JP5058169B2 - Intestinal permeation inhibitor of allergen - Google Patents
Intestinal permeation inhibitor of allergen Download PDFInfo
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- JP5058169B2 JP5058169B2 JP2008531966A JP2008531966A JP5058169B2 JP 5058169 B2 JP5058169 B2 JP 5058169B2 JP 2008531966 A JP2008531966 A JP 2008531966A JP 2008531966 A JP2008531966 A JP 2008531966A JP 5058169 B2 JP5058169 B2 JP 5058169B2
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Description
本発明はアレルゲンの腸管透過抑制剤に関する。 The present invention relates to an allergen intestinal permeation inhibitor.
食物アレルギーの発症までには、食物由来のタンパクが循環血中に移行する過程が含まれる。その過程には、（１）消化管腔内でのタンパク消化不良、（２）分泌型ＩｇＡの産生不足、（３）腸管粘膜バリアの変化、（４）リソゾームの機能不全などの原因が考えられる（非特許文献４）。このうち、腸管粘膜バリアの変化、すなわち、腸管における物質の透過の変化としては、腸管の上皮細胞間に存在するタイトジャンクションやエンドサイトーシス等の細胞内トランスポートが関与していると考えられている。アミノ酸などの低分子物質は、タイトジャンクションの間を通り抜けて吸収されるが、場合として細菌やウイルス、タンパク質などの巨大分子もこのタイトジャンクションを通過して吸収され、感染症や食物アレルギーを引き起こすと考えられている。従って、タイトジャンクションにおける吸収を抑制すれば、このような感染症や食物アレルギーを防止することができる。もっとも、エンドサイトーシス等の細胞内トランスポートを抑制することによっても、同様に腸管における吸収をコントロールすることも考えられる。 By the onset of food allergy, a process in which food-derived proteins move into the circulating blood is included. The process may be caused by (1) poor protein digestion in the digestive tract cavity, (2) insufficient production of secretory IgA, (3) changes in intestinal mucosal barrier, (4) lysosomal dysfunction, etc. (Non-Patent Document 4). Of these, changes in the intestinal mucosal barrier, that is, changes in permeation of substances in the intestinal tract, are thought to involve intracellular transport such as tight junctions and endocytosis that exist between epithelial cells of the intestinal tract. Yes. Low-molecular substances such as amino acids are absorbed through tight junctions, but in some cases, macromolecules such as bacteria, viruses, and proteins are absorbed through these tight junctions, causing infections and food allergies. It is considered. Therefore, if the absorption at the tight junction is suppressed, such infection and food allergy can be prevented. However, it is also conceivable to control absorption in the intestinal tract in the same manner by suppressing intracellular transport such as endocytosis.
これまで、タイトジャンクションにおける透過抑制を制御する物質として、例えば、特開平８−７３３７５号公報には、β−ラクトグロブリンのようなホエイタンパク質やその分解物が、また、特開２００４−１０４８８号公報や非特許文献１には、トリプトファンエステル、フェニルアラニンエステル又はチロシンエステルなどのアミノ酸のアルカノールエステル、ＷＳ、ＷＧ、ＷＳＮなどのトリプトファン、フェニルアラニン又はチロシンを含む２〜５のアミノ酸からなるペプチドが、さらに、特開２００２−２５７８１４号公報や非特許文献１，２には、β−カゼインタンパク質の分解物であるＤＫＩＨＰＦのアミノ酸配列を有するペプチドが示されている。そして、特開２００３−８１８６７号公報や非特許文献１には、ＷＳＮＳＧＮＦＶＧＧのアミノ酸配列を有するペプチドが、特開２００４−２６２８１５号公報にはＳＮＡＬＶＳＰＤＷＳＭＴＧＨのアミノ酸配列を有するペプチドが示されている。また、非特許文献３には、トリプトファンが卵白アミノ酸の透過制御に好ましいことが示されている。このように、これまでに種々のペプチドがアレルゲンの腸管透過作用を抑制することが見出されている。なお、特開２００４−１０４８８号公報には、トリプトファンを含む２〜５個のアミノ酸からなるペプチドとして、ＷＧ、ＷＳＮＳＧ、ＷＳＮ、ＷＳの４つのペプチドしか具体的に示されておらず、本願におけるＮＰＷＤＱを含む配列を有するペプチドが腸管透過抑制を示すかどうかは当該公報から予期できるものではない。
Until now, as substances that control permeation suppression in tight junctions, for example, JP-A-8-73375 discloses whey proteins such as β-lactoglobulin and degradation products thereof, and JP-A-2004-10488. And
ところで、本発明の起因物質であるＥＭＣ（酵素処理チーズ：Enzyme Modified Cheese）は、グリーンチーズや熟成後あるいは熟成途中のナチュラルチーズ、プロセスチーズにリパーゼや酸性及び中性プロテアーゼなどの微生物、動物由来の各種酵素を添加して熟成を促進させたものであって、元来チーズその他乳製品の風味の改良や強化を目的として、フレーバー、呈味剤として使用されてきたものである。このＥＭＣからは、これまでアンジオテンシン変換酵素阻害活性を有するペプチドしか見出されているにすぎず（ＷＯ２００４／４７５４３号参照）、その他の医薬用途に用いられる可能性については全く未知のものであった。 By the way, EMC (Enzyme Modified Cheese) which is the causative substance of the present invention is green cheese, natural cheese after ripening or during ripening, processed cheese, microorganisms such as lipase, acidic and neutral protease, and animal-derived It has been ripened by adding various enzymes, and has been originally used as a flavor and a flavoring agent for the purpose of improving and enhancing the flavor of cheese and other dairy products. From this EMC, only a peptide having an angiotensin converting enzyme inhibitory activity has been found so far (see WO 2004/47543), and the possibility of being used for other pharmaceutical uses was completely unknown. .
本発明は上記の背景技術に基づいてなされたものであって、本願発明者らはＥＭＣの新たな作用効果を見出すべく鋭意努力したところ、これまでに見出されたものとは異なるアミノ酸配列を有するペプチドが腸管におけるアレルゲンの透過性を抑制することを見出し、本願発明を完成するに至った。 The present invention has been made on the basis of the above-mentioned background art. The inventors of the present application have made an intensive effort to find out a new action effect of EMC, and as a result, an amino acid sequence different from those previously found. It has been found that the peptides possessed suppress the permeability of allergens in the intestinal tract, and the present invention has been completed.
本発明のアレルゲン腸管透過抑制剤は、例えばＧＰＩＶＬＮＰＷＤＱなど、ＮＰＷＤＱのアミノ酸配列を有する数個乃至十数個のアミノ酸からなるペプチド、または、アミノ酸配列の一部のアミノ酸が置換・欠失・付加されたからなるペプチドからなることを特徴としている。 Since the allergen intestinal permeation inhibitor of the present invention is a peptide consisting of several to a dozen amino acids having the amino acid sequence of NPWDQ, such as GPIVLNPWDQ, or a part of the amino acid sequence is substituted, deleted, or added. It is characterized by comprising the following peptide.
すなわち、本発明は、
[１] ＮＰＷＤＱのアミノ酸配列からなるペプチドであるアレルゲン腸管透過抑制剤、
[２] ＧＰＩＶＬＮＰＷＤＱのアミノ酸配列からなるペプチドであるアレルゲン腸管透過抑制剤、
[３] [１]又は[２]に記載のアレルゲン腸管透過抑制剤と無毒である担体を含む医薬組成物、
[４] [１]又は[２]に記載のアレルゲン腸管透過抑制剤が人工的に添加された食品（ただしＥＭＣが添加された乳製品を除く）若しくは飲料水であることを特徴とする食品組成物、
[５] 食物アレルゲンと当該食物アレルゲンと拮抗しうる量の[１]又は[２]に記載のアレルゲン腸管透過抑制剤が人工的に添加された食品（ただしＥＭＣが添加された乳製品を除く）若しくは飲料水であることを特徴とする食品組成物、
[６] 食物アレルゲンを含む食品又は飲料水と[１]又は[２]に記載のアレルゲン腸管透過抑制剤とが組み合わされたことを特徴とする食物アレルギー患者向けの食品組成物、
[７] 前記食物アレルゲンは、卵白アルブミンである[５]又は[６]の何れかに記載の食品組成物又食物アレルギー患者向けの食品組成物、
[８］ [１]又は[２]に記載のアレルゲン腸管透過抑制剤の、医薬組成物又は食品組成物の製造のための使用、
を提供するものである。
That is, the present invention
[1] allergen intestinal permeation inhibitor is a peptide comprising the amino acid sequence of NPWDQ,
[2] an allergen intestinal permeation inhibitor, which is a peptide consisting of the amino acid sequence of GPIV LNPWDQ,
[3] A pharmaceutical composition comprising a carrier that is non-toxic and an allergen intestinal permeation inhibitor according to [1] or [2] ,
[4] A food composition characterized by being a food to which the allergen intestinal permeation inhibitor according to [1] or [2] is artificially added (excluding dairy products to which EMC is added) or drinking water object,
[5] Food allergens and foods to which the allergen intestinal permeation inhibitor described in [1] or [2] that can antagonize the food allergens is artificially added (except for dairy products to which EMC is added) Or a food composition characterized by being drinking water,
[6] A food composition for food allergy patients, comprising a combination of a food or drinking water containing a food allergen and the allergen intestinal permeation inhibitor according to [1] or [2] ,
[7] The food composition according to any one of [5] or [6] , wherein the food allergen is ovalbumin, or a food composition for patients with food allergies,
[8] Use of the allergen intestinal permeation inhibitor according to [1] or [2] for production of a pharmaceutical composition or a food composition,
Is to provide.
本発明によると、新たな食物アレルギー抑制剤が提供される。特に本発明のアレルゲン腸管透過抑制剤は、ＥＭＣ中から見出されることにより、ＥＭＣを用いたチーズの摂取により食物アレルギーの発症を抑えることが期待される。また、本発明のペプチドを例えば卵製品など、食物アレルゲンである卵白アルブミンなどを含む食品や飲料水に添加したり、これらの食品等と共に摂取したりすれば、これらの食品中に含まれるアレルゲンの吸収が抑制される。この結果、食物アレルギー患者にとっても安心して食することができる新たな食品が提供される。 According to the present invention, a new food allergy inhibitor is provided. In particular, when the allergen intestinal permeation inhibitor of the present invention is found in EMC, it is expected to suppress the development of food allergy by ingestion of cheese using EMC. In addition, if the peptide of the present invention is added to a food or drinking water containing egg white albumin, which is a food allergen, such as an egg product, or taken together with these foods, the allergen contained in these foods Absorption is suppressed. As a result, a new food that can be safely eaten by patients with food allergies is provided.
本発明のアレルゲン腸管透過抑制剤は、配列番号１に示されるＮＰＷＤＱのアミノ酸配列を有する数個乃至十数個のアミノ酸からなるペプチドからなるものである。本発明のペプチドはＮＰＷＤＱのアミノ酸配列を有していればよく、この配列はペプチドのＮ末端あるいはＣ末端側のいずれに位置していても差し支えない。つまり、この配列のＮ末端側あるいはＣ末端側にさらに数個のアミノ酸が結合しているペプチドであってもよい。本発明のペプチドは数個乃至十数個のアミノ酸がペプチド結合したものであるが、結合するアミノ酸数は多くても２０個、好ましくは１０個程度までのものがよく、例えば配列番号２に示されるＧＰＩＶＬＮＰＷＤＱのアミノ酸配列からなるペプチドが挙げられ、アミノ酸数は少ないことが望まれる。もっとも、アレルゲン腸管透過抑制作用を奏する限り、配列番号１のアミノ酸配列のうち、いずれか１個又は数個のアミノ酸が置換、欠失又は挿入されたペプチドであってもよい。 The allergen intestinal permeation inhibitor of the present invention comprises a peptide consisting of several to a dozen amino acids having the amino acid sequence of NPWDQ shown in SEQ ID NO: 1. The peptide of the present invention only needs to have the amino acid sequence of NPWDQ, and this sequence may be located on either the N-terminal side or the C-terminal side of the peptide. That is, it may be a peptide in which several amino acids are further bonded to the N-terminal side or C-terminal side of this sequence. The peptide of the present invention is a peptide in which several to a dozen amino acids are peptide-bonded, but the number of amino acids to be bonded is at most 20, preferably about 10, and is shown in SEQ ID NO: 2, for example. The peptide consisting of the amino acid sequence of GPIV LNPWDQ is preferred, and it is desirable that the number of amino acids is small. However, as long as the allergen intestinal permeation inhibitory effect is exhibited, a peptide in which any one or several amino acids in the amino acid sequence of SEQ ID NO: 1 are substituted, deleted, or inserted may be used.
これらのペプチドは、実質的に無毒である担体と共に各種の組成物として提供される。ここで、実質的に無毒とはヒト（その他の動物等摂取が想定される動物等を含む）が摂取しても好ましくない作用が発揮されず、ペプチドの作用を低減するおそれのないことを意味し、製剤学的又は衛生上許容される各種の担体が用いられる。このような担体として、例えば、コーンスターチなどの各種デンプン、ラクトース、結晶セルロース、デキストロース、マンニトール、スクロース、ソルビトール、ゼラチン、アラビアゴム、リン酸二カルシウム、リン酸三カルシウム、リン酸一カルシウム、リン酸ナトリウム、炭酸ナトリウムなどの賦形剤、ステアリン酸、ステアリン酸亜鉛、ステアリン酸カルシウムまたはステアリン酸マグネシウムなどの滑沢剤、スクロース、ポリエチレングリコール、ポリビニルピロリドンなどの結合剤、安息香酸、クエン酸、酒石酸、コハク酸、リン酸、塩酸などの適当な有機酸・無機酸又はそのアルカリ金属（例えばナトリウム又はカリウム）塩などのｐＨ調整剤、着香剤、矯臭剤、着色剤、崩壊剤、溶解補助剤、懸濁剤、コーティング剤などが例示される。そしてこれらの担体とともに、散剤、顆粒剤、細粒剤、ドライシロップ剤、錠剤、カプセル剤、注射剤、液剤、軟膏剤、坐剤、貼付剤など経口または非経口的に投与される医薬組成物として提供される。さらに適当量のビタミン、ミネラル、有機酸、糖類、アミノ酸、ペプチド類など他の成分を添加してもよい。 These peptides are provided in various compositions with carriers that are substantially non-toxic. Here, “substantially non-toxic” means that undesirable effects are not exerted even if ingested by humans (including animals assumed to be ingested by other animals, etc.), and there is no possibility of reducing the action of peptides. In addition, various carriers that are pharmaceutically or hygienically acceptable are used. Examples of such carriers include various starches such as corn starch, lactose, crystalline cellulose, dextrose, mannitol, sucrose, sorbitol, gelatin, gum arabic, dicalcium phosphate, tricalcium phosphate, monocalcium phosphate, sodium phosphate. , Excipients such as sodium carbonate, lubricants such as stearic acid, zinc stearate, calcium stearate or magnesium stearate, binders such as sucrose, polyethylene glycol, polyvinylpyrrolidone, benzoic acid, citric acid, tartaric acid, succinic acid PH adjusting agents, flavoring agents, flavoring agents, coloring agents, disintegrating agents, solubilizing agents, suspensions, etc., suitable organic acids and inorganic acids such as phosphoric acid and hydrochloric acid or alkali metal (for example, sodium or potassium) salts thereof Agent, coating agent, etc. It is shown. In addition to these carriers, as pharmaceutical compositions administered orally or parenterally such as powders, granules, fine granules, dry syrups, tablets, capsules, injections, solutions, ointments, suppositories, patches, etc. Provided. Further, other components such as vitamins, minerals, organic acids, saccharides, amino acids and peptides may be added in appropriate amounts.
本発明のアレルゲン腸管透過抑制剤は、医薬組成物（医薬品）または食品組成物（飲食品）いずれの形態でも利用することができる。例えば、医薬品として直接投与することにより、または特定保健用食品等の特別用途食品、つまり特定の用途に用いられること若しくは特定の作用や効能を期待できる食品や栄養機能食品として直接摂取することにより各種のアレルギーの予防および／または治療をすることが期待される。また、液状、ペースト状、固形、粉末等の形態を問わず、各種食品（牛乳、加工乳、乳飲料、清涼飲料、発酵乳、ヨーグルト、チーズ、パン、ビスケット、クラッカー、ピッツァクラスト、アイスクリーム、キャンディ、調製粉乳、流動食、病者用食品、幼児用粉乳等食品、授乳婦用粉乳等食品、栄養食品、冷凍食品、加工食品その他の市販食品等）に添加し、これを摂取してもよい。なお、本発明において医薬組成物とは、一般的に医薬と観念されるものであって、上記のごとく本発明のアレルゲン腸管透過抑制剤に種々の担体が混合された組成物を意味する。また、本発明において食品組成物とは、一般的に食品として観念される組成物だけでなく、いわゆる健康食品と称させるように医薬品に類似させた組成物を含む意味で用いられる。また、本発明において特定保健用食品とは、特定の機能や効能を標榜可能にまで本発明の有効成分が配合若しくは含有されている食品、または食品に健康表示（健康への効用をしめす表現）を具体的に表示することが公に許可された食品を意味する。 The allergen intestinal permeation inhibitor of the present invention can be used in any form of a pharmaceutical composition (medicine) or a food composition (food or beverage). For example, it can be administered directly as a pharmaceutical product, or as a special-purpose food such as a food for specified health use, that is, used for a specific use or directly taken as a food that can be expected to have a specific action or effect, or a functional nutritional food. It is expected to prevent and / or treat allergies. In addition, various foods (milk, processed milk, milk drinks, soft drinks, fermented milk, yogurt, cheese, bread, biscuits, crackers, pizza crusts, ice cream, Candy, prepared milk powder, liquid food, foods for the sick, infant milk powder, foods such as infant milk powder, nutritional foods, frozen foods, processed foods and other commercial foods) Good. In the present invention, the pharmaceutical composition is generally regarded as a medicine, and means a composition in which various carriers are mixed with the allergen intestinal permeation inhibitor of the present invention as described above. In the present invention, the term “food composition” is used to mean not only a composition generally considered as a food but also a composition similar to a pharmaceutical product so as to be called a health food. Further, in the present invention, the food for specified health use is a food that contains or contains the active ingredient of the present invention to the extent that a specific function or effect can be advocated, or a health indication on the food (an expression that indicates its effect on health). This means food that is publicly permitted to be specifically labeled.
本発明のアレルゲン腸管透過抑制剤を含有する飲食品又は食品組成物は、水、タンパク質、糖質、脂質、ビタミン類、ミネラル類、有機酸、有機塩基、果汁、フレーバー類等を混合して使用することができる。タンパク質としては、例えば全脂粉乳、脱脂粉乳、部分脱脂粉乳、カゼイン、ホエイ粉、ホエイタンパク質、ホエイタンパク質濃縮物、ホエイタンパク質分離物、α−カゼイン、β−カゼイン、κ−カゼイン、β−ラクトグロブリン、α−ラクトアルブミン、ラクトフェリン、大豆タンパク質、鶏卵タンパク質、肉タンパク質等の動植物性タンパク質、これらの分解物；バター、乳性ミネラル、クリーム、ホエイ、非タンパク態窒素、シアル酸、リン脂質、乳糖等の各種乳由来成分などが挙げられる。カゼインホスホペプチド、アルギニン、リジン等のペプチドやアミノ酸を含んでいてもよい。糖質としては、例えば、糖類、加工澱粉（デキストリンのほか、可溶性澱粉、ブリティッシュスターチ、酸化澱粉、澱粉エステル、澱粉エーテル等）、食物繊維などが挙げられる。脂質としては、例えば、ラード、魚油等、これらの分別油、水素添加油、エステル交換油等の動物性油脂；パーム油、サフラワー油、コーン油、ナタネ油、ヤシ油、これらの分別油、水素添加油、エステル交換油等の植物性油脂などが挙げられる。ビタミン類としては、例えば、ビタミンＡ、カロチン類、ビタミンＢ群、ビタミンＣ、ビタミンＤ群、ビタミンＥ、ビタミンＫ群、ビタミンＰ、ビタミンＱ、ナイアシン、ニコチン酸、パントテン酸、ビオチン、イノシトール、コリン、葉酸などが挙げられ、ミネラル類としては、例えば、カルシウム、カリウム、マグネシウム、ナトリウム、銅、鉄、マンガン、亜鉛、セレンなどが挙げられる。有機酸としては、例えば、リンゴ酸、クエン酸、乳酸、酒石酸などが挙げられる。本発明のアレルゲン腸管透過抑制剤を含有する飲食品の製造において、これらは合成品であっても天然物由来品のいずれでもよく、またはこれらを含む食品を原材料として用いてもよい。これらの成分は、２種以上を組み合わせて使用することができる。食品の形態としては、固体でも液体でもかまわない。またゲル状などであってもよい。特に、ヨーグルトやチーズなどの乳製品、プリンやケーキ、卵焼きなど卵を使用した卵製品、スポーツドリンク、果汁ドリンク、コーヒー飲料、茶飲料などの各種飲料、パンやベーグル、お好み焼き、うどんなど麦粉を使用した食品など、アレルギー症状を呈する可能性の高い食品に好適である。 The food or drink or food composition containing the allergen intestinal permeation inhibitor of the present invention is used by mixing water, protein, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, flavors, etc. can do. Examples of the protein include whole milk powder, skim milk powder, partially skimmed milk powder, casein, whey powder, whey protein, whey protein concentrate, whey protein isolate, α-casein, β-casein, κ-casein, β-lactoglobulin , Α-lactalbumin, lactoferrin, soy protein, chicken egg protein, meat protein and other animal and vegetable proteins, degradation products thereof; butter, milk minerals, cream, whey, non-protein nitrogen, sialic acid, phospholipid, lactose, etc. And various milk-derived components. It may contain peptides and amino acids such as casein phosphopeptide, arginine and lysine. Examples of the saccharide include saccharides, processed starch (in addition to dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, and the like. Examples of the lipid include animal oils such as lard, fish oil, etc., fractionated oils, hydrogenated oil, transesterified oil, etc .; palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, fractionated oils thereof, Examples include vegetable oils such as hydrogenated oils and transesterified oils. Examples of vitamins include vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline. Examples of minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, and selenium. Examples of the organic acid include malic acid, citric acid, lactic acid, and tartaric acid. In the production of foods and drinks containing the allergen intestinal permeation inhibitor of the present invention, these may be either synthetic products or products derived from natural products, or foods containing these may be used as raw materials. These components can be used in combination of two or more. The form of food may be solid or liquid. It may be in the form of a gel. In particular, dairy products such as yogurt and cheese, egg products using eggs such as pudding and cakes, fried eggs, sports drinks, fruit juice drinks, coffee drinks, tea drinks, and other beverages, bread and bagels, okonomiyaki, udon flour It is suitable for foods that have a high possibility of exhibiting allergic symptoms.
本発明のペプチドは、卵、小麦、そば、魚など各種の食物アレルギーを有する患者に対して与えられるが、その投与量は概ね１日あたりペプチドとして０．１μｇ〜１００ｍｇが目安である。この投与量は、アレルギー患者の年齢、体重、症状や対象となるアレルゲン、摂取対象となる食品の種類や量に応じて適宜変更される。また、食品中に添加する場合には、添加対象中に含まれるアレルゲンと拮抗可能な量（アレルギーの症状を抑制可能な量）が添加されるのが好ましく、この場合も対象となる食品の種類や存在するアレルゲン、例えば卵白アルブミンの量などによっても異なるが、食品・飲料水中に０.００１〜１０重量％を目安に添加される。もっとも、本発明のペプチドは必ずしも食品中に添加する必要もなく、食品と共に摂取可能なように、目的の食品とセット商品にして食物アレルギー患者向けに提供されることもある。もっとも、本発明のアレルゲン腸管透過抑制剤や医薬組成物等はヒトのみを対象とするのでなく、犬や猫等のペットに代表されるようなヒト以外の動物も対象にできる。 The peptide of the present invention is given to patients having various food allergies such as eggs, wheat, buckwheat and fish, and the dosage is generally 0.1 μg to 100 mg as a peptide per day. This dosage is appropriately changed according to the age, weight, symptoms, allergen to be treated, and the type and amount of food to be ingested. In addition, when added to food, it is preferable to add an amount capable of antagonizing the allergen contained in the addition target (amount capable of suppressing allergic symptoms). Depending on the amount of allergens present, such as ovalbumin, etc., 0.001 to 10% by weight is added to food / drinking water as a guide. However, the peptide of the present invention does not necessarily need to be added to food, and may be provided for food allergy patients as a set product together with the target food so that it can be taken together with the food. However, the allergen intestinal permeation inhibitor and the pharmaceutical composition of the present invention are not only intended for humans, but can also be used for non-human animals such as dogs and cats.
本発明のアレルゲン腸管透過抑制剤が抑制するアレルギーの種類は特に限定されないが、例えば花粉症、アトピー性皮膚炎、気管支喘息、アレルギー性結膜炎、アレルギー性鼻炎、アレルギー性胃腸炎、アナフィラキシー反応、薬物アレルギー、じんましん、血清病、溶血性貧血、接触性皮膚炎、重症筋無力症、グッドパスチェア症候群、糸球体腎炎等を挙げることができる。対象とするアレルゲンも特に限定されないが、例えば食品（小麦、大麦、オーツ麦、ライ麦、そば、卵、乳、チーズ、落花生、米、トウモロコシ、アワ、キビ、ヒエ、大豆、じゃがいも、やまいも、にんにく、たまねぎ、ニンジン、パセリ、セロリ、トマト、オレンジ、もも、りんご、キウイフルーツ、メロン、イチゴ、バナナ、くるみ、ゴマ、まつたけ、あわび、いか、いくら、えび、かに、さけ、さば、アジ、イワシ、タラ、イカ、タコ、ホタテ、牛肉、鶏肉、豚肉、ゼラチン等）、動物（イヌ、ネコ、マウス、ラット、ハト等やその皮膚、体毛、糞、羽毛等）、昆虫（蛾、ユスリカ、スズメバチなど、およびこれら昆虫の分泌物、鱗粉）、ダニ、寄生虫（アニサキス、回虫など）、草木（スギ、ヒノキ、ブタクサ、イネ科植物、ヨモギ、ウルシ、ハンノキ等やその花粉、樹液等）、かび、ほこり、ハウスダスト、ゴム、金属、化学物質、医薬品、等を挙げることができる。 The type of allergy that the allergen intestinal permeation inhibitor of the present invention suppresses is not particularly limited. For example, hay fever, atopic dermatitis, bronchial asthma, allergic conjunctivitis, allergic rhinitis, allergic gastroenteritis, anaphylactic reaction, drug allergy , Urticaria, serum sickness, hemolytic anemia, contact dermatitis, myasthenia gravis, Goodpath chair syndrome, glomerulonephritis and the like. The target allergen is not particularly limited. For example, food (wheat, barley, oats, rye, buckwheat, egg, milk, cheese, peanut, rice, corn, millet, millet, millet, soy, potato, yam, garlic, Onion, carrot, parsley, celery, tomato, orange, peach, apple, kiwi fruit, melon, strawberry, banana, walnut, sesame, matsutake, abalone, squid, how much, shrimp, crab, salmon, mackerel, horse mackerel, sardines , Cod, squid, octopus, scallops, beef, chicken, pork, gelatin, etc.), animals (dogs, cats, mice, rats, pigeons, etc. and their skin, hair, feces, feathers, etc.), insects (spider, chironomid, wasp And these insect secretions, scales, mites, parasites (anisakis, roundworms, etc.), vegetation (cedar, cypress, ragweed, grass family, mugwort) Sumac, alder etc. and their pollen, sap, etc.), can be cited molds, dust, house dust, rubber, metal, chemical, pharmaceuticals, and the like.
次に本発明のアレルゲン腸管透過抑制剤について、下記実施例に基づいて詳細に説明する。なお、本発明は下記実施例に限られないのは言うまでもない。 Next, the allergen intestinal permeation inhibitor of the present invention will be described in detail based on the following examples. Needless to say, the present invention is not limited to the following embodiments.
まず、複数の酵素の組み合わせによって得られた３種類のＥＭＣについて、腸管透過抑制作用を調べた。 First, the intestinal permeation inhibitory action of three types of EMC obtained by combining a plurality of enzymes was examined.
１）ＥＭＣの調製
まず、異なる酵素の組み合わせによって３種類のＥＭＣを調製した。
（スターター培養液の調製）
２０ｇのスキムミルク粉末（生化学用、和光純薬工業）を２００ｍｌの蒸留水に溶解、滅菌して、１０w/v％の脱脂粉乳培地を調製した。ここに約０．１ｇのMM Culture（MM100：Lactococcus lactis subsp. lactis、Lactococcus lactis subsp. cremoris、Lactococcus lactis subsp. lactis biovar diacetylactisの３種類の菌を含む、Dairy Connection Inc (Wisconsin, USA)）を接種し、３７℃で１６時間培養した。1) Preparation of EMC First, three types of EMC were prepared by combining different enzymes.
(Preparation of starter culture)
20 g of skim milk powder (for biochemistry, Wako Pure Chemical Industries) was dissolved in 200 ml of distilled water and sterilized to prepare a 10 w / v% skim milk medium. About 0.1 g of MM Culture (MM100: Dairy Connection Inc (Wisconsin, USA) containing three types of bacteria, Lactococcus lactis subsp. Lactis, Lactococcus lactis subsp. Cremoris, Lactococcus lactis subsp. Lactis biovar diacetylactis) And cultured at 37 ° C. for 16 hours.
(ＥＭＣの調製方法)
ミートチョッパーにて粉砕したデンマークスキムチーズ（熟成期間６ヶ月）に蒸留水を５０w/v％となるように添加し、さらに上記の方法で調製したスターター培養液（最終濃度０.１５〜０.３wt％）、塩化ナトリウム及び表１中の酵素Ａを添加した。このものを発酵温度３４℃で２日間撹拌し、加水分解した（発酵開始時のｐＨは５.５であった。）。次に、クエン酸水溶液でｐＨを４.１に調整し、表１中の酵素Ｂ及び酵素Ｃを添加した。さらに、３４℃で５日間、撹拌して分解した。分解終了後、水酸化ナトリウム水溶液にてｐＨを５.０に調整し、１１０℃にて１０分間加熱して酵素を失活させ、凍結乾燥してＥＭＣ約６０ｇを得た。ＥＭＣの調製に用いた配合表を表１に示す。また、用いられた酵素の性質等を表２に示す。(Method for preparing EMC)
Distilled water was added to Danish skim cheese crushed with meat chopper (ripening period 6 months) so that it might become 50 w / v%, and also the starter culture solution prepared by the above method (final concentration 0.15-0.3 wt. %), Sodium chloride and enzyme A in Table 1 were added. This was stirred for 2 days at a fermentation temperature of 34 ° C. and hydrolyzed (pH at the start of fermentation was 5.5). Next, the pH was adjusted to 4.1 with an aqueous citric acid solution, and enzymes B and C in Table 1 were added. Furthermore, it decomposed | disassembled by stirring for 5 days at 34 degreeC. After completion of the decomposition, the pH was adjusted to 5.0 with an aqueous sodium hydroxide solution, the enzyme was deactivated by heating at 110 ° C. for 10 minutes, and freeze-dried to obtain about 60 g of EMC. Table 1 shows the recipe used for the preparation of EMC. Table 2 shows the properties of the enzyme used.
このようにして得られた各ＥＭＣ（ＥＭＣ−１、ＥＭＣ−２又はＥＭＣ−３）を５０w/v％となるようにそれぞれ水に懸濁し、遠心分離した後（３,０００ｇ×１０分）、その上清液（最終的にＥＭＣ濃度が０.０１ｍｇ／ｍｌとなる。）について以下の方法で腸管透過抑制作用を調べた。 Each EMC (EMC-1, EMC-2 or EMC-3) thus obtained was suspended in water so as to be 50 w / v% and centrifuged (3,000 g × 10 minutes). The intestinal permeation inhibitory effect of the supernatant liquid (final EMC concentration of 0.01 mg / ml) was examined by the following method.
２）Caco-2細胞を用いた腸管透過抑制作用
（Caco-2細胞を用いた腸管透過モデルの作成）
Caco-2細胞(ATCC HTB-37、American Type Culture Collection(ATCC))は３０〜１１０継代のものを用いた。細胞培養用の培地として、２０％牛胎子血清（以下「FCS」ともいう、ICN Biochemicals, Inc.）、１％非必須アミノ酸（Gibco Life Technologies）、１００ＩＵ／ｍｌペニシリン（Gibco Life Technologies）、１００μｇ／ｍｌストレプトマイシン（Gibco Life Technologies）及び５０μｇ／ｍｌゲンタマイシン（Gibco Life Technologies）を含有するダルベッコ改変イーグル培地（以下「DMEM」とも言う、Gibco Life Technologies）を用いた。まずCaco-2細胞を、７５ｃｍ２の組織培養フラスコで約７０〜８０％コンフルエンスになるまで培養した。次いで、１２穴トランズウェル細胞培養チャンバー（直径１２ｍｍ、孔径０.４μｍ、透過性膜を有する）にCaco-2細胞を２×１０５cells／ｃｍ２の濃度で接種し、５％ＣＯ２雰囲気下、３７℃にて約１０〜２１日間培養してCaco-2単層細胞を得た。このようにして得たCaco-2単層細胞のapical（管腔）側には微細柔突起が多く小腸のbrush borderやtight junctionの特徴に類似している。また、前記培養期間でトランスポーターやペプチダーゼなどの代謝酵素も発現することから小腸膜モデルとして使用することができる。2) Intestinal permeation inhibition using Caco-2 cells (Creation of intestinal permeation model using Caco-2 cells)
Caco-2 cells (ATCC HTB-37, American Type Culture Collection (ATCC)) from 30 to 110 passages were used. As a medium for cell culture, 20% fetal calf serum (hereinafter also referred to as “FCS”, ICN Biochemicals, Inc.), 1% non-essential amino acid (Gibco Life Technologies), 100 IU / ml penicillin (Gibco Life Technologies), 100 μg / Dulbecco's Modified Eagle's Medium (hereinafter also referred to as “DMEM”, Gibco Life Technologies) containing ml streptomycin (Gibco Life Technologies) and 50 μg / ml gentamicin (Gibco Life Technologies) was used. First, Caco-2 cells were cultured in a 75 cm 2 tissue culture flask until they were about 70-80% confluent. Next, Caco-2 cells were inoculated at a concentration of 2 × 10 5 cells / cm 2 into a 12-well Transwell cell culture chamber (diameter 12 mm, pore diameter 0.4 μm, having a permeable membrane) in a 5% CO 2 atmosphere. The cells were cultured at 37 ° C. for about 10 to 21 days to obtain Caco-2 monolayer cells. The apical (lumen) side of the Caco-2 monolayer cells obtained in this way has many fine soft processes similar to the characteristics of the brush border and tight junction of the small intestine. Further, since metabolic enzymes such as transporters and peptidases are also expressed during the culture period, they can be used as a small intestinal membrane model.
（腸管透過モデルの検証）
上記の方法にて作成したCaco-2単層細胞において、充分にタイトジャンクションが形成されているかどうかを検証するために、Caco-2単層細胞の経上皮電気抵抗(TEER)を測定した。Ａｇ／ＡｇＣｌ電極を用いた抵抗値測定システム（Millicell-ERS、Millipore）を用い、経上皮電気抵抗が約３００Ω・ｃｍ２以上のものをアッセイに用いた。各ウェルはクラスタープレート上に設置し、外側培養液（基底側、１.５ｍｌ）と内側培養液（管腔側、０.５ｍｌ）を満たした（図１参照）。Caco-2単層細胞は２４時間毎に新鮮な培地に交換して培養した。(Verification of intestinal permeation model)
In order to verify whether a tight junction was sufficiently formed in the Caco-2 monolayer cells prepared by the above method, the transepithelial electrical resistance (TEER) of the Caco-2 monolayer cells was measured. A resistance value measurement system (Millicell-ERS, Millipore) using an Ag / AgCl electrode was used for the assay with a transepithelial electrical resistance of about 300 Ω · cm 2 or more. Each well was placed on a cluster plate and filled with an outer culture (basal side, 1.5 ml) and an inner culture (luminal side, 0.5 ml) (see FIG. 1). Caco-2 monolayer cells were cultured by replacing with fresh medium every 24 hours.
（FITC-OVAの調製）
アレルゲンには、卵白アルブミン（以下「OVA」とも言う）をfluorescein isothiocyanate（以降FITCともいう）で標識したFITC-OVAを用いた。FITC-OVAの調製は、Maedaらの方法に従って行った（Maeda H, Ishida N, Kawauchi H, Tsujimura K、「Reaction of fluorescein-isothiocyanate with proteins and amino acids. I. Covalent and non-covalent binding of fluorescein-isothiocyanate and fluorescein to proteins.」、J Biochem (Tokyo)、65(5)、pp.777-783(1969)）。反応液を流水に対して１晩透析を行い、さらにSephadex G-50 カラム（Aldrich）で未反応のFITCを除去して非吸着画分（FITC-OVA）を濃縮した。これを凍結乾燥して以下の腸管透過抑制試験に用いた。(Preparation of FITC-OVA)
As the allergen, FITC-OVA in which ovalbumin (hereinafter also referred to as “OVA”) was labeled with fluorescein isothiocyanate (hereinafter also referred to as FITC) was used. FITC-OVA was prepared according to the method of Maeda et al. (Maeda H, Ishida N, Kawauchi H, Tsujimura K, “Reaction of fluorescein-isothiocyanate with proteins and amino acids. I. Covalent and non-covalent binding of fluorescein- isothiocyanate and fluorescein to proteins. ", J Biochem (Tokyo), 65 (5), pp.777-783 (1969)). The reaction solution was dialyzed overnight against running water, and unreacted FITC was removed with a Sephadex G-50 column (Aldrich) to concentrate the non-adsorbed fraction (FITC-OVA). This was freeze-dried and used for the following intestinal permeation inhibition tests.
（腸管透過抑制試験）
FITC-OVA透過性測定の１日前から、被検物質（ＥＭＣ−１、ＥＭＣ−２又はＥＭＣ−３）０．０１ｍｇ／ｍｌを含有した培地で上記Caco-2単層細胞を培養した。また、被検物質を含有しない培地で培養したものをコントロールとした。試験当日に、Caco-2単層細胞をHanks' balanced salt solution（以下「HBSS」ともいう、Gibco Life Technologies）で２回洗浄し、ＨＢＳＳに被検物質０．００５ｍｇ（最終濃度０．０１ｍｇ／ｍｌ）とFITC-OVA ０.５ｍｇを添加した液０.５ｍｌを管腔側に、ＨＢＳＳに被検物質０．０１５ｍｇ（最終濃度０．０１ｍｇ／ｍｌ）を添加した液１.５ｍｌを基底側に入れた。この条件でCaco-2単層細胞を５％ＣＯ２雰囲気下、３７℃にて３０分インキュベーションし、基底側の培地を採取した。また、コントロールについても同様に操作したが、ＨＢＳＳには被検物質を添加しなかった。(Intestinal permeation inhibition test)
One day before the FITC-OVA permeability measurement, the Caco-2 monolayer cells were cultured in a medium containing 0.01 mg / ml of a test substance (EMC-1, EMC-2, or EMC-3). Moreover, what was cultured with the culture medium which does not contain a test substance was set as control. On the day of the test, Caco-2 monolayer cells were washed twice with Hanks' balanced salt solution (hereinafter also referred to as “HBSS”, Gibco Life Technologies), and 0.005 mg of test substance (final concentration 0.01 mg / ml) in HBSS. ) And 0.5 ml of FITC-OVA added 0.5 mg on the luminal side, and 1.5 ml of HBSS added with 0.015 mg test substance (final concentration 0.01 mg / ml) on the basal side It was. Under these conditions, Caco-2 monolayer cells were incubated at 37 ° C. for 30 minutes in a 5% CO 2 atmosphere, and the basal medium was collected. Moreover, although it operated similarly about control, the test substance was not added to HBSS.
基底側のFITC-OVA濃度を、分光蛍光光度計（RF-1500、島津製作所）で励起波長４９５ｎｍ、測定波長５２０ｎｍの蛍光強度で測定した。その結果を図２に示す。腸管透過阻害活性（％）は以下の数式にて算出した。
腸管透過阻害活性（％）＝(１−Ａ／Ｂ)×１００
Ａ：被検物質添加時のFITC-OVA濃度
Ｂ：コントロールのFITC-OVA濃度
この結果、ＥＭＣ−２の腸管透過阻害活性はコントロールに対して有意（ｐ＜０．０５）に低い値を示した。The FITC-OVA concentration on the basal side was measured with a spectrofluorophotometer (RF-1500, Shimadzu Corporation) at an excitation wavelength of 495 nm and a measurement intensity of 520 nm. The result is shown in FIG. Intestinal permeation inhibitory activity (%) was calculated by the following formula.
Intestinal permeation inhibitory activity (%) = (1−A / B) × 100
A: FITC-OVA concentration when test substance is added
B: FITC-OVA concentration of control As a result, the intestinal permeation inhibitory activity of EMC-2 was significantly lower (p <0.05) than the control.
次に、腸管透過抑制作用が強かったＥＭＣ−２を用いて、腸管透過抑制作用物質の分離・同定を行った。 Next, the intestinal permeation inhibitory substance was separated and identified using EMC-2, which had a strong intestinal permeation inhibitory effect.
１）ＥＭＣ−２からの粗分画
３０ｍｇのＥＭＣ−２を０.１％ＴＦＡ（buffer A）に溶解し、Ｃ１８Sep-Pack（Waters, Milford MA）に吸着させた。その後、buffer Aと８０％のアセトニトリルを含む０.０８５％ＴＦＡ（buffer B）の混合液３種類（容量比７５：２５，５０：５０，２５：７５）を各８ｍｌずつ用いて順次溶出させた。これらの溶出液（Ｆｒ１：アセトニトリル濃度２０％、Ｆｒ２：アセトニトリル濃度４０％、Ｆｒ３：アセトニトリル濃度６０％）を凍結乾燥した後、実施例１に記載の透過抑制試験に用いた。得られた被検物質（Ｆｒ１、Ｆｒ２またはＦｒ３）をＥＭＣ換算で０．６ｍｇ／ｍｌの濃度で腸管透過抑制試験に供した。その結果を図３に示す。これらの画分はいずれも腸管透過抑制作用を示した。特に、Ｆｒ２に強い腸管透過抑制作用が認められた。1) Crude fraction from EMC-2 30 mg of EMC-2 was dissolved in 0.1% TFA (buffer A) and adsorbed on C18 Sep-Pack (Waters, Milford MA). Thereafter, three kinds of mixed solutions (volume ratio 75:25, 50:50, 25:75) of 0.085% TFA (buffer B) containing buffer A and 80% acetonitrile were sequentially eluted using 8 ml each. . These eluates (Fr1:
２）ＨＰＬＣによる分画
腸管透過抑制作用が強く現れたＦｒ２についてさらに分画を進めた。分画は、逆相カラム（CAPCELL PAK C18 MG(φ4.6×250mm、資生堂製)）を用いたＨＰＬＣにより、次の溶出条件下で行った。その結果を図４に示す。
溶出条件
溶媒Ａ：０.１％ＴＦＡ溶液
溶液Ｂ：０.０８５％ＴＦＡを含む８０%アセトニトリル溶液
溶出：６０分でＡ１００％からＢ１００％へ直線的に上昇させた
流速：１.０ｍｌ／ｍｉｎ
検出波長：２２０ｎｍ2) Fractionation by HPLC Fr2 in which the intestinal permeation inhibitory action appeared strongly was further fractionated. Fractionation was performed by HPLC using a reverse phase column (CAPCELL PAK C18 MG (φ4.6 × 250 mm, manufactured by Shiseido)) under the following elution conditions. The result is shown in FIG.
Elution conditions Solvent A: 0.1% TFA solution Solution B: 80% acetonitrile solution containing 0.085% TFA Elution: linearly increased from A100% to B100% in 60 minutes Flow rate: 1.0 ml / min
Detection wavelength: 220 nm
ＨＰＬＣ分画により得られた１０個以上のピークを分取し、凍結乾燥した後、実施例１に記載の腸管透過抑制試験に供試したところ、図４に示すピークＰ１〜Ｐ３の分画が透過抑制作用を示した。その結果を図５に示す。特に、ピークＰ１の分画に強い腸管透過抑制作用が認められた。得られた被検物質（図４に示す各ピーク）をＥＭＣ換算で０．６ｍｇ／ｍｌの濃度で腸管透過抑制試験に供した。 Ten or more peaks obtained by HPLC fractionation were collected, lyophilized, and then subjected to the intestinal permeation inhibition test described in Example 1. As a result, the fractions of peaks P1 to P3 shown in FIG. Permeation suppression effect was shown. The result is shown in FIG. In particular, a strong intestinal permeation inhibitory effect was observed on the peak P1 fraction. The obtained test substance (each peak shown in FIG. 4) was subjected to an intestinal permeation suppression test at a concentration of 0.6 mg / ml in terms of EMC.
２）アミノ配列の決定
腸管透過抑制試験で作用が強く表れたＰ１フラクションの分画から得られたペプチドのN末端アミノ酸配列解析をしたところ、そのアミノ酸配列は、配列番号２に示すＧＰＩＶＬＮＰＷＤＱであることが決定された。N末端アミノ酸配列解析、プロテインシーケンサー（Ｇ１００５Ａ、Ｈｅｗｌｅｔｔ−Ｐａｃｋａｒｄ Ｃｏ．）を用いて行った。なお、このアミノ酸配列は、αｓ２カゼイン（ＮＣＢＩ登録番号 ＮＰ７７６９５３）のアミノ酸番号１０２−１１１のアミノ酸配列を一致することが確認された。2) Determination of amino sequence When the N-terminal amino acid sequence analysis of the peptide obtained from the fraction of the P1 fraction that showed strong action in the intestinal permeation inhibition test was conducted, the amino acid sequence was GPIVLNPWDQ shown in SEQ ID NO: 2. Was decided. N-terminal amino acid sequence analysis was performed using a protein sequencer (G1005A, Hewlett-Packard Co.). Incidentally, this amino acid sequence was confirmed to match the amino acid sequence of amino acid numbers 102-111 of .alpha.s 2 Casein (NCBI Registration No. NP776953).
３）活性部位の決定
次に、これらのアミノ酸配列を元に、Ｆｍｏｃ−固相合成法によってＧＰＩＶＬ（配列番号３：αｓ２カゼインのアミノ酸番号１０２−１０６）及びＮＰＷＤＱ（配列番号１：αｓ２カゼインのアミノ酸番号１０７−１１１）のそれぞれ５つのアミノ酸からなるペプチド並びに配列番号２に示す１０個のアミノ酸配列からなるペプチド（αｓ２カゼインのアミノ酸番号１０２−１１１）を合成し、それぞれのペプチドについて腸管透過抑制作用を調べた。試験方法は実施例１に記載の腸管透過抑制に準ずる。その結果を図６及び図７に示す。この結果、配列番号２に示す１０個のアミノ酸からなるペプチドは、１０−４〜１０−６Ｍの濃度でコントロールに対して有意（ｐ＜０．０５）に抑制作用を示した。また、１０個のアミノ酸配列のうち、Ｃ末側の５つのアミノ酸配列ＮＰＷＤＱを有するペプチドが、アミノ酸配列１で示されたペプチドと同程度の作用を示し、この配列が活性部位と考えられた。3) Determination of the active site then based on these amino acid sequences, Fmoc-solid phase synthesis method by GPIVL (SEQ ID NO 3: .alpha.s 2 casein amino acid numbers 102-106) and NPWDQ (SEQ ID NO 1: .alpha.s 2 Casein was synthesized in the amino acid number 107-111) of each of the five consisting of the amino acid peptide and peptide consisting of 10 amino acid sequence shown in SEQ ID NO: 2 (.alpha.s 2 casein amino acid number 102-111), for each peptide intestinal permeability The inhibitory action was examined. The test method conforms to the intestinal permeation suppression described in Example 1. The results are shown in FIGS. As a result, the peptide consisting of 10 amino acids shown in SEQ ID NO: 2 showed a significant (p <0.05) inhibitory effect on the control at a concentration of 10 −4 to 10 −6 M. Further, among the 10 amino acid sequences, a peptide having 5 amino acid sequences NPWDQ on the C-terminal side showed the same effect as the peptide represented by
〔トランスセルラールート阻害条件における腸管透過抑制試験〕
Soderholmら（Soderholm JD et al、Epithelial permeability to proteins in the noninflamed ileum of Crohn's disease?、Gastroenterology、117(1)、pp.65-72 (1999)）は、OVAは細胞間隙経路（パラセルラールート）と細胞内経路（トランスセルラールート）の両方で腸管を透過すると報告している。パラセルラールートによる透過は、腸管のタイトジャンクション（ＴＪ）の状態で左右される。一方、トランスセルラールートによる透過はエネルギー依存的輸送であるため、アジ化ナトリウムや2,4-dinitrophenolなどの特異的透過阻害剤（Ma TY 、Mechanism of extracellular calcium regulation of intestinal epithelial tight junction permeability: role of cytoskeletal involvement.、Microsc Res Tech. 、51(2)、pp.156-68(2000))によってトランスセルラールートによる透過が遮断されることになる。そこで、ＮＰＷＤＱの配列からなるペプチドがいずれの、あるいは双方の透過ルートを抑制しているのかを明らかにするためにアジ化ナトリウムを添加し、ＮＰＷＤＱ存在下および非存在下での透過抑制を比較した。[Intestinal permeation suppression test under transcellular route inhibition conditions]
Soderholm et al. (Soderholm JD et al, Epithelial permeability to proteins in the noninflamed ileum of Crohn's disease ?, Gastroenterology, 117 (1), pp.65-72 (1999)), OVA It has been reported to penetrate the intestinal tract by both intracellular pathways (transcellular route). Permeation through the paracellular route depends on the tight junction (TJ) state of the intestinal tract. On the other hand, since permeation through the transcellular route is energy-dependent transport, specific permeation inhibitors such as sodium azide and 2,4-dinitrophenol (Ma TY, Mechanism of extracellular calcium regulation of intestinal epithelial tight junction permeability: role of Cytoskeletal involvement., Microsc Res Tech., 51 (2), pp.156-68 (2000)) will block the transcellular route. Therefore, sodium azide was added to clarify which or both of the peptides comprising the NPWDQ sequence suppressed the permeation route, and the permeation suppression in the presence and absence of NPWDQ was compared. .
（腸管透過抑制試験）
FITC-OVAの調製および腸管透過抑制試験は実施例１に準じて行った。また、被検物質（ＮＰＷＤＱ（最終濃度１０−４Ｍ））添加の１０分前および被検物質（ＮＰＷＤＱ）の添加と同時に、アジ化ナトリウム（和光純薬）を、添加後の最終濃度１０ｍＭになるように添加した。(Intestinal permeation inhibition test)
Preparation of FITC-OVA and intestinal permeation inhibition test were performed according to Example 1. Also, 10 minutes before the addition of the test substance (NPWDQ (final concentration 10 −4 M)) and simultaneously with the addition of the test substance (NPWDQ), sodium azide (Wako Pure Chemical Industries) was added to a final concentration of 10 mM after the addition. It added so that it might become.
その結果を図８に示す。ここに示すように、トランスセルラールート透過抑制剤であるアジ化ナトリウムの添加によってOVAの透過が抑制されたため、OVAのCaco-2単層膜透過にはトランスセルラールートが関与していることが明らかとなった（図８グラフ中）。さらに、ＮＰＷＤＱの同時添加により、アジ化ナトリウム単独添加よりもOVAの透過がさらに抑制された（図８グラフ右）。アジ化ナトリウム単独添加によってOVAのトランスセルラールートが抑制されているのにもかかわらず（図８グラフ左）、ＮＰＷＤＱ同時添加により、OVAの透過がさらに抑制されていることから、ＮＰＷＤＱは、少なくともOVAのパラセルラールートを抑制することが分かった。 The result is shown in FIG. As shown here, OVA permeation was suppressed by the addition of sodium azide, a transcellular route permeation inhibitor, and it was clear that transcellular route was involved in OVA Caco-2 monolayer membrane permeation. (In the graph of FIG. 8). Furthermore, the simultaneous addition of NPWDQ further suppressed the permeation of OVA more than the addition of sodium azide alone (right graph in FIG. 8). Although the transcellular route of OVA is suppressed by the addition of sodium azide alone (left graph in FIG. 8), the permeation of OVA is further suppressed by the simultaneous addition of NPWDQ, so that NPWDQ is at least OVA It was found to suppress the paracellular route.
〔FITC-dextran及びHRPの透過抑制試験〕
次に、ＮＰＷＤＱが双方の透過経路に及ぼす影響をより明確にするために、FITC-dextran(FD-4)およびHRPの透過抑制について調べた。FD-4はパラセルラールートのみを透過するモデル化合物として、HRPはトランスセルラールートのみを透過するモデル化合物として広く用いられている（Wang Q et al.、Intestinal permeability is reduced and IL-10 levels are increased in septic IL-6 knockout mice、Am J Physiol Regul Integr Comp Physiol、281(3)、R1013-1023(2001)および、Heyman M et al.、Horseradish peroxidase transport across adult rabbit jejunum in vitro、Am J Physiol、242(6)、G558-564(1982)及び上野川ら編、生物化学実験法 50 腸管細胞機能実験法、学会出版センター発行、2005年）。[Permeation inhibition test of FITC-dextran and HRP]
Next, in order to clarify the influence of NPWDQ on both permeation paths, the permeation suppression of FITC-dextran (FD-4) and HRP was examined. FD-4 is widely used as a model compound that permeates only the paracellular route, and HRP is widely used as a model compound that permeates only the transcellular route (Wang Q et al., Intestinal permeability is reduced and IL-10 levels are increased. in septic IL-6 knockout mice, Am J Physiol Regul Integr Comp Physiol, 281 (3), R1013-1023 (2001) and Heyman M et al., Horseadish peroxidase transport across adult rabbit jejunum in vitro, Am J Physiol, 242 (6), G558-564 (1982) and edited by Uenogawa et al.,
（腸管透過抑制試験）
実施例１の方法にて、アレルゲンをFITC-OVAの代わりにFITC-dextran(FD-4, 分子量4300, SIGMA)及びhorseradish peroxidase(HRP, 分子量40kDa, SIGMA)を用いて試験を行った。また、透過液中のアレルゲン濃度は以下に示す方法にて算出した。(Intestinal permeation inhibition test)
By the method of Example 1, allergens were tested using FITC-dextran (FD-4, molecular weight 4300, SIGMA) and horseradish peroxidase (HRP,
＜FD-4透過量の測定＞
FITC-dextranの測定、腸管透過阻害活性（％）の算出は実施例１のFITC-OVAに準じて行った。
<HRP透過量の測定＞
HRPの透過量は、透過液のペルオキシダーゼ活性から求めた。４０倍希釈した透過液（200μL）に100μLの基質発色液（o-phenylenediamine(OPD)１３mg、過酸化水素２００μLを含む０.１Mクエン酸緩衝液(pH5.0, 50mL)）を加えて反応させた後、マイクロプレートリーダーにより４９０ｎｍの吸光度を測定した。HRPの透過抑制活性は、（試料無添加時の吸光値−試料添加時の吸光値）/試料無添加時の吸光値×１００で表した。<Measurement of FD-4 transmission>
Measurement of FITC-dextran and calculation of intestinal permeation inhibition activity (%) were performed according to FITC-OVA in Example 1.
<Measurement of HRP permeability>
The permeation amount of HRP was determined from the peroxidase activity of the permeate. To the permeate (200 μL) diluted 40 times, add 100 μL of the substrate coloring solution (0.1 M citrate buffer (pH 5.0, 50 mL) containing 13 mg of o-phenylenediamine (OPD) and 200 μL of hydrogen peroxide). Thereafter, the absorbance at 490 nm was measured with a microplate reader. The permeation inhibiting activity of HRP was expressed as (absorbance value when no sample was added−absorbance value when the sample was added) / absorbance value when no sample was added × 100.
測定はそれぞれ測定開始時、測定開始１時間後、測定開始２時間後に行い、FD-4透過量、HRP透過量ともに、測定開始〜測定開始後１時間の透過量の差（透過時間０−１）並びに測定開始後１時間〜測定開始後２時間の透過量の差（透過時間１−２）を求め、それらの結果を表３及び表４に示した。表３及び表４に示すように、ＮＰＷＤＱの添加によって、HRPおよびFITC-dextranのいずれの透過も顕著に抑制したことから、ＮＰＷＤＱは、トランスセルラールート及びパラセルラールートの双方の物質透過を抑制することが示された。 Each measurement is performed at the start of measurement, 1 hour after the start of measurement, and 2 hours after the start of measurement. Both the FD-4 transmission amount and the HRP transmission amount are the difference in transmission amount between the measurement start and the measurement start for 1 hour (transmission time 0-1). ) And the difference in permeation amount (transmission time 1-2) from 1 hour after the start of measurement to 2 hours after the start of measurement, and the results are shown in Tables 3 and 4. As shown in Table 3 and Table 4, the addition of NPWDQ markedly suppressed the permeation of both HRP and FITC-dextran, so NPWDQ suppresses the material permeation of both the transcellular route and the paracellular route. It was shown that.
本発明によると、新規なアレルゲン腸管透過抑制剤が提供され、食物アレルギーの治療や予防に寄与する。また、これらのペプチドを乳製品や卵製品に併存させると食物アレルギーの症状を軽減させることができるので、食物アレルギー患者にとっても安心して食することができる食品が提供される。 According to the present invention, a novel allergen intestinal permeation inhibitor is provided, which contributes to the treatment and prevention of food allergies. In addition, when these peptides coexist with dairy products and egg products, the symptoms of food allergies can be reduced, so that foods that can be eaten with peace of mind by food allergic patients are provided.
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