JP2007091656A - New peptide with opioid activity and new anxiolytic peptide - Google Patents
New peptide with opioid activity and new anxiolytic peptide Download PDFInfo
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- JP2007091656A JP2007091656A JP2005284602A JP2005284602A JP2007091656A JP 2007091656 A JP2007091656 A JP 2007091656A JP 2005284602 A JP2005284602 A JP 2005284602A JP 2005284602 A JP2005284602 A JP 2005284602A JP 2007091656 A JP2007091656 A JP 2007091656A
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Abstract
Description
本発明はＴｙｒ−Ｐｒｏ−Ｐｈｅ−Ｖａｌ−Ｖａｌで表される新規ペプチド、および該ペプチドを有効成分として含有する抗不安剤に関する。更に本発明はＴｙｒ−Ｐｒｏ−Ｐｈｅ−Ｖａｌ−Ｖａｌ−Ａｓｎ−Ａｌａで表される新規ペプチド、および該ペプチドを有効成分として含有する鎮痛剤に関する。 The present invention relates to a novel peptide represented by Tyr-Pro-Phe-Val-Val and an anxiolytic agent containing the peptide as an active ingredient. Furthermore, the present invention relates to a novel peptide represented by Tyr-Pro-Phe-Val-Val-Asn-Ala, and an analgesic containing the peptide as an active ingredient.
オピオイド活性を有するペプチド（いわゆるオピオイドペプチド）であるβ-カゾモルフィン４を、カゼインペプトンから単離したことが報告された。これは食品蛋白質から生理活性ペプチドが派生するという最初の例である（Brandl,V. et al.,Hoppe-Seyler’s Z.Physiol.Chem.,360,1211-1216(1979)）。また本発明者らは人乳β-カゼインの部分的な一次配列を決定し、その中に牛乳β-カゾモルフィンに類似した配列であるＴｙｒ−Ｐｒｏ−Ｐｈｅ−Ｖａｌ−Ｇｌｕ−Ｐｒｏ−Ｉｌｅ−Ｐｒｏを見出した。なおこの配列は、決定された人乳β-カゼインの全一次構造の第５１−５８残基に相当する。そして本発明者らは人乳β-カゼインのＴｙｒ51をＮ末端にもつ種々の長さのペプチドを化学合成し、オピオイド受容体に対するそれらの評価を行なったことを報告している（食品・医薬分野における蛋白質テーラリング、学会出版センター発行、R.E.フィニー、J.R.ウィテーカー編、p101-105）。 It was reported that β-casomorphin 4 which is a peptide having opioid activity (so-called opioid peptide) was isolated from casein peptone. This is the first example in which a bioactive peptide is derived from a food protein (Brandl, V. et al., Hoppe-Seyler's Z. Physiol. Chem., 360, 1211-1216 (1979)). In addition, the present inventors determined a partial primary sequence of human milk β-casein, among which Tyr-Pro-Phe-Val-Glu-Pro-Ile-Pro, which is a sequence similar to bovine milk β-casomorphin, was included. I found it. This sequence corresponds to residues 51-58 of the determined primary structure of human milk β-casein. The present inventors have reported that peptides of various lengths having Tyr 51 of human milk β-casein at the N-terminus were chemically synthesized and evaluated for opioid receptors (food / pharmaceuticals). Protein tailoring in the field, published by Academic Publishing Center, edited by RE Finney, JR Whitaker, p101-105).
しかしながら、人乳カゼイン由来のペプチドを大量に調製することは困難である。そこで本発明者らは食品蛋白質の一次構造データベース中でヒトβ-カゾモルフィン４（Ｔｙｒ−Ｐｒｏ−Ｐｈｅ−Ｖａｌ：ＹＰＦＶ）配列について検索を行なったところ、該配列がダイズβ-コングリシニンのβ-サブユニット中に存在することを見出した。 However, it is difficult to prepare a large amount of peptides derived from human milk casein. Therefore, the present inventors searched for the human β-casomorphin 4 (Tyr-Pro-Phe-Val: YPFV) sequence in the primary structure database of food proteins, and the sequence was the β-subunit of soybean β-conglycinin. I found that it exists in.
ところでダイズおよびダイズの加工食品はわが国の伝統食品として多く食されており、ダイズに含有される成分の生理活性に関する研究が行なわれている。しかし、ダイズ蛋白質又はその酵素分解物であるペプチドが脳機能に及ぼす影響についての報告はまだ少ない。ダイズおよびその加工食品が多く食されている現状を考えると、ダイズ由来の蛋白質又はペプチドの脳機能調節作用について検討することは非常に意義が大きいと考えられる。そしてそのような検討の一環として近年ダイズ蛋白質を摂取することにより、ヒトの精神的なストレスが緩和されたり、学習や記憶などの脳の高次機能の向上に資することが知られている（畠山英子ら、大豆たん白質研究、Vol.6,147-152(2003)）。 By the way, soybeans and processed foods of soybeans are often eaten as traditional foods in Japan, and studies on physiological activities of components contained in soybeans are being conducted. However, there are still few reports on the effects of soy protein or its enzymatic degradation peptide on brain function. Considering the current situation where soybeans and their processed foods are eaten in large quantities, it is considered to be very significant to examine the brain function-regulating action of soybean-derived proteins or peptides. In recent years, taking soy protein as part of such studies has been known to reduce human mental stress and to improve higher brain functions such as learning and memory. Eiko et al., Soy Protein Research, Vol. 6,147-152 (2003)).
ダイズ由来の新たな生理活性ペプチドを得ることの需要は未だにある。そこでＹＰＦＶ配列を基にしてその誘導体である新規ペプチドを取得し、その生理活性を検討することにより、疼痛の改善やストレスが原因の不安症などに有用な、ダイズ蛋白質由来の新規な機能性ペプチドを得ることが本発明の課題である。 There is still a need to obtain new bioactive peptides derived from soybean. Therefore, by obtaining a novel peptide that is a derivative based on the YPFV sequence and examining its physiological activity, a novel functional peptide derived from soybean protein that is useful for improving pain and anxiety caused by stress, etc. It is an object of the present invention to obtain the above.
そこで上記課題を解決するべく、本発明者らはＹＰＦＶ配列を含むＴｙｒ−Ｐｒｏ−Ｐｈｅ−Ｖａｌ−Ｖａｌ（ＹＰＦＶＶ）からなる新規ペプチド、およびＴｙｒ−Ｐｒｏ−Ｐｈｅ−Ｖａｌ−Ｖａｌ−Ａｓｎ−Ａｌａ（ＹＰＦＶＶＮＡ）からなる新規ペプチドを合成し、それらのペプチドの生理活性についても検討した。その結果本発明者らは、ＹＰＦＶＶＮＡがオピオイド活性を有することと、ＹＰＦＶＶが抗不安活性を有することを見出した。 Therefore, in order to solve the above problems, the present inventors have developed a novel peptide consisting of Tyr-Pro-Phe-Val-Val (YPFVV) containing a YPFV sequence, and Tyr-Pro-Phe-Val-Val-Asn-Ala (YPFVVNA). ) Were synthesized, and the physiological activity of these peptides was also examined. As a result, the present inventors have found that YPFVVNA has opioid activity and that YPFVV has anxiolytic activity.
本発明の新規ペプチドであるＴｙｒ−Ｐｒｏ−Ｐｈｅ−Ｖａｌ−Ｖａｌ（ＹＰＦＶＶ）は抗不安活性を有している。また本発明の新規ペプチドであるＴｙｒ−Ｐｒｏ−Ｐｈｅ−Ｖａｌ−Ｖａｌ−Ａｓｎ−Ａｌａ（ＹＰＦＶＶＮＡ）はオピオイド活性を有している。本発明により得られた生理活性ペプチドは、ダイズのコングリシニンの酵素分解によって得られるので、容易に大量に取得できるという利点を有する。またＹＰＦＶＶの抗不安活性は腹腔内投与のみならず経口投与をした場合でも認められたので、ＹＰＦＶＶは経口投与が可能な抗不安薬となり得るという利点を有する。 Tyr-Pro-Phe-Val-Val (YPFVV), a novel peptide of the present invention, has anxiolytic activity. Moreover, Tyr-Pro-Phe-Val-Val-Asn-Ala (YPFVVNA) which is a novel peptide of the present invention has opioid activity. Since the bioactive peptide obtained by the present invention is obtained by enzymatic degradation of soybean conglycinin, it has the advantage that it can be easily obtained in large quantities. Further, since the anxiolytic activity of YPFVV was observed not only when administered intraperitoneally but also when administered orally, YPFVV has the advantage that it can be an anxiolytic drug that can be administered orally.
本発明は、Ｔｙｒ−Ｐｒｏ−Ｐｈｅ−Ｖａｌ−Ｖａｌで表される新規ペプチドを提供するものである。Ｔｙｒ−Ｐｒｏ−Ｐｈｅ−Ｖａｌ−Ｖａｌで表されるペプチドは抗不安活性を有するので、本発明により当該ペプチドを有効成分として含有する抗不安剤が提供される。また当該ペプチドを用いて不安を緩和する方法も本発明の範囲内である。なお下記の実施例で示すように、Ｔｙｒ−Ｐｒｏ−Ｐｈｅ−Ｖａｌ−Ｖａｌで表されるペプチドはオピオイド活性も有していた。 The present invention provides a novel peptide represented by Tyr-Pro-Phe-Val-Val. Since the peptide represented by Tyr-Pro-Phe-Val-Val has anxiolytic activity, the present invention provides an anxiolytic agent containing the peptide as an active ingredient. A method for alleviating anxiety using the peptide is also within the scope of the present invention. In addition, as shown in the following Example, the peptide represented by Tyr-Pro-Phe-Val-Val also had opioid activity.
更に本発明は、Ｔｙｒ−Ｐｒｏ−Ｐｈｅ−Ｖａｌ−Ｖａｌ−Ａｓｎ−Ａｌａで表される新規ペプチドを提供するものである。Ｔｙｒ−Ｐｒｏ−Ｐｈｅ−Ｖａｌ−Ｖａｌ−Ａｓｎ−Ａｌａで表されるペプチドはオピオイド活性を有するので、本発明により当該ペプチドを有効成分として含有する鎮痛剤が提供される。また当該ペプチドを用いて疼痛を緩和する方法も本発明の範囲内である。なお上記でいうＴｙｒはチロシン、Ｐｒｏはプロリン、Ｐｈｅはフェニルアラニン、Ｖａｌはバリン、Ａｓｎはアスパラギン、Ａｌａはアラニンを示す。かかるアミノ酸はいずれもＬ−体である。 Furthermore, this invention provides the novel peptide represented by Tyr-Pro-Phe-Val-Val-Asn-Ala. Since the peptide represented by Tyr-Pro-Phe-Val-Val-Asn-Ala has opioid activity, the present invention provides an analgesic containing the peptide as an active ingredient. Moreover, the method of relieving pain using the said peptide is also within the scope of the present invention. In the above, Tyr is tyrosine, Pro is proline, Phe is phenylalanine, Val is valine, Asn is asparagine, and Ala is alanine. All of these amino acids are L-forms.
上記のペプチドはダイズ由来の蛋白質であるβ-コングリシニンの酵素分解によって、簡便に且つ大量に得ることができる。なおＴｙｒ−Ｐｒｏ−Ｐｈｅ−Ｖａｌ（以下ＹＰＦＶと記載する）からなるβ-カゾモルフィン４は人乳のβ−カゼイン由来のペプチドであるが、人乳カゼインを大量に入手することは困難である。 The above peptides can be obtained easily and in large quantities by enzymatic degradation of β-conglycinin, a protein derived from soybean. Β-casomorphin 4 composed of Tyr-Pro-Phe-Val (hereinafter referred to as YPFV) is a peptide derived from β-casein of human milk, but it is difficult to obtain human milk casein in large quantities.
なおβ-コングリシニンの部分配列であるＶＩＰＡＡＹＰＦＶＶＮＡＴＳＮＬから、膵臓エラスターゼで消化することによりＡＹＰＦＶＶを得ることが可能であり、これを更にロイシンアミノペプチダーゼで消化したところ、本発明のＴｙｒ−Ｐｒｏ−Ｐｈｅ−Ｖａｌ−Ｖａｌ（以下ＹＰＦＶＶと記載する）を得ることができた。そのようにして本発明者らがＹＰＦＶＶを得たところ、その収率は36.5%であった。更にＹＰＦＶＶからＴｙｒ−Ｐｒｏ−Ｐｈｅ−Ｖａｌ−Ｖａｌ−Ａｓｎ−Ａｌａ（以下ＹＰＦＶＶＮＡと記載する）を得ることも可能である。なおダイズペプチドから酵素分解によりＹＰＦＶＶを得る方法は上記において述べたものに限定されるものではない。エラスターゼ以外のエンドペプチダーゼを使用することも、ロイシンアミノペプチダーゼ以外のアミノペプチダーゼを使用することも可能である。 It is possible to obtain AYPFVV by digesting with pancreatic elastase from VIPAAYPFVVNATSNL which is a partial sequence of β-conglycinin, and when this is further digested with leucine aminopeptidase, Tyr-Pro-Phe-Val- Val (hereinafter referred to as YPFVV) could be obtained. Thus, when the present inventors obtained YPFVV, the yield was 36.5%. Further, Tyr-Pro-Phe-Val-Val-Asn-Ala (hereinafter referred to as YPFVVNA) can be obtained from YPFVV. The method for obtaining YPFVV from soybean peptides by enzymatic degradation is not limited to that described above. Endopeptidases other than elastase can be used, and aminopeptidases other than leucine aminopeptidase can be used.
更に分離ダイズ蛋白質を上記と同様に膵臓エラスターゼおよびロイシンアミノペプチダーゼで消化することによりＹＰＦＶＶが生成することを、LC/MS（液体クロマトグラフィー質量分析）により確認した。なお原料中のβ-コングリシニンβ-サブユニットの含量が不明であるために収率の計算はできなかった。 Further, it was confirmed by LC / MS (Liquid Chromatography Mass Spectrometry) that digested soybean protein was digested with pancreatic elastase and leucine aminopeptidase as described above. The yield could not be calculated because the content of β-conglycinin β-subunit in the raw material was unknown.
また本発明のペプチドは、ペプチド合成法で取得することもできる。即ち、ペプチド合成に通常用いられる方法である液相法または固相法で、ペプチド結合の任意の位置で二分される２種のフラグメントの一方に相当する反応性カルボキシル基を有する原料と、他方のフラグメントに相当する反応性アミノ基を有する原料とを、2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate（ＨＢＴＵ）等の活性エステルを用いた方法や、カルボジイミドを用いた方法等を用いて縮合させることができる。生成する縮合物が保護基を有する場合、その保護基を除去することによっても製造し得る。 The peptide of the present invention can also be obtained by peptide synthesis. That is, in a liquid phase method or a solid phase method, which is a method usually used for peptide synthesis, a raw material having a reactive carboxyl group corresponding to one of two kinds of fragments bisected at an arbitrary position of the peptide bond, and the other A method using an active ester such as 2- (1H-Benzotriazole-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) with a raw material having a reactive amino group corresponding to the fragment, or carbodiimide The condensation can be carried out using a method using When the resulting condensate has a protecting group, it can also be produced by removing the protecting group.
この反応工程において反応に関与すべきでない官能基は、保護基により保護される。アミノ基の保護基としては、例えばベンジルオキシカルボニル（Ｂｚ）、ｔ−ブチルオキシカルボニル（Ｂｏｃ），ｐ−ビフェニルイソプロピロオキシカルボニル、９ーフルオレニルメチルオキシカルボニル（Ｆｍｏｃ）等が挙げられる。カルボキシル基の保護剤としては例えばアルキルエステル、ベンジルエステル等を形成し得る基が挙げられるが、固相法の場合は、Ｃ末端のカルボキシル基はクロロトリチル樹脂、クロルメチル樹脂、オキシメチル樹脂、Ｐ−アルコキシベンジルアルコール樹脂等の担体に結合している。縮合反応は、カルボジイミド等の縮合剤の存在下にあるいはＮ−保護アミノ酸活性エステルまたはペプチド活性エステルを用いて実施する。 Functional groups that should not participate in the reaction in this reaction step are protected by protecting groups. Examples of the amino-protecting group include benzyloxycarbonyl (Bz), t-butyloxycarbonyl (Boc), p-biphenylisopropyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc) and the like. Examples of the carboxyl group-protecting agent include groups capable of forming alkyl esters, benzyl esters and the like. In the solid phase method, the C-terminal carboxyl group is a chlorotrityl resin, chloromethyl resin, oxymethyl resin, P- It is bound to a carrier such as an alkoxybenzyl alcohol resin. The condensation reaction is carried out in the presence of a condensing agent such as carbodiimide or using an N-protected amino acid active ester or peptide active ester.
縮合反応終了後、保護基は除去されるが、固相法の場合はさらにペプチドのＣ末端と樹脂との結合を切断する。更に、本発明のペプチドは通常の方法に従い精製される。例えばイオン交換クロマトグラフィー、逆相液体クロマトグラフィー、アフィニティークロマトグラフィー等が挙げられる。合成したペプチドの合成はエドマン分解法でＣ−末端からアミノ酸配列を読み取るプロティンシークエンサー、ＧＣ−ＭＳ等で分析される。 After completion of the condensation reaction, the protecting group is removed, but in the solid phase method, the bond between the C-terminal of the peptide and the resin is further cleaved. Furthermore, the peptides of the present invention are purified according to conventional methods. Examples thereof include ion exchange chromatography, reverse phase liquid chromatography, affinity chromatography and the like. Synthesis of the synthesized peptide is analyzed by a protein sequencer that reads the amino acid sequence from the C-terminal by the Edman degradation method, GC-MS, or the like.
次に医薬品として用いる場合について説明する。本発明のペプチドであるＹＰＦＶＶＮＡはオピオイド活性を有するので、そのペプチドを鎮痛剤として用いることができる。 Next, the case where it uses as a pharmaceutical is demonstrated. Since YPFVVNA, which is a peptide of the present invention, has opioid activity, the peptide can be used as an analgesic.
現在オピオイド活性を有する化合物はいくつも知られているが、代表的な化合物としてはモルヒネやコデインなどのアヘンアルカロイドを挙げることができる。これらの物質は麻薬性鎮痛物質（オピエート）ともいわれ、オピオイド受容体を認識して結合し、中枢神経系を介して強力な鎮痛効果を発揮するために、末梢に作用する解熱性鎮痛薬では抑えられない痛みを抑えることができる。そのためにオピオイド活性を有する化合物の臨床的な意義は大きい。 A number of compounds having opioid activity are currently known, but typical compounds include opium alkaloids such as morphine and codeine. These substances, also called narcotic analgesics (opiates), recognize and bind to opioid receptors and exert powerful analgesic effects via the central nervous system, so they are suppressed by antipyretic analgesics acting on the periphery. Can suppress the pain that can not be. Therefore, the clinical significance of compounds having opioid activity is great.
また脳内にはオピエートを認識する受容体（オピオイド受容体）があることが見出されている。そして、そのオピオイド受容体に対する内因性リガンドとして、エンケファリン、ダイノルフィン、β-エンドルフィンなどのオピオイドペプチドが存在することが知られている。本発明のＹＰＦＶＶＮＡはペプチドであり、脳内麻薬といわれているオピオイドペプチドと同じくペプチド性のオピオイド活性物質が本発明により提供されるので、本発明のペプチドは副作用の少ない鎮痛剤となり得る。 It has also been found that there are receptors (opioid receptors) that recognize opiates in the brain. It is known that opioid peptides such as enkephalin, dynorphin, and β-endorphin exist as endogenous ligands for the opioid receptor. The YPFVVNA of the present invention is a peptide, and since the peptide-like opioid active substance is provided by the present invention in the same manner as an opioid peptide called an intracerebral narcotic, the peptide of the present invention can be an analgesic with few side effects.
またＹＰＦＶＶは抗不安活性を有するので、そのペプチドを抗不安剤として用いることもできる。現在医薬品として市販されている主要な抗不安剤はベンゾジアゼピン系誘導体の物質である。これらのベンゾジアゼピン系誘導体の抗不安剤は安全性が高く、優れた性質を有する薬物であるが、抗痙攣作用、筋弛緩作用、鎮静催眠作用や麻酔増強作用などの薬理作用を同時に有する。そこでベンゾジアゼピン系誘導体以外に、穏やかでかつ安全性が高く、経口で手軽に服用することができる抗不安剤を求める需要は未だに大きい。それを考えると、食品であるダイズに由来し且つ経口投与で有効であるという特徴を有する、本発明により提供される抗不安剤の意義は大きい。 Since YPFVV has anxiolytic activity, the peptide can also be used as an anxiolytic agent. The main anti-anxiety agents currently marketed as pharmaceuticals are benzodiazepine derivatives. These anxiolytic agents of benzodiazepine derivatives are drugs having high safety and excellent properties, but have pharmacological actions such as anticonvulsant action, muscle relaxing action, sedative hypnotic action and anesthetic enhancing action at the same time. Therefore, in addition to benzodiazepine derivatives, there is still a great demand for an anxiolytic agent that is mild and highly safe and can be easily taken orally. In view of that, the anxiolytic agent provided by the present invention, which is characterized by being derived from soybean as a food and effective by oral administration, has great significance.
本発明のペプチドの投与経路は特に限定されるものではなく、経口投与、非経口投与、直腸内投与のいずれを採用することも可能であり、経口的あるいは非経口的に投与することができる。本ペプチドの投与量は化合物の種類、投与方法、投与される者の状態や年齢等により異なるが、１日あたり通常は０．１ｍｇ／ｋｇ〜１０００ｍｇ／ｋｇ、好ましくは１ｍｇ／ｋｇ〜１００ｍｇ／ｋｇである。本発明のペプチドは通常、製剤用担体と混合して調製した製剤の形で投与される。製剤用担体としては、製剤分野において常用され、かつ本発明のペプチドと反応しない物質が用いられる。 The administration route of the peptide of the present invention is not particularly limited, and any of oral administration, parenteral administration, and rectal administration can be adopted, and it can be administered orally or parenterally. The dosage of this peptide varies depending on the type of compound, the administration method, the condition and age of the administered person, etc., but is usually 0.1 mg / kg to 1000 mg / kg, preferably 1 mg / kg to 100 mg / kg per day. It is. The peptide of the present invention is usually administered in the form of a preparation prepared by mixing with a pharmaceutical carrier. As a pharmaceutical carrier, a substance that is commonly used in the pharmaceutical field and does not react with the peptide of the present invention is used.
具体的には、その様な物質の例として乳糖、ブドウ糖、マンニット、デキストリン、シクロデキストリン、デンプン、蔗糖、メタケイ酸アルミン酸マグネシウム、合成ケイ酸アルミニウム、カルボキシメチルセルロースナトリウム、ヒドロキシプロピルデンプン、カルボキシメチルセルロースカルシウム、イオン交換樹脂、メチルセルロース、ゼラチン、アラビアゴム、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、ポリビニルピロリドン、ポリビニルアルコール、軽質無水ケイ酸、ステアリン酸マグネシウム、タルク、トラガント、ベントナイト、ビーガム、酸化チタン、ソルビタン脂肪酸エステル、ラウリル硫酸ナトリウム、グリセリン、脂肪酸グリセリンエステル、精製ラノリン、グリセロゼラチン、ポリソルベート、マクロゴール、植物油、ロウ、流動パラフィン、白色ワセリン、フルオロカーボン、非イオン性界面活性剤、プロピレングルコール、水等が挙げられる。 Specifically, examples of such substances are lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminate metasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, carboxymethylcellulose calcium. , Ion exchange resin, methylcellulose, gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, bee gum, titanium oxide, sorbitan fatty acid ester, Sodium lauryl sulfate, glycerin, fatty acid glycerin ester, purified lanolin, glycerogelatin, polyso Bate, macrogol, vegetable oils, waxes, liquid paraffin, white petrolatum, fluorocarbons, nonionic surfactants, propylene glycol, water and the like.
剤型としては、錠剤、カプセル剤、顆粒剤、散剤、シロップ剤、懸濁剤、座剤、軟膏、クリーム剤、ゲル剤、貼付剤、吸入剤、注射剤等が挙げられる。これらの製剤は常法に従って調製される。尚、液体製剤にあっては、用時、水又は他の適当な溶媒に溶解または懸濁する形であってもよい。また錠剤、顆粒剤は周知の方法でコーティングしてもよい。注射剤の場合には、本発明のペプチドを水に溶解させて調製されるが、必要に応じて生理食塩水あるいはブドウ糖溶液に溶解させてもよく、また緩衝剤や保存剤を添加してもよい。 Examples of the dosage form include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, injections, and the like. These preparations are prepared according to a conventional method. The liquid preparation may be dissolved or suspended in water or other appropriate solvent at the time of use. Tablets and granules may be coated by a known method. In the case of injection, it is prepared by dissolving the peptide of the present invention in water, but it may be dissolved in physiological saline or glucose solution as necessary, and a buffer or preservative may be added. Good.
これらの製剤は、本発明のペプチドを０．０１％〜１００重量％、好ましくは１〜９０重量％の割合で含有することができる。これらの製剤はまた、治療上価値のある他の成分を含有していてもよい。 These preparations can contain the peptide of the present invention in a proportion of 0.01% to 100% by weight, preferably 1 to 90% by weight. These formulations may also contain other therapeutically valuable ingredients.
経口投与用の固形製剤を製造するには、有効成分と賦形剤成分例えば乳糖、澱粉、結晶セルロース、乳酸カルシウム、無水ケイ酸などと混合して散剤とするか、さらに必要に応じて白糖、ヒドロキシプロピルセルロース、ポリビニルピロリドンなどの結合剤、カルボキシメチルセルロース、カルボキシメチルセルロースカルシウムなどの崩壊剤などを加えて湿式又は乾式造粒して顆粒剤とする。錠剤を製造するには、これらの散剤及び顆粒剤をそのまま或いはステアリン酸マグネシウム、タルクなどの滑沢剤を加えて打錠すればよい。これらの顆粒又は錠剤はヒドロキシプロピルメチルセルロースフタレート、メタクリル酸−メタクリル酸メチルポリマーなどの腸溶剤基剤で被覆して腸溶剤製剤、あるいはエチルセルロース、カルナウバロウ、硬化油などで被覆して持続性製剤とすることもできる。また、カプセル剤を製造するには、散剤又は顆粒剤を硬カプセルに充填するか、有効成分をそのまま或いはグリセリン、ポリエチレングリコール、ゴマ油、オリーブ油などに溶解した後ゼラチン膜で被覆し軟カプセルとすることができる。 In order to produce a solid preparation for oral administration, an active ingredient and excipient components such as lactose, starch, crystalline cellulose, calcium lactate, anhydrous silicic acid and the like are mixed to form a powder, or if necessary, sucrose, Add a binder such as hydroxypropylcellulose and polyvinylpyrrolidone, a disintegrant such as carboxymethylcellulose and carboxymethylcellulose calcium, and wet or dry granulate to form granules. In order to produce tablets, these powders and granules may be tableted as they are or after adding a lubricant such as magnesium stearate or talc. These granules or tablets should be coated with an enteric solvent base such as hydroxypropylmethylcellulose phthalate or methacrylic acid-methyl methacrylate polymer and coated with an enteric solvent preparation, or with ethylcellulose, carnauba wax, hydrogenated oil, etc. You can also. In order to produce capsules, powders or granules are filled into hard capsules, or active ingredients are dissolved as they are or dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, etc., and then coated with a gelatin film to form soft capsules. Can do.
経口投与用の液状製剤を製造するには、有効成分と白糖、ソルビトール、グリセリンなどの甘味剤とを水に溶解して透明なシロップ剤、更に精油、エタノールなどを加えてエリキシル剤とするか、アラビアゴム、トラガント、ポリソルベート８０、カルボキシメチルセルロースナトリウムなどを加えて乳剤又は懸濁剤としてもよい。これらの液状製剤には所望により矯味剤、着色剤、保存剤などを加えてもよい。 In order to produce a liquid preparation for oral administration, an active ingredient and a sweetener such as sucrose, sorbitol, and glycerin are dissolved in water to add a transparent syrup, further essential oil, ethanol, etc. to make an elixir, Gum arabic, tragacanth, polysorbate 80, sodium carboxymethyl cellulose and the like may be added to form an emulsion or suspension. These liquid preparations may contain a flavoring agent, a coloring agent, a preservative and the like as desired.
注射剤を製造するには、有効成分を必要に応じて塩酸、水酸化ナトリウム、乳糖、乳酸、ナトリウム、リン酸一水素ナトリウム、リン酸二水素ナトリウムなどのｐＨ調整剤、塩化ナトリウム、ぶどう糖などの等張化剤と共に注射用蒸留水に溶解し、無菌濾過してアンプルに充填するか、更にマンニトール、デキストリン、シクロデキストリン、ゼラチンなどを加えて真空凍結乾燥し、用事溶解型の注射剤としてもよい。また、有効成分にレチシン、ポリソルベート８０、ポリオキシエチレン硬化ヒマシ油などを加えて水中で乳化せしめ注射剤用乳剤とすることもできる。 In order to produce injections, active ingredients such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate, sodium dihydrogen phosphate, pH adjusters, sodium chloride, glucose etc. It can be dissolved in distilled water for injection together with an isotonic agent, filtered aseptically and filled into ampoules, or further lyophilized by adding mannitol, dextrin, cyclodextrin, gelatin, etc. . In addition, reticin, polysorbate 80, polyoxyethylene hydrogenated castor oil and the like may be added to the active ingredient and emulsified in water to give an emulsion for injection.
直腸投与剤を製造するには、有効成分をカカオ脂、脂肪酸のトリ、ジ及びモノグリセリド、ポリエチレングリコールなどの座剤用基材と共に加湿して溶解し型に流し込んで冷却するか、有効成分をポリエチレングリコール、大豆油などに溶解した後、ゼラチン膜で被覆すればよい。 To produce a rectal dosage form, the active ingredient is moistened with a suppository base material such as cacao butter, fatty acid tri, di- and monoglycerides, polyethylene glycol, etc., dissolved, poured into a mold and cooled, or the active ingredient is made of polyethylene. What is necessary is just to coat | cover with a gelatin film | membrane after melt | dissolving in glycol, soybean oil, etc.
皮膚用外用剤を製造するには、有効成分を白色ワセリン、ミツロウ、流動パラフィン、ポリエチレングリコールなどに加えて必要ならば加湿して練合し軟膏剤とするか、ロジン、アクリル酸アルキルエステル重合体などの粘着剤と練合した後ポリアルキルなどの不織布に展延してテープ剤とする。 In order to produce an external preparation for skin, the active ingredient is added to white petrolatum, beeswax, liquid paraffin, polyethylene glycol, etc., and if necessary, moistened and kneaded to make an ointment, or rosin, alkyl acrylate polymer After being kneaded with an adhesive such as polyalkyl, it is spread on a non-woven fabric such as polyalkyl to obtain a tape.
次に実例を挙げて本発明を更に具体的に説明する。しかし下記の実施例は本発明の範囲を限定するものではない。 Next, the present invention will be described more specifically with examples. However, the following examples do not limit the scope of the present invention.
まずβ−コングリシニン中に含まれているＹＰＦＶ、ＹＰＦＶＶ、ＹＰＦＶＶＮＡをペプチド合成により取得した。そしてそれらのオピオイド活性をモルモット回腸アッセイの系で測定した。 First, YPFV, YPFVV, and YPFVVNA contained in β-conglycinin were obtained by peptide synthesis. Their opioid activity was measured with a guinea pig ileal assay system.
（モルモット回腸アッセイ）
モルモット回腸縦走筋を剥離すると、Auerbach神経叢が付随した形で得られる。このようにして得られた縦走筋神経叢標本はオピオイドに対して丸ごとの回腸よりも高い感度を示す。この標本を栄養液中に吊るして電気刺激を与えると、神経終末からアセチルコリンが放出され、平滑筋は収縮する。オピオイドアゴニストは電気刺激によるアセチルコリンの放出を阻害して収縮の抑制を抑制するので、かかるモルモット回腸の収縮を検出することによってオピオイド活性を評価するのが、モルモット回腸アッセイの系である。
(Guinea pig ileal assay)
When the longitudinal muscle of the guinea pig ileum is removed, the Auerbach plexus is obtained. The longitudinal muscle plexus specimen thus obtained is more sensitive to opioids than the whole ileum. When this specimen is suspended in a nutrient solution and applied with electrical stimulation, acetylcholine is released from nerve endings and smooth muscle contracts. Since opioid agonists inhibit the suppression of contraction by inhibiting the release of acetylcholine by electrical stimulation, it is the guinea pig ileal assay system that evaluates opioid activity by detecting such contraction of the guinea pig ileum.
モルモット回腸アッセイにおいては、まずモルモットを屠殺してその回腸を摘出し、縦走筋標本の一方を支持棒に、もう一方についてはリングを介してトランスジューサーに接続する。そしてその標本を、栄養液を満たしたマグヌス管にセットする。標本に電気刺激を与えて収縮させ、その電気刺激による収縮が安定したらオピオイド活性を評価する対象である薬物を添加し、平滑筋の収縮による張力の変化からその薬物のオピオイド活性を検出する。なお張力の変化はトランスジューサーによって電気信号に変換されてレコーダーに記録される。モルモット回腸アッセイは本技術分野で繁用されている方法であり、その詳細は例えば川岸舜朗編著、学会出版センター、生物化学実験法３８「食品中の生体機能調節物質研究法」p99-116などに記載されている。 In the guinea pig ileal assay, the guinea pig is first sacrificed and the ileum is removed and one of the longitudinal muscle specimens is connected to a support rod and the other is connected to the transducer via a ring. The specimen is set in a Magnus tube filled with nutrient solution. The specimen is contracted by applying electrical stimulation, and when the contraction by the electrical stimulation is stabilized, a drug which is an object for evaluating opioid activity is added, and the opioid activity of the drug is detected from a change in tension due to contraction of smooth muscle. The change in tension is converted into an electrical signal by a transducer and recorded on a recorder. The guinea pig ileal assay is a method that is frequently used in this technical field. For details, see, for example, edited by Goro Kawagishi, Academic Publishing Center, Biochemical Experimental Method 38 “Research Methods for Biofunctional Modulators in Foods” It is described in.
（結果）
上記のモルモット回腸アッセイの系を用いて以下の５つのペプチドについてオピオイド活性を評価し、それらのペプチドのオピオイド活性についてIC50値を求めた。その結果を表１に示す。
（１）ＹＰＦＶ（Ｔｙｒ−Ｐｒｏ−Ｐｈｅ−Ｖａｌ）
（２）ＹＰＦＶＶ（Ｔｙｒ−Ｐｒｏ−Ｐｈｅ−Ｖａｌ−Ｖａｌ）
（３）ＹＰＦＶＶＮＡ（Ｔｙｒ−Ｐｒｏ−Ｐｈｅ−Ｖａｌ−Ｖａｌ−Ａｓｎ−Ａｌａ）
（４）人乳β-カゾモルフィン５（Ｔｙｒ−Ｐｒｏ−Ｐｈｅ−Ｖａｌ−Ｇｌｕ）
（５）人乳β-カゾモルフィン７（Ｔｙｒ−Ｐｒｏ−Ｐｈｅ−Ｖａｌ−Ｇｌｕ−Ｐｒｏ−Ｉｌｅ）
(result)
Using the above guinea pig ileal assay system, the following five peptides were evaluated for opioid activity, and IC 50 values were determined for the opioid activity of these peptides. The results are shown in Table 1.
(1) YPFV (Tyr-Pro-Phe-Val)
(2) YPFVV (Tyr-Pro-Phe-Val-Val)
(3) YPFVVNA (Tyr-Pro-Phe-Val-Val-Asn-Ala)
(4) Human milk β-casomorphin 5 (Tyr-Pro-Phe-Val-Glu)
(5) Human milk β-casomorphin 7 (Tyr-Pro-Phe-Val-Glu-Pro-Ile)
表１に示されるように、ＹＰＦＶのIC50は20μM、ＹＰＦＶＶのIC50は8μM、ＹＰＦＶＶＮＡのIC50は3μM、人乳β-カゾモルフィン５のIC50は14μM、人乳β-カゾモルフィン７のIC50は25μMであった。よってＹＰＦＶＶのオピオイド活性はＹＰＦＶ（人乳β-カゾモルフィン４）の2.5倍であり、また人乳β-カゾモルフィン５の1.8倍であった。そしてＹＰＦＶＶＮＡのオピオイド活性は更に強かった。この結果により、本発明のペプチドであるＹＰＦＶＶとＹＰＦＶＶＮＡの両者は、いずれも強いオピオイド活性を有することが示された。 As shown in Table 1, IC 50 of YPFV is 20 [mu] M, IC 50 of YPFVV is 8 [mu] M, IC 50 of YPFVVNA is 3 [mu] M, IC 50 of human milk β- casomorphins 5 14 [mu] M, IC 50 of human milk β- casomorphin 7 Was 25 μM. Therefore, the opioid activity of YPFVV was 2.5 times that of YPFV (human milk β-casomorphin 4) and 1.8 times that of human milk β-casomorphin 5. And the opioid activity of YPFVVNA was even stronger. From these results, it was shown that both YPFVV and YPFVVNA, which are peptides of the present invention, have strong opioid activity.
（高架十字迷路実験）
高架十字迷路（Eleveted plus maze：EPM）は、２つのオープンアーム（24x5x0.5）と２つのクローズアーム（24x5x13）からなり、それらのアームは床から50cm高くなった中央プラットフォームと結合している。高い位置にあるにも関わらず、クローズアームの周りには囲いがあるために、マウスは安全に歩行する事ができる。一方オープンアームの周囲は開放されていて囲いがないために、オープンアームを歩行するマウスは高い位置から転落するという不安感を感じる。そのために、マウスがオープンアームにいる時間が長いほどマウスの不安感は緩和されており、抗不安活性の指標となる。
(Elevated cross maze experiment)
The elevated plus maze (EPM) consists of two open arms (24x5x0.5) and two closed arms (24x5x13), which are connected to a central platform 50cm above the floor. Despite being in a high position, the mouse can safely walk because of the enclosure around the close arm. On the other hand, since the surroundings of the open arm are open and there is no enclosure, the mouse walking on the open arm feels anxiety that it falls from a high position. Therefore, the longer the mouse stays in the open arm, the less the anxiety of the mouse, which becomes an index of anti-anxiety activity.
オープンアームの一つに面している中央プラットフォーム上にマウスを置いて試験を開始した。５分の試験時間の間、オープンアーム内で過ごした累積時間を記録した。オープンアーム内で過ごした時間のパーセンテージを不安の指標として計算した。 The test was initiated by placing the mouse on a central platform facing one of the open arms. The cumulative time spent in the open arm during the 5 minute test time was recorded. The percentage of time spent in the open arm was calculated as an indicator of anxiety.
（統計解析）
高架十字迷路試験で得たデータを、平均とSEMで表した。データを１方向または２方向ANOVAにより解析し、引き続いて多重比較のためのダネット試験を行った。
(Statistical analysis)
Data obtained in the elevated plus maze test were expressed as mean and SEM. Data were analyzed by one-way or two-way ANOVA, followed by Dunnett's test for multiple comparisons.
（結果）
生理食塩水溶水に溶解したＹＰＦＶＶを、マウスを高架十字迷路上に置く前に腹腔内投与（i.p）した（n=12）。そしてＹＰＦＶＶの投与群（3mg/kg、10mg/kg、30mg/kg）と非投与群（0mg/kg）において、オープンアーム内で過ごした時間のパーセンテージを比較した。その結果を図１に示す。図１に示されるように、ＹＰＦＶＶを3mg/kgまたは10mg/kg腹腔内投与投与した群ではオープンアーム内で過ごした時間が有意に（*はp<0.05の有意差、**はp<0.01の有意差を示す）増加した。この結果は、ＹＰＦＶＶが腹腔内投与で抗不安活性を有することを示すものである。
(result)
YPFVV dissolved in saline solution was administered intraperitoneally (ip) before placing the mouse on the elevated plus maze (n = 12). The percentage of time spent in the open arm was compared between the YPFVV administration group (3 mg / kg, 10 mg / kg, 30 mg / kg) and the non-administration group (0 mg / kg). The result is shown in FIG. As shown in FIG. 1, in the group administered YPFVV 3 mg / kg or 10 mg / kg intraperitoneally, the time spent in the open arm was significantly different (* is a significant difference of p <0.05, ** is p <0.01). Increased). This result indicates that YPFVV has anxiolytic activity when administered intraperitoneally.
更にマウスを高架十字迷路上に置く３０分前に、生理食塩水に溶解したＹＰＦＶＶを経口投与（p.o）した（n=12）。そしてオープンアーム内で過ごした時間のパーセンテージを、ＹＰＦＶＶの投与群（10mg/kg、30mg/kgおよび100mg/kg）と非投与群（0mg/kg）で比較した。その結果を図２に示す。図２に示されるように、ＹＰＦＶＶを10mg/kgまたは30mg/kgの用量で経口投与した群では、オープンアーム内で過ごした時間が有意に増加した（***はP<0.001の有意差を示す）。この結果は、ＹＰＦＶＶが経口投与でも抗不安活性を有することを示すものである。 Furthermore, 30 minutes before placing the mouse on the elevated plus maze, YPFVV dissolved in physiological saline was orally administered (p.o) (n = 12). The percentage of time spent in the open arm was compared between the YPFVV administration group (10 mg / kg, 30 mg / kg and 100 mg / kg) and the non-administration group (0 mg / kg). The result is shown in FIG. As shown in FIG. 2, the time spent in the open arm was significantly increased in the group that was orally administered YPFVV at a dose of 10 mg / kg or 30 mg / kg (*** indicates a significant difference of P <0.001). Show). This result indicates that YPFVV has anxiolytic activity even when administered orally.
本発明の新規ペプチドであるＹＰＦＶＶは抗不安活性を有しているので、当該ペプチドを有効成分と含有する抗不安剤を得ることが可能である。ＹＰＦＶＶの抗不安活性は経口投与でも認められたので、ＹＰＦＶＶは経口投与が可能な抗不安薬となり得るという利点を有する。更に本発明の新規ペプチドであるＹＰＦＶＶＮＡはオピオイド活性を有しているので、当該ペプチドを有効成分として含有する鎮痛剤を得ることも可能である。本発明により得られた生理活性ペプチドは、ダイズのコングリシニンの酵素分解によって大量に得ることが可能である。 Since YPFVV, which is a novel peptide of the present invention, has an anxiolytic activity, an anxiolytic agent containing the peptide as an active ingredient can be obtained. Since the anxiolytic activity of YPFVV was also observed after oral administration, YPFVV has the advantage that it can be an anxiolytic drug that can be administered orally. Furthermore, since YPFVVNA, which is a novel peptide of the present invention, has opioid activity, it is also possible to obtain an analgesic containing the peptide as an active ingredient. The physiologically active peptide obtained by the present invention can be obtained in large quantities by enzymatic degradation of soybean conglycinin.
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