JPH11255647A - Medicine for disease caused by smooth muscle dedifferentiation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a medicine having a differentiation-inducing action into smooth muscle cells, having a low mol.wt. and useful for preventing or treating diseases caused by smooth muscle dedifferentiation by compounding a specific ascorbic acid or its derivative as an active ingredient. SOLUTION: This medicine contains ascorbic acid of the formula [R<1> and R<2> are each H, a (substituted) lower alkyl, a (substituted) lower alkenyl or the like; R<3> and R<4> are each H, a (substituted) lower alkyl, a (substituted) lower alkylsulfonyl or the like] or its derivative or its pharmacologically acceptable salt as an active ingredient. The compound of the formula is preferably a compound selected from the group consisting of L-ascorbic acid, L-ascorbic acid 2-phosphoric acid, 5,6-O-isopropylidene-L-ascorbic acid, etc., and is preferably administered at a daily dose of, for example, 50-300 mg for an adult with one or more portions.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a therapeutic agent for diseases caused by smooth muscle dedifferentiation. More specifically, the present invention relates to the treatment of smooth muscle cells which dedifferentiate and abnormally proliferate by administration of a component exhibiting smooth muscle differentiation inducing activity. The present invention relates to a therapeutic agent for a disease caused by smooth muscle dedifferentiation for inducing differentiation and withdrawing from abnormal proliferation.

[0002]

2. Description of the Related Art Smooth muscle cells are differentiated into muscle cells in tissues such as the circulatory, digestive, respiratory and genitourinary tracts, and their homeostasis is maintained by their contraction and relaxation. ing. Such smooth muscle cells, unlike other muscle cells such as skeletal muscle cells and cardiomyocytes, have plasticity in the state of differentiation. That is, when skeletal muscle cells and cardiomyocytes differentiate to the final stage, they do not dedifferentiate again, whereas smooth muscle cells easily dedifferentiate under various conditions. For example, in the case of vascular smooth muscle cells, vascular endothelial cells are damaged, platelet adhesion occurs, and macrophages and lymphocytes infiltrate into the vascular intima, resulting in dedifferentiation.

[0003] The dedifferentiated smooth muscle cells are called a synthetic type, and have enhanced migration and proliferation ability. Abnormal proliferation and thickening of synthetic smooth node cells occur, resulting in exacerbation of diseases such as narrowing of vascular lumen, arteriosclerosis, or restenosis after percutaneous coronary angioplasty (PTCA). Will be. Such diseases caused by abnormal proliferation and thickening due to dedifferentiation of smooth muscle cells include restenosis after percutaneous angioplasty (hereinafter also referred to as PTA) in addition to arteriosclerosis and restenosis after PTCA. , Coronary spasm, hypertensive nephropathy, glomerulonephritis, bronchial asthma,
Dysuria and leiomyomas or leiomyosarcoma, including fibroids and gastric sarcomas, are known. Cell proliferative vasculitis seen in transplanted organs is also characterized by thickening of the vascular intima due to dedifferentiation of smooth muscle cells. In particular, coronary atherosclerosis, which frequently occurs early after heart transplantation, is an important factor that determines the prognosis or survival period of transplantation, and the lack of effective preventive and ameliorating agents has become an important problem.

[0004] As described above, although dedifferentiation of smooth muscle cells causes serious diseases that are life-threatening, there is no effective preventive or therapeutic drug. The reason is that little is known about the mechanism of controlling smooth muscle cell differentiation. Drugs that suppress the growth of smooth muscle cells are being actively searched for, but there are problems with cell selectivity, and no clinically effective drug has been known so far. As described above, as a therapeutic agent for various diseases caused by dedifferentiation of smooth muscle cells, development of an agent that induces differentiation into smooth muscle cells has been desired.

Recently, it has been reported that hexamethylene bisacetamide, an ADP-ribosylation inhibitor, and genistein, a tyrosine kinase inhibitor, have an effect of delaying the dedifferentiation of cultured smooth muscle cells (Grainger, D., et al.
J. et al., Biochem. J. 283 , 403-408, 1992. and Heidin, U.
Et al., Arterioscler.Thromb.Vasc.Biol. 17, 1977-1984,199
7.). However, it is unclear whether these compounds have the effect of inducing dedifferentiated synthetic smooth muscle cells to differentiate.

[0006] On the other hand, it has been already confirmed that ascorbic acid and a pharmaceutically acceptable salt thereof have an activity of inducing differentiation of skeletal muscle cells and osteoblasts. (Hata Ryuichiro, Senoo Haruki, Vitamin, 69 , 675-688, 1995). However, it is not known at all that it has a smooth muscle differentiation inducing effect. Also, it has been reported that ascorbic acid has an effect of suppressing restenosis after PTCA (Tomoda H. et al., Am. J.
Cardio1. 78, 1284-1286,1996). It has not been reported whether this inhibitory effect acts directly on smooth muscle cells.
Moreover, it has not been reported at all whether it is effective for other diseases caused by smooth muscle dedifferentiation. No low molecular compound other than ascorbic acid having a smooth muscle differentiation-inducing action has been reported at all.

[0007]

DISCLOSURE OF THE INVENTION The present invention has been made to find a low molecular weight compound having an activity of inducing differentiation into smooth muscle cells, and to use it as an active ingredient for the prevention or treatment of the above-mentioned diseases caused by smooth muscle dedifferentiation. To provide a remedy for the disease.

[0008]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that ascorbic acid or a derivative thereof has a smooth muscle differentiation inducing action, and completed the present invention. That is, the present invention provides a compound represented by the general formula (I):

[0009]

Embedded image

Wherein R ¹ and R ² are the same or different and are hydrogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkanoyl, substituted or unsubstituted lower alkoxy Carbonyl, substituted or unsubstituted aralkyl, sulfo,
Phospho, or represents a Pirohosuho may represent R ¹ and a lower alkylene or a substituted or unsubstituted Kishireniren of R ² is a substituted or unsubstituted together, R ³ and
R ⁴ is the same or different and is hydrogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkylsulfonyl, substituted or unsubstituted arylsulfonyl, substituted or unsubstituted lower alkanoyl, or substituted or unsubstituted lower alkoxy Represents carbonyl or R ³ and R ⁴
May be substituted or unsubstituted lower alkylidene or substituted or unsubstituted cycloalkylidene) ascorbic acid or a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient Provided is a therapeutic agent for a disease caused by dedifferentiation.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to the preparation of ascorbic acid or a derivative thereof represented by the general formula (I) or a pharmaceutically acceptable salt thereof (hereinafter, also simply referred to as "ascorbic acid or a derivative thereof"). A therapeutic agent for diseases caused by smooth muscle dedifferentiation, characterized in that it is contained as a muscle differentiation-inducing active ingredient.

In the definition of each group of the formula (I), lower alkyl includes straight-chain or branched C1-8, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, sec-pentyl, tert
-Pentyl, hexyl, isohexyl, heptyl, octyl, isooctyl and the like are included, and the lower alkyl part of lower alkanoyl, lower alkoxycarbonyl and lower alkylsulfonyl has the same meaning as the above lower alkyl. Lower alkylene is formed by removing one hydrogen from the lower alkyl, and lower alkylidene is formed by removing one hydrogen from the α carbon of the lower alkyl. Examples of cycloalkylidene include those having 3 to 8 carbon atoms, such as cyclopropylidene, cyclobutylidene, cyclopentylidene, cyclohexylidene, cycloheptylidene, cyclooctylidene, and the like. Examples include branched C 2 -C 6, such as vinyl, allyl, propenyl, methacryl, butenyl, crotyl, pentenyl, hexenyl and the like. As aralkyl,
C7 to C15 benzyl, phenethyl, benzhydryl, naphthylmethyl and the like are included, and the aryl moiety of arylsulfonyl includes phenyl, naphthyl and the like.

Substituted lower alkyl, substituted lower alkenyl,
Substituents in substituted lower alkanoyl, substituted lower alkoxycarbonyl, substituted lower alkylene, substituted lower alkylidene, substituted cycloalkylidene may be the same or different and have 1 to 3 substituents, such as hydroxy, lower alkoxy, aralkyloxy, carboxy, lower alkoxy. Includes carbonyl, aryl, amino, mono-lower alkyl substituted amino, di-lower alkyl substituted amino, nitro, halogen, sulfonamide, trifluoromethyl and the like. Here, the alkyl moiety in lower alkoxy, lower alkoxycarbonyl, mono-lower alkyl-substituted amino and di-lower alkyl-substituted amino has the same meaning as the lower alkyl. Aryl is as defined above, and the aralkyl moiety in aralkyloxy is also as defined above. Further, halogen means each atom of fluorine, chlorine, bromine and iodine.

The substituents of the substituted aralkyl, substituted arylsulfonyl, substituted lower alkylsulfonyl and substituted xylenylene may be the same or different and have 1 to 3 substituents such as hydroxy, lower alkyl, lower alkoxy, lower alkanoyloxy, nitro, halogen and aralkyl. Oxy,
Sulfonamide, trifluoromethyl and the like are included.
Here, the alkyl moiety in lower alkyl, lower alkoxy, and lower alkanoyloxy has the same meaning as the lower alkyl. In the aralkyloxy, the aralkyl moiety and halogen are as defined above.

Some of the ascorbic acid represented by the general formula (I) or a derivative thereof may have a positional isomer, a geometric isomer, an optical isomer, a diastereomer, a tautomer and the like. However, the invention embraces all these isomers and their mixtures.

Ascorbic acid or a derivative thereof represented by the general formula (I) is preferably L-ascorbic acid, L-ascorbic acid 2-phosphate, or 5,6-O
-Isopropylidene-L-ascorbic acid, L-ascorbic acid 2-sulfate, (+)-5,6-O-cyclohexylidene-L-ascorbic acid, D-isoascorbic acid, D-isoascorbic acid 2-phosphate Acid or 5,6
-O-isopropylidene-D-isoascorbic acid, D
-Isoascorbic acid 2-sulfuric acid, (-)-5,6-O-
Cyclohexylidene-D-ascorbic acid. Examples of the metal contained in the pharmaceutically acceptable salt of ascorbic acid or a derivative thereof represented by the general formula (I) include, for example, sodium, potassium, calcium, barium, iron, zinc, chromium and the like.

In the present invention, diseases caused by dedifferentiation of smooth muscle cells include arteriosclerosis, restenosis after PTCA, restenosis coronary spasm after PTA, and mammals including humans.
Diseases such as coronary spasm, hypertensive nephropathy, glomerulonephritis, bronchial asthma, dysuria, leiomyoma, leiomyosarcoma, and cell proliferative vasculitis found in transplanted organs are exemplified.
The present invention is not limited to this, and can be applied to any disease that can induce and treat smooth muscle differentiation. That is, the therapeutic agent of the present invention induces differentiation of smooth muscle cells that are dedifferentiated and abnormally proliferates, and is effective against diseases caused by the dedifferentiation of these smooth muscle cells by inducing differentiation and withdrawal from abnormal proliferation. Useful as a prophylactic or therapeutic agent. The therapeutic agent of the present invention is a drug for the purpose of treating, reducing (improving symptoms), maintaining (preventing deterioration), or preventing the above diseases.

The differentiation-inducing agent of the present invention comprises, in addition to the ascorbic acid derivative represented by the general formula (I) or a pharmaceutically acceptable salt thereof, if necessary, a pharmaceutically acceptable carrier or a known carrier. It can be formulated into a formulation suitable for oral or parenteral administration depending on the purpose, together with the drug and other additives. Furthermore, it is also possible to prepare a sustained-release preparation by a known technique. Examples of the oral preparation include solid preparations such as powders, granules, capsules, and tablets, and liquid preparations such as syrups, elixirs, and emulsions. Powders can be obtained, for example, by mixing with excipients such as lactose, starch, crystalline cellulose, calcium lactate, calcium hydrogen phosphate, magnesium metasilicate aluminate, and silicic anhydride.

Granules may be used in addition to the above-mentioned excipients, if necessary.
For example, it can be obtained by further adding a binder such as sucrose, hydroxypropylcellulose, polyvinylpyrrolidone, starch and the like, and a disintegrant such as carboxymethylcellulose, carboxymethylcellulose calcium, starch and the like, and granulating by wet or dry method. Tablets, the powder or granules as they are, or magnesium stearate,
It can be obtained by adding a lubricant such as talc and tableting.
Dragees can be prepared using hydroxypropylcellulose phthalate, titanium oxide, gelatin, sucrose and the like. Further, the tablets or granules are coated with an enteric base such as hydroxypropylcellulose phthalate, methyl methacrylate copolymer, hydroxypropylmethylcellulose acetate succinate, cellulose acetate phthalate, or with ethylcellulose, carnauba wax, hardened oil and the like. It can be coated and made into enteric or sustained release formulations.

A hard capsule can be obtained by filling the above powder or granule into a hard capsule. Soft capsules can be obtained by dissolving ascorbic acid or a derivative thereof in glycerin, polyethylene glycol, sesame oil, olive oil, or the like, and coating this with a gelatin film. The syrup can be obtained by dissolving a sweetener such as sucrose, sorbitol, glycerin and ascorbic acid or a derivative thereof in water. An elixir may be prepared by adding an essential oil, ethanol, etc. in addition to the sweetener and water, or gum arabic, tragacanth, polysorbate 8
0, carboxymethylcellulose sodium, lecithin, crystalline cellulose / carmellose sodium, macrogol, polyethylene glycol and the like can be added to make an emulsion or suspension. In addition, flavoring agents, coloring agents, preservatives, and the like can be added to these liquid preparations as needed.

Parenteral preparations include injections, rectal preparations, pessaries, skin external preparations, inhalants, aerosols,
Eye drops and the like can be mentioned. Injectables may be added to ascorbic acid or a derivative thereof, as necessary, with hydrochloric acid, sodium hydroxide, lactic acid, sodium lactate, acetic acid, sodium acetate, sodium monohydrogen phosphate, sodium dihydrogen phosphate, potassium monohydrogen phosphate, phosphorus PH adjusters such as potassium dihydrogenate, tonicity agents such as sodium chloride and glucose,
After addition of distilled water for injection and sterile filtration or heat steam sterilization (autoclave), it can be obtained by filling into an ampoule. Further, a sugar alcohol such as mannitol, dextran, gelatin or the like is further added, followed by freeze-drying under vacuum to obtain a dissolution type injection for use. In addition, ascorbic acid or a derivative thereof is added with an emulsifier such as lecithin, polysorbate 80, polyoxyethylene hydrogenated castor oil, macrogol, a solubilizer, or a solubilizing agent, and then emulsified in water to form an emulsion for injection. Can also.

The preparation for rectal administration is prepared by adding a suppository base such as mono-, di- or triglyceride of cocoa fatty acid, polyethylene glycol or the like to ascorbic acid or a derivative thereof, and then dissolving the mixture by heating, and pouring it into a mold. It can be obtained by cooling or dissolving ascorbic acid or a derivative thereof in polyethylene glycol, soybean oil and the like, and then coating with a soft rectal capsule, a gelatin film or the like.

External preparations for the skin include ascorbic acid or derivatives thereof, white petrolatum, beeswax, liquid paraffin,
Add polyethylene glycol etc. and heat if necessary,
It can be obtained by kneading. A poultice or a tape can be obtained by kneading ascorbic acid or a derivative thereof with a pressure-sensitive adhesive such as rosin or an alkyl acrylate polymer, and spreading this on a nonwoven fabric or the like. Inhalants can be obtained by dissolving or dispersing ascorbic acid or a derivative thereof in a propellant such as a pharmaceutically acceptable inactivating gas (nitrogen gas, carbon dioxide gas, etc.), and filling this in a pressure-resistant container. it can.

The administration method of the differentiation-inducing agent of the present invention is not particularly limited, and an administration method according to the target disease and the above-mentioned dosage form is used. The dosage is appropriately determined depending on the disease to be treated, the age, weight, health condition, and condition of the patient, but is generally 50 mg to 3000 mg per adult per day,
It is preferably administered in the range of 300 mg to 2000 mg once a day or more.

[0025]

The present invention will be described below with reference to examples. Example 1 Differentiation-Inducing Action on Bone Marrow Stromal Cells Having Pluripotency The bone marrow stromal cells are cells that exist in the bone marrow and maintain the proliferation ability of hematopoietic cells. It has been reported that among these bone marrow stromal cells, cells which differentiate in a smooth muscle-like manner when cultured in vitro are present (Li, J. et al. Exp. Hemat. 23 , 133-141, 1995).

Then, established bone marrow stromal cell lines TBRB and TBR10-1 established from temperature-sensitive SV40 large T antigen gene transgenic mice (Okuyama, R. et al., Exp. Cell Res. 218 , 424-429, 1995). ,
Kameoka, J et al., J. Cell. Physiol. 164 , 55-64, 1995)
Indicates the passage medium, that is, 2% fetal bovine serum (FCS, Hyclo
ne Laboratories lnc., Logan, UT, USA) 10 μg
/ Ml bovine transferrin (Canadian Bioclinical Ltd.
Scarborough), Canada) 1 μg / ml insulin (Canadian Bioclinical Limited (Ca)
nadian Bioclinical Ltd. Scarborough), Canada),
RIT containing 10 ng / ml human EGF (Yukinaga Pharmaceutical)
33 ° C., 5% CO _{2 in} C80-7 medium (Kyokuto Pharmaceutical)
It is known that culturing under conditions allows passage while maintaining an undifferentiated state. However, these cells were transformed into α-MEM (Life Technologies, Inc., Grandls) containing a differentiation medium (10% FCS, 50 μM β-mercaptoethanol (Nacalai Tesque)).
land, NY, USA) at 33 ° C and 5% CO ₂
It has been reported that when cultured for more than a week, it differentiates into smooth muscle and expresses smooth muscle-specific proteins, calponin, SM22α, α-actin, and high-molecular-weight caldesmon (Arakawa et al., No. 20). Annual Meeting of the Molecular Biology Society of Japan, p-50, 1997).

Therefore, the undifferentiated TBRB strain and the TBR10-1 strain cultured in the subculture medium are cultured in the subculture medium containing ascorbic acid or a derivative thereof,
Using the expression of calponin, SM22α, α-actin as an index, the inducing action of ascorbic acid and its derivatives on smooth muscle differentiation was evaluated. Specifically, 50 μg / ml L
The TBRB strain was cultured for 9 days in a subculture medium to which -ascorbic acid or L-ascorbic acid 2-phosphate was added, and the expression of smooth muscle-specific protein was examined. The cells cultured in a 35 mm culture dish (Iwaki Glass) were added to Dulbecco's PBS.
(Dainippon Pharmaceutical), 250 μl of RlPA buffer (20 mM Tris-HCl, pH 7.5, 0.1%
A cell extract was prepared with SDS, 1% Triton X-100, 1% sodium deoxycholate). Lae
mmli method (Laemmli, UK, Nature 227 , 680-685,
1970), SDS-polyacrylamide gel electrophoresis was performed to separate proteins. Gel to ClearBlot
-P membrane (Ato) to Tobin's method (Towbin, H.
Natl. Acad. Sci. USA. 76 , 4350-4354, 197.
In 9), the protein was transcribed. In addition, ECL Western Blotting Detection System (Amersham International plc, Buckinghamshire),
(UK) was used to detect α-actin protein.
The anti-α-actin antibody used was from Sigma.

FIG. 1A shows the results of the Western blotting. Lane 1 shows a system in which L-ascorbic acid was added to the subculture medium, and lane 2 shows a system in which L-ascorbic acid 2-phosphate was added to the subculture medium. Lane 3 shows the results in the passage medium, and Lane 4 shows the results in the differentiation medium. It can be seen that treatment with 50 μg / ml L-ascorbic acid or L-ascorbic acid 2-phosphate induces the expression of α-actin protein as in the case of culture in a differentiation medium.

In addition, 30 μM 5,6-O-
Isopropylidene-L-ascorbic acid, 30 μM L
-Ascorbic acid 2-sulfuric acid, 300 μM (+)-5
6-O-cyclohexylidene-L-ascorbic acid was added, and the TBR10-1 strain was cultured for 5 days, and the expression of α-actin was examined in the same manner as described above. As the secondary antibody, an alkaline phosphatase-conjugated antibody (Promega Corp. Madison, USA) was used, and a Vistra ECF Western blotting detection system (Amersham International (A)
mersham International plc, Buckinghamshire), UK) using a fluoroimager (FluorImager,
Molecular dynamics, Inc. (Molecular Dyna
mics lnc. Sunnyvale), USA).

FIG. 1B shows the result of the Western blotting. In FIG. 1B, lane 1 is a subculture medium, lane 2 is a differentiation medium, and lane 3 is a subculture medium containing 5,6-O-
A system to which isopropylidene-L-ascorbic acid was added, lane 4 was a system to which L-ascorbic acid 2-sulfate was added to the subculture medium, and a lane 5 was (+)-5, 6 to the subculture medium.
It is the result of the system which added -O-cyclohexylidene-L-ascorbic acid. From these results, 5,6-O-
When any of isopropylidene-L-ascorbic acid, L-ascorbic acid 2-sulfate, and (+)-5,6-O-cyclohexylidene-L-ascorbic acid is added, the cells are cultured in a differentiation medium. It can be seen that the expression level increases to the same extent or more.

Furthermore, the mRNA transcript level of the smooth muscle-specific gene was determined using reverse transcriptase-PC.
It examined using the R (RT-PCR) method. 50 in subculture medium
μg / ml L-ascorbic acid or L-ascorbic acid 2-phosphate was added, and the total R was determined from the TBRB strain cultured for 9 days using RNeasy (Qiagen GmbH, Germany).
NA was extracted. From 1 μg of this total RNA, a Superscript Preamplification System (Su
perScript Preamplification System, Life Technologi
es, Inc., Gaitherburg, USA). Using this cDNA, PCR was performed by a known method. The composition of the PCR reaction solution was 1 mM Tris-HC
1, pH 8.3, 0.15 mM MgCl ₂ , 5 mM
KCl, 200 mM dATP, 200 mM dCT
P, 200 mM dGTP, 200 mM dTTP, 2
50 nM primer, 2.5 U Taq DNA polymerase (Boehringer Mannheim)
nheim GmbH, Mannheim), Germany).
I went in. The primers used were calponin sense primer (SEQ ID NO: 1), calponin antisense primer (SEQ ID NO: 2), SM22α sense primer (SEQ ID NO: 3), SM22α antisense primer (SEQ ID NO: 4), α-actin And an antisense primer for α-actin (SEQ ID NO: 6). Further, as a control, glyceraldehyde triphosphate dehydrogenase (GAPD)
H) sense primer (SEQ ID NO: 7) and GAPDH antisense primer (SEQ ID NO: 8) were used.

The PCR was performed after denaturation at 95 ° C. for 5 minutes, followed by 9
These conditions were repeated for 20 cycles, with one cycle consisting of 45 ° C. for 5 seconds, 1 minute at 60 ° C., and 2 minutes at 72 ° C. The resulting reaction product was subjected to agarose gel electrophoresis, and a band was detected. The result is shown in FIG. In FIG. 2, lane 1 shows L-ascorbic acid added to the subculture medium, and lane 2
Added L-ascorbic acid 2-phosphate to the subculture medium, 3
Indicates the results of the passage medium and 4 indicates the results of the differentiation medium. GADPH
Is considered to have a substantially constant expression level regardless of the differentiation state. Therefore, looking at the expression levels of calponin, SM22α, and α-actin with respect to the expression level of GADPH, L-ascorbic acid or L-ascorbic acid 2-
It can be seen that when treated with phosphoric acid, the expression of calponin, SM22α, α-actin was induced more strongly than when cultured in a differentiation medium. From the above results, in the undifferentiated bone marrow stromal cell line, the action of inducing differentiation of ascorbic acid and its derivatives into smooth muscle cells was confirmed.

Example 2 Differentiation-Inducing Effect on Cultured Rat Smooth Muscle Cells SM1 is one of smooth muscle-specific proteins, and smooth muscle cells dedifferentiated at disease sites such as arteriosclerosis and restenosis after PTCA. It is known that its expression is reduced in M. (Machinori Aikawa, Ryozo Nagai, Mol. Med. 33, 141)
4-1422, 1996). Therefore, the differentiation-inducing effect of ascorbic acid and its derivatives on synthetic rat cultured smooth muscle cells was evaluated using the increased expression of SM1 as an index.

From rat thoracic and abdominal aortas, a known method (Campbell, JH et al., Arteriosclerosis. 9, 633-643, 1).
989). Culture was performed using Medium 199 with Earle's containing Earle's salt containing 10% FCS.
salt, Life Technologies lnc. Grand Island NY USA)
In 37 ° C., 5% CO ₂ . As a culture dish, collagen TYPEI coated shear (Iwaki Glass) was used. Spread the cells on a 12-well flat bottom plate, 0.3m
M ascorbic acid derivative was added and cultured for 2 weeks. The cells are subjected to the Vistra ECF Western Blotting System (Amersham International plc, Bucking hamsh).
Western blotting was performed according to the protocol of ire), UK) and smooth muscle type myosin heavy chain (SM
1) The expression of the protein was examined. Primary antibodies are known (Ha
segawa K et al., J. Mol. CeI. Cardiol. 29 , 2177-2186, 1997)
Was used. The secondary antibody is an alkaline phosphatase conjugated antibody (Promega Corp.
on), USA) using a fluoroimager (FluorImag
er, Molecular Dynamics Inc. (Molecular
Dynamics lnc. Sunnyvale), USA).

FIG. 3 shows the results of the examination by Western blotting. In FIG. 3, 1 is no addition, 2 is L
-Ascorbic acid addition, 3 shows the results of dehydroascorbic acid addition, 4 shows the results of 5,6-O-isopropylidene-L-ascorbic acid addition. It can be seen that treatment with 0.1 mM L-ascorbic acid and 5,6-O-isopropylidene-L-ascorbic acid increases the expression level of SM1 protein. Also, dehydroascorbic acid, which is an oxidized form of L-ascorbic acid, had no effect, suggesting that the reduced form is important for smooth muscle differentiation induction.

Next, in order to examine the time-dependent change in the smooth muscle differentiation-inducing action, 0.3 mM L-ascorbic acid 2-phosphate was added and cultured for 4, 7, 10 and 14 days to examine the expression of SM1 protein. Was. As a result, it was shown that the expression was maintained at a high level even on day 7 and thereafter, and that the action was sustained (FIG. 4).

Example 3 Induction of Differentiation of Rat Balloon Injured Blood Vessels on Smooth Muscle Cells Male SD weighing 360-440 g
Rats (Charles River Japan) were anesthetized with Nembutal, and then exposed the left common carotid artery and left external carotid artery.
A 2F balloon catheter (Edwards Laboratories) was inserted retrograde into the left common carotid artery from the left external carotid artery. Endothelial injury was performed by inflating the balloon with air and pulling to the catheter insertion site. After repeating this operation seven times, the catheter was removed, the external carotid artery was ligated and the wound was sutured. L-ascorbic acid was suspended in distilled water and orally administered at a dose of 3 g / kg once a day immediately before balloon injury and up to 6 days after injury.

On day 7 of the injury, the left and right carotid arteries were removed and
Frozen under nitrogen and crushed in a mortar. The RIPA bag
Far [20 mmol / L Tris-HCl (pH 7.
5), 0.1% (w / v) SDS, 0.01% (w / v)
v) Triton X-100, 0.01% (w / v)
Sodium oxycholate, 0.1 mmol / L pA
PMSF], and extraction operation was performed. every
The supernatant was collected by centrifugation at 14,000 rpm, and
Used for Park quantification, the remainder is SDS-polyacrylamide
It was subjected to gel electrophoresis. 5% poly for SM1
Krylamide gel [Atosha] for calponin
Is a 10% gel [Atto] using electrophoresis buffer
[25 mmol / L Tris, 250 mmol / L glyci
, 0.1% (w / v) SDS]. Swimming
After running, blot the gel on a nitrocellulose membrane and blot.
After performing the locking operation,
Was served. Anti-SM1 antibody (Hasegawa K.
 J. Mol. Cell. Cardiol. 29, 2177-2186, 1997),
Using anti-calponin antibody [Sigma] as secondary antibody
Therefore, alkaline phosphatase-labeled anti-
IgG antibody [Sigma], calponin is alkaline
Phosphatase-labeled anti-mouse IgG antibody [Sigma]
used. Attophos [Molecular dynamics and
Amersham] Fluorescence obtained using solution as substrate
FluorImager, Molecular Dyna
Mix Inc. (Molecular Dynamics lnc. Sunnyval
e), US)), and bands were digitized at the same time. Ma
To compare the fluorescence intensity between different gels,
The extract prepared separately from the rat carotid artery was used as the standard sample.
Run on each gel with other samples and digitized
The fluorescence intensity of each band is compared to the fluorescence intensity of the standard sample.
It was displayed as a ratio and compared. The results are shown in FIG.
You. In FIG. 5, A is the result of SM1 and B is the result of calponin.
Where VI is the control group and AI is L-asco, respectively.
The values of the rubic acid administration group are shown. By causing injury
Smooth muscle cells dedifferentiate and express SM1 and calponin
Administration of L-ascorbic acid.
Decrease in their expression is suppressed, and L-ascorbic acid
Was confirmed to have a differentiation-inducing effect.

[0039]

According to the present invention, effective treatment for various diseases caused by dedifferentiation of smooth muscle cells based on the smooth muscle differentiation inducing activity of ascorbic acid and its derivatives represented by the general formula [1]. Can provide medicine.

[0040]

[Sequence List] SEQ ID NO: 1 Sequence length: 20 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: TAACCGAGGT CCTGCCTACG 20

SEQ ID NO: 2 Sequence length: 20 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: TGTGGGTGGG CTCACTCAGC 20

SEQ ID NO: 3 Sequence length: 40 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: ATCAAGCTTC GCTACTCTCC TTCCAGTCCA CAAACGACCA 40

SEQ ID NO: 4 Sequence length: 33 Sequence type: number of nucleic acid chains: single stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence: ATCGGATCCC TTCCCTTTCT AACTGATGAT CTG 33

SEQ ID NO: 5 Sequence length: 20 Sequence type: Number of nucleic acid chains: Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: GGCATCCACG AAACCACCTA 20

SEQ ID NO: 6 Sequence length: 20 Sequence type: number of nucleic acid chains: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence: CACGAGTAAC AAATCAAAGC 20

SEQ ID NO: 7 Sequence length: 20 Sequence type: number of nucleic acid chains: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence: TTCCAGTATG ACTCCACTCA 20

SEQ ID NO: 8 Sequence length: 20 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Type of sequence: Other nucleic acid Synthetic DNA Sequence: ATCACGCCAC AGCTTTCCAG 20

[Brief description of the drawings]

FIG. 1 shows that L-bone stromal cell line TBRB has L-
It is a photograph of the western blotting which examined the expression of (alpha) -actin protein when adding ascorbic acid or L-ascorbic acid 2-phosphate and culture | cultivating for 9 days. B
Is used to convert bone marrow stromal cell line TBR10-1 to 5,6-
When cultured for 5 days in a subculture medium supplemented with O-isopropylidene-L-ascorbic acid, L-ascorbic acid 2-sulfate, or (+)-5,6-O-cyclohexylidene-L-ascorbic acid 4 is a photograph of Western blotting in which the expression of α-actin protein was examined.

FIG. 2: Calponin, α-actin, S when bone marrow stromal cell line TBRB was cultured for 9 days after adding L-ascorbic acid or L-ascorbic acid 2-phosphate
It is a photograph of agarose gel electrophoresis in which the expression of M22α mRNA was examined.

[FIG. 3] Western blotting showing the expression of SM1 protein when L-ascorbic acid, 5,6-O-isopropylidene L-ascorbic acid and dehydroascorbic acid were added to rat cultured smooth muscle cells and cultured for 14 days. It is a photograph of.

FIG. 4 is a graph showing the relationship between the time after addition of L-ascorbic acid 2-phosphate to rat cultured smooth muscle cells and the expression level of SM1 protein.

FIG. 5: SM1 of smooth muscle cells at the site of injury when L-ascorbic acid was orally administered (AI) or not administered (VI) to rats with carotid artery balloon injury after 6 days. A) is a graph showing the expression levels of calponin (B).

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 31/375 ADU A61K 31/375 ADU AED AED // C07D 307/62 C07D 307/62

Claims (3)

[Claims] 1. A compound of the general formula (I) (Wherein R ¹ and R ² are the same or different and are hydrogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkanoyl, substituted or unsubstituted lower alkoxycarbonyl,
A substituted or unsubstituted aralkyl, sulfo, phospho, or represents a Pirohosuho may represent R ¹ and a lower alkylene or a substituted or unsubstituted Kishireniren of R ² is a substituted or unsubstituted together, R ³ and R ⁴ is the same or different and is hydrogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkylsulfonyl, substituted or unsubstituted arylsulfonyl, substituted or unsubstituted lower alkanoyl, or substituted or unsubstituted lower alkoxy Ascorbic acid or a derivative thereof or a pharmacologically acceptable derivative thereof, which may represent carbonyl, or R ³ and R ⁴ may together represent a substituted or unsubstituted lower alkylidene or a substituted or unsubstituted cycloalkylidene) Of diseases caused by smooth muscle dedifferentiation containing salt as active ingredient Medicine. 2. Ascorbic acid or a derivative thereof or a pharmaceutically acceptable salt thereof is L-ascorbic acid,
L-ascorbic acid 2-phosphate, 5,6-O-isopropylidene-L-ascorbic acid, L-ascorbic acid 2
-Sulfuric acid, (+)-5,6-O-cyclohexylidene-L
2. The therapeutic agent for a disease caused by smooth muscle dedifferentiation according to claim 1, which is a compound selected from the group consisting of ascorbic acid and a pharmaceutically acceptable salt thereof. 3. The method according to claim 1, wherein the ascorbic acid or a derivative thereof or a pharmaceutically acceptable salt thereof is L-ascorbic acid 2
-Phosphoric acid, 5,6-O-isopropylidene-L-ascorbic acid, L-ascorbic acid 2-sulfate, (+)-5
3. A compound selected from the group consisting of 6-O-cyclohexylidene-L-ascorbic acid and a pharmaceutically acceptable salt thereof, resulting from smooth muscle dedifferentiation according to claim 2. Drugs for treating diseases.