JP2016210709A - Vascular endothelial cell growth factor (VEGF) inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a vascular endothelial cell growth factor (VEGF) inhibitor.SOLUTION: The vascular endothelial cell growth factor (VEGF) inhibitor contains a pinecone lignin glucoside as an active ingredient.SELECTED DRAWING: None

Description

  The present invention relates to a vascular endothelial growth factor (VEGF) inhibitor.

  Vascular endothelial growth factor (VEGF) is known as a protein involved in angiogenesis and increased vascular permeability.

  Currently, anti-VEGF antibodies and molecules that inhibit the binding of VEGF to the VEGF receptor (VEGFR) are being actively developed as therapeutic agents for diseases involving angiogenesis and diseases caused by angiogenesis. For example, McGen (trade name), Lucentis (registered trademark), and the like are commercially available as VEGF binding inhibitors, and are used for the treatment of macular degeneration and the like (see, for example, Non-Patent Document 1).

Anne E. Fung et al., 2007 Am J of Ophthalmol vol. 143, No. 4, pp.566-583

  An object of the present invention is to provide a vascular endothelial growth factor (VEGF) inhibitor.

  One embodiment of the present invention is a vascular endothelial growth factor (VEGF) inhibitor containing lignin glycoside as an active ingredient, wherein the lignin glycoside has the following properties: It is an endothelial cell growth factor (VEGF) inhibitor.

(1) Lignin and polysaccharide are bound (2) Molecular weight is 8000 to 10,000
(3) The binding ratio between lignin and polysaccharide is 1: 1 to 2: 1 (molecular ratio).
(4) The polysaccharide is composed of 10-20% uronic acid and 80-90% neutral sugar.

  The lignin glycoside may be isolated by the following steps.

(1) A process of hydrothermally treating pine cones of pine needles (2) A step of ethanol-treating the pine cones treated with hot water (3) A step of drying the pine cones subjected to ethanol treatment (4) A sodium hydroxide solution of the dried pine cones (5) Step of neutralizing the extract (6) Step of adding ethanol to the extract neutralized in (5) and recovering the precipitate The lignin glycoside May have the following structural formula as a partial structure.

  The lignin glycoside may be isolated in the following steps.

(1) A process of hydrothermally treating pine cones of pine needles (2) A step of ethanol-treating the pine cones treated with hot water (3) A step of drying the pine cones subjected to ethanol treatment (4) A sodium hydroxide solution of the dried pine cones (5) Step of neutralizing the extract (6) Step of adding ethanol to the extract neutralized in (5) and recovering the precipitate One embodiment is a medicament used as one or more of a therapeutic agent and a preventive agent for a disease in which angiogenesis is involved in a disease state, and a therapeutic agent and a preventive agent for a disease in which an increase in vascular permeability is associated with a disease state. And any one of the above lignin glycosides as an active ingredient.

Diseases caused by angiogenesis, or diseases caused by increased vascular permeability are diabetic retinopathy, macular edema due to retinal vein occlusion, neovascular glaucoma, proliferative diabetic retinopathy, myopic blood vessels due to choroidal neovascularization Neonatal maculopathy or idiopathic choroidal neovascularization, age-related macular degeneration, retinal ischemia, Crow-Fukase syndrome (aka POEMS syndrome), peripheral arterial occlusion disease, Casabach-Merit syndrome, cerebral edema, regional cerebral ischemia, polycyst It may be at least one selected from ovarian ovary syndrome and endometriosis. (Andreoli CM and Miller JW 2007 Curr Opin Ophthalmol vol.18, No.6, pp.502-508)
The lignin glycoside is preferably not used in combination with Ganoderma. The lignin glycoside may be used as an antitumor agent in combination with a cell growth inhibitor.

  The present invention has made it possible to provide a vascular endothelial growth factor (VEGF) inhibitor.

In one Example of this invention, it is the graph which showed the presence or absence of the cytotoxicity and VEGF inhibitory effect of a pine cone lignin glycoside.

  The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention, and are shown for illustration or explanation. It is not limited. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

(1) Pine cone lignin glycoside The compound contained in the vascular endothelial growth factor (VEGF) inhibitor of the present invention is a kind of lignin glycoside contained in a pine family plant, and has the following properties.
(I) Lignin and polysaccharide are bound (II) Molecular weight is 8000 to 10,000
(III) The binding ratio between lignin and polysaccharide is 1: 1 to 2: 1 (molecular ratio).
(IV) The polysaccharide is composed of 10 to 20% uronic acid and 80 to 90% neutral sugar. Examples of neutral sugars include glucose, galactose, mannose, and arabinose.

  This lignin glycoside can be identified by the following extraction method from pine cones.

(I) Hot water treatment First, a pine cone of Goyo pine is hydrothermally treated. The amount of water may be appropriately determined according to the amount of pine cones. Although the temperature of water is not specifically limited, 90 degreeC or more is preferable and 100 degreeC is more preferable. The treatment time is not particularly limited, and the treatment may be performed several times from 1 hour to 1 day, but it is preferable to perform the treatment for 3 hours.
(II) Ethanol treatment Next, the hot water-treated pine cone is treated with ethanol. The amount of ethanol may be appropriately determined according to the amount of pinecone. Pine cones are preferably immersed in 100% ethanol in a dry state, and may be allowed to stand for 1 hour to 1 day, but are preferably allowed to stand at room temperature for 2 hours.
(III) Drying treatment The ethanol-treated pine cone is washed with acetone to remove the ethanol. The amount of acetone may be appropriately determined according to the amount of pine cone. Then leave it to dry the pine cones.
(IV) Sodium hydroxide treatment The dried pine cone is treated with sodium hydroxide to obtain an extract. The amount of sodium hydroxide may be appropriately determined according to the amount of pine cone. The concentration of sodium hydroxide is not particularly limited, but is preferably 0.1N to 1N, and more preferably 0.3N. The treatment time may be left for 1 hour to 1 day, but is preferably left at room temperature for 1 day. Thereafter, the pine cone is removed and the extract is recovered.
(V) Neutralization treatment The extract thus obtained is neutralized. The acid to be used is not particularly limited, but hydrochloric acid or acetic acid is preferable. The final pH is preferably 5-6, and more preferably 5.5.
(VI) Ethanol precipitation Ethanol is added to the neutralized extract, preferably left at 4 ° C. for 1 hour to 1 day to precipitate lignin glycosides, and suction filtered to remove the supernatant.
(VII) Purification Finally, the precipitate is purified. For example, fractionation can be performed by gel filtration, and the VEGF inhibitory action can be evaluated for each fraction by a known method to obtain a VEGF-inhibited fraction.

The lignin glycoside extracted in this way (referred to herein as pine cone lignin glycoside) is presumed to have the following structural formula as a partial structure.

  The above-mentioned method for producing a pine cone lignin glycoside is only a method for identifying the pine cone lignin glycoside used in the present invention, and the extraction method when this pine cone lignin glycoside is actually used is not particularly limited. May be used. For example, the extraction methods described in Japanese Patent Publication No. 278260 and Japanese Patent Publication No. 2784605 can be used.

(2) Vascular Endothelial Cell Growth Factor (VEGF) Inhibitor Pine Lignin Glycoside has an inhibitory action on vascular endothelial cell growth factor (VEGF) and can therefore be used as a VEGF inhibitor. Here, the origin of VEGF is not particularly limited, but it may be human or vertebrate other than human.

  Since VEGF has functions such as inducing angiogenesis and increasing vascular permeability, inhibiting VEGF in vivo can exert an anti-angiogenic effect, an increase in vascular permeability, etc. it can. Therefore, a VEGF inhibitor containing a pinecone lignin glycoside as an active ingredient is responsible for angiogenesis and / or increased vascular permeability through pathological conditions through angiogenesis inhibitory action, vascular permeability increase inhibitory action, etc. Inhibition of increased vascular permeability can be used to treat or prevent various diseases that lead to treatment. Examples of diseases include macular edema due to diabetic retinopathy, retinal vein occlusion, neovascular glaucoma, proliferative diabetic retinopathy, myopic neovascularization or idiopathic choroidal neovascularization due to choroidal neovascularization, age-related macular degeneration, retina Can be used for ischemia, Crow-Fukase syndrome (also known as POEMS syndrome), peripheral arterial occlusion disease, Casabach-Merit syndrome, cerebral edema, regional cerebral ischemia, polycystic ovary syndrome, endometriosis, tumors, etc. .

(3) Pharmaceutical use of pine cone lignin glycosides Pine pine lignin glycosides have an inhibitory action on vascular endothelial growth factor (VEGF), so that they can be used, for example, in pharmaceutical compositions (pharmaceutical compositions and reagent product compositions). Can be used to produce a medicament or reagent using methods well known to those skilled in the art. As a raw material of such a composition, a purified pine cone lignin glycoside may be used, but an extract such as pine cone mixed with impurities may be used. For example, in the purification method, An extract that has been subjected to ethanol precipitation may be used. Here, the pine derived from the pine cone is not particularly limited as long as it contains a pine cone lignin glycoside, but particularly Japanese pine containing a large amount of the pine cone lignin glycoside is preferable.

  As a medicine containing a pine cone lignin glycoside, any of the diseases described in (2) can be targeted. Medicines containing pine cone lignin glycosides are preferably used excluding anticancer agents, antitumor agents, anti-inflammatory agents, antiviral agents, antiparasitic agents, antibacterial agents, immune enhancers, and blood flow improvers. .

  The pharmaceutical preparation may be carried out by a method well known to those skilled in the art, and the shape is not particularly limited, and may be a tablet, capsule, bagging, syrup, suppository, bottling, ointment, eye drop or nasal drop.

  Reagents containing pine cone lignin glycosides can also be used as various research reagents in vitro or in vivo. The purpose is not limited, and may be, for example, drug development or basic research. Moreover, the object to be used is not limited, and for example, it may be extracted from a cell, a cell, or a living body.

(4) Combined use of lignin glycoside and other pharmaceuticals The lignin glycoside of the present invention may be used in combination with known VEGF inhibitors represented by anti-VEGF antibodies such as Lucentis (registered trademark). Can be used to produce the drug using methods well known to those skilled in the art. Preferably, the use combined with Ganoderma is excluded. Specific embodiments thereof are the same as those described in (3).

  On the other hand, pine cone lignin glycoside has a vascular endothelial growth factor (VEGF) inhibitory action, so it can exhibit a synergistic effect with a drug that does not have a vascular endothelial growth factor (VEGF) inhibitory action, and is particularly preferably used in combination. Is possible. For example, a synergistic effect is expected when used in combination with an antitumor agent that is a cell growth inhibitor as an antitumor agent.

(5) Method of using the VEGF inhibitor of the present invention The VEGF inhibitor of the present invention can be used for all purposes for the purpose of inhibiting VEGF.

For example, when used for a pharmaceutical, the administration target of a pharmaceutical containing a pinecone lignin glycoside is a human or a non-human vertebrate suffering from the disease described in (2) or (3). The administration method is not limited, and it may be oral, parenteral, systemic administration, or local administration.
Reagent products containing pine cone lignin glycosides can be used in vivo such as in mammals other than humans and in vitro such as cultured cells and cultured tissues.

(1) Preparation of pine cone lignin glycoside 100 g of pine cone of pine needle pine was placed in 1 L of hot water, left for 2 hours, the hot water was changed, and the same treatment was repeated twice. In a semi-dry state, it was immersed in 1 L of 100% ethanol and allowed to stand at room temperature for 2 hours. Then, it wash | cleaned with 300 mL acetone, and left still at room temperature all day and night and dried.
The pine cone pretreated in this way was immersed in 1 L of 0.3N sodium hydroxide and allowed to stand at room temperature all day and night, and then the pine cone was filtered and removed. 2N hydrochloric acid was added dropwise to the resulting extract to adjust the pH to 5.5. An equal amount of 100% ethanol was added to this solution and allowed to stand at 4 ° C. overnight. The generated precipitate was collected by suction filtration, dissolved in an equal amount of water at a weight / volume ratio, dialyzed against a large amount of water for 24 hours, and then freeze-dried to obtain a powder. .

  This powder was dissolved in 1 mL of water and purified by column chromatography (Sepharose CL-4B).

  This powder was dissolved in 1 mL of 10% ethanol and purified by column chromatography (Toyopearl HW40-F) to obtain a powder containing pine lignin glycosides.

(2) VEGF inhibitory effect of pine cone lignin glycoside (PLG) An experiment for confirming the cytotoxicity of pine cone lignin glycoside and an inhibitory effect on angiogenesis induced by VEGF was conducted as an example of the VEGF inhibitory effect.

  CS-C medium (without serum and growth factor, Cell Systems Corporation), 1/10 volume of FBS, 1/100 volume of heparin (50 IU / mL), and 1/100 volume of penicillin -A medium supplemented with streptomycin (100 U / mL-100 μg / mL) was prepared. Hereinafter, this medium is referred to as “experimental medium”.

Then, the prepared laboratory medium was poured into 96-well plates, human retinal endothelial cells (Human Retinal Endothelial Cells (HREC) , Cell manufactured by Systems Corporation) were seeded 10 ⁴ cells per well, temperature 37 ° C., 5% Cultivated in CO ₂ for 2 days.

Next, the experimental medium used for the culture for 2 days was removed from the wells and replaced with the experimental medium supplemented with the substances shown in Table 1. The concentration of VEGF in the experimental medium was 3 ng / mL. In addition, each culture medium for exchange used what was preincubated at 37 degreeC for 1 hour. After exchanging the medium, the cells were further cultured at 37 ° C. and 5% CO ₂ for 2 days. Here, in Table 1, groups 1 to 4 are media for confirming cytotoxicity, and groups 5 to 9 are media for confirming the VEGF inhibitory effect. In addition, each group was cultured using 4 wells.

In addition, as a control experiment, fibroblast growth factor (aFGF), which is one of vascular endothelial growth factors, was used instead of VEGF, and the same conditions were used using an experimental medium containing the test substance shown in Table 2. Was cultured. The concentration of aFGF in the experimental medium was 10 ng / mL.

Next, the absorbance (OD ₄₉₀ ) at ₄₉₀ nm of the medium containing HREC in each well was measured. For each group, an average value of four wells was determined, and this average value was used as an index of the number of viable cells.

  The above results are shown in FIG. In the upper diagram of FIG. 1, the results of groups 1 to 9 in Table 1 are shown in order from the left, and in the lower diagram of FIG. 1, the results of groups 1 to 6 in Table 2 are shown in order from the left.

(3) Results As shown in the upper diagram of FIG. 1, a conventionally known anti-VEGF antibody, that is, a VEGF inhibitor, Lucentis (registered trademark), inhibited VEGF-induced vascular endothelial cell proliferation. Similarly, the pinecone lignin glycoside of the present invention also inhibited VEGF-induced vascular endothelial cell proliferation. The higher the concentration of pine cone lignin glycoside, the higher the growth inhibitory effect. Pine cone lignin glycosides did not show cytotoxicity even at 100 μg / mL.

On the other hand, as shown in the lower diagram of FIG. 1, the pine cone lignin glycoside did not inhibit the proliferation of vascular endothelial cells induced by aFGF.
Thus, the pinecone lignin glycoside is effective as a VEGF inhibitor that specifically inhibits VEGF.

Claims (8)

A vascular endothelial growth factor (VEGF) inhibitor containing lignin glycoside as an active ingredient,
The lignin glycoside has the following properties: a vascular endothelial growth factor (VEGF) inhibitor.
(1) Lignin and polysaccharide are bound (2) Molecular weight is 8000 to 10,000
(3) The binding ratio between lignin and polysaccharide is 1: 1 to 2: 1 (molecular ratio).
(4) The polysaccharide is composed of 10-20% uronic acid and 80-90% neutral sugar. 2. The vascular endothelial growth factor (VEGF) inhibitor according to claim 1, wherein the lignin glycoside is isolated by the following step.
(1) A process of hydrothermally treating pine cones of pine needles (2) A step of ethanol-treating the pine cones treated with hot water (3) A step of drying the pine cones subjected to ethanol treatment (4) A sodium hydroxide solution of the dried pine cones (5) Step of neutralizing the extract (6) Step of adding ethanol to the extract neutralized in (5) and recovering the precipitate 3. The vascular endothelial growth factor (VEGF) inhibitor according to claim 1 or 2, wherein the lignin glycoside has the following structural formula as a partial structure.
  The vascular endothelial growth factor (VEGF) inhibitor according to any one of claims 1 to 3, which is not used in combination with ganoderma. A therapeutic agent and a preventive agent for a disease in which angiogenesis is involved in a disease state, and a medicament used as any one or more of a therapeutic agent and a preventive agent for a disease in which an increase in vascular permeability is involved in a disease state,
The pharmaceutical which contains the lignin glycoside of any one of Claims 1-3 as an active ingredient.   Diseases caused by angiogenesis or diseases caused by increased vascular permeability are diabetic retinopathy, macular edema due to retinal vein occlusion, neovascular glaucoma, proliferative diabetic retinopathy, choroidal neovascularization Neonatal maculopathy or idiopathic choroidal neovascularization, age-related macular degeneration, retinal ischemia, Crow-Fukase syndrome (aka POEMS syndrome), peripheral arterial occlusion disease, Casabach-Merit syndrome, cerebral edema, regional cerebral ischemia, polycyst The medicament according to claim 5, which is at least one selected from ovarian ovary syndrome and endometriosis.   The pharmaceutical which contains the lignin glycoside of any one of Claims 1-3 characterized by not being used together with Ganoderma as an active ingredient.   The antitumor agent which contains the lignin glycoside of any one of Claims 1-3 used together with a cell growth inhibitor as an active ingredient.