JP2006008713A - Vascular endothelial cell growth inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new medicine for inhibiting vascular endothelial cell growth. <P>SOLUTION: This vascular endothelial cell growth inhibitor contains astaxanthin and/or its ester as an active ingredient. The vascular endothelial cell growth inhibitor is used as a therapeutic agent for various diseases originated from neovascularization, such as tumor growth, rheumatoid arthritis, and diabetic retinopathy, and is especially able to be useful for cancer cell growth inhibition, solid tumor contraction, and the like. In addition, the vascular endothelial cell growth inhibitor is low toxic and is hence highly safe. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a vascular endothelial cell growth inhibitor. More specifically, the present invention relates to a vascular endothelial cell growth inhibitor containing astaxanthin and / or an ester thereof as an active ingredient.

  Vascular endothelial cells are a single layer of cell that forms the intima by lining the lumen of a blood vessel. Vascular endothelial cells mediate the transport of necessary components such as nutrients in the blood to the tissue and prevent an unnecessary large amount of components from passing; the blood smoothly circulates without coagulation; Has the effect of preventing bleeding when a blood vessel is disconnected; the effect of regulating blood vessel tension; the effect of controlling inflammation by producing factors involved in inflammation; the effect of controlling the pathology of blood vessels, etc. Yes.

  Vascular endothelial cells are also important factors in angiogenesis. Angiogenesis occurs in the order of digestion of the basement membrane by proteases produced by vascular endothelial cells, migration / proliferation of vascular endothelial cells, and then lumen formation of vascular endothelial cells. Angiogenesis is thought to be caused by various growth factors, cytokines, arachidonic acid metabolites, monobutyrin and the like. Known growth factors that cause angiogenesis include fibroblast growth factor, tumor growth factor, vascular endothelial growth factor (VEGF), and vascular permeability factor. Among them, VEGF is considered to be the most important angiogenic factor because the receptor exists only in the vascular endothelium and acts selectively on the vascular endothelium.

  Angiogenesis is known to be closely associated with various diseases. Diseases involving angiogenesis include growth and metastasis of malignant tumors, diabetic retinopathy, neovascular glaucoma, age-related macular degeneration, inflammatory skin diseases, rheumatoid arthritis, osteoarthritis and the like.

  For example, when a malignant tumor grows, the tumor cell itself induces angiogenesis by an angiogenic factor in order to obtain nutrients and oxygen necessary for the growth of the tumor cell. When a malignant tumor metastasizes to another organ or site, angiogenesis is induced, and tumor cells move along the bloodstream. In diabetic retinopathy, diabetic blood clogs capillaries, causing bleeding and edema in the retina, which becomes chronic and causes oxygen and nutrient deficiencies in the retina. A new blood vessel is generated in this, and a fibrous tissue is formed around it. This fiber tissue pulls up the retina (retinal detachment), tears the blood vessels of the retina, causes bleeding (vitreous hemorrhage), and eventually leads to severe visual impairment and blindness.

  As described above, angiogenesis caused by proliferation of vascular endothelial cells is deeply involved in the onset and progression of various diseases. Therefore, for the purpose of treating these diseases, the search for substances that inhibit angiogenesis and vascular endothelial cell proliferation. A lot has been done so far. Examples of substances and drugs having an action of inhibiting angiogenesis include sulfated polysaccharides (Patent Document 1), ascorbic ether and related compounds (Patent Document 2), thiazole derivatives (Patent Document 3), cartilage cartilage extract (chondroitin) And mucopolysaccharide) (Patent Document 4). However, the substances proposed as substances exhibiting the action of inhibiting angiogenesis have not been able to exert practically satisfactory effects. For this reason, it is required to develop a highly safe substance that more strongly inhibits the proliferation of vascular endothelial cells. Various inhibitors targeting the above-mentioned VEGF and VEGF receptors have been developed for the purpose of inhibiting vascular endothelial cell proliferation (for example, Patent Documents 5 to 7).

  As a method for treating cancer, which is one of diseases associated with vascular endothelial cell proliferation or 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 cancers 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); Tumor formation inhibitory action by a kind of astaxanthin derivative (Non-Patent Document 4) ); Immune response enhancing action (Non-patent Document 5) has been reported. However, there is no report about the inhibition of vascular endothelial cell proliferation or angiogenesis as described above.
JP-A-63-119500 Japanese Patent Laid-Open No. 58-131978 Japanese Examined Patent Publication No. 6-62413 JP-A-10-147534 JP-A-10-324625 JP 2002-220340 A JP 2005-506288 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 the present invention is to provide a new drug that inhibits vascular endothelial cell proliferation in order to suppress angiogenesis.

  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 vascular endothelial cell growth inhibitory activity and completed the present invention. did.

  That is, the present invention provides a vascular endothelial cell growth inhibitor containing astaxanthin and / or an ester thereof as an active ingredient.

  The present invention also provides a therapeutic agent for diseases associated with vascular endothelial cell proliferation, comprising astaxanthin and / or an ester thereof as an active ingredient.

  According to the present invention, a novel vascular endothelial cell proliferation inhibitor is provided. This vascular endothelial cell growth inhibitor can be used as a therapeutic agent for various diseases derived from angiogenesis such as tumor growth, rheumatoid arthritis, diabetic retinopathy and the like. The vascular endothelial cell growth inhibitor of the present invention may be particularly useful for suppressing the growth of cancer cells, shrinking solid tumors, and the like. Furthermore, since the vascular endothelial cell growth inhibitor of the present invention has low toxicity, it is highly safe.

  Astaxanthin and / or its ester, which is an active ingredient of the vascular endothelial cell growth 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 vascular endothelial cell growth inhibitor of the present invention is useful for the treatment of cancer derived from angiogenesis and rheumatoid arthritis, and can be used as a therapeutic agent for diseases associated with vascular endothelial cell proliferation. Diseases derived from angiogenesis include malignant tumor growth and metastasis, diabetic retinopathy, neovascular glaucoma, age-related macular degeneration, inflammatory skin diseases, rheumatoid arthritis, osteoarthritis, and the like.

  The administration route of the vascular endothelial cell growth inhibitor or the therapeutic agent for diseases associated with vascular endothelial cell proliferation of the present invention may be either oral administration or parenteral administration. 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 vascular endothelial cell growth inhibitor or the therapeutic agent for diseases associated with vascular endothelial cell growth of the present invention varies depending on the purpose of administration and the situation (sex, age, 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 vascular endothelial cell growth 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 and 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: Measurement of growth inhibition on vascular endothelial cells)
Changes in the number of human umbilical vein endothelial cells (HUVEC) were measured based on the ability of viable cells to change alamarBlue from a non-fluorescent oxidized form (blue) to a fluorescent reduced form (red).

  First, 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% (v / v)% DMSO. A stock test solution containing 2.5 μM astaxanthin monoester was prepared.

On the other hand, HUVEC (ATCC CRL-1730) was obtained from American Type Culture Collection, and Endothelial Cell Growth Medium (CELL APPLICATIONS, containing 10% fetal bovine serum supplemented with 1% Antibiotic-Antimycotic (GIBCO BRL, USA). USA)), and precultured at 37 ° C. in a 5% CO ₂ atmosphere.

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. A mitomycin (Kyowa Hakko Kogyo Co., Ltd.), a standard reference material, was also tested at the same time.

  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. The percentage of cell proliferation was calculated based on the following formula.

  The relationship between the concentrations of astaxanthin monoester and mitomycin and the calculated growth rate is shown in FIG. Compared to the control, astaxanthin monoester showed significant growth inhibition at a concentration of 250 μM. In addition, the ratio of 50% or less with respect to the control was considered significant. In the control mitomycin, significant growth inhibition was less than 10 μM.

Next, nonlinear regression analysis was performed using GraphPad Prism (GraphPad Software, USA) to calculate 50% inhibitory concentration (IC ₅₀ ), total growth inhibitory concentration (TGI), and 50% lethal concentration (LC ₅₀ ). IC ₅₀ is a concentration at which the increase in the number of cells from the start of the test is 50% of the number of cells at the end of the test in the control. TGI is a concentration at which the number of cells at the end of the test is the same as the number of cells at the start of the test. LC ₅₀ is a concentration at which the number of cells at the end of the test is half that at the start of the test. The results are shown in Table 1.

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

(Reference Example 1: Angiogenesis inhibitory activity)
HUVECs differentiate and form capillary-like structures in Matrigel in the presence of endothelial cell proliferation supplements. Therefore, whether or not the astaxanthin monoester confirmed to have a vascular endothelial cell growth inhibitory action in Example 1 actually inhibits angiogenesis was evaluated by observing the non-construction of the vascular network continuum. .

  First, the Matrigel matrix (BD Biosciences, USA) was thawed and kept on ice at 4 ° C., and 50 μL of the 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.

Next, 200 μL of HUVEC suspension (approximately 1.5 × 10 ⁴ cells / well) was placed in a 96-well Matrigel plate. Next, 2 μL of each stock test solution or vehicle (40 (v / v)% DMSO) prepared in the same manner as in Example 1 above was added to each of the two wells, and the mixture was added at 37 ° C. under 5% CO ₂ atmosphere. Incubate for hours. The final concentration of DMSO was 0.4 (v / v)%, and the final concentration of astaxanthin monoester was 250, 25, 2.5, 0.25, and 0.025 μM. The standard reference material suramin (Sigma, USA) was also tested at the same time.

  At the end of the incubation, the endothelial cell tube morphology of individual wells was evaluated by micrographs. The results are shown in FIG. As can be seen from FIG. 2, in the presence of astaxanthin monoester, the formation of a continuous network between cell bodies formed in the control vehicle was insufficient.

  Next, the length of the formed blood vessel was measured for each micrograph. A tube having a length of 30% or more shorter than that of the control was determined to have a significant inhibitory effect. The results are shown in FIG. As a result, astaxanthin monoester significantly inhibited angiogenesis compared to the vehicle-treated control group, and the minimum inhibitory concentration (MIC) was 250 μM. For the suramin, a standard reference material tested at the same time, the MIC was 10 μM.

  According to the present invention, a novel vascular endothelial cell proliferation inhibitor is provided. This vascular endothelial cell growth inhibitor can be used as a therapeutic agent for various diseases derived from angiogenesis such as tumor growth, rheumatoid arthritis, diabetic retinopathy and the like. The vascular endothelial cell growth inhibitor of the present invention may be particularly useful for suppressing the growth of cancer cells, shrinking solid tumors, and the like. Furthermore, since the vascular endothelial cell growth inhibitor of the present invention has low toxicity, it is highly safe.

It is a graph which shows the relationship between the density | concentration of an astaxanthin monoester (□) and mitomycin (*), and the growth rate of HUVEC. It is a microscope picture (* 40) about the blood vessel formation by HUVEC on Matrigel in the presence of astaxanthin monoester or suramin. It is a graph which shows the inhibition rate with respect to the angiogenesis of HUVEC by various concentrations of astaxanthin monoester (A) or suramin (B).

Claims (2)

  A vascular endothelial cell growth inhibitor comprising astaxanthin and / or an ester thereof as an active ingredient.   A therapeutic agent for diseases associated with proliferation of vascular endothelial cells, comprising astaxanthin and / or an ester thereof as an active ingredient.

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