WO1996003148A1

WO1996003148A1 - Method and drug for treating diabetes

Info

Publication number: WO1996003148A1
Application number: PCT/JP1995/001484
Authority: WO
Inventors: Koichiro Kitagawa; Atsushi Hirano; Hiroyuki Ohno
Original assignee: Ono Pharmaceutical Co., Ltd.
Priority date: 1994-07-27
Filing date: 1995-07-26
Publication date: 1996-02-08
Also published as: JPH1135479A

Abstract

A novel protein p82; a method and drug for treating diabetes by tyrosinephosphorylating the protein; a drug for treating diabetes containing a derivative capable of tyrosine-phosphorylating the protein, such as a thiazoline derivative, a benzoquinone derivative, a naphthoquinone derivative or vanadic acid, as the active ingredient; and a method of screening a drug for treating diabetes by using the protein. The tyrosinephosphorylation of protein p82 is an indispensable step for glycogen synthesis and the above treatment method and drug and screening method are useful for the amelioration of hyperglycemia and the prevention, treatement, diagnosis and/or measurement of diabetes, particularly non-insulin-dependent diabetes mellitus.

Description

Description Treatment method and therapeutic agent for diabetes

The present invention relates to a novel protein, a method for treating diabetes and a therapeutic agent using the protein. More specifically, p82, which is a key protein of the insulin signal transduction system in the liver, and tyrosine phosphorylation of that protein (hereinafter sometimes simply referred to as phosphorylation in the present specification). A therapeutic method for diabetes characterized by promoting glycogen synthesis; a therapeutic agent for the therapeutic method; a therapeutic agent for diabetes containing a substance that tyrosine phosphorylates protein P82 as an active ingredient; and a protein p. A method for screening a therapeutic agent for diabetes, which comprises using 8 2. Background art

Diabetes is a disease in which the metabolic mechanism of sugar is impaired due to a lack of insulin action. The basic characteristics are diminished glucose tolerance and chronic hyperglycemia. Diabetes is divided into two categories depending on the cause of the disorder.

One type is a type in which insulin is not secreted or its secretion level is extremely low even when the blood sugar level rises due to food intake, and is called insulin-dependent diabetes mellitus (IDDM or type I diabetes). Insulin-dependent diabetes has been shown to be caused by the destruction of S cells in the arm. As a treatment, insulin is supplied from outside the body.

The other is that, despite the normal secretion of insulin, the response mechanisms in peripheral tissues are not working properly, such as muscle uptake in muscle. It is a type in which blood glucose levels do not decrease due to a decrease in promoting action, suppression of daricogen synthesis in the liver, and enhancement of gluconeogenesis, etc., and are called insulin-independent diabetes (NIDDM or type II diabetes). In the United States, 5% of the population over the age of 40 is so common that non-insulin-dependent diabetes is said to be common, but the cause has not yet been elucidated.

Glucose metabolism in a normal state is performed by the following mechanism.

That is, carbohydrates ingested as food are digested into glucose in the small intestine and taken up in the blood. The S / C cells of the kidneys sense the rise in blood sugar and secrete insulin. Insulin acts on the target peripheral tissues (muscle and liver) in the bloodstream, lowering blood glucose by promoting the absorption of glucose in the blood and accumulating it as glycogen.

Specifically, in muscle, a glucose transporter 4 (G1ut4) is specifically expressed in muscle, and G1ut4 is converted from the intracellular to the cell membrane by insulin signal transduction. It promotes sugar uptake activity by trans-cases and increasing its number. On the other hand, in the liver, there is G 1 ut 2 as a sugar transporter, whose number is said to remain unchanged. In the liver, enzyme-induced promotion of glycolysis-based key proteins such as dalcokinase, phosphofructokinase, and pyruvate kinase is observed with insulin signal transduction. As a result, the liver increases glucose absorption and suppresses the release of new sugars into the blood. In the liver, the action of insulin promotes the activity of glycogen synthase and suppresses phosphorylase kinase, and glycogen accumulates in tissues. Z b

In particular, the insulin-mediated regulation of dalycogen synthesis is one of the areas that are currently receiving the most attention.

The mechanism of action of insulin on the promotion of glycogen synthesis in the liver has been studied mainly on the serine-threonine kinase cascade system.

Ras Raf 1 MAPKK ISPK

PP1G GSK3 glycogen synthase

(MAPKK is mitogen-activated protein kinase kinase ^ IS PK Is insul in— stimulated protein kinase ゝ PP 1 G means protein phosphatase 1G, GSK 3 means glycogen synthase kinase 3 Completion of the transduction pathway described in (1) has made great strides in elucidating the mechanism by which insulin regulates glycogen synthesis in the liver.

On the other hand, when insulin binds to the α-subunit of the insulin receptor, the subunit is autolysinated, and at the same time, the tyrosin kinase site in the receptor is activated. Six proteins have been identified as substrates that are phosphorylated by this kinase.

One is the insulin receptor substrate-1 (IRS-1) with a molecular weight of 18.5 kD, consisting of 1235 amino acids. Tyrosine phosphorylation of IRS-1 has been shown to bind and activate PI 3-kinase, an enzyme that phosphorylates phosphatidylinositol.

It is also believed that activation of Ras occurs through IRS-1. Ash / Grb2 (molecular weight 25 kD) and S os (molecular weight 18 k The pathway via the conjugate of D) has been reported but has not yet been proven.

In 1988, Cooper et al. (Cooper, G. J. S.) and others discovered a hormone secreted from the spleen S cells as well as insulin due to hyperglycemia, and named it amylin. They pointed out that amirin is a causative substance of insulin resistance because it abolishes insulin action as a physiological action of amylin.

In 1991, there was a definitive report that overexpression of amylin in transgenic mice causes insulin-independent diabetes. More recently, it has been reported that amilin inhibits the action of insulin to stimulate glycogen synthesis in the liver. However, it is currently unknown how amylin is involved in the insulin signaling system. Disclosure of the invention

The present inventors have found that amylin does not affect insulin in the liver.

> if

Focusing on the inhibition of the stimulatory action, we investigated the site of inhibition of the amylin phosphorylation cascade system and succeeded in identifying fq5 ({(I white (p82).

As a result, the p82 protein was involved in the synthesis of glyogen in the liver ⁽ ^ r), and the present invention was completed.

That is, the present invention

(1) a method for treating diabetes, comprising tyrosine phosphorylating protein p82;

(2) a therapeutic agent for diabetes characterized by tyrosine phosphorylation of protein p82,

(3) an antidiabetic agent comprising, as an active ingredient, a substance that tyrosine phosphorylates protein p82;

(4) a method for screening a therapeutic agent for diabetes, which comprises using protein p82;

(5) a protein with a molecular weight of 82 kD that activates the synthesis of dalycogen by being tyrosine phosphorylated, and

(6) The present invention relates to a protein having a molecular arrangement of 82 kD which is isolated with an anti-phosphotyrosine antibody by adding insulin to rat F a0 cells.

Insulin promotes glycogen synthesis in the liver as previously reported. On the other hand, amilin inhibits the action of insulin to promote glycogen synthesis in a concentration-dependent manner. The present inventors suspect that this may be because amylin inhibits the insulin signaling system, and developed the insulin-dependent tyrosine phosphorylation cascade system as an anti-phosphotyrosine antibody (PY69 antibody from ICN, Mouse Anti-phosphotyrosine Monoc Cat. No. 69-138 (Ronal antibody). As a result, a protein having a molecular weight of 82 kD, whose tyrosine phosphorylation was specifically inhibited by amirin treatment, was successfully identified.

Hereinafter, this protein is called P82 based on its molecular weight, and the tyrosine phosphorylated protein is called Pp82. In the liver, several proteins that are phosphorylated on tyrosine by insulin have been reported, but no protein with a molecular weight of 82 kD has been reported. From the viewpoint of molecular weight, this is presumed to be a completely novel protein that has not been known so far.

Further, since the protein p82 of the present invention does not react with an antibody against AshZGrbS and Sos, the protein P82 is presumed to be a novel protein completely different from AshZGrb2 and Sos. .

Next, the present inventors examined the correlation between the degree of tyrosine phosphorylation of P82 and the degree of acceleration of glycogen synthesis using various compounds.

(±) — 5— [4- (6-hydroxy 2,5,7,8—tetramethyl chroman-2-ylmethoxy) benzyl] 1,2,4-thiazolidinedione (CS-045), vitamin K5 and vanadic acid have been reported to promote glycogen synthesis in the liver. Thus, when the promotion of tyrosine phosphorylation of p82 by these compounds was examined, all compounds promoted the phosphorylation. These results revealed that activation of P82 protein by tyrosine phosphorylation is essential for the activation of glycogen synthase by insulin and is a protein involved in signal transduction following the insulin receptor.

Accordingly, the present invention relates to a method for treating diabetes, particularly insulin-independent diabetes, which is characterized by tyrosine phosphorylation of protein P82.

Furthermore, the present invention relates to a therapeutic agent for diabetes, particularly insulin-independent diabetes, which is characterized by tyrosine phosphorylation of protein P82. Here, a method for treating diabetes and a therapeutic agent for diabetes, which is characterized by tyrosine phosphorylation of protein p82, are defined as all diabetes mellitus whose main action mechanism is tyrosine phosphorylation of protein P82. It includes a therapeutic method and a therapeutic agent for diabetes.

In addition, the present invention relates to a therapeutic agent for diabetes containing a substance that tyrosine phosphorylates protein P82 as an active ingredient.

Here, the substance that tyrosine phosphorylates the protein p82 is not only a substance that is currently confirmed to have that activity, but also a substance that will be confirmed to have a new activity in the future. Substances. At present, substances that have been confirmed to have cinnacin oxidation activity include, for example,

(1) — General formula (I)

(In the formula, X represents an oxygen atom, a sulfur atom or an imino group, n represents an integer of 1 to 3, R ¹ represents a hydrogen atom or a C 1-4 alkyl group,

R 2

R ³ and R ⁵ are each independently hydrogen atom, C 1 represents 4 Al kill or alkoxy group C 1 to 4, R 4 is a hydrogen atom, Arukiru group of C 1 ~ 4,

0

Base (Wherein, R ⁶ is a C 1-6 alkyl group, C 2-6 alkenyl group, C 4-7 cycloalkyl group, phenyl group, naphthyl group, or nitro group, halogen atom, NR ′ R ⁸ group (Wherein, R ⁷ and R ° represent a hydrogen atom or a C 1-4 alkyl group.), One or two groups selected from a C 1-4 alkyl group, a C 1-4 alkoxy group, and a hydroxyl group Represents a fuunyl or naphthyl group, a 4- to 7-membered heterocyclic ring containing one oxygen atom, sulfur atom or nitrogen atom, or a C 6 to 11 aralkyloxy group. Gin derivatives, their non-toxic and acid addition salts,

(2) General formula (Π)

(lib)

(In the formula, m Rs are each independently a hydrogen atom, a C 1-4 alkyl group, a hydroxy group, an amino group, or a COOR ^{1 ()} group (wherein, R ^1G is a hydrogen atom Or m represents an alkyl group of 1 to 4), and m represents an integer of 1 to 3. Benzene or naphthalene derivative represented by), a non-toxic salt and an acid addition salt thereof,

(3) Vanadic acid.

More specifically, as the compound represented by the general formula (I),

5— [4— (6-hydroxy 2,5,7,8—tetramethylchroman—2-ylmethoxy) benzyl] 1-2,4-thiazolidinedione (CS-045),

5- [4- [2- (6-hydroxy-1,2,5,7,8-tetramethylchroman-2-yl) ethoxy] benzyl] — 2,4-thiazolidindio ,

5-[4-(6-hydroxy2,5,7,8-tetramethylchroman- 1-ylmethoxy) benzyl]

5— [4— [2- (6-hydroxy-1,2,5,7,8—tetramethylchroman-1-yl) ethoxy] benzyl] —rhodanine. — As the compound represented by the general formula (II),

4Amino 2—Hydroxybenzoic acid,

(4) Amino (11) Naphthol,

4-Amino-2-naphthol,

1-ami nonaphthalene,

1,4—dihydroxynaphthalene,

4Amino 2-methyl-1 naphthol (vitamin Κ),

0

1,4-dihydroxy-2-naphthoic acid and the like.

Examples of suitable non-toxic salts include salts of alkali metal (such as potassium and sodium), salts of alkaline earth metals (such as calcium and magnesium), ammonium salts, and pharmaceutically acceptable salts. Acceptable organic amines (tetramethylammonium, triethylamine, methylamine, dimethylamine, cyclobetylamine, benzylamine, phenethylamine, pyridin, monoethylamine) Salts of min, diethanolamine, tris (hydroxymethyl) amine, lysine, arginine, N-methyl-D-glucamine, etc. are mentioned.

Suitable acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, phosphate, and nitrate, or citrate, trifluoroacetate, lactate, tartrate, and the like. Oxalate, fumarate, maleate, citrate, benzoate, methyl sulfonate, ethane sulfonate, benzene sulfonate, toluene sulfonate, isethionate, glucuronic acid And organic acid salts such as dalconate.

—The compounds represented by the general formulas (I) and (Π) are known per se or can be easily produced by known methods. Industrial applicability

The substance that tyrosine phosphorylates the protein P82 used in the present invention is useful for ameliorating hyperglycemia, preventing diabetes, and in particular, preventing or treating insulin-independent diabetes.

In order to use the respective active ingredients of the substance that tyrosine phosphorylates the protein P82 according to the present invention and the non-toxic salts and acid addition salts thereof for the above-mentioned purposes, generally, systemically or locally, orally or parenterally It is administered in the form of The dose varies depending on the age, body weight, symptoms, therapeutic effect, administration method, treatment time, and the like, but is usually orally administered once to several times a day or parenteral (preferably intravenously). Administration) or continuous intravenous administration for 1 hour to 24 hours per day.

When the compound of the present invention is administered, it is used as a solid composition, a liquid composition and other compositions for oral administration, an injection, an external preparation, a suppository and the like for parenteral administration.

Solid compositions for oral administration include tablets, pills, capsules, powders, granules and the like.

Capsules include hard capsules and soft capsules. In such a solid composition, the one or more active substances include at least one inert diluent, such as lactose, mannitol, mannite, glucose, hydroxypropylcellulose, microcrystalline cellulose. , Starch, polyvinylpyrrolidone, mixed with magnesium metasilicate aluminate.

0 one The composition may be formulated according to the usual practice with additives other than inert diluents, e.g. lubricants such as magnesium stearate, disintegrants such as calcium cellulose dalycholate, glutamate or aspartate. Such a solubilizing agent may be contained.

Tablets or pills may be coated with a film of gastric or enteric substance such as sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, etc., if necessary. It may be coated with a layer. Also included are capsules of absorbable materials, such as gelatin.

Liquid compositions for oral administration include pharmaceutically acceptable emulsions, solutions, syrups, elixirs and the like. In such liquid compositions, one or more active substances are contained in commonly used inert diluents (eg, purified water, ethanol). The composition may contain, in addition to the inert diluent, adjuvants such as wetting agents and suspending agents, sweetening agents, flavoring agents, fragrances, and preservatives.

Other compositions for oral administration include sprays which contain one or more active substances and are formulated in a manner known per se. This composition contains, in addition to the inert diluent, a stabilizer such as sodium bisulfite to provide isotonicity, an isotonic agent such as sodium chloride, sodium citrate or citric acid. May be. The method for producing the spraying agent is described in detail in, for example, US Pat. Nos. 2,868,691 and 3,095,355.

Injections for parenteral administration according to the present invention include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Aqueous solutions and suspensions include, for example, distilled water for injection and physiological saline. Non-aqueous solutions and suspensions include, for example, propylene glycol, polyethylene Glycols, vegetable oils such as olive oil, alcohols such as ethanol, polysorbate 80 (registered trademark), and the like. Such compositions may also contain adjuvants such as preserving, wetting, emulsifying, dispersing, stabilizing, and solubilizing agents (eg, glutamic acid, aspartic acid). .

These are sterilized by filtration through a bacteria-retaining filter, blending of a bactericide or irradiation. They can also be used to produce sterile solid compositions which are sterilized or dissolved in sterile distilled water for injection or other solvents before use.

Other compositions for parenteral administration include topical solutions containing one or more active substances and are formulated in a conventional manner, such as topical, topical, topical, suppository and vaginal preparations for rectal administration. Includes accessories for internal administration. It is presumed that the protein P82 of the present invention has an action mechanism as described above. Therefore, the protein P82 of the present invention itself can be used as an agent for improving hyperglycemia, preventing diabetes, particularly insulin-independent diabetes and as a Z or therapeutic agent.

The protein of the present invention includes those obtained by binding a sugar chain and phosphate to the protein obtained by the present invention. That is, the present invention includes a sugar chain-bound p82 protein and a tyrosine-phosphorylated P82 protein (pp82).

Furthermore, the present invention relates to a method for screening a therapeutic agent for diabetes, which comprises using the protein P82.

From the above information transmission mechanism, primary evaluation of darikogen synthesis activity can be performed by compound screening using the degree of tyrosine phosphorylation of the P82 protein as an index. For example, F a 0 rat bets hepatocytes using Darube Tsu co Modified Eagle Medium (1 0% fetal calf serum-containing), 5% C 0 ₂ culture Incubate at 37 ° C for 8-10 days in a culture vessel. After that, the cells are treated with serum-free Dulbecco's modified Eagle's medium for 18 hours, and then amylin is added, followed by incubation for 30 minutes. After adding insulin, culturing for a certain period of time, and washing with ice-cold phosphate buffer. After homogenizing the cells, collect the cytosol fraction centrifugally. An anti-phosphotyrosine antibody is added to this fraction, reacted, and recovered with Protein G Sepharose. After eluting the pp 82 protein with a phenyl phosphate solution, solubilize it in SDS sample buffer and perform electrophoresis. After the protein is transferred to a dinitrocellulose membrane, the protein is detected by an estanbut method using an anti-phosphotyrosine antibody, and the degree of phosphorylation of p82 protein is quantified.

Therefore, according to the present invention, a therapeutic agent for diabetes, particularly insulin-independent diabetes, can be screened by using protein P82.

Further, according to the present invention, diabetes can be diagnosed by using the protein p82. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail with reference to examples, but these do not limit the present invention. Example 1: Purification method of p82

F ao rats using preparative hepatocyte cells preparative Rei ⁽² 2 5 cm 2), in 5% CO _n incubator, and cultured 7-1 0 days at 37. The medium used was Dulbecco's modified Eagle medium (containing 10% fetal bovine serum), and the medium was changed every three days. The cells were treated with serum-free Dulbecco's modified Eagle's medium for 2 hours, and then 200 mM vanadate and 100 nM insulin were added. Reacted for 10 minutes. The cells were suspended in 25 mM Tris buffer (200 mM vanadate, containing a protease inhibitor), solubilized, and the supernatant was collected by centrifugation. A final concentration of 0.1% octa (ethylene glycol) ether (C12E8) was added, and the mixture was subjected to Millipore membrane filtration.

A protein G sepharose gel to which an anti-phosphotyrosine antibody (I 丫 1 ^ ?? 69 antibody, mouse anti-phosphotyrosine monoclonal antibody of Catalog No. 69-138) was conjugated was prepared, and the sample solution treated above was filled. . Tyrosine phosphorylated protein (PP82) was adsorbed on the gel. After washing the column with 25 mM Tris buffer, pp82 was eluted with 1 OmM phenyl phosphate solution. The eluate was concentrated with Centricon 30 and then precipitated by acetone precipitation. The purified PP82 was separated by electrophoresis, transferred to a PVDF membrane, treated with trypsin, and separated by liquid chromatography. The phosphoric acid was removed from the thus purified pp82 by a known method to obtain purified p82. Example 2: Glycogen synthesis in F ao hepatocytes

F a0 rat hepatocytes (10 ⁵ cells) were transformed into 5% CO using Dulbecco's modified Figur medium (containing 10% fetal calf serum). The cells were cultured at 37 ° C for 8 days in an incubator. After that, the cells were treated with a modified Dulbecco's Eagle's medium without serum for 18 hours, and then washed with Krebs-Ringer buffer. Subsequently, amylin was added, and the cells were further cultured for 30 minutes. Insulin was added thereto, followed by culturing for 10 minutes, and C-glucose (5 mM, 0.25 / zC i) was added thereto, followed by culturing for 1 hour at 37 ° C. Thereafter, the cells were washed with the same buffer and treated with a 1 N aqueous solution of sodium hydroxide for 1 hour. The cells were collected by centrifugation, left overnight in 75% ethanol, and the recovered glycogen was measured with a liquid scintillation counter. The results are shown in Table 1.

^, π rm Ma ¹⁴ C emissions amount

Caro cause factor

(dpm / dish) Control 250 Insulin (100 425 Insulin + Amylin (10 nM) 220 Insulin + Amylin (1 nM) 250 Insulin + Amylin (100 pM) 300

From Table 1, it was confirmed that amylin inhibited the glycogen synthesis promoting action of insulin in a concentration-dependent manner. Example 3: Tyrosine phosphorylation of p82 protein

F a 0 rat hepatocytes (10 ⁵ Zd ish) with dull Beck co variant Igu Le medium (10% fetal calf serum-containing), and in 5% C0 ₉ incubator, 8 days of culture at 37 ° C for did. After that, the cells were treated with a modified Dulbecco's Eagle's medium containing no serum for 18 hours. After adding insulin, incubate for 10 minutes, and ice-cold phosphate buffer Washed with liquid. After homogenizing the cells, the cytosol fraction was collected by centrifugation. The p82 protein was recovered by Sepharose beads to which an anti-phosphorotyrosine antibody was bound, then eluted with phenylphosphite, and detected by Western blotting. Using purified PP82 as a standard, the amount of pP82 in the band was measured with a densitometer. The results are shown in Table 2.

Table 2

From Table 2, it was confirmed that amylin suppresses the action of insulin to promote the tyrosine phosphorylation of p82 protein.

Example 4: Correlation between the degree of phosphorylation of p82 protein and glycogen synthesis activity CS—045, vitamin K5, and vanadic acid were performed in the same manner as in Examples 2 and 3, and The results shown in 3 were obtained. Table 3

From Table 3, it was confirmed that a compound that promotes tyrosine phosphorylation of p82 protein promotes glycogen synthesis. Formulation Example 1

The following components were mixed by a conventional method, and tabletted to obtain 10 σ tablets containing 5 _mg of the active ingredient in one tablet.

• (±) — 5— [4- (6-hydroxy-1,2,5,7,8-tetramethyl roman-1-2-ylmethoxy) benzyl] 1,2,4-thiazolidine …… 500 mg

• Calcium carboxymethylcellulose 200 mg · magnesium stearate ... 100 mg

* Microcrystalline cellulose …… 9.2g Formulation Example 2

The following components were mixed in a conventional manner, and the mixture was tableted to give 100 tablets each containing 5 mg of the active ingredient.

• Vitamin K 550 mg

• Calcium carboxymethylcellulose 200 mg

• Magnesium stearate 100 mg

'Microcrystalline cellulose …… 9. 2 g

Claims

The scope of the claims

1) A method for treating diabetes, comprising tyrosine phosphorylating protein P82.

2) A therapeutic agent for diabetes characterized by tyrosine phosphorylation of protein p82.

3) An antidiabetic agent comprising, as an active ingredient, a substance that tyrosine phosphorylates protein P82.

4) A method for screening a therapeutic agent for diabetes, which comprises using protein p82.

5) A protein with a molecular weight of 82 kD that activates glycogen synthesis by being tyrosine phosphorylated.

6) A protein with a molecular weight of 82 kD that is isolated with an anti-phosphotyrosine antibody by adding insulin to rat Fa0 cells.