JPH08193026A - Amyloid beta-protein coagulation/deposition inhibitor - Google Patents

Abstract

PURPOSE: To obtain an amyloid β-protein coagulation and/or deposition inhibitor useful for preventing and treating Alzheimer's disease, containing, as active ingredient, a hydroquinone or quinone compound. CONSTITUTION: This inhibitor contains, as active ingredient, 1-70 (pref. 5-50)wt.% of a hydroquinone or quinone compound of formula I, II or III. Using an appropriate vehicle etc., this inhibitor can be manufactured into pharmaceutical preparations in the form of an oral agent such as capsules, tablets, granules, powder, suspension, liquid or syrup, an injection, suppository, or agent for external use. The hydroquinone or quinone compound has activity to inhibit amyloid β-protein coagulation and/or deposition and also has action of suppressing the neurocytotoxicity due to amyloid β-protein.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an inhibitor of amyloid β protein aggregation and / or deposition. For more information,
The present invention relates to an aggregation and / or deposition inhibitor of amyloid β protein containing hydroquinones or quinones as an active ingredient.

[0002]

2. Description of the Related Art Amyloid β protein is a major component that forms an amyloid core in senile plaques, which is one of the characteristic pathological changes observed in the brain of Alzheimer's disease patients.
A peptide consisting of 43 amino acids, which is a transmembrane amyloid precursor protein APP (Amyloid Protein Precursor)
Abbreviation) (for example, MORI, H.
Et al (1992) THE JOURNAL OF BIOLOGICAL CHEMISTR
Y 267: 17082-17086, LANSBURY, P.
T., Jr. (1992) BIOCHEMISTRY Volume 31 6865
Page 6870, SISODIA, SS et al. (1990) SCIENCE
Vol. 248, pp. 492-495, MULLAN, M. et al. (1993)
Year) TRENDS IN NEUROSCIENCE, Vol. 16, pp. 398-403 etc.). From experiments using chemically synthesized amyloid β protein and the like, such amyloid β protein has a strong self-aggregating property (eg, JARRETT, JT et al. (1993) CELL No. 73).
Volume 1055-1058, BURDICK, D. et al. (1992)
Year) THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 267, pages 546-554, FRASER, PE et al. (1992) BIOC
HEMISTRY, Vol. 31, p. 10716-10723, etc.), and it has been reported that aggregated amyloid β protein may directly show cytotoxicity to nerve cells and enhances susceptibility to cytotoxicity caused by excitatory amino acids. (For example, PIKE, CJ et al. (1993) THE JOUR
NAL OF NEUROSCIENCE Vol. 13, pp. 1676-1687, M
ATTSON, MP et al. (1993) TRENDS IN NEUROSCIENC
E Vol. 16, pp. 409-414 etc.). In particular,
The partial peptide β25-35 of the amyloid β protein (amino acid sequence GSNKGAIIGLM) exhibits neurotoxicity, and one of the action points of β25-35 is the mitochondrial electron transfer system of neurons, and the MTT (3-
(4,5-dimethylthiazol-2-yl) -2,5-diph
phenyltetrazolium bromide) The ability to know the intensity of cytotoxicity by measuring its reducing activity.
Have already been reported (Yankner et al., Science, 250,
279-282, 1990; Kaneko et al., Neurochemistry, 32,
148-149, 1993). Furthermore, amyloid β
It is known that senile plaque formation due to protein deposition in the brain is a pathological change that appears earlier than neurofibrillary tangles, which is another characteristic pathological change in the brain of Alzheimer's disease patients ( For example, SEJKOE, DJ (1991)
NEURON Vol. 6, pp. 487-498, RUMBLE, B. et al. (198
9th) THE NEW ENGLAND JOURNAL OF MEDICENE No. 320
Pp. 1446-1452). That is, in Alzheimer's disease, the mechanism of onset that amyloid β protein is aggregated / deposited in brain tissues to form senile plaques, resulting in neuronal cell death and dementia is predominant.

Therefore, a drug which inhibits aggregation and / or deposition of amyloid β protein is expected to be useful as a therapeutic drug and / or a preventive drug for Alzheimer-type dementia. There are reports of rifamycins as drugs having such inhibitory activity (eg, TOMIYAM, T. et al. (1994) BIOCHEMICAL AND BIOPHYSICAL RESEARCH
(See COMMUNICATIONS Vol. 204, pages 76-83, etc.)
However, it has not been reported that hydroquinone, quinone and the like have an activity of inhibiting aggregation and / or deposition of amyloid β protein.

[0004]

DISCLOSURE OF THE INVENTION The present invention relates to amyloid β
It is intended to provide a drug that inhibits protein aggregation and / or deposition. Further, the present invention relates to amyloid β
It is an object of the present invention to provide a therapeutic or prophylactic agent for Alzheimer's disease based on the action of inhibiting protein aggregation and / or deposition.

[0005]

Means for Solving the Problems As a result of diligent studies on the possibility of a drug that inhibits aggregation and / or deposition of amyloid β protein, the present inventors have found that hydroquinones or quinones of the present invention are amyloid β protein. The present inventors have found that they have an activity of inhibiting aggregation and / or deposition, and further have an action of suppressing neuronal toxicity caused by amyloid β protein, and arrived at the present invention.

[0006]

That is, the present invention has the following formula [I], [II] or [III]

[0007]

Embedded image

An amyloid β protein aggregation and / or deposition inhibitor containing a hydroquinone or a quinone represented by the formula (1) and an active ingredient represented by the above formulas [I], [II] or [III]. It is a therapeutic or prophylactic agent for Alzheimer-type dementia, which contains hydroquinones or quinones and is based on the amyloid β protein aggregation and / or deposition inhibitory action.

The hydroquinones and quinones relating to the present invention are all known compounds, and for example, hydroquinone, P-benzoquinone, 1,4-dihydroxynaphthalene and the like are available as reagents.

The aggregation and / or deposition inhibitor of amyloid β protein of the present invention is preferably used as a aggregation and / or deposition inhibitor of amyloid β protein of the present invention by blending a pharmaceutically acceptable carrier. Can be used. Examples of the pharmaceutically acceptable carrier in this case include the same as the below-mentioned excipients. In this case, the compounding amount of the compound of the present invention and the carrier depends on the dose of the active ingredient as described below, but is not particularly limited and may be selected in a wide range. Usually, the hydroquinone or quinone of the present invention is the whole composition. 1 to 70% by weight, preferably 5 to 5
0% by weight. The obtained composition is further prepared by a known method using suitable excipients and the like, oral capsules such as soft capsules, hard capsules, tablets, granules, powders, suspensions, solutions, syrups, and injections. It is provided as an agent, a suppository or an external preparation. Such excipients include vegetable oils (eg corn oil, cottonseed oil, coconut oil, almond oil, peanut oil,
Olive oil), oily esters such as medium-chain fatty acid glyceride oil, mineral oil, glycerin esters such as tricaprylin and triacetin, alcohols such as ethanol, physiological saline, propylene glycol, polyethylene glycol, petrolatum, animal fats and oils, cellulose derivatives (Crystalline cellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), polyvinylpyrrolidone, dextrin, lactose, mannitol, sorbitol, starch and the like.

The hydroquinones or quinones of the present invention have an activity of inhibiting aggregation and / or deposition of amyloid β protein, and are therapeutic agents for Alzheimer's dementia and / or
It is also useful as a preventive drug.

Various test methods and measuring methods used in the present invention are as follows.

(1) Test for amyloid β protein aggregation inhibitory activity
Test method It was carried out by a method using Thioflavin T.

The dye called thioflavin T (ThT) is
A new fluorescence (4) that was not shown in the free state was bound to the β-sheet structure of aggregated proteins such as amyloid β protein.
It is reported to emit light of 82 nm) (Harry LeVi
ne III. 1993, ProteinScience 2, 404-41
0). The intensity of fluorescence is proportional to the degree of aggregation of bound proteins. The amyloid β protein aggregation inhibitory activity of a drug can be examined by measuring the degree of aggregation of the drug-containing amyloid β protein by measuring the fluorescence intensity of ThT binding thereto.

Specifically, amyloid β containing a drug
10 μl is taken from the protein sample solution, and this is added to 1 ml of a ThT solution dissolved in 50 mM sodium phosphate buffer (pH 6.0) at a concentration of 3 μM and stirred. After stirring, the spectrofluorometer (JAS
CO), excitation wavelength 450 nm, fluorescence wavelength 48
The fluorescence of the solution is measured at 2 nm. If the increase in fluorescence is suppressed for a certain period of time as compared with a solution containing only amyloid β protein that does not contain a drug, the drug is determined to have amyloid β protein aggregation inhibitory activity.

(2) Amyloid β using PC12 cells
Method for assaying protein neurocytotoxicity (a) Preparation of cells PC12 cells (RCB0009, Lot6) distributed from the cell bank of RIKEN were used. This cell is a clonal cell derived from rat adrenal medulla, and differentiates into a sympathetic ganglion cell-like by culturing in the presence of NGF (Gree
ne and Tischler, Proc. Natl. Acad. Sci. USA, 73:
2424 to 2428, 1976), it is widely used as a nerve cell model system.

RPMI containing 10% horse serum (Gibco) and 5% quasi-fetal bovine serum (Mitsubishi Kasei)
Using 1640 (manufactured by Gibco) as a medium,
Performed in the presence of 5% CO ₂ . 5,000 cells / well
96-well culture plate (Sumitomo Bakelite, SUMI
LON CELL LIGHT C-1), the next day, D-glucose-free, 50 ng / ml / NGF
Replace with medium containing (from Sigma, rat submandibular gland)
After 4 days, the medium is exchanged and the culture is continued. After 7 days, the culture is used for the following measurements.

(B) Measurement of MTT reducing activity of PC12 cells by amyloid β1-40 peptide The above-mentioned (a) PC12 cells on the 7th day of culture were exchanged with a medium (1).
After 80 μl / well, phosphate buffered saline (PB)
2) Amyloid β1-40 peptide solution dissolved in S)
Add 0 μl / well. 40 hours later, 25 μl / wel of 5 mg / ml PBS solution of MTT (manufactured by Sigma) was used.
l of each is added and the culture is continued at 37 ° C. in the presence of 5% CO ₂ . After 4 hours, the medium was sucked up, and 0.04N HCl /
100 μl / well of isopropanol was added to dissolve the formazan, and colorimetric determination (57) was performed using a microplate reader (Molecular Devices, Vmax).
0 to 650 nm), and changes in MTT reducing ability of PC12 cells are measured.

[0019]

EXAMPLES The present invention will be described in detail below with reference to examples and examples. In addition, unless otherwise specified,% indications in Examples and the like indicate weight / volume%.

Reference Example 1 Synthesis of Amyloid β1-40 Peptide In the following examples, the amyloid β protein used to examine the inhibitory activity against amyloid β protein aggregation was:
Amyloid β1-40 peptide was synthesized.

The peptide was synthesized using Fmoc-amino acid as a raw material by a solid phase method using a peptide synthesizer (manufactured by Applied Biosystems). After completion of the synthesis, the peptide was cleaved from the resin and C ₁₈
This was purified by reverse phase high performance liquid chromatography (manufactured by Waters) using a column. It was confirmed by a peptide sequencer (manufactured by Applied Biosystems) that the obtained amyloid β1-40 peptide had a target amino acid sequence. The amyloid β1-40 peptide thus obtained was freeze-dried and used in the following examples.

[Example 1] β1-4 obtained in Reference Example 1
The 0 peptide was dissolved twice in deionized water to obtain a 40 μM peptide solution. An equivalent amount of 2 x PBS (Phosphate
Buffered Saline) solution was added to make a 20 μM peptide solution, and 100 μl of each was dispensed into an Eppendorf tube. Next, hydroquinone, P-benzoquinone and 1,4-dihydroxynaphthalene were added at 10 mM and 1 mM.
Alternatively, it was dissolved in DMSO to have a concentration of 0.1 nM. 1 μl of each of these compound solutions was added to the above peptite solution (3 tubes / group) and stirred (final concentrations of these compounds will be 100 μM, 10 μM or 1 μM). As a control, 1 μl of DMSO alone was added to the above peptide solution. These were incubated at 37 ° C., and after one week, β
Aggregation of 1-40 peptides was measured. As a result, as shown in FIG. 1, hydroquinone, P-benzoquinone and 1,4-dihydroxynaphthalene were β1-
The aggregation of 40 peptides was suppressed.

[Embodiment 2] In the above Embodiment 1, 37
Neuronal toxicity was measured according to the above-mentioned test method (2) for each sample incubated at 1 ° C for 1 week. As a result, as shown in FIG. 2, hydroquinone, P-benzoquinone and 1,4-dihydroxynaphthalene were compared to compound 1 for 20 μM of amyloid β1-40 peptide.
When incubated at 00 μM (final concentrations of β1-40 peptide and compound are 2 μM and 10 μM, respectively)
Furthermore, the toxicity of amyloid β1-40 peptite was completely suppressed.

[Brief description of drawings]

1 shows the inhibitory effect on aggregation of β1-40 peptide by the inhibitor of amyloid β protein aggregation and deposition of the present invention in Example 1. FIG. In the bar graphs in the figure, for the control of the compound (-) of the present invention, hydroquinone, p-benzoquinone and 1,4-dihydroxynaphthalene, the final concentration was 100 μM (black bar graph), 10 respectively.
μM (white bar graph) and 1 μM (dot bar graph) are shown.

[Fig. 2] Fig. 2 shows suppression of neurotoxicity (MT by the amyloid β protein aggregation / deposition inhibitor of the present invention in Example 2).
T reducing ability lowering action) effect. In the bar graphs in the figure, for the control compound (-), hydroquinone, p-benzoquinone and 1,4-dihydroxynaphthalene, the final concentrations were 10 μM (black bar graph), 1 μM (white bar graph) and 0. 1 μM (dotted bar graph) is shown.

Claims (2)

[Claims] 1. A compound represented by the following formula [I], [II] or [III]: An amyloid β protein aggregation and / or deposition inhibitor containing a hydroquinone or a quinone represented by the following as an active ingredient. 2. The above formulas [I] and [II] as the active ingredient.
Alternatively, a therapeutic or prophylactic agent for Alzheimer's dementia based on the amyloid β protein aggregation and / or deposition inhibitory effect, which comprises a hydroquinone or quinone represented by [III].