JPH07238035A - Agent for decomposing beta-amyloid protein - Google Patents

Abstract

PURPOSE:To provide a betaA-decomposing agent useful for treating and preventing diseases caused by the deposition of a beta-amyloid protein (betaA), such as Alzheimer's disease. CONSTITUTION:A beta-amyloid protein-decomposing agent contains an insulin- decomposing enzyme as an active ingredient.

Description

Detailed Description of the Invention

[0001]

The present invention relates to βA useful for treating and preventing diseases caused by deposition of β-amyloid protein (hereinafter referred to as βA) such as Alzheimer's disease.
Regarding decomposing agents.

[0002]

2. Description of the Related Art βA is considered to be a causative substance that causes symptoms such as dementia by depositing on nerve cells or blood vessels in the brain of patients with certain neurological diseases such as Alzheimer's disease. βA
Is derived from the transmembrane precursor protein (βAPP) 39-
It consists of 43 amino acids and is said to occur during the normal metabolic process of βAPP in the cytoplasm. The accumulation of βA in the brain of Alzheimer's disease patients is thought to be due to a defect in the βA degradation system. Therefore, substances involved in βA degradation are considered to be useful for treating and preventing diseases caused by βA deposition such as Alzheimer's disease. Conventionally, a method for removing βA or a method for preventing βA accumulation has not been known.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a βA-degrading agent useful for treating and preventing diseases caused by βA deposition such as Alzheimer's disease.

[0004]

The present invention relates to a βA degrading agent containing an insulin degrading enzyme as an active ingredient. Insulin-degrading enzyme is, for example, International Enzyme Commission Classification No. EC3.4.2
It is an enzyme represented by 2.11, rat liver, human red blood cells,
Purify from human placenta, or use the corresponding cDNA [Sc
ience, 242 , 1415 (1988)] in a microorganism, a plant cell or an animal cell and then isolated and purified.

Human erythrocyte insulin degrading enzyme is Scienc
e, 242 , 1415 (1988), human placental insulin-degrading enzyme, Biochem. Biophys. Res. Comm., 18
It can be obtained by the method described in 1, 1398 (1991).
The rat liver insulin-degrading enzyme can be obtained by the method described in Test Example 1 described later. Also, cd
As a method for expressing NA, for example, human insulin-degrading enzyme cDNA [Science, 242 , 141] in which a nucleotide sequence encoding several histidine residues was previously ligated to the terminal
5 (1988)] to silkworm nuclear polyhedrosis virus (BmNPV).
The vector is ligated to the vector pBK283 having the promoter of the polyhedrin gene, and the silkworm cells (BmN4) are co-infected with BmNPV DNA. A recombinant virus that has undergone homologous recombination is cloned from infected cells, and this is infected with Bombyx mori larvae to produce human insulin-degrading enzyme. This is subjected to zinc affinity column chromatography to purify human insulin degrading enzyme.

Next, the pharmacological action of insulin-degrading enzyme will be described with reference to test examples. Test Example 1 βA Degradation Action Rat cytoplasmic fraction was subjected to 4-step chromatography [diethylaminoethyl-Toyopearl (registered trademark, Tosoh Corporation), hydroxylapatite, pentyl agarose, TSK G3000SW high performance liquid chromatography (Tosoh Corporation)]. Thus, the insulin-degrading enzyme was purified. 0.2 μg of this purified enzyme and βA
_1-40 (Bachem California) 5 μg
Or βA _1-28 (corresponding to residues _{1-28 of} βA _1-40 , B
Achem California) 11 μg to 5
0 mM Tris-HCl buffer (pH 7.0) 70 μ
Incubated in 1 at 37 ° C. Samples were taken over time, and a 0.06% trifluoroacetic acid aqueous solution was added to the sample.
The reaction was stopped by adding 7 ml. The reaction solution was subjected to RP-318 reverse phase column chromatography (Bio Rad; particle size 5 μm, 250 × 4.6 mm; 0-80% acetonitrile-0.06% trifluoroacetic acid aqueous solution linear concentration gradient;
The flow rate was 0.5 ml / min), and the absorbance of the eluate at 220 nm was measured. The results are shown in FIG.

[0007]

FIG. 1 shows that insulin-degrading enzyme decomposed βA into small fragments almost completely.

Test Example 2 Insulin-degrading enzyme¹²⁵I-
βA crosslinking test Tissue homogenate was centrifuged (100,000 xg, 6
0 min) rat brain or rat liver cells
50 μg of mass fraction and¹²⁵I-βA_1-28[ΒA _1-28To
Na¹²⁵Chloramine using I (Amersham)
T method [Biochem.J.,164, 607 (1977)]
And purified by reverse phase high performance liquid chromatography]
2 mM 1,10-phenanthroline and 5 mM
150 mM Natri chloride in the presence of tolylenediaminetetraacetic acid
In 20 mM phosphate buffer (pH 7.4) containing um (total
50 μl) and incubated at 24 ° C. 10 minutes later,
Cross-linking agent disuccinimidyl suberate (1 mM / 25%
Add 3 μl of methyl sulfoxide) and continue for another 15 minutes.
React at temperature and add to this 5% sodium dodecyl sulfate
(SDS) and 25% 2-mercaptoethanol
The reaction was stopped by adding 14 μl of the buffer solution. 100
After heating at ℃ for 3 minutes, the product was treated with SDS-polyacrylic acid.
Dogel gel electrophoresis, dried, imaging plate
Exposed to BAS2000 (Fuji Film) and analyzed
It was The results are shown in FIG.

[0009]

FIG. 2 clearly shows that ¹²⁵ I-βA _1-28
Specifically bound to a protein having a molecular weight of 110,000 (lane 1, brain; lane 5, liver). further,
The cross-linking reaction with a protein having a molecular weight of 110,000 was inhibited by the addition of an excessive amount of unlabeled βA _1-28 (lane
2), and it was also inhibited by addition of unlabeled βA _1-40 (lane 3) or insulin (lane 4).

The above facts indicate that among the intracellular proteolytic enzymes, the one having βA degrading activity is only the insulin degrading enzyme. The insulin-degrading enzyme can be used as it is or in various pharmaceutical forms. The pharmaceutical composition of the present invention can be produced by uniformly mixing, as an active ingredient, an effective amount of insulin-degrading enzyme with a pharmaceutically acceptable carrier. These pharmaceutical compositions are preferably in unit dose form suitable for administration by injection.

The injection can be prepared by using a carrier made of distilled water, salt solution, glucose solution or a mixture of salt water and glucose solution. At this time, according to a conventional method, it is prepared as a solution, suspension or dispersion using an appropriate solubilizing agent or suspending agent. Alternatively, the solution can be freeze-dried to prepare a freeze-drying agent. The conditions for freeze-drying are not particularly limited, but usually freeze at −50 ° C. or lower for 1 to 5 hours, shelf temperature −20 to 0 ° C., vacuum degree 50 to 1
Dry at 50 mTorr for 24-48 hours, then shelf temperature 1
16 ~ 2 at 0 ~ 30 ℃, vacuum degree 50 ~ 100mTorr
Dry for 4 hours to obtain a lyophilized product.

The βA-degrading agent of the present invention may contain various conventional pharmaceutical carriers, excipients, diluents, stabilizers or anti-adsorption agents. Insulin-degrading enzyme can be administered as an injection in the above-mentioned pharmaceutical form, and its effective dose and the number of administrations vary depending on the administration form, age, weight and symptoms of the patient, but usually 1.5 per adult.
μg to 15 mg, preferably 15 μg to 5 mg is administered 1 to 7 times per week. As the administration method, intravenous injection,
Subcutaneous injection, intramuscular injection, intracerebral administration and the like are used.

Examples will be shown below.

[0014]

【Example】

Example 1 Injection The injection having the following composition is prepared by a conventional method. Insulin-degrading enzyme purified from human erythrocytes [Science, 242 ,
1415 (1988)] 0.125 mg / ml in distilled water for injection 80
It is dissolved in ml, 5.0 g of D-mannitol is added thereto, and the volume is adjusted to 100 ml with distilled water for injection. The resulting solution is aseptically filtered using a 0.22 μm disposable membrane filter and then placed in a glass vial.
Aseptically fill each ml to obtain an injection (containing 0.2 mg of active ingredient per vial).

Prescription human erythrocyte insulin degrading enzyme 0.2 mg D-mannitol 100 mg Distilled water for injection Appropriate amount 2.0 ml

Example 2 Injection Preparation An injection preparation having the following composition is prepared by a conventional method. Insulin-degrading enzyme purified from human placenta [Biochem. Biophy
s. Res. Comm., 181, 1398 (1991)] 0.125 mg / m
1 is dissolved in 80 ml of distilled water for injection, 5.0 g of D-mannitol is added thereto, and the volume is 100 m with distilled water for injection.
Adjust to l. The obtained solution is subjected to aseptic filtration using a disposable membrane filter of 0.22 μm,
Aseptically fill 2 ml each into glass vials to obtain an injection (containing 0.2 mg of active ingredient per vial).

Formulation Human placental insulin degrading enzyme 0.2 mg D-mannitol 100 mg Distilled water for injection Appropriate amount 2.0 ml

Example 3 Injectable Agent An injectable agent having the following composition is prepared by a conventional method. Insulin-degrading enzyme purified from rat liver (see Test Example 1)
0.125 mg / ml is dissolved in 80 ml of distilled water for injection, 5.0 g of D-mannitol is added thereto, and the volume is adjusted to 100 ml with distilled water for injection. The resulting solution was subjected to aseptic filtration using a disposable membrane filter of 0.22 μm, and then aseptically filled in glass vials in an amount of 2 ml at a time, and an injectable solution (active ingredient per vial of 0.
Containing 2 mg).

Formulation Rat liver insulin degrading enzyme 0.2 mg D-mannitol 100 mg Distilled water for injection Appropriate amount 2.0 ml

Example 4 Injectable Solution An injectable solution having the following composition is prepared by a conventional method. Insulin-degrading enzyme purified from rat liver (see Test Example 1)
0.625 mg / ml is dissolved in 80 ml of distilled water for injection, 5.0 g of D-mannitol is added thereto, and the volume is adjusted to 100 ml with distilled water for injection. The obtained solution was subjected to aseptic filtration using a disposable membrane filter of 0.22 μm, and aseptically filled into glass vials in an amount of 2 ml each, and an injection (active ingredient 1.
Containing 0 mg).

Formulation Rat liver insulin degrading enzyme 1.0 mg D-mannitol 100 mg Distilled water for injection Appropriate amount 2.0 ml

[0022]

INDUSTRIAL APPLICABILITY The present invention provides a βA-degrading agent useful for treating and preventing diseases caused by βA deposition such as Alzheimer's disease.

[Brief description of drawings]

FIG. 1 is a high-performance liquid chromatogram showing the time course of βA decomposition by an insulin-degrading enzyme.

FIG. 2 is a diagram in which the results of an insulin-degrading enzyme- ¹²⁵ I-βA crosslinking test are analyzed by electrophoresis.

[Explanation of symbols] a: Results when using βA _1-40 b: Results when using βA _1-28 1: Results when using rat brain cytoplasmic fraction 2: Rat brain cytoplasmic fraction Use and excess unlabeled βA
Result when _1-28 was added 3: Result when rat brain cytoplasmic fraction was used and result when unlabeled βA _1-40 was added 4: Result when rat brain cytoplasmic fraction was used and insulin was added 5: Results when using rat liver cytoplasmic fraction

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Claims (1)

[Claims] 1. β containing insulin-degrading enzyme as an active ingredient
Amyloid proteolytic agent.