JP2012041283A - Neovascularization inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a neovascularization inhibitor which acts effectively, and is relatively inexpensive and novel.SOLUTION: Provided is a neovascularization inhibitor, a main active constituent of which is at least either one of 5,8,11-eicosatrienoic acid (n-9) and 11,14,17-eicosatrienoic acid (n-3). The inhibitor is available as a liquid medicine, an emulsion, an ointment, a lotion agent or a fomentation. It may be used also as an eye drop, an eye injection, and a cosmetic.

Description

  The present invention relates to an angiogenesis inhibitor that suppresses the formation of blood vessels in the human body and can be used as a therapeutic agent for various diseases caused by angiogenesis.

  Human angiogenesis is a physiological phenomenon, and occurs in limited areas such as embryonic angiogenesis and tissue organization, luteinization, follicle formation, and endometrial formation in the human body. Furthermore, it is found in repair processes such as wounds and inflammation.

  On the other hand, angiogenesis may be associated with a pathological situation. The pathological conditions that cause rapid proliferation and expansion of capillaries, causing damage and other adverse effects on tissues include trachoma, herpes corneal infection, surgical invasion and inflammation in corneal transplantation, and chemicals such as alkali Examples include corneal disorders such as corneal damage, diabetic retinopathy, glaucoma, and eye tumors. Especially in the cornea, corneal neovascularization is a beneficial reaction for the living body in terms of promoting the healing of corneal injury, but the newly vascularized blood vessel may continue to proliferate even after its role is over. When blood vessels proliferate, the transparency of the cornea is not maintained, and there is a risk of causing corneal damage that causes visual impairment and the like. In addition, angiogenesis is indispensable for cancer growth and metastasis because cancer is rapidly metabolized and requires a large amount of nutrition.

  Other examples of diseases caused by angiogenesis include psoriasis and pyogenic granulomas in the dermatology area, and hemangiomas in the pediatric area. In the surgical area, there are hypertrophic scars and granulation. Examples of internal medicine include rheumatoid arthritis and edema sclerosis, and atherosclerosis, which is a heart disease.

Thus, development of compounds useful as therapeutic and preventive drugs for diseases associated with abnormal growth of angiogenesis has been promoted, and some are disclosed in Patent Documents 1 to 3. Furthermore, as an angiogenesis inhibitor, anti-vascular endothelial growth factor (VEGF: Vascular Endothelial)
Growth Factor) antibody, partially mutated VEGF that has lost its signal transduction ability, solubilized VEGF receptor, microbial metabolite fumagillin analog, tetracycline antibiotics that inhibit collagenase activity, heparin-binding angiogenic factor receptor Drugs such as D-gluco-galactan sulfate derived from microorganisms that have a binding-inhibiting action are known. In addition, polysulfur oxides such as suramin, carboxymethyl chitin sulfate, polysulfated pentosan, and interferon are also known.

JP 2002-201138 A Special table 2003-238441 gazette JP 2010-90036 A

  However, these conventionally known angiogenesis inhibitors have side effects or have problems with stability. Further, there is a problem that anti-VEGF antibodies and the like are very expensive.

  The present invention has been made in view of the above problems of the background art, and an object thereof is to provide a relatively inexpensive angiogenesis inhibitor that acts effectively and has no side effects.

  In order to solve the above problems, the present inventors have studied various unsaturated fatty acids, and in particular, unsaturated fatty acids such as 5,8,11-cis-eicosatrienoic acid (mead acid) cause angiogenesis. It has been found to suppress.

  The present invention relates to an angiogenesis inhibitor comprising at least one of 5,8,11-eicosatrienoic acid (n-9) and 11,14,17-eicosatrienoic acid (n-3) as a main active ingredient. It is.

  Furthermore, it suppresses the formation of capillaries in cancer tissue, and suppresses the formation of capillaries in the cornea of the eye or in the retina. Furthermore, it suppresses the formation of capillaries of skin tissue on the body surface, and suppresses the formation of capillaries of muscle tissue in the body.

  The pharmaceutical form is a solution, emulsion, ointment, lotion, or poultice. In addition, the dosage form may be an encapsulated solution, an ampoule sealed for injection, a bottle filled for drinking, or an eye drop or eye drop. Furthermore, it can also be used as a cosmetic having an anti-angiogenic function.

  The angiogenesis inhibitor of the present invention can be easily taken into the body, has no side effects, and can effectively inhibit angiogenesis.

Cultured with human umbilical vein endothelial cells (HUVECs) and fibroblasts mixed with mead acid at various concentrations with or without vascular endothelial growth factor (VEGF-A) It is a graph which shows Control as 1 about the blood vessel area of the picked-up image of what was culture | cultivated similarly without adding anything (Control). Same as that without adding anything to mead acid culture with human umbilical vein endothelial cells (HUVECs) and fibroblasts with or without VEGF-A. It is a graph which shows Control as 1 about the blood vessel length of what was cultured (Control). Same as that without adding anything to the mixed culture system of human umbilical vein vascular endothelial cells (HUVECs) and fibroblasts, with or without VEGF-A and mead acid added at each concentration It is a graph which shows Control as 1 about the blood vessel branch number of what was cultured in (Control). It is a photograph which shows what was cultured without adding anything to the mixed culture system of human umbilical vein endothelial cells (HUVECs) and fibroblasts. It is a photograph which shows what was cultured with the culture solution which added mead acid (10 < ^-5 > mol / L) to the mixed culture system of human umbilical vein endothelial cells (HUVECs) and a fibroblast. It is a photograph which shows what cultured by adding VEGF-A which does not contain mead acid to the mixed culture system of human umbilical vein endothelial cells (HUVECs) and fibroblasts. It is a photograph which shows what culture | cultivated by adding mead acid (10 < ^-7 > mol / L) and VEGF-A to the mixed culture system of human umbilical vein endothelial cells (HUVECs) and a fibroblast. It is a photograph which shows what culture | cultivated by adding mead acid (10 < ^-6 > mol / L) and VEGF-A to the mixed culture system of a human umbilical vein endothelial cell (HUVECs) and a fibroblast. It is a photograph which shows what culture | cultivated by adding mead acid (10 < ^-5 > mol / L) and VEGF-A to the mixed culture system of a human umbilical vein endothelial cell (HUVECs) and a fibroblast. A mixture of human umbilical vein endothelial cells (HUVECs) and fibroblasts cultured with dihomo-γ-linolenic acid (DGLA) added at various concentrations, with or without VEGF-A It is a graph which shows Control as 1 about the blood vessel area of the picked-up image of what was culture | cultivated similarly without adding anything (Control). Similar to the case of cultivated human umbilical vein endothelial cells (HUVECs) and fibroblasts with or without VEGF-A and DGLA added at each concentration. It is a graph which shows Control as 1 about the blood vessel length of what was cultured (Control). Similar to the case of cultivated human umbilical vein endothelial cells (HUVECs) and fibroblasts with or without VEGF-A and DGLA added at each concentration. It is a graph which shows Control as 1 about the blood vessel branch number of what was cultured (Control). It is a photograph which shows what was cultured without adding anything to the mixed culture system of human umbilical vein endothelial cells (HUVECs) and fibroblasts. It is a photograph which shows what culture | cultivated by adding DGLA ( ^10-5 mole / L) to the mixed culture system of human umbilical vein endothelial cells (HUVECs) and a fibroblast. It is a photograph which shows what cultured by adding VEGF-A which does not contain DGLA to the mixed culture system of a human umbilical vein endothelial cell (HUVECs) and a fibroblast. It is a photograph which shows what DGLA ( ^10-7 mole / L) and VEGF-A were added and cultured to the mixed culture system of human umbilical vein endothelial cells (HUVECs) and fibroblasts. It is a photograph which shows what DGLA ( ^10-6 mole / L) and VEGF-A were added and cultured to the mixed culture system of human umbilical vein endothelial cells (HUVECs) and fibroblasts. It is a photograph which shows what DGLA (10 < ^-5 > mol / L) and VEGF-A were added and cultured to the mixed culture system of human umbilical vein endothelial cells (HUVECs) and a fibroblast. In a mixed culture system of human umbilical vein endothelial cells (HUVECs) and fibroblasts, VEGF-A was added and cultured in the same manner without adding anything (Control), and suramin was added. It is a graph which shows Control as 1 about the blood vessel area of the picked-up image of what was cultured by adding mead acid. In a mixed culture system of human umbilical vein endothelial cells (HUVECs) and fibroblasts, VEGF-A was added and cultured in the same manner without adding anything (Control), and suramin was added. It is a graph which shows Control as 1 about the blood vessel length of what was added and mead acid was cultured. In a mixed culture system of human umbilical vein endothelial cells (HUVECs) and fibroblasts, VEGF-A was added and cultured in the same manner without adding anything (Control), and suramin was added. It is a graph which shows Control as 1 about the blood vessel branch number of what was added and mead acid cultured. 11, 14, 17-eicosatriene at each concentration with or without the addition of vascular endothelial growth factor (VEGF-A) to a mixed culture system of human umbilical vein vascular endothelial cells (HUVECs) and fibroblasts It is a graph which shows Control as 1 about the blood vessel area of the imaging | photography image of what was culture | cultivated by adding an acid (n-3), and what was culture | cultivated similarly without adding anything (Control). 11, 14, 17-eicosatrienoic acid (n-3) is added at each concentration with or without VEGF-A added to a mixed culture system of human umbilical vein endothelial cells (HUVECs) and fibroblasts. It is a graph which shows Control as 1 about the blood vessel length of what was cultured in the same way, and what was cultured similarly without adding anything (Control). 11, 14, 17-eicosatrienoic acid (n-3) at each concentration with or without VEGF-A added to a mixed culture system of human umbilical vein endothelial cells (HUVECs) and fibroblasts It is a graph which shows Control as 1 about the blood vessel branch number of what was culture | cultivated in addition and what was culture | cultivated similarly without adding anything (Control).

  Hereinafter, embodiments of the present invention will be described in detail. 5,8,11-cis-eicosatrienoic acid (mead acid), which is an active ingredient of the present invention, is an omega-9 (or n-9) unsaturated fatty acid. An omega-9 unsaturated fatty acid has a double bond closest to the methyl end of the fatty acid between the 9th and 10th carbons, counting from the methyl group, and has two or more double bonds. It means what you have. The angiogenesis inhibiting function preferably has 18 to 22 carbon atoms. Since all naturally occurring omega-9 unsaturated fatty acids are cis type, it is preferable to use mead acid which is a cis type omega-9 unsaturated fatty acid in the present invention. In addition, it was found that the omega-3 polyunsaturated fatty acid 11,14,17-eicosatrienoic acid (n-3) also has the same function.

  Further, the mead acid and 11,14,17-eicosatrienoic acid (n-3) (hereinafter referred to as mead acid) of the present invention can be used in the form of free fatty acid, but can be acceptable as a drug. Salts such as sodium, potassium, lithium or other alkali metal salts, zinc salts, calcium salts, magnesium salts such as mono-, di-, triglycerides, esters of lower alcohols, It may be used in various forms such as phospholipid, glycolipid, amide and the like, and ethyl ester and triglyceride are particularly preferable.

  Any source may be used for the medic acid used in the present invention. It may be produced by microorganisms capable of producing mead acid, animal tissues deficient in essential fatty acid deficiency, cultured animal cells deficient in essential fatty acid deficiency, synthesized chemically or enzymatically Or natural products such as those extracted, separated and purified from animal cartilage. Specifically, microorganisms capable of producing mead acid and the like are microorganisms having Δ5 desaturase activity and Δ6 desaturase activity and having reduced or lacked Δ12 desaturase activity, for example, Mortierella alpina SAM1861 (FERM BP-3590) can be used.

  Extraction / separation / purification of free medeic acid or the like from these microorganisms is first performed on the fats and oils obtained from the cells by organic solvent extraction or supercritical carbon dioxide extraction treatment with, for example, n-hexane. Hydrolysis and esterification operations are performed to obtain a free fatty acid mixture or a fatty acid ester mixture. Thereafter, the free fatty acid or fatty acid ester of 5,8,11-cis-eicosatrienoic acid, which is the target omega-9 unsaturated fatty acid, is obtained by urea fractionation, liquid-liquid partition chromatography, column chromatography, and the like. Can be obtained with a purity of 80% or more.

  In addition, the mead acid and the like which are the active ingredients of the present invention are not necessarily limited to high-purity purified products, and oils and fats containing mead acid and the like (in these fats and oils triglycerides such as mead acid, diglycerides, monoglycerides, phosphorus Lipids, glycolipids, free mead acid, and the like), and free fatty acid mixtures or fatty acid ester mixtures containing mead acid and the like can be used. As described above, fats and oils containing mead acid and the like can destroy cells from cultured cells of microorganisms capable of producing mead acid and the like. Moreover, the free fatty acid mixture or fatty acid ester mixture containing mead acid etc. can be obtained by performing hydrolysis and esterification operation to this fats and oils.

  When the mead acid or the like of the present invention is used as a pharmaceutical product, the dosage form may be any dosage form such as oral administration or parenteral administration. For example, there are injection liquids, infusions, eye drops, and eye drops as liquids to be injected into the desired affected area. When used on the skin, it is used as a suspension, emulsion, ointment, lotion, or poultice. When taken internally, it can be used as a powder, granule, tablet, capsule, intestinal solvent, troche, liquid for internal use, syrup and the like, and these are used alone or in combination depending on the symptoms. Further, it can be used in cosmetics.

  These various preparations are prepared by using known adjuvants that can be usually used in the pharmaceutical preparation technical field such as excipients, binders, disintegrants, lubricants, and corrigents according to the purpose according to conventional methods. Can be The dose varies depending on the purpose of administration and the situation of the administration subject (gender, age, body weight, etc.), but is usually 1 to 3000 mg per day, preferably 1 to 2000 mg when administered orally to an adult. In the case of parenteral administration, the dosage can be adjusted appropriately within the range of 0.1 to 2000 mg, preferably 0.1 to 1000 mg per day. It is known that mead acid and the like, which are active ingredients of the present invention, are always synthesized in a very small amount even in vivo, and are biosynthesized to the same extent as other polyunsaturated fatty acids when essential fatty acids are deficient. ing. In addition, in an experiment in which 2 g / day / Kg was continuously administered (oral administration) for 2 weeks to 7-week-old IRC male mice, there was a result that no abnormal symptom was observed, which is excellent in terms of safety. ing.

  When the mead acid of the present invention is used in the form of a food or drink, it may be in the form of the above-mentioned preparation, but essentially contains the required amount of medic acid or the like as a raw material for food or drink, particularly the mead acid of the present invention. In addition to raw materials for foods and beverages that are not processed, they can be processed and produced by general production methods. Although the blending amount varies depending on the dosage form and the morphological properties of the food, it is generally 0.001 to 50% by mass with respect to the total amount of the food, but is not particularly limited.

  In particular, for intake as health foods and functional foods, for example, proteins (proteins such as milk proteins with high amino acid balance, soy protein, and egg albumin are most widely used as protein sources. Products, egg white oligopeptides, soybean hydrolysates, etc., as well as mixtures of amino acids alone), sugars, fats, trace elements, vitamins, emulsifiers, fragrances, etc., the medic acid of the present invention was incorporated Processing forms such as natural liquid foods, semi-digested nutritional foods and component nutritional foods, and drinks can be mentioned. Moreover, under the management of a dietitian based on a doctor's meal note, the fatty acid of the present invention can be added to any food during cooking in a hospital meal and given to the patient in the form of a functional food adjusted on the spot.

  The form of food and drink includes solid or liquid foods or luxury products such as bread, noodles, rice, confectionery (biscuits, cakes, candy, chocolate, Japanese confectionery), agricultural products such as tofu and processed products thereof, and sake. , Medicinal liquor, mirin, vinegar, soy sauce, miso, dressing, and other fermented foods, yoghurt, ham, bacon, sausage, mayonnaise and other livestock foods, kamaboko, fried tempura, marinated foods such as hampen, fruit juice drinks, soft drinks And beverages such as sports drinks, alcoholic drinks, and tea.

  The angiogenesis inhibitor of the present invention is not limited to the above embodiment, and may be an unsaturated fatty acid such as mead acid that can be easily taken into the body, has no side effects, and can effectively inhibit angiogenesis. It is sufficient if the molecular arrangement is similar. The same can be said for 11,14,17-eicosatrienoic acid (n-3) as for the above-mentioned mede acid.

The experimental results relating to the anti-angiogenic effect of mead acid of the present invention are shown below. In this experiment, an angiogenesis measurement kit (Kurashiki Boseki Co., Ltd.) was used. This kit is a mixed culture system of human umbilical vein vascular endothelial cells (HUVECs) and fibroblasts, and nothing is added to this as control, and vascular endothelial growth factor: VEGF-A (10 ng / ml) is added. These were added and mead acid-free (10 ^-5 mole / L) and cultured for 10 days. Three kinds of mede acids (10 ^-7 mole / L), (10 ^-6 mole / L), (10 ^-5 mole / L) are added with mead acid containing VEGF-A (10 ng / ml). It is. The number of samples is 4 each.

  After completion of the cell culture, the cell group was fixed with 70% ethanol. The primary antibody (mouse anti-human CD-31) and secondary antibody (goat anti-mouse IgG alkaline phosphatase binding) were each immunoreacted at 37 ° C. for 1 hour.

After completion of immune reaction, 5-buromo-4-chloro-3-indolyl
Stained with phosphate / nitro-blue-tetrazolium solution. After staining, a digital camera was attached to the microscope and images were taken. The obtained image was subjected to image analysis with angiogenesis quantification software Ver. 2 (Kurashiki Boseki Co., Ltd.), and the stained site was quantified.

  The quantitative items are an area, a length, and the number of branches. As a control for mead acid, Dihomo-γ-linolenic acid (DGLA), 20: 3 (n-6, with the same carbon number and the same double bond number (only the position of the double bond is different) ) It was used.

The results are shown in FIGS. As shown in FIGS. 1 to 3, cells cultured in a culture solution in which Mead acid (10 ⁻⁵ mole / L) is added to Control have no difference in neovascularization. No significant difference was observed in the micrographs showing the blood vessel images of the above, and angiogenesis was slightly suppressed in the case of adding mead acid. On the other hand, when the vascular endothelial growth factor: VEGF-A (10 ng / ml) was added without mead acid, the blood vessel area, the length of the blood vessel, and the number of branches of the blood vessel were compared with Control. A value of 2 to 4 times is shown by angiogenesis. Furthermore, in the culture solution containing vascular endothelial growth factor, the addition of medic acid suppresses the increase in the vascular area, the length of the vasculature, and the number of branch of the vasculature compared to the one without added mead acid. It can be seen that the higher the concentration of mead acid, the more the growth of new blood vessels is suppressed. Even when the microphotographs of FIGS. 6 to 9 are observed, the number of blood vessels that appear to be black and linear is suppressed as the concentration of mead acid is higher, and there is a clear difference.

  Therefore, in the mixed culture system of human umbilical vein vascular endothelial cells and fibroblasts, angiogenesis is activated by adding vascular endothelial growth factor, but when mead acid is added to this, vascularity depends on its concentration. It became clear that the newborn was suppressed.

  In contrast, with dihomo-γ-linolenic acid (DGLA), as shown in FIGS. 10 to 18, dihomo-γ-instead of mede acid under the same conditions as described above. In the culture test in which linolenic acid was added, the values of the blood vessel area, the length of the blood vessel, and the number of branches of the blood vessel were increased even when only dihomo-γ-linolenic acid was added. Furthermore, as shown in FIGS. 10 to 12, no inhibitory effect was observed against vascular endothelial cell growth factor. This is clear from the fact that the number of blood vessels that appear to be black and linear is not suppressed even in the micrographs shown in FIGS.

Next, an experimental example in which the anti-angiogenic function of mead acid is compared with suramin, which is an existing angiogenesis inhibitor, is shown. As shown in FIGS. 19 to 21, vascular endothelial cell growth factor: VEGF-A (10 ng / ml) is added to a mixed culture system of human umbilical vein vascular endothelial cells (HUVECs) and fibroblasts, and nothing is added. Control and suramin (2.5 × 10 ^-5 mole / L) added, mead acid not added, mead acid (10 ^-7 mole / L), (10 ^-6 mole / L) added Compared. As a result, the blood vessel area, the length of the blood vessel, and the number of branches of the blood vessel added mead acid (10 ⁻⁶ mole / L) showed an angiogenesis inhibitory effect equivalent to 25 times the amount of suramin.

Next, an experiment similar to that described above was performed on 11,14,17-eicosatrienoic acid (n-3), an omega-3 polyunsaturated fatty acid, which was estimated to have the same effect as mead acid. It was. As shown in FIGS. 22 to 24, in the culture test in which 11, 14, 17-eicosatrienoic acid (n-3) was added instead of mead acid, only mead acid was added compared to Control. The values of the blood vessel area, the length of the blood vessel, and the number of branches of the blood vessel are slightly reduced, and angiogenesis is slightly suppressed. Furthermore, as shown in FIG. 22 to FIG. 24, the blood vessel area, the length of the blood vessel, and the number of branches of the blood vessel are also added to those added with the vascular endothelial cell growth factor, as in the case of adding mead acid. An inhibitory effect on angiogenesis was observed.

Claims (9)

  Angiogenesis comprising at least one of 5,8,11-eicosatrienoic acid (n-9) and 11,14,17-eicosatrienoic acid (n-3) as a main active ingredient Inhibitor.   The angiogenesis inhibitor of Claim 1 which suppresses the neovascularization of the cancer tissue.   The angiogenesis inhibitor according to claim 1, which inhibits the formation of capillaries in the cornea of the eye.   The angiogenesis inhibitor according to claim 1, which inhibits the formation of capillaries in the retina of the eye.   The angiogenesis inhibitor according to claim 1, which inhibits the formation of capillaries in skin tissue on the body surface.   The angiogenesis inhibitor according to claim 1, which inhibits the formation of capillaries in muscle tissue in the body.   The angiogenesis inhibitor according to claim 1, wherein the form is a liquid, emulsion, ointment, lotion, or poultice.   The angiogenesis inhibitor according to claim 1, which is an eye drop or an eye drop. A cosmetic comprising the angiogenesis inhibitor according to claim 1 or 7, and having a function of inhibiting angiogenesis.