JPH0940693A - Inhibitor of prolyl endopeptidase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new peptide, having a specific amino acid sequence, derived from a rice protein, excellent in inhibiting ability against prolyl endopeptidases, administrable for a long period due to its high safety and useful for prevention, treatment, etc., of dementia. SOLUTION: This inhibitor of prolyl endopeptidases is a new peptide having an amino acid sequence of the formula. The peptide has a high inhibiting ability against the prolyl endopeptidases and is excellent in safety due to its origin from a natural substance and useful as an active ingredient of the inhibitor of the prolyl endopeptidases effectively used in prevention, treatment, etc., of dementia. The peptide is obtained by suspending SAKE (Japanese rice wine) lees produced in brewing SAKE from rice according to a method for brewing the SAKE with a liquefied liquor in water, adding a pepsin thereto, carrying out the reaction at 37 deg.C and pH 1.5 for 1hr, neutralizing the resultant reactional mixture, then heating the neutralized mixture in a boiling water bath for 10min, terminating the reaction, collecting a supernatant by centrifugation, passing the collected supernatant through an adsorbent column and purifying the adsorbed fraction by using the ion exchange chromatography, gel filtration and reversed phase chromatography, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a prolyl endopeptidase inhibitor comprising a peptide derived from rice protein and / or a derivative thereof as an active ingredient.

[0002]

2. Description of the Related Art The importance of geriatric medicine has been greatly emphasized in the aging society. Among them, prevention and treatment of dementia is a major problem not only for the person himself, but also for his family and society. Representative examples of dementia include cerebrovascular dementia and Alzheimer-type dementia, and the cause of the latter has not been completely elucidated compared to the former.
In addition, Alzheimer's dementia is also called amnesia, and since there is not much impairment in other motor skills other than a short-term loss of memory, a heavy burden is placed on nursing care and it has become a problem. It is urgent. As such, the background and mechanism of onset are still largely unknown, but therapeutic drugs are being actively developed and some are progressing to clinical trials.

[0003] Prolyl endopeptidase (hereinafter referred to as PE
P (sometimes referred to as P) (EC 3.4.21.26)
It is a serine protease that has specificity for proline and cleaves the peptide at the carboxyl group side, and is known to have high activity in brain, testis and liver (protein nucleic acid enzyme,
25, 513-523, (1980), protein nucleic acid enzyme, 29, 127-133, (1984)). PEP is known to degrade neurotransmitter substance P and THR (thyroid stimulating hormone) and vasopressin, which are thought to be involved in memory. Vasopressin is a peptide hormone that acts on reabsorption of water in the kidney, but is also involved in learning and memory processes in the brain, and there is a fact that amnesia progresses when this hormone is degraded and inactivated . In addition, it is known that the amount of vasopressin in dementia patients is lower than that in normal persons (BI
OINDUSTRY, 4, 788-796, (198
7)).

The decomposition of vasopressin by PEP is shown in the following chemical formula 1. In healthy people, PEP in the brain works normally, but if the regulatory mechanism is lost for some reason, vasopressin is degraded more than necessary, causing damage to memory retention. Therefore, studies have been made to inhibit the activity of this enzyme to treat dementia. For example, prolyl prolinal derivatives (protein nucleic acid enzymes, 29, 127, (1
984)), N-acylpyrrolidine derivatives (JP-A-61
-37764, JP-A-61-183297, JP-A-61-61297
238775, JP-A-61-238799, JP-A-6-1987
2-114957) have been screened.

[0005]

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However, since these are synthetic products, there is a problem in safety, and since their structures are similar, structure-activity relationships have already been clarified (ABC, 55,
37-43, (1991)). In order to overcome this situation, there is a need for inhibitors that are safe and have unique structures not found in synthetic inhibitors.

[0007]

SUMMARY OF THE INVENTION The present invention has been made to meet such needs in the industry, and has been made in order to develop a particularly safe and excellent PEP inhibitor. .

[0008]

Means for Solving the Problems The present invention has been made to achieve the above object, and the present inventors have intensively searched for a prolyl endopeptidase inhibitor having no side effects in natural substances. As a result, the presence of this inhibitor in rice protein was confirmed, and the substance was identified as Leu-Leu-Ser-Pro-
The present invention was completed by finding out that the peptide has a structure of Phe-Trp-Asn-Ile-Asn-Ala.

[0009] The peptide of the present invention can be obtained from rice protein, but can also be obtained from rice protein-containing substances in addition to rice protein. Rice protein-containing substances include brown rice, white rice,
It can be obtained not only from rice bran and brewed foods made from rice (such as sake, miso phosphorus, miso, and soy sauce), but also from its by-products (such as bran and lees).

The peptide of the present invention is produced by decomposing rice protein (containing substance), and a known proteolytic method such as enzymatic decomposition, chemical decomposition, or physical decomposition is appropriately used. Enzyme digestion is performed under mild conditions and has the advantage of low denaturation of the produced peptide. do it.

[0011] In this case, when a fermented product such as sake is used as a raw material, it is not necessary to treat with a protease, and if necessary, it may be concentrated and then immediately proceed to the extraction step. When the raw material is undegraded rice protein such as untreated rice protein, or when the raw material is fermentation by-products such as lees and bran that are not sufficiently decomposed, the enzyme may be used. Processing is preferred.

The peptide of the present invention can be used not only from rice,
Isolation and purification from other cereals, meat, other natural products and their degradation products, peptide synthesis using reverse reaction of hydrolase, genetic engineering, and organic synthesis Can be manufactured.

[0013] Separation and purification from natural products is carried out by extracting as it is or decomposed by an acid, an alkali or an enzyme, if necessary, extracting and concentrating it if necessary, and then, after extracting and concentrating the adsorbed affinity to various adsorbents. Differences in solubility in various solvents,
A method usually used for separation and purification, such as a difference in elution rate due to the molecular sieve effect, or a combination thereof may be used.

For example, in order to separate and purify the present peptide from rice protein, sake lees obtained when sake is brewed from rice by the sake brewing method using a liquefied liquid (Imayasu et al., Nohka, 63, 971 (1989)) are used. It is used as a protein source, which is decomposed with a proteolytic enzyme and concentrated to use prolyl endopeptidase inhibitory activity as an index, and various chromatographies such as SP-Sephadex C-25 (Pharmacia) and SP-Toyopearl 650S (Tosoh Corporation). Ion exchange chromatography, manufactured by Sephadex G-15 (manufactured by Pharmacia), gel filtration by Toyopearl HW-40 (manufactured by Tosoh Corporation), CAPCELL PAKC18 (manufactured by Shiseido), Cosmosil C18 (manufactured by Nacalai Tesque).
It can be carried out by reverse phase chromatography or the like.

The peptide thus obtained has excellent PEP inhibitory activity and is derived from natural products and is safe, as is apparent from the examples described later. In a 10-day acute toxicity test using, no death was observed even with oral administration of 1000 mg / kg, and high safety was confirmed. Therefore, the present peptide is extremely useful as a PEP inhibitor and a preventive or therapeutic agent for human dementia. In the present invention, in addition to the above-mentioned peptide, the above-mentioned peptide is used as a basic skeleton, and any amino acid is removed from the N-terminal and / or C-terminal of the peptide and / or substituted with another substance such as another amino acid. The peptide derivative can be used in the same manner as the above peptide.

For the above purpose, the present peptide may be administered parenterally or orally, but it can be formulated into a form suitable for the method of administration. The injection form may be obtained by aseptically dissolving the present peptide together with pharmaceutical auxiliaries (pH adjuster, isotonic agent, preservative, etc.). Oral dosage forms may be in the form of drugs (tablets, capsules, granules, etc.) together with pharmaceutical auxiliaries. In addition, it can be used as an inhalant or an external preparation. In addition, since this peptide contains only natural amino acids, it has extremely high stability and can be taken orally continuously, so it can be included in existing foods to prevent or treat dementia. It may be eaten as a sex food, food for specified health use, nutritional supplement, or health food.

To apply the present peptide to humans,
It is preferably administered intravenously, intramuscularly or orally. The pharmaceutically effective dose of the present peptide varies depending on the age of the patient and the condition of the individual. Usually, in the case of intravenous administration, the present peptide is used in an amount of 0.01 to 1000 m / day per human.
g / kg, intramuscularly, 0.01 to 1000 mg / kg of the present peptide per human per day, and oral administration of 0.5 to 0.5 mg of the present peptide per human per day.
~ 2000 mg / kg, preferably 1-1000 mg / kg
Administer kg.

[0018]

Example 1 Measurement of prolyl endopeptidase inhibitory activity and purification of inhibitory peptides were performed as follows.

1) Method of measuring PEP inhibitory activity In accordance with the method shown in Table 1 below, the inhibitory activity of PEP was measured.

[0020]

[Table 1]

That is, a PEP (derived from a genus Flavobacterium) solution, a buffer solution and a sample were mixed (30 ° C.,
5 minutes). Then, a synthetic substrate dissolved in dioxane (Z-
Gly-Pro-pNA, Z-Gly-Pro-ZN
Nap and / or Z-Gly-Pro-MCA) were added and reacted at 30 ° C. for 10 minutes. After adding hydrochloric acid, the absorbance was measured at OD 410 nm.

2) Purification of PEP-inhibiting peptide 100 g of sake lees (water content 36%) when sake is brewed from rice by a brewing method using a liquefied solution are suspended in 2 L of water,
40 mg of pepsin (1: 60,000, manufactured by Sigma)
In addition, the mixture was reacted at 37 ° C. and pH 1.5 for 1 hour, neutralized, and then heated in a boiling water bath for 10 minutes to stop the reaction.
A lysate was obtained by centrifugation at 5,000 rpm for 10 minutes and freeze-dried to obtain 5.8 g of a decomposed product. Next, 0.1% T
A liquefied lees pepsin degradation product (lyophilized product) dissolved in the same buffer was adsorbed to Chromatolex ODS (DM1020T) manufactured by Fuji Silysia equilibrated with 20% acetonitrile containing FA. Then 60% acetonitrile
Was eluted.

The adsorbed fraction was subjected to Prep Nova-Pak
TFA, acetonitrile (A: 10% CH ₃ CN) by HRC18 (6 μm 60 A 25 × 100 mm × 2)
in 0.1% TFA, B: 60% CH ₃ CN in
B. 0.1% TFA 5 ml / min conc 20
Fractionation was performed at -50% / 20 min). Next, the active fraction was further subjected to Shodex Asahipak GS 320
Fractionation was performed using 2F × 21.5 × 300 mm. (A: wa
ter, B: CH ₃ CN ₅ ml / min con
c. 10%)

Subsequently, μBondasphere 5 μm
Purification proceeded with C4, 100A 19 x 150 mm (Waters). (A: 10% CH ₃ CN in
0.1% TFA, B: 60% CH ₃ CN in 0.1
% TFA 5 ml / min, B.I. conc. 40-70
% / 20min) Then, Capsellpak C18 5 μm, AG
120A 15 × 250 mm (Shiseido), alkaline (A: 5% CH ₃ CN in 10
mM (NH ₄ ) ₂ CO ₃ , B: 35% CH ₃ CN in 1
0 mM (NH ₄ ) ₂ CO ₃ 3 ml / min. con
c. Reversed phase HPLC at 20-80% / 20 min)
Was used for purification. Finally, the previously active fraction was
ndasphere 5 μm C4, 100A 19 × 1
The fraction was again fractionated by 50 mm (Waters) (A: 10
% CH ₃ CN in 0.02% TFA, B: 60% C
H ₃ CN in 0.02% TFA 5 ml / min,
B. conc. 60-70% / 20 min), the inhibitor peak was isolated.

Next, the amino acid sequence of the inhibitory peptide was performed. As a result, the sequence of the inhibitory peptide was as follows. Leu-Leu-Ser-Pro-Phe-Trp-A
sn-Ile-Asn-Ala

[0026]

Example 2 Synthesis of a prolyl endopeptidase inhibitory peptide was performed as follows.

A peptide obtained by removing 1 or 2 residues of Leu from the above peptide and the N-terminal of the peptide was synthesized by a peptide synthesizer (Pepticoupler 2200, manufactured by Vega) and purified by HPLC. Prolyl endopeptidase inhibitory activity was measured. The inhibition rate was calculated by the following equation, and the obtained results are shown in Table 2 below. Inhibition rate = (AB) × 100 / A A = 410 nm absorption value without inhibitor B = 410 nm absorption value with inhibitor

[0028]

[Table 2]

[0029]

According to the present invention, an excellent PEP inhibitor can be obtained. This inhibitor is not only excellent in PEP inhibitory ability but also derived from natural products and extremely safe, so that it can be administered for a long period of time, especially for prevention and treatment of dementia. Useful.

Claims (3)

[Claims] 1. A novel peptide having the following sequence. Leu-Leu-Ser-Pro-Phe-Trp-A
sn-Ile-Asn-Ala 2. Leu-Leu-Ser-Pro-Ph
A prolyl endopeptidase inhibitor comprising a peptide represented by e-Trp-Asn-Ile-Asn-Ala as an active ingredient. 3. Leu-Leu-Ser-Pro-Ph
Using e-Trp-Asn-Ile-Asn-Ala as a basic skeleton, a peptide derivative obtained by removing any amino acid from the N-terminal and / or C-terminal of this peptide or substituting it with another substance is used as an active ingredient. Prolyl endopeptidase inhibitor.