JPH0441426A - Enzyme activator - Google Patents

Abstract

PURPOSE:To obtain a safe enzymic activator, containing an N-myristoylamino acid (derivative) as an active ingredient, capable of remarkably enhancing enzymic activity of ingensin with low toxicity and useful as a medicine for preventing and treating Alzheimer diseases. CONSTITUTION:An enzymic activator containing a compound expressed by the formula (A is amino acid residue) or a derivative thereof as an active ingredient. Glycine, D-serine, L-serine, D-alanine and L-alanine are preferred as the amino acid in the compound expressed by the formula. The compound expressed by the formula is capable of promoting decomposition of A4 (beta-protein) which is a principal ingredient of amyloids accumulated in the brain in Alzheimer diseases by activating ingensin.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention relates to an enzyme activator useful as a medicine such as a therapeutic agent for Alzheimer's disease, and more specifically to an enzyme activator that is useful as a medicine such as a therapeutic agent for Alzheimer's disease. This invention relates to a synthetic enzyme activator.

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

Ishiura et al. (FEBS Lett, Vol. 189, p. 119, 1985) discovered that succinyl-leucyl- is a substance that binds to DEAE cellulose and is eluted at high salt concentrations during screening for artificial substrates for Ca-dependent proteases. They discovered a substance that exhibits the activity of decomposing leucyl-halyl-tyrosine-methylcoumarylamide, and named this enzyme ingensin (multicatalytic protein).

This enzyme activity shows properties such as being recovered into a polymer with more than 500,000 molecules by gel filtration and being activated by linoleic acid. It is stated that all enzymes are composed of about 20 subunits with a molecular weight of 35,000 to 25,000.

The effects of ingensin have not been clear for a long time, but Ishiura et al. (FEBS Lett, Vol. 201, p. 87, 1)
(986) revealed that ATP-dependent protein degradation in rabbit reticulocytes was suppressed by an inhibitor of ingensin, indicating that ingensin is involved in the degradation of abnormal proteins. In particular, it is known that amyloid accumulates in the brain in Alzheimer's disease, and during the production process of A4 (β-protein), the main component of amyloid, A4, which is 3 peptides smaller than A4,
It is known that this ingensin acts on the production of A4, which is an abnormal protein.
t, vol. 257, p. 388, 1989).

It is also stated that ingensin is the same substance as 198 particles (prosomes) that bind to free mRNA that is not bound to polysomes of erythroblasts and HeLa cells. mRNA bound to these 193 particles
A is inactivated, but when the protein is removed from this complex it becomes translatable, meaning that the prosome is
It is thought to suppress protein translation by RNA (J, Mo1. Biol.

187, p. 479, 1986).

In addition to ingensin, multicatalytic proteins include proteasome, macropain, and prosome, but it has been revealed that these substances are the same substance (Biochem, J.
, + vol. 255, p. 750, 1988).

(Problems to be Solved by the Invention) It has been known that fatty acids such as linoleic acid and myristic acid each increase the enzyme activity of ingensin by 5 to 10 times. The degree of activation is still insufficient (Biochem, B
iophys, Acta+ volume 882, page 305, 1
986).

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

(Means for Solving the Problems) The present invention provides an ingensin enzyme activator containing N-myristoyl amino acid and its derivatives represented by the general formula: % formula % (wherein A represents an amino acid residue) as an active ingredient. It is.

In the above general formula, A represents a natural or synthetic amino acid residue, and preferable examples of the amino acid include α-amino acids such as glycine, serine, alanine, phenylalanine, threonine, glutamic acid, aspartic acid, valine, leucine, and proline. In particular, glycine, D-serine, L-serine, D-alanine, L-alanine, etc. are preferred. N-Myristoyl amino acid includes all compounds in which an amino acid is bonded to myristic acid.
The following are exemplified as representative compounds.

N-Myristoyl-Glycine N-Myristoyl-S-Serine N-Myristoyl-S-Alanine N-Myristoyl-S-Phenylalanine N-Myristoyl-S-Threonine N-Myristoyl-S-Glutamic acid N-Myristoyl-S-Aspartic acid N- Myristoyl-S-valine N-Myristoyl-S-Leucine N-Myristoyl-S-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 The N-myristoyl amino acid derivatives also include salts of the above compounds. The most commonly used salts are sodium, potassium, calcium, etc. salts.

The compound represented by the above general formula can be synthesized by reacting a natural fatty acid with an amino acid in the presence of a catalyst such as 4-hydroxyphenyldimethylsulfomethylsulfate or dicyclohexylcarbodiimide.

The enzyme activator of the present invention containing the compound represented by the above general formula as an active ingredient can be administered orally or parenterally. That is, it can be administered orally in the form of commonly used dosage forms such as tablets, capsules, syrups, suspensions, etc., or it can be administered by injection in the form of solutions, emulsions, suspensions, etc. etc., can be administered parenterally. Conventional excipients, disintegrants, binders, lubricants, dyes, diluents, etc. are used in their preparation.

Dose and frequency of administration vary depending on symptoms, age, body weight, administration form, etc., but are usually 0.1 to 500 doses per adult per day.
The dose can be administered in the range of 0 µ, preferably 10 to 1000 µ.

The extent to which the obtained activator according to the present invention activates the ingensin enzyme depends on succinyl-leucyl, which is enzymatically known to directly correspond to the enzymatic decomposition of amyloid A4 (β-protein). It can be easily measured using an enzymatic decomposition reaction of -leucyl-valyl-tyrosine-methylcoumarylamide (SLLVT).

(Effects of the Invention) The enzyme activator of the present invention has a remarkable action of activating the ingensin enzyme. In addition, the main component, N-myristoyl amino acid, is composed of myristic acid, a naturally occurring fatty acid, and amino acids, so it is safe and has little toxicity, and by activating ingensin, it has an effect on the brain in Alzheimer's disease. It promotes the decomposition of A4 (β-protein), which is the main component of accumulated amyloid, and is useful as a medicine for the prevention and treatment of Alzheimer's disease.

(Example) Hereinafter, the present invention will be explained in more detail with reference to Examples.

[Manufacturing example]

First, a production example of N-myristoyl amino acid and its derivatives will be shown.

Production Example I L-serine 2. Og (0.02 mol) and triethylamine 2.8 yd (0.02 mol) were dissolved in 60-ml water, and myristic acid ester of 4-hydroxyphenyl dimethyl sulfomethyl sulfate (trade name, Sunceller My-DSP (Sanshin Manufactured by Kagaku Kogyo Co., Ltd.))9
．． 52 g (0.02 mol) was added and stirred at room temperature for 12 hours.

The reaction mixture was adjusted to pH 2-3 with IN-H (J) and the reaction mixture was extracted with ethyl acetate. The ethyl acetate layer was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The product was extracted with ethyl acetate-methanol (3 : lv/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 2.0 g (0.02 mol) of D-serine and 2.8 d (0.02 mol) of triethylamine were added to 60
d was dissolved in water, 9.52 g (0.02 mol) of Suncellar My-DSP (Sanshin Chemical Industry Co., Ltd.) was added, and the mixture was stirred at room temperature for 12 hours.

The reaction solution was brought to p) 12-3 with IN-HCZ and the reaction mixture was extracted with ethyl acetate. After drying the ethyl acetate layer over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure. The product was dissolved in ethyl acetate-methanol (3: lv/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 added to 60 m
1 was dissolved in water, 9.52 g (0.02 mol) of Suncellar My-DSP (Sanshin Chemical Industry Co., Ltd.) was added, and the mixture was stirred at room temperature for 12 hours.

The reaction mixture was brought to IN-H (J with p) 12-3 and the reaction mixture was extracted with ethyl acetate. (The ethyl acetate layer is diluted with anhydrous sulfuric acid)
After drying with IJum, 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-di-alanine.

Production Example 4 2.66 g (0.01 mol) of 4-hydroxyphenyldimethylsulfomethyl sulfate (trade name, Suncellar DSP (manufactured by Sanshin Kagaku Kogyo Co., Ltd.)) was heated and dissolved in 40-acetonitrile, and 2.28 g of myristic acid was dissolved. (
0.01 mol) was added thereto, and the mixture was stirred at 0°C. Add 2.06 g (0.01 g) of dicyclohexylcarbodiimide to this solution.
mol) was gradually added and incubated at 0°C for 2 hours, then at room temperature for 2 hours.
Stir for hours. The reaction solution was filtered to remove dicyclohexylurea, and the filtrate was distilled off under reduced pressure to obtain an oil.

0.75 g (0.01 mol) of glycine in 10-10% of water
and triethylamine 1.4 yd (0.01 mo
1) was added and while stirring, an acetonitrile solution of the oil was added and stirred at room temperature for 10 hours. After the reaction, the reaction solution was concentrated under reduced pressure, adjusted to pH 2-3 by adding 6N-HCj, 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 dissolved in ethyl acetate-methanol (3: lv/v), concentrated under reduced pressure, and the crystallized product was washed with ethyl acetate and hexane to give N-myristoyl-glycine 1
．． 1g was obtained.

Production example 5 Suncellar DSP (Sanshin Chemical Industry Co., Ltd.) 2.6
6 g (0.01 mol) was heated and dissolved in 40-acetonitrile, and 2.28 g (0.01 mol) of myristic acid was dissolved under nitrogen stream.
, 01 mol) and stirred at 0°C. To this solution was added 2.06 g of dicyclohexylcarbodiimide (0.01 mo
1) was gradually added, and the mixture was stirred at 0°C for 2 hours and then at room temperature for 2 hours. The reaction solution was filtered to remove by-produced dicyclohexylurea, and the filtrate was distilled off under reduced pressure to obtain an oily substance. water 1
〇- L-serine 1. While Og (0.01 mol) and 1.4 wrl (0.01 mol) of triethylamine were added and stirred, an acetonitrile solution of the oil was added and stirred at room temperature for 10 hours. After the reaction, the reaction solution was concentrated under reduced pressure, adjusted to pH 2-3 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 dissolved in ethyl acetate-methanol (3: lv/v), adjusted to pH 1l with 10% sodium hydroxide solution, and after confirming the formation of a precipitate, condensed under reduced pressure to obtain the crystallized product. was washed with ethyl acetate and hexane to obtain 1.9 g of N-myristoyl-serine sodium salt.

[Formulation example]

The following formulations were prepared using N-myristoyl amino acid and its derivatives obtained in each production example as active ingredients.

Formulation example 1 (tablet) One tablet (150cm) contains the following ingredients.

Active ingredient (manufacturing example 1) 50 ■Milk tJ!
' 48 ■Starch
50 ■Polyvinylpyrrolidone
1.5 ■ Magnesium stearate
0.5 ■ Total 150 Akebono Preparation Example 2 (Capsule) One gelatin capsule contains the following ingredients (150 ■).

Active ingredient (manufacturing example 2) 50 ■Lactose
59.5 ■ Starch
40 ■Light silicic anhydride
0.5 ■ Total 150 ■ Formulation Example 3 (Granules) 1 g of granules contains the following ingredients.

Active ingredient (Manufacturing Example 3) 200 ■ Lactose 450 ■ Starch 300 ■ Hydroxypropylcellulose 50 ■ Total 1000 mg [Enzyme Activity Measurement] Enzyme activity of kidney sinus was measured as follows. Ingensin, 50mM Tris-HCl buffer, 0.1m
M succinyl-leucyl-leucyl-valyltyrosine-
Add a predetermined amount of each enzyme activator to methylcoumarylamide (SLLVT), incubate at 37°C for 30 minutes, and then stop the reaction by adding 10% sodium dodecyl sulfate. Aminomethylcoumarin was measured using a fluorescence absorbance meter. (Excitation: 380 nm, Fluorescence: 460 nm). The results are shown in FIGS. 1 and 2. In the figure, ○ indicates an enzyme activator containing N-myristoyl-d-serine, . and the mouth represents a control containing myristic acid in place of these compounds.

〔result〕

As shown in Figure 1, N-myristoyl-L serine (○
), the enzyme activity of ingensin was activated from 5.5 to 128.0 (23 times) with an added amount of 0.25 w/d. On the other hand, the same amount of myristic acid (b) activated the enzyme activity only five times.

For N-myristoyl-D-serine (・), 0.75■
/-, the enzyme activity was activated 21 times.

In addition, as shown in Figure 2, N-myristoyl-thi-alanine (△) reduced the enzyme activity by 2 at an addition amount of 0.1■/i.
Activated 6 times, N-myristoylroalanine (mu)
In this case, it was activated 32 times at 0.5■/+d. This proved that N-myristoyl amino acid activates ingensin more than the previously known myristic acid.

[Brief explanation of drawings]

FIG. 1 is a graph showing the relationship between the amount of N-myristoylserine added and enzyme activity. FIG. 2 is a graph showing the relationship between the amount of N-myristoylalanine added and enzyme activity. In the figure, ○ is N-myristoyl-d-serine, . is N-myristoyl-D-serine, and △ is N-myristoyl-d-serine.
Alanine and Mu indicate enzyme activators containing N-myristoylroalanine, respectively, and Mou indicates a control containing myristic acid instead of these compounds.

Claims (1)

[Claims] (1) Ingensin enzyme activator containing N-myristoyl amino acid and its derivatives as an active ingredient represented by the general formula ▲ Numerical formula, chemical formula, table, etc. ▼ (In the formula, A represents an amino acid residue).