JP3727383B2 - Prolyl endopeptidase inhibitor - Google Patents

Description

【０００６】
しかし、これらは合成品のため安全性に問題があり、また構造が類似であるため既に構造活性相関が明らかとなっているのが現状である（ＡＢＣ、５５、３７−４３、（１９９１））。このような現状を打破するために、安全でしかも合成阻害剤にないユニークな構造を持った阻害物質が求められている。
【０００７】
【発明が解決しようとする課題】
本発明は、このような業界のニーズに応えるためになされたものであって、特に安全性の高いすぐれたＰＥＰ阻害剤を開発する目的でなされたものである。
【０００８】
【課題を解決するための手段】
本発明は、上記目的を達成するためになされたものであって、本発明者らは、天然物質中に副作用のないプロリルエンドペプチダーゼ阻害物質を鋭意検索した結果、米蛋白中に本阻害物質の存在を認め、該物質がＬｅｕ−Ｌｅｕ−Ｓｅｒ−Ｐｒｏ−Ｐｈｅ−Ｔｒｐ−Ａｓｎ−Ｉｌｅ−Ａｓｎ−Ａｌａという構造のペプチドであることを見いだし本発明を完成した。
【０００９】
本発明のペプチドは、米蛋白質から得られるが、米蛋白質のほか、米蛋白質含有物質からも得ることができる。米蛋白質含有物質としては、玄米、白米、糠、また、米を原料とする醸造食品（清酒、味りん、味噌、醤油など）のほか、その副産物（糠、粕など）からも得ることができる。
【００１０】
本発明のペプチドは、米蛋白質（含有物質）を分解することによって製造し、酵素分解、化学的分解、物理的分解等既知の蛋白分解法が適宜使用される。酵素分解法はマイルドな条件で行われるので生成ペプチドの変性が低い等の利点があり、ペプシン、トリプシン、キモトリプシン、フイシンといった動植物起源の蛋白質分解酵素のほか、微生物起源の蛋白質分解酵素を用いて処理すればよい。
【００１１】
この場合、清酒等発酵が充分に行われたものを原料とする場合には、蛋白質分解酵素で処理する必要はなく、必要あれば濃縮した後、直ちに抽出工程に入ればよい。また、未処理の米蛋白質のように蛋白質分解が行われていないものを原料としたり、粕や糠といった発酵副産物であって分解が充分に行われていないものを原料とする場合には、酵素処理することが好適である。
【００１２】
本発明のペプチドは、米だけからでなく、その他の穀類、肉類、その他の天然物やその分解物から単離精製することや、加水分解酵素の逆反応を利用したペプチド合成法、遺伝子工学的手法、有機化学的手法でのペプチド合成法により製造することができる。
【００１３】
天然物中からの分離精製は、有姿のままか必要ならば酸、アルカリ、または酵素により分解したものを、必要ならば抽出、濃縮したのち種々の吸着剤に対する吸着親和性の差、種々の溶剤に対する溶解性の差、分子篩効果による溶出速度の差などの通常分離精製に用いられる方法や、それらを適宜組み合わせておこなえばよい。
【００１４】
例えば、米蛋白質から本ペプチドを分離精製するには、液化液による清酒醸造法（今安聰ら：農化，６３，９７１（１９８９））により米から清酒を醸造したときの酒粕を蛋白源とし、それを蛋白分解酵素で分解、濃縮しプロリルエンドペプチダーゼ阻害活性を指標とし、種々のクロマトグラフィー、例えば、ＳＰ−セファデックスＣ−２５（ファルマシア社製）、ＳＰ−トヨパール６５０Ｓ（東ソー社製）によるイオン交換クロマトグラフィー、セファデックスＧ−１５（ファルマシア社製）、トヨパールＨＷ−４０（東ソー社製）によるゲル濾過、ＣＡＰＣＥＬＬ ＰＡＫＣ１８（資生堂製）、ＣｏｓｍｏｓｉｌＣ１８（ナカライテスク社製）などによる逆相クロマトグラフィーなどにより実施することができる。
【００１５】
このようにして得られたペプチドは、後記する実施例からも明らかなように、卓越したＰＥＰ阻害能を有し、しかも天然物由来であって安全であり、現にウィスター系マウス１０匹を用いた１０日間の急性毒性試験においても、１０００ｍｇ／ｋｇの経口投与でも死亡例は認められず、高い安全性が確認された。
したがって、本ペプチドは、ＰＥＰ阻害剤、ヒトの痴呆症の予防、治療剤としてきわめて有用である。
また本発明においては、上記ペプチドのほか、上記ペプチドを基本骨格とし、このペプチドのＮ末端及び／又はＣ末端から任意のアミノ酸を除いたり、及び／又は、別のアミノ酸等他の物質に置換してなるペプチド誘導体も、上記ペプチドと同様に使用することができる。
【００１６】
上記目的のために、本ペプチドは非経口的又は経口的に投与すればよいが、それらの投与方法に適した形態に製剤することができる。注射剤としての形態は本ペプチドを製薬補助剤（ｐＨ調節剤、等張剤、保存剤など）と共に無菌的に溶解すればよい。経口投与剤は製薬補助剤と共に薬剤の形態（錠剤、カプセル、顆粒剤など）をとれば良い。その他、吸入剤、外用剤としての利用も可能である。また本ペプチドは天然型のアミノ酸のみを含むので安定性が極めて高く、継続的に経口摂取可能であることから、既存の食品に含有させて痴呆症の予防、または治療の機能を持たせた機能性食品、特定保健用食品、栄養剤、または健康食品として食してもよい。
【００１７】
本ペプチドをヒトに対して適用するには、静脈、筋肉、又は経口投与するのが好ましい。本ペプチドの薬学的に有効な投与量は、患者の年令および個人個人の症状等によって異なるが、通常、静脈投与の場合はヒト１人当り１日に本ペプチドを０．０１〜１０００ｍｇ／ｋｇ投与し、筋肉投与の場合はヒト１人当り１日に本ペプチドを０．０１〜１０００ｍｇ／ｋｇ投与し、経口投与の場合はヒト１人当り１日に本ペプチドを０．５〜２０００ｍｇ／ｋｇ、望ましくは１〜１０００ｍｇ／ｋｇ投与する。
【００１８】
【実施例１】
プロリルエンドペプチダーゼ阻害活性の測定と阻害ペプチドの精製を次のようにして行った。
【００１９】
１）ＰＥＰ阻害活性測定法
下記表１の手法にしたがい、ＰＥＰの阻害活性を測定した。
【００２０】
【表１】

【００２１】
すなわち、ＰＥＰ（フラボバクテリウム属菌由来）溶液、緩衝液、サンプルを混合した（３０℃、５分間）。次いでジオキサンにとかした合成基質（Ｚ−Ｇｌｙ−Ｐｒｏ−ｐＮＡ、Ｚ−Ｇｌｙ−Ｐｒｏ−Ｚ−ＮＮａｐ、及び／又はＺ−Ｇｌｙ−Ｐｒｏ−ＭＣＡ）を加えて、３０℃で１０分間反応させた。そして塩酸を加えた後、ＯＤ４１０ｎｍで吸光度を測定した。
【００２２】
２）ＰＥＰ阻害ペプチドの精製
液化液による清酒醸造法により米から清酒を醸造したときの酒粕（水分３６％）１００ｇを２Ｌの水に懸濁し、ペプシン（１：６０，０００、ジグマ社製）を４０ｍｇ加え、３７℃、ｐＨ１．５で１時間反応させ、中和したのち、沸騰水浴中で１０分間加熱し、反応を停止した。５０００回転１０分間の遠心分離により溶解物を得て凍結乾燥により分解物５．８ｇを得た。次に、０．１％ＴＦＡ含有２０％アセトニトリルで平衡化したフジシリシア製クロマトレックスＯＤＳ（ＤＭ１０２０Ｔ）に、同様のバッファーに溶解した液化粕ペプシン分解物（凍結乾燥品）を吸着させた。その後、アセトニトリル６０％で溶出させた。
【００２３】
吸着画分をＰｒｅｐ Ｎｏｖａ−Ｐａｋ ＨＲＣ１８（６μｍ６０Ａ２５×１００ｍｍ×２）によりＴＦＡ、アセトニトリル系（Ａ：１０％ＣＨ₃ＣＮ ｉｎ０．１％ＴＦＡ、Ｂ：６０％ＣＨ₃ＣＮ ｉｎ ０．１％ＴＦＡ ５ｍｌ／ｍｉｎ Ｂ．ｃｏｎｃ ２０−５０％／２０ｍｉｎ）で分画した。
次に、活性画分を更にＳｈｏｄｅｘ Ａｓａｈｉｐａｋ ＧＳ ３２０ ２Ｆ２１．５×３００ｍｍにより分画した。（Ａ：ｗａｔｅｒ、Ｂ：ＣＨ₃ＣＮ ５ｍｌ／ｍｉｎ Ｂ．ｃｏｎｃ．１０％）
【００２４】
続いてμＢｏｎｄａｓｐｈｅｒｅ ５μｍＣ４，１００Ａ １９×１５０ｍｍ（Ｗａｔｅｒｓ）により精製を進めた。（Ａ：１０％ＣＨ₃ＣＮ ｉｎ ０．１％ＴＦＡ、Ｂ：６０％ＣＨ₃ＣＮ ｉｎ ０．１％ＴＦＡ ５ｍｌ／ｍｉｎ，Ｂ．ｃｏｎｃ．４０−７０％／２０ｍｉｎ）
ついで、Ｃａｐｓｅｌｌｐａｋ Ｃ１８ ５μｍ，ＡＧ １２０Ａ １５×２５０ｍｍ（Ｓｈｉｓｅｉｄｏ）によって、アルカリ性（Ａ：５％ＣＨ₃ＣＮ ｉｎ １０ｍＭ（ＮＨ₄）₂ＣＯ₃，Ｂ：３５％ＣＨ₃ＣＮ ｉｎ １０ｍＭ（ＮＨ₄）₂ＣＯ₃ ３ｍｌ／ｍｉｎ，Ｂ．ｃｏｎｃ．２０−８０％／２０ｍｉｎ）における逆相ＨＰＬＣにより精製をおこなった。
最後に先の活性画分をμＢｏｎｄａｓｐｈｅｒｅ ５μｍＣ４，１００Ａ １９×１５０ｍｍ（Ｗａｔｅｒｓ）により再度分画し（Ａ：１０％ＣＨ₃ＣＮ ｉｎ ０．０２％ＴＦＡ、Ｂ：６０％ＣＨ₃ＣＮ ｉｎ ０．０２％ＴＦＡ ５ｍｌ／ｍｉｎ，Ｂ．ｃｏｎｃ．６０−７０％／２０ｍｉｎ）、阻害物質のピークを単離した。
【００２５】
次に、阻害ペプチドのアミノ酸シーケンスをおこなった。その結果阻害ペプチドの配列は以下のようであった。
Ｌｅｕ−Ｌｅｕ−Ｓｅｒ−Ｐｒｏ−Ｐｈｅ−Ｔｒｐ−Ａｓｎ−Ｉｌｅ−Ａｓｎ−Ａｌａ
【００２６】
【実施例２】
プロリルエンドペプチダーゼ阻害ペプチドの合成を次のようにして行った。
【００２７】
上記のペプチドとそのペプチドのＮ末から、Ｌｅｕを１又は２残基除いたペプチドをペプチド合成機（ベガ社製、ペプチカプラー２２００）により合成し、ＨＰＬＣにより精製した。プロリルエンドペプチダーゼ阻害活性を測定した。阻害率は次の式により算出し、得られた結果は、下記表２に示した。
阻害率＝（Ａ−Ｂ）×１００／Ａ
Ａ＝阻害剤を含まない場合の４１０ｎｍ吸収値
Ｂ＝阻害剤を含む場合の４１０ｎｍ吸収値
【００２８】
【表２】

【００２９】
【発明の効果】
本発明によって、すぐれたＰＥＰ阻害剤が得られる。本阻害剤は、ＰＥＰ阻害能にすぐれているだけでなく、天然物由来であってきわめて安全性が高いため、長期間に亘って投与することが可能であり、特に痴呆症の予防、治療に有用である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a prolyl endopeptidase inhibitor containing a peptide derived from rice protein and / or a derivative thereof as an active ingredient.
[0002]
[Prior art]
The importance of geriatric medicine is greatly highlighted for today's aging society. Among them, prevention and treatment of dementia is a big problem not only for the person but also for the family and society. Representative examples of dementia include cerebrovascular dementia and Alzheimer's dementia, and the cause of the latter has not been fully elucidated compared to the former. Alzheimer-type dementia is also referred to as amnesia, and other than the loss of memory for a short period of time, there are not many obstacles in other motor skills, which creates a heavy burden on nursing care and is a problem. It is an urgent need. As described above, there are still many unclear points about the background and the onset mechanism, but the development of therapeutic drugs is being actively carried out, and there are some that have progressed to clinical trials.
[0003]
Prolyl endopeptidase (hereinafter also referred to as PEP) (EC 3.4.21.26) is a serine protease that has specificity for proline and cleaves peptides on its carboxyl side, and it is found in the brain, testis, and liver. Is known to have high activity (protein nucleic acid enzyme, 25, 513-523, (1980), protein nucleic acid enzyme, 29, 127-133, (1984)). PEP is known to degrade THR (thyroid stimulating hormone) and vasopressin, which are thought to be related to neurotransmitter substance P and memory. Vasopressin is a peptide hormone that plays a role in reabsorption of water in the kidney, but it is also involved in the learning and memory processes in the brain, and there is a fact that amnesia progresses when degradation and inactivation of this hormone occurs. . Moreover, it is known that the amount of vasopressin in patients with dementia is less than that of normal persons (BIOINDUSTRI, 4, 788-796, (1987)).
[0004]
Degradation of vasopressin by PEP is shown in Chemical Formula 1 below. In healthy people, PEP in the brain works normally, but if the regulation mechanism is lost for some reason, vasopressin is degraded more than necessary, and memory retention appears injured. Therefore, research aimed at inhibiting the activity of this enzyme and treating dementia has been made. For example, prolylprolinal derivatives (protein nucleic acid enzyme, 29, 127, (1984)), N-acylpyrrolidine derivatives (Japanese Patent Laid-Open Nos. 61-37764, 61-183297, 61-238775, and JP-A-61-238775). Sho 61-238799, JP-A 62-114957) and the like have been screened.
[0005]
[Chemical 1]

[0006]
However, since these are synthetic products, there are problems in safety, and since the structures are similar, the structure-activity relationship is already clear (ABC, 55, 37-43, (1991)). . In order to overcome this situation, there is a need for an inhibitor having a unique structure that is safe and not found in synthetic inhibitors.
[0007]
[Problems to be solved by the invention]
The present invention has been made to meet the needs of the industry, and has been made for the purpose of developing an excellent PEP inhibitor with particularly high safety.
[0008]
[Means for Solving the Problems]
The present invention has been made to achieve the above object, and as a result of intensive search for a prolyl endopeptidase inhibitor having no side effects in natural substances, the present inventors have found that the inhibitor is present in rice protein. The present invention was completed by finding that the substance was a peptide having a structure of Leu-Leu-Ser-Pro-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 can be obtained from brown rice, white rice, koji, and brewed foods made from rice (sake, miso, miso, soy sauce, etc.) and by-products (koji, koji, etc.). .
[0010]
The peptide of the present invention is produced by degrading rice protein (containing substance), and known proteolytic methods such as enzymatic degradation, chemical degradation, and physical degradation are appropriately used. Enzymatic degradation is performed under mild conditions and has advantages such as low denaturation of the peptide produced. It is treated with proteolytic enzymes of animal and plant origin such as pepsin, trypsin, chymotrypsin and huisin, and proteolytic enzymes of microbial origin. do it.
[0011]
In this case, in the case of using a material that has been sufficiently fermented with sake or the like as a raw material, it is not necessary to treat with a proteolytic enzyme. In addition, when using unprocessed rice protein such as untreated rice protein as a raw material, or using fermentation by-products such as koji and koji that have not been sufficiently decomposed as raw materials, It is preferable to process.
[0012]
The peptide of the present invention can be isolated and purified from not only rice, but also other cereals, meats, other natural products and their degradation products, peptide synthesis methods utilizing the reverse reaction of hydrolases, genetic engineering It can be produced by a peptide synthesis method using a technique or an organic chemical technique.
[0013]
Separation and purification from natural products can be carried out as it is or after degradation with acid, alkali, or enzyme, if necessary, and if necessary, after extraction and concentration, the difference in adsorption affinity for various adsorbents, What is necessary is just to carry out combining the method normally used for isolation | separation refinement | purification, such as the difference of the solubility with respect to a solvent, the difference of the elution rate by a molecular sieve effect, and those.
[0014]
For example, in order to separate and purify this peptide from rice protein, the sake source is sake lees produced when sake is brewed from rice by a sake brewing method using a liquefied liquid (Imayasu et al .: Noka, 63, 971 (1989)). Then, it is decomposed and concentrated with a proteolytic enzyme, and prolyl endopeptidase inhibitory activity is used as an index, and various chromatographies such as SP-Sephadex C-25 (Pharmacia), SP-Toyopearl 650S (Tosoh) Ion exchange chromatography by Sephadex G-15 (Pharmacia), gel filtration by Toyopearl HW-40 (Tosoh), CAPCELL PAKC18 (Shiseido), Cosmosil C18 (Nacalai Tesque), etc. Etc. can be implemented.
[0015]
The peptide thus obtained has an excellent PEP inhibitory ability, is derived from a natural product, and is safe, as will be apparent from Examples described later, and actually 10 Wistar mice were used. Even in a 10-day acute toxicity test, no death was observed even after oral administration of 1000 mg / kg, and high safety was confirmed.
Therefore, this 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 peptide, the peptide is used as a basic skeleton, and an arbitrary amino acid is removed from the N-terminal and / or C-terminal of the peptide and / or replaced with another substance such as another amino acid. The peptide derivative can be used in the same manner as the above peptide.
[0016]
For this purpose, the peptide may be administered parenterally or orally, but can be formulated in a form suitable for the method of administration. The form of the injection may be aseptically dissolved together with the pharmaceutical adjuvant (pH adjuster, isotonic agent, preservative, etc.). Orally administered drugs may be in the form of drugs (tablets, capsules, granules, etc.) together with pharmaceutical adjuvants. In addition, it can be used as an inhalant and an external preparation. In addition, since this peptide contains only natural amino acids, it is extremely stable and can be taken orally continuously, so it can be used in existing foods to prevent or treat dementia. It may be eaten as a sex food, a food for specified health use, a nutrient, or a health food.
[0017]
In order to apply this peptide to humans, it is preferably administered intravenously, intramuscularly or orally. The pharmaceutically effective dosage of this peptide varies depending on the age of the patient and individual symptoms, etc., but in the case of intravenous administration, the peptide is usually administered in an amount of 0.01 to 1000 mg / kg per person per day. In the case of intramuscular administration, the peptide is administered in an amount of 0.01 to 1000 mg / kg per person per day. In the case of oral administration, the peptide is administered in an amount of 0.5 to 2000 mg / kg per person per day. Desirably, 1 to 1000 mg / kg is administered.
[0018]
[Example 1]
Measurement of prolyl endopeptidase inhibitory activity and purification of the inhibitory peptide were performed as follows.
[0019]
1) Method for measuring PEP inhibitory activity PEP inhibitory activity was measured according to the method shown in Table 1 below.
[0020]
[Table 1]

[0021]
That is, a PEP (flavobacterium genus) solution, a buffer solution, and a sample were mixed (30 ° C., 5 minutes). Subsequently, a synthetic substrate (Z-Gly-Pro-pNA, Z-Gly-Pro-Z-NNap, and / or Z-Gly-Pro-MCA) dissolved in dioxane was added and reacted at 30 ° C. for 10 minutes. And after adding hydrochloric acid, the light absorbency was measured by OD410nm.
[0022]
2) 100 g of sake lees (water 36%) when brewing sake from rice by the sake brewing method using a purified liquefied liquid of PEP inhibitory peptide was suspended in 2 L of water and pepsin (1: 60,000, manufactured by Sigma) was suspended. 40 mg was added, 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 5000 rpm for 10 minutes, and 5.8 g of a degradation product was obtained by lyophilization. Next, a liquefied coconut pepsin degradation product (lyophilized product) dissolved in the same buffer was adsorbed onto Chromalex ODS (DM1020T) manufactured by Fujisilicia equilibrated with 20% acetonitrile containing 0.1% TFA. Thereafter, elution was performed with 60% acetonitrile.
[0023]
TFA by the adsorbed fraction Prep Nova-Pak HRC18 (6μm60A25 × 100mm × 2), acetonitrile system _{(A: 10% CH 3 CN} in0.1% TFA, B: 60% CH 3 CN in 0.1% TFA 5ml / min B. conc 20-50% / 20 min).
Next, the active fraction was further fractionated by Shodex Asahipak GS 320 2F 21.5 × 300 mm. (A: water, B: CH ₃ CN 5 ml / min B. conc. 10%)
[0024]
Subsequently, purification proceeded with μBondasphere 5 μm C4, 100A 19 × 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. conc. 40-70% / 20 min)
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 10 mM (NH ₄ ) ₂ CO _Three 3 ml / min, B.I. conc. Purification was performed by reverse phase HPLC at 20-80% / 20 min).
Finally, the previous active fraction was fractionated again with μBondasphere 5 μm C4, 100A 19 × 150 mm (Waters) (A: 10% CH ₃ CN in 0.02% TFA, B: 60% CH ₃ CN in 0.02% TFA 5 ml / min, B. conc. 60-70% / 20 min), peak of inhibitor was isolated.
[0025]
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-Asn-Ile-Asn-Ala
[0026]
[Example 2]
Prolyl endopeptidase-inhibiting peptide was synthesized as follows.
[0027]
A peptide obtained by removing one or two residues of Leu from the above peptide and the N terminus of the peptide was synthesized with a peptide synthesizer (Pegacoupler 2200, manufactured by Vega) and purified by HPLC. Prolyl endopeptidase inhibitory activity was measured. The inhibition rate was calculated by the following formula, and the obtained results are shown in Table 2 below.
Inhibition rate = (A−B) × 100 / A
A = 410 nm absorption value without inhibitor B = 410 nm absorption value with inhibitor
[Table 2]

[0029]
【The invention's effect】
The present invention provides excellent PEP inhibitors. This inhibitor is not only excellent in PEP inhibitory activity, but also derived from natural products and extremely safe. Therefore, it can be administered over a long period of time, especially for the prevention and treatment of dementia. Useful.

Claims (3)

A novel peptide having the following sequence:
Leu-Leu-Ser-Pro-Phe-Trp-Asn-Ile-Asn-Ala   A prolyl endopeptidase inhibitor comprising a peptide represented by Leu-Leu-Ser-Pro-Phe-Trp-Asn-Ile-Asn-Ala as an active ingredient.   Prolyl endopeptidase inhibition with Leu-Leu-Ser-Pro-Phe-Trp-Asn-Ile-Asn-Ala as the basic skeleton, and a peptide obtained by removing one or two amino acids at the N-terminal of this peptide. Agent.