JP3486730B2 - Vascular endothelial growth factor inhibitor - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a vascular endothelial growth factor inhibitor containing a mercaptoacyl amino acid derivative as an active ingredient.

[0002]

BACKGROUND OF THE INVENTION Blood vessel homeostasis is maintained by various functions of endothelial cells. The vascular endothelial cells 1) act as an intermediary for transporting necessary components such as nutrients in blood to tissues, and prevent an unnecessarily large amount of components from passing therethrough,
2) It has an effect of allowing blood to circulate smoothly without coagulation, 3) has an effect of preventing bleeding when the blood vessel is disconnected, and 4) has an adjusting effect of keeping the blood vessel tension constant.

[0003] Angiogenesis occurs at the stages of decomposition of basement membrane by protease produced in vascular endothelial cells, migration / proliferation of vascular endothelial cells, lumen formation of vascular endothelial cells, formation of basement membrane and surrounding peripheral cells. Angiogenesis is closely associated with various diseases, particularly retinal thrombotic diseases such as retinal vein occlusion and retinal artery occlusion and retinal diseases such as macular degeneration, neovascular glaucoma, and tumors such as hemangiomas.
Angiogenesis is thought to be caused by various growth factors, cytokines, arachidonic acid metabolites, monobutyrin and the like. Among these, growth factors are considered to be the most important angiogenic factors (Ayumi, 170, 536-539 (1994)).

Growth factors that cause angiogenesis include
Fibroblast growth factor, tumor growth factor, vascular endothelial growth factor (VEGF), vascular permeability factor, etc. are known,
Among them, the receptor of VEGF exists only in the vascular endothelium, and it has been reported that VEGF selectively acts on the vascular endothelium (J. Clin. Invest., 89, 244-253 (1992)). Therefore, inhibition of VEGF is expected to treat diseases associated with angiogenesis.

On the other hand, as an action of the mercaptoacyl amino acid derivative, an antirheumatic action (Japanese Patent Publication No. 60-11888).
No.), sputum lysing action (Japanese Patent Publication No. 56-5388), hepatic disorder inhibitory action (Japanese Patent Publication No. 62-13922, Japanese Pharmacological Journal, 75, 433-445 (1979)), cataract treatment action (Japanese Patent Publication No. Sho). 63-13964, Invest. Ophthalmo.
L.Vis. Sci., 25, 1051-1055 (1984)), therapeutic action for uveitis (JP-A-2-96521), therapeutic action for diabetes (JP-A-4-154721), therapeutic effect for osteoporosis (special feature). (Kaihei 4-154722), renal disease treatment (JP-A-4-342524), cystinuria treatment (JP-A-5-186341, J. Urol., 117,
628-630 (1977)) has already been reported to be useful, and its safety is known to be high. However, there is no report on the effect on VEGF.

[0006]

It was a very interesting subject to find out a new pharmacological action of the mercaptoacyl amino acid derivative useful as a medicine.

[0007]

Means for Solving the Problems In order to find a new pharmacological action of a mercaptoacyl amino acid derivative, the present inventors have an action on VEGF-mRNA expression and release of VEGF from human glioblastoma cells (human Glioblastoma cells). The effect on was investigated. As a result, the compound represented by the following general formula [〓] or a salt thereof (hereinafter referred to as the present compound) has an inhibitory effect on VEGF-mRNA expression and an inhibitory effect on VEGF release from human Glioblastoma cells. It is suggested that it is also useful as a therapeutic agent for diseases involved, particularly retinal thrombotic diseases such as retinal vein occlusion and retinal artery occlusion and retinal diseases such as macular degeneration, neovascular glaucoma and tumors such as hemangiomas. It was

[0008]

[Chemical 2]

[In the formula, R1 represents a hydrogen atom, a lower alkyl group, an amino lower alkyl group, a hydroxy lower alkyl group, a mercapto lower alkyl group or a carboxy lower alkyl group. A represents a lower alkylene group. same as below. ]

The above-defined groups will be explained in more detail. The lower alkyl means a straight-chain or branched alkyl having 1 to 6 carbon atoms such as methyl, ethyl, propyl, hexyl and isopropyl. What is a lower alkylene?
A straight-chain or branched alkylene having 1 to 6 carbon atoms such as methylene, ethylene, (dimethyl) methylene, (diethyl) methylene and the like is shown.

The salt in the present compound is not particularly limited as long as it is a pharmaceutically acceptable salt, and a salt with an inorganic acid such as hydrochloric acid, nitric acid or sulfuric acid, or an alkali metal or alkaline earth such as sodium, potassium or calcium. Salts with similar metals,
Examples thereof include salts with organic amines such as ammonium salts, diethylamine and triethanolamine salts. In addition, the compound may be in the form of a hydrate.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The compound represented by the general formula [I] has diastereoisomers and optical isomers.
They are all included in the present invention. A single diastereoisomer and an optical isomer can be obtained by using an optically active raw material, but when a racemic body is used as a raw material, each isomer can be obtained by a commonly used method such as a method using an optical resolving agent. The body can be separated.

Among the present compounds, particularly preferred examples are bucillamine (the following formula [〓]), thiopronin (the following formula [〓]) or N- (2,2-dimethyl-3-mercaptopropynyl) -L-cysteine. (The following formula [〓]) is mentioned.

[0014]

[Chemical 3]

[Chemical 4]

[Chemical 5]

Details of the action of this compound on VEGF will be described in the Examples section. However, this compound remarkably suppresses the expression of VEGF-mRNA, and also human Gl.
It was found that the release of VEGF from ioblastoma cells was significantly suppressed.

The compound can be administered orally or parenterally. Dosage forms include tablets, capsules,
Granules, powders, injections and the like can be mentioned, and they can be formulated using a commonly used technique. For example, oral agents such as tablets, capsules, granules, and powders include bulking agents such as lactose, crystalline cellulose, starch, and vegetable oils, lubricants such as magnesium stearate and talc, hydroxypropyl cellulose, polyvinylpyrrolidone, and the like. A binder, a disintegrating agent such as carboxymethylcellulose calcium and low-substituted hydroxypropylmethylcellulose, a coating agent such as hydroxypropylmethylcellulose, macrogol, and a silicone resin, a film forming agent such as a gelatin film may be added as necessary.

The dose of the present compound can be appropriately selected according to the symptoms, age, dosage form, etc., but in the case of an oral preparation, it is usually 0.1 to 5000 mg, preferably 1 to 1000 mg, once or divided into several times. Be administered.

The results of the pharmacological test are shown below, but this is for better understanding of the present invention and does not limit the scope of the present invention.

[0019]

【Example】

[1. Effect of suppressing VEGF-mRNA expression] Glial cells isolated from bovine retina (Virchows Arch., 426, 479-486).
(1995)) and cultured glial cells of rat (Nature,
359, 843-845 (1992)) in hypoxia.
It has been reported that enhanced expression of GF-mRNA is observed. Therefore, using this rat-derived cultured glial cell (hereinafter referred to as C6 glial cell), VE of the present compound
The effect on the expression of GF-mRNA was examined.

In order to confirm whether or not the function of the cell itself is affected, glyceraldehyde-3-phosphate dehydrogenase (G3PDH) -which is involved in the pentose-phosphate cycle, which is one of the glucose oxidation pathways, is confirmed. The effect on the expression of mRNA was also examined.

(Experimental Method) C6 glial cells that had been cultured to confluence were cultured in a culture medium containing a test compound under conditions of 95% N2.5% CO2. 18
After a while, from this C6 glial cell, Quick Prep yo mRNA Pu
mRNA was separated and purified using a rification Kit (Pharmacia). Then, mRNA was converted into cDNA using mRNA PCR Kit (Takara), and VEGF was added.
Alternatively, PCR was performed using the G3PDH primer.
Agarose electrophoresis was performed on 10 μl of the PCR product. Amount of VEGF-mRNA and G3PDH-mR
The amount of NA was analyzed by an image analyzer.

(Results) As an example of the experimental results, when bucillamine (500 μM) was used as the test compound, the expression of VEGF-mRNA was reduced to 1/5 by the addition of bucillamine.

Bucillamine had no effect on the expression of G3PDH-mRNA. This indicates that the VEGF-mRNA expression-suppressing effect observed with bucillamine was not caused by suppressing the function of C6 glial cells themselves.

From this fact, bucillamine was found to be VEGF-m
It was revealed that the expression of RNA was specifically suppressed.

[2. VEG from human Glioblastoma cells
F release inhibitory effect] Human Glioblastoma cell (U87MG cell)
Release of VEGF in the non-stimulated state, Proc. Natl.
Acad. Sci., 93,8502-8507 (1996). VE
To confirm the inhibitory effect of GF release, the above human Gliobl
astoma cells and Human VEGF measurement kit- (IB
L) (manufactured by Immune Biology Research Institute) was used to examine the VEGF release inhibitory effect of the mercaptoacylamino derivative.

(Preparation of human Glioblastoma cells) 1. 25 ml of a mixed solution of 10% fetal bovine serum and D-MEM / high glucose medium (hereinafter referred to as a culture solution) and human Glioblas
75mm2 of 1ml toma cell suspension (U87MG; ATCC)
Add to the culture container of and incubate overnight. 2. Discard the supernatant of the culture and replace with 25 ml of fresh culture. 3. After that, exchange the culture medium within 5 days. When the cells grow and become near confluence,
Subculture is performed by the following procedure. 4. The supernatant of the culture solution is discarded from the culture container and washed with 10 ml of phosphate buffered saline. 5. At room temperature, 5 ml of trypsin / ethylenediaminetetraacetic acid solution is added and human Glioblastoma cells are detached from the wall surface of the container. 6. Add 10 ml of the culture solution to this and transfer to a centrifuge tube. Centrifuge for 5 minutes at 1000 rpm. 7. The supernatant is discarded and 5 ml of phosphate buffered saline is added to suspend. 8. Centrifuge again at 1000 rpm for 5 minutes, discard the supernatant, and add 5 ml of the culture medium to suspend it to give a cell suspension.

(Preparation of test compound solution) 1. 2.6 × 1 was prepared by dissolving 5.2 × 10 −5 mol of the test compound in 2 ml of 25% ethanol / phosphate buffered saline.
Make a 0-2M solution.

(Experimental Method) 0.5 ml of culture solution and cell suspension (5.0 × 104 cells) were placed on a culture plate.
0.1 ml of s / ml) was added and cultured for 2 days. Each well
Remove the supernatant of the culture broth of 95% N2.5
0.5 ml of the culture solution replaced with% CO2 mixed gas was added. To this, 10 µl of a 2.6 × 10 -2 M test compound solution
Was added to give a final concentration of 500 μM.

After culturing this for 24 hours in a flex sampler filled with 95% N2.5 / 5% CO2 mixed gas, the supernatant of each well was separated and the VEGF concentration in the supernatant was determined by the Human VEGF measurement kit- ( IBL) (manufactured by Immune Biology Institute) was used for measurement.

As a control, 25% ethanol / phosphate buffered saline 10 was used instead of the test compound solution.
μl was added, and other operations were performed in the same manner as above.

The VEGF release inhibition rate was calculated by the following (formula 1).

(Formula 1) VEGF release inhibition rate (%) = [1- (VEGF concentration when using test compound solution / VEGF concentration when controlling)] × 100

(Results) As an example of the experimental results, as test compounds, bucillamine, thiopronin and N- (2,2
Table 1 shows the experimental results using -dimethyl-3-mercaptopropionyl) -L-cysteine (Compound A).

[0034]

[Table 1]

As shown in Table 1, the compound was VEGF.
The amount of liberation of was significantly reduced with respect to the control.

[0036]

EFFECTS OF THE INVENTION From the results of the above-mentioned pharmacological test, the present compound shows that VEGF-mRNA expression inhibitory action and human Glioblas
It has an inhibitory effect on VEGF release from toma cells, and
Excellent as a therapeutic agent for diseases involving EGF, particularly retinal thrombotic diseases such as retinal vein occlusion and retinal artery occlusion and retinal diseases such as macular degeneration, neovascular glaucoma and tumors such as hemangiomas There is expected. -1- Reference number 98J03E

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI A61P 43/00 111 A61P 43/00 111

Claims (4)

(57) [Claims] 1. A vascular endothelial growth factor inhibitor comprising a compound represented by the following general formula [〓] or a salt thereof as an active ingredient. [Chemical 1] [In the formula, R1 represents a hydrogen atom, a lower alkyl group, an amino lower alkyl group, a hydroxy lower alkyl group, a mercapto lower alkyl group or a carboxy lower alkyl group.
A represents a lower alkylene group. ] 2. The inhibitor according to claim 1, wherein the vascular endothelial growth factor is a vascular endothelial growth factor existing in the retina. 3. A vascular endothelial growth factor inhibitor containing bucillamine or a salt thereof as an active ingredient. 4. The inhibitor according to claim 3, wherein the vascular endothelial growth factor is a vascular endothelial growth factor existing in the retina.