KR20100056212A - A pharmaceutical composition for preventing or treating transglutaminase-related disease comprising sulfarlem, and a method for preventing or treating transglutaminase-related disease by using thereof - Google Patents

Abstract

PURPOSE: A transglutaminase inhibitor containing sulfarlem is provided to apply to a patient with abnormal transglutaminase overexpression and to suppress transglutaminase without side effects. CONSTITUTION: A pharmaceutical composition for preventing or treating diseases caused by transglutaminase activation contains sulfarlem or pharmaceutically acceptable salt thereof. The diseases are inflammatory diseases, cancers, or neurological disorders. The composition has anti-metastasis effect. The inflammatory diseases are chronic inflammatory disease such as degenerative arthritis, septicemia, or autoimmune myositis. The neurological disorders are Alzheimer diseases, Huntington disease, or Parkinson's disease.

Description

Composition for the prevention or treatment of transglutaminase-related diseases, including sparparme, and a method for preventing or treating transglutaminase-related diseases using the same, and a method for treating transglutaminase-related disease comprising sulfarlem, and a method for preventing or treating transglutaminase-related disease by using skilled}

The present invention relates to a transglutaminase inhibitor comprising a chaparem or a pharmaceutically acceptable salt thereof, and more particularly to the action of transglutaminase involved in the pathological mechanism of disease in the case of aberrant expression. It relates to transglutaminase inhibitors and novel uses thereof, including sampharem or a pharmaceutically acceptable salt thereof for inhibition.

Transglutaminase is an enzyme that cross-links proteins and is widely distributed in nature such as animals, plants, and microorganisms. When transglutaminase acts on protein molecules, cross-links are formed by acyl exchange reactions, cross-reactions and deaminoation reactions, and these bonds have particular effects on the gel formation, thermal stability and hydration ability of proteins. Transglutaminase is an enzyme that acts as a protective agent in blood clotting and wound healing. However, if its 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).

In particular, the expression of transglutaminase is increased in diseases with inflammation, and examples of such diseases are chronic inflammatory diseases such as degenerative arthritis, diabetes mellitus, autoimmune myositis, arteriosclerosis, stroke, cirrhosis, malignant breast cancer, and Alzheimer's disease. , Parkinson's disease, Huntington's disease, meningitis, inflammatory gastric ulcer and sepsis. In addition, increased expression of NF-κB and 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).

The relationship between the chemical resistance of cancer and transglutaminase is not known yet.However, if the expression of transglutaminase is inhibited in breast cancer cell lines with chemical resistance, the cancer cells are sensitive to the anticancer agent and die. (Antonyak et al., Augmentation of tissue tansglutaminase expression and activation by epidermal growth factor inhibit doxorubicin-induced apoptsis 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 molecular pathology of transglutaminase has recently been revealed, further validating its 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, 2004).

The complex mechanisms of inflammation have not been fully understood, but to date, the main pathway of inflammation is due to the activation of a transcriptional substance called NF-κB. The pathway of activation of NF-κB was known to be the activation of kinase following the signal transduction system, but the activation of kinase inhibitors was reduced as it was confirmed that activation of NF-κB could occur without the help of kinase (Tergaonkar et al., IkappaB kinase-independent IkappaBalpha degradation pathway: functional NF-kappaB activity and implications for cancer therap.Mol Cell Biol. 2003 Nov; 23 (22): 8070-83).

The inventors found that the expression of NF-κB and transglutaminase also increased in inflammation, and the mechanisms were revealed in the inflammation model. The main mechanism is that transglutaminase can cross-link I-κBα to activate NF-κB without the help of kinases (IKK, NAK) (Jongmin Lee, et al. Transglutaminase 2 induces NF-κB activation via a novel pathway in BV-2 microglia.J. Biol. Chem. 279, 53725-53735, 2004). In addition, since transglutaminase is a calcium dependent enzyme, NF-κB can be activated only by increasing calcium in cells.

Inflammatory responses were first made by the activity of the NF-κB transcription factor, which is the inflammatory factor including transglutaminase and its inhibitor, I-κBα, which normally inhibits NF-κB by I-κBα. No continuous activity occurs. However, 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 retaining more activated NF-κB. Suggested. In general, the activation of NF-κB is believed to produce I-κBα to inhibit NF-κB and induce transglutaminase inhibitors to prevent a vicious cycle of NF-κB activation by transglutaminase. However, when such homeostasis is broken, inflammation may be continuously induced by inflammatory cells due to continuous activation of NF-κB by transglutaminase. In addition, these malignant circuits may cause cancer metastasis and drug resistance in cancer tissues (Jongmin Lee, et al. Transglutaminase 2 induces NF-κB activation via a novel pathway in BV-2 microglia. J. Biol Chem. 279, 53725-53735, 2004).

Therefore, the inhibitor of transglutaminase may act as an important substance that breaks the continuous ring of NF-κB to improve inflammatory diseases, and transglutamina prepared by the present inventors with the peptide reported in the above-mentioned literature. It is assumed that the effect of replacing the steroidal agent of the inhibitor is based on this fact (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).

An amine compound is known to inhibit transglutaminase activity. Representatively, cystamine (nature genetics 18, 111-117, 1998; Nature Medicine 8, 143-149, 2002) and Putrescine. See also 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) have been developed. Toxicity is known which causes nonspecific inhibition of other enzymes.

Therefore, there is a need for the development of safe and effective transglutaminase specific inhibitors. Recently, Son et al. Have been successful in administering steroid-supplied transglutaminase inhibitors in eye allergy models induced by pollen in guinea pigs (Sohn, J., Kim, T.- I., Yoon, Y.-H., and Kim, S.-Y .; Transglutaminase inhibitors: A New Anti-Inflammatory Aproach in Allergic Cojunctivitis.J. Clin. Invest. 111, 121-8, 2003). Elastase inhibitor elafin is a new type proteinase inhibitor which has a transglutaminase-mediated anchoring seuence termed "cememtoin". 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 inflammation-related diseases. Examples of such diseases include chronic inflammatory diseases such as degenerative arthritis, diabetes mellitus, autoimmune myositis, arteriosclerosis, stroke, liver cirrhosis, malignant breast cancer, and meningitis. , Inflammatory gastric ulcer and sepsis.

In addition to the above compounds, other chemical inhibitors have been developed, but all are known to cause toxicity when 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 there are still many problems to be solved in practical use in terms of cost and safety. have.

Szaparem is anesole dithiolethione, 5- (para-methoxyphenyl) -3H-1,2-dithiol-thione (5- (p-methoxyphenyl) -3H-1,2-dithiole- 3-thione) and belongs to the group of cyclic compounds containing sulfur known as 1,2-dithiole-3-thione. These cyclic compounds are known to be present in vegetables belonging to cruciferous plants such as cabbage (Christen, Methods Enzymol 252, 316-323, 1995). Seloparem has been used clinically as an antagonist and salivary secretion for decades without major side effects.

Therefore, the present inventors attempted to select drugs that do not know the mechanism among drugs already recognized for safety and commercialized, to identify the mechanism and to find a new use using them, and to screen useful substances among them as transglutaminase inhibitors. As a result, a new discovery of the activity of transpar glutaminase inhibitors was brought to the present invention.

It is a further object of the present invention to provide a pharmaceutical composition for treating a disease caused by an increase in transglutaminase activity, including stoolarem or a pharmaceutically acceptable salt thereof.

It is an object of the present invention to provide a method for inhibiting transglutaminase, characterized by using sparparem or a pharmaceutically acceptable salt thereof as the active ingredient.

It is another object of the present invention to provide a method for treating a disease caused by an increase in transglutaminase activity using stoolarem or a pharmaceutically acceptable salt thereof.

As one aspect for achieving the above object, the present invention is a pharmaceutical composition, active ingredient for preventing or treating a disease caused by the increase of the activity of transglutaminase, including spararem or a pharmaceutically acceptable salt thereof The present invention relates to a method of inhibiting the activity of transglutaminase by using chaparem and a method of treating a disease caused by an increase in the activity of transglutaminase.

Szaparem belongs to the group of cyclic compounds containing sulfur known as 1,2-dithiole-3-thione, as described above. Its chemical name is 5- (para-methoxyphenyl) -3H-1,2-dithiol-3-thione (5- (p-methoxyphenyl) -3H-1,2-dithiole-3-thione) and anesol dithi It is also called Orthion.

The molecular formula of the Suleparem is C ₁₀ H ₈ OS ₃ and the molecular weight is 240.36 and the structural formula is as follows.

Sullparem used in the present invention can be prepared by synthesis, and the synthesis and the resulting separation purification can be obtained according to known methods. These preparations can be used as a commercially available drug in addition to being manufactured directly.

In addition, the transglutaminase inhibitor according to the present invention may include a pharmaceutically acceptable salt of szaparem.

In one specific embodiment of the present invention, the inventors have determined that transglutaminase binds [1,4, ^-14 C] putrescine to succinylate casein, thereby allowing Schalparem to react. Was observed to inhibit. The transglutaminase activity was determined by the concentration of suparem, when Schepharm was added to the transglutaminase reaction that binds ¹⁴ C-labeled putrescine with succinylated casein in vitro. Decreased with increasing (FIG. 1). Therefore, it can be seen that chaparem is an inhibitor of transglutaminase, and it can be seen that using chaparam reduces the activity of increased transglutaminase when transglutaminase is overexpressed.

As used herein, the term "prevention" means any action that inhibits or delays the development of any disease due to an increase in the activity of transglutaminase by administration of a composition comprising szaparem or a pharmaceutically acceptable salt thereof. As used herein, "treatment" means any action that improves or beneficially alters all diseases caused by the increase of the activity of transglutaminase by administration of the pharmaceutical composition.

In the present invention, the disease caused by the increase of transglutaminase activity includes all diseases caused by increased activity such as overexpression of transglutaminase, but specifically includes inflammatory diseases, neurological diseases, and cancer.

The expression of transglutaminase is increased in diseases with inflammation, such as chronic inflammatory diseases such as degenerative arthritis, autoimmune myositis, arteriosclerosis, stroke, liver cirrhosis, malignant breast cancer, meningitis, inflammatory gastric ulcer And sepsis.

In the case of neurological diseases, diseases related to the 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, hypothyroidism, alcoholic dementia Alzheimer's, Huntington's disease Central nervous system diseases caused by injury and death of cells of the nervous system are representative. The main symptoms of these diseases include impaired cognitive function, language, judgment, abstraction, spatial and visual strength, and other new skills acquisition, and symptoms of personality change and emotional instability eventually lead to death. . Among them, the pharmaceutical composition of the present invention may 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 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 are aggregated by transglutaminase, but is not limited thereto. It can be used for the treatment of any disease in which transglutaminase is overexpressed in nervous tissue.

For cancer, inhibition of transglutaminase is important in terms of cancer prevention and treatment, with a marked increase in the expression of transglutaminase in cancers with cancer metastasis, chemical and radiological resistance. Specific cancers that can be prevented or treated using the pharmaceutical composition comprising the chaparem of the present invention are cancers that show an increase in transglutaminase, specifically colon 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, brain tumor, bladder cancer, ovarian cancer and gallbladder cancer, and the like.

Compositions and methods of treatment comprising the chaparem or pharmaceutically acceptable salts thereof of the present invention are cattle, horses, sheep, pigs, goats, camels that can cause diseases due to increased activity of transglutaminase as well as humans. It can also be used in mammals such as, nutrition, dogs and cats.

The pharmaceutical composition comprising the chaparem of the present invention or a pharmaceutically acceptable salt thereof may be used as a single agent or may be prepared and used as a complex including a pharmaceutical composition approved for the treatment of transglutaminase-related diseases. have.

Pharmaceutical compositions comprising stoolarem or pharmaceutically acceptable salts thereof can be prepared by filling the capsules including stoolarem without excipients or uniformly and sufficiently in contact with particulate solid carriers, liquid carriers 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, as disclosed in Remington's Pharmaceutical Science, 19 ^th Ed., 1995, Mack Publishing Company, Easton PA, et al. As can be formulated, it may be formulated into a suitable formulation known in the art.

The pharmaceutical composition comprising the chaparem or pharmaceutically acceptable salts of the present invention is applicable to any formulation comprising the chaparem or a pharmaceutically acceptable salt thereof as an active ingredient, oral or parenteral It can also be prepared. Parenteral formulations may be in the form of sprays, such as injection, application or aerosol, preferably in the form of sprays such as injectable or aerosol, and oral formulations are also preferred.

Oral dosage forms comprising the pharmaceutical composition of the invention may be formulated, for example, in tablets, troches, lozenges, water-soluble or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs. have. 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, magnesium stearate It may include a lubricating oil, such as calcium stearate, sodium stearyl fumarate or polyethylene glycol wax, and in the case of a capsule, it may further contain a liquid carrier such as fatty oil in addition to the above-mentioned materials.

In the case of parenteral administration of the pharmaceutical composition of the present invention, 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 may be formulated. To formulate injectable formulations, spararem, its pharmaceutically acceptable salts or derivatives thereof are mixed in water with stabilizers or buffers to prepare solutions or suspensions which are formulated for unit administration of ampoules or vials. For injection into the suppository, it may be formulated into a composition for rectal administration 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.

The pharmaceutical compositions of the present invention may be administered in a conventional manner via rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, nasal, inhalation, intraocular or intradermal routes. Parenteral administration means administration modes including intravenous, intramuscular, intraperitoneal, intrasternal, transdermal and intraarterial injection and infusion. Parenteral administration of the pharmaceutical composition comprising the chaparem of the present invention is admixed with a pharmaceutically acceptable carrier, i.e., a carrier which is nontoxic to the receptor and compatible with other formulation ingredients at the concentrations and dosages employed, under the desired purity. It is preferred to formulate in dosage unit form. In particular, the formulation preferably does not contain oxidizing agents and other compounds known to be harmful to the human body.

The chaparem or pharmaceutically acceptable salts thereof of the present invention may be administered in a pharmaceutical composition together 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 patient's age, weight, general health, sex and diet, time of administration, It is desirable to apply differently depending on the route of administration and the rate of release of the composition, the duration of treatment, the use of or with the specific composition, and various factors including drugs and similar factors well known in the medical arts. Suitable formulations known in the art are described in Remington's Pharmaceutical Science, 19 ^th Ed., 1995, Mack Publishing Company, Easton PA. Therefore, it is preferable to determine the effective amount of sulpareme or a pharmaceutically acceptable salt thereof suitable for the purpose of the present invention in consideration of the foregoing.

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.

According to the present invention, a composition for preventing or treating a disease related to transglutaminase, a method for inhibiting transglutaminase, and transglutamin, comprising sageparem or a pharmaceutically acceptable salt thereof as an active ingredient Provided are methods for the treatment of aminase-related diseases.

The novel method of inhibiting transglutaminase by using sageparem or a pharmaceutically acceptable salt thereof according to the present invention is a stable method for a subject having a disease in which transglutaminase is overexpressed, and is easily available without side effects. It can be applied to obtain the effect of inhibiting transglutaminase.

Example 1 Transglutaminase  Activity inhibition in in vitro  Experiment

Transglutaminase measured the binding of [1,4, ^-14 C] putrescine to succinylated casein, and observed that Supparem inhibited the response.

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). Store this solution in the Deep freezer before use. [1,4, ^-14 C] putrescine dihydrochloride was purchased from GE HEalthcare (Cat. No. CFA301) and the stock solution may have 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. Stock solutions of sparparm were prepared by dissolving sparparm in DMSO at a concentration of 10 mM and then diluted them with DMSO to each concentration.

The substrate solution was prepared by mixing 450 μl of succinylated casein solution and 50 μl of [1,4, ^-14 C] putrescine dihydrochloride solution. Each sample was prepared by mixing 96 μl of reaction buffer, 3 μl of stockplot of Scheparem and 1 μl of transglutaminase stock solution. After incubating the samples at 37 ° C. for 10 minutes, 500 μl of substrate solution β 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 Counler) and corrected with the activity of DMSO-control as a standard. The measured value is shown as the activity of transglutaminase. This experiment (assay) was repeated three times under the same conditions and the values are shown in Table 1 below.

density Assay 1 Assay 2 Assay 3 Average SD (standard deviation) 0.0μM 1.0000 1.0000 1.0000 1.0000 0.0000 0.5 μM 0.3608 0.4222 0.3822 0.3884 0.0312 2.0 μM 0.0478 0.0496 0.0484 0.0486 0.0009 10.0 μM 0.0200 0.0233 0.0184 0.0206 0.0025 50.0 μM 0.0118 0.0162 0.0133 0.0138 0.0022

In addition, the average value is shown with respect to the concentration of a slareparem. IC ₅₀ Values were calculated by normal nonlinear regression, which was determined to be 0.3870 (± 0.0175) μM.

Relative transglutaminase inhibitory activity measured according to the concentration of sparparem is shown in FIG. As shown in FIG. 1, it can be seen that the activity of transglutaminase is inhibited in concentration-dependent manner.

The method of inhibiting transglutaminase inhibitor and transglutaminase comprising a chaparem or a pharmaceutically acceptable salt thereof according to the present invention is a method of inhibiting transglutaminase such as a neurological disease or cancer. It can be easily applied to a subject having a disease to be expressed in a more stable manner without side effects, thereby obtaining an effect of inhibiting transglutaminase.

Figure 1 shows the transglutamina of szaparem, which measured that transglutaminase binds [1,4, ^-14 C] putrescine to succinylated casein, and observed that shalparem inhibited the response. This is a graph of in vitro testing of inhibitory activity.

Claims (11)

A pharmaceutical composition for treating or preventing a disease selected by Scheparem or a pharmaceutically acceptable salt thereof, caused by increased activity of transglutaminase. The pharmaceutical composition of claim 1, wherein the disease is an inflammatory disease, a cancer, or a nervous system disease. The pharmaceutical composition of claim 2, wherein the composition has an effect of inhibiting cancer metastasis. The pharmaceutical composition according to claim 2, wherein the inflammatory disease is selected from chronic inflammatory diseases such as degenerative arthritis, sepsis, autoimmune myositis. The pharmaceutical composition of claim 2, wherein the neurological disease is selected from Alzheimer's disease, Huntington's disease and Parkinson's disease. A method of inhibiting the activity of transglutaminase using a substance selected from stoolarem or a pharmaceutically acceptable salt thereof. A method of treating or preventing a disease caused by increased activity of transglutaminase, comprising administering the pharmaceutical composition of any one of claims 1 to 5. 8. The method of claim 7, wherein the disease is an inflammatory disease, a cancer or a nervous system disease. The method of claim 8, wherein the method has an effect of inhibiting cancer metastasis. The method of claim 8, wherein the inflammatory disease is selected from chronic inflammatory diseases such as degenerative arthritis, sepsis, autoimmune myositis. The method of claim 8, wherein the neurological disease is selected from Alzheimer's disease, Huntington's disease and Parkinson's disease.

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