JPH06183963A - Inhibitor of tyrosine-specific phosphoenzyme and carcinostatic agent - Google Patents

Abstract

PURPOSE:To obtain a medicine having low toxicity and remarkable inhibitary activity against a tyrosine-specific phosphoenzyme and carcinostatic action. CONSTITUTION:This inhibitor of a tyrosine-specific phosphoenzyme as carcinostatic agent comprise tyrosine sulfate or its physiologically permissible salt as an active ingredient.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a tyrosine-specific phosphatase inhibitor and a carcinostatic agent containing sulfated tyrosine or a physiologically acceptable salt thereof as an active ingredient.

[0002]

BACKGROUND OF THE INVENTION Cancers caused by radiation, chemical carcinogens or viruses are often accompanied by chromosomal abnormalities, that is, genetic abnormalities.
The crazy gene that causes cancer is called an oncogene, and it exists in normal cells as its protooncogene (PROTO-ONCOGENE). PROTO-ONCOGENE is not carcinogenic as it is. Up to now, several dozen types of oncogenes have been identified. Among these oncogenes, there is a gene group that encodes tyrosine-specific phosphatase, and these oncogenes are associated with carcinogenesis and proliferation of cancer cells. It is considered (Fukushima Medical Journal Vol. 41 No. 4 (1991) 379-395). Such oncogenes include src (Rous sarcoma virus), yes (Y73 sarcoma virus), fps (Fujinami sarcoma virus), mos.
(Moloney sarcoma virus), fes (feline sarcoma virus), erb B (avian acute erythroblastosis virus) and many others are known. Thus, tyrosine-specific phosphatase derived from oncogenes is considered to be involved in the canceration of normal cells and the growth of cancer cells. Therefore, a drug capable of specifically inhibiting the enzyme activity is to prevent carcinogenesis. It is also useful for treating cancer. JP-A-62-106017 discloses 5,7-dihydroxy-4′-substituted-isoflavone-2 having tyrosine-specific phosphatase inhibitory activity.
-A carcinostatic agent containing a carboxylic acid or its ethyl ester as an active ingredient is disclosed.

[0003]

Accordingly, it is an object of the present invention to provide a tyrosine-specific kinase inhibitor capable of inhibiting the activity of tyrosine-specific kinase. Another object of the present invention is to provide an anticancer agent containing a drug having a tyrosine-specific phosphatase inhibitory activity as an active ingredient.

[0004]

[Means for Solving the Problems] In order to achieve the above object, the present inventors have searched for compounds having tyrosine-specific phosphorylase inhibitory activity, and as a result, sulfated tyrosine and its salts were found to be specific for oncogene-derived tyrosine. The inventors have found that they have a strong inhibitory action on selective phosphatase and completed the present invention. The present invention provides a tyrosine-specific kinase inhibitor containing sulfated tyrosine or a physiologically acceptable salt thereof as an active ingredient. The present invention also provides an anticancer agent containing sulfated tyrosine or a physiologically acceptable salt thereof as an active ingredient. Hereinafter, the present invention will be described in detail. The tyrosine in the sulfated tyrosine of the present invention may be D-form, L-form or DL-form. As the physiologically acceptable salt of this sulfated tyrosine, sodium salt, potassium salt, lithium salt, calcium salt, magnesium salt, barium salt,
Known metal salts, inorganic acid salts, organic acid salts and the like such as tetrabutylammonium salt, hydrochloride, sulfate and acetate can be mentioned.

Sulfated tyrosine is described in 19 by Reitz et al.
Its synthesis was reported in 1946 (HC Reitz et a
L., J. Am. Chem. Soc., Vol. 68, p1024 (1946)). 1
It was first discovered in 954 as a protein component (F.
R. Bettelheim. J. Am. Chem. Soc., Vol. 76, p2838 (19
54))), and sulfated tyrosine is known to exist in the urine of mammals in a free state (RA John, et al.,
Biochem. J. Vol. 94, p37 (1965)). Thus, the sulfate esterification of tyrosine and its peptides and tyrosine residues in proteins is generally carried out in vivo in animals, plants, microorganisms, etc. for detoxification and expression of physiological effects, and sulfated tyrosine and It is known that peptides and proteins containing it exist as biological components (Review; WB
Huttner, Method in Enzymology (F. Wold and K. Molda
ve, eds.) Vol.107, pp200-223 (1984)). Since then, there have been numerous reports, but nothing was known about the tyrosine-specific inhibitory action of sulfated tyrosine.

The sulfated tyrosine of the present invention can be produced by treating tyrosine enzymatically or chemically. For example, as an enzymatic method, (1) Saidh
a, T, Arch. Biochem. Biophys., 272, 237-244 (198
9); and (2) Llu, MC, Biochem. Int., 21, 815-821.
(1991) is known. Examples of the chemical method include a method of degrading a protein containing sulfated tyrosine and a method of converting tyrosine into a sulfate ester. In the latter case, a known sulfuric acid esterification reagent can be used, but sulfuric acid, chlorosulfonic acid, sulfur trioxide, trimethylsilylsulfonic acid chloride, etc. are suitable.
eitz and others have been reported (HC Reitz et al., J. Am. Che
m. Soc., Vol. 68, p1024 (1946)). As the salt of sulfated tyrosine, potassium salt (FR Jevons, Bioche
m. J. 89,621 (1963)), barium salt (L. Moroder, Hopp
e-Seyler's Z. Physiol. Chem., 360,787 (1979)), sodium salt (WB Huttner, Method in Enzymology (F.
Wold and K. Moldave, eds.) Vol. 107, pp200-223 (1
984)) etc. are known.

The tyrosine-specific phosphatase inhibitor of the present invention is used as an active ingredient of an anticancer agent, as well as as a drug for treating and preventing various diseases caused by tyrosine-specific phosphatase, and as a tyrosine-specific inhibitor. It can also be used as a reagent for research on phosphorylase. The anticancer agent of the present invention includes, in addition to its active ingredient, sulfated tyrosine or a physiologically acceptable salt thereof, lactose, starch, dextrin, calcium phosphate, calcium carbonate, synthetic and natural aluminum silicate, magnesium oxide, and hydroxide. Pharmaceutical additives such as excipients, auxiliaries and additives such as aluminum, magnesium stearate and sodium bicarbonate may be included. The type and addition amount of these agents can be appropriately selected depending on the administration method and dosage form of the anticancer agent. The administration method of the anticancer agent of the present invention includes intravenous administration,
Infusion, oral administration, subcutaneous administration, intraperitoneal administration and the like can be mentioned, and the dosage forms include injections, infusions, tablets, capsules,
Granules and the like can be mentioned. These formulations can be manufactured according to a conventional method. The content of sulfated tyrosine or a physiologically acceptable salt thereof, which is an active ingredient in the anticancer drug of the present invention, can be appropriately selected according to the symptoms, administration route, dosage form and the like. Further, the dose of the anticancer agent of the present invention varies greatly depending on the progressing state of the disease and the administration form.
However, in the case of an adult, the active ingredient is about 1 mg to 10 g /
The day is appropriate.

The present invention will be described in more detail with reference to synthesis examples, test examples and examples. Synthesis Example Synthesis of L-Sulfated Tyrosine Sodium 2 g of L-tyrosine, 5 g of sulfur trioxide trimethylammonium salt, and 20 ml of pyridine were reacted at 55 ° C. for 93 hours under a nitrogen atmosphere. After the reaction was completed, two layers were formed, so the pyridine layer of the supernatant was removed, and the pyridine was washed with methylene chloride. This was poured into 200 ml of water containing 12 g of cooled sodium hydrogen carbonate, and insoluble matter was removed by filtration. Then, 100 ml of water containing 6.8 g of tetrabutylammonium hydrogensulfate and 1.8 g of sodium hydrogencarbonate was added to the filtrate, and the mixture was extracted with methylene chloride. The methylene chloride layer was dried over magnesium sulfate and then concentrated and dried. Amberlite IR 120B (Na ⁺ ) was added to the residue.
200 ml and 200 ml of water were added, and the mixture was stirred at room temperature overnight. After removing the amberlite by filtration, the aqueous solution was washed with methylene chloride and freeze-dried. As a result, 2 g of white or pale yellowish white powder of L-sulfated tyrosine sodium was obtained.

The IR and UV spectra of the L-sulfated sodium tyrosine thus synthesized were measured. The results are shown below. IR: ν _max, cm ^-1 (KBr) 3475, 1660, 1510, 1250, 1060, 1020, 880 UV: λ _max, nm (H ₂ O) 263 (ε = 2.6 × 10 ² ) λ _max, nm (0.01 N hydrochloric acid) 263 (ε = 5.0 × 10 ² ). Furthermore, when this sample was heated in a 0.1N hydrochloric acid solution at 80 ° C. for 10 minutes, λ _max, 2 peculiar to the phenolic hydroxyl group,
UV absorption at 76 nm appeared and showed the same absorption peak as that of L-tyrosine. When the hydrochloric acid hydrolyzate was analyzed by high performance liquid chromatography, it showed a single peak, which was consistent with the retention time of L-tyrosine. As is clear from the above UV measurement, UV absorption derived from the phenolic hydroxyl group near 280 nm disappeared in this sample. Therefore, the L-sulfated sodium tyrosine synthesized here is considered to be a sodium salt of a sulfuric acid ester of the phenolic hydroxyl group of tyrosine represented by the following formula. _{^{R 1 OOC-CH (NH 2}} ) -CH 2 -C 6 H 4 -OSO
₃ R ^{2 In the} formula, at least one of R ¹ and R ² is a sodium atom, and the other is a hydrogen atom or a sodium atom.

(Test Example) Oncogene-derived tyrosine-specific reagent
Method for measuring oxidase inhibitory activity : method for measuring tyrosine-specific phosphatase activity of human epidermal cancer cell growth factor receptor (EGF receptor, A431 cells) (S. Choen et al., J. Biol. Chem., Vol. 2)
55, 4834-4842 (1980)) A cell membrane prepared from human epithelial cancer cells (A431 cells) known to contain a large amount of EGF receptor was used as an enzyme source. 50 mM Hepes in 30 μl
-NaOH pH7.2,10mM MgCl _2, 3.
3 mM MnCl ₂ , 1 mM DTT, various concentrations of L-
Sodium sulfate tyrosine, and [γ- ³² P] AT
The reaction solution containing the cell membrane of P, A431 cells was reacted at 0 ° C. for 30 minutes, and then the reaction was stopped. The reaction solution was analyzed by 8% polyacrylamide gel electrophoresis-autoradiography, the degree of phosphorylation of the EGF receptor having a molecular weight of 160,000 was measured by a densitometer, and the inhibitory concentration of sodium L-sulfated tyrosine was determined. . The cell membrane preparation from A431 cells was performed as follows. A431 cells cultured in a Dulbecco's MEM (manufactured by Nippon Suisan Co., Ltd.) medium containing 7% fetal bovine serum (manufactured by Gibco) were collected and the method of Cohen et al. (S. Choen et al., J. Biol. Chem., Vol. . twenty five
7, 1523-1531 (1982)). As a result, the 50% inhibitory concentration of L-sulfated sodium tyrosine was 16 μg / ml. Thus, the sulfated tyrosine of the present invention showed a strong oncogene-derived tyrosine-specific kinase inhibitory activity.

(Toxicity) A) Effect on T Cell Proliferation The effect of a drug on the growth of HTLV-IIIB non-infected MT-4 cells was observed in the same manner as in Test Example. The results are shown in Table 1.

[Table 1] B) Suppression of proliferation of mouse leukemia cell L1210 cells To the mouse leukemia cell L1210 cell solution, L-sulfated tyrosine sodium was added at a concentration of 250 μg / ml, and 4
Cultured for a day. Compared to the control without addition, it showed almost no growth inhibition. C) Acute toxicity test using mice When 500 mg / Kg sodium L-sulfated tyrosine sodium was intraperitoneally administered to 4-week-old ICR male mice, no remarkable toxicity was observed.

[0012]

[Example] Formulation example 1 (injection / drip) 500 mg of L-sulfated sodium tyrosine which is an active ingredient
Powdered glucose (5 g) is added so as to be contained, and the mixture is aseptically distributed into vials, sealed, and sealed with an inert gas such as nitrogen or helium, and stored in a cool dark place. Before use, add 500 ml of 0.85% physiological saline to prepare an intravenous injection for 10 to 50 days a day.
Administer 0 ml by intravenous injection or infusion depending on the symptoms. Formulation Example 2 (Injection / Drip) An intravenous injection for mild disease is produced in the same manner as in Formulation Example 1 except that 50 mg of L-sulfated tyrosine sodium, which is an active ingredient, is used. Administer 500 ml by intravenous injection or infusion depending on the symptoms. Formulation Example 3 (Injection, Capsule) 30 mg of L-sulfated tyrosine sodium as an active ingredient, 1 g of refined sesame oil and 100 mg of aluminum stearate gel
It is then dissolved in, sealed and sealed with an inert gas such as nitrogen or helium and stored in a cool dark place to prepare a subcutaneous injection preparation. Depending on the symptom, 1 to 10 ml is subcutaneously administered once a day. Also, 0.5 ml each of the above-mentioned preparation is dispensed into capsules to give oral capsules, and 1 to 10 capsules are orally administered daily depending on the symptoms.

Formulation Example 4 (Enteric-coated tablet) 1,000 enteric-coated tablets for adults (a) and dwarf (b) were prepared with the following composition. [A] (a) (b) L-sulfated sodium tyrosine 100 (g) 50 (g) Lactose 99.4 49.7 Hydroxypropyl cellulose 0.6 0.3 Magnesium stearate 2.0 1.0 [B] (b) (b) Cellulose acetate phthalate 6.0 (g) 4.0 (g) Hydroxypropylmethyl 6.0 4.0 Cellulose phthalate [A] components are taken and mixed well, and the mixture is directly pressurized or kneaded well, and then the screen of extrusion granulator. The granules were molded through and the sufficiently dried product was pressed to produce tablets. Next, the formed tablet is coated with a well-dissolved base material [B] to form an enteric-coated tablet.

Formulation Example 5 (Enteric coated granules) 1,000 g of enteric coated granules were produced with the following components. [A] L-Sulfated Tyrosine Sodium 100 (g) Lactose 737 Hydroxypropyl Cellulose 3 [B] Cellulose Acetate Phthalate 80 (g) Hydroxypropyl Methyl 80 Cellulose Phthalate [A] components were taken and mixed well, According to a conventional method, it was formed into granules, dried well, and sieved to produce granules suitable for bottles, heat-sealed packaging and the like. Next, while the granules are allowed to flow in suspension, the dissolved [B] base material is coated to form an enteric-coated granule.

Formulation Example 6 (Enteric Coated Capsules) 1,000 enteric coated capsules were prepared with the following ingredients.
(A) is for adults, (b) is for dwarfs. [A] (a) (b) L-Sulfated tyrosine sodium 100 (g) 50 (g) Lactose 24.6 74.4 Hydroxypropyl cellulose 0.4 0.4 [B] (b) (b) Cellulose acetate phthalate 10 (g) 10 ( g) Hydroxypropylmethyl 10 10 cellulose phthalate An enteric-coated granule suitable for a capsule was prepared in the same manner as described in Formulation Example 5 using the above components, and the composition was filled into a capsule to form an enteric-coated capsule. And

[0016]

INDUSTRIAL APPLICABILITY The sulfated tyrosine and physiologically acceptable salts thereof, which are the active ingredients of the present invention, have low toxicity, and have a remarkable tyrosine-specific phosphatase inhibitory activity and anticancer activity.

Claims (2)

[Claims] 1. A tyrosine-specific kinase inhibitor containing sulfated tyrosine or a physiologically acceptable salt thereof as an active ingredient. 2. An anticancer agent containing sulfated tyrosine or a physiologically acceptable salt thereof as an active ingredient.