JPH08268889A - Arterialization inhibitor - Google Patents

Abstract

PURPOSE: To obtain an arterialization inhibitor containing a specific compound as an active ingredient, excellent in arterialization-inhibiting action, having low toxicity and capable of safely using and useful for preventing and treating diabetic retinopathy, chronic inflammation, psoriasis and arteriosclerosis. CONSTITUTION: This inhibitor contains 3-hydroxymethyl-6-methoxy-8-hydroxy-1 H-2-benzopyran-1-one of the formula as an active ingredient. The compound of the formula is obtained by culturing, e.g. Streptoverticillium eurocidicum M-143-37Fl1 strain (FERM-BP-2783) and collecting the compound from the culture solution. The content of the compound of the formula is preferably 5-50wt.% based on the total composition when the inhibitor is a solid agent such as tablet and the content of the compound is usually preferably 0.1-10wt.% when the inhibitor is a liquid agent such as injection. Furthermore, the compound of the formula is preferably administered in a daily dose of 0.3-300mg/kg in the case of oral administration or 0.03-30mg/kg in the case of parenteral administration.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a low toxicity angiogenesis inhibitor which is expected to be applied to various diseases.

[0002]

The compound of the present invention, 3-hydroxymethyl-6-methoxy-8-hydroxy-1H-2-benzopyran-1-one, is described in JP-A-3-2177 and JP-A-6-183966, respectively. Although it is disclosed that it exhibits an antitumor action and an immunomodulatory action, it is not described in the specification that it has an angiogenesis inhibiting action. On the other hand, as substances that inhibit angiogenesis so far,
Combinations of protamine, heparin and cortisone acetate, fumagillin and its derivatives, interferon, etc. have been reported. However, these angiogenesis inhibitors have problems such as weak inhibitory activity, lack of sustained activity, and side effects such as cytotoxicity, fever and bleeding.

[0003]

DISCLOSURE OF THE INVENTION The present invention provides an angiogenesis inhibitor which is excellent in angiogenesis inhibitory action, has low toxicity, and can be used safely.

[0004]

The present inventors have found that 3-hydroxymethyl-6-methoxy-8-hydroxy-1H-
As a result of intensive studies on the pharmacological activity of 2-benzopyran-1-one, it was found that this substance has a strong angiogenesis-inhibiting activity and few side effects, and completed the present invention. The present invention provides 3-hydroxymethyl-6-methoxy-8-hydroxy-1H represented by the following formula (I).
It is an angiogenesis inhibitor containing 2-benzopyran-1-one as an active ingredient.

[0005]

Embedded image

The compound (I) of the present invention is a microorganism belonging to the genus Streptoverticillium and having an ability to produce the compound (I), for example, Streptoverticillium eurosidicum MI43-37F11 strain (FERM BP-
2783) is cultured in a nutrient medium and collected from the culture solution (JP-A-3-2177). The compound (I) of the present invention is a 2-benzopyran derivative,
For example, it can also be produced by reacting 3-benzyloxymethyl-6,8-dimethoxy-1H-2-benzopyran-1-one with BBr ₃ under a nitrogen stream (JP-A-4-112884).

The compound (I) of the present invention exhibits an action of inhibiting the lumen formation of vascular endothelial cells obtained from bovine carotid arteries, and is a disease in which angiogenesis plays an important role, that is, diabetic retinopathy, various types. It is expected to be applied to the prevention and treatment of chronic inflammation, psoriasis, angiogenesis associated with corneal transplantation, and arteriosclerosis. In addition, it shows almost no toxicity and can be used effectively and safely.

The compound (I) of the present invention is useful as an angiogenesis inhibitor, and its pharmaceutical preparation includes known fillers, fillers, binders, humectants, disintegrants, disintegration inhibitors, and surface active agents. It can be prepared by using a diluent such as a lubricant, or an excipient. Various forms of this pharmaceutical preparation can be selected according to the therapeutic purpose, and typical examples thereof include tablets, pills, powders, solutions, suspension syrups, emulsions, granules, capsules, suppositories, and injections. Examples include agents (solutions, suspensions, etc.).

In the case of molding into tablets, known carriers can be widely used, and examples thereof include lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose and silicic acid. Agent,
Water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, binders such as polyvinylpyrrolidone, dry starch, sodium alginate, agar powder, laminaran powder, hydrogen carbonate Sodium, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, disintegrants such as starch and lactose, sucrose, cacao butter, disintegration inhibitors such as hydrogenated oil, quaternary ammonium salt base, lauryl Absorption promoters such as sodium sulfate, humectants such as glycerin and starch, adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid, purified talc, stearate, boric acid powder, lubricants such as polyethylene glycol Examples are agents It can be.

Further, the tablets can be formed into tablets coated with a usual coating, if necessary, such as sugar-coated tablets, gelatin-coated tablets, film-coated tablets, double tablets and multilayer tablets. When molding in the form of pills, those conventionally known in this field can be widely used as carriers, for example, glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, excipients such as kaolin and talc, gum arabic powder, Examples thereof include tragacanth powder, gelatin, binders such as ethanol, and disintegrants such as laminaran agar powder.

In the case of molding in the form of suppositories, those conventionally known in this field can be widely used as carriers, such as polyethylene glycol, cocoa butter higher alcohols, esters of higher alcohols, gelatin and semisynthetic glycerides. You can

When prepared as an injection, it is preferable that the solution and suspension are sterilized and isotonic with blood. When the solution and suspension are molded into the form of these solutions and suspensions, they are used as diluents. All those commonly used in the field can be used, and examples thereof include water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. In this case, a sufficient amount of sodium chloride, glucose or glycerin to prepare an isotonic solution may be contained in the formulation, and a usual solubilizing agent, buffer, soothing agent, etc. may be used. Good. Further, for oral administration, a coloring agent, a preservative, a flavoring agent, a flavoring agent, a sweetening agent and the like and other pharmaceuticals may be contained in the preparation, if necessary.

The amount of the compound (I) of the present invention to be contained in the angiogenesis inhibitor of the present invention is not particularly limited and can be selected within a wide range, but it is usually solid such as tablets, granules and capsules. In the case of agents, it is 1 to 70% by weight, preferably 5 to 50% by weight of the total composition, and in the case of solutions such as solutions, injections and suspensions, it is 0.1 to 10% by weight.

The administration method of (I) of the compound of the present invention is not particularly limited, and it is administered according to various formulation forms, age and sex of patients, other conditions, and degree of disease. For example, tablets, pills, solutions, suspension syrups, emulsions, granules and capsules are orally administered. In the case of an injection, it may be administered intravenously alone or in a mixture with a normal replacement fluid such as glucose or amino acid, and if necessary, may be administered intramuscularly, intradermally, subcutaneously or intraperitoneally alone. In the case of suppositories, they are administered rectally.

The dose of the compound (I) of the present invention is as follows:
The dose may be adjusted depending on the patient's age, sex, other conditions, degree of disease, etc., but is 0.3 to 300 mg / kg per day for oral administration, and 0.03 for parenteral administration.
-30 mg / kg is suitable.

Hereinafter, the effects of the present invention will be described more specifically with reference to examples, but the present invention is not limited to these.

[0017]

【Example】

Example 1 Effect on proliferation of vascular endothelial cells The effect on proliferation of vascular endothelial cells obtained from bovine carotid artery was examined. That is, endothelial cells (1 × 10 ⁴ ce
lls / well) containing 10% fetal bovine serum (FBS) (DMEM, Dulbecco's mo)
Place in a multi-dish containing dified Eagle's medium) and incubate at 37 ° C (5
% Carbon dioxide, 95% oxygen) (Day)
0). After 24 hours, some well cells were trypsinized, and the number of cells was counted. Then, phosphate buffer (PBS) was added to the remaining wells to wash the cells, and then 3%
The culture solution was exchanged for DMEM containing FBS, and the cells were cultured in the above incubator for 20 to 30 minutes. When the temperature and pH were adjusted, various concentrations of compound (I) or the control agent fumagillin was added to each well, and then bFGF (basi) was added.
c fibroblast growh facto
r) was added to give a final concentration of 10 ng / ml (Day 1). After culturing for 72 hours and treating the cells with trypsin, the number of cells was counted (Day 4). The test compound was added at various concentrations with and without the addition of bFGF, and the effect of the addition was determined by the number of cells / we.
It was evaluated as 11 Table 1 shows the results.

[0018]

[Table 1]

Compound (I) exhibited a vascular endothelial cell growth inhibitory effect at a concentration of 0.01 to 0.1 mM when bFGF was not added and at a concentration of 0.1 to 1 mM when added. The inhibitory effect of compound (I) was relatively remarkable when bFGF was not added. On the other hand, the control drug fumagillin is bF
When GF was not added, the concentration was 0.005-0.1 mM, and when GF was added, the concentration was 0.001-0.1 mM, showing strong growth inhibitory effects, respectively. The inhibitory action of fumagillin is the addition of bFGF,
It was almost the same regardless of no addition. Compound (I)
The inhibitory effect on the growth of vascular endothelial cells was milder than that of the control drug fumagillin.

Example 2 Effect of Vascular Endothelial Cells on Lumen Formation The details of the test method are described in Journal of Clini.
cal Investigation, vol 92:
page 54-61, 1993. That is, type I collagen solution (manufactured by Nitta Gelatin Co., Ltd.), 10 × DMEM (sodium bicarbonate is not added, but 10 times amount of antibiotics is added, and filter sterilized DME is added.
M) and reconstitution buffer (2.2 g of sodium bicarbonate to 100 ml of 0.05 N sodium hydroxide solution,
A solution prepared by dissolving 4.77 g of HEPES and sterilized by filtration was prepared, and the respective liquids were mixed at a ratio of 8: 1: 1 while cooling and dispensed in a 35 mm plastic dish. This petri dish was incubated in an incubator at 37 ° C. for 5 to 10 minutes to cause gelation. 10% F on top of this gel
DMEM containing BS was added, and trypsinized bovine vascular endothelial cells were added. After culturing for 2-3 days, when the cells became confluent, DMEM containing 10% FBS was replaced. After culturing for 20 to 30 minutes, when the temperature and pH were adjusted, various concentrations of compound (I) were added, then bFGF was added so that the final concentration was 10 ng / ml, and the cells were cultured in an incubator at 37 ° C for 3 days. The culture medium was exchanged on the 3rd day, compound (I) and bFGF were added by the same procedure as above, and the cells were further cultured for 3 days. After that, four arbitrary visual fields around the gel were observed and recorded with a phase contrast microscope connected to a video camera, and the average sum of the lengths of the lumens in one visual field was obtained using an image analyzer. Table 2 shows the results. The compound (I) of the present invention strongly inhibited the lumen formation of vascular endothelial cells at a concentration of 0.01 to 0.1 mM.

[0021]

[Table 2]

Example 3 Subacute toxicity Three SD rats were treated with the compound (I) of the present invention at 1000 m.
g / kg was orally administered for 1 month, but there were no deceased individuals,
No toxic symptoms were observed. The toxicity of the compound (I) of the present invention to warm-blooded animals is very low.

[0023]

INDUSTRIAL APPLICABILITY As described above, the compound (I) of the present invention
Is a disease in which angiogenesis plays an important role because it strongly suppresses the luminal formation of vascular endothelial cells compared with the inhibitory effect on the proliferation of vascular endothelial cells, namely diabetic retinopathy, various chronic inflammations, psoriasis, and cornea. It is expected to be applied to angiogenesis associated with transplantation and prevention and treatment of arteriosclerosis. Moreover, it is particularly excellent as an angiogenesis inhibitor in terms of toxicity and can be used effectively and safely.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location // C07D 311/76 C07D 311/76

Claims (1)

[Claims] (1) Formula (1) 3-hydroxymethyl-6-methoxy-8-
An angiogenesis inhibitor containing hydroxy-1H-2-benzopyran-1-one as an active ingredient.