JP3318622B2 - Prolyl endopeptidase inhibitor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a prolyl endopeptidase inhibitor and, more particularly, to a drug or food useful for the prevention and / or treatment of dementia, which has been increasing in recent years and for which measures are desired. Are concerned with prolyl endopeptidase inhibitors which are expected.

[0002]

BACKGROUND ART Prolyl endopeptidase (EC 3.4.2)
1.26) was initially used as an oxytocin inactivating enzyme.
Discovered in the human uterus by Walter et al. In 1971, and subsequently purified solely from lamb and bovine brain (Science
173 , 827 (1971), Molecular & Cellular Biochemistry
30 , 111 (1980), Journal of the Japanese Society of Agricultural Chemistry 58 , 1147 (198
Four)). Similar enzymes have also been found in bacteria of the genus Flavobacterium (J. Biol. Chem. 255 , 4786 (1980)).
On the other hand, the presence of vasopressin and vasopressin derivative [pGlu ⁴ , Cyt ⁶ ] AVP- (4-9) in the brain has been confirmed, but these peptides have memory retention activity by the step-through passive avoidance learning method. (Science 221,
1310 (1983), Nature 308, 276 (1984)).

[0003] Yoshimoto and Tsuru reported that high activity of prolyl endopeptidase was observed in the hippocampus of the brain, which is a place where memory was fixed, and that prolyl endopeptidase in the brain was less than about 10 amino acids such as vasopressin. Note that prolyl endopeptidase normally functions normally in the brain, but if for some reason the regulatory mechanism is lost, vasopressin is degraded more than necessary and memory retention is impaired. Thought to appear, Z-Gly-Pro-CH ₂ Cl,
Z-Pro-prolinal, Z-Val-prolinal, Boc-Pro-prolinal
We synthesized prolyl endopeptidase inhibitors and confirmed that they show anti-amnesic effect (Journal of the Japanese Society of Agricultural Chemistry 58 (111), 1147 (1984), Chemistry and Biology 25 (9),
554 (1987)). Recently, prolyl endopeptidase inhibitors derived from microorganisms and food components have also been discovered (Agric. Biol. Chem. 55 , 825 (1991);
298, Abstracts of Annual Meeting of the Pharmaceutical Society of Japan, p.119).

[0004] On the other hand, plants such as corn, soybeans and carrots produce proteins containing a large amount of proline.
They include Pro-Pro-Pro-Val-His-Leu, Pro-Pro-Val-Ty
r-Lys, Pro-Pro-Ile-Tyr-Lys, Pro-Pro-Val-Tyr-Thr,
A peptide having 5 to 6 amino acids such as Pro-Pro-Val-His-Lys is present as a repeating sequence (The Journalof
Biological Chemistry 26 2, 8367 (1987)). In particular, corn protein contains 50-60% prolamin and 35-40% glutelin, and the main component prolamin is zein (zei).
n). Zein is classified into three types, α, β, and γ (J. Cereal Sci. 5, 117 (1987)).
o-Pro-Pro-Val-His-Leu as the basic unit, a six-repeat structure, and a sequence in which every other proline is arranged Pro-Arg-Pro
-Gln-Pro-His-Pro-Gln-Pro-His-Pro is included (Nu
cleic Acids Res. 13 (5), 1493 (1985)).

[0005]

DISCLOSURE OF THE INVENTION The present invention provides a functional food having a prolyl endopeptidase inhibitory activity, and therefore, as a therapeutic and / or prophylactic agent for dementia, or for preventing symptoms such as dementia through daily ingestion. An object of the present invention is to provide a specific peptide-based prolyl endopeptidase inhibitor which is expected to be useful as a drug.

[0006]

SUMMARY OF THE INVENTION Many prolyl endopeptidase inhibitors have proline (Pro) in the molecule, and some edible plants produce proline-rich proteins, Is Pro-Pro-Pro-Val-His-Leu, P
ro-Pro-Val-Tyr-Lys, Pro-Pro-Ile-Tyr-Lys, Pro-Pro-Va
A peptide of 5 to 6 amino acids such as l-Tyr-Thr, Pro-Pro-Val-His-Lys is present as a repeating sequence. Based on these facts, the present inventors consider that there is a possibility that a prolyl endopeptidase inhibitor may be mass-produced inexpensively by treating a plant protein with a protease, and the present inventors have found that the above-mentioned repetitive sequence has been considered. Various peptides were chemically synthesized and their effects on prolyl endopeptidase were investigated. As a result, the following peptides were found to inhibit prolyl endopeptidase.

That is, the present invention provides a prolyl endopeptidase inhibitor comprising at least one of the following peptides composed of L-form amino acids and an acid addition salt thereof as an active ingredient: Val-His-Leu-Pro-Pro- Pro, Leu-Pro-Pro-Pro-Val-His,
His-Leu-Pro-Pro-Pro-Val, His-Leu-Pro-Pro-Pro-Val-H
is-Leu-Pro-Pro-Pro-Val, Leu-Pro-Pro-Pro-Val, Pro-Pr
o-Pro-Val, Pro-Arg-Pro-Gln-Pro-His-Pro-Gln-Pro-His
-Pro, Lys-Pro-Pro-Val, Lys-Pro-Pro-Ile, and Thr-P
About ro-Pro-Val.

Here, the acid addition salt is a pharmaceutically acceptable acid (inorganic acid or organic acid) addition salt, for example, hydrochloride, hydrobromide, sulfate, nitrate, acetate, benzoate, maleic acid. Salts, fumarate, succinate, tartrate, citrate, oxalate, methanesulfonate, toluenesulfonate, aspartate, glutamate and the like are included. The equivalent of the acid to be added can be greater than 0 equivalent to an equivalent of the total number of basic amino acid residues, His, Arg and Lys in the peptide + 1.

Although the peptides in the claims of the present application are present in the sequence of known plant proteins, they are fragments thereof and have a previously unknown utility.
In view of this, the following peptides, except those known as angiotensin converting enzyme inhibitors and the like, are novel compounds to the best of our knowledge: His-Leu-Pro-Pro-Pro-Val-His-Leu-Pro- Pro-Pro-Val, L
eu-Pro-Pro-Pro-Val, Pro-Arg-Pro-Gln-Pro-His-Pro-Gl
n-Pro-His-Pro, Lys-Pro-Pro-Val, Lys-Pro-Pro-Ile and Thr-Pro-Pro-Val. Incidentally, in the peptides of the claims of the present application, Val-His-Le
u-Pro-Pro-Pro, Leu-Pro-Pro-Pro-Val-His and His-Leu-
Pro-Pro-Pro-Val is publicly known (JP-A-2-3612)
7, Agric. Biol. Chem. 53 , 1077 (1989)).

[0010] The peptide used in the present invention is usually a method of introducing amino acids stepwise by an organic chemical synthesis method,
Alternatively, it is produced by an enzymatic hydrolysis method of a natural protein, but it can also be produced by a peptide synthesis method using a reverse reaction of a hydrolase, a genetic engineering method, or the like. Many peptide synthesis methods are known so far, for example, Nobuo Izumiya, Tetsuo Kato, Higashihiko Aoyagi, Michinori Waki,
"Basics and experiments of peptide synthesis" are described in detail in Maruzen Co., Ltd. The peptide used in the present invention can be produced by any of these synthesis methods, for example, by so-called solid phase peptide synthesis or solution phase peptide synthesis.

[0011] Liquid phase peptide synthesis is described in the above "Basic and Experimental Peptide Synthesis",
For example, an amino acid to be located at the C-terminus of the present peptide, whose carboxyl group is protected by a benzyl group (Bzl), a t-butyl group (t-Bu) or the like, and an amino acid to be located adjacent to the C-terminal amino acid. Amino acids whose α-amino group is protected with a t-butyloxycarbonyl group (Boc), a benzyloxycarbonyl group (z), etc. are dissolved in dimethylformamide (DMF), dimethylacetamide, etc., and these are dissolved in dicyclohexyl. Carbodiimide (DC
C) and reaction in the presence of 1-hydroxybenzotriazole (HOBT) at room temperature overnight. The dipeptide obtained after removal of the amino-protecting group of the product by a conventional method is reacted in the same manner as the amino-protected third amino acid to remove the amino-protecting group, and the same procedure is repeated to obtain the present peptide. If the amino acid to be reacted has amino, guanidino or imidazolyl groups other than the α-position which should not participate in the reaction, these groups should generally be protected prior to the reaction. Protecting group for alcoholic hydroxyl group Bzl, etc., protecting group for amino group other than α-position includes those described as protecting group for α-amino group, and protecting group for guanidino group is tosyl group.
(Tos) and the like, and the protecting group for the imidazolyl group includes Tos and the like. Introduction of these protecting groups can be carried out by a conventional method, for example, as described in the above-mentioned "Basic and Experimental Peptide Synthesis". After completion of the final reaction, these protecting groups are removed, and these deprotection can be carried out by a conventional method, for example, as described in the above-mentioned “Basic and Experimental Peptide Synthesis”. For example, for the amino protecting group, Boc is with trifluoroacetic acid (TFA) or formic acid, for Z with catalytic reduction, for the carboxyl protecting group, Bzl is, for example, with catalytic reduction.
-Bu can be removed by TFA or HCl / dioxane. Further, for alcoholic hydroxyl groups, Bzl may be, for example, by catalytic reduction or HF.
For guanidino and imidazolyl protecting groups Tos is Na
Can be removed by / NH ₃ or HF.

On the other hand, for solid phase peptide synthesis, synthesis using a peptide synthesizer (for example, a 430A type peptide synthesizer produced by Applied Biosystems) has recently been widely used. That is, in this method, basically, an α-amino acid whose amino group is protected by Boc (Boc-amino acid) is used as a starting material, for example, a phenylacetamidomethyl (PAM) resin to which L-Pro is bound, That is, L-Pro-O-CH ₂ -PAM
N (available from Applied Biosystems)
-Stepwise extension by repeated removal of peptide and Boc from the side. Boc-Arg (Mts) (Mts is mesitylene-2-sulfonyl) and Boc-L-Gln are subjected to an elongation reaction via 1-hydroxybenzotriazole (HOBT) as an intermediate, while other Boc-amino acids are Subject to an elongation reaction via a symmetrical anhydride using DCC as an intermediate. In the above Boc-amino acids, if there is a reactive functional group, it should generally be protected by a suitable protecting group. An example of a protected Boc-amino acid in the context of the present invention is Boc-L-Arg (Mts)
, Boc-L-Lys (Cl-Z) (Cl-Z is 4-chlorobenzyloxycarbonyl), Boc-L-Thr (Bzl), Boc-L-His (DNP)
(DNP is 2,4-dinitrophenyl) and the like.

In the synthesis system using the 430A peptide synthesizer, the following reagents and solvents are used in addition to the amino acid substance: N, N-diisopropylethylamine (TFA neutralizer), TFA (Boc cleavage), MeOH (produced) Dissolution and removal of urea compounds), HOBT (0.5M HOBT / D
MF), DCC (0.5M DCC / dichloromethane (DCM), DCM and
DMF (solvent, neutralizer (70% ethanolamine, 29.5% methanol) (neutralization of waste liquid). Equip amino acid substances and their reagents and solvents at designated places.
The use of these substances is done automatically by the peptide synthesizer. The reaction temperature and time are also set automatically, but the reaction temperature is usually room temperature. Peptide in which the reactive functional group in the peptide is protected by the above means-
O-CH ₂ -PAM is obtained. The operation of the solid phase peptide synthesis experiment is described in Applied Biosystems 430A peptide synthesizer user's manual (Part N
umber 900066, Version 1.3B, July 1, 1988).

The obtained reactive functional group-protected peptide-O-CH ₂ -PAM is prepared according to a conventional method, for example, as described in the above-mentioned “Basic and Experimental Peptide Synthesis” or the 430A peptide synthesizer user's manual. And then
For example, treating resin and trifluoromethanesulfonic acid (TFMSA) with TFA as a diluent for TFMSA in the presence of thioanisole and / or ethanedithiol as scavengers to capture cations resulting from cleavage of protecting groups. Cleavage of the protecting group, thereby obtaining the desired peptide. In the TFMSA method, when the protected peptide -O-CH ₂ -PAM has a L-His (DNP) residues, which is subjected to the process after the DNP removal by thiophenol. Further, for example, a peptide having a reactive functional group protected by a hydrogen fluoride device manufactured by Peptide Research Institute
-O-CH ₂ -PAM, anisole and hydrogen fluoride are introduced, and -2
After performing the reaction at 1 ° C. for 1 hour, the target peptide can be obtained by removing hydrogen fluoride and washing with ether.

[0015] Among the peptides which can be used as an active ingredient in the prolyl endopeptidase inhibitor of the present invention, His-Leu-
Pro-Pro-Pro-Val, His-Leu-Pro-Pro-Pro-Val-His-Leu-P
ro-Pro-Pro-Val, Leu-Pro-Pro-Pro-Val, Pro-Pro-Pro-V
al, and Pro-Arg-Pro-Gln-Pro-His-Pro-Gln-Pro-His-
Pro corn protein γ-zein subtilisin,
It can also be obtained by enzymatic or acid hydrolysis with a protease having pepsin, papain or a similar substrate. The enzymatic reaction is usually performed simply in water or a buffer (for example, Tris-HCl buffer or phosphate buffer).

The γ-zein used may be γ-zein alone or a mixture containing α-zein and β-zein in addition to γ-zein. These zeins may be commercially available, zein separated from corn protein obtained in the process of producing corn starch, or zein separated therefrom by a known method (Plant Physiol., 80 62).
3 (1986)) may be γ-zein. In addition, Japanese Unexamined Patent Publication
Reference Example 1 of No. 36127 shows a production example of γ-zein. The enzyme used is subtilisin, papain or pepun.

The concentration of the protein substrate is not particularly limited as long as stirring and mixing can be performed, but is preferably in the range of 2 to 20% (w / v) for facilitating stirring. The amount of the enzyme thermolysin to be added varies depending on its titer, but it is usually at least 0.01% by weight, preferably 0.1 to 10% by weight, based on the protein. It is also possible to add a part of the enzyme during the reaction. The pH and temperature of the reaction may be near the optimum temperature of the enzyme used. Subtilisin is suitably at pH 6-9, temperature 30-70 ° C, and papain and pepsin at pH 5-8, temperature 30-60 ° C. The reaction time varies depending on the type of the enzyme, the amount added, the reaction temperature and the reaction pH, and is not constant, but is usually about 1 to 40 hours.
Termination of the hydrolysis reaction by a known method, for example, by heating the hydrolyzate or citric acid, organic acids such as malic acid, hydrochloric acid, inorganic acids such as phosphoric acid or sodium hydroxide,
It can be carried out by inactivating the enzyme by changing the pH by adding an alkali such as potassium hydroxide, or by separation by filtration using an ultrafiltration membrane or the like.

The resulting hydrolyzate solution is then subjected to solid-liquid separation (eg, centrifugation or filtration), and the resulting liquid is fractionated by ultrafiltration, gel filtration, or the like to obtain a liquid having a molecular weight of 10,000 or less. obtain. This liquid contains the target peptide of the present invention, and the liquid or a concentrate thereof (for example,
(Lyophilized product) is further fractionated to obtain each target peptide.
In the present invention, the freeze-dried product is referred to as γ-zein freeze-dried product, and the case where it is used will be further described.
First, the lyophilized γ-zein is subjected to an anion exchange resin, for example, a weakly basic anion exchange resin (for example, D
EAE (Toyopearl 650M) or a cation exchange resin, for example, a weakly acidic cation exchange resin (for example, SP-Toyopearl 650M manufactured by Tosoh Corporation).

In the fractionation method in which the lysate of γ-zein is first passed through an anion exchange resin, each peptide is eluted with a linear gradient (for example, in the case of using DEAET Toyopearl 650M, a tris buffer → an appropriate concentration). In Tris buffer
(NaCl). The eluate is collected in several fractions, each of which is subjected to gel filtration (for example, using Sephadex LH-20), cation exchange resin treatment (for example, SP-Toyopearl)
650M), reverse-phase HPLC (for example, Capsule Pack _C18 , a trade name of octadecylsilane manufactured by Shiseido Co., Ltd., or Sensupak 1251-Y, a trade name of octadecylsilane manufactured by Senshu Chemical Co., Ltd.) And fractionated into individual peptides. In brief, as exemplified in Example 2 below, the above-mentioned liquid having a molecular weight of 10,000 or less is concentrated and the peptide eluted at the same position as the present peptide chemically synthesized by HPLC is fractionated. Each peptide can also be obtained.

The acid addition salt of the present peptide can be produced by a conventional method. For example, an acid addition salt is obtained by reacting the present peptide with an acid in an amount of from greater than 0 equivalent to the total number of base amino acid residues contained therein to +1 equivalent in water, and then lyophilizing the product. Obtainable. Further, it may be obtained as an acid addition salt in the production process in the present invention.

The present peptide and its acid addition salt have a prolyl endopeptidase inhibitory activity and are expected to be effective in treating and preventing dementia in humans. The present peptide and its acid addition salt are used as they are or usually in a pharmaceutical composition with at least one pharmaceutical auxiliary. The present peptide and its acid addition salt can be administered parenterally (that is, intravenous injection, rectal administration, etc.) or orally, and can be formulated into a form suitable for each administration method.

The preparation form as an injection usually includes a sterilized aqueous solution. Formulations of the above form also include buffer pH adjusters (sodium hydrogen phosphate, citric acid, etc.), tonicity agents (sodium chloride, glucose, etc.), preservatives (methyl parahydroxybenzoate, propyl p-hydroxybenzoate, etc.) And other pharmaceutical auxiliaries other than water. The preparation can be sterilized by filtration through a bacteria-retaining filter, by mixing a bactericide into the composition, or by irradiating or heating the composition. The preparation can be produced as a sterilized solid composition or dissolved in sterile water or the like before use.

[0023] Oral formulations are formulated in a form suitable for absorption by the gastrointestinal tract. Tablets, capsules, granules, fine granules and powders are commonly used pharmaceutical auxiliaries such as binders (syrup, gum arabic, gelatin, sorbit, tragacanth, polyvinylpyrrolidone, hydroxypropylcellulose, etc.), excipients (lactose) Sucrose, corn starch, calcium phosphate, sorbite, glycine, etc.), lubricants (magnesium stearate, talc, polyethylene glycol, silica, etc.), disintegrants (potato starch,
Carboxymethylcellulose) and wetting agents (such as sodium lauryl sulfate). Tablets can be coated by conventional methods. The oral solution can be made into an aqueous solution or the like or a dry product.
Such an oral solution is prepared by using a conventional additive such as a preservative (p-
Methyl or propyl hydroxybenzoate, sorbic acid, etc.).

The amount of the present peptide or its acid addition salt in the present prolyl endopeptidase inhibitor can be varied, but is usually 5 to 100% (w / w), particularly 10 to 60% (w / w). Is appropriate. The dose of the present prolyl endopeptidase inhibitor is suitably from 10 to 200 mg / kg / day as an active ingredient. Incidentally, the acute toxicity of the peptides are LD ₅₀ (ICR strain mice, oral administration)> 3g / kg.

Further, since the present peptide has the advantage that it does not adversely affect the living body even when ingested in large amounts,
Alternatively, it may be added to various nutrients or the like or contained in foods and drinks to be eaten as a functional food or health food having an anti-dementia effect and a function of preventing dementia. That is, for example, by adding nutrients such as various vitamins and minerals,
For example, liquid foods such as nutritional drinks, soy milk, and soups, solid foods of various shapes, and powders can be used as they are or added to various foods. The content and intake of the active ingredient in the present prolyl endopeptidase inhibitor as such a functional food or health food may be the same as those in the above-mentioned pharmaceuticals.

[0026]

Next, the present invention will be described with reference to examples. Example 1 Synthesis of His-Leu-Pro-Pro-Pro-Val and prolyl endopeptidase inhibitory activity a) Synthesis of His-Leu-Pro-Pro-Pro-Val Peptide synthesizer (Model 430A) manufactured by Applied Biosystems ) To 0.5 mmol of Boc-Val-O-CH ₂ -PAM resin and 2 mmol each of Boc-His (Tos), Boc-Leu, Boc-Pro
, And His- (Tos) -Leu
The -Pro-Pro-Pro-Val- O-CH 2 -PAM were synthesized. Note that Tos represents a tosyl group. Next, the synthetic peptide resin was introduced into a hydrogen fluoride device manufactured by Peptide Research Institute, and anisole 1.5 ml was added.
After the addition, 10 ml of hydrogen fluoride were introduced. After the reaction at -2 ° C for 1 hour, hydrogen fluoride was removed under reduced pressure, the peptide was washed three times with anhydrous ether and chloroform alternately, and the peptide was dissolved in 60 ml of 2N acetic acid and freeze-dried. By this method, 130 mg of His-Leu-Pro-Pro-Pro-Val white powder was obtained. Next, the present peptide was subjected to high performance liquid chromatography (H
(PLC).

The conditions for purification by HPLC are shown below. Column: Merck LiChosorb RP-SelectB (250 x
Eluent: gradient of 3.5 to 67% acetonitrile containing 0.1% trifluoroacetic acid Flow rate: 6 ml / min Various analytical values of this peptide are shown in Table 1 below. Amino acid analysis was performed by hydrolyzing 6N hydrochloric acid at 110 ° C for 24 hours.
The analysis was performed using a 35-type amino acid analyzer. The mass spectrometry was performed by the FAB-MS method using a JEOL HX-110 mass spectrometer.

B) Val-His-Leu-Pro-Pro-Pro, Leu-Pro-P
ro-Pro-Val-His, His-Leu-Pro-Pro-Pro-Val-His-Leu-Pr
o-Pro-Pro-Val, Leu-Pro-Pro-Pro-Val, Pro-Pro-Pro-Va
l, Pro-Arg-Pro-Gln-Pro-His-Pro-Gln-Pro-His-Pro, Ly
Synthesis of s-Pro-Pro-Val, Lys-Pro-Pro-Ile, Thr-Pro-Pro-Val As in the synthesis of His-Leu-Pro-Pro-Pro-Val,
Biosystems Peptide Synthesizer (Type 430A)
Was synthesized by an anhydrous symmetric method using DCC, and the protecting group and the resin were cleaved with hydrogen fluoride. The conditions for peptide purification are the same as described above. Various analytical values of these peptides are shown in Table 1 below.

C) Measurement of prolyl endopeptidase inhibitory activity The prolyl endopeptidase inhibitory activity of the peptide obtained as described above was measured as follows. c-1) Measurement of inhibitory activity against microbial prolyl endopeptidase F. meningosepticum- derived prolyl endopeptidase purchased from Seikagaku Corporation was dissolved in 0.1 M phosphate buffer at pH 7.0, and 0.1 unit / ml of enzyme solution. Also, 2mM
Z-Gly-Pro-pNA (manufactured by Bachem, Z represents a benzyloxycarbonyl group, and pNA represents paranitroanilide) was dissolved in the above phosphate buffer (containing 40% dioxane) to prepare a substrate solution. 1.5 ml of aqueous solution of each of the above peptides
Put 40 μl in a volume of plastic tube, add 80 μl of phosphate buffer and 40 μl of the substrate solution, and incubate at 30 ° C. for 10 minutes, then add the prolyl endopeptidase solution 40
μl was added, mixed well, and reacted at 30 ° C. for 10 minutes. Thereafter, the reaction was stopped by adding 200 μl of 1N HCl. After the reaction was stopped, paranitroaniline released by the enzyme reaction was quantified by HPLC. The HPLC measurement conditions are as follows.

HPLC measurement conditions Column: μBondasphere 5 μC8-, manufactured by Waters
300A (150 x φ3.9mm) Elution: 53% acetonitrile containing 0.1% trifluoroacetic acid Detection: absorbance at 410nm These experiments were performed multiple times, and the inhibition rate was calculated by the following formula. A: Peak area of paranitroaniline without an inhibitor B: Peak area of paranitroaniline with an inhibitor added The results are shown in Table 2 below. The concentration of the peptide at an inhibition rate of 50% was defined as the IC ₅₀ value, and is shown in Table 3 below.

C-2) Measurement of Inhibitory Activity against Bovine Brain Prolyl Endopeptidase 25 g of bovine brain acetone powder (manufactured by Sigma) containing 20 mM Tris-HCI containing 10 mM EDTA and 10 mM 2-mercaptoethanol
It was dissolved in 200 ml of buffer (pH 7.0), centrifuged at 16,000 rpm for 20 minutes, and the supernatant was collected. Next, prolyl endopeptidase was partially purified by column chromatography using DEAE-Toyopearl (Tosoh) to obtain a 0.1 unit / ml enzyme solution. In addition, 2 mM Z-Gly-Pro-pNA was added to the above Tris-HCl
It was dissolved in a buffer (containing 40% dioxane) to obtain a substrate solution. 40 μl of each of the aqueous solutions of the various peptides described above was placed in a 1.5 ml plastic tube, 80 μl of a phosphate buffer solution and 40 μl of a substrate solution were added thereto, and the mixture was incubated at 37 ° C. for 10 minutes, and then the prolyl endopeptidase solution 40 μl was added.
Was added and mixed well, and reacted at 37 ° C. for 10 minutes. The following measurement conditions were the same as those for the measurement of the inhibitory activity on microbial prolyl endopeptidase, and the results are shown in Table 4 below.

Example 2 His-Leu-Pro- from γ-zein
Production of Pro-Pro-Val 0.2 g of γ-zein was dispersed in 12 ml of 50 mM Tris-HCl (pH 8.2), and 10 mg of subtilisin Carlsberg (manufactured by Sigma) was added thereto. After reaction at 37 ° C for 18 hours, molecular weight 10,000
And passed through an ultrafiltration membrane (Millipore Co., Ltd., Morcut) to recover the low-molecular-weight peptide passing therethrough. Concentrate this, HP
In LC, peptides eluted at the same position as the chemically synthesized His-Leu-Pro-Pro-Pro-Val were collected and collected. HP
The LC conditions are as follows. HPLC separation column: Merck Superspher RP-8 (125 x φ4m
m) Elution: 7-63% acetonitrile gradient containing 0.1% trifluoroacetic acid Detection: ultraviolet absorption at 210 nm The collected peptide solution was dried under reduced pressure to obtain a final sample.

[0033]

[Table 1] Analysis value of synthetic peptide ──────────────────────────────────── [α] _D ²⁵ (°) Amino acid analysis value Mass analysis value Peptide (H ₂ O) (Composition ratio) FAB-MS (m / z) ─────────────────────── ───────────── Val-His-Leu-Pro-Pro-Pro -192 Val 1.00, His 0.98, 659 (M + H) ⁺ (C = 0.4) Leu 1.04, Pro 3.11 Leu -Pro-Pro-Pro-Val-His -228 Leu 1.00, Pro 3.05, 659 (M + H) ⁺ (C = 0.2) Val 1.00, His 1.03 His-Leu-Pro-Pro-Pro-Val -218 His 1.00 , Leu 1.08, 659 (M + H) ⁺ (C = 0.3) Pro 2.92, Val 1.00 His-Leu-Pro-Pro-Pro-Val- -242 His 0.81, Leu 1.09, 1299 (M + H) ⁺ His- Leu-Pro-Pro-Pro-Val (C = 0.1) Pro 3.09, Val 1.00 Leu-Pro-Pro-Pro-Val -238 Leu 1.00, Pro 3.08, 522 (M + H) ⁺ (C = 0.3) Val 1.04 Pro-Pro-Pro-Val -242 Pro 2.94, Val 1.00 409 (M + H) ⁺ (C = 0.4) Pro-Arg-Pro-Gln-Pro-His- -203 Pro 6.3, Arg 0.8, 1287 (M + H) ⁺ Pro-Gln-Pro-His-Pro (C = 0.3) Glu 2.0, His 1.7 Lys-Pro-Pro-V al -169 Lys 0.89, Pro 2.06 440 (M + H) ⁺ (C = 0.4) Val 1.00 Lys-Pro-Pro-Ile -167 Lys 1.00, Pro 1.94 454 (M + H) ⁺ (C = 0.3) Ile 1.12 Thr-Pro-Pro-Val -192 Thr 1.00, Pro 2.07 413 (M + H) ⁺ (C = 0.5) Val 1.00 ────────────────────── ──────────────

[0034]

[Table 2] Inhibitory activity against prolyl endopeptidase derived from F. meningosepticum ─ Peptide concentration (μM) Inhibition rate (%) ──────────────────────────────────── Val-His-Leu-Pro-Pro-Pro 800 31 Leu-Pro-Pro-Pro-Val-His 800 12 His-Leu-Pro-Pro-Pro-Val 400 80 His-Leu-Pro-Pro-Pro-Val -His-Leu-Pro-Pro-Pro-Val 400 88 Leu-Pro-Pro-Pro-Val 400 39 Pro-Pro-Pro-Val 400 26 Pro-Arg-Pro-Gln-Pro-His- Pro-Gln- Pro-His-Pro 400 14 Lys-Pro-Pro-Val 400 65 Lys-Pro-Pro-Ile 400 52 Thr-Pro-Pro-Val 400 12 ──────────────── ────────────────────

[0035]

[Table 3]

[0036]

[Table 4] Inhibitory activity against bovine brain-derived prolyl endopeptidase Peptide concentration (μM) Inhibition rate (%) ──────────────────────────────────── Val -His-Leu-Pro-Pro-Pro 400 65 Leu-Pro-Pro-Pro-Val-His 400 68 His-Leu-Pro-Pro-Pro-Val 400 28 His-Leu-Pro-Pro-Pro-Pro-Val- His-Leu-Pro-Pro-Pro-Val 400 64 Pro-Arg-Pro-Gln-Pro-His- Pro-Gln-Pro-His-Pro 400 71 ────────────── ──────────────────────

[0037]

According to the present invention, a novel and useful prolyl endopeptidase inhibitor is provided.

Continued on the front page (51) Int.Cl. ⁷ Identification code FI C07K 7/06 C07K 7/08 7/08 A61K 37/64 (72) Inventor Hidekatsu Maeda 1-3-1 Higashi, Tsukuba, Ibaraki Pref. Inside the Research Institute of Microorganisms (72) Inventor Shinsuke Miyoshi 2-20-2 Hinode, Funabashi-shi, Chiba Pref. Showa Sangyo Co., Ltd. Examiner at the Research Institute Seiko Tamura (56) References JP-A-2-36127 (JP, A) JP-A-3-31298 (JP, A) JP-A-4-208299 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 38/55 CA (STN) REGISTRY ( STN)

Claims (1)

(57) [Claims] 1. A prolyl endopeptidase inhibitor comprising at least one of the following peptides composed of L-form amino acids and acid addition salts thereof as an active ingredient: Val-His-Leu-Pro-Pro-Pro, Leu -Pro-Pro-Pro-Val-His,
His-Leu-Pro-Pro-Pro-Val, His-Leu-Pro-Pro-Pro-Val-H
is-Leu-Pro-Pro-Pro-Val, Leu-Pro-Pro-Pro-Val and Pro
-Pro-Pro-Val.