JPH0253735A - Agent for alleviating dementia - Google Patents

Abstract

NEW MATERIAL:A peptide shown by the formula (Asn is asparagine; Gys is cysteine; Pro is proline; Arg is arginine), a derivative or a salt thereof. USE:An agent for alleviating dementia having excellently improving action on dementia and low toxicity useful for preventing and treating presbyophrenia, cerebrovascular dementia, Alzheimer's disease, Pick's disease, etc. PREPARATION:A peptide shown by the formula is obtained by an ordinary peptide synthesis by either of liquid phase method or solid phase method. Azide method, acid chloride method or acid anhydride method is used as a condensation method to form azide bond. Before the condensation reaction, carboxyl, amino, etc., not participating in the reaction may be protected and carboxyl, amino, etc., taking part in the reaction may be activated.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to an anti-dementia agent containing an effective amount of a peptide having nootropic activity.

[Prior Art] It has been known for a long time that Pap pressin has a nootropic effect, but recently peptides that can be considered as fragments of Pap pressin, such as H-Asn-Cys-Pro-Arg-OHH-Cys, have recently been investigated.
It has been reported that -OH etc. also have nootropic effects similar to pap pressin [Plain Research (Braine-Re5
371.17 (I986)].

[Problems to be Solved by the Invention] The present invention aims to provide an anti-dementia agent containing an effective amount of a novel peptide that has an even better nootropic effect than the Pap pressin and Pap pressin fragment peptides. This is the purpose.

[Means for Solving the Problems] The present invention provides an effective amount of a peptide represented by formula (I):
and an anti-dementia agent containing a pharmacologically acceptable carrier or diluent.

The functional group derivatives of the peptide of formula (I) above mean the following.

a) N-acyl derivatives derived from aliphatic carboxylic acids with 1 to 6 carbon atoms, preferably acetic acid; b) amides or 1,000- or di-alkyl derivatives with alkyl groups of 1 to 6 carbon atoms; Esters derived from alkyl-substituted amides and c) alcohols having 1 to 18 carbon atoms, preferably aliphatic alcohols having 1 to 6 carbon atoms.

The pharmacologically acceptable salts of the peptide group or its functional group derivatives include acid addition salts and basic salts. Such acid addition salts include inorganic acids (e.g., hydrochloric acid, sulfuric acid, phosphoric acid) or organic acids (e.g., acetic acid,
Examples include salts of propionic acid, citric acid, tartaric acid, malic acid, oxalic acid, methanesulfonic acid, and the like. Further, examples of the basic salt include sodium salt, potassium salt, triethylamine salt, and the like.

In this specification, amino acids, peptides, protecting groups, solvents, etc. are referred to by abbreviations commonly used in the art, or IU
Abbreviations adopted by the PAC-IUB nomenclature committee are used. For example, the following abbreviations are used: In addition, amino acids shall mean L-type amino acids.

^Sn: to asparagine rg: arginine Cys Nijistin Pro Nibroline Boc: t-butoxycarbonyl Z: penzyloxycarbonyl Mbs: P-methoxybenzenesulfonyl MBzl
: p-methoxybenzyl 0Bzl : benzyl ester O5u : N-hydroxysuccinimide ester DCC
: N,N'-dicyclohexylcarbodiimide DCU
rea: N,N'-dicyclohexylurea 110
Bt: 1-hydroxybenzotriazole NMM:
N-Methylmorpholine TF Nitrifluoroacetic acid MS 8 hemethanesulfonic acid Ac0EL: Ethyl acetate DMF: N,N-dimethylformamide Me011: Methanol THF: Tetrahydrofuran The peptide that is the active ingredient of the anti-dementia agent of the present invention can be used in peptide chemistry. Commonly used methods, e.g. 5chr
``The Peptide (T
he Peptides)” Volume 1, Acad
emic Press, New York, U.S.
, A, (1965), "Basics and Experiments of Peptide Synthesis" by Nobuo Izumiya et al. can also be manufactured.

Condensation methods for forming peptide bonds include azide method, acid chloride method, acid anhydride method, mixed acid anhydride method, N
, N'-dicyclohexylcarbodiimide method, N,N''
-dicyclohexylcarbodiimide-additive method,
Examples include an active ester method, a carbonyldiimidazole method, a redox method, and a method using a Woodward reagent.

Before carrying out the condensation reaction, carboxyl groups, amino groups, etc. that do not participate in the reaction may be protected, or carboxyl groups, amino groups that participate in the reaction may be activated by means known per se.

Examples of the carboxyl protecting group include esters such as methyl, ethyl, benzyl, p-nitrobenzyl, t-butyl, and cyclohexyl.

Examples of protecting groups for amino groups include benzyloxycarbonyl group, t-butoxycarbonyl group, isobornyloxycarbonyl group, and 9-fluorenylmethyloxycarbonyl group.

Examples of the protecting group for the guanidino group include a nitro group, a benzyloxycarbonyl group, a tosyl group, a p-methoxybenzenesulfonyl group, and a mesitylenesulfonyl group.

Examples of protecting groups for mercapto groups include trityl group,
Examples include an acetamidomethyl group, a benzyl group, a P-methoxybenzyl group, and a 3-nitro-2-pyridinesulfenyl group.

Examples of activated carboxyl groups include:
Corresponding acid anhydrides, azides, active esters [alcohols (e.g., pentachlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, N-hydroxy-5-norbornene-2,3-dicarboximide) , N-hydroxysuccinimide, N-
esters with hydroxyphthalimide, 1-hydroxybenzotriazole), and the like. Examples of activated amino groups include the corresponding phosphoric acid amides.

The reaction is usually carried out in a solvent such as chloroform, dichloromethane, ethyl acetate, N.

The reaction can be carried out in a solvent such as N-dimethylformamide, dimethylsulfoxide, pyridine, dioxane, tetrahydrofuran, water, methanol, or a mixture thereof.

The reaction temperature can be within the commonly used range of about -30°C to about 50°C.

The protecting group removal reaction for the peptide of the present invention varies depending on the type of protecting group used, but it is necessary that the protecting group be removed without affecting the peptide bond.

Examples of the method for removing the protecting group include acid treatment with hydrogen chloride, hydrogen bromide, anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid, or a mixture thereof. Other examples include reduction with sodium in liquid ammonia and palladium on carbon. In the deprotection reaction by acid treatment, it is effective to add a cation scavenger such as anisole, phenol, and thioanisole.

After the reaction, the peptide in the anti-dementia agent of the present invention produced in this way can be obtained by a known peptide separation method such as extraction, distribution, reprecipitation, recrystallization, column chromatography, etc. Can be done.

Furthermore, the peptide in the anti-dementia agent of the present invention can be made into a derivative in its functional group or a pharmacologically acceptable salt thereof, as described above, by a method known per se.

The peptide in the anti-dementia agent of the present invention exhibits a strong nootropic effect in a passive avoidance test in rats.

Examples of useful target diseases for the anti-dementia agent of the present invention include senile dementia (Alzheimer's dementia), cerebrovascular dementia, Alzheimer's disease, Bick's disease, Huntington's chorea, Creutzfeldt-Jakob disease, Parkinson's disease,
Myelin degeneration of the cerebellum, dementia, etc. can be mentioned, and it can be used for the prevention or treatment of these diseases.

The toxicity of the peptide in the anti-dementia agent of the present invention is extremely low, and there have been no cases of death even at doses far exceeding the medicinally effective dose.

The peptide in the anti-dementia agent of the present invention can be administered as a free form, a derivative at its functional group, or a salt thereof. Regardless of the dosage, the amount of the free form is generally in the range of 1 ng to 1 mg/kg body weight per day. In particular, for parenteral administration and nasal administration, 10 ng to 100 μg/kg/day is preferable, and for oral administration and rectal administration, it is preferable to administer 10 to 100 times the parenteral administration. The peptide in the anti-dementia agent of the present invention is mainly administered parenterally (e.g., intravenous or subcutaneous injection, intraventricular or intrathecal administration, nasal administration, rectal administration).
However, in some cases, it may also be administered orally.

Examples of dosage forms include injections, suppositories, powders, nasal sprays,
Examples include pills and tablets. The peptide in the anti-dementia agent of the present invention can be stored as a solution in physiological saline, but it can also be prepared into a freeze-dried ampoule by adding mannitol and sorbitol, and dissolved at the time of use.

Examples of synthesis of peptides used in the anti-dementia agent of the present invention are shown below.

In the synthesis example, the developing solvent for thin layer chromatography was as follows, and Merck & Co. TLC plate silica gel 80 F 254 was used.

R,': Chloroform-methanol-acetic acid-water (80:
20:2.5:5) Lower layer R, 2: Chloroform-methanol-water (70:30:
5) Rf': n-butanol-acetic acid-water (2:1:]'
) Also, for purification by high performance liquid chromatography, column: μBondapak C, 81, 9x15c
m mobile phase: A) 0.05! tTFA, B) 7 Setonitrile.

[Synthesis Example 1] Asn-Cys-Pro-Arg-011 acetate (
I) Boc-Pro-Arg(Mbs)-0Bzlt
l-Arg(Mbs)-0Bzl hydrochloride 14.2 g in 200 mfl THF solution with NMM 3.1 m
l, add 9.4 g of Boc-Pro-O5u,
Stirred at room temperature for 18 hours.

THF was distilled off, and the residue was dissolved in AcOEt, washed with diluted hydrochloric acid, saturated sodium bicarbonate, and water, and dried over anhydrous sodium sulfate.

The solvent was distilled off to obtain the title compound as an oil.

Yield = 18g R,': 0.69 Rr": 0.86 [a] o
: 29.6″(c-0,5,DMF)(2) B
oc-Cys(MBzl)-Pro-Arg(Mbs)
-0BzlBoc-Pro-Arg(Mbs)-0Bz
l 3.7 g to 15 m of 4 N HClAc0Et
After standing for 30 minutes at room temperature in L.l., the solvent was removed. After drying the residue under reduced pressure, 50 mI of DMF was added. 1.4 ml of NMM, Boc-Cys (MBz
l)-0H2,2g, )lOBtO,95g and 0
1.3 g of CC was added. After stirring at room temperature for 18 h, DC
Urea was filtered off and DMF was distilled off.

The residue was diluted with 2-butanol-C)12c12 (5:1
v/v), washed with saturated aqueous sodium bicarbonate, dilute aqueous hydrochloric acid saturated with sodium chloride, and saturated brine, and dried over anhydrous sodium sulfate.

After evaporating the solvent, the residue was purified with a silica gel column using (:lI(:I3-Me) to obtain the title compound as an oil.

Yield: 4g Re': 0. 82 Re2: 0. 8
8 [α]. Knee 25.0° (c-0,5,DMF
)(3) Z-Asn-Cys(MBzl)-Pro
-8rg(I4bs)-0BzlBoc-Cys(MB
zl)-Pro-Arg(Mbs)-0Bzl 1.7
After standing for 30 minutes at room temperature in 5 m 11 of 4 N HCl-Ac0Et, the solvent was removed. 2- to the residue
Add butanol (:1I2C12 (5:1 v/v) and saturated sodium bicarbonate water, separate the organic layer,
After washing with saturated brine, it was dried over anhydrous sodium sulfate.

The solvent was distilled off, the residue was dissolved in 30 ml of DMF, and under ice cooling, 58 g of Z-Asn-OHO, 1 l of OBt O,3
4 g and 0.45 g of DCG were added. After stirring at room temperature for 18 hours, DCUrea was filtered off and DMF was distilled off.

The residue was dissolved in 2-butanol-(:JClz (5:1
v/v), washed with saturated aqueous sodium bicarbonate, dilute aqueous hydrochloric acid saturated with sodium chloride, and saturated brine, and dried over anhydrous sodium sulfate.

After distilling off the solvent, ether was added to crystallize and collect by filtration to obtain the title compound.

Yield: 1.8g Melting point: 98-100°C Rr': 0.70 Rr2: o, 82[α]
n knee 29.2° (cJ, 5, DMF) (4
) II-Asn-Gys-Pro-Arg-OH acetate Z-As+r-Cys (MBzl)-Pro-Ar
After stirring 1,00 mg of g(Mbs)-0Bz in 4 ml of MSA and 0.4 m of anisole in JZ at room temperature for 1.5 hours, ether was added and the supernatant was removed. The precipitate was dissolved in water, and after treatment with DowexlX2 (acetate type), water was distilled off.

The residue was dissolved in 0.05% TFA and 12 ml 1/min (
Flow rate), 0 to 10% B) 20 minutes linear gradient (mobile phase), high performance liquid chromatography loading, Do
After treatment with wexlX2 (acetate form), the title compound was obtained by lyophilization.

Yield: 47 mg Rr3 (containing 1% ethanedithiol): 0.18 [a]o
Knee 54.6° (c-0,5, water) Next, pharmacological test examples showing the effectiveness of the peptide of the present invention will be shown.

[Pharmacological test example] The effect on memory consolidation was determined using Wistar male rats by Burbach et al.
Science), 221.1310-1312 (I
This study was conducted using a trial passive avoidance experiment based on the method of [983]. The experimental setup consisted of a light room and a dark room, and the floor was made of stainless steel grids. Rats placed in a light room are allowed to freely move to a dark room, and when they enter the dark room, they receive a single electric shock. The maintenance of passive avoidance behavior in response to electric shock is determined by the time it takes for a rat placed in a light room to enter a dark room after a certain period of time (response latency).
It was determined by.

1) Examination of memory promoting effect Immediately after experiencing an electric shock (0.25 mA), the peptide or physiological saline in the anti-dementia agent of the present invention obtained in Synthesis Example 1 was subcutaneously administered, and 24 hours later, the electric shock was applied. A shock memory retention test was conducted. Control group rats that received saline alone generally exhibited response latencies after the 50 second face. The number of rats used for testing each group is 6-8. The maximum measurement time was 600 seconds.

For the peptide obtained in Synthesis Example 1, the dosage was 1 n
At g/kg, the extension of response latency over the control group was 316%.

2) Cycloheximide
) Examination of the effect of improving experimental retrograde amnesia by subcutaneously administering the peptide or physiological saline in the anti-dementia agent of the present invention, and 1 hour later, an electric shock (0.5 mA) was administered, and immediately thereafter, cycloheximide 2 was administered subcutaneously. .7~3.0
mg/kg or physiological saline was administered subcutaneously, and a memory retention test was performed 48 hours later. Rats administered only with physiological saline generally exhibited a response latency of around 300 seconds, while rats in the control group administered only with cycloheximide exhibited a response latency of around 50 seconds and developed retrograde amnesia.

The average response latency of the peptide-administered group and that of the control group in the anti-dementia drug of the present invention were compared. The number of rats used for testing each group is 6-8. Maximum measurement time is 6
00 seconds.

For the peptide obtained in Synthesis Example 1, the dosage was 1 n
g/kg, the response latency was increased by 320% compared to the control group.

From the above test results, it is clear that the peptide in the anti-dementia agent of the present invention exhibits an excellent memory promoting effect and an improving effect on retrograde amnesia.

Next, Examples of the anti-dementia agent of the present invention will be shown.

[Example 1] (Injection) 1 mg of the peptide O obtained in Synthesis Example 1 and 0.9 g of sodium chloride were contained in 100 mff1 of distilled water for injection, and the pH was adjusted to 6.0 to 8 with sodium hydroxide. An aqueous solution adjusted to .0 was prepared. After bacterial filtration, 1 ml of this
The mixture was filled into ampoules, sealed and heat sterilized to produce an injection.

[Example 2] (Lyophilized preparation) 5 mg of the peptide obtained in Synthesis Example 1 and 5 g of D-mannitol were added to 100 ml of distilled water for injection, and p
An aqueous solution in which H was adjusted to 6.0 to 8.0 with a phosphate buffer was prepared. Filter this for bacteria and put it in a vial with 1mf.
After dispensing, freeze-drying was performed to produce a freeze-dried injection.

[Example 3] (Nasal drop) 100ml of physiological saline! 10 mg of the peptide obtained in Synthesis Example 1 was added to the solution, and the pH was adjusted to 3.0 with citric acid buffer.
Adjusted to 0-6.0, 50μ in one dose 0.5mf
A nasal spray containing g was produced.

[Example 4] (Planning) Hard fat (triglyceride of saturated fatty acids) 98
．． After adding 0.5 g of egg yolk lecithin to 5 g and melting at 40 to 45 ° C., a solution prepared by dissolving 5 mg of the peptide obtained in Synthesis Example 1 in 1 g of PEG400 was added thereto and stirred and dispersed. 1 g of it was poured into a planing mold, and after solidification, it was separated from the mold to produce a planing.

[Effects of the Invention] The peptide in the anti-dementia agent of the present invention is a novel compound and has an excellent nootropic effect, and the anti-dementia agent of the present invention is an excellent anti-dementia agent.

Patent applicant: Nippon Chemifa Co., Ltd. Patent applicant: Fujirebio Co., Ltd.

Claims (1)

[Claims] 1. An effective amount of a peptide represented by formula (I): [There is a gene sequence] (I) or a derivative in its functional group, or a pharmacologically acceptable salt thereof;
and an anti-dementia agent comprising a pharmacologically acceptable carrier or diluent.