를 포함하는 SDS 샘플 완충액을 첨가하여 수행하였다. I-κBα 모노머 및 폴리머의 양을 확인하기 위하여 샘플을 I-κBα에 대한 웨스턴 블롯팅으로 분석하였다(도 2). 도 2에 나타난 바와 같이 트란스글루타미나제만을 첨가한 경우 I-κBα 모노머의 양이 현저하게 감소하였으나, NDGA를 처리하는 경우 I-κBα 모노머의 양이 회복됨을 확인할 수 있다. 또한 이러한 회복량은 NDGA의 양 의존적임을 알 수 있다.Analyzes whether compounds of the present invention inhibit polymer formation of I-κBα by transglutaminase 2 and restore monomer I-κBα levels with reduced I-κBα polymer levels In order to pre-incubate 0.2mU of guinea pig transglutaminase 2 with reaction buffer (0.1M Tris, pH8.0, 0.15M NaCl, 10mM CaCl 2 , 1.0mM EDTA) at 37 ° C for 10 minutes ( preincubation). After pre-incubation, 20ng of purified recombinant human I-κBα was added to confirm the amount of monomer in the control group, while NDGA was treated by concentration, followed by incubation at 37 ° C. for 1 hour, thereby recovering I-κBα monomer by NDGA. Whether or not was observed. Reaction termination was performed by adding SDS sample buffer containing 10 mM EDTA. Samples were analyzed by western blotting for I-κBα to confirm the amount of I-κBα monomer and polymer (FIG. 2). As shown in FIG. 2, when only transglutaminase was added, the amount of the I-κBα monomer was significantly decreased, but the amount of the I-κBα monomer was recovered when NDGA was treated. It can also be seen that the amount of recovery is dependent on the amount of NDGA.
본 발명의 NDGA를 포함하는 약제학적 조성물 및 이를 이용한 질환 치료 및 예방 방법은, 염증성 질환, 신경계 질환 및 암 질환과 같은 트란스글루타미나제의 비정상적 활성에 의해 유도된 질환을 효과적으로 예방 및 치료할 수 있다.The pharmaceutical composition comprising the NDGA of the present invention and a method for treating and preventing diseases using the same can effectively prevent and treat diseases induced by abnormal activity of transglutaminase, such as inflammatory diseases, neurological diseases and cancer diseases. .

Claims (7)

  1. NDGA를 유효성분으로 포함하는 트란스글루타미나제 활성 억제용 조성물.Transglutaminase activity inhibitory composition comprising NDGA as an active ingredient.
  2. NDGA를 유효성분으로 포함하는, 트란스글루타미나제 활성 증가에 의해 초래되는 질병인 신경계 질환, 염증성 질환 또는 암 질환을 예방 또는 치료하기 위한 약제학적 조성물.A pharmaceutical composition for preventing or treating a nervous system disease, an inflammatory disease or a cancer disease which is a disease caused by an increase in transglutaminase activity, comprising NDGA as an active ingredient.
  3. 제2항에 있어서, 상기 신경계 질환은 알츠하이머 병, 다경색 치매, 저갑상선증, 알코올성 치매, 헌팅턴 병 또는 파킨슨씨 병인 약제학적 조성물.The pharmaceutical composition of claim 2, wherein the neurological disease is Alzheimer's disease, multi-infarct dementia, hypothyroidism, alcoholic dementia, Huntington's disease or Parkinson's disease.
  4. 제2항에 있어서, 상기 염증성 질환은 퇴행성 관절염, 당뇨병, 자가면역 근육염, 동맥 경화, 뇌졸증, 간경화, 뇌막염, 염증성 위궤양, 담낭 결석, 신장 결석, 부비강염, 비염, 결막염, 천식, 피부염증성 질환, 염증성 장질환, 류마티스 염증성 질환, 염증성 콜라겐 혈관 질환, 골반 염증성 질환, 사구체신염, 염증성 피부 질환, 또는 유육종증인 약제학적 조성물. The method of claim 2, wherein the inflammatory disease is degenerative arthritis, diabetes mellitus, autoimmune myositis, arteriosclerosis, stroke, cirrhosis, meningitis, inflammatory gastric ulcer, gallbladder stones, kidney stones, sinusitis, rhinitis, conjunctivitis, asthma, dermatitis, inflammatory A pharmaceutical composition that is enteric disease, rheumatic inflammatory disease, inflammatory collagen vascular disease, pelvic inflammatory disease, glomerulonephritis, inflammatory skin disease, or sarcoidosis.
  5. 제2항에 있어서, 상기 암 질환은 대장암, 소장암, 직장암, 항문암, 식도암, 췌장암, 위암, 신장암, 자궁암, 유방암, 폐암, 임파선암, 갑상선암, 전립선암, 백혈병, 피부암, 결장암, 뇌종양, 방광암, 난소암, 또는 담낭암인 약제학적 조성물.According to claim 2, wherein the cancer disease, colorectal cancer, small intestine cancer, rectal cancer, anal cancer, esophageal cancer, pancreatic cancer, stomach cancer, kidney cancer, uterine cancer, breast cancer, lung cancer, lymph gland cancer, thyroid cancer, prostate cancer, leukemia, skin cancer, colon cancer, A pharmaceutical composition that is brain tumor, bladder cancer, ovarian cancer, or gallbladder cancer.
  6. 제1항의 조성물을 투여하는 것을 포함하는 트란스글루타미나제의 활성 억제 방법.A method of inhibiting activity of transglutaminase comprising administering the composition of claim 1.
  7. 제2항의 약제학적 조성물을 투여하는 것을 포함하는, 트란스글루타미나제의 활성 증가에 의해 초래되는 신경계 질환, 염증성 질환 또는 암 질환의 치료 또는 예방 방법.A method of treating or preventing a nervous system disease, an inflammatory disease or a cancer disease caused by increased activity of transglutaminase, comprising administering the pharmaceutical composition of claim 2.
PCT/KR2009/004175 2008-07-25 2009-07-27 Ndga-containing composition for the suppression of transglutaminase WO2010011117A2 (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107449848A (en) * 2017-09-22 2017-12-08 完美（中国）有限公司 A kind of method of La Ruia extracts in discriminating cosmetic material

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