JP6562416B2 - Angiogenesis inhibitor - Google Patents

Description

  The present invention relates to an angiogenesis inhibitor comprising a naturally occurring substance as an active ingredient.

  It is known that angiogenesis is involved in the onset and progression of solid tumors, ophthalmic diseases such as diabetic retinopathy, glaucoma and macular degeneration, inflammation, rheumatoid arthritis, psoriasis vulgaris, and periodontal disease. It has been. Therefore, while research and development of angiogenesis inhibitors have been actively conducted for a long time as drugs for preventing and treating such diseases (for example, Patent Document 1), in recent years, highly safe naturally-derived substances are effective. An angiogenesis inhibitor as a component is desired.

Japanese Patent No. 3135616

  Therefore, an object of the present invention is to provide an angiogenesis inhibitor containing a naturally occurring substance as an active ingredient.

  As a result of intensive studies in view of the above points, the present inventors have found that proteoglycan contained in salmon cartilage and its enzymatic degradation product have an excellent angiogenesis inhibitory action.

  As described in claim 1, the angiogenesis inhibitor of the present invention made based on the above findings contains proteoglycan contained in salmon cartilage and / or its enzymatic degradation product as an active ingredient.

  ADVANTAGE OF THE INVENTION According to this invention, the angiogenesis inhibitor which uses as an active ingredient the proteoglycan contained in a salmon cartilage and its enzyme degradation product as a naturally derived substance can be provided.

2 is a photograph showing that salmon nasal cartilage-derived proteoglycan in Example 1 has an excellent inhibitory effect on the formation of human vascular endothelial cells. FIG. 6 is a graph showing that the inhibitory effect of salmon nasal cartilage-derived proteoglycan on the lumen formation of human vascular endothelial cells is concentration-dependent. In comparative example 1, it is a graph which shows that the inhibitory effect with respect to the luminal formation of a human vascular endothelial cell is superior to the chondroitin sulfate derived from a shark cartilage by the proteoglycan derived from a salmon nasal cartilage. It is a graph which shows that the inhibitory effect with respect to the luminal formation of the human vascular endothelial cell of the salmon nasal cartilage proteoglycan in Example 2 does not fall by heat processing. It is a graph which shows that the salmon nasal cartilage origin proteoglycan in Example 3 does not affect the growth of a human vascular endothelial cell. It is a graph which shows that the salmon nasal cartilage origin proteoglycan in Example 4 has the effect | action which reduces the expression of a matrix metalloprotease gene. It is a graph which shows that the inhibitory effect with respect to the luminal formation of a human vascular endothelial cell increases by decomposing | disassembling the salmon nasal cartilage proteoglycan in Example 5. FIG.

  The angiogenesis inhibitor of the present invention comprises proteoglycan contained in salmon cartilage and its enzymatic degradation product as active ingredients. Proteoglycan is a main component constituting animal cartilage together with collagen and hyaluronic acid, and has been known for a long time to have excellent water retention. In addition, the research group of the present inventors has found that proteoglycans contained in salmon cartilage have a cell growth promoting action and a hyaluronic acid synthesis promoting action (Japanese Patent No. 5194253). However, the full pharmacological action of proteoglycans and their enzymatic degradation products contained in salmon cartilage has not been clarified yet.

  The proteoglycan contained in salmon cartilage as an active ingredient of the angiogenesis inhibitor of the present invention may be separated and purified from salmon nasal cartilage, for example. The method for separating and purifying proteoglycan from salmon nasal cartilage is not particularly limited. For example, according to the method described in Japanese Patent No. 3731150, crude proteoglycan using acetic acid as an elution solvent from minced salmon nasal cartilage. After elution, the obtained eluate is filtered and centrifuged, and the semi-solid precipitate containing crude proteoglycan obtained by adding sodium chloride saturated ethanol to the supernatant and centrifuging is dissolved in acetic acid, Subsequently, it may be prepared by separation and purification by dialysis. The proteoglycan derived from salmon nasal cartilage thus prepared is highly purified having a molecular weight of about 250 to 450 kDa, and is suitable as an active ingredient of the angiogenesis inhibitor of the present invention. Note that proteoglycans derived from salmon nasal cartilage prepared according to the method described in Japanese Patent No. 3731150 are already commercially available as lyophilized powders. In addition, proteoglycan contained in salmon cartilage is obtained by crushing frozen salmon nasal cartilage according to the method described in Japanese Patent No. 5252623, adding water thereto, at a temperature of 0 to 20 ° C., and a pH of 4.8 to 7. The step of processing, the obtained solid-liquid mixture is centrifuged, the uppermost lipid layer and the aqueous layer of the intermediate layer are removed, the precipitate is recovered, the obtained precipitate is dried and micronized The obtained dry fine powder may be prepared by adding ethanol as a solvent to extract and remove residual lipids, and finally removing ethanol.

  In addition, examples of the enzymatic degradation product of proteoglycan contained in salmon cartilage include degradation products of proteoglycan contained in salmon cartilage by sugar chain degrading enzymes and proteolytic enzymes. Here, examples of sugar chain degrading enzymes include known enzymes capable of degrading glycosaminoglycans possessed by proteoglycans contained in salmon cartilage, such as chondroitinase ABC, chondroitinase AC, and testicular hyaluronidase. . Examples of proteolytic enzymes include known enzymes capable of degrading core proteins possessed by proteoglycans contained in salmon cartilage, such as actinase E, proteinase K, trypsin, and papain. Degradation of proteoglycan contained in salmon cartilage using such an enzyme may be performed under optimum conditions known for the enzyme used. The resulting enzymatic degradation product may or may not be purified (for example, degradation in which the core protein is contained in the glycosylation product or glycosaminoglycan is contained in the proteolysis product) A mixture of products).

  As will be apparent from the examples described later, proteoglycan contained in salmon cartilage and its enzymatic degradation product have an angiogenesis inhibitory action superior to chondroitin sulfate derived from shark cartilage. The effect is not reduced by heat treatment and does not affect the growth of vascular endothelial cells. In addition, when the proteoglycan contained in salmon cartilage is enzymatically degraded, the angiogenesis inhibitory action is enhanced. Therefore, the angiogenesis inhibitor of the present invention is useful for the prevention and treatment of solid tumors, ophthalmic diseases such as diabetic retinopathy, glaucoma and macular degeneration, inflammation, rheumatoid arthritis, psoriasis vulgaris, and periodontal disease. Can be used.

  Administration of the angiogenesis inhibitor of the present invention to the administration subject can be performed by oral administration or parenteral administration. What is necessary is just to formulate in the dosage form suitable for each administration method in the case of administration. Examples of the dosage form include tablets, capsules, granules, powders, fine granules, pills, troches, sublingual tablets, suppositories, ointments, emulsions, suspensions, syrups, eye drops, eye ointments, etc. The preparation of these formulations includes non-toxic excipients, binders, lubricants, disintegrants, preservatives, isotonic agents, stabilizers, dispersants, antioxidants, colorants, taste masking It can be carried out by a method known per se using additives such as an agent and a buffer. Non-toxic additives include, for example, starch, gelatin, glucose, lactose, fructose, maltose, magnesium carbonate, talc, magnesium stearate, methylcellulose, carboxymethylcellulose, gum arabic, polyethylene glycol, propylene glycol, petrolatum, glycerin, ethanol Syrup, sodium chloride, sodium sulfite, sodium phosphate, citric acid, polyvinylpyrrolidone, water and the like. The content of the active ingredient in the preparation varies depending on the dosage form, but it is generally desirable that the concentration is 0.01 to 100% by weight. The dosage of the preparation can be widely adjusted according to the gender, age and weight of the administration subject, the severity of symptoms, the diagnosis of a doctor, etc., but in general, it should be 0.01 to 300 mg / Kg per day. it can. The above dose may be administered once or divided into several times a day. In addition, the angiogenesis inhibitor of the present invention can be eaten as various forms of food (including supplements).

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is limited to the following description and is not interpreted. In the experiment, lyophilized powder of proteoglycan separated and purified from salmon nasal cartilage according to the method described in Japanese Patent No. 3731150 as a proteoglycan contained in salmon cartilage (molecular weight 344 kDa, hexosamine: uronic acid: sulfuric acid = 1. 00: 0.99: 0.67, Wako Pure Chemical Industries, Ltd., hereinafter referred to as “salmon nasal cartilage-derived proteoglycan”).

Example 1: Effect of salmon nasal cartilage-derived proteoglycan on the luminal formation of human vascular endothelial cells, an angiogenesis model (experimental method)
Luminogenesis was induced by culturing an immortalized human umbilical vein endothelial cell line (EA.hy926) on a gel made of basement membrane component (BME) (Trevigen Inc. MD, USA). Specifically, 0.05 mL of BME was placed in each well of a 96-well plate and kept at 37 ° C. for 30 minutes for gelation, and then 0.1 mL of 199 medium was used for EA. The liquid in which hy926 was suspended was layered on the gel in each well. At this time, salmon nasal cartilage-derived proteoglycan dissolved in water was added to 199 medium so as to have various concentrations of 0 to 1 mg / mL in terms of uronic acid. After culturing at 37 ° C. for 16 to 24 hours, a photograph of the blood vessel-like tubular network formed on the gel was taken with a phase contrast microscope, and the image analysis program ImageJ (NIH, USA) and the Angiogenesis-Analyzer plug-in ( The total number of tube-like structures and the total extension were measured using Gilles Carpentier.ImageJ contribution: Angiogenesis Analyzer.ImageJ News, 5 October 2012.).

(Experimental result)
FIG. 1A shows a photograph of a blood vessel-like tubular network when no salmon nasal cartilage-derived proteoglycan is added, and FIG. 1B shows a case where salmon nasal cartilage-derived proteoglycan is added to a concentration of 1 mg / mL in terms of uronic acid. Show photos. In addition, the total number of tube-like structures and the measurement results of the total length when salmon nasal cartilage-derived proteoglycan is added at various concentrations from 0 to 1 mg / mL in terms of uronic acid are shown in FIGS. (Relative value with the measurement result when the test substance is not added as 100%). As is clear from FIG. 1, salmon nasal cartilage-derived proteoglycan effectively inhibited the formation of a blood vessel-like tubular network. In addition, as is apparent from FIG. 2, the inhibitory effect of salmon nasal cartilage-derived proteoglycan on the lumen formation of human vascular endothelial cells is concentration-dependent, and when added to a concentration of 1 mg / mL in terms of uronic acid Inhibited angiogenesis by about 70%.

Comparative Example 1: Action of chondroitin sulfate derived from shark cartilage on luminal formation of human vascular endothelial cells (experimental method)
Chondroitin sulfate derived from shark cartilage (chondroitin sulfate C: Seikagaku Corporation Cat. No. 400675) was examined for angiogenesis inhibitory activity in the same manner as in Example 1.

(Experimental result)
The right side of FIG. 3 shows the total number of tube-like structures and the total extension measurement results when chondroitin sulfate derived from shark cartilage is added to a concentration of 1 mg / mL in terms of uronic acid (column of “chondroitin sulfate”) ). In addition, the total number of tube-like structures and the measurement results of the total extension when salmon nasal cartilage proteoglycan is added at a concentration of 1 mg / mL in terms of uronic acid are shown in the center of FIG. 3 ("Proteoglycan" column). As apparent from FIG. 3, salmon nasal cartilage-derived proteoglycan inhibited angiogenesis by about 70%, but chondroitin sulfate derived from shark cartilage inhibited angiogenesis by only about 35% (when no test substance was added). The relative measurement value was 100% (left “untreated” column).

Example 2: Effect of heat-treated salmon nasal cartilage-derived proteoglycan on the lumen formation of human vascular endothelial cells Salmon nasal cartilage-derived proteoglycan dissolved in water and boiled for 5 minutes has an angiogenesis inhibitory effect in the same manner as in Example 1. When examined, no decrease in the effect due to heat treatment was observed, and the effect was similar to that of salmon nasal cartilage-derived proteoglycan that had not been heat-treated (FIG. 4).

Example 3: Safety of salmon nasal cartilage-derived proteoglycan against human vascular endothelial cells (experimental method)
In each well of a 96-well plate, an EA. hy926 was cultured in 199 medium containing 10% fetal bovine serum (FBS) at 37 ° C. for 24 hours. After removing the culture medium from each well, salmon nasal cartilage-derived proteoglycan dissolved in water was added to each well at a concentration of 0 to 1 mg / mL in terms of uronic acid, and further added to each well at 37 ° C. Cultured for 24 hours. Thereafter, 0.01 mL of Cell counting reagent SF (Nacalai) was added to each well, and further cultured at 37 ° C. for 1 hour, and the absorbance at 450 nm was measured with a microplate reader.

(Experimental result)
FIG. 5 shows the cell viability when salmon nasal cartilage-derived proteoglycan is added to various concentrations of 0 to 1 mg / mL in terms of uronic acid (the cell viability without adding the test substance is 100%) Relative value). As is clear from FIG. 5, even when salmon nasal cartilage-derived proteoglycan was added to a concentration of 1 mg / mL in terms of uronic acid and cultured, human vascular endothelial cells were not affected. .

Example 4: Effect of salmon nasal cartilage-derived proteoglycan on expression of matrix metalloprotease gene (experimental method)
In each well of a 12-well plate, an EA. hy926 was cultured in 199 medium containing 10% fetal bovine serum (FBS) at 37 ° C. for 24 hours. After removing the medium from each well, salmon nasal cartilage-derived proteoglycan dissolved in water was added to a concentration of 1 mg / mL in terms of uronic acid, 0.5% FBS and 0.5% bovine serum albumin A 199 medium containing (BSA) was placed in each well and further cultured at 37 ° C. for 24 hours. Thereafter, total RNA was extracted from the cells using RNeasy mini kit (QIAGEN). CDNA was synthesized from the total RNA using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems), and FastStart Universal Probe Master (Roche) and TaqMan prob using the cDNA as a template. And the expression levels of various matrix metalloproteinase (MMP) genes were estimated using the GAPDH gene as an internal standard gene. In addition, ID of TaqMan probe used for experiment is as follows.
MMP-1: Hs00899658_m1
MMP-2: Hs01554827_m1
MMP-3: Hs00968305_m1
MMP-7: Hs01042796_m1
MMP-9: Hs00957555_m1
MMP-14: Hs01037309_g1
GAPDH: Hs02758991_g1

(Experimental result)
FIG. 6 shows the expression levels of mRNA of MMP-1, MMP-2, and MMP-14 (relative values where the expression level when no test substance is added is 1). As is clear from FIG. 6, salmon nasal cartilage-derived proteoglycan reduced the expression levels of MMP-1, MMP-2, and MMP-14 mRNA by 20 to 25%. The expression levels of MMP-3, MMP-7, and MMP-9 mRNA were very small and difficult to analyze.

Example 5: Effect of enzymatic degradation product of salmon nasal cartilage-derived proteoglycan on luminal formation of human vascular endothelial cells (experimental method)
Proteoglycan derived from salmon nasal cartilage of 0.3 mg was used for 18 hours at 37 ° C. with actinase E (1 mg / mL actinase E (Kaken Pharmaceutical), 100 mM Tris-HCl, pH 8.0, 10 mM CaCl ₂ ) as a proteolytic enzyme. After processing and degrading the core protein, the enzymatic reaction was stopped by boiling for 5 minutes to obtain a proteolytic product. Also, 0.3 mg of salmon nasal cartilage-derived proteoglycan was added to chondroitinase ABC (300 mU chondroitinase ABC (Sigma-Aldrich), 50 mM Tris-HCl, pH 8.0, 60 mM CH ₃ COONa, 0, which is a glycolytic enzyme. (.02% BSA) at 37 ° C. for 48 hours to decompose glycosaminoglycan and then boiled for 5 minutes to stop the enzymatic reaction to obtain a sugar chain degradation product. In addition, 0.3 mg salmon nasal cartilage-derived proteoglycan was treated with chondroitinase ABC at 37 ° C. for 48 hours to decompose the glycosaminoglycan and then boiled for 5 minutes before stopping the enzyme reaction. Subsequently, the actinase E was treated at 37 ° C. for 18 hours to decompose the core protein, followed by boiling for 5 minutes to stop the enzymatic reaction, thereby obtaining a sugar chain / proteolysis product. About each degradation product, it carried out similarly to Example 1, and investigated the angiogenesis inhibitory effect.

(Experimental result)
Measurement results of total number of tube-like structures and total length when salmon nasal cartilage-derived proteoglycan was added at a concentration corresponding to 1 mg / mL in terms of uronic acid, FIG. 7 shows the total number of tube-like structures and the total extension when the nasal cartilage-derived proteoglycan is added to a concentration of 1 mg / mL in terms of uronic acid (measurement results when no test substance is added). Relative value with 100%). As is clear from FIG. 7, it was found that the angiogenesis inhibitory action can be enhanced by enzymatic degradation of salmon nasal cartilage-derived proteoglycan (the action is not reduced by heat treatment to stop the enzyme reaction). The degree of increase in the angiogenesis inhibitory effect was greater for the sugar chain degradation product than for the protein degradation product. The angiogenesis inhibitory action of the sugar chain / protein degradation product was similar to the angiogenesis inhibitory action of the sugar chain degradation product.

Formulation Example 1: Tablets Produced by tableting after thoroughly mixing each component of salmon nasal cartilage-derived proteoglycan, lactose, starch, carboxymethylcellulose, talc, and magnesium stearate.

Formulation Example 2: Capsules Produced by thoroughly mixing each component of salmon nasal cartilage-derived proteoglycan, lactose, starch, and magnesium stearate before filling into capsules.

  INDUSTRIAL APPLICABILITY The present invention has industrial applicability in that it can provide an angiogenesis inhibitor containing a proteoglycan contained in salmon cartilage and its enzyme degradation product as an active ingredient as a naturally derived substance.

Claims (1)

  An angiogenesis inhibitor comprising a proteoglycan contained in salmon cartilage and / or its enzymatic degradation product as an active ingredient.