JP2004346020A - Protease inhibitor - Google Patents

Abstract

An object of the present invention is to provide a cysteine protease inhibitor which can be widely used as a food material, and which can be used as a prophylactic / therapeutic agent for diseases involving cysteine protease, and which can be used in various foods and drinks and feeds.
The present invention relates to a cysteine protease inhibitor containing, as an active ingredient, one or more mixtures selected from the group consisting of lactoferrin, a partial peptide of lactoferrin, and transferrin, osteoporosis, malignant neoplastic hypercalcemia. A cysteine protease inhibitor which can be used for a prophylactic / therapeutic agent for diseases, food and drink, feed, and the like.
[Selection] Figure 2

Description

【００７４】
［試験例４］
本試験は、ヒトラクトフェリンのシステインプロテアーゼに対する阻害効果を測定するために行った。
【００７５】
（１）試験方法
試験試料として市販のヒトラクトフェリン（シグマ社製）を使用し、システインプロテアーゼとしてパパイン、カテプシンＢ、カテプシンＬ、及びカテプシンＳ（以上いずれも市販品：和光純薬工業社製）に対して、試験例３に記載のシステインプロテアーゼ阻害活性測定方法と同様の方法によりシステインプロテアーゼ阻害活性を測定した。
【００７６】
（２）試験結果
本試験の結果は、図２に示すとおりである。図２は、パパイン、カテプシンＢ、カテプシンＬ、及びカテプシンＳに対するヒトラクトフェリンのシステインプロテアーゼ阻害効果を示す。その結果、パパイン及びカテプシンＬのシステインプロテアーゼ活性は、ヒトラクトフェリンの濃度が１０^−６Ｍに達したときにほぼ完全に阻害された。また、カテプシンＢ及びカテプシンＳのシステインプロテアーゼ活性は、ヒトラクトフェリンの濃度が１０^−５Ｍに達したときに８０％以上阻害され、１０^−４Ｍの濃度でほぼ完全に阻害された。従って、ヒトラクトフェリンは、パパイン、カテプシンＢ、カテプシンＬ、及びカテプシンＳに対してシステインプロテアーゼ阻害活性を有することが判明した。
【００７７】
［試験例５］
本試験は、ラクトフェリンの部分ペプチドのシステインプロテアーゼに対する阻害効果を測定するために行った。
【００７８】
（１）試験方法
試験試料として、後記する実施例１で製造した、配列表の配列番号１に記載のアミノ酸配列のうち、アミノ酸番号６７９〜６９５のアミノ酸配列を有するペプチド〔以下、ヒトラクトフェリンペプチドＹ_６７９−Ｋ_６９５（図３を参照）と記載する。〕を使用し、システインプロテアーゼとしてパパイン、カテプシンＢ、及びカテプシンＬ（以上いずれも市販品：和光純薬工業社製）に対して、試験例３に記載のシステインプロテアーゼ阻害活性測定方法と同様の方法によりシステインプロテアーゼ阻害活性を測定した。
【００７９】
（２）試験結果
本試験の結果は、図４に示すとおりである。図４は、パパイン、カテプシンＢ、及びカテプシンＬに対するヒトラクトフェリンペプチドＹ_６７９−Ｋ_６９５のシステインプロテアーゼ阻害効果を示す。その結果、パパイン及びカテプシンＬのシステインプロテアーゼ活性は、ペプチドの濃度が１０^−４Ｍに達したときに５０％以上阻害され、１０^−３Ｍの濃度でほぼ完全に阻害された。また、カテプシンＢのシステインプロテアーゼ活性は、ペプチドの濃度が１０^−３Ｍに達したときに６０％阻害された。従って、ヒトラクトフェリンペプチドＹ_６７９−Ｋ_６９５は、パパイン、カテプシンＢ、及びカテプシンＬに対してシステインプロテアーゼ阻害活性を有することが判明した。また、後記する実施例１で製造した、配列表の配列番号２に記載のアミノ酸配列のうち、アミノ酸番号６７６〜６９２のアミノ酸配列を有するペプチド〔以下、ウシラクトフェリンペプチドＹ_６７６−Ｋ_６９２（図３を参照）と記載する。〕について同様の阻害活性測定試験を行ったところ、ヒトラクトフェリンペプチドＹ_６７９−Ｋ_６９５とほぼ同等の活性を示すことが明らかとなった。
【００８０】
［試験例６］
本試験は、ウシラクトフェリンの部分ペプチドのシステインプロテアーゼに対する阻害効果を測定するために行った。
【００８１】
（１）試験方法
試験試料として、後記する実施例１と同様の方法で製造した、配列表の配列番号２に記載のアミノ酸配列のうち、アミノ酸番号３６〜６０のアミノ酸配列を有するペプチド〔以下、ウシラクトフェリンペプチドＦ_３６−Ｆ_６０と記載する。〕を使用し、システインプロテアーゼとしてパパイン（市販品：和光純薬工業社製）に対して、試験例３に記載のシステインプロテアーゼ阻害活性測定方法と同様の方法によりシステインプロテアーゼ阻害活性を測定した。
【００８２】
（２）試験結果
本試験の結果は、表２に示すとおりである。表２は、パパインに対するウシラクトフェリンペプチドのシステインプロテアーゼ阻害効果を示す。その結果、パパインのシステインプロテアーゼ活性は、ウシラクトフェリンペプチドＦ_３６−Ｆ_６０の濃度が１０^−４Ｍに達したときに８８％阻害された。従って、ウシラクトフェリンペプチドＦ_３６−Ｆ_６０は、パパインに対してシステインプロテアーゼ阻害活性を有することが判明した。
【００８３】
【表２】

【００８４】
［試験例７］
本試験は、トランスフェリンのシステインプロテアーゼに対する阻害効果を測定するために行った。
【００８５】
（１）試験方法
試験試料として市販のウシトランスフェリン（日本製薬社製）を使用し、システインプロテアーゼとしてパパイン、カテプシンＢ、及びカテプシンＬ（以上いずれも市販品：和光純薬工業社製）に対して、試験例３に記載のシステインプロテアーゼ阻害活性測定方法と同様の方法によりシステインプロテアーゼ阻害活性を測定した。
【００８６】
（２）試験結果
本試験の結果は、図５に示すとおりである。図５は、パパイン、カテプシンＢ、及びカテプシンＬに対するトランスフェリンのシステインプロテアーゼ阻害効果を示す。その結果、パパインのシステインプロテアーゼ活性は、トランスフェリンの濃度が１０^−４Ｍに達したときに４０％阻害され、１０^−３Ｍの濃度で完全に阻害された。また、カテプシンＢ及びカテプシンＬのシステインプロテアーゼ活性は、トランスフェリンの濃度が１０^−４Ｍに達したときに３０％以上阻害された。従って、ウシトランスフェリンは、パパイン、カテプシンＢ、及びカテプシンＬに対してシステインプロテアーゼ阻害活性を有することが判明した。
【００８７】
【実施例】
次に実施例を示して本発明を更に詳細に説明する。尚、本発明は以下の実施例に限定されるものではない。
［実施例１］
配列表の配列番号１に記載のアミノ酸配列のうち、アミノ酸番号６７９〜６９５のアミノ酸配列を有するペプチド（図３を参照：ヒトラクトフェリンペプチドＹ_６７９−Ｋ_６９５）を、以下の方法により製造した。
【００８８】
尚、前記本発明のペプチドは、自動アミノ酸合成装置（アプライド・バイオシステムズ社製。Ｍｏｄｅｌ ４３３Ａ）を用いて合成を行い製造を行った。
【００８９】
２０％ピペリジン含有Ｎ−メチルピロリドン（アプライド・バイオシステムズ社製。以下、Ｎ−メチルピロリドンをＮＭＰと略記する）により、ペプチド合成用固相樹脂であるＨＭＰ樹脂（アプライド・バイオシステムズ社製）のアミノ保護基であるＦｍｏｃ基を切断除去し、ＮＭＰで洗浄した後、 Ｆｍｏｃ−スレオニン［具体的には、合成するペプチドのＣ末端アミノ酸に相当するＦｍｏｃ−アミノ酸（アプライド・バイオシステムズ社製）］をＦａｓｔＭｏｃ（登録商標）リージェントキット（アプライド・バイオシステムズ社製）を使用して縮合させ、ＮＭＰで洗浄した。次に、前記Ｆｍｏｃ基の切断、続いて、Ｃ末端から２番目のアミノ酸に相当するＦｍｏｃ−アラニンの縮合、及び洗浄を行い、さらにＦｍｏｃ−アミノ酸の縮合及び洗浄を繰り返し、保護ペプチド樹脂を作製し、樹脂より粗製ペプチドを回収した。
【００９０】
前記粗製ペプチドから、高速液体クロマトグラフィー（以下、ＨＰＬＣと略記する。）によりペプチドの精製を行った。カラムは逆相系のＣ１８−ＯＤＳ（メルク社製。Ｌｉｃｈｒｏｓｐｈｅｒ１００）を使用した。得られた精製ペプチドはＨＰＬＣ分析を行い、精製物が単一であることを更に確認した。また、精製ペプチドのアミノ酸配列を、気相式自動アミノ酸シーケンサー（アプライド・バイオシステムズ社製。Ｍｏｄｅｌ ４７３Ａ）を用いて決定した結果、配列番号１のアミノ酸番号６７９〜６９５のアミノ酸配列を有していた。
【００９１】
尚、同様の方法により配列番号２に記載のアミノ酸配列のうち、アミノ酸番号６７６〜６９２のアミノ酸配列を有するペプチド（図３を参照：ウシラクトフェリンペプチドＹ_６７６−Ｋ_６９２）も製造した。
【００９２】
［実施例２］
（ラクトフェリンを配合した錠剤の調製）
次の組成からなる錠剤のシステインプロテアーゼ阻害剤を次の方法により製造した。
ウシラクトフェリン（森永乳業社製） ４０．０（％）
乳糖（森永乳業社製） １８．５
トウモロコシ澱粉（日清製粉社製） ３０．７
ステアリン酸マグネシウム（太平化学産業社製） １．４
カルボキシメチルセルロ−スカルシウム（五徳薬品社製） ９．４
【００９３】
ウシラクトフェリン、乳糖、トウモロコシ澱粉及びカルボキシメチルセルロ−スカルシウムの混合物に、滅菌精製水を適宜添加しながら均一に混練し、５０℃で３時間乾燥させ、得られた乾燥物にステアリン酸マグネシウムを添加して混合し、常法により打錠し、錠剤を得た。
【００９４】
［実施例３］
（カプセル入りラクトフェリンの調製）
乳糖（和光純薬工業社製）６００ｇ、トウモロコシデンプン（日清製粉社製）４００ｇ、結晶セルロース（和光純薬工業社製）４００ｇ及びウシラクトフェリン（森永乳業社製）６００ｇを、５０メッシュ篩（ヤマト科学社製）により篩分けし、厚さ０．５ｍｍのポリエチレン製の袋にとり、転倒混合し、全自動カプセル充填機（Ｃｅｓｅｒｅ Ｐｅｄｉｎｉ社製。プレス式）を用い、前記粉末をカプセル（日本エランコ社製。１号ゼラチンカプセル、Ｏｐ．Ｙｅｌｌｏｗ Ｎｏ．６ Ｂｏｄｙ、空重量は７５ｍｇ）に内容量２７５ｍｇで充填し、ウシラクトフェリン８２ｍｇ入りのカプセル剤７，０００個を得た。
【００９５】
［実施例４］
（ウシラクトフェリンを添加した飲料の調製）
脱脂粉乳（森永乳業社製）９０ｇを５０℃の温湯８００ｍｌに溶解し、砂糖（日新製糖社製）３０ｇ、インスタントコーヒー粉末（ネスレ社製）１４ｇ、カラメル（昭和化工社製）２ｇ、及びコーヒーフレーバー（三栄化学社製）０．０１ｇ、を攪拌しながら順次添加して溶解し、１０℃に冷却し、ウシラクトフェリン（森永乳業社製）１ｇを添加し、ウシラクトフェリン約０．１％を含むシステインプロテアーゼ阻害効果を有する乳飲料を調製した。
【００９６】
［実施例５］
（ウシラクトフェリンを添加した経腸栄養食粉末の調製）
ホエー蛋白酵素分解物（森永乳業社製）１０．８ｋｇ、デキストリン（昭和産業社製）３６ｋｇ、及び少量の水溶性ビタミンとミネラルを水２００ｋｇに溶解し、水相をタンク内に調製した。これとは別に、大豆サラダ油（太陽油脂社製）３ｋｇ、パーム油（太陽油脂社製）８．５ｋｇ、サフラワー油（太陽油脂社製）２．５ｋｇ、レシチン（味の素社製）０．２ｋｇ、脂肪酸モノグリセリド（花王社製）０．２ｋｇ、及び少量の脂溶性ビタミンを混合溶解し、油相を調製した。タンク内の水相に油相を添加し、攪拌して混合した後、７０℃に加温し、更にホモゲナイザーにより１４．７ＭＰａの圧力で均質化した。次いで、９０℃で１０分間殺菌した後に、濃縮し、噴霧乾燥して、中間製品粉末約５９ｋｇを調製した。この中間製品粉末５０ｋｇに、蔗糖（ホクレン社製）６．８ｋｇ、アミノ酸混合粉末（味の素社製）１６７ｇ、及びウシラクトフェリン（森永乳業社製）６０ｇを添加し、均一に混合して、ウシラクトフェリンを含有するシステインプロテアーゼ阻害効果を有する経腸栄養食粉末約５７ｋｇを製造した。
【００９７】
【発明の効果】
以上詳記したとおり、本発明はラクトフェリン、ラクトフェリンの部分ペプチド、及びトランスフェリンからなる群より選択される１種又は複数種の混合物を有効成分として含有するシステインプロテアーゼ阻害剤に関するものであり、本発明により奏される効果は次のとおりである。
（１）食品素材として利用することができるタンパク質であるので、安全性に優れ日常的に長期間投与又は摂取が可能である。
（２）幅広いシステインプロテアーゼに対して阻害活性スペクトルを有する。
（３）システインプロテアーゼが関与する疾患の予防・治療剤として使用することが可能である。
【００９８】
【配列表】

【図面の簡単な説明】
【図１】図１は、ウシ乳タンパク質の逆ザイモグラフィーの検出を示す図（写真）である。
【図２】図２は、システインプロテアーゼに対するヒトラクトフェリンのシステインプロテアーゼ阻害効果を示す図である。
【図３】図３は、実施例１で製造したヒトラクトフェリンの部分ペプチド（ヒトラクトフェリンペプチドＹ_６７９−Ｋ_６９５）、及びウシラクトフェリンの部分ペプチド（ウシラクトフェリンペプチドＹ_６７６−Ｋ_６９２）、並びに配列表の配列番号３に記載のアミノ酸配列のうち、アミノ酸番号６６６〜６８２のアミノ酸配列を有するペプチド（ヒトトランスフェリンペプチドＹ_６６６−Ｒ_６８２）、及び配列表の配列番号４に記載のアミノ酸配列のうち、アミノ酸番号６７２〜６８８のアミノ酸配列を有するペプチド（ウシトランスフェリンペプチドＹ_６７２−Ｒ_６８８）のアミノ酸配列を示す図である。
【図４】図４は、システインプロテアーゼに対するヒトラクトフェリンペプチドＹ_６７９−Ｋ_６９５のシステインプロテアーゼ阻害効果を示す図である。
【図５】図５は、システインプロテアーゼに対するトランスフェリンのシステインプロテアーゼ阻害効果を示す図である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a cysteine protease inhibitor containing, as an active ingredient, one or more kinds of mixtures selected from the group consisting of lactoferrin, lactoferrin partial peptide, and transferrin, osteoporosis, malignant neoplastic hypercalcemia It is a cysteine protease inhibitor that can be used as a prophylactic / therapeutic agent for diseases and the like, foods and drinks, feeds and the like.
[0002]
[Prior art]
Proteolytic enzymes having a thiol group at the active center are collectively referred to as cysteine proteases (thiol proteases). Cathepsin L and cathepsin B are one of the representative cysteine proteases together with calcium-dependent neutral protease (CAMP), papain, ficin, promelain and the like. Substances having an inhibitory effect on these cysteine proteases are diseases in which cysteine proteases are involved, for example, muscular dystrophy, muscular atrophy, myocardial infarction, stroke, Alzheimer's disease, impaired consciousness and movement at the time of head trauma, multiple occurrences For the treatment of sclerosis, peripheral nerve neuropathy, cataract, inflammation, allergy, fulminant hepatitis, osteoporosis, hypercalcemia, breast cancer, prostate cancer, benign prostatic hyperplasia, or for suppressing cancer growth, preventing metastasis, platelets Promising as an aggregation inhibitor. In recent years, Katsunuma et al. Have elucidated the relationship between cathepsin L and cathepsin B and osteoporosis or malignant hypercalcemia. Attention has been focused on application to medicines as a therapeutic agent for bloodemia (for example, see Non-Patent Document 1). In bone tissue, bone formation by osteoblasts and bone resorption by osteoclasts are performed throughout the life, and during growth, bone weight increases due to bone formation exceeding bone resorption. On the other hand, in old age, on the other hand, bone resorption exceeds bone formation, so that bone weight decreases and osteoporosis develops. Although there are various causes of these osteoporosis, bone breakdown (bone resorption) can be particularly cited as one of the main causes. This can be further divided into two causes as follows. That is, one is caused by insufficient absorption and deposition of calcium, and more specifically, it relates to the supply, transfer, absorption, and deposition of calcium. Vitamin D derivatives, female hormones (estrogens), etc. Probably involved. The other is to promote the degradation of collagen, which is a bone-supporting tissue, and is mainly due to the degradation of bone collagen by cysteine proteases secreted from lysosomes in osteoclasts, especially cathepsin L and cathepsin B. is there. These cathepsins L and B, secreted from lysosomes in osteoclasts, promote the degradation of collagen in bone tissue, thereby lysing old bone and releasing calcium along with hydroxyproline into the blood. Therefore, by inhibiting the degradation of cathepsins L and B by collagen, excessive bone breakdown can be prevented, and thus osteoporosis can be treated. As therapeutic agents for these osteoporosis, estrogen, anabolic hormone, calcium, vitamin D, calcitonin, bisphosphonate and the like are known. As for osteoporosis therapeutic agents having the mechanism of action of so-called cysteine protease inhibition of cathepsin L inhibition and cathepsin B inhibition, development of osteoporosis treatment agents using several cysteine protease inhibitors has been promoted. The development of is desired.
[0003]
On the other hand, hypercalcemia is a metabolic disorder in which the calcium concentration in serum is higher than a normal value, and is often found in tumor patients. If left unchecked, the life of the patient is said to be about 10 days. Many of the causes are bone metastases of the tumor. When the tumor spreads to bone, bone destruction occurs and calcium is released into the blood. This calcium is processed in the kidney, but when the speed of bone destruction exceeds the processing capacity of the kidney, hypercalcemia develops. As a treatment method, a method of promoting excretion of calcium from the kidney by using an infusion of physiological saline combined with furosemide, a method of using calcitonin which is a therapeutic agent for osteoporosis, and the like are known. That is, it can be said that a therapeutic agent for osteoporosis such as suppressing bone resorption is also effective as a therapeutic agent for malignant neoplastic hypercalcemia.
[0004]
The following publications have already been disclosed by the present inventors as cysteine protease inhibitors that can be used for such a purpose.
(1) Cathepsin L-specific inhibitory polypeptide (Patent Document 1)
(2) Thiol protease inhibitor (Patent Document 2)
(3) Valine derivative and its use (Patent Document 3)
(4) Thiol protease inhibitor (Patent Document 4)
(5) FA-70C1 substance (Patent Document 5)
(6) FA-70D substance, its production method and its use (Patent Document 6)
However, further development of a cysteine protease inhibitor that has no antigenicity and can be used as a safe material has been desired.
[0005]
On the other hand, it is known that protease inhibitors exist in breast milk. Known protease inhibitors in breast milk include α ₁ -Antichymotrypsin, α ₁ Anti-trypsin, inter-alpha ₂ -Trypsin inhibitor, α ₂ -Antiplasmin, α ₂ -Including trace amounts of inhibitors such as macroglobulin, antithrombin III, and antileucoprotease (for example, see Non-Patent Document 2).
[0006]
The following publications have already been disclosed for proteins having cysteine protease inhibitory activity in milk.
(1) A novel cysteine protease inhibitor having a sugar chain derived from bovine colostrum and having a molecular weight of about 57 kDa (Patent Document 7)
(2) A novel cysteine protease inhibitor having a molecular weight of 16 ± 2 kDa or 13 ± 2 kDa derived from bovine colostrum (Patent Document 8)
(3) Lactoferrin and β-casein are examples of novel proteins derived from human milk having a molecular weight of 16 ± 2 kDa or 13 ± 2 kDa and proteins contained in large amounts in mammalian milk. Lactoferrin (lactoferrin: hereinafter may be abbreviated as Lf) is an iron-binding glycoprotein having a molecular weight of 80 kDa mainly contained in breast milk, and against harmful microorganisms such as Escherichia coli, Candida, Clostridium, and Staphylococcus. Is known to exhibit antibacterial action (for example, see Non-Patent Document 3 and Non-Patent Document 4). Also, as a milk protein having various actions, it is widely used as a therapeutic agent for diseases. Lactoferrin is a protein derived from milk, which is highly safe, can be used for a long period of time, is almost tasteless and odorless, and is a highly versatile protein as an additive for various foods, pharmaceuticals and feeds. .
[0007]
In addition, as an example of applying lactoferrin to a therapeutic agent for diseases,
(1) Antitumor agent (Patent Document 10)
(2) Anti-rheumatic agent (Patent Document 11)
(3) IgA production promoter (Patent Document 12)
(4) Infant formula for improving constipation (Patent Document 13)
(5) Angiogenic therapeutic agent (Patent Document 14)
(6) Oral cancer metastasis inhibitor (Patent Document 15)
(7) IgE production inhibitor (Patent Document 16)
Are disclosed.
[0008]
In addition, regarding the decomposition product of Lf,
(8) Antibacterial activity and inhibition of thyroxinase activity (Patent Document 17)
(9) Prevention of adhesion of pathogenic bacteria to cells (Patent Document 18)
(10) Antiviral action (Patent Document 19)
Are disclosed.
[0009]
Further, it has been disclosed that a bone enhancer containing iron-lactoferrin as an active ingredient is effective for prevention and treatment of bone diseases (Patent Document 20). In this technology, iron-lactoferrin in which iron and lactoferrin are bound is administered as an active ingredient, and iron, which is a coenzyme of enzymes involved in hydroxylation of proline and lysine, which are precursors of collagen, which is a bone matrix, is supplied. It enhances collagen production.
[0010]
As described above, cysteine protease inhibitors have been found so far, and some cysteine protease inhibitors have also been found in animal cells, blood, urine, milk and the like. Proteinaceous cysteine protease inhibitors are collectively referred to as cystatins. The cystatin family is known to have a common active site, and its sequence has been clarified (Non-Patent Document 5).
[0011]
However, it is not known that lactoferrin and its partial peptide, and transferrin have a cysteine protease inhibitory action, and that lactoferrin and transferrin have a region having homology to the common active site sequence of the cystatin family. Was not known. Furthermore, lactoferrin and its partial peptide, and a preventive / therapeutic agent for a bone disease whose mechanism of action is cysteine protease inhibition by transferrin have not been known.
[0012]
[Patent Document 1]
JP-A-7-179496
[Patent Document 2]
JP-A-9-221425
[Patent Document 3]
JP 2001-139534 A
[Patent Document 4]
JP-A-7-242600
[Patent Document 5]
JP-A-2000-72797
[Patent Document 6]
WO 97/31122 pamphlet
[Patent Document 7]
JP-A-7-2896
[Patent Document 8]
JP-A-7-126294
[Patent Document 9]
JP-A-10-80281
[Patent Document 10]
Japanese Patent Publication No. 5-86932
[Patent Document 11]
JP-A-5-186368
[Patent Document 12]
JP-A-6-32743
[Patent Document 13]
JP-A-6-205640
[Patent Document 14]
JP-A-9-194388
[Patent Document 15]
JP-A-10-59864
[Patent Document 16]
JP 2001-158748 A
[Patent Document 17]
European Patent Publication No. 438750
[Patent Document 18]
JP-A-3-220130
[Patent Document 19]
JP-A-1-233226
[Patent Document 20]
JP 2000-281586 A
[0013]
[Non-patent document 1]
Nobuhiko Katsunuma, "BIO media", Vol. 7, No. 6, pp. 73-77, 1992
[Non-patent document 2]
Isao Kiyozawa, Nutrition of Breast Milk, Kanehara Publishing, pp. 80-81
[Non-Patent Document 3]
Journal of Pediatrics, vol. 94, page 1, 1979
[Non-patent document 4]
Journal of Daily Science, vol. 67, p. 60, 1984
[Non-Patent Document 5]
Osamu Hayaishi, "Protease and its Inhibitors", Medical View, 1st edition, 1st edition, pp. 104-115, 1993
[0014]
[Problems to be solved by the invention]
The present invention can be widely used as a food material, osteoporosis, preventive and therapeutic agents for malignant neoplastic hypercalcemia, and various versatile foods and feeds, etc. It is intended to provide a high cysteine protease inhibitor.
[0015]
[Means for Solving the Problems]
The present inventors have conducted a thorough search for cysteine protease inhibitors that can be used as a safe material without antigenicity, and as a result, lactoferrin and lactoferrin-derived peptide fragments that are milk-derived proteins, and transferrin have cysteine protease inhibition. They have found that they have activity, and have completed the present invention.
[0016]
The gist of the present invention is as follows (1) to (8).
(1) A cysteine protease inhibitor containing, as an active ingredient, one or a mixture of lactoferrin, a partial peptide of lactoferrin, and transferrin.
[0017]
(2) The cysteine protease inhibitor according to the above (1), wherein the lactoferrin and / or transferrin is one or a mixture of one or more selected from the group consisting of a metal saturated type, a partially saturated metal type, and an apo type.
[0018]
(3) The cysteine protease according to (1) or (2), wherein the lactoferrin and / or the partial peptide of lactoferrin is a mixture of one or more of the following proteins or peptides shown in (a) to (d): Inhibitors.
(A) A protein or peptide having at least the amino acid sequence of amino acids 679 to 695 among the amino acid sequences described in SEQ ID NO: 1 in the sequence listing.
(B) a peptide having at least the amino acid sequence of amino acids 679 to 695 in the amino acid sequence of SEQ ID NO: 1 in the sequence listing, wherein one or more amino acids are substituted, deleted, added, or inverted; A protein or peptide comprising cysteine protease inhibitory activity.
(C) a protein or peptide having at least the amino acid sequence of amino acids 676 to 692 among the amino acid sequences described in SEQ ID NO: 2 in the sequence listing;
(D) a peptide having at least the amino acid sequence of amino acids 676 to 692 in the amino acid sequence of SEQ ID NO: 2 in the sequence listing, wherein one or more amino acids are substituted, deleted, added, or inverted; A protein or peptide comprising cysteine protease inhibitory activity.
[0019]
(4) The cysteine protease according to (1) or (2), wherein the lactoferrin and / or the partial peptide of lactoferrin is a mixture of one or more of the following proteins or peptides shown in (e) to (j): Inhibitors.
(E) a protein or peptide having at least the amino acid sequence of amino acids 20 to 30 in the amino acid sequence of SEQ ID NO: 1 in the sequence listing;
(F) A peptide having at least the amino acid sequence of amino acids 20 to 30 in the amino acid sequence of SEQ ID NO: 1 in the sequence listing, wherein one or more amino acids are substituted, deleted, added, or inverted. A protein or peptide comprising cysteine protease inhibitory activity.
(G) A protein or peptide having at least the amino acid sequence of amino acids 31 to 66 among the amino acid sequences described in SEQ ID NO: 1 in the sequence listing.
(H) a peptide having at least the amino acid sequence of amino acids 31 to 66 in the amino acid sequence of SEQ ID NO: 1 in the sequence listing, wherein one or more amino acids are substituted, deleted, added, or inverted; A protein or peptide comprising cysteine protease inhibitory activity.
(I) A protein or peptide having at least the amino acid sequence of amino acid numbers 20 to 66 among the amino acid sequences described in SEQ ID NO: 1 in the sequence listing.
(J) a peptide having an amino acid sequence of at least amino acids 20 to 66 in the amino acid sequence of SEQ ID NO: 1 in the sequence listing, wherein one or more amino acids are substituted, deleted, added, or inverted; A protein or peptide comprising cysteine protease inhibitory activity.
[0020]
(5) The cysteine protease according to (1) or (2), wherein the lactoferrin and / or the partial peptide of lactoferrin is one or a mixture of proteins or peptides shown in the following (k) to (n): Inhibitors.
(K) A protein or peptide having at least the amino acid sequence of amino acids 36 to 60 among the amino acid sequences described in SEQ ID NO: 2 in the sequence listing.
(L) a peptide having at least the amino acid sequence of amino acids 36 to 60 in the amino acid sequence of SEQ ID NO: 2 in the sequence listing, wherein one or more amino acids are substituted, deleted, added, or inverted; A protein or peptide comprising cysteine protease inhibitory activity.
(M) A protein or peptide having at least the amino acid sequence of amino acids 39 to 44 among the amino acid sequences described in SEQ ID NO: 2 in the sequence listing.
(N) a peptide having at least the amino acid sequence of amino acids 39 to 44 in the amino acid sequence of SEQ ID NO: 2 in the sequence listing, wherein one or more amino acids are substituted, deleted, added, or inverted; A protein or peptide comprising cysteine protease inhibitory activity.
[0021]
(6) The cysteine protease inhibitor according to any of (1) to (5) above, which is a prophylactic / therapeutic agent for a disease involving cysteine protease.
[0022]
(7) The cysteine protease inhibitor according to any of (1) to (6), wherein the disease involving cysteine protease is osteoporosis or malignant hypercalcemia.
[0023]
(8) A food or drink composition or feed composition to which the cysteine protease inhibitor according to any of (1) to (7) is added.
[0024]
BEST MODE FOR CARRYING OUT THE INVENTION
Next, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the following preferred embodiments, and can be freely modified within the scope of the present invention. In addition, in this specification, the percentage is represented by mass unless otherwise specified.
[0025]
The present invention is a cysteine protease inhibitor containing, as an active ingredient, one or a mixture of lactoferrin, a partial peptide of lactoferrin, and transferrin.
[0026]
The lactoferrin used in the present invention is commercially available lactoferrin, colostrum, transitional milk, normal milk, terminal milk, or processed milk of mammals (eg, human, cow, goat, sheep, horse, etc.). Lactoferrin obtained by separating from the above-mentioned raw materials using non-fat milk, whey or the like as a raw material, for example, by a conventional method such as ion exchange chromatography. Among them, it is preferable to use commercially available lactoferrin manufactured on an industrial scale (for example, manufactured by Morinaga Milk Industry Co., Ltd.). Furthermore, it is also possible to use lactoferrin produced in microorganisms, animal cells, transgenic animals, etc. by genetic engineering techniques.
[0027]
In the effect of the cysteine protease inhibitor of the present invention, the metal content in lactoferrin is not particularly limited, and in the present invention, apo-lactoferrin obtained by removing lactoferrin with hydrochloric acid, citric acid, or the like; A metal-saturated lactoferrin with a degree of saturation of 100%, obtained by chelating with a metal such as copper, zinc, manganese, etc .; and a metal-saturated lactoferrin with a metal bound at a degree of saturation of less than 100%. Any one or a mixture of selected ones can be used.
[0028]
The method for producing the partial peptide of lactoferrin used in the present invention includes a method for producing the lactoferrin by hydrolysis with an acid or a protease by a known method, and a method for synthesizing a recombinant peptide using a genetic engineering technique. And a method for producing a synthetic peptide by chemical synthesis. When the method of production by hydrolysis is used, the partial peptide of lactoferrin of the present invention may be used in a form contained as a mixture in the hydrolyzate, or used in a form purified by HPLC or the like according to a conventional method. May be.
[0029]
As a preferred form of lactoferrin or a partial peptide of lactoferrin used in the present invention, at least amino acid numbers 679 to 695 and amino acid numbers 20 to 20 of the amino acid sequence described in SEQ ID NO: 1 in the amino acid sequence of human lactoferrin. Among them, a protein or peptide having any amino acid sequence among 30 and / or amino acid numbers 31 to 66 and amino acid numbers 20 to 66 can be exemplified. In addition, among the amino acid sequences described in SEQ ID NO: 2 in the sequence listing, which is the amino acid sequence of bovine lactoferrin, any one of at least amino acids 676 to 692, amino acids 36 to 60, and amino acids 39 to 44 And the like. In addition, in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing and the amino acid sequence described in SEQ ID NO: 2 in the sequence listing, amino acid numbers 1 to 19 are signal sequences.
[0030]
Among them, among the amino acid sequences described in SEQ ID NO: 1, amino acid numbers 679 to 695, and amino acid numbers 20 to 30 and / or amino acid numbers 31 to 66, and amino acid numbers 20 to 66 Has a cysteine protease inhibitory activity, and can be used as the cysteine protease inhibitor of the present invention. Furthermore, in the amino acid sequence of SEQ ID NO: 2, a peptide having any one of amino acid numbers 676 to 692, amino acids 36 to 60, and amino acids 39 to 44 has a cysteine protease inhibitory activity. However, it can be used for the cysteine protease inhibitor of the present invention.
[0031]
In addition, since lactoferrin itself has a cysteine protease inhibitory activity, any of amino acid numbers 679 to 695, amino acid numbers 20 to 30 and / or amino acid numbers 31 to 66, and amino acid numbers 20 to 66 of SEQ ID NO: 1 in the sequence listing can be used. And a peptide having an amino acid sequence having an extended sequence on the N-terminal side or the C-terminal side or on both sides thereof is also considered to have cysteine protease inhibitory activity. Furthermore, an amino acid comprising any one of amino acids 676 to 692, amino acids 36 to 60, and amino acids 39 to 44 of SEQ ID NO: 2 in the sequence listing and having the sequence extended to the N-terminal side or the C-terminal side or both. A peptide having a sequence is also considered to have cysteine protease inhibitory activity.
[0032]
These proteins and peptides can be obtained, for example, by chemical synthesis based on the amino acid sequence containing the cysteine protease inhibitory region according to the present invention since the region has been clarified by the present invention, or can be obtained by genetic recombination technology or the like. You can also. For example, an appropriate primer is prepared based on a base sequence encoding an amino acid sequence containing the region, and the base sequence is amplified by PCR or the like using the primer and a cDNA containing the target base sequence as a template. The expressed nucleotide sequence can be obtained by expressing the nucleotide sequence using an appropriate expression system.
[0033]
In addition, in ordinary genes, mutations such as substitution, deletion, insertion, addition, or inversion of one or more bases at one or more positions naturally exist depending on differences in species, genera, individuals, and the like. In some cases, mutations may occur in amino acids of proteins encoded by genes having such mutations. Lactoferrin and partial peptides of lactoferrin that can be used in the present invention include those containing such mutations as long as the cysteine protease inhibitory activity is not impaired.
[0034]
Here, lactoferrin or a partial peptide of lactoferrin that can be used in the present invention includes at least amino acid numbers 679 to 695, amino acid numbers 20 to 30, and amino acid numbers 31 to 31 in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing. 66 and a protein or peptide having any one of the amino acid sequences of amino acid numbers 20 to 66, and among the amino acid sequences of SEQ ID NO: 2, at least amino acid numbers 676 to 692, amino acid numbers 36 to 60, and amino acid number 39 A protein or peptide having any one of amino acids 44 to 44, including one or more amino acid substitutions, deletions, insertions, additions or inversions, and having a cysteine protease inhibitory activity. . Incidentally, what is possible as “substitution, deletion, insertion, addition or inversion of one or more amino acids” is a steric interaction between a protein or a peptide having at least the amino acid sequence of the amino acid number and a cysteine protease. It can be arbitrarily set within a range that does not affect the action and does not impair the cysteine protease inhibitory activity. For example, the term “plurality” refers to any one of amino acids 679 to 695, amino acids 20 to 30, amino acids 31 to 66, and amino acids 20 to 66 described in SEQ ID NO: 1 in the sequence listing; Among the amino acids 676 to 692, amino acids 36 to 60, and amino acids 39 to 44 of SEQ ID NO: 2, the amino acid residues vary depending on the position and type in the three-dimensional structure of the protein. For example, 2 to 5, preferably 2 to 3, and more preferably 2.
[0035]
Furthermore, in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing, any one of amino acids 679 to 695, amino acids 20 to 30, amino acids 31 to 66, and amino acids 20 to 66, and the sequence listing In any of amino acids 676 to 692, amino acids 36 to 60, and amino acids 39 to 44 described in SEQ ID NO: 2, it is desirable to substitute the amino acid within a range that does not change the type of amino acid. That is, it is desirable that the amino acid in the sequence and the amino acid that substitutes for the same amino acid are amino acids of similar type in structural classification. Specifically, when the amino acid of the sequence is an acidic amino acid or an acidic amino acid amide, substitution with any of aspartic acid, glutamic acid, asparagine, or glutamine, and when the amino acid is a basic amino acid, lysine, histidine, or arginine Substitution with any one of them, phenylalanine when it is an aromatic amino acid, tyrosine, or substitution with any one of tryptophan, when it is an aliphatic amino acid or an oxyamino acid, glycine, alanine, valine, leucine, isoleucine, serine Alternatively, substitution with any of threonine, and in the case of amino acids other than the above (cysteine, methionine, proline, etc.), it is desirable to arbitrarily substitute as long as the cysteine protease inhibitory activity is not impaired. In addition, among the amino acid sequences described in SEQ ID NO: 1 in the sequence listing, amino acids in the range other than amino acids 679 to 695, amino acids 20 to 30, amino acids 31 to 66, and amino acids 20 to 66, or Among the amino acids in the range other than the amino acids 676 to 692, amino acids 36 to 60, and amino acids 39 to 44 in the description of SEQ ID NO: 2 in the sequence listing, the positions and types of the amino acid residues in the three-dimensional structure of the protein For example, the number is 2 to 10, preferably 2 to 5, and more preferably 2 to 3.
[0036]
The nucleotide sequence encoding a protein or peptide substantially the same as the lactoferrin protein or a partial peptide of lactoferrin as described above, for example, by site-directed mutagenesis, substitution of amino acid residues at a specific site, deletion, insertion, It can be obtained by modifying the base sequence to include addition or inversion. The modified base sequence as described above can also be obtained by a conventionally known mutation treatment.
[0037]
By expressing the nucleotide sequence having the above mutation in an appropriate cell, and examining the cysteine protease inhibitory activity by the method for measuring the cysteine protease inhibitory activity described in Test Examples or Examples of the present invention, the lactoferrin protein or lactoferrin protein A nucleotide sequence encoding a protein or peptide substantially identical to the partial peptide is obtained.
[0038]
The transferrin used in the present invention may be a commercially available transferrin or a mammal (eg, human, cow, goat, sheep, horse, etc.) milk or blood, etc., and separated by, for example, column chromatography. Can be used. Furthermore, it is also possible to use transferrin produced by microorganisms, animal cells, transgenic animals, etc. by genetic engineering techniques.
[0039]
In the effect of the cysteine protease inhibitor of the present invention, the metal content in transferrin is not particularly limited, and in the present invention, apo-type transferrin in which transferrin is deferred with hydrochloric acid, citric acid, or the like; A metal-saturated transferrin having a degree of saturation of 100% obtained by chelating with a metal such as copper, zinc, or manganese; and a metal partially-saturated transferrin having a metal bound at various degrees of saturation of less than 100%. Any one or a mixture of selected ones can be used.
[0040]
In the present invention, a partial peptide of transferrin can also be used. A comparison of the amino acid sequence (amino acids 679 to 695 of SEQ ID NO: 1 and amino acids 676 to 692 of SEQ ID NO: 2) deduced to be the cysteine protease inhibitory active region of lactoferrin with the amino acid sequence of transferrin resulted in SEQ ID NO: 3 in the amino acid sequence of human transferrin (amino acid number 666 to 682) (human transferrin peptide Y ₆₆₆ -R ₆₈₂ ) And the amino acid sequence of bovine transferrin shown in SEQ ID NO: 4 (amino acid numbers 672 to 688 (bovine transferrin peptide Y) ₆₇₂ -R ₆₈₈ ) Showed high homology with the cysteine protease inhibitory active region of lactoferrin (see FIG. 3). Since this and transferin showed cysteine protease inhibitory activity as shown in the Examples below, partial peptides of human transferrin and bovine transferrin containing the above-mentioned respective regions also have cysteine protease inhibitory activity. Is strongly suggested.
[0041]
The method for producing a partial peptide of transferrin used in the present invention includes a method of producing the transferrin by hydrolyzing the transferrin with an acid or a protease by a known method, and a method of synthesizing a recombinant peptide using a genetic engineering technique. And a method for producing a synthetic peptide by chemical synthesis. When the method of production by hydrolysis is used, the partial peptide of transferrin of the present invention may be used in the form of a mixture in the hydrolyzate, or used in a form purified by HPLC or the like according to a conventional method. May be.
[0042]
A preferred form of transferrin or a partial peptide of transferrin used in the present invention is a protein having at least the amino acid sequence of amino acid numbers 666 to 682 among the amino acid sequences described in SEQ ID NO: 3 in the amino acid sequence of human transferrin. Alternatively, among the amino acid sequences described in SEQ ID NO: 4 in the Sequence Listing, which is the amino acid sequence of bovine transferrin, include peptides and proteins or peptides having at least the amino acid sequence of amino acids 672 to 688. In addition, among the amino acid sequences described in SEQ ID NO: 3 in the sequence listing and the amino acid sequences described in SEQ ID NO: 4 in the sequence listing, the amino acid sequences of amino acids 1 to 19 are signal sequences.
[0043]
The peptide consisting of the amino acid sequence of amino acids 666 to 682 in the amino acid sequence of SEQ ID NO: 3 and the peptide consisting of the amino acid sequence of 672 to 688 in the amino acid sequence of SEQ ID NO: 4 are the present invention. Among the amino acid sequences described in SEQ ID NO: 1 in the sequence listing, which is the amino acid sequence of human lactoferrin, which is a cysteine protease inhibitory active region, the amino acid sequence of amino acids 679 to 695 and the amino acid sequence of bovine lactoferrin Among the amino acid sequences described in SEQ ID NO: 2 in the sequence listing, the amino acid sequence is considered to have cysteine protease inhibitory activity based on homology with the amino acid sequences of amino acids 676 to 692. In addition, since transferrin itself has a cysteine protease inhibitory activity, of the amino acid sequence described in SEQ ID NO: 3, it includes the amino acid sequence of amino acids 666 to 682 and extends the sequence to the N-terminal side or the C-terminal side or both. A peptide having the amino acid sequence of SEQ ID NO: 4 and a peptide having an amino acid sequence having an extended amino acid sequence on the N-terminal side or the C-terminal side or on both of them, including cysteine protease inhibitor It is considered to have activity.
[0044]
These proteins and peptides can be obtained by chemical synthesis based on the amino acid sequence containing the cysteine protease inhibitory region, for example, since the present invention has revealed the region presumed to be the cysteine protease inhibitory region by the present invention. It can also be obtained by technology. For example, an appropriate primer is prepared based on a base sequence encoding an amino acid sequence containing the region, and the base sequence is amplified by PCR or the like using the primer and a cDNA containing the target base sequence as a template. The expressed nucleotide sequence can be obtained by expressing the nucleotide sequence using an appropriate expression system.
[0045]
In addition, in ordinary genes, mutations such as substitution, deletion, insertion, addition, or inversion of one or more bases at one or more positions naturally exist depending on differences in species, genera, individuals, and the like. In some cases, mutations may occur in amino acids of proteins encoded by genes having such mutations. Transferrin and partial peptides of transferrin that can be used in the present invention include those containing such mutations as long as the cysteine protease inhibitory activity is not impaired.
[0046]
Here, transferrin or a partial peptide of transferrin that can be used in the present invention includes, among the amino acid sequences of SEQ ID NO: 3, a peptide consisting of at least the amino acid sequence of amino acids 666 to 682, and SEQ ID NO: 4. A protein or peptide having at least the amino acid sequence of amino acid numbers 672 to 688 in the amino acid sequence, comprising one or more amino acid substitutions, deletions, insertions, additions or inversions, and having a cysteine protease inhibitory activity Or a peptide. Incidentally, what is possible as “substitution, deletion, insertion, addition or inversion of one or more amino acids” is a steric interaction between a protein or a peptide having at least the amino acid sequence of the amino acid number and a cysteine protease. It can be arbitrarily set within a range that does not affect the action and does not impair the cysteine protease inhibitory activity. For example, the term “plurality” refers to the amino acid sequence of amino acid residues 666 to 682 in the amino acid sequence described in SEQ ID NO: 3 and the amino acid sequence of amino acids 672 to 688 in SEQ ID NO: 4 in the sequence listing. Although it differs depending on the position and type in the three-dimensional structure of the protein, it is, for example, 2 to 5, preferably 2 to 3, and more preferably 2.
[0047]
Furthermore, in the amino acid sequence of SEQ ID NO: 3 in the amino acid sequence of SEQ ID NO: 3, and in the amino acid sequence of SEQ ID NO: 4 in the amino acid sequence of SEQ ID NO: 4, in the amino acid sequence of amino acid number 679-683, It is desirable to substitute the amino acid within a range that does not change the type. That is, it is desirable that the amino acid in the sequence and the amino acid that substitutes for the same amino acid are amino acids of similar type in structural classification. Specifically, when the amino acid of the sequence is an acidic amino acid or an acidic amino acid amide, substitution with any of aspartic acid, glutamic acid, asparagine, or glutamine, and when the amino acid is a basic amino acid, lysine, histidine, or arginine Substitution with any one of them, phenylalanine when it is an aromatic amino acid, tyrosine, or substitution with any one of tryptophan, when it is an aliphatic amino acid or an oxyamino acid, glycine, alanine, valine, leucine, isoleucine, serine Alternatively, substitution with any of threonine, and in the case of amino acids other than the above (cysteine, methionine, proline, etc.), it is desirable to arbitrarily substitute as long as the cysteine protease inhibitory activity is not impaired. In addition, in the amino acid sequence of SEQ ID NO: 3, amino acids in the range other than amino acids 666 to 682, and in the sequence list of SEQ ID NO: 4, amino acids in the range other than amino acids 672 to 688 In the above, the number varies depending on the position and type of the amino acid residue in the three-dimensional structure of the protein, but is, for example, 2 to 10, preferably 2 to 5, and more preferably 2 to 3.
[0048]
The nucleotide sequence encoding a protein or peptide substantially the same as the transferrin protein or partial peptide of transferrin as described above, for example, by site-directed mutagenesis, substitution of amino acid residues at specific sites, deletion, insertion, It can be obtained by modifying the base sequence to include addition or inversion. The modified base sequence as described above can also be obtained by a conventionally known mutation treatment.
[0049]
By expressing the nucleotide sequence having the above mutation in an appropriate cell, and examining the cysteine protease inhibitory activity by the method for measuring the cysteine protease inhibitory activity described in the Test Examples or Examples of the present invention, the transferrin protein or transferrin A nucleotide sequence encoding a protein or peptide substantially identical to the partial peptide is obtained.
[0050]
In the cysteine protease inhibitor of the present invention, lactoferrin, a partial peptide of lactoferrin, transferrin or a partial peptide of transferrin can be used alone or in combination of two or more. In addition, a partial peptide of lactoferrin can be used alone or in combination of two or more. Furthermore, the partial peptide of transferrin can be used alone or in combination of two or more.
[0051]
Lactoferrin, a partial peptide of lactoferrin, or transferrin that can be used in the present invention has inhibitory activity against cysteine proteases such as cathepsins B, L, S, and papain. Cysteine protease inhibitory activity can be measured according to the method of Barrett et al. In the test examples or examples of the present invention, the measurement method will be described in detail.
[0052]
The cysteine protease inhibitor of the present invention is administered orally or parenterally to mammals, including humans, in combination with lactoferrin, a partial peptide of lactoferrin, or transferrin, or a combination thereof with a pharmaceutically acceptable pharmaceutical carrier. can do. The dosage unit form of the preparation of the present invention is not particularly limited and can be appropriately selected depending on the purpose of treatment. Specifically, tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, syrups Preparations, suppositories, injections, ointments, patches, eye drops, nasal drops and the like. Upon formulation, additives such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, diluents, surfactants, and solvents for injections are commonly used as pharmaceutical carriers for ordinary drugs. Can be used.
[0053]
The amount of lactoferrin, a partial peptide of lactoferrin, or transferrin contained in the preparation of the present invention is not particularly limited, and may be appropriately selected. For example, each is usually 0.005 to 60% by mass, preferably 0. The content is preferably from 0.5 to 50% by mass.
[0054]
The administration method of the preparation of the present invention is not particularly limited, and is determined according to various preparation forms, the age and sex of the patient, other conditions, the degree of symptoms of the patient, and the like. The dose of the active ingredient in the preparation of the present invention is appropriately selected depending on the usage, the age, sex, degree of disease, and other conditions of the patient. Usually, the amount of lactoferrin, a partial peptide of lactoferrin, or transferrin as an active ingredient is preferably 0.1 to 1200 mg / kg / day, preferably 10 to 500 mg / kg / day. It can be administered once a day or divided into a plurality of doses.
[0055]
Cysteine protease inhibitors of the present invention, cysteine protease involved diseases, such as allergy, muscular dystrophy, muscular atrophy, myocardial infarction, stroke, Alzheimer's disease, multiple sclerosis, cataract, osteoporosis, malignant hypercalcemia, Prophylactic / therapeutic agents for benign prostatic hyperplasia, breast cancer, prostate cancer, etc., or cancer cell growth or metastasis inhibitors, or bacterial (eg, Staphylococcus aureus V8) or viral (eg, poliovirus, herpes virus) growth inhibition Useful as an agent. Although the cysteine protease inhibitor of the present invention may be used alone, it can also be used in combination with the known agent for preventing or treating the above-mentioned disease, or the above-mentioned agent for inhibiting the growth of bacteria and viruses. When used in combination, the effect of preventing or treating the disease or the effect of suppressing the growth of bacteria and viruses can be enhanced. The preventive / therapeutic agent for the disease or the bacterial / viral growth inhibitor used in combination may be contained as an active ingredient in the composition of the present invention or may be separately contained without being contained in the composition of the present invention. May be commercialized as a combination of the above-mentioned medicines and combined at the time of use.
[0056]
The food and drink composition of the present invention can be produced by adding lactoferrin, a partial peptide of lactoferrin, or transferrin to a food or beverage, and can be taken orally. Examples of the form of the food and drink composition include drinks such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, and lactic acid bacteria drinks (including concentrated undiluted solutions of these drinks and powder for adjustment); ice creams, sorbets, shaved ice, and other ice confections Candy, chewing gum, candy, gum, chocolate, tablet confectionery, snacks, biscuits, jellies, jams, creams, baked confectioneries, etc .: dairy products such as processed milk, milk drinks, fermented milk, butter; bread; Nutritional foods, liquid foods, milk for childcare, sports drinks, and other functional foods are exemplified.
[0057]
In the food and drink composition of the present invention, the amount of lactoferrin, a partial peptide of lactoferrin, or transferrin to be added is appropriately set depending on the form of the food and drink composition, but 0.005 to 60% by mass in a normal food or drink. , Preferably 0.05 to 50% by mass.
[0058]
The feed composition of the present invention can be produced by adding lactoferrin, a partial peptide of lactoferrin, or transferrin to the feed, and orally administered to general mammals, livestock, fish farming, and pets. Is possible. Examples of the form of the feed composition include pet food, livestock feed, fish feed, and the like, and are mixed with cereals, meals, bran, fish meal, bone meal, fats and oils, skim milk powder, whey, mineral feed, yeasts, and the like. Thus, the feed composition of the present invention can be produced.
[0059]
In the feed composition of the present invention, the amount of lactoferrin, a partial peptide of lactoferrin, or transferrin to be added is appropriately set depending on the form of the feed composition, but is usually 0.005 to 60% by mass, preferably 0% by mass in a normal feed. What is necessary is just to add so that it may become 0.05 to 50 mass%.
[0060]
Next, the present invention will be described in detail with reference to test examples.
[Test Example 1]
This test was performed to detect cysteine protease inhibitors in milk.
[0061]
(1) Detection method
The present inventor used a technique called "reverse zymography" as a method for detecting a protease inhibitor, and detected a protease inhibitor present on a gel of SDS polyacrylamide gel electrophoresis. Inverse zymography is based on the reverse of ordinary zymography, and its basic principle is as follows. That is, a sample containing a protease inhibitor is applied to an SDS polyacrylamide gel containing gelatin, and after electrophoresis, the gel is immersed in a protease solution to degrade proteins in the gel. As a result of this operation, the portion where the inhibitor is present inhibits the activity of the protease, so that gelatin is prevented from being decomposed by the protease, and the gelatin is stained with a staining solution, whereby the inhibitor can be identified.
[0062]
(2) Test method
The method of inverse zymography in the present invention is as follows.
Using as a sample the total protein in milk and bovine lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd.), electrophoresis (hereinafter referred to as SDS polyacrylamide gel electrophoresis) was performed using 12.5% SDS polyacrylamide gel containing 0.1% gelatin. -PAGE may be abbreviated). After electrophoresis, the gel was washed by immersing it in a 2.5% Triton X-100 solution for 45 minutes, and then immersed in distilled water for another 45 minutes three times to wash the gel. This gel was immersed in 100 ml of 0.025 M acetate buffer (pH 5.5) containing 1 mg papain (31 units / ml), and incubated at 37 ° C. for 10 hours to digest gelatin. After the gel was washed with distilled water, the gel was stained with a staining solution (0.025% Coomassie Brilliant Blue (CBB) R-250, 40% methanol, 7% acetic acid aqueous solution) for 1 hour, and then decolorized solution (40% methanol, (10% acetic acid aqueous solution).
[0063]
Separately, reverse zymography was performed as above using a 12.5% SDS polyacrylamide gel containing no gelatin as a control test. Further, normal 12.5% SDS-PAGE (CBB staining) was performed.
[0064]
(3) Test results
The results of this test are as shown in FIG. FIG. 1 is a result showing an inverse zymography pattern. 1 is a normal SDS-PAGE pattern of total protein in milk, 2 lane is a reverse zymography pattern of total protein in milk, and 3 lane is a gel of total protein in milk without gelatin. Reverse zymography (control) pattern, lane 4 shows the pattern of bovine lactoferrin reverse zymography, and lane 5 shows the pattern of bovine lactoferrin gel containing no gelatin (control). The arrow in the figure indicates the migration position of bovine lactoferrin (molecular weight 72 kDa) by SDS-PAGE. Note that lanes 6 and 7 have no direct relation to this test example.
[0065]
As is clear from FIG. 1, a positive band of reverse zymography was confirmed in almost the same position as the bovine lactoferrin migration position (72 kDa) in two lanes. This confirmed the presence of a substance having cysteine protease inhibitory activity in milk. In addition, a positive band was confirmed in reverse zymography using papain in which bovine lactoferrin migrated in four lanes.
[0066]
These results suggested that bovine lactoferrin has cysteine protease inhibitory activity.
[0067]
[Test Example 2]
This test was performed to determine the N-terminal amino acid sequence of the 72 kDa band for which the cysteine protease inhibitory activity was suggested in Test Example 1.
[0068]
(1) Test method
After performing SDS-PAGE using the total protein sample in the milk used in Test Example 1 in the same manner, transferring to a polyvinylidene difluoride (PVDF) membrane, and staining the PVDF membrane with CBB, around 72 kDa The electrophoresed staining band was cut out. The N-terminal amino acid sequence of this band was determined using a G1005A protein sequencing system manufactured by Hewlett-Packard.
[0069]
(2) Test results
As a result of determining the amino acid sequence of the 72 kDa staining band in milk, the N-terminal amino acid sequence of the 72 kDa staining band was completely identical to that of bovine lactoferrin described in SEQ ID NO: 2 in the sequence listing. Therefore, both the results of this test and the results of Test Example 1 revealed that bovine lactoferrin has cysteine protease inhibitory activity.
[0070]
[Test Example 3]
This test was performed to measure the inhibitory effect of bovine lactoferrin on cysteine protease.
[0071]
(1) Test method
A commercially available bovine lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd.) was used as a test sample, and cysteine protease was used for papain, cathepsin B, cathepsin L, and cathepsin S (all commercially available products: manufactured by Wako Pure Chemical Industries, Ltd.). The inhibitory activity was measured. The inhibitory activity was measured by the method described below by Barrett et al. [Methods in Enzymology, Vol. 80, pp. 535-561, 1981]. That is, Z-Phe-Arg-MCA (final concentration: 20 mM: manufactured by Peptide Institute) was added as a substrate to samples dissolved at various concentrations in 0.1 M acetate buffer pH 5.5, and cysteine protease (this test). Then, for each sample, a solution selected from papain, cathepsin B, cathepsin L and cathepsin S (manufactured by Wako Pure Chemical Industries, Ltd.) (final concentration: 15 units / ml) is added and mixed, and the mixture is mixed at 37 ° C. for 10 minutes. After the reaction, the fluorescence intensity (excitation wavelength: 370 nm, emission wavelength: 460 nm) of AMC released from the digested substrate was measured using a fluorescence spectrophotometer (manufactured by Hitachi, Ltd.).
[0072]
(2) Test results
The results of this test are as shown in Table 1. Table 1 shows the cysteine protease inhibitory effect of bovine lactoferrin on papain, cathepsin B, cathepsin L, and cathepsin S. As a result, the cysteine protease activities of papain and cathepsin L were determined when bovine lactoferrin concentration was 10%. ^-6 It was completely inhibited when M was reached. The cysteine protease activity of cathepsin B and cathepsin S was determined when bovine lactoferrin concentration was 10%. ^-5 M is inhibited by 70% or more when M is reached. ^-4 It was completely inhibited at the M concentration. Thus, bovine lactoferrin was found to have cysteine protease inhibitory activity on papain, cathepsin B, cathepsin L, and cathepsin S.
[0073]
[Table 1]

[0074]
[Test Example 4]
This test was performed to measure the inhibitory effect of human lactoferrin on cysteine protease.
[0075]
(1) Test method
Using a commercially available human lactoferrin (manufactured by Sigma) as a test sample, papain, cathepsin B, cathepsin L, and cathepsin S (all of which are commercially available products: manufactured by Wako Pure Chemical Industries, Ltd.) were used as cysteine proteases. The cysteine protease inhibitory activity was measured by the same method as the cysteine protease inhibitory activity measurement method described in 3.
[0076]
(2) Test results
The results of this test are as shown in FIG. FIG. 2 shows the cysteine protease inhibitory effect of human lactoferrin on papain, cathepsin B, cathepsin L, and cathepsin S. As a result, the cysteine protease activities of papain and cathepsin L were determined when the concentration of human lactoferrin was 10 ^-6 M was almost completely inhibited when M was reached. In addition, the cysteine protease activities of cathepsin B and cathepsin S were determined when human lactoferrin concentration was 10%. ^-5 80% or more when M ^-4 It was almost completely inhibited at the M concentration. Therefore, human lactoferrin was found to have cysteine protease inhibitory activity on papain, cathepsin B, cathepsin L, and cathepsin S.
[0077]
[Test Example 5]
This test was performed to measure the inhibitory effect of a partial peptide of lactoferrin on cysteine protease.
[0078]
(1) Test method
As a test sample, a peptide having an amino acid sequence of amino acid numbers 679 to 695 among the amino acid sequences described in SEQ ID NO: 1 in the sequence listing and manufactured in Example 1 described below [hereinafter, human lactoferrin peptide Y ₆₇₉ -K ₆₉₅ (See FIG. 3). And using papain, cathepsin B, and cathepsin L as cysteine proteases (all commercially available products: manufactured by Wako Pure Chemical Industries, Ltd.) in the same manner as the method for measuring the cysteine protease inhibitory activity described in Test Example 3. The cysteine protease inhibitory activity was measured according to
[0079]
(2) Test results
The results of this test are as shown in FIG. FIG. 4 shows human lactoferrin peptide Y against papain, cathepsin B and cathepsin L. ₆₇₉ -K ₆₉₅ 3 shows the cysteine protease inhibitory effect of the present invention. As a result, the cysteine protease activities of papain and cathepsin L were measured at a peptide concentration of 10%. ^-4 50% or more when reaching M ^-3 It was almost completely inhibited at the M concentration. In addition, the cysteine protease activity of cathepsin B was determined when the peptide concentration was 10%. ^-3 When M was reached, it was inhibited by 60%. Therefore, human lactoferrin peptide Y ₆₇₉ -K ₆₉₅ Was found to have cysteine protease inhibitory activity on papain, cathepsin B and cathepsin L. In addition, among the amino acid sequences described in SEQ ID NO: 2 in the sequence listing and manufactured in Example 1 described below, a peptide having an amino acid sequence of amino acids 676 to 692 [hereinafter, bovine lactoferrin peptide Y ₆₇₆ -K ₆₉₂ (See FIG. 3). ], The same inhibitory activity measurement test was carried out. As a result, human lactoferrin peptide Y ₆₇₉ -K ₆₉₅ It was found that they exhibited almost the same activity as.
[0080]
[Test Example 6]
This test was performed to measure the inhibitory effect of partial bovine lactoferrin peptide on cysteine protease.
[0081]
(1) Test method
As a test sample, a peptide having the amino acid sequence of amino acids 36 to 60 [hereinafter referred to as bovine lactoferrin peptide F] in the amino acid sequence of SEQ ID NO: 2 prepared in the same manner as in Example 1 described later. ₃₆ -F ₆₀ It is described. And the cysteine protease inhibitory activity was measured for papain (commercial product: Wako Pure Chemical Industries, Ltd.) as the cysteine protease by the same method as the cysteine protease inhibitory activity measurement method described in Test Example 3.
[0082]
(2) Test results
The results of this test are as shown in Table 2. Table 2 shows the cysteine protease inhibitory effect of bovine lactoferrin peptide on papain. As a result, the cysteine protease activity of papain was reduced to bovine lactoferrin peptide F ₃₆ -F ₆₀ Concentration of 10 ^-4 88% was inhibited when M was reached. Therefore, bovine lactoferrin peptide F ₃₆ -F ₆₀ Was found to have cysteine protease inhibitory activity on papain.
[0083]
[Table 2]

[0084]
[Test Example 7]
This test was performed to measure the inhibitory effect of transferrin on cysteine protease.
[0085]
(1) Test method
A commercially available bovine transferrin (manufactured by Nippon Pharmaceutical Co., Ltd.) was used as a test sample, and papain, cathepsin B, and cathepsin L (all commercially available products: manufactured by Wako Pure Chemical Industries) were used as cysteine proteases in Test Example 3. The cysteine protease inhibitory activity was measured by the same method as the cysteine protease inhibitory activity measurement method described above.
[0086]
(2) Test results
The results of this test are as shown in FIG. FIG. 5 shows the cysteine protease inhibitory effect of transferrin on papain, cathepsin B and cathepsin L. As a result, the cysteine protease activity of papain showed that ^-4 40% inhibition when M ^-3 It was completely inhibited at the M concentration. In addition, the cysteine protease activities of cathepsin B and cathepsin L were determined when transferrin concentration was 10%. ^-4 When M was reached, inhibition was 30% or more. Therefore, bovine transferrin was found to have cysteine protease inhibitory activity on papain, cathepsin B, and cathepsin L.
[0087]
【Example】
Next, the present invention will be described in more detail with reference to examples. Note that the present invention is not limited to the following embodiments.
[Example 1]
Among the amino acid sequences described in SEQ ID NO: 1 in the sequence listing, a peptide having the amino acid sequence of amino acids 679 to 695 (see FIG. 3: human lactoferrin peptide Y ₆₇₉ -K ₆₉₅ ) Was produced by the following method.
[0088]
The peptide of the present invention was synthesized and produced using an automatic amino acid synthesizer (manufactured by Applied Biosystems, Model 433A).
[0089]
N-methylpyrrolidone containing 20% piperidine (manufactured by Applied Biosystems; hereinafter, N-methylpyrrolidone is abbreviated as NMP) is used to prepare amino acids of HMP resin (manufactured by Applied Biosystems) which is a solid phase resin for peptide synthesis. After the Fmoc group which is a protecting group is cleaved off and washed with NMP, Fmoc-threonine [specifically, Fmoc-amino acid corresponding to the C-terminal amino acid of the peptide to be synthesized (manufactured by Applied Biosystems)] is used as FastMoc. It was condensed using a (registered trademark) Regent kit (manufactured by Applied Biosystems) and washed with NMP. Next, the Fmoc group was cleaved, followed by condensation of Fmoc-alanine corresponding to the second amino acid from the C-terminal and washing, and further, condensation and washing of Fmoc-amino acid were repeated to prepare a protected peptide resin. The crude peptide was recovered from the resin.
[0090]
The peptide was purified from the crude peptide by high performance liquid chromatography (hereinafter abbreviated as HPLC). The column used was a reversed-phase C18-ODS (manufactured by Merck Ltd., Lichrospher 100). The obtained purified peptide was analyzed by HPLC, and it was further confirmed that a single purified product was obtained. The amino acid sequence of the purified peptide was determined using a gas-phase automatic amino acid sequencer (Applied Biosystems, Model 473A). As a result, the peptide had the amino acid sequence of amino acid numbers 679 to 695 of SEQ ID NO: 1. .
[0091]
A peptide having the amino acid sequence of amino acids 676 to 692 out of the amino acid sequence of SEQ ID NO: 2 by the same method (see FIG. 3: bovine lactoferrin peptide Y ₆₇₆ -K ₆₉₂ ) Was also manufactured.
[0092]
[Example 2]
(Preparation of tablets containing lactoferrin)
A tablet cysteine protease inhibitor having the following composition was produced by the following method.
Bovine lactoferrin (manufactured by Morinaga Milk Products) 40.0 (%)
Lactose (Morinaga Milk Products) 18.5
Corn starch (manufactured by Nisshin Seifun KK) 30.7
Magnesium stearate (manufactured by Taihei Chemical Industry Co., Ltd.) 1.4
Carboxymethylcellulose calcium (Gotoku Pharmaceutical Co., Ltd.) 9.4
[0093]
The mixture of bovine lactoferrin, lactose, corn starch and carboxymethylcellulose calcium is uniformly kneaded while appropriately adding sterilized purified water, dried at 50 ° C. for 3 hours, and magnesium stearate is added to the obtained dried product. Then, the mixture was mixed and tableted by a conventional method to obtain a tablet.
[0094]
[Example 3]
(Preparation of lactoferrin in capsule)
600 g of lactose (manufactured by Wako Pure Chemical Industries, Ltd.), 400 g of corn starch (manufactured by Nisshin Seifun Co., Ltd.), 400 g of crystalline cellulose (manufactured by Wako Pure Chemical Industries, Ltd.) and 600 g of bovine lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd.) were treated with a 50 mesh sieve (Yamato). The powder was placed in a 0.5 mm thick polyethylene bag, mixed by inversion, and the powder was converted into a capsule (Nippon Elanco Co., Ltd.) using a fully automatic capsule filling machine (Cesere Pedini Co., press type). No. 1 gelatin capsule, Op. Yellow No. 6 Body, empty weight: 75 mg) was filled with an inner volume of 275 mg to obtain 7,000 capsules containing 82 mg of bovine lactoferrin.
[0095]
[Example 4]
(Preparation of beverage containing bovine lactoferrin)
90 g of skim milk powder (manufactured by Morinaga Milk Industry Co., Ltd.) is dissolved in 800 ml of hot water at 50 ° C., 30 g of sugar (manufactured by Nissin Sugar Co., Ltd.), 14 g of instant coffee powder (manufactured by Nestlé), 2 g of caramel (manufactured by Showa Kako), and coffee 0.01 g of a flavor (manufactured by Sanei Chemical Co., Ltd.) is added successively while stirring, and the mixture is dissolved. A milk beverage having a cysteine protease inhibitory effect was prepared.
[0096]
[Example 5]
(Preparation of Enteral Nutrition Food Powder with Bovine Lactoferrin)
10.8 kg of whey protein enzyme digest (manufactured by Morinaga Milk Industry Co., Ltd.), 36 kg of dextrin (manufactured by Showa Sangyo Co., Ltd.), and small amounts of water-soluble vitamins and minerals were dissolved in 200 kg of water, and an aqueous phase was prepared in the tank. Separately, 3 kg of soybean salad oil (manufactured by Taiyo Yushi), 8.5 kg of palm oil (manufactured by Taiyo Yushi), 2.5 kg of safflower oil (manufactured by Taiyo Yushi), 0.2 kg of lecithin (manufactured by Ajinomoto), An oil phase was prepared by mixing and dissolving 0.2 kg of fatty acid monoglyceride (manufactured by Kao Corporation) and a small amount of fat-soluble vitamin. The oil phase was added to the water phase in the tank, mixed with stirring, heated to 70 ° C., and further homogenized with a homogenizer at a pressure of 14.7 MPa. Next, after sterilizing at 90 ° C. for 10 minutes, the mixture was concentrated and spray-dried to prepare about 59 kg of an intermediate product powder. To 50 kg of the intermediate product powder, 6.8 kg of sucrose (manufactured by Hokuren), 167 g of mixed powder of amino acid (manufactured by Ajinomoto Co.), and 60 g of bovine lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd.) are added and uniformly mixed, and bovine lactoferrin is mixed. About 57 kg of enteral nutritional food powder having a cysteine protease inhibitory effect was produced.
[0097]
【The invention's effect】
As described above in detail, the present invention relates to a cysteine protease inhibitor containing, as an active ingredient, one or more mixtures selected from the group consisting of lactoferrin, a partial peptide of lactoferrin, and transferrin. The effects achieved are as follows.
(1) Since the protein can be used as a food material, it is excellent in safety and can be administered or taken daily for a long period of time.
(2) It has an inhibitory activity spectrum for a wide range of cysteine proteases.
(3) It can be used as a prophylactic / therapeutic agent for diseases involving cysteine protease.
[0098]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a diagram (photograph) showing detection of bovine milk protein by reverse zymography.
FIG. 2 is a diagram showing the cysteine protease inhibitory effect of human lactoferrin on cysteine protease.
FIG. 3 shows a partial peptide of human lactoferrin produced in Example 1 (human lactoferrin peptide Y). ₆₇₉ -K ₆₉₅ ) And a partial peptide of bovine lactoferrin (bovine lactoferrin peptide Y ₆₇₆ -K ₆₉₂ ) And a peptide having an amino acid sequence of amino acid numbers 666 to 682 among the amino acid sequences described in SEQ ID NO: 3 in the sequence listing (human transferrin peptide Y ₆₆₆ -R ₆₈₂ ) And a peptide having an amino acid sequence of amino acids 672 to 688 among the amino acid sequences described in SEQ ID NO: 4 in the sequence listing (bovine transferrin peptide Y ₆₇₂ -R ₆₈₈ FIG. 3 shows the amino acid sequence of FIG.
FIG. 4. Human lactoferrin peptide Y against cysteine protease. ₆₇₉ -K ₆₉₅ It is a figure which shows the cysteine protease inhibitory effect of.
FIG. 5 is a graph showing the cysteine protease inhibitory effect of transferrin on cysteine protease.

Claims (8)

A cysteine protease inhibitor comprising, as an active ingredient, one or a mixture of lactoferrin, a partial peptide of lactoferrin, and transferrin. The cysteine protease inhibitor according to claim 1, wherein the lactoferrin and / or transferrin is one or a mixture of at least one selected from the group consisting of a metal saturated type, a partially saturated metal type, and an apo type. The cysteine protease inhibitor according to claim 1 or 2, wherein the lactoferrin and / or the partial peptide of lactoferrin is one or a mixture of proteins or peptides shown in the following (a) to (d).
(A) A protein or peptide having at least the amino acid sequence of amino acids 679 to 695 among the amino acid sequences described in SEQ ID NO: 1 in the sequence listing.
(B) a peptide having at least the amino acid sequence of amino acids 679 to 695 in the amino acid sequence of SEQ ID NO: 1 in the sequence listing, wherein one or more amino acids are substituted, deleted, added, or inverted; A protein or peptide comprising cysteine protease inhibitory activity.
(C) a protein or peptide having at least the amino acid sequence of amino acids 676 to 692 among the amino acid sequences described in SEQ ID NO: 2 in the sequence listing;
(D) a peptide having at least the amino acid sequence of amino acids 676 to 692 in the amino acid sequence of SEQ ID NO: 2 in the sequence listing, wherein one or more amino acids are substituted, deleted, added, or inverted; A protein or peptide comprising cysteine protease inhibitory activity. The cysteine protease inhibitor according to claim 1 or 2, wherein the lactoferrin and / or the partial peptide of lactoferrin is one or a mixture of proteins or peptides shown in the following (e) to (j).
(E) a protein or peptide having at least the amino acid sequence of amino acids 20 to 30 in the amino acid sequence of SEQ ID NO: 1 in the sequence listing;
(F) A peptide having at least the amino acid sequence of amino acids 20 to 30 in the amino acid sequence of SEQ ID NO: 1 in the sequence listing, wherein one or more amino acids are substituted, deleted, added, or inverted. A protein or peptide comprising cysteine protease inhibitory activity.
(G) A protein or peptide having at least the amino acid sequence of amino acids 31 to 66 among the amino acid sequences described in SEQ ID NO: 1 in the sequence listing.
(H) a peptide having at least the amino acid sequence of amino acids 31 to 66 in the amino acid sequence of SEQ ID NO: 1 in the sequence listing, wherein one or more amino acids are substituted, deleted, added, or inverted; A protein or peptide comprising cysteine protease inhibitory activity.
(I) A protein or peptide having at least the amino acid sequence of amino acid numbers 20 to 66 among the amino acid sequences described in SEQ ID NO: 1 in the sequence listing.
(J) a peptide having an amino acid sequence of at least amino acids 20 to 66 in the amino acid sequence of SEQ ID NO: 1 in the sequence listing, wherein one or more amino acids are substituted, deleted, added, or inverted; A protein or peptide comprising cysteine protease inhibitory activity. The cysteine protease inhibitor according to claim 1 or 2, wherein the lactoferrin and / or the partial peptide of lactoferrin is one or a mixture of proteins or peptides shown in the following (k) to (n).
(K) A protein or peptide having at least the amino acid sequence of amino acids 36 to 60 among the amino acid sequences described in SEQ ID NO: 2 in the sequence listing.
(L) a peptide having at least the amino acid sequence of amino acids 36 to 60 in the amino acid sequence of SEQ ID NO: 2 in the sequence listing, wherein one or more amino acids are substituted, deleted, added, or inverted; A protein or peptide comprising cysteine protease inhibitory activity.
(M) A protein or peptide having at least the amino acid sequence of amino acids 39 to 44 among the amino acid sequences described in SEQ ID NO: 2 in the sequence listing.
(N) a peptide having at least the amino acid sequence of amino acids 39 to 44 in the amino acid sequence of SEQ ID NO: 2 in the sequence listing, wherein one or more amino acids are substituted, deleted, added, or inverted; A protein or peptide comprising cysteine protease inhibitory activity. The cysteine protease inhibitor according to any one of claims 1 to 5, which is an agent for preventing or treating a disease associated with cysteine protease. The cysteine protease inhibitor according to any one of claims 1 to 6, wherein the disease associated with cysteine protease is osteoporosis or malignant hypercalcemia. A food or drink composition or feed composition comprising the cysteine protease inhibitor according to any one of claims 1 to 7.

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