JP2006008714A - Matrix metalloproteinase inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new medicine for inhibiting matrix metalloproteinase (MMP), and to provide an agent for inhibiting / treating diseases including the growth and metastasis of tumor, in which MMP is estimated to participate. <P>SOLUTION: The matrix metalloproteinase (MMP) inhibitor, the tumor growth and metastasis inhibitor, and the therapeutic agent for diseases in which MMP participates, each, contains astaxanthin and/or its ester as an active ingredient. The MMP inhibitor is able to be useful for treating diseases in which MMP is estimated to participate. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a matrix metalloproteinase inhibitor. More specifically, the present invention relates to a matrix metalloproteinase inhibitor containing astaxanthin and / or an ester thereof as an active ingredient, and a therapeutic agent for diseases involving matrix metalloproteinases including tumor growth and metastasis inhibitors.

  Matrix metalloproteinases (MMPs) are a superfamily of zinc endopeptidases that exhibit proteolytic activity using extracellular matrix components (eg, collagen, fibronectin, laminin, gelatin, proteoglycan, etc.) as substrates. MMP-1 (fibroblast collagenase), MMP-2 (72 kDa gelatinase), MMP-9 (92 kDa gelatinase), MMP-3 (Stromelysin 1), MMP-10 (Stromelysin 2), MMP-11 (S Tromelysin 3), MMP-7 (matrilysin), MMP-8 (neurotrophic collagenase), MMP-13 (collagenase 3), metalloelastase (MMP-12) and the like.

Properties common to these MMPs are: (1) having Zn ²⁺ at the active center and requiring calcium ion (Ca ²⁺ ) for enzyme activity; (2) secreted as a latent enzyme and active extracellularly (3) having high homology to the amino acid sequence, (4) having a resolution of various extracellular matrix components existing in the living body, and (5) a tissue metalloproteinase inhibitor (common inhibitor) ( It is known that the activity is inhibited by TIMP).

  Many of the known MMP inhibitors are peptide derivatives based on natural amino acids and are analogs to cleavage sites in the natural substrate of MMP. Among them, the substrate (Gly-Ile-Ala-Gly or Gly-Leu-Ala-Gly) near the collagen cleavage point has a high affinity for collagenase, and has a zinc affinity group at the cleavage site of this substrate. Many substrate analog matrix metalloproteinase inhibitors chemically modified in this way have been studied (for example, Patent Document 1). However, these substrate analog inhibitors are expected to have various problems because they are peptide analogs. Accordingly, pseudopeptides or peptide-like substances having a smaller peptide structure include, for example, sulfonamide compounds (for example, Patent Document 2), hydroxamic acid compounds (for example, Patent Document 3), aminobutanoic acid derivatives (for example, Patent Documents). 4), carboxylic acid derivatives (for example, Patent Document 5) and the like. On the other hand, some MMP inhibitors derived from natural products are also known (for example, Patent Documents 6 and 7).

  MMP is greatly involved in both physiological and pathological tissue degradation. For example, the skin maintains its structure and normal metabolism by various proteins such as collagen and elastin, and retains moisture. However, the degradation of these proteins by MMP causes sagging, wrinkles, and drying. In addition, diseases related to MMP include rheumatoid arthritis, osteoarthritis, osteoporosis, periodontal disease, multiple sclerosis, gingivitis, corneal epidermis and gastric ulcer, atherosclerosis, and worsening of the disease state. There are numerous diseases resulting from the destruction of connective tissue including angiogenesis and diseases such as tumor growth and metastasis. In particular, in tumor growth / metastasis, not only tumor cells infiltrate connective tissue, but also help angiogenesis and cause degradation of the vascular basement membrane. As a result, it causes metastasis.

  As a method for treating cancer, which is one of the diseases associated with angiogenesis, surgical methods, radiation therapy, chemotherapy (drug administration) and the like are generally performed. Among these, as chemotherapy, treatment methods that administer drugs that act directly on tumor cells and kill tumor cells are widely applied, and proposals for antitumor agents to be applied to such treatment methods are Many. However, since such a drug kills tumor cells and also acts on normal cells, it has a high therapeutic effect on cancer, but has a drawback of having extremely strong side effects. In addition, even if such a drug works on a cell-by-cell basis, it does not reach the deep part of the tumor mass, so it is not a fundamental solution. Furthermore, cancers have different properties depending on their organs, and it is not uncommon for the selection and administration method of antitumor agents to differ, and antitumor agents that are effective against various types of cancer are desired.

Carotenoids (carotenoids) are fat-soluble biological pigments that are widely distributed in animals, plants, and microorganisms, and have about 600 to yellow to orange to red colors. Astaxanthin, which is one type, is contained in crustaceans such as krill, shrimp and crabs, salmon and trout muscles and eggs (such as salmon roe), and body surfaces such as Thai, carp and goldfish. Astaxanthin is known not only to be provitamin A and to have a remarkable antioxidant effect, but also as a potential antitumor agent. For example, tumor formation inhibitory action of bladder cancer, oral cancer, and colon cancer (Non-patent Document 1); anti-stress action (Patent Document 8); apoptosis-inducing action (Patent Document 9); topoisomerase inhibitory action (Patent Document 10); Protein kinase inhibitory action (Patent Document 11); Skin protective action from UV irradiation (Non-Patent Document 2); Breast cancer growth inhibitory action (Non-Patent Document 3); ); Immune response enhancing action (Non-patent Document 5) has been reported. However, there is no report about MMP inhibition and tumor metastasis suppression as described above.
Special Table 2004-529874 JP 2002-284686 A JP 2003-321435 A JP 2003-212831 A JP-A-10-130217 JP-T-2004-520294 JP 2005-8539 A JP-A-9-124470 JP 2001-114673 A Japanese Patent Laid-Open No. 2001-114674 JP 2001-114683 A T. Tanaka et al., Carcinogenesis, 1995, 16 (12), pp.2957-2963 NM Lyons and NM O'Brien, J. Dermatol. Sci., 2002, 30, pp. 73-84 BP Chew et al., Anticancer Res., 1999, 19, pp.1849-1853 LM Hix et al., Cancer Lett., 2004, 211, pp. 25-37 H. Jyonouchi et al., Nutr. Cancer, 2000, 36, 1, pp.59-65

  An object of this invention is to provide the novel chemical | medical agent which inhibits a matrix metalloproteinase (MMP). It is another object of the present invention to provide an inhibitor of tumor growth and metastasis that is considered to involve MMP, and a therapeutic agent for diseases and symptoms that are considered to involve MMP.

  As a result of various studies on astaxanthin, which has been reported as an antitumor agent as described above, the present inventors have found that astaxanthin has a matrix metalloproteinase (MMP) inhibitory activity. Was completed.

  That is, the present invention provides a matrix metalloproteinase inhibitor containing astaxanthin and / or an ester thereof as an active ingredient.

  In one embodiment, the matrix metalloproteinase is involved in the degradation of epidermal and dermal constituent proteins.

  The present invention also provides a tumor growth and metastasis inhibitor comprising astaxanthin and / or an ester thereof as an active ingredient.

  The present invention further provides a therapeutic agent for diseases involving matrix metalloproteinases, which contains astaxanthin and / or an ester thereof as an active ingredient.

  According to the present invention, a novel matrix metalloproteinase (MMP) inhibitor is provided. This MMP inhibitor can be used as a therapeutic agent for conditions associated with tissue degradation and inflammation, for example, diseases such as tumor growth, rheumatoid arthritis, diabetic retinopathy and the like. In particular, according to the present invention, a tumor metastasis inhibitor is also provided. Furthermore, since the MMP inhibitor and tumor growth and metastasis inhibitor of the present invention have low toxicity, they are highly safe. In addition, the MMP inhibitor of the present invention inhibits MMPs involved in degradation of epidermal and dermal constituent proteins, that is, collagenase, elastase, and the like, thus giving skin skin, suppressing wrinkle formation, and moisturizing the skin. The effect is also obtained.

  Astaxanthin and / or its ester, which is an active ingredient of the matrix metalloproteinase (MMP) inhibitor of the present invention, has the following formula:

(Here, R ¹ and R ² are each independently a hydrogen atom or a fatty acid residue). The ester of astaxanthin is not particularly limited. For example, saturated fatty acids such as palmitic acid and stearic acid, or oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, bishomo-γ-linolenic acid, arachidonic acid, Examples thereof include monoesters or diesters of unsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid. These can be used alone or in appropriate combination. Astaxanthin has a structure having extra oxo groups and hydroxy groups at both ends of the skeleton of β-carotene, and therefore has low molecular stability unlike β-carotene. On the other hand, the ester body (for example, krill extract) in which the hydroxyl groups at both ends are esterified with unsaturated fatty acid or the like is more stable.

  Astaxanthin and / or its ester used in the present invention may be either chemically synthesized or derived from natural products. Examples of the latter natural products include red yeast containing astaxanthin and / or an ester thereof; shellfish shells such as tigliopath (red daphnia) and krill; and microalgae such as green algae. In the present invention, an extract containing astaxanthin and / or its ester produced by any method can be used as long as the characteristics of astaxanthin and / or its ester can be utilized. In general, extracts from these natural products are used, and the extract may be in the form of an extract or may be appropriately purified as necessary. In the present invention, such a crude extract or crushed powder containing astaxanthin and / or an ester thereof, or an appropriately purified or chemically synthesized product containing astaxanthin and / or its ester may be used alone or in appropriate combination. Can do. Considering the stability in the body, an ester form is preferably used.

  The MMP inhibitors of the present invention can be used for conditions associated with tissue degradation and inflammation, such as rheumatoid arthritis, osteoarthritis, osteopenia (eg osteoporosis), periodontitis, gingivitis, corneal epidermis or gastric ulcer, skin aging, and tumors May be useful for treating or preventing metastasis, invasion and proliferation of (but not limited to). The MMP inhibitors of the present invention include neuroinflammatory diseases (including diseases involving myelin degradation), such as multiple sclerosis, and angiogenesis-dependent diseases (arthritic conditions and solid tumor growth as well as psoriasis, proliferative retinopathy) , Including neovascular glaucoma, intraocular tumors, hemangiofibromas and hemangiomas).

  The tumor growth and metastasis inhibitor of the present invention and the therapeutic agent for diseases involving MMP contain astaxanthin and / or an ester thereof as an active ingredient, similar to the MMP inhibitor of the present invention described above. In particular, it is useful for suppressing metastasis of metastatic tumors such as certain types of breast cancer.

  The route of administration of the MMP inhibitor of the present invention or tumor growth and metastasis inhibitor or therapeutic agent for a disease involving MMP may be either oral or parenteral. The dosage form is appropriately selected depending on the administration route. For example, injections, infusions, powders, granules, tablets, capsules, pills, enteric solvents, troches, liquids for internal use, suspensions, emulsions, syrups, liquids for external use, poultices, nasal drops, ear drops Agents, eye drops, inhalants, ointments, lotions, suppositories, enteral nutrients and the like. This can be used alone or in combination depending on the symptoms. In these preparations, auxiliary agents usually used in the pharmaceutical preparation technical field such as excipients, binders, preservatives, oxidation stabilizers, disintegrants, lubricants, and corrigents are used as necessary. .

  The dose of the MMP inhibitor of the present invention or tumor growth and metastasis suppressor or therapeutic agent for a disease involving MMP varies depending on the purpose of administration and the situation (sex, age, body weight, etc.) of the administration subject. Usually, for adults, 0.1 mg to 2 g per day, preferably 4 mg to 500 mg per day in the case of oral administration, in terms of astaxanthin free body, while 0.01 mg to 1 g per day for parenteral administration, Preferably it can be administered at 0.1 mg to 500 mg.

  The MMP inhibitor of the present invention can be used not only as a pharmaceutical as described above, but also as a quasi-drug, cosmetic, functional food, nutritional supplement, food or drink, and the like. When used as a quasi-drug or cosmetic, it can be used together with various adjuvants commonly used in the technical field such as quasi-drug or cosmetic, if necessary. Or, when used as a functional food, nutritional supplement, or food and drink, it is usually used in foods such as sweeteners, spices, seasonings, preservatives, preservatives, bactericides, and antioxidants as necessary. You may use with the additive used. Further, it may be used in a desired shape such as solution, suspension, syrup, granule, cream, paste, jelly, etc., or as necessary. The ratio contained in these is not specifically limited, It can select suitably according to a use purpose, a usage form, and a usage-amount.

(Preparation Example 1: Preparation of astaxanthin monoester)
Astaxanthin monoester was prepared as follows. The Haematococcus pulvialis strain K0084 was cultured at 25 ° C. under irradiating gas containing 3% CO ₂ under light irradiation conditions, under nutrient stress (nitrogen source deficiency), and cysted. The cysted cells were crushed by means commonly used by those skilled in the art, and the oily fraction was extracted with ethanol. The extract contained lipids such as triglycerides in addition to astaxanthins. The extract was subjected to column chromatography using a synthetic resin adsorbent to obtain a purified product containing an astaxanthin monoester. This purified product was analyzed by HPLC, and it was confirmed that this purified astaxanthin monoester contained a monoester having a molecular weight of 858 as a main component, did not contain free and diester forms of astaxanthin, and contained a slight amount of diglyceride. .

(Example 1: Effect on various enzymes)
The astaxanthin monoester obtained in Preparation Example 1 was examined for effects on the activities of various enzymes. Astaxanthin monoester was dissolved in dimethyl sulfoxide (DMSO), added to the assay system for each enzyme shown in Tables 1 and 2 below, and incubated at 250 μM.

  Table 3 summarizes the inhibition rate of each enzyme activity by 250 μM astaxanthin monoester.

  Astaxanthin monoester showed a very high inhibition rate with respect to the matrix metalloproteinase (MMP) used for the test. From this, it was found that astaxanthin monoester can inhibit the activity on the entire MMP group.

(Reference Example 1: Measurement of 50% lethal concentration and inhibitory concentration against HUVEC)
Human umbilical vein endothelial cells (HUVEC) (ATCC CRL-1730) were obtained from American Type Culture Collection and endothelial cell growth containing 10% fetal bovine serum supplemented with 1% Antibiotic-Antimycotic (GIBCO BRL, USA) Medium (CELL APPLICATIONS, USA)) was pre-cultured at 37 ° C. in a 5% CO ₂ atmosphere.

  Matrigel matrix (BD Biosciences, USA) was thawed and kept on ice at 4 ° C., and 50 μL of matrix was transferred to each well of a 96-well tissue culture plate. The plate was incubated at 37 ° C. for at least 1 hour to solidify the matrix solution.

  On the other hand, the astaxanthin monoester obtained in Preparation Example 1 above was dissolved in dimethyl sulfoxide (DMSO) and then diluted with distilled water to obtain 25000, 2500, 250, 25, and 40% in 40 (v / v)% DMSO. A stock test solution containing 2.5 μM astaxanthin monoester was prepared.

100 μL of HUVEC suspension (approximately 2.5 × 10 ³ cells / well) was placed in a 96-well Matrigel plate at 37 ° C. in a 5% CO ₂ atmosphere. After 24 hours, 100 μL of growth medium and 2 μL of each of the above stock test solutions or vehicles (40 (v / v)% DMSO) were added to each of the two wells and incubated for a further 72 hours. The final concentrations of DMSO and astaxanthin monoester were 250, 25, 2.5, 0.25, and 0.025 μM.

  At the end of the incubation, 20 μL of 90% alamarBlue reagent was added to each well and incubated for an additional 6 hours. Next, the fluorescence intensity of each well was measured at an excitation wavelength of 530 nm and an emission wavelength of 590 nm using a Spectrafluor Plus plate reader, and the number of viable cells was counted. This is based on the ability of viable cells to change alamarBlue from a non-fluorescent oxidized form (blue) to a fluorescent reduced form (red). The 50% lethal concentration is calculated as the concentration that gives 50% of the number of cells at the start of the experiment, and the 50% inhibitory concentration is 50% of the number of cells in the case of the control vehicle. Concentration was calculated.

As a result, the 50% lethal concentration (LC ₅₀ ) of astaxanthin monoester with respect to HUVEC was 250 μM (maximum dissolution concentration in DMSO), indicating that the toxicity was low. Furthermore, the 50% inhibitory concentration (IC ₅₀ ) was 51 μM, and it was also found that the cell growth inhibitory effect was obtained at a relatively low concentration.

  According to the present invention, a new MMP inhibitor is provided. This MMP inhibitor is a condition involving tissue degradation and inflammation, such as rheumatoid arthritis, osteoarthritis, osteopenia (eg osteoporosis), periodontitis, gingivitis, corneal epidermis or gastric ulcer, skin aging, and tumor metastasis Can be useful for treating or preventing, but not limited to, invasion and proliferation. In addition, neuroinflammatory diseases (including diseases involving myelin degradation), such as multiple sclerosis, and angiogenesis-dependent diseases (arthritic symptoms and solid tumor growth and psoriasis, proliferative retinopathy, angiogenic glaucoma, eye It may also be useful for the treatment of internal tumors, including angiofibromas and hemangiomas). According to the present invention, a novel tumor growth and metastasis inhibitor is also provided, which is useful for suppressing the growth and metastasis of metastatic tumors such as certain breast cancers. In addition, if the MMP inhibitor of the present invention is used, it is useful as a cosmetic because it provides skin elasticity, suppresses wrinkle formation, and moisturizes the skin.

Claims (4)

  A matrix metalloproteinase inhibitor comprising astaxanthin and / or an ester thereof as an active ingredient.   The inhibitor according to claim 1, wherein the matrix metalloproteinase is involved in degradation of epidermal and dermal constituent proteins.   A tumor growth and metastasis inhibitor comprising astaxanthin and / or an ester thereof as an active ingredient.   A therapeutic agent for diseases involving matrix metalloproteinases, comprising astaxanthin and / or an ester thereof as an active ingredient.