JPH10236953A - P21 inducer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prepare a p21 inducer capable of treating cancers and immunopathies by including a carbostyril derivative therein. SOLUTION: This medicine comprises a carbostyril derivative represented by the formula (R is benzoyl which may have a lower alkoxy as a substituent group on the phenyl ring; the C bond between the 3- and the 4-positions of the carbostyril skeleton is a single or a double bond) or its salt as an active ingredient. 6[4-(3,4-Dimethoxybenzoyl)-1-piperazinyl]-3,4-dihydropcarbostyril is exemplified as the compound represented by the formula. The compound represented by the formula in an amount of 1-70wt.% is contained in the composition and orally, perrectally or intrarectally administered. The daily dose thereof is 0.5-30mg based on 1kg body weight and 10-1,000mg active ingredient is contained in the administration unit form and daily administered in one to three divided portions. The medicine is effective in prophylaxis and treatment of Sjogren syndrome, autoimmune pancreatitis, systemic lupus erythematosus(SLE), etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a p21 (p21 ^WAF1 ) inducer containing a specific carbostyril derivative as an active ingredient.

[0002]

2. Description of the Related Art The tumor suppressor gene p53 (hereinafter referred to as "p53 gene") is one of the tumor suppressor genes attracting attention because it frequently mutates in many human tumors. And has been shown to play an important role in cell differentiation and induction of apoptosis (Proc. Natl. Acad. Sci., USA, 89 , 7491-7495 (1.
992); Cancer Res., 51 , 6304-6311 (1991); Nature, 3
52 , 345-347 (1991); Mol. Cell. Biol., 13 , 1415-1423.
(1993); Proc. Natl. Acad. Sci., USA, 89 , 4495-4499 (1
992); Cell, 74 , 777-779 (1993); Proc. Natl. Acad. Sc
i., USA, 88 , 8982-8986 (1991); Oncogene, 7 , 1853-1.
857 (1992); Blood, 83 , 2230-2237 (1994); Cell Grow
th & Differentiation, 7 , 21-30 (1996)). These functions of the wild-type p53 gene are inactivated by the mutant p53 gene (Cell, 70 , 523-52).
6 (1992)).

On the other hand, induction of p21 gene expression is
Has been found to be involved in cell growth arrest and apoptosis caused by E. coli and differentiation induction of certain cancer cells (Cell, 75 , 817-825 (1993); Cancer Res., 5 ).
4 , 1169-1174 (1994); Cell, 76 , 1013-1023 (1994); Pr
oc.Natl.Acad.Sci., USA, 89 , 7491-7495 (1992)). The p21 gene is located on human chromosome 6p21.2 and has a p53 DNA binding site of 2.p21 of the p21 coding sequence.
4 kb upstream (Cell, 75 , 817-825 (199
3)).

[0004] The 21-kD nucleoprotein encoded by the p21 gene has a G1-cyclin-dependent kinase inhibitory activity.
vity) (Cell, 75 , 805-816 (1993)), and p21 is a proliferating cell
It is known to directly inhibit DNA replication depending on nuclear antigen) (Nature, 369 , 574-578 (199
Four)).

[0005] Further, cell differentiation depends on G1 in the cell cycle.
Therefore, if G1 arrest can be induced in cancer cells, it is considered that differentiation and apoptosis can be induced in the cancer cells. Induction of the p21 gene is more likely to induce G1 arrest in such cells. It is thought to have a favorable effect on the treatment and treatment of cancer and immune disorders.

[0006] Furthermore, in autoimmune diseases, C is
Activation of D4 lymphocytes is thought to be involved, and p2
The expression of 1 acts to suppress the proliferation of this lymphocyte,
In addition, for example, it can suppress the production of cytokines that activate Th1 and Th2 lymphocytes and the like, and it is thought that they can treat various autoimmune diseases or abnormalities such as Sjogren's syndrome, autoimmune pancreatitis, and SLE. Can be

[0007] Thus, there is a need in the art to provide means for enabling p21 induction in these cells, and thereby enabling G1 arrest.

[0008]

SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide a p21 inducing means or a G1 arresting means for cells which meets the needs of the art.

The inventors of the present invention have conducted intensive studies for the above purpose, and as a result, have found that the carbostyril derivative represented by the following general formula (1) and a salt thereof induce the desired p21 gene expression. Here, the present invention has been completed.

[0010]

That is, according to the present invention, at least one compound selected from carbostyryl derivatives represented by the following general formula (1) and salts thereof is contained as an active ingredient. A p21 inducer is provided.

[0011]

Embedded image

According to the present invention, in particular, the p21 derivative described above, wherein the carbostyril derivative is 6- [4- (3,4-dimethoxybenzoyl) -1-piperazinyl] -3,4-dihydrocarbostyril or a salt thereof. An agent is provided.

In the p21 inducer of the present invention, the carbostyril derivative represented by the above general formula (1), a salt thereof, and a production method thereof as an active ingredient are described in, for example, Japanese Patent Publication No. 43747/1989. I have. The publication also describes that the carbostyril derivative is useful as a cardiotonic agent. Furthermore, for example, WO 93/00902 teaches that the carbostyril derivative has an ability to regulate apoptosis and an ability to induce cell differentiation and can be adapted to various diseases based on these actions. However, the p21 induction effect to G1 arrest induction effect according to the present invention are not related to the known effects described for the carbostyril derivative,
Of course, it cannot be predicted from these known functions.

The inducing agent of the present invention is useful for the treatment or treatment of cancer and various immune disorders, for example, Sjogren's syndrome, SLE, etc., based on the effect of inducing the expression of the p21 gene.

The inducing agent of the present invention induces the expression of the above-mentioned desired p21 gene via the induction of the p53 gene or directly without through the induction of the p21 gene, as shown in the following Examples. , The desired G1 arrest in the target cell is induced. Therefore, the present invention p
According to the use of the 21-inducing agent, a desired p21-inducing effect can be exhibited even in a target cell having a mutant p53,
Since it is considered that there is no such induction in normal cells, it can be used very satisfactorily for the above various diseases.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION Each group represented by the above general formula (1) is more specifically as follows. That is, as the lower alkoxy group, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-
Examples thereof include linear or branched alkoxy groups having 1 to 6 carbon atoms such as butoxy, pentyloxy and hexyloxy groups.

Examples of the benzoyl group which may have a lower alkoxy group as a substituent on the phenyl ring include, for example, benzoyl, 2-methoxybenzoyl, 3-methoxybenzoyl, 4-methoxybenzoyl, 2-ethoxybenzoyl, 3-ethoxybenzoyl , 4-ethoxybenzoyl, 3-isopropoxybenzoyl, 4-butoxybenzoyl, 2-pentyloxybenzoyl, 3-hexyloxybenzoyl, 3,4-dimethoxybenzoyl,
A phenyl ring such as 2,5-dimethoxybenzoyl, 3,4,5-trimethoxybenzoyl or the like may have 1 to 3 straight or branched alkoxy groups having 1 to 6 carbon atoms as substituents. A benzoyl group can be exemplified.

Among the above-mentioned carbostyryl derivatives as active ingredients in the present invention, particularly preferred are, for example, 6- [4- (3,4-dimethoxybenzoyl) -1-piperazinyl] -3,4-dihydrocarbostyril ( Hereinafter, "vesnarinone") can be exemplified.

The above-mentioned salts of the carbostyril derivatives include pharmacologically acceptable acid addition salts formed using ordinary acids. Specific examples of the acidic compound that forms the salt include inorganic acids such as sulfuric acid, phosphoric acid, nitric acid, hydrochloric acid, and hydrobromic acid, acetic acid, oxalic acid, maleic acid, fumaric acid, malic acid, tartaric acid, and citric acid. And organic acids such as succinic acid, ethanesulfonic acid, p-toluenesulfonic acid, and benzoic acid.

Formula (1) which is an active ingredient of the drug of the present invention
Is generally used in the form of general pharmaceutical preparations. Such a preparation is prepared by using a diluent or excipient such as a filler, a bulking agent, a binder, a humectant, a disintegrant, a surfactant and a lubricant which are usually used. Various forms can be selected as the pharmaceutical preparation depending on the purpose of treatment, and typical examples are tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, suppositories,
Examples include injections (solutions, suspensions, etc.).

In the case of forming into tablets, carriers conventionally known in this field can be widely used, such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, and silicic acid. Excipients such as water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose,
Binders such as potassium phosphate and polyvinylpyrrolidone, dried starch, sodium alginate, agar powder, laminaran powder, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, monoglyceride stearate, starch, lactose, etc. Disintegrants, sucrose, stearin, cocoa butter, disintegration inhibitors such as hydrogenated oil, quaternary ammonium bases, absorption promoters such as sodium lauryl sulfate, glycerin, humectants such as starch, starch, lactose, kaolin,
Examples thereof include adsorbents such as bentonite and colloidal silicic acid, and lubricants such as purified talc, stearates, powdered boric acid, and polyethylene glycol. Further, the tablets can be made into tablets coated with a usual coating, if necessary, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets or double tablets or multilayer tablets.

In the form of pills, carriers conventionally known in the art can be widely used, such as excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, talc, and the like. Examples thereof include rubber powder, tragacanth powder, binders such as gelatin and ethanol, and disintegrants such as laminaran agar.

For shaping in the form of suppositories, conventionally known carriers can be widely used, and examples thereof include polyethylene glycol, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides. it can.

When prepared as an injection, the solution and suspension are preferably sterilized and isotonic with blood. When formed into the form of these solutions, emulsions and suspensions, they may be diluted. All the agents commonly used in this field can be used as the agent, and examples thereof include water, ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. In this case, a sufficient amount of salt, glucose, glycerin, etc., for preparing an isotonic solution may be contained in the pharmaceutical preparation, and ordinary solubilizing agents, buffers, soothing agents, etc. are added. May be.

The G1 arrest inducer of the present invention may contain a coloring agent, a preservative, a fragrance, a flavoring agent, a sweetener, and other pharmaceuticals as necessary.

The amount of the active ingredient compound contained as an active ingredient in the medicament of the present invention is not particularly limited and may be appropriately selected from a wide range, but is usually about 1 to 70% by weight, preferably about 1 to 30% by weight of the whole composition. It is appropriate to set it in the range of about% by weight.

The method of administering the pharmaceutical preparation of the present invention thus obtained is not particularly limited, and is determined according to various preparation forms, the age and sex of the patient, other conditions, and the degree of the disease. For example, a pharmaceutical preparation in the form of an injection can be administered by intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal administration, or the like. It can also be administered. Tablets, pills, granules,
The pharmaceutical preparation of the present invention in a solid form such as a capsule or a liquid form for oral administration can be administered orally or enterally. Suppositories can be administered rectally.

The dose of the drug of the present invention can be appropriately selected from a wide range and is not particularly limited. Usually, the carbostyril derivative of the general formula (1) (and a salt thereof) is used in an amount of about 0 to 1 kg / body weight per day. The amount is preferably selected from the range of about 0.5 to 30 mg, and it is appropriate that the dosage unit form contains about 10 to 1000 mg of the active ingredient. The administration of the p21 inducer of the present invention can be divided into once a day or 3 to 4 times a day.

[0029]

According to the present invention, there is provided a p21 inducer containing a carbostyril derivative represented by the general formula (1) and / or a salt thereof as an active ingredient.

[0030]

The present invention will be described in more detail with reference to Formulation Examples and Pharmacological Test Examples.

[0031]

Formulation Example 1 Vesnarinone 5 mg Starch 132 mg Magnesium stearate 18 mg Lactose 45 mg Total 200 mg A tablet of the above composition was produced in one tablet.

[0032]

Formulation Example 2 Vesnarinone 150 g Avicel (trade name, manufactured by Asahi Kasei Corporation) 40 g Corn starch 30 g Magnesium stearate 2 g Hydroxypropyl methylcellulose 10 g Polyethylene glycol-6000 3 g Castor oil 40 g Methanol 40 g The above-mentioned active ingredient compound, Avicel, corn starch and magnesium stearate After mixing and polishing, sugar coating R10mm
Tablet with kine. The obtained tablets were treated with hydroxypropyl methylcellulose, polyethylene glycol-600.
0, coating with a film coating agent consisting of castor oil and methanol to produce film coated tablets.

[0033]

Formulation Example 3 Vesnarinone 150.0 g Citric acid 1.0 g Lactose 33.5 g Dicalcium phosphate 70.0 g Pluronic F-68 30.0 g Sodium lauryl sulfate 15.0 g Polyvinylpyrrolidone 15.0 g Polyethylene glycol (Carbowax 1500) 4.5 g Polyethylene glycol (Carbowax 6000) 45.0 g Corn starch 30.0 g Dry sodium lauryl sulfate 3.0 g Dry magnesium stearate 3.0 g Ethanol Appropriate amount Active compound, citric acid, lactose, dicalcium phosphate, Pluronic F Mix -68 and sodium lauryl sulfate.

The above mixture was designated as No. 60 screen, polyvinyl pyrrolidone, carbowax 1500
And wet granulation with an alcoholic solution containing Carbowax 6000. If necessary, alcohol is added to make the powder into a peat-like mass. Add corn starch and continue mixing until uniform particles are formed. No. Pass through 10 screens, place in trays and dry in oven at 100 ° C. for 12-14 hours. The dried particles were no. The mixture is sieved through 16 screens, dried sodium lauryl sulfate and dried magnesium stearate are added, mixed, and compression-molded into a desired shape using a tableting machine.

The core is treated with varnish, and talc is sprayed to prevent moisture absorption. An undercoat layer is coated around the core. Apply varnish a sufficient number of times for internal use. An additional subbing layer and smooth coating are applied to make the tablet completely round and smooth. Color coating is carried out until the desired hue is obtained. After drying, the coated tablets are polished to prepare tablets of uniform gloss.

[0036]

[Pharmacological Test Example 1] (1) Cells and their culture: human salivary gland squamous cell carcinoma cells T
YS [Am. J. Pathol., 124 , 496-509 (1986)] is 10
The cells were cultured at 37 ° C. in the presence of 5% CO ₂ using Dulbecco's modified Eagle medium (DMEM; GIBCO Labs.) Supplemented with 5% newborn calf serum (FCS; Bio-Whittaker).

(2) Treatment of TYS cells with compound: 96
Place 2 TYS cells in a well plate (Falcon Labware).
The cells were sowed at 10 ³ cells / well (Day 0). The next day (Day 1), vesnarinone 10mg / ml DMSO
By adding the solution to the medium, various concentrations (5, 10, 30
Or 50 μg / ml) of vesnarinone. Thereafter, the medium was changed daily with a medium containing the same concentration of vesnarinone. Cell proliferation was determined by the MTT method [Cancer Res., 47 , 936-942 (198
7)].

(3) Isolation of RNA Cytoplasmic total RNA was prepared by lysing cells in a hypotonic buffer containing Nonidet p-40 (Sigma) and removing nuclei.

(4) Cloning of human p21 ^WAF1 cDNA: 5 μg of the above cytoplasmic total RNA which was isolated from TYS cells treated with 50 μg / ml of vesnarinone for 3 days
, Reverse transcriptase (Molony murine leukemia virus; GIBC
O) and a random primer (GIBCO) were used for reverse transcription, and the resulting cDNA was subjected to PCR. In PCR, the final concentrations of dNTPs and primers in the reaction mixture were 200 μM and 1 μM, respectively. TaqDNA
Polymerase (Takara Shuzo Co., Ltd.) was added to the mixture at a final concentration of 0.05 U / μl, and the reaction was performed in a temperature controller (Takara) after 3 minutes at 94 ° C, 1 minute at 94 ° C, 60 ° C.
The test was performed with 30 cycles of 1.5 minutes, 2.5 minutes at 72 ° C, and an extension of 4 minutes at 72 ° C.

The sequences of the primers used are shown in the following table.

[0041]

[Table 1]

The obtained PCR product (875 bp) was converted into pst1
And the digest (208 bp) was subcloned into the same restriction enzyme site of the pUC19 vector. The DNA sequence of the cloned human p21 ^WAF1 cDNA was obtained by using a fluorescent labeled primer and Takara kit (Takara Taq Cycle Seq.).
uencing Kit; Takara) [Proc. Natl. Acad. Sci., USA, 74 , 5463-9567]
(1977)].

Electrophoresis and scanning were performed using a Shimadzu DNA sequencer (Shimadzu DSQ-500 DNA sequencer; Shimadzu Corse).
p.).

(5) PCR-SSCP and p53 sequencing: Chromosomal DNA of TYS cells was purified using a DNA extraction kit (Stratagene).

Exons 5, 6, 7 and 8 of the p53 gene
Fluorescence PCR-SSCP using a DNA sequencer was performed for the detection of the mutation existing in [Biomedical Res., 1] .
6 , 273-279 (1995)]. These exons are known to contain hot spots for p53 mutations [Oncoge
ne, 10 , 6681-6688 (1995)].

For direct sequencing of exon 8, a 2.8 kb fragment containing exons 5-8 was ligated in a temperature controller (Gene Amp PCR System PJ2000; Perkin Elmer).
Amplified using APCR kit (Takara)
Min, 58 ° C for 1 min, 70 ° C for 4 cycles and 70 ° C for 7 min; primers: P3 and P4 or P5 shown in Table 2
use).

[0047]

[Table 2]

The PCR product (2.8 kb) was purified by agarose gel electrophoresis. The DNA sequence of the PCR product was analyzed using a DNA sequencer (ALFred DNA sequencer; Phar
maciaFine Chemical Co.) (ALFred
The primers were determined by the dideoxy chain termination method using the Auto Cycle Sequencing Kit (Pharmacia) (as primers, a Cy5-labeled P6 primer having the nucleotide sequence shown in Table 3 and a Cy5-labeled M13 reverse sequence primer (Cy5
labeled-M13 reverse sequencing primer) was used).

[0049]

[Table 3]

(6) Northern blot analysis: cytoplasmic RNA
(20 μg) was electrophoresed on a formaldehyde / 1.0% agarose gel, and this was subjected to a nylon filter (Hybo
nd-N; Amersham). 50% formamide, 5 × salt-sodium phosphate-EDTA (saline-sodium phosphate-EDTA),
42 ° C. in the presence of 0.1% SDS, 5 × Denhardt's solution and 100 μg / ml salmon sperm DNA
Hybridization was performed with a 32P-labeled cDNA probe for 15 to 20 hours. 0.1 × SSC (standard
The cells were sufficiently washed twice with 0.5% SDS at room temperature and once at 50 ° C. for 40 minutes. Exposure to X-ray film was performed at -70 ° C using an intensifying screen.

The probes used were human p21 ^WAF1 cDNA (875 bp) having a complete open reading frame cloned from TYS cells and human TGF-β.
No. 1 (ATCC No. 59955) and Sma1-Smal fragment (592 bp) and human β-actin (ATCC No. 77644:
HFBCA46) is a Kho1-Xho1 fragment (2.1 kb) of pHFβA-1.

Also, the densitometric analysis of the signal is performed by NIH Image 1.44 program and / or BA
S-2000II image analysis system (Fiji photo fil
m).

(7) Immunoblot: TYS cells were isolated from SD
Mix in S-gel electrophoresis loading buffer and boil for 10 minutes. The obtained extract was subjected to electrophoresis on a 12.5% polyacrylamide gel (50 μg protein / lane), and Immobilon-P membrane (Millimobil-P membrane; Millimeter) was used.
pore Corp.) [Nature, 227 , 680
-685 (1970)]. Peroxidase-conjugated second antibody (horse
radish peroxydase-conjugated second antibodies; Am
ersham) and a chemiluminescent reagent (Amersham).
A protein immunoreactive with 21 ^WAF1 monoclonal antibody 6B6 (PharMingen) was detected [Anal. Biochem., 151 , 205-.
209 (1985)].

(2) Results (1) Proliferation of TYS cells The effect of vesnarinone on the proliferation of TYS cells was determined by MTT.
As a result of the test, vesnarinone significantly suppressed the cell growth in a concentration-dependent manner.

(2) Expression of p53 gene in TYS cells PCR-SSCP analysis confirmed the presence of a mutant band having a different mobility in exon 8 of p53 gene in TYS cells. As a result of direct DNA sequencing of the site, TY
It was confirmed that the S cell had a mutant p53 gene in which the codon Asp (GAC) at position 281 was replaced with His (CAC).

(3) Induction of p21 ^WAF1 gene The expression of p21 ^WAF1 mRNA in TYS cells treated with vesnarinone was evaluated by Northern blot analysis. The results are shown in FIGS.

In FIG. 1, “A” indicates that in vesnarinone-treated (+) or untreated cells (−: control)
p21 ^WAF1 , TGF-β1 and β-actin (β-acti
The autoradiography from the first day (Day 1) to the fourth day (Day 4) of n) is shown by “B” in FIG.
Shows the results of their densitometric analysis as mRNA levels relative to β-actin.
to β-actin).

FIG. 2 shows vesnarinone treatment or TGF-β
The same result as above for one treated cell is shown.

The expression of p21 ^WAF1 and TGF-β1 was 5
Enhanced by one day treatment with 0 μg / ml vesnarinone. Treatment of TYS cells with vesnarinone
Although the expression of GF-β1 mRNA was slightly enhanced,
p21 ^WAF1 mRNA expression was ^observed in the first and third
And on day 4, significant enhancement was noted. 5ng / ml
Of TGF-β1 alone in TYS cells
^{Induces WAF1} expression (FIG. 2). Vesnarinone or TGF
Induction of p21 ^WAF1 by -β1 alone showed a time-dependent enhancement at 24 hours tested (except TG
P21 ^WAF1 expression in F-β1 treated cells showed a transient decrease by 6 hours). Further, according to FIG.
GF-β is found to be a stronger inducer of p21 ^WAF1 expression than vesnarinone under the conditions of the present experiment.
An increase in p21 ^WAF1 mRNA is due to vesnarinone or TGF
Less than 2 hours after -β treatment, this induction is rapid. The steady-state level of p21 ^WAF1 mRNA continued to increase over 24 hours after addition of vesnarinone or TGF-β. On the other hand, the amount of β-actin was not constant (FIG. 2).
Vesnarinone-treated cells increased at 2 hours and then
It decreased from 2 hours to 24 hours. This is TG
Constant β up to 12 hours in F-β1 treated cells
-Extremely symmetric compared to actin levels. This suggests that β-actin expression may be affected for at least 6 hours after treatment with vesnarinone.

[0060] (4) p21 ^WAF1 expression of p21 ^WAF1 proteins in expression vesnarinone processing TYS cell protein, Fig. 3 the results of evaluation by immunoblot analysis
Shown in

In FIG. 3, lane 1 shows a control (untreated with vesnarinone, Vesnarinone 0 μg / ml), and lanes 2 to 4 show 10 μg / ml and 30 μg / ml of vesnarinone sequentially.
Shown are the results for cells treated with ml and 50 μg / ml, respectively.

According to the result of the immunoblot analysis, the expression of p21 ^WAF1 protein by vesnarinone was enhanced in a concentration-dependent manner of the added compound.

[0063]

[Pharmacological test example 2] (1) Experimental method BALB / c of 6 weeks old purchased from CLEA Japan
AJcl-nu nude mice (female) were used. The breeding was performed in a clean rack, and feed and water were sterilized.

The human gastric cancer cell line MKN-45 (poorly differentiated adenocarcinoma) was cultured in a plastic culture dish of 10 cm in diameter in 15 ml of PRMI1640 medium (manufactured by Nissui Pharmaceutical) containing 10% FBS. Confluent (conf
When the cells have become luten), the cells are washed with a PBS (-) solution (manufactured by Nissui Pharmaceutical Co., Ltd.), and then washed with 0.01% EDTA (Dojindo Laboratories) containing 0.125% trypsin (manufactured by Difco). The cells were detached from the flask, and the cells were suspended in a 1: 1 mixture of a Hanks solution (manufactured by Gibco) and a PBS (-) solution to prepare a cell suspension.

The above cell suspension was transplanted subcutaneously to the abdomen of 10 nude mice at 1.6 × 10 ⁷ cells (0.3 ml). When the tumor mass was formed, the mice were sacrificed,
The removed tumor was washed with Hank's solution after removing the capsule and the necrotic part, and a cubic tumor piece of 2-3 mm square was formed as uniform as possible, and a trocar was placed subcutaneously on the back of 120 nude mice. Implanted with a needle.

In the nude mice, the day when the tumor volume reaches around 100 mm ³ from the day of transplantation of the tumor (day 0) (day 8).
Were grouped according to tumor weight (n = 7).

Vesnarinone is 0.5% C under aseptic conditions.
It was dissolved in distilled water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) containing MC (manufactured by Katayama Chemical Industry Co., Ltd.) to prepare 5 mg / ml,
0.1 ml per 10 g of mouse body weight was orally administered every day for 4 weeks using oral Zonte (dose: 50 mg / k)
g, vesnarinone administration group).

The control group contained 0.5% CM
C-containing distilled water for injection (0.2 ml / 20 g, oral) was administered.

The tumor mass was fixed in 10% formalin and then embedded in paraffin. The specimen was cut into sections, deparaffinized, and immersed in a methanol solution containing 3% H ₂ O ₂ for 10 minutes. After washing with PSB (-), 10% normal goat serum (Hist
ofine, Nichirei) at room temperature for 30 minutes. PBS
After washing with (-), the cells were stained with a 100-fold diluted anti-p53 rabbit polyclonal antibody (CM1, 1: 100; Medac) for 90 minutes at room temperature. After washing with PBS (-), peroxidase-labeled streptavidin (streptoavidine,
Histofine, Nichirei) for 45 minutes at room temperature. 0.
6 mg / ml of 3,3'-diaminobenzidine hepcidin Tris containing (Dojin Chemical Laboratories) and 0.3% H ₂ O ₂ - was developed by immersion for 10 minutes in HCl buffer (pH 7.4).

(2) Results The results are shown in FIG.

This graph shows the percentage of each p53-positive cell in the vesnarinone-administered group and the control group with the p53-positive cells (%) on the vertical axis (* indicates a significant difference by the Student's t-test, p <0.01), which shows that the expression of wild-type p53 was significantly enhanced in the vesnarinone administration group.

The following can be understood from the above test results. That is, the expression of p21 in cells induces G1 arrest. In addition, cells are thought to differentiate from G1 and enter the process of differentiation, resulting in the cessation of cell proliferation and suppression of CD4 activation.

[Brief description of the drawings]

FIG. 1. TY treated with Vesnarinone
¹⁵ is a photograph instead of a drawing, showing the result of evaluating the expression of p21 ^WAF1 mRNA in S cells by Northern blot analysis.

FIG. 2. Ts treated with vesnarinone or TGF-β1
¹⁵ is a photograph replacing a drawing, showing the result of evaluating the expression of p21 ^WAF1 mRNA in YS cells by Northern blot analysis.

FIG. 3. p21 in vesnarinone-treated TYS cells
⁴ is a photograph replacing a drawing, showing the result of evaluating the expression of ^WAF1 protein by immunoblot analysis.

FIG. 4 is a graph showing that administration of vesnarinone significantly enhances the expression of wild-type p53.

Claims (2)

[Claims] 1. A compound of the general formula [In the formula, R represents a benzoyl group which may have a lower alkoxy group as a substituent on the phenyl ring. The carbon-carbon bond between the 3-position and the 4-position of the carbostyril skeleton indicates a single bond or a double bond. ] A p21 inducer comprising at least one compound selected from the carbostyril derivative represented by the formula (1) and a salt thereof as an active ingredient. 2. The method of claim 1, wherein the carbostyril derivative is 6- [4-
The p21 inducer according to claim 1, which is (3,4-dimethoxybenzoyl) -1-piperazinyl] -3,4-dihydrocarbostyril or a salt thereof.