JP2900526B2 - Enzyme activator - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an enzyme activator useful as a medicament such as a therapeutic agent for Alzheimer's disease, and more particularly to a kidney bean containing an N-myristoyl amino acid and a derivative thereof as an active ingredient. It relates to a syn enzyme activator.

(Prior Art) Ingensin enzyme is known to be an enzyme that degrades A4 (β-protein) accumulated in the brain in Alzheimer's disease.

Ishiura et al. (FEBS Lett., 189, 119, 1985)
During screening of artificial substrates for dependent proteases,
A substance that binds to DEAE cellulose and is eluted at a high salt concentration.
A substance having an activity of decomposing succinyl-leucyl-leucyl-valyl-tyrosine-methylcoumarylamide was found, and this enzyme was named ingensin (multicatalytic protein). This enzyme activity is determined by gel filtration.
Shows properties such as being recovered by more than 500,000 high molecular substances, activated by linoleic acid, rat liver, human placenta,
It is described that this enzyme isolated and purified from rabbit reticulocytes is composed of 20 or more subunits each having a molecular weight of 35-25,000.

The action of ingensin was not clear for a long time,
Ishiura et al. (FEBS Lett., 201, 87, 1986) show that ATP-dependent proteolysis in rabbit reticulocytes is inhibited by inhibitors of ingensin. It indicates that it is related to decomposition. In particular, in Alzheimer's disease, it is known that amyloid accumulates in the brain. In the process of producing A4 (β-protein), which is a main component of this amyloid, the generation of A4 ', which is three peptides smaller than A4, causes this bean. It is known that syn is acting, and it is suggested that it may help to degrade A4, an abnormal protein (FEBS Lett., 257, 388, 1989).

It is also described that ingensin is the same substance as 19S particles (prosomes) bound to free mRNA that is not bound to polysomes of erythroblasts and Hela cells. Although the mRNA to which the 19S particles are bound is inactivated, translation is possible when the protein is removed from this complex, and it is considered that the prosome suppresses protein translation by mRNA (J. Mol. Biol., 187, 4
79, 1986).

In addition to multigenetic protein, besides ingensin, proteasome, macropain, prosome and the like are known, and it has been revealed that these substances are the same substance (Biochem. J., 255 volumes,
750, 1988).

(Problems to be Solved by the Invention) So far, it has been known that fatty acids such as linoleic acid and myristic acid are activated 5 to 10 times as substances that increase the enzyme activity of ingensin. The degree of activation is still inadequate (Biochem. Biophys.
a, 882, 305, 1986).

An object of the present invention is to provide an enzyme activator comprising a low-toxicity compound close to a natural product, for remarkably increasing the enzyme activity of ingensin.

(Means for Solving the Problems) The present invention has a general formula: (Wherein, A represents an amino acid residue) An activator of ingensin comprising an N-myristoyl amino acid and its derivative represented by the formula:

In the above general formula, A represents a natural or synthetic amino acid residue, and the amino acid is preferably an α-amino acid such as glycine, serine, alanine, phenylalanine, threonine, glutamic acid, aspartic acid, valine, leucine, or proline. , Especially glycine, D-
Serine, L-serine, D-alanine, L-alanine and the like are preferred. Examples of the N-myristoyl amino acid include all compounds in which an amino acid is bound to myristic acid, and representative compounds include the following.

N-myristoyl-glycine N-myristoyl-L-serine N-myristoyl-L-alanine N-myristoyl-L-phenylalanine N-myristoyl-L-threonine N-myristoyl-L-glutamic acid N-myristoyl-L-aspartic acid N- Myristoyl-L-valine N-myristoyl-L-leucine N-myristoyl-L-proline N-myristoyl-D-serine N-myristoyl-D-alanine N-myristoyl-D-phenylalanine N-myristoyl-D-threonine N-myristoyl -D-glutamic acid N-myristoyl-D-aspartic acid N-myristoyl-D-valine N-myristoyl-D-leucine N-myristoyl-D-proline Further, as the N-myristoyl amino acid derivative, Salts are also included. The most commonly used salts are
Salts such as sodium, potassium and calcium.

The compound represented by the above general formula can be synthesized by reacting a fatty acid, which is a natural product, with an amino acid in the presence of a catalyst such as, for example, 4-hydroxyphenyldimethylsulfomethyl sulfate or dicyclohexylcarbodiimide.

The enzyme activator of the present invention containing the compound represented by the general formula as an active ingredient can be administered orally or parenterally. That is, tablets, capsules, syrups, suspensions and the like, which are commonly used dosage forms, can be orally administered, or a solution thereof,
Parenteral administration, such as injection, can be carried out in the form of a liquid such as an emulsion or suspension. These preparations include the usual excipients, disintegrants,
Binders, lubricants, dyes, diluents and the like are used.

The dose and frequency of administration vary depending on symptoms, age, body weight, dosage form, etc., but are usually 0.1 to 5000 m per adult per day.
g, preferably in the range of 10-1000 mg.

The extent to which the obtained activator of the present invention activates the ingensin enzyme is determined by the succinyl-leucyl enzymologically known to directly correspond to the enzymatic degradation of amyloid A4 (β-protein). -Leucyl-valyl-tyrosine-methylcoumarylamide (SLLVT) can be easily measured using an enzymatic decomposition reaction.

(Effect of the Invention) The enzyme activator of the present invention has a remarkable action of ingensin enzyme activation. In addition, N-myristoyl amino acid, which is a main component, is composed of naturally occurring fatty acids, myristic acid and amino acids, so it is safe with low toxicity, and activates ingensin to induce brain damage in Alzheimer's disease. It promotes the degradation of A4 (β-protein), a major component of accumulated amyloid, and is useful as a medicament for preventing and treating Alzheimer's disease.

(Examples) Hereinafter, the present invention will be described more specifically with reference to examples.

(Production example)

First, production examples of N-myristoyl amino acids and derivatives thereof will be described.

Production Example 1 2.0 g (0.02 mol) of L-serine and triethylamine
2.8 ml (0.02 mol) is dissolved in 60 ml of water, and 9.52 g (0.02 mol) of myristic acid ester of 4-hydroxyphenyldimethylsulfomethyl sulfate (trade name, Suncellar My-DSP (manufactured by Sanshin Chemical Industry Co., Ltd.)) At room temperature
Stir for 12 hours. The reaction was adjusted to pH 2-3 with 1N HCl and the reaction mixture was extracted with ethyl acetate. After the ethyl acetate layer was dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure. The product was dissolved in ethyl acetate-methanol (3: 1 v / v), concentrated under reduced pressure, and the crystallized product was washed with ethyl acetate and hexane to obtain 3.6 g of N-myristoyl-L-serine.

Production Example 2 D-serine 2.0 g (0.02 mol) and triethylamine
Dissolve 2.8 ml (0.02 mol) in 60 ml of water,
-9.52 g (0.02 mol) of DSP (Sanshin Chemical Industry Co., Ltd.) was added, and the mixture was stirred at room temperature for 12 hours. The reaction solution is adjusted to pH 2-3 with 1N-HCl.
And the reaction mixture was extracted with ethyl acetate. After the ethyl acetate layer was dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure. The product was dissolved in ethyl acetate-methanol (3: 1 v / v), concentrated under reduced pressure, and the crystallized product was washed with ethyl acetate and hexane to obtain 3.8 g of N-myristoyl-D-serine.

Production Example 3 1.78 g (0.02 mol) of L-alanine and 2.8 ml (0.02 mol) of triethylamine were dissolved in 60 ml of water.
9.52 g (0.02 mol) of My-DSP (Sanshin Chemical Industry Co., Ltd.) was added, and the mixture was stirred at room temperature for 12 hours. 1N-HCl to pH 2 ~
3 and the reaction mixture was extracted with ethyl acetate. After the ethyl acetate layer was dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure. The product was dissolved in ethyl acetate-methanol (3: 1 v / v), concentrated under reduced pressure, and the crystallized product was washed with ethyl acetate and hexane and washed with 3.6 g of N-myristoyl-L-alanine.
I got

Production Example 4 2.66 g (0.01 mol) of 4-hydroxyphenyldimethylsulfomethyl sulfate (trade name, Suncellar DSP (manufactured by Sanshin Chemical Industry Co., Ltd.)) was dissolved by heating in 40 ml of acetonitrile, and 2.28 g (0.01 mol) of myristic acid was dissolved. At 0 ° C
And stirred. To this solution, 2.06 g (0.01 mol) of dicyclohexylcarbodiimide was gradually added, and the mixture was stirred at 0 ° C. for 2 hours and further at room temperature for 2 hours. The reaction solution was filtered to remove dicyclohexylurea, and the filtrate was evaporated under reduced pressure to obtain an oil. 0.75 g (0.01 mol) of glycine and 1.4 ml (0.01 mol) of triethylamine were added to 10 ml of water, and while stirring, a solution of the above oily substance in acetonitrile was added, followed by stirring at room temperature for 10 hours. After the reaction, the reaction solution was concentrated under reduced pressure, adjusted to pH 2 to 3 by adding 6N-HCl, and extracted with ethyl acetate. After washing the ethyl acetate layer with water, it was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The product was treated with ethyl acetate-methanol (3: 1 v /
v), concentrated under reduced pressure, and the crystallized product was washed with ethyl acetate and hexane to obtain 1.1 g of N-myristoyl-glycine.

Production Example 5 Suncellar DSP (Sanshin Chemical Industry Co., Ltd.) 2.66 g (0.
01mol) was dissolved in 40 ml of acetonitrile by heating, and 2.28 g (0.01 mol) of myristic acid was added under a nitrogen stream, followed by stirring at 0 ° C. To this solution was added dicyclohexylcarbodiimide 2.06
g (0.01 mol) was gradually added, and the mixture was stirred at 0 ° C. for 2 hours and further at room temperature for 2 hours. The reaction solution was filtered to remove dicyclohexylurea produced as a by-product, and the filtrate was distilled off under reduced pressure to obtain an oil. To 10 ml of water, 1.0 g (0.01 mol) of L-serine and 1.4 ml (0.01 mol) of triethylamine were added, and while stirring, a solution of the above oily substance in acetonitrile was added, followed by stirring at room temperature for 10 hours. After the reaction, the reaction solution was concentrated under reduced pressure, adjusted to pH 2 to 3 by adding 6N-HCl, and extracted with ethyl acetate. After washing the ethyl acetate layer with water, it was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The product was treated with ethyl acetate-methanol (3: 1 v
/ v), adjusted to pH 11 with a 10% sodium hydroxide solution, and after confirming that a precipitate had formed, concentrated under reduced pressure. The crystallized product was washed with ethyl acetate and hexane, and
-1.9 g of myristoyl-L-serine sodium salt was obtained.

(Formulation example)

The following preparations were prepared using the N-myristoyl amino acids and derivatives thereof obtained in each of the production examples as active ingredients.

Formulation Example 1 (Tablets) One tablet (150 mg) contains the following components.

Active ingredient (Preparation example 1) 50 mg Lactose 48 mg Starch 50 mg Polyvinylpyrrolidone 1.5 mg Magnesium stearate 0.5 mg Total 150 mg Formulation example 2 (capsule) One gelatin capsule contains the following components (150 mg).

Active ingredient (Production example 2) 50 mg Lactose 59.5 mg Starch 40 mg Light anhydrous silicic acid 0.5 mg Total 150 mg Formulation example 3 (granules) 1 g of granules contains the following ingredients.

Active ingredient (Production Example 3) 200 mg Lactose 450 mg Starch 300 mg Hydroxypropylcellulose 50 mg Total 1000 mg [Enzyme activity measurement] The enzyme activity of ingencin was measured as follows.
A predetermined amount of each enzyme activator was added to ingensin, 50 mM Tris-HCl buffer, 0.1 mM succinyl-leucyl-leucyl-valyl-tyrosine-methylcoumatorilamide (SLLVT), and the mixture was incubated at 37 ° C. for 30 minutes. ,
The reaction was stopped by adding 10% sodium dodecyl sulfate. Aminomethylcoumarin released from SLLVT by enzymatic degradation is analyzed by a fluorescence spectrophotometer (excitation 380 nm,
Fluorescence 460 nm). The results are shown in FIG. 1 and FIG. In the figure, ○ indicates N-myristoyl-L-serine, ● indicates N-myristoyl-D-serine, Δ indicates N-myristoyl-L-alanine, ▲ indicates N-myristoyl-D-serine.
An enzyme activator containing alanine is shown, and □ shows a control containing myristic acid instead of these compounds.

〔result〕

As shown in FIG. 1, N-myristoyl-L-serine (○) activated the enzyme activity of ingencin from 5.5 to 128.0 (23-fold) at the added amount of 0.25 mg / ml. On the other hand, with myristic acid (□), the enzyme activity was activated only 5-fold with the same amount. N-myristoyl-D-serine (●) activated the enzyme activity 21-fold at 0.75 mg / ml.

Also, as shown in FIG. 2, N-myristoyl-L-
Alanine (△) activated the enzyme activity 26-fold at the added amount of 0.1 mg / ml, and N-myristoyl-D-alanine (▲) activated the enzyme activity 32-fold at 0.5 mg / ml. This gives N
-It has been demonstrated that myristoyl amino acids activate ingensin more than hitherto known myristic acid.

[Brief description of the drawings]

FIG. 1 is a graph showing the relationship between the amount of N-myristoylserine added and the enzyme activity. FIG. 2 is a graph showing the relationship between the amount of N-myristoylalanine added and the enzyme activity. In the figure, ○ indicates N-myristoyl-L-serine, ● indicates N-myristoyl-D-serine, and Δ indicates N-myristoyl-L-.
Alanine and ▲ indicate enzyme activators containing N-myristoyl-D-alanine, respectively, and □ indicates a control containing myristic acid in place of these compounds.

Claims (1)

(57) [Claims] (1) General formula (In the formula, A represents an amino acid residue) An ingensin enzyme activator comprising, as an active ingredient, an N-myristoyl amino acid represented by the formula: