JPH05331072A - Prolyl endopeptidase inhibitor - Google Patents

Abstract

PURPOSE:To provide a new and useful prolyl endopeptidase inhibitor promising its use in medicines and/or foods useful for preventing and/or curing dementia. CONSTITUTION:The inhibitor containing, as active ingredient, at least one kind of the peptides each composed of L-amino acids described blow and their acid- addition salts thereof; namely: Val-His-Leu-Pro-Pro-Pro, Leu-Pro-Pro-Pro-Val-His, His-Leu-Pro-Pro-Pro-Val, His-Leu-Pro-Pro-Pro-Val-His-Leu-Pro-Pro-Pro-Val, Leu-Pro-Pro-Pro-Val, Pro-Pro-Pro-Val, Pro-Arg-Pro-Gln-Pro-His-Pro-Gln-Pro-His- Pro, Lys-Pro-Pro-Val, Lys-Pro-Pro-Ile, and Thr-Pro-Pro-Val.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a prolyl endopeptidase inhibitor, more specifically, it can be used as a pharmaceutical or food useful for the prevention and / or treatment of dementia, which has been increasing in recent years and for which countermeasures are desired. Relates to a prolyl endopeptidase inhibitor that is expected.

[0002]

[Prior Art] Prolyl Endopeptidase (EC 3.4.2
1.26) was originally used as an oxytocin inactivating enzyme, 19
Discovered in the human uterus by Walter et al. In 71 and subsequently purified singly from lamb and bovine brain (Science
173 , 827 (1971), Molecular & Cellular Biochemistry
30 , 111 (1980), Journal of Japanese Society of Agricultural Chemistry 58 , 1147 (198)
Four)). Similar enzymes have also been found in Flavobacterium bacteria (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 showed memory retention activity by the step-through type passive avoidance learning method. (Science 221,
1310 (1983), Nature 308, 276 (1984).

In Yoshimoto and Tsuru, high activity of prolyl endopeptidase was found in the hippocampus of the brain, which is a fixed place for memory, and that prolyl endopeptidase in the brain was about 10 amino acids or less like vasopressin. It is noted that it normally acts on the peptide of erythrocyte, and normally, the prolyl endopeptidase in the brain works normally, but if the regulatory mechanism is lost for some reason, vasopressin is decomposed more than necessary and memory retention is impaired. Think that it will appear, Z-Gly-Pro-CH ₂ Cl,
Z-Pro-prolinal, Z-Val-prolinal, Boc-Pro-prolinal
We synthesized prolyl endopeptidase inhibitors such as and confirmed that they have anti-amnestic effects (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 ingredients have also been discovered (Agric. Biol. Chem. 55 , 825 (1991), JP-A-3-31).
298, 1990 Annual Meeting of the Pharmaceutical Society, p.119).

On the other hand, plants such as corn, soybean and carrot produce a protein 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,
Peptides of 5 to 6 amino acid residues such as Pro-Pro-Val-His-Lys exist as repeating sequences (The Journalof
Biological Chemistry 26 2, 8367 (1987)). In particular, corn protein contains 50 to 60% prolamin and 35 to 40% glutelin, and the main component prolamin is zein (zei).
n). Zein is divided into three types, α, β, and γ (J. Cereal Sci. 5, 117 (1987)), and γ-zein contains Pr.
6-repeat structure with o-Pro-Pro-Val-His-Leu as the basic unit and a sequence of prolines arranged every other pair Pro-Arg-Pro
-Gln-Pro-His-Pro-Gln-Pro-His-Pro included (Nu
cleic Acids Res. 13 (5), 1493 (1985)).

[0005]

The present invention has a prolyl endopeptidase inhibitory action, and is therefore a functional food for treating and / or preventing dementia or for preventing symptoms such as dementia through daily intake. It is an object of the present invention to provide a specific peptide-based prolyl endopeptidase inhibitor that is expected to be useful as

[0006]

Many prolyl endopeptidase inhibitors have proline (Pro) in the molecule, and some edible plants produce proline-rich proteins and Is Pro-Pro-Pro-Val-His-Leu, P
ro-Pro-Val-Tyr-Lys, Pro-Pro-Ile-Tyr-Lys, Pro-Pro-Va
Peptides of 5 to 6 amino acid residues such as l-Tyr-Thr and Pro-Pro-Val-His-Lys are present as repeating sequences. Based on these facts, the present inventors consider that there is a possibility that a prolyl endopeptidase inhibitor can be mass-produced at low cost by treating a plant protein with a proteolytic enzyme (protease). 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 is a prolyl endopeptidase inhibitor containing, as an active ingredient, at least one of the following peptides composed of L-amino acids and their acid addition salts: 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
Regarding ro-Pro-Val.

Here, the acid addition salt is a pharmaceutically acceptable acid (inorganic acid and organic acid) addition salt, for example, hydrochloride, hydrobromide, sulfate, nitrate, acetate, benzoate, maleic acid. It includes salts, fumarates, succinates, tartrates, citrates, oxalates, methanesulfonates, toluenesulfonates, aspartates, glutamate and the like. The equivalent weight of the acid to be added can be from more than 0 equivalent to the equivalent of the total number of basic amino acid residues in the peptide, His, Arg and Lys + 1.

The peptides claimed in the present application are present in the sequences of known plant proteins, but are fragments thereof and have as yet unknown utility.
From this point of view, the following peptides, excluding those known as angiotensin converting enzyme inhibitors, are novel compounds as far as the inventors know: 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. 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 known (Japanese Patent Laid-Open No. 2-3612)
7, Agric. Biol. Chem. 53 , 1077 (1989)).

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 utilizing the 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, Tohiko Aoyagi, Michinori Waki,
"Basics and experiments of peptide synthesis" are described in detail in Maruzen Co., Ltd. The peptides used in the present invention can be produced by any of these synthetic methods, for example by so-called solid phase peptide synthesis or liquid phase peptide synthesis.

Solution-phase peptide synthesis is described above in "Peptide Synthesis Fundamentals and Experiments", and accordingly,
For example, an amino acid to be located at the C-terminal of the present peptide, the carboxyl group of which should be located adjacent to a C-terminal amino acid and an amino acid protected by a benzyl group (Bzl), t-butyl group (t-Bu), etc. An amino acid whose α-amino group is protected by a t-butyloxycarbonyl group (Boc), a benzyloxycarbonyl group (z), etc. is dissolved in dimethylformamide (DMF), dimethylacetamide, etc., and these are dissolved in dicyclohexyl. Carbodiimide (DC
C) and 1-hydroxybenzotriazole (HOBT) in the presence of the reaction at room temperature overnight. Then, 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 then the same procedure is repeated to obtain the present peptide. If the amino acid to be reacted has an amino group other than the α-position, a guanidino group or an imidazolyl group, which should not be involved in the reaction, these groups should generally be protected prior to the reaction. Protecting groups for alcoholic hydroxyl groups such as Bzl, etc., protecting groups for amino groups other than α-position include those mentioned as protecting groups for α-amino groups, and protecting groups for guanidino groups include tosyl groups.
(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 "Basics and Experiments of Peptide Synthesis". After the end of the final reaction, these protecting groups are removed, but their deprotection can be carried out by conventional methods, for example as described in "Peptide synthesis basics and experiments" above. For example, for amino protecting groups, Boc is trifluoroacetic acid (TFA) or formic acid, Z is for catalytic reduction, and for carboxyl protecting groups, Bzl is, for example, by catalytic reduction, t
-Bu can be removed by TFA or HCl / dioxane. Further, for alcoholic hydroxyl groups, Bzl is, for example, by catalytic reduction or HF,
For guanidino and imidazolyl protecting groups Tos is Na
It can be removed by / NH ₃ or HF.

On the other hand, for solid phase peptide synthesis, synthesis using a peptide synthesizer (for example, 430A type peptide synthesizer produced by Applied Biosystems) has been widely used recently. That is, in this method, basically, an α-amino acid having a Boc-protected amino group (Boc-amino acid) is used as a starting material, for example, L-Pro-bound phenylacetamidomethyl (PAM) resin, That is, L-Pro-O-CH ₂ -PAM
N (available from Applied Biosystems)
-Prolonged stepwise 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 extension reaction via 1-hydroxybenzotriazole (HOBT) as an intermediate, and other Boc-amino acids are Subject to an extension 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 according to the 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 synthetic system using the 430A type peptide synthesizer, the following reagents and solvents are used in addition to the amino acid substance: N, N-diisopropyl pyramine (TFA neutralizer), TFA (Boc cleavage), MeOH (generated). 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) (waste liquid neutralization). Equipped with amino acid substances and these reagents and solvents at designated places.
The use of these substances is done automatically by the peptide synthesizer. Although the reaction temperature and time are automatically set, the reaction temperature is usually room temperature. Peptide whose 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 described above is performed by Applied Biosystems, Inc. 430A type 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 was prepared according to a conventional method, for example as described in the above "Basics and Experiments of Peptide Synthesis" or 430A Peptide Synthesizer User's Manual. And then
For example, treatment of trifluoromethanesulfonic acid (TFMSA) with TFA as a diluent for TFMSA in the presence of thioanisole and / or ethanedithiol as a scavenger to capture cations resulting from cleavage of the protecting group The protecting group is cleaved to obtain the desired peptide. In the above TFMSA method, when the protected peptide —O—CH ₂ —PAM has an L-His (DNP) residue, this is subjected to the above treatment after removal of DNP with thiophenol. In addition, for example, a peptide with a reactive functional group protected in a hydrogen fluoride device manufactured by Peptide Research Co., Ltd.
-O-CH ₂ -PAM, introduced anisole and hydrogen fluoride, -2
It is also possible to obtain the target peptide by carrying out the reaction at 1 ° C. for 1 hour and then removing hydrogen fluoride and washing with ether.

Among the peptides that can be used as the 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 is a subtilisin of the maize protein γ-zein,
It can also be obtained enzymatically or by acid hydrolysis with a protease having pepsin, papain or a similar substrate. The enzymatic reaction is usually performed simply in water or a buffer (eg, 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, or zein separated from corn protein obtained in the production process of corn starch, or separated from it by a known method (Plant Physiol., 80 62).
3 (1986)) γ-zein. In addition, JP-A-2-
Reference Example 1 of 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 it is preferably in the range of 2-20% (w / v) which facilitates stirring. The amount of enzyme thermolysin added varies depending on its titer, but it is usually 0.01% by weight or more, 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 in the vicinity of the optimum temperature of the enzyme used, and subtilisin preferably has a pH of 6 to 9 and a temperature of 30 to 70 ° C, and papain and pepsin have a pH of 5 to 8 and a temperature of 30 to 60 ° C. The reaction time is not constant because it depends on the type of enzyme, the amount added, the reaction temperature, and the reaction pH, but is usually about 1 to 40 hours.
The hydrolysis reaction can be stopped by a known method, for example, by heating the hydrolyzate or citric acid, an organic acid such as malic acid, hydrochloric acid, an inorganic acid such as phosphoric acid or sodium hydroxide,
It can be carried out by inactivating the enzyme due to a pH change due to the addition of 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 (for example, centrifugation or filtration), and the obtained 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 peptide of interest of the present invention, and this liquid or its concentrate (for example,
The freeze-dried 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 of using it will be further described.
The γ-zein freeze-dried product was first subjected to an anion exchange resin, for example, a weakly basic anion exchange resin (for example, Tosoh Corp. D
EAE Toyopearl 650M) or a cation exchange resin, for example, a weakly acidic cation exchange resin (eg Tosoh Corp. SP-Toyopearl 650M).

In the fractionation method in which the freeze-dried product 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, Tris buffer → the appropriate concentration, In Tris buffer
Fractionation by NaCl). Take the eluate in several fractions and filter each by gel filtration (eg using Sephadex LH-20), cation exchange resin treatment (eg SP-Toyopearl).
650M), reverse-phase HPLC (for example, Capsule Pack C ₁₈ which is the trade name of octadecylsilane manufactured by Shiseido Co., Ltd. or Senshu Pack 1251-Y which is the trade name of octadecylsilane manufactured by Senshu Chemical Co., Ltd.) And then fractionated into individual peptides. In addition, briefly, as illustrated in Example 2 below, by concentrating a liquid having a molecular weight of 10000 or less and collecting a peptide eluted at the same position as the chemically synthesized peptide by HPLC, 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 prepared by reacting the peptide with water in an amount of more than 0 equivalent to the total number of basic amino acid residues contained therein + 1 equivalent, and then freeze-drying the product. Obtainable. Further, it may be obtained as an acid addition salt in the production process of the present invention.

The present peptide and its acid addition salt have a prolyl endopeptidase inhibitory action and are expected to be effective for the treatment and prevention of human dementia. The peptide and its acid addition salt are used as they are, or usually in the form of a pharmaceutical composition with at least one pharmaceutical adjuvant. The 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 dosage form as an injection usually includes a sterile aqueous solution. The above-mentioned preparations also include buffers pH adjusters (sodium hydrogen phosphate, citric acid, etc.), tonicity agents (sodium chloride, glucose, etc.), preservatives (methyl paraoxybenzoate, propyl p-hydroxybenzoate, etc.) Other pharmaceutical adjuvants other than water can be included. The preparation can be sterilized by filtration through a bacteria-retaining filter, incorporation of a bactericide into the composition, irradiation of the composition, and heating. The preparation can also be produced as a sterilized solid composition and dissolved in sterilized water before use.

The orally administered drug is formulated into 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, sorbit, glycine, etc.), lubricants (magnesium stearate, talc, polyethylene glycol, silica, etc.), disintegrants (potato starch,
Carboxymethyl cellulose and the like) and wetting agents (sodium lauryl sulfate and the like) can be included. The tablets can be coated by a conventional method. The oral solution can be made into an aqueous solution or a dry product.
Such oral liquid preparations are prepared by using conventional additives such as preservatives (p-
(Methyl or propyl hydroxybenzoate, sorbic acid, etc.) may be included.

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

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

[0026]

EXAMPLES 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 manufactured by Applied Biosystems (type 430A) ) 0.5 mmol Boc-Val-O-CH ₂ -PAM resin and 2 mmol each Boc-His (Tos), Boc-Leu, Boc-Pro.
And His- (Tos) -Leu by the anhydrous symmetry method with DCC.
The -Pro-Pro-Pro-Val- O-CH 2 -PAM were synthesized. In addition, Tos represents a tosyl group. Next, the synthetic peptide resin was introduced into a hydrogen fluoride device manufactured by Peptide Institute, and 1.5 ml of anisole was added.
After adding, 10 ml of hydrogen fluoride was introduced. After the reaction at −2 ° C. for 1 hour, hydrogen fluoride was removed under reduced pressure, the peptide was washed with anhydrous ether and chloroform alternately three times, and the peptide was dissolved in 60 ml of 2N acetic acid and freeze-dried. By this method, 130 mg of white powder of His-Leu-Pro-Pro-Pro-Val was obtained. This peptide was then analyzed by high performance liquid chromatography (H
PLC).

The purification conditions by HPLC are shown below. Column: Merck LiChosorb RP-SelectB (250 x
φ25 mm) Eluent: Gradient of 3.5-67% 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 carried out after hydrolysis with 6N hydrochloric acid at 110 ° C for 24 hours, after which Hitachi 8
The analysis was performed using a 35-type amino acid analyzer. Further, mass spectrometry was performed by FAB-MS method using a JEOL HX-110 type 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 Similar to the synthesis of His-Leu-Pro-Pro-Pro-Val
Biosystems peptide synthesizer (430A type)
Was synthesized by the anhydrous symmetry method using DCC, and the protecting group and the resin were cleaved with hydrogen fluoride. Peptide purification conditions 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-derived prolyl endopeptidase F. meningosepticum- derived prolyl endopeptidase purchased from Seikagaku Co., Ltd. was dissolved in 0.1 M phosphate buffer of pH 7.0 to give 0.1 unit / ml of enzyme solution. 2mM
Z-Gly-Pro-pNA (manufactured by Backchem, Z represents benzyloxycarbonyl group, pNA represents paranitroanilide) was dissolved in the above phosphate buffer solution (containing 40% dioxane) to obtain a substrate solution. 1.5 ml each of the above various peptide aqueous solutions
Place 40 μl in a plastic tube of volume, add 80 μl of phosphate buffer solution and 40 μl of substrate solution to it, and incubate at 30 ° C for 10 minutes. Then, add the above prolyl endopeptidase solution 40
μl was added and mixed well, and the reaction was carried out at 30 ° C. for 10 minutes. The reaction was then stopped by adding 200 μl of 1N HCl. After the reaction was stopped, paranitroaniline liberated by the enzymatic reaction was quantified by HPLC. The HPLC measurement conditions are as follows.

HPLC measurement conditions Column: Waters μBondasphere 5 μC8-
Elution of 300A (150 x φ3.9 mm): 53% acetonitrile containing 0.1% trifluoroacetic acid Detection: Absorption at 410 nm A plurality of such experiments were performed, and the inhibition rate was calculated from the following formula. A: Peak area of para-nitroaniline in the absence of inhibitor B: Peak area of para-nitroaniline in the case of adding inhibitor The results are shown in Table 2 below. The concentration of the peptide when the inhibition rate was 50% was defined as the IC ₅₀ value, which is shown in Table 3 below.

C-2) Measurement of inhibitory activity against bovine brain-derived prolyl endopeptidase 20 g of bovine brain acetone powder (manufactured by Sigma) containing 10 mM EDTA and 10 mM 2-mercaptoethanol was added to 20 mM Tris-HCI
It was dissolved in 200 ml of a buffer solution (pH 7.0), centrifuged at 16,000 rpm for 20 minutes, and the supernatant was collected. Then, the prolyl endopeptidase was partially purified by column chromatography with DEAE-Toyopearl (Tosoh) to prepare an enzyme solution of 0.1 unit / ml. In addition, 2 mM Z-Gly-Pro-pNA was added to the above Tris-HCl.
It was dissolved in a buffer solution (containing 40% dioxane) to obtain a substrate solution. Put 40 μl of each of the above-mentioned various peptides in a 1.5 ml capacity plastic tube, add 80 μl of phosphate buffer and 40 μl of substrate solution, and incubate at 37 ° C for 10 minutes, and then add 40 μl of the above prolyl endopeptidase solution.
Was added, mixed well, and reacted at 37 ° C. for 10 minutes. The following measurement conditions are the same as in the case of measuring the inhibitory activity against microbial-derived 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 ℃ for 18 hours, molecular weight 10,000
The low-molecular-weight peptide passing through the ultrafiltration membrane (Morpor cut manufactured by Millipore) was recovered. Concentrate this, HP
In LC, the peptide eluted at the same position as the chemically synthesized His-Leu-Pro-Pro-Pro-Val was collected and collected. HP
The LC conditions are as follows. HPLC separation conditions 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 Absorbed peptide solution was dried under reduced pressure to give the final preparation.

[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-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 F. meningosepticum- derived prolyl endopeptidase ──────────────────────────────────── ─ 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-Val- His-Leu-Pro-Pro-Pro-Val 400 64 Pro-Arg-Pro-Gln-Pro-His- Pro-Gln-Pro-His-Pro 400 71 ─────────────── ──────────────────────

[0037]

INDUSTRIAL APPLICABILITY The present invention provides a novel and useful prolyl endopeptidase inhibitor.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Internal reference number FI Technical display location C07K 99:00 (72) Inventor Hideoki Tanaka 1-3-3 Higashi, Tsukuba-shi, Ibaraki Industrial Technology Institute Inside the Institute for Microbial Technology (72) Hidekatsu Maeda 1-3-3 Higashi, Tsukuba, Ibaraki Prefecture Inside Institute for Microbial Technology, Industrial Technology Institute (72) Shinsuke Miyoshi 2-20-2 Hinode, Funabashi, Chiba Showa Sangyo Co., Ltd.

Claims (1)

[Claims] 1. A prolyl endopeptidase inhibitor containing, as an active ingredient, at least one of the following peptides composed of L-form amino acids and acid addition salts thereof: 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-Pro
-Pro-Val.