JP2628106B2 - Apoptosis regulator - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel apoptosis regulator.

BACKGROUND ART The apoptosis regulator of the present invention comprises at least one selected from carbostyryl derivatives represented by the following general formula (1) (hereinafter, referred to as “compound (1)”) and salts thereof.
The seed is the active ingredient.

(1) 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. The above compound (1) and its production method are described in, for example, Japanese Patent Publication No. 43747/1989, and it is also known that this compound is useful as a cardiotonic agent.

The present inventor has further studied the compound (1), and as a result, it has been found that the compound (1) has an anticancer effect, a cell differentiation inducing ability, etc. which are difficult to predict from the above-mentioned known effects. (Suppression or promotion).

Thus, cell death has hitherto been attributed to two types of mechanisms. One of them is classic cell death, which has been called necrosis. It is morphologically characterized by marked mitochondrial swelling, cytoplasmic swelling, nuclear degeneration followed by cell disruption and autolysis, and occurs passively and accidentally. Tissue necrosis is generally recognized by physical trauma to cells, chemical poisons, and the like.

Another type is called apoptosis (programmed cell death) (Kerr, JFRand Wyllie, AH, B
rJCancer, 265, 239 (1972)). It is believed that this occurs under various physiological conditions. Its morphological features include lack of contact with surrounding cells, cytoplasmic enrichment, chromatin condensation and nuclear condensation associated with endonuclease activity, nuclear segmentation, and the like. Loss of microvilli and smoothing of the cell surface (membrane blebbing) are also observed, and the phenomenon that the nucleosome unit of DNA is fragmented into DNA of 180 to 200 bases in length due to endonuclease activity. Are also observed, and the final fragment of apoptotic cells has been discussed as a mechanism for phagocytosis by neighboring cells (Duvall, Ea
nd Wyllie, AH, Immunology Today, 7 (4), 115-119
(1986): Science, 245, 301-305 (1989)). The Willie also reports that apoptosis of thymocytes induced by glucocorticoids is accompanied by activation of intracellular endonucleases (Wyllie, AH, Nature, 2).
84, 555-556 (1986)). The DNA of cells undergoing apoptosis due to endonuclease activity is fragmented to the oligonucleotide level, which can be easily confirmed by agarose gel electrophoresis.

Apoptosis is thought to be pre-programmed cell death seen during development, differentiation and tissue turnover (Wyllie, AH, et al., Int. Rev. Cyt.
ol. , 68, 251-306 (1980)).

In addition, increasing the concentration of calcium or increasing the cAMP concentration of thymocytes with calcium ionophore promotes the DNA fragmentation characteristic of the apoptosis (Wyllie, AH, et al., J. Pathol. ., 142, 67-77 (198
4)) Therefore, it is speculated that the involvement of calcium ions and cAMP in the mechanism of apoptosis. HL induced differentiation by retinoic acid and calcium ionophore
Apoptosis of -60 cells has been reported as an example above. (Martin, SJ, et al., J. Immunol., 145, 1859-18
67 (1990): Martin, SJ, et al., Clin. Exp. Immunol. , 7,
9, 448-453 (1990)).

The above-mentioned apoptosis may be caused by physiological cell death observed in cells undergoing embryogenesis and normal cell rotation (eg, liver, adrenal cortex, prostate, etc.), glucocorticoid treatment, cytotoxicity by cytotoxic-T cells, and hormone dependence. It has been reported that it is also induced by atrophy of sexual tissues, irradiation, NK cells, killer cells, cytokines such as tumor necrosis factor (TNF) and lymphotoxin (LT) (Wyllie,
AH, et al., Int. Rev. Cytol. , 68, 251 (1980): Duvall,
E. and Wyllie, AH, Immunology Today, 7 , 115-119 (19
86): Sellins, KS, et al., J. Immunol., 139, 3199 (198
7): Yamada, T., et al., Int. J. Radiat. Biol., 53 , 65 (198
8): Wyllie, AH, Nature, 284 , 555 (1980): Schmid, D.
S., et al., Proc. Natl. Acad. Sci., USA, 83 , 1881-1885 (1
986): John, C., et al., J. Immunol., 129 (4), 1782-17
87 (1982): Howell, DM, et al., J. Immunol., 140, 689-
692 (1988): Gillian.B., Et al., Eur.J.Immunol., 17 , 68.
9-693 (1987)). In addition, certain antibodies, such as anti-
CD3 antibody, anti-APO-I antibody, anti-Fas antibody, etc. (Trauth, BC, e
t al., Science, 245, 301-305 (1989): Smith, CA, et a.
l., Nature, 337, 181-184 (1989): Tadakuma, T., et al.,
Apoptosis is also induced in Eur. J. Immunol. , 20, 779 (1990), and the findings of spontaneous regression in malignant tumors (Yasuo Nakamura et al., Clinical Dermatology, 35 (4), 289-295 (1981)) Apoptosis has also been confirmed.

On the other hand, those that suppress the apoptosis include RN
Actinomycin D (A)
D), a protein synthesis inhibitor cycloheximide (Cyclo
heximide), calcium ion (Ca ²⁺ ) chelating agents, etc., and cyclosporin A, an immunosuppressant, hematopoietic cytokines (IL-3, GM-CSF, G
-CSF etc.), IL-2, bcl-2 gene products, etc. are also reported to suppress apoptosis (Cohen, JJJ Immuno).
l., 132 , 38 (1984): Wyllie, AH, et al., J. Pathol., 14
2, 67 (1984): Shi, Y., et al., Nature, 339, 625 (1989):
Williams, GT, et al., Nature, 343, 76 (1990): Nielo,
MA, J. Immunol., 143, 4166 (1989): Vaux, DL, et al.,
Nature, 335, 1440 (1988)). However, the above cycloheximide induces apoptosis in acute leukemia cells, actinomycin D induces apoptosis in small intestinal crypt cells, and both induce apoptosis in HL-60 cells (Martin, SJ, et al., J. Immunol., 145 , 18).
59-1867 (1990). Cycloheximide, on the other hand,
It has been reported that apoptosis of lymphocytic tumor cells, which is present before X-ray irradiation and increased by X-ray irradiation, and that actinomycin D increases the above-mentioned apoptosis. Involvement of types, conditions, and other mechanisms has also been suggested (Tadahiko Igarashi et al., Journal of the Japanese Society of Hematology, 51 (2), 144 (1988)). In any case, the differentiation, proliferation, and maturation of cells are closely related to apoptosis, and it is considered that substances having an action related to the differentiation, proliferation, etc. of such cells are also involved in apoptosis.

Recently, anti-apoptotic treatments
Treatment of cancer with Apo-I antibodies has also been attempted. Among myelodysplastic syndromes (MDS), refractory anemia (RA) and pancreatic anemia (RARS), mainly due to pancytopenia, have retinoic acid and active vitamins as hematopoietic cell differentiation inducers.
And D _3, GM-CSF and IL-3 in combination is preferable as suppressing apoptosis modifier excess apoptosis platelet producing cells, also of the same MDS, proliferation of blasts predominant RAEB and said R
In the transition period AEB (RAEB-t), the retinoic acid or an active vitamin D ₃ is, as a differentiation inducing agent to induce differentiation into hematopoietic cells of blasts, also Etopojido (etoposide) and aclarubicin (aclarubicin) bud It is said that each acts as an apoptosis regulator that suppresses the proliferation of spheres (promotes apoptosis) (Shibuya, T., J. Clinical and Ex
perimental Medicine, 160 (5), 319-323 (1992)).

Murakami and colleagues also report that about half of transgenic mice expressing anti-erythrocyte autoantibodies develop autoimmune diseases due to loss of self-tolerance, and the reaction between autoantigens and autoantibody-producing cells as in normal mice. It has been reported that it is due to the lack of ability to remove autoantibody-producing cells by inducing apoptosis (Murakami, M., et al., Nature, 357 ,
77-80 (1992)).

Watanabe-Fukunaga et al. Found that in MRLlpr / lpr mice, the Fas molecule involved in apoptosis was abnormal, and the mechanism of negative selection (apoptosis) of autoreactive T cells in the thymus did not work, resulting in the development of autoimmune diseases. (Watanabe-Fukuna
ga, R., et al., Nature, 356, 314-317 (1992)).

Montagnier et al. Have observed an apoptotic band of DNA in T lymphocyte extracts from HIV-infected patients, a phenomenon that is 90% of asymptomatic HIV-infected patients, AIDS and ADS.
It is observed in 100% of RC patients and reports that induction of apoptosis is also enhanced in HIV-infected patients (Montag
nier, L., et al., Sixieme Colloque des Cent Gardes, 9
-17 (1991)).

During cell development during chick development, NGF is added to chicken embryos.
Pre-administration (nerve growth factor: a protein that promotes cell hypertrophy and nerve fiber elongation in ganglia of nerve cells)
Neuronal death during this development is completely suppressed (Hamburge
r, V., et al., J. Neurosci., 1 , 60 (1981)). Conversely, when an antibody against NGF is administered, about 90% of young sympathetic nerve cells are administered.
Has been reported to be lost (Levi-Montalchin
i, R.and Booker, B., Proc.Natl.Acad.Sci ., USA, 46, 384
(1960)).

Clark distinguishes spontaneous neuronal cell death into three types, in which the morphological characteristics of type I are consistent with apoptosis, and cell death by growth factor removal results in type I cell death, Since fragmentation also occurs, this is considered apoptosis (Clark, PGH, Anat Em
bryol., 181 , 195 (1990): J. Neurosci., 1 , 60 (1981): P
. roc.Natl.Acad.Sci, USA, 46, 384 (1960): Rawson, CL,
et al., J. Cell. Biol., 113, 671 (1991)).

Edwards et al. Report that NGF can suppress programmed death of sympathetic nerve cells, and that apoptosis may be suppressed by NGF (Edwards, SN, et al., J. Med.
Neurochemistry, 57 (6), 2140-2143 (1991)).

According to a report by Fischer et al., When NGF is administered to aging and learning-impaired rats, the NGF acts on cholinergic neurons in the basal forebrain, which is known to be damaged by Alzheimer's disease. Recovery of learning disabilities is observed (Fischer, W., et al., Nature, 329, 65 (1987): Bard
e Y-A, Neuron, 2, 1525 (1989): Hatanaka, H., Develop B
rain Res., 30 , 47 (1986): Hatanaka, H., et al., Develop
Brain Res., 39 , 85 (1988)). Hatanaka and colleagues said that NGF is effective in differentiation, maturation, survival maintenance, prevention of aging, protection and recovery action against nerve cell damage, neurological diseases associated with brain aging,
In particular, they suggest that they may have a protective effect on neuronal cell death in Alzheimer's disease (Hatanaka Hiroshi, Metabolism, 28 , 891-899 (1).
991)).

Furthermore, in the liver damage of drug-resistant viral hepatitis, it is thought that the apoptosis enhancement directly or through an immune mechanism is involved in the liver damage by drug or virus infection (Bursh, W., et al.). al., TiPS, 13 , 245-251 (199
2)).

On the other hand, it is known that mitogen causes liver cells to proliferate in the liver, resulting in a hyperplastic state, and this state is normalized by hepatocyte shedding and necrosis, that is, apoptosis (Kerr, JF et al., Br. J. .Cancer, 26 , 239-257 (1
972)). The apoptosis occurs in the liver in a hyperplastic liver,
It has been observed in hyperplastic nodules and liver cancer (Columbano,
A., et al., Lab. Invest., 52, 670-675 (1985): Columban
o, A. et al., Am. J. Pathol., 116 , 441-446 (1984)), Keller et al. state that apoptosis is not associated with inflammation or fibrosis (Kerr, JF, et al., Lancet, 2, 827-828 (197
9)). From these facts, if it is possible to suppress apoptosis for acute and chronic hepatitis, it is considered that such hepatitis can be treated. In the process of transition of chronic hepatitis to liver cirrhosis and liver cancer, the above-mentioned apoptosis is in a suppressed state, which is considered to progress to fibrosis and cirrhosis following inflammation of hepatocytes by cytotoxic T cells. Can be considered to be able to suppress hepatitis and prevent progression to cirrhosis.

DISCLOSURE OF THE INVENTION According to the present invention, there is provided an apoptosis-modulating agent containing as an active ingredient at least one selected from compound (1) and salts thereof.

The apoptosis-regulating agent of the present invention has apoptosis-regulating ability, and based on this action, for example, as described above, an anticancer agent, an antiretroviral agent, a therapeutic agent for an autoimmune disease, a therapeutic agent for thrombocytopenia, a therapeutic agent for Alzheimer's disease Drugs, hepatitis, liver disease treatment such as cirrhosis, cancer metastasis inhibitor, etc.
Effective in the pharmaceutical field.

In the general formula representing the compound (1), the benzoyl group which may have a lower alkoxy group as a substituent on the phenyl ring includes benzoyl, 2-methoxybenzoyl, 3-methoxybenzoyl, 4-methoxybenzoyl, Ethoxybenzoyl, 3-ethoxybenzoyl, 4-ethoxybenzoyl, 4-isopropoxybenzoyl, 4-hexyloxybenzoyl, 3,4-dimethoxybenzoyl, 3,4-diethoxybenzoyl, 3,4,5
Benzoyl groups which may have 1 to 3 carbon atoms having 1 to 3 straight or branched alkoxy groups as substituents on the phenyl ring such as -trimethoxybenzoyl, 2,5-dimethoxybenzoyl and the like. .

Among the above-mentioned compound (1) used as an active ingredient in the present invention, particularly preferred are, for example, 6- [4
-(3,4-Dimethoxykibenzoyl) -1-piperazinyl] -3,4-dihydrocarbostyril and salts thereof.

The above-mentioned active ingredient compound can easily form a pharmacologically acceptable salt using an ordinary acid, and these salts can be used as the active ingredient compound similarly to the free form compound. Examples of the acid include inorganic acids such as sulfuric acid, nitric acid, hydrochloric acid, and hydrobromic acid, and acetic acid, p-toluenesulfonic acid, ethanesulfonic acid, oxalic acid, maleic acid, fumaric acid, citric acid, succinic acid, benzoic acid, and the like. Can be exemplified.

The compound (1) which is an active ingredient of the apoptosis regulator of the present invention is generally used in the form of a general pharmaceutical preparation. Such a preparation is prepared using a commonly used diluent or excipient such as a filler, a bulking agent, a binder, a humectant, a disintegrant, a surfactant, and a lubricant. As the pharmaceutical preparation, various forms can be selected according to the purpose of treatment, and typical examples thereof include tablets, pills, powders, solutions, suspensions, emulsions,
Examples include granules, capsules, suppositories, injections (solutions, suspensions, etc.), eye drops and the like.

For molding into tablets, those conventionally known in the art can be widely used as carriers, for example, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, etc. Excipient, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, binder for polyvinylpyrrolidone sugar, dried starch, sodium alginate, agar powder, laminaran powder Disintegrating agents such as sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, monoglyceride stearate, starch, lactose, disintegrating inhibitors such as sucrose, stearin, cocoa butter, hydrogenated oil, Quaternary ammonium bases, absorption promoters such as sodium lauryl sulfate, humectants such as glycerin and starch, adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid, purified talc, stearates, boric acid powder, Lubricants such as polyethylene glycol can be exemplified. 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 molding into a pill form, those conventionally known in the art can be widely used as carriers, for example, excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, talc, gum arabic powder, Examples include binders such as tragacanth powder, 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.

When prepared as an injection, the solution and suspension are preferably sterilized and isotonic with blood, and when formed into the form of these solutions, emulsions and suspensions, they may be used as diluents. Any of those commonly used in the field can be used, 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 or glycerin to prepare an isotonic solution may be contained in the pharmaceutical preparation, and a usual solubilizing agent, buffer, soothing agent and the like may be added. You may.

Furthermore, the apoptosis-controlling agent of the present invention may contain a coloring agent, a preservative, a flavor, a flavoring agent, a sweetening agent, and other pharmaceuticals as necessary.

The amount of the active ingredient compound contained in the pharmaceutical preparation is not particularly limited and may be appropriately selected in a wide range, but it is usually 1 to 70% by weight, preferably 1 to 30% by weight in the whole composition. Is appropriate.

The administration method of the above pharmaceutical preparation is not particularly limited, and is determined according to various preparation forms, patient age, gender and other conditions, degree of disease, and the like. For example, tablets, pills, solutions, suspensions,
Emulsions, granules and capsules are orally administered. In the case of an injection, it is administered intravenously, alone or as a mixture with a normal replenisher such as glucose or amino acid, and if necessary, intramuscularly, intradermally, subcutaneously or intraperitoneally. In the case of suppositories it is administered rectally and in the case of eye drops it is applied to the eye.

The dose of the above pharmaceutical preparation is appropriately selected depending on the usage, the age of the patient, gender and other conditions, the degree of the disease, and the like. Usually, the amount of the compound (1) which is the active ingredient is about 0.5 to 30 mg / kg of body weight per day. It is good to do. The dosage unit form should preferably contain about 10-1000 mg of the active ingredient.

The apoptosis controlling agent of the present invention can be applied to various diseases based on its apoptosis controlling ability, cell differentiation inducing ability, and the like, and a desired pharmacological effect can be expected. More specifically, as the applicable disease, for example, cancer, AIDS, ARC (AIDS
Related diseases), ATL (Adult T-cell leukemia: Adult T-cell leukemia)
ukemia), Hairy cell leukemi
a), HTLV-I-related diseases such as myelopathy (HAM / TSP), respiratory disorder (HAB / HABA), arthropathy (HAAP), uveitis (HAU), and autoimmune diseases such as SLE (systemic lupus erythematosus) ), Collagen diseases such as rheumatoid arthritis (RA), ulcerative colitis, Sjogren's syndrome, primary biliary cirrhosis, idiopathic thrombocytopenic purpura (Idiopathic Thrombocytopenic Pu)
rapura: ITP), autoimmune hemolytic anemia, myasthenia gravis,
Examples include Hashimoto's disease and insulin-dependent (type I) diabetes. The apoptosis-modulating agent of the present invention is also applied to various diseases associated with thrombocytopenia, such as myelodysplastic syndrome, periodic thrombocytopenia, aplastic anemia, idiopathic thrombocytopenia, and generalized intravascular coagulation. it can. Further, the modifier of the present invention
Various types of hepatitis such as C, A, B, and F, Alzheimer's disease, Alzheimer's senile dementia, myocarditis, ARDS (adult respiratory distress syndrome) infection, cirrhosis, benign prostatic disease, uterine fibroids, bronchial asthma , Arteriosclerosis, various congenital malformations,
Nephritis, senile cataract, chronic fatigue syndrome (Chronic Fatigi
u Syndrome, muscular dystrophy (Myotonic dystroph)
It is applicable to various diseases such as y).

When the apoptosis-modulating agent of the present invention is used, for example, as an anti-cancer agent, administration thereof can promote or suppress induction of apoptosis after or directly without differentiation of cancer cells, and thus exert an anti-cancer effect. In addition, in this case, the agent of the present invention can be used, for example, in combination with various other anticancer agents and radiation therapy known as chemotherapeutic agents for cancer, regardless of the formulation form and administration route. Thus, since the active ingredient compound of the present invention can exhibit an excellent anticancer effect, it can further promote the effects of other anticancer agents used in combination and exert a synergistic effect. Therefore,
Even when the amount of the combined anticancer agent is considerably smaller than the amount usually used, a sufficient cancer therapeutic effect can be obtained, and thereby the side effects of the combined anticancer agent can be reduced. Examples of such chemotherapeutic agents include 5-fluorouracil (5-FU, manufactured by Kyowa Hakko Kogyo Co., Ltd.), mitomycin (Mitomycin-C, manufactured by Dojo Co., Ltd.), and furtafurol (FT-20).
7. Taiho Pharmaceutical Co., Ltd.), Endoxa
n, Shionogi & Co., Ltd.), Toyomycin (Toyomici)
n, manufactured by Takeda Pharmaceutical Co., Ltd.).

When the apoptosis-modulating agent of the present invention is used for treating thrombocytopenia, for example, in MDS patients such as RA and RARS, the administration of the agent exerts an apoptosis-inhibiting effect together with a cell differentiation-inducing accelerating effect, thus increasing the proliferation of hematopoietic cells. Can stimulate and cause normal differentiation, maturation. RAEB and RA
In MDS patients such as EB-t, the administration of the modulator of the present invention induces blast differentiation and suppresses blast proliferation,
Thus, growth of mature cells can occur. In addition, the agent of the present invention acts on megakaryocyte progenitor cells and megakaryocyte cells to promote their differentiation and maturation, so that an effect of promoting platelet production can be expected.

When the apoptosis-modulating agent of the present invention is used for the treatment of thrombocytopenia, it can be used in combination with other drugs such as conventionally known thrombocytopenia, thereby promoting the effect of the concomitant drug, and thus reducing the amount of the concomitant drug. Even in very small amounts, a sufficient therapeutic effect can be achieved, and side effects can be reduced in some cases.

The modulator of the present invention is also useful as an agent for treating and preventing Alzheimer's disease. In this case, for example, in a patient with classical Alzheimer's disease or Alzheimer's senile dementia, the modulator of the present invention exhibits an NGF-like action by suppressing apoptosis, and thus exerts the above therapeutic and preventive effects.
Further, in this case, the preparation of the present invention can be used in combination with a conventionally known therapeutic agent for Alzheimer's disease such as a cerebral circulation improving agent or a cerebral metabolism improving agent, which may promote the effects thereof and reduce the side effects caused by the agents. is there.

Further, by controlling apoptosis, the modulator of the present invention can treat hepatitis or prevent fibrosis of hepatocytes in patients with drug-induced and viral hepatitis, and thus can be used as an agent for preventing cirrhosis.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a photograph showing the results of DNA fragmentation in the apoptosis regulating effect test described in Pharmacological Test Example 1 described below.

FIG. 2 is a graph showing the results of a growth inhibitory effect test on M12 melanoma cells described in Pharmacological Test Example 5 described below.

FIG. 3 is a graph showing the results of a life extension test for an autoimmune disease described in Pharmacological Test Example 8 below.

4 to 7 are photographs showing the results of the thrombocytopenia improving effect test described in Pharmacological Test Example 9 below.

FIG. 8 is a photograph showing the results of an autoimmune disease therapeutic effect test described in Pharmacological Test Example 10 below.

9 and 10 are photographs showing the results of an effect test on PC12 cells described in Pharmacological Test Example 11 described later.

FIG. 11 is a graph showing the results of an effect test on liver failure described in Pharmacological Test Example 12 below.

FIG. 12 is a graph showing the results of an effect test on an experimental lung metastasis model of B16 melanoma cells described in Pharmacological Test Example 13 below.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, preparation examples of the apoptosis regulator of the present invention and the results of pharmacological tests performed on the active ingredient compound of the apoptosis regulator will be described.

Formulation Example 1 6- [4- (3,4-dimethoxybenzoyl) -1-piperazinyl] -3,4-dihydrocarbostyril 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 After mixing and polishing the above active ingredient compound, Avicel, corn starch and magnesium stearate, the mixture is tableted with sugar coated R10 mm kine. The obtained tablets are coated with a film coating agent consisting of hydroxypropylmethylcellulose, polyethylene glycol-6000, castor oil and methanol to produce film-coated tablets.

Formulation Example 2 6- [4- (3,4-Dimethoxybenzoyl) -1-piperazinyl] -3,4-dihydrocarbostyril 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 Polyvinyl pyrrolidone 15.0 g Polyethylene glycol (Carbowax 1500) 4.5 g Polyethylene glycol (Carbowax 6000) 45.0 g Corn starch 3.0 g Dry sodium lauryl sulfate 3.0 g Dry magnesium stearate 3.0 g Ethanol Mix acid, lactose, dicalcium phosphate, Pluronic F-68 and sodium lauryl sulfate.

The above mixture was sieved with a No. 60 screen, and polyvinylpyrrolidone, carbowax 1500 and carbowax
Wet granulation with an alcoholic solution containing 6000. If necessary, alcohol is added to make the powder into a pasty mass. Add corn starch and continue mixing until uniform particles are formed. Pass through a No. 10 screen, place in a tray and dry in an oven at 100 ° C. for 12-14 hours. The dried particles are sieved through a No. 16 screen, and dried sodium lauryl sulfate and dried magnesium stearate are added.
The mixture is 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.

Using the test compounds shown below, the following pharmacological tests were performed.

(Test compound)

1. 6- [4- (3,4-dimethoxybenzoyl) -1-
Piperazinyl] -3,4-dihydrocarbostyril 2.6- [4- (3,4,5-trimethoxybenzoyl)-
1-piperazinyl] -3,4-dihydrocarbostyril 3.7- [4- (3,4,5-trimethoxybenzoyl)-
1-piperazinyl] -3,4-dihydrocarbostyril 4.7- [4- (3,4-dimethoxybenzoyl) -1-
Piperazinyl] -3,4-dihydrocarbostyril 5.6- [4- (4-Exibenzoyl) -1-piperazinyl] -3,4-dihydrocarbostyril 6.5- [4- (3,4-dimethoxybenzoyl) ) -1-
Piperazinyl] -3,4-dihydrocarbostyril 7.6- [4- (3,4-dimethoxybenzoyl) -1-
Piperazinyl] carbostyril 8.8- [4- (3,4-dimethoxybenzoyl) -1-
Piperazinyl] -3,4-dihydrocarbostyril Pharmacological test example 1 Apoptosis regulating effect test CMK cells were cultured in RPMI1640 medium containing 10% fetal calf serum.
After adjusting the concentration to × 10 ⁵ cells / ml, compound 1 was added to the mixture at 30 μl.
g / ml at 37 ° C for 2 and 4 days in culture flasks. In addition, what added only the solvent as a control was cultured similarly.

Remove cells from culture flask and 200x for 10 minutes at 4 ° C
Centrifuged at G. The pellet was dissolved in a hypotonic buffer (10 mM Tris-HCl, 1 mM EDTA and 0.2% Triton X100, pH 7.5) and centrifuged at 13,000 × G at 4 ° C. for 10 minutes. After collecting the supernatant, the mixture was allowed to stand at -20 ° C overnight in 50% isopropyl alcohol-0.5M NaCl, and the resulting precipitate was collected at 4 ° C for 10 minutes.
After centrifugation at 13000 × G for 1 minute, the supernatant was discarded and dried. Next, the cells were resuspended in a 10 mM Tris-HCl-1 mM EDTA (pH 7.4) solution, RNase was added to the supernatant to a final concentration of 0.1 mg / ml, and the mixture was incubated at 37 ° C. for 1 hour.

Furthermore, for a sample amount corresponding to 1 × 10 ⁶ cells, 15 mM EDTA, 2% SDS, 50% glycerol and 0.05%
Loading buffer containing bromophenyl blue was added to 1/5 volume of the sample and incubated at 65 ° C for 10 minutes.

The obtained sample was subjected to electrophoresis in a 1.5% agarose gel at 100 V for 100 minutes. DNA was stained with ethidium bromide.

 The results obtained are shown in FIG.

In the figure, lanes on both sides are markers (marker 1 = 120, 600 and 1350 bp, marker 2 = 72, 420 and 9), respectively.
30bp), and the second lane from the left is Compound 1 addition 2
The third lane from the left shows 2 days after the control, the fourth lane from the left shows 4 days after the addition of Compound 1, and the fifth lane from the left shows 4 days after the control.

From the figure, it is apparent that the addition of Compound 1 causes the expression of DNA fragments to appear in an electrophoretic manner in a time-dependent manner, indicating that Compound 1 promotes apoptosis of CMK cells. You can see that

Pharmacological test example 2 Cancer cell growth inhibitory effect test (1) Preparation of human promyelocytic leukemia cells (HL-60) Human promyelocytic leukemia cells (HL-60)
Gallo) et al., A human leukemia cell line established by the literature [Gallo, RC, et al., Blood, 54 , 713].
(1979)] and has been deposited with the American Type Culture Collection (ATCC) under the deposit number "ATCC No. CCL-240".

The HL-60 was adjusted to 1 × 10 ⁵ cells / ml in RPMI-1640 medium (Gibco) supplemented with 10% FCS (fetal calf serum, Gibco).

(2) Preparation of human gastric cancer cell line (KATO-III) The medium was adjusted to 1 × 10 ⁵ cells / ml in the same medium as in the above (1).

(3) Preparation of test compound Test compound 1 was dissolved in acetic acid, diluted with the same medium as in the above (1), neutralized with 2N-NaOH, and then dissolved in 100 μg / ml, 10 μg / ml.
The concentrations were adjusted to μg / ml and 1.0 μg / ml.

(4) Preparation of Giemsa staining solution Giemsa reagent (Merck) in phosphate buffer (pH 6.4)
Then, it was diluted 50 to 100 times to obtain a Giemsa staining solution.

(5) Preparation of esterase staining solution A reagent for esterase staining by naphthol AS · D chloroacetate method (Muto Chemical Co., Ltd.) was used.

(6) Preparation of anti-CD33 monoclonal antibody My9 Monoclonal antibody that specifically reacts with immature granulocytes
My9 (Coulter) was used after dissolving 0.5 ml of distilled water per vial and dissolving.

(7) Results a) To the HL-60 prepared in the above (1), the test compound prepared in the above (3) was added to a final concentration of 10 μg / ml and 1.0 μg / ml, respectively, and cultured in 12 wells. Using a plate (Coaster), the cells were cultured in a 5% carbon dioxide incubator at 37 ° C. for 3 to 4 days. As a control, a medium containing no test compound was used. After culturing, remove the cell suspension from each well and add 0.2%
The cells were mixed with a trypan blue-containing phosphate buffer, and the number of unstained viable cells was counted under a microscope.
Readjusted to 10 ⁵ cells / ml, and the incubation was continued in the same manner.

Table 1 shows the cell growth inhibitory effects on HL-60 on days 27 and 33.

The KATO-III prepared in the above (2) was cultured in the same manner, and the cell growth inhibitory effects on KATO-III on the 24th and 33rd days are shown in Table 2.

From Tables 1 and 2, it is clear that the growth of tumor cells is suppressed depending on the concentration of the test compound.

b) Differentiation of HL-60 The HL-60 prepared in the above (1) was cultured in the same manner as in a), the cells on day 27 were added to a slide glass, and Giemsa staining was performed. Morphological observations under a microscope showed that when the test compound was added, it differentiated into cells with granules as morphological changes compared to the case without the test compound. Admitted. The results of esterase staining by the naphthol AS-D chloroacetate method also confirmed that 70 to 85% of HL-60 had differentiated into granulocytes.

c) Reactivity with anti-CD33 monoclonal antibody My9 The HL-60 prepared in (1) above was cultured in the same manner as in a), and the cells on the 27th day were adjusted to 1 × 10 ⁶ cells / ml. Then, 100 μl of the mixture was reacted with 10 μl of fluorescence-labeled (FITC) My9, and measured by flow cytometry. The positive rate is shown in Table 3.

Table 3 shows that when the test compound was added, the number of HL-60 undifferentiated cells decreased and the ratio of differentiated cells increased.

From the above results, the active ingredient compound of the present invention was confirmed to have a growth inhibitory effect on tumor cells and an ability to induce differentiation.

Fluorescence labeled (FITC) to a monoclonal antibody recognizing FH-6 adjustments sialyl Le ^X Pharmacological Test Example 3 Differentiation inducing test (8) a monoclonal antibody FH-6, the present invention the active ingredient compound and reactivity with FH-6 Was examined by a flow cytometry method as follows.

(9) Reactivity with monoclonal antibody FH-6 The test compound prepared in the above (3) was added to the HL-60 prepared in the above (1) at final concentrations of 1.0 μg / ml and 10 μg / ml, respectively. The mixture was added and cultured at 37 ° C. in a 5% carbon dioxide incubator for 2 hours. Thereafter, the number of cells was adjusted to 1 × 10 ⁶ cells / ml, and 100 μl of the mixture was reacted with 10 μl of fluorescently labeled FH-6, and measured by flow cytometry. The positive rate is shown in Table 4.

From the above results, the addition of the active ingredient compound of the present invention, HL
It was confirmed that sialic acid was reduced from the cell surface of −60. This indicates that HL-60 was differentiated and sialidase activity was increased.

Pharmacological test example 4 Effect on human promyelocytic leukemia cells (HL-60) HL-60 in RPMI-1640 medium supplemented with 10% FCS at 37 ° C, 5% C
The cells were cultured under O ₂ to prepare a cell concentration of 5 × 10 ⁴ cells / ml.

Next, the above-mentioned medium containing 30 μg / ml of each test compound was added to each well of a 6-well microplate (manufactured by Coaster), and then cultured at 37 ° C. under 5% CO ₂ for 3 days. As a control group, there was provided a group in which only the above-mentioned medium was added and culture was performed in the same manner. After culturing, each cultured cell suspension was placed in an Eppendorf tube, stained with 0.2% trypan blue-containing phosphate buffer, and the number of viable cells was counted using a hemocytometer.

The above cells were prepared at 1 × 10 ⁷ cells / ml, and 100 μl of the cells were added to fluorescein isothiocyanate (FITC) -labeled anti-human CD11b.
Add 5 μl of antibody (Mol, manufactured by Coulter), and place on ice for 3 hours.
The reaction was performed in the dark for 0 minutes. After the reaction, the plate was washed twice with PBS (phosphate buffer, manufactured by Nissui Pharmaceutical Co., Ltd.) containing 0.1% BSA (bovine serum albumin, manufactured by Sigma) and finally suspended in 500 μl, followed by flow cytometry. By profile
The fluorescence intensity was measured by II (manufactured by Coulter).

 The results are shown in Tables 5 and 6.

In each of the groups using each test compound from each table, a cell growth inhibitory effect was observed, and an increase in CD11b expression resulted in
It was recognized that the induction of differentiation into granulocytes, monocytes and macrophages was promoted.

Pharmacological Test Example 5 M12 melanoma cell growth inhibitory effect test This test was performed using 10 Balb / c nude mice per group.

On the test start day (day 0), 2 × 10 ⁶ M12 melanoma cells
Individuals were transplanted into test group mice. When the tumor volume reached 100 mm ³ , mice in the test group were orally administered 10 mg / kg / day of test compound 1. The above administration was performed 30 times from the 25th day to the 55th day. After transplantation of the tumor cells, the diameter of the tumor was measured daily using a capillary. Tumor volume was calculated by the following formula.

Tumor volume (mm ³ ) = (major axis) × (minor axis) ² × 1/2 The results are shown in FIG.

FIG. 2 also shows the same results of the control group (control, no test compound administered) mice. In the figure, * indicates a significant difference p <0.05 according to the Student's T test. FIG. 2 shows that the administration of the test compound can significantly suppress the growth of M12 melanoma cells.

Pharmacological Test Example 6 ATL-infected cell growth inhibitory effect test The following pharmacological test was performed using Compound 1 as a test compound.

(A) Human peripheral blood acute lymphocytic leukemia cells (Molt-4)
Preparation of human peripheral blood acute lymphocytic leukemia cells (Molt-4) [J.
Minowada, et al., J. Natl. Cancer Inst., 49 , 891-895 (1
972): ATCC CRL1582] in RPMI-1640 medium (Gibco) supplemented with 10% FCS (fetal calf serum, Gibco) at 1 × 10 ⁵
ATL virus-uninfected cells.

(B) Preparation of human adult T-cell leukemia cells (5S) Human adult T-cell leukemia cells (5S) were added to 10% FCS (manufactured by Gibco) in RPMI-1640 medium (manufactured by Gibco) at 1 × 10 ^5.
Cells / ml and used as ATL virus-infected cells.

(C) Preparation of test compound After dissolving compound 1 in hydrochloric acid, dilute with the same medium as above, and add 2N
After neutralization with NaOH, the solution was adjusted to concentrations of 1 μg / ml, 10 μg / ml and 30 μg / ml and used.

(D) Pharmacological test Each cell prepared in the above (a) and (b) is put into each well of a 24-well culture plate (manufactured by Nunc), and the test compound prepared in the above (c) is added thereto ( Final concentration 1μg / m
1, 10 μg / ml and 30 μg / ml) and cultured at 37 ° C. in a 5% carbon dioxide incubator for 4 days. As a control, a medium containing no test compound was used. After the completion of the culture, the cell suspension in each well was taken, and the number of viable cells was counted.

 Table 7 shows the results.

Table 7 shows that the compound of the present invention (Compound 1)
It is clear that the proliferation of ATL-infected cells is strongly inhibited at a concentration of 1 μg / ml without affecting the cells not infected with the LV virus.

Pharmacological Test Example 7 Test of antiretroviral activity (a) Preparation of normal human transformed T cells (MT-4) Normal human transformed T cells (MT-4) [Nagum
o, T. and Hoshino, H., Jpn. J. Cancer Res., 79 , 9-11 (18
8)] was adjusted to 2 × 10 ⁵ cells / ml in RPMI-1640 medium (manufactured by Gibco) supplemented with 10% FCS (manufactured by Gibco).

(B) Preparation of test compound After dissolving compound 1 in hydrochloric acid, dilute with the same medium as above, and add 2N
After neutralization with NaOH, 1 μg / ml, 10 μg / ml, 30 μg / ml and 100 μg / ml
Each concentration of μg / ml was prepared and used.

(C) Pharmacological test In each well of a 12-well culture plate (manufactured by Coaster),
Each cell prepared in the above (a) and the test compound prepared in the above (b) (final concentration: 1 μg / ml, 10 μg / ml, 30 μg / ml and 100 μg / ml) were added, and further, 50 μl / well of the HIV suspension Was added to each well to infect cells, and the cells were cultured at 37 ° C. in a 5% carbon dioxide incubator for 3 days. As a control, a medium containing no test compound was used. After the completion of the culture, the cell suspension in each well was taken, and the number of viable cells was counted.

Next, the cell suspension in each well was centrifuged (1500
(rpm × 5 minutes) to obtain 750 μl of the culture supernatant.

750 μl of the culture supernatant obtained above is placed in an Eppendorf tube (1.8 ml), 4 M NaCl and PEG-6000 (manufactured by Sigma) are further added, and the mixture is allowed to react at 0 ° C. for 2 hours to sediment the virus. (15000 rpm × 20 minutes) to obtain the virus precipitate. Solution I [50% glycerin (manufactured by Wako Pure Chemical Industries, Ltd.), 25 mM Tris-hydrochloric acid (pH 7.5), 50 m
M KCl, 0.025% Triton X-100 (manufactured by Sigma) and 5 mM
DTT (manufactured by Sigma)] and solution II [0.9% Triton X-100
(Manufactured by Sigma) and 1.5 M KCl], and dissolved at 0 ° C. for 30 minutes. 5 mg / 10 μl of the obtained virus lysis solution
ml BSA (bovine serum albumin: Sigma), 1M Tris
Hydrochloric acid, 100 mM DTT (manufactured by Sigma), 1 M KCl, 10 mM poly A
(Manufactured by Sigma), 0.15 mM TTP (manufactured by Sigma), 200 mM Mg
Cl ₂ , 0.15 mM oligo dT (manufactured by NEN) and [ ³ H] -TTP (NEN
Was added and reacted at 37 ° C. for 1 hour. Immediately after the reaction, the reaction was stopped by placing on ice and the filter (DE-81 F
ilter; manufactured by Whatman) and dried. After drying, the plate was washed three times with 0.5 M Na ₂ HPO ₄ , rinsed in ethanol, dried, and the radioactivity of [ ³ H] -TTP incorporated into the DNA on the membrane was measured using a liquid scintillation counter. This is used as the reverse transcriptase activity (unit: cp
m).

Table 8 shows the results of the number of living cells (× 10 ⁴ ) and the reverse transcriptase activity (cpm) determined above.

Table 8 shows that the active ingredient compound of the present invention (Compound 1) was 30
It was found that the doses of μg / ml and 100 μg / ml strongly suppressed reverse transcriptase activity as well as cell proliferation.

From the results of the above pharmacological test examples 6 and 7, it is clear that the compound used as an active ingredient in the present invention has strong antiretroviral activity.

Pharmacological Test Example 8 autoimmune disease model effects on animals _{W / BF 1: (NZW ×} BXSB) F 1 mice are spontaneous autoimmune disease mouse, not only lupus nephritis, hypertension and myocardial infarction frequently It is a model animal that merges [Han
g, LM, et al., J. Exp. Med., 154 , 216-221 (1981)].
In addition, anti-platelet antibodies appeared with age and marked thrombocytopenia was observed, which is also a model animal for ITP (Oyaizu,
N., et al., J. Exp. Med., 167 , 2017-2077 (1988)]. The model has been reported to kill more than 90% of myocardial infarction or renal failure within 8 months of age [Susumu Ikehara, Metabolism,
Special issue, 26 , 169-176 (1989)].

The effect of prolonging the life of W / BF ₁ : (NZW × BXSB) F ₁ mice and the effect of administering Compound 1 of the present invention on blood cells and anti-platelet antibodies were examined.

_{W / BF 1: (NZW ×} BXSB) F 1 mice were used from 14 weeks of age (Kiwa purchased from experimental animal, Inc.). For each experiment, 10 mice were used per group.

The mice in each of the above groups were fed with food (Oriental Yeast Co., Ltd.) and water which are usually given to mice of this type.

Compound 1 (Lot No. 9D87M) was suspended in a 0.5% carboxymethylcellulose (CMC: Carboxy Methyl Cellulose; manufactured by Cellogen) solution and used for the experiment. Compound 1 was administered at a dose of 100 mg / kg / day and 300 mg / kg / day
Administration was started at the age of week, and gavage administration was performed for 5 weeks and 2 rests for 6 weeks until 20 weeks of age. A non-administration group was provided as a control group.

(1) As observation items, the number of survivors from the age of 14 weeks to the age of 20 weeks was observed as a life-prolonging effect, and the statistical processing was performed by using the non-administered group as a control to test a significant difference by a Generalized Wilcoxon test. FIG. 3 shows the results.

In the figure, the vertical axis indicates the percentage (%) of the number of living and dying, and the horizontal axis indicates the age (14 to 20 weeks).

According to the figure, in the group administered with Compound 1 at 100 mg / kg / day, no deaths were observed until the age of 20 weeks. In the 300 mg / kg / day administration group, two animals died at the age of 18 weeks. In the non-administration group, 2 animals at 17 weeks of age, 1 animal at 18 weeks of age, and 3 animals at 19 weeks of age were confirmed, and the active ingredient compound of the present invention (Compound 1) showed a prolonged survival effect on autoimmune disease model mice. confirmed.

(2) Blood cell analysis was performed by collecting blood samples by fundus blood sampling at 14, 16, 18, and 20 weeks of age, and using an automatic blood cell analyzer (Coulter).
The change in blood cell count (white blood cell count, red blood cell count, platelet count) was measured using JR; In addition, the number of leukocyte fractions (the number of neutrophils and the number of lymphocytes) was determined after preparing a blood smear and staining with Wright Giemsa. Statistical processing was performed by the Student's t-test using the non-administration group on each blood collection day as a control.

 Table 9 shows the results.

W / BF ₁ mice hematological characteristics decrease of leukocytosis and platelets is observed with aging as in the report of the Ikehara. The leukocyte count tended to increase with age in the non-administration group and the compound 1 administration group, and the effect of the compound of the present invention was not observed. In the non-administration group, the platelet count decreased from 14 weeks of age (1144.5 ± 121.3 / mm ³ ), and at the age of 20 weeks, it was about one fifth (228.0
± 90.0 / mm ³ ). However, in the group administered with 300 mg / kg / day of the active ingredient compound of the present invention (compound 1), the effect of suppressing the decrease in platelet count appeared from the start of administration, and at the age of 20 weeks (823.
3 ± 24.5 mm ³ ), a significant inhibitory effect was observed in the non-administration group (228.0 ± 90.0 / mm ³ ). Similar results were obtained for the red blood cell count.

(3) The W / BF ₁ mice, Oyaizu (see, Oyaizu) et the cause of thrombocytopenia is reported to occur by the appearance of anti-platelet antibodies, as described above (2), the present compound 1
Administration reduced the decrease in platelets.
Antiplatelet antibodies at the age of one week were identified.

The measurement of anti-platelet antibody was performed by incubating platelets collected from Balb / c mice with W / BF ₁ plasma administered up to 20 weeks of age at room temperature for 30 minutes, washing twice, and FITC-labeled anti-mouse Ig.
G (manufactured by Tago; code No. 6250) and stained.
Analysis was performed using a CS analyzer (EPICS-Profile II, manufactured by Coulter Corporation). The negative control was set so that the plasma of the Balb / c mouse was 98% negative, and the positive rate of each sample was determined.

 Table 10 shows the results.

In the table, NT indicates that measurement was impossible because it died at the time of measurement, and the numbers indicate percent positive rate (%).

According to the table, in the non-administration group, the positive rate was 19.6 ± 5.5%, whereas the active ingredient compound of the present invention (Compound 1) was 100 m
In the g / kg / day group, the positive rate was 13.2 ± 2.7%, and in the 300 mg / kg / day group, the positive rate was 10.7 ± 2.2%, indicating a dose-dependent suppression of antiplatelet antibodies.

W / BF ₁ mice are spontaneous mouse model of the aforementioned as human chronic ITP, develop lupus nephritis than age 3 months, 8 months earlier 90% or more but die in myocardial infarction or renal insufficiency, the It has been reported that mice can be treated by transplanting bone marrow of normal Balb / c mice [Ikehara, S., et al., Proc. Natl. Acad. Sci. U.
SA, 82 , 2483-2487 (1985); Yasumizu, R., et al., Proc. N
atl.Acad.Sci.USA, 84, 6555-6557 (1987) ; Ikehara, S,.
et al., Proc. Natl. Acad. Sci. USA, 56 , 3306-53310 (198
9)]. The most effective treatment for ITP in actual clinical practice is currently bone marrow transplantation.

Carbostyril derivative of the present invention the active ingredient compound, the (1) showed a significant life-prolonging effect on the W / BF ₁ mice As can be seen from the results to (3), is simultaneously hematological characteristics of the mouse It also suppresses thrombocytopenia due to the appearance of anti-platelet antibodies, indicating that it has an autoimmune disease ameliorating effect.

Pharmacological Test Example 9 Thrombocytopenia-Improving Effect Test 1) Preparation of Test Compound 1 Compound 1 was dissolved in 1N hydrochloric acid and diluted with FCS (fetal bovine serum, Gibco) to prepare a 1 mg / ml solution. 10
% FCS-added RPMI-1640 medium (manufactured by Flow) and 1N
After neutralization with -NaOH, the solution was adjusted to a concentration of 30 µg / ml and used.

2) Preparation of CMK cells CMK cells were established from patients with acute megakaryoblastic leukemia by Sato and colleagues, and anti-platelet antibodies (anti-glycoprotein II b / I
IIa antibody, anti-glycoprotein Ib antibody, Plt-1 antibody), and has megakaryocyte-like properties such as formation of α-granules and platelet separation membranes in the cytoplasm.・ Used for analysis of differentiation [Sato, T., et al., Ex
p.Hematol., 15 , 495 (1987); Sunami, S., et al., Blood, 7
0 , 368 (1987); Komatsu, N., et al., Blood, 74 (1) 42-
48 (1989); Fuse, A., et al., British J. Haematology, 7
7 , 32-36 (1991); Journal of the Japanese Society of Hematology, 53 (2) 294-2
95,317-318 (1990)].

CMK cells were provided by Dr. Takeyuki Sato of the Department of Pediatrics, Chiba University School of Medicine, and 5 × 10 ⁴ cells / ml were added to 10% FCS-containing RPMI-164.
The cells were cultured in medium 0 and passaged every 4 days based on the number of cells.

3) Effect of Compound 1 on expression of platelet-associated antigen in CMK cells 1 × 10 ⁵ CMK cells / ml in 10% F containing 30 μg / ml of Compound 1
After culturing in CS-containing RPMI-1640 medium for 4 days, expression of platelet-related antigen on the cell surface was determined by flow cytometry (manufactured by Flow Co.) using FITC-labeled Plt-1 antibody (manufactured by Coulter).
Was measured by In addition, only a medium containing no compound 1 was used as a solvent control.

For analysis of cell surface antigen, 1 × 10 ⁶ CMK cells / 100 μl were taken in a tube (Fisher), and FITC-labeled Plt-1 was collected.
After adding 5 μl of the antibody and reacting at 4 ° C. for 30 minutes, 0.1
After washing three times with a phosphate buffer containing 5% bovine serum albumin, the percentage of positive cells and the fluorescence intensity were measured.

The Plt-1 antibody (manufactured by Coulter) is an antibody that recognizes glycoprotein IIb / IIIa, a platelet-associated antigen, and is CM
It has been reported that K cells increase with differentiation or maturation [Komatsu, N., et al., Blood, 74 (1) 42-
48 (1989)].

 Table 11 shows the results.

According to the table, about 96% of the CMK cells used were positive for platelet-related antigen recognized by the Plt-1 antibody.
No change in the percentage of positive cells was observed even with the addition of 30 μg / ml.

However, when comparing the average fluorescence intensity representing the amount of antigen expression per cell, the average fluorescence intensity of the solvent control was 100%
As a result, 191% was obtained in cells to which 30 μg / ml of compound 1 was added.
It can be seen that the addition of Compound 1 increased the expression level of the platelet-related antigen recognized by the Plt-1 antibody on the surface of the CMK cells about 2-fold.

4) Morphological observation of CMK cells 1 × 10 ⁵ CMK cells / ml containing compound 1 and RP containing 10% FCS
The cells were cultured in MI-1640 medium for 4 days, and the morphology of the cells was observed by a phase contrast photograph. In addition, a part of the cells was collected, a cytospin specimen was prepared, and the cell morphology was observed by Wright Giemsa staining.

The results are shown in FIGS. 4 and 6 show a solvent control, and FIGS. 5 and 7 show a phase contrast diagram and a staining diagram of cells by administration of Compound 1 at 30 μg / ml.

From the phase contrast diagram, it was confirmed that the cells to which the compound 1 of the present invention was added at 30 μg / ml showed fragmentation of cells in a manner similar to the platelet production process. From the staining diagram, germination of the cell membrane and lobulation of the nucleus were observed in the cells to which the compound 1 of the present invention was added.

From the above results, the carbostyril derivative, which is an active ingredient of the present invention, increases the expression level of platelet-related antigen on the surface of CMK cells, causes fragmentation of cells in a platelet production process-like manner, and further germinates cell membranes of cells. And nucleation of the nucleus, it is thought to promote the differentiation or maturation of CMK cells. Therefore, it can be expected that this unique action acts on megakaryocyte progenitor cells or megakaryocyte cells to promote their differentiation and maturation, thereby promoting platelet production and improving thrombocytopenia.

Pharmacological Test Example 10 Autoimmune Disease Therapeutic Effect Test In this test, female MRL / Mp-lpr mice (7 weeks old, purchased from Charles River Japan, Inc.) from 19 weeks of age, 5 per group
They were used individually.

The test mouse is a mouse spontaneously developing an autoimmune disease, and its pathological condition is characterized by human SLE (Systemic
Known lupus erythematosus-like immune complex glomerulonephritis, lymphoma, vasculitis, polyarthritis, etc. (Theo
filopolos, A.and Dixon, FJ, Adv.Immunology , 37, 269
(1985): Murphy, EDRoths, JB, Autocommunity and l
ymphoproliferation, Elsevier North Holland, New yor
k, 207-221 (1978): Yoshihiro Taniguchi, Modern Medicine, 21 , 131 (19
89)).

Each mouse of the compound 1 administration group and the control group (untreated group) was supplied with mouse feed (manufactured by Oriental Yeast) and water.

Compound 1 is 0.5% carboxymethylcellulose (CMC,
(Daiichi Kogyo Seiyaku Co., Ltd.) solution.

Administration of Compound 1 was started at the age of 19 weeks at a dose of 300 mg / kg / day per 1 kg of mouse body weight, and gavage administration was carried out 5 weeks and 2 days a week until 25 weeks of age (Compound 1 administration group).

For each group of mice, changes in urine protein, lymphoma diameter, vasculitis of the pinna, and the presence or absence of hair loss were examined.

As a result, the compound 1 administration group tended to suppress the increase in urine protein level as compared with the control group. The lymphoma diameter was 7.8 compared to 9.0 ± 0.8 mm in the control group.
It was ± 1.3 mm, and its hypertrophy was slightly suppressed.

FIG. 8 shows the results of comparing the degree of vasculitis, alopecia and lymphoma.

As shown in the figure, in the compound 1 administration group, vasculitis and hair loss were improved and the lymphoma diameter was reduced compared to the control group.

From the above results, it can be seen that administration of Compound 1 can be expected to improve the autoimmune disease based on the apoptosis-modulating effect.

Pharmacological Test Example 11 Effect test on PC12 cells PC12 cells subcultured in Dulbecco's modified MEM (D-MEM) medium containing 5% heat-inactivated (56 ° C, 30 minutes) horse serum and 10% fetal calf serum (FCS) Was coated with collagen and had a diameter of 35
The cells were transplanted into a mm plastic Petri dish at a concentration of 6 × 10 ⁴ cells / 3 ml medium. On the second day of transplantation, the cells were replaced with D-MEM (control) containing various concentrations of compound 1, nerve growth factor (NGF, manufactured by Wako Pure Chemical Industries) and FCS, and culturing was continued for each of them for 3 days. The morphological change of the eye cells was observed with a phase contrast microscope.

 The results are shown in FIGS.

In FIG. 9, the upper row shows the control group to which only FCS was added, and the lower row shows Compound 1
As shown in the figure, formation of nerve fiber-like projections, which is a typical morphological change, was observed in the lower row compared to the upper row. It can be seen that it is observed under coexistence (lower left). When the co-existing FCS concentration is further increased, cells with different morphologies also appear (lower middle and right). Thus, morphological changes were induced in 30-50% of all cells.

In Fig. 10, the upper row is the group to which only NGF is added (however, in the presence of 0.3% FCS)
And the lower row is 30 μg / ml of Compound 1 in the presence of 0.3% FCS.
This is a group to which NGF (0 to 30 ng / ml) was added. From the figure,
In addition to the NGF-stimulated neurite formation seen in the upper row, the lower row shows that the coexistence of compound 1 and NGF (but in the presence of 0.3% FCS) promotes the elongation of the above-mentioned projections more than those alone. Admitted.

From each of the above figures, it can be seen that the active ingredient compound 1 of the present invention exhibits an NGF-like action based on an apoptosis-modulating action, which is useful as a therapeutic agent for Alzheimer's disease.

Pharmacological test example 12 Effect test on liver failure Male BALB / c mice (7 weeks old, purchased from SLC, Inc.) were subjected to heat-killed P.acnes (Osaka City University Medical School No. 3)
3 mg / body was intravenously administered.
Seven days later, 10 mg / kg or 100 mg / kg of Compound 1 was administered together with the vehicle (0.5% CMC, manufactured by Daiichi Kogyo Seiyaku Co., Ltd.) administration group and the non-administration group.
Was administered orally. 1 of the above compound 1 administration
5 hours after LPS (Lipopoly saccharide, Sigma)
Acute liver injury was induced by intravenous administration of μg / body.

The survival rate of each group of mice was observed up to 7 days after LPS administration.

 The results are shown in FIG.

From the figure, the survival rate of the 10 mg / kg administration group of Compound 1 (test group) was 30%, and the survival rate of the 100 mg / kg administration group of Compound 1 (test group) was 70%. In contrast, the survival rate in the non-administration group was 0%, and that in the vehicle administration group was 10%. From this,
Compound 1 was found to exert an excellent life-prolonging effect in an acute hepatitis model in a dose-dependent manner, and thus was confirmed to be effective as a therapeutic agent for hepatitis based on an apoptosis-modulating effect.

Pharmacological Test Example 13 Effect test of B16 melanoma cells on experimental lung metastasis model 2 × 10 ⁶ B16 mouse melanoma cells (Koren, S.an
d Fleischmann, WR, J. Interferon Reseach, 6 , 473-48
2 (198) was suspended in an Eagle-MEM medium (manufactured by Nippon Pharmaceutical Co., Ltd.) supplemented with 10% FBS (manufactured by Gibco) with or without the addition of 3 μg / ml or 10 μg / ml of compound 1. In a 75 cm ² flask (manufactured by Corning Incorporated) under a 5% carbon dioxide gas atmosphere at 37 ° C (National Appliance, Model 53)
00 (National appliance, model 5300)) for 4 days. After the culture, the cells were washed with Dulbecco's PBS ^(-) (Nissui Pharmaceutical Co., Ltd.) solution, added with a 0.05% trypsin (Flow Co., Ltd.) solution and detached from the flask. After washing twice by centrifugation at 1200 rpm for 5 minutes (using 05PR-22, manufactured by Hitachi, Ltd.), 0.2% trypan blue (manufactured by Wako Pure Chemical Industries) solution was added, and a hemocytometer (No. J7796, KAYAGAKI WORKS) The number of viable cells was counted, and the number of cells was diluted to 1.5 × 10 ⁶ cells / ml with Hanks' (manufactured by Flow Corporation) solution.

Next, the treated cells obtained above were combined with 7 C57BL /
Six mice (7 weeks old, female, purchased from Charles River Japan, Inc.) were transplanted 3 × 10 ⁵ cells each from the tail vein, and 12 days later, the mice were killed by dislocation of the cervical spine and the number of colonies metastasized to the lung was counted. .

 The results are shown in FIG. 12 and Table 12 below.

From the above figures and tables, metastasis was clearly inhibited in both the group transplanted with cells cultured in the medium containing 3 μg / ml of Compound 1 and the group transplanted with cells cultured in the medium containing 10 μg / ml of Compound 1. The effect was recognized. This indicates that the active ingredient compound of the present invention is useful as a cancer metastasis inhibitor based on the apoptosis-modulating effect.

INDUSTRIAL APPLICABILITY The apoptosis regulating agent of the present invention can be effectively used as a chemotherapeutic agent for cancer based on its unique apoptosis regulating ability and cell differentiation inducing ability. Also, as a therapeutic agent for AIDS, ARC, ATL and other related diseases and HTLV-I related disease due to anti-retroviral effect, as a therapeutic agent for autoimmune disease due to prolonged effect of autoimmune disease It is effective as a therapeutic agent for thrombocytopenia, as a therapeutic agent for Alzheimer's disease due to an NGF-like effect, as a therapeutic agent for hepatitis due to a prolonged effect of liver failure, and as a cancer metastasis inhibitor due to a cancer metastasis suppressing effect.

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical indication location // C07D 215/22 C07D 215/22 (31) Priority claim number Japanese Patent Application No. 4-100585 (32 ) Priority Date Hei 4 (1992) March 25 (33) Priority Country Japan (JP) (72) Inventor Hiroyuki Ichikawa 3-11 Nakadori-cho, Tokushima City, Tokushima Prefecture (72) Inventor Seiji Akamatsu Tokushima Prefecture 223-1 Kawasaki, Omamachi, Naruto (72) Inventor Shiro Saito 2294-80 Yamanamachi, Takasaki City, Gunma Prefecture

Claims (8)

(57) [Claims] (1) 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. ] An apoptosis-modulating agent comprising as an active ingredient at least one compound selected from the carbostyril derivative represented by the formula (1) and a salt thereof together with a pharmacologically acceptable carrier. 2. The apoptosis regulator according to claim 1, which is used as an anticancer agent. 3. The apoptosis regulator according to claim 1, which is used as an antiretroviral agent. 4. The apoptosis regulator according to claim 1, which is used as a therapeutic agent for an autoimmune disease. 5. The apoptosis regulator according to claim 1, which is used as a therapeutic agent for thrombocytopenia. 6. The apoptosis-modulating agent according to claim 1, which is used as a therapeutic agent for Alzheimer's disease. 7. The apoptosis-modulating agent according to claim 1, which is used as a therapeutic agent for liver disease. 8. The apoptosis regulator according to claim 1, which is used as a cancer metastasis inhibitor.