JPS6351335A - Degranulation inhibitor for mast cell - Google Patents
Degranulation inhibitor for mast cellInfo
- Publication number
- JPS6351335A JPS6351335A JP61196347A JP19634786A JPS6351335A JP S6351335 A JPS6351335 A JP S6351335A JP 61196347 A JP61196347 A JP 61196347A JP 19634786 A JP19634786 A JP 19634786A JP S6351335 A JPS6351335 A JP S6351335A
- Authority
- JP
- Japan
- Prior art keywords
- bbi
- soybean
- inhibitor
- degranulation
- trypsin inhibitor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000003112 inhibitor Substances 0.000 title claims abstract description 23
- 210000003630 histaminocyte Anatomy 0.000 title claims abstract description 19
- 235000010469 Glycine max Nutrition 0.000 claims abstract description 17
- 244000068988 Glycine max Species 0.000 claims abstract description 17
- 239000002753 trypsin inhibitor Substances 0.000 claims abstract description 9
- 229940122618 Trypsin inhibitor Drugs 0.000 claims abstract description 8
- 101710162629 Trypsin inhibitor Proteins 0.000 claims abstract description 8
- 239000004480 active ingredient Substances 0.000 claims abstract description 4
- 230000002401 inhibitory effect Effects 0.000 abstract description 11
- 239000000126 substance Substances 0.000 abstract description 5
- 101710202338 Bowman-Birk type trypsin inhibitor Proteins 0.000 abstract description 3
- 238000002523 gelfiltration Methods 0.000 abstract description 3
- 238000001556 precipitation Methods 0.000 abstract description 3
- 208000026935 allergic disease Diseases 0.000 abstract description 2
- 239000012736 aqueous medium Substances 0.000 abstract description 2
- 238000000605 extraction Methods 0.000 abstract description 2
- 238000005194 fractionation Methods 0.000 abstract description 2
- 239000012528 membrane Substances 0.000 abstract description 2
- 239000003495 polar organic solvent Substances 0.000 abstract description 2
- 238000000926 separation method Methods 0.000 abstract description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract 1
- RXKJFZQQPQGTFL-UHFFFAOYSA-N dihydroxyacetone Chemical compound OCC(=O)CO RXKJFZQQPQGTFL-UHFFFAOYSA-N 0.000 abstract 1
- 108090000227 Chymases Proteins 0.000 description 15
- 102000003858 Chymases Human genes 0.000 description 15
- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 description 14
- 230000000694 effects Effects 0.000 description 12
- 108060005989 Tryptase Proteins 0.000 description 8
- 102000001400 Tryptase Human genes 0.000 description 8
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- 238000000034 method Methods 0.000 description 8
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- 229940088598 enzyme Drugs 0.000 description 4
- 239000008187 granular material Substances 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- MRXDGVXSWIXTQL-HYHFHBMOSA-N (2s)-2-[[(1s)-1-(2-amino-1,4,5,6-tetrahydropyrimidin-6-yl)-2-[[(2s)-4-methyl-1-oxo-1-[[(2s)-1-oxo-3-phenylpropan-2-yl]amino]pentan-2-yl]amino]-2-oxoethyl]carbamoylamino]-3-phenylpropanoic acid Chemical compound C([C@H](NC(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C=O)C1NC(N)=NCC1)C(O)=O)C1=CC=CC=C1 MRXDGVXSWIXTQL-HYHFHBMOSA-N 0.000 description 3
- 108010039627 Aprotinin Proteins 0.000 description 3
- OLVPQBGMUGIKIW-UHFFFAOYSA-N Chymostatin Natural products C=1C=CC=CC=1CC(C=O)NC(=O)C(C(C)CC)NC(=O)C(C1NC(N)=NCC1)NC(=O)NC(C(O)=O)CC1=CC=CC=C1 OLVPQBGMUGIKIW-UHFFFAOYSA-N 0.000 description 3
- 206010061218 Inflammation Diseases 0.000 description 3
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- 241000700159 Rattus Species 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
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- 229960004405 aprotinin Drugs 0.000 description 3
- 108010086192 chymostatin Proteins 0.000 description 3
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- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 3
- 238000005342 ion exchange Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- DIOSHTLNZVXJOF-UHFFFAOYSA-N 2,5-bis(3-oxobutanoylamino)benzenesulfonic acid Chemical compound CC(=O)CC(=O)NC1=CC=C(NC(=O)CC(C)=O)C(S(O)(=O)=O)=C1 DIOSHTLNZVXJOF-UHFFFAOYSA-N 0.000 description 2
- 108090000317 Chymotrypsin Proteins 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 108090000190 Thrombin Proteins 0.000 description 2
- 239000005862 Whey Substances 0.000 description 2
- 102000007544 Whey Proteins Human genes 0.000 description 2
- 108010046377 Whey Proteins Proteins 0.000 description 2
- 229960002376 chymotrypsin Drugs 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000009931 harmful effect Effects 0.000 description 2
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- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 2
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 229960004072 thrombin Drugs 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- 102100022524 Alpha-1-antichymotrypsin Human genes 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 101000796737 Homo sapiens Tryptase delta Proteins 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- GDBQQVLCIARPGH-UHFFFAOYSA-N Leupeptin Natural products CC(C)CC(NC(C)=O)C(=O)NC(CC(C)C)C(=O)NC(C=O)CCCN=C(N)N GDBQQVLCIARPGH-UHFFFAOYSA-N 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 108010073771 Soybean Proteins Proteins 0.000 description 1
- 102100032760 Tryptase delta Human genes 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- 230000009285 allergic inflammation Effects 0.000 description 1
- 108010091628 alpha 1-Antichymotrypsin Proteins 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 238000002306 biochemical method Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- -1 butyroxycarbonyl Chemical group 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000003601 chymase inhibitor Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 230000002900 effect on cell Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- YQDHCCVUYCIGSW-LBPRGKRZSA-N ethyl (2s)-2-benzamido-5-(diaminomethylideneamino)pentanoate Chemical group NC(=N)NCCC[C@@H](C(=O)OCC)NC(=O)C1=CC=CC=C1 YQDHCCVUYCIGSW-LBPRGKRZSA-N 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 description 1
- 108010052968 leupeptin Proteins 0.000 description 1
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Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
Description
【発明の詳細な説明】
(産業上の利用分野)
この発明は、キマーゼ及びトリブターゼ阻害剤殊に、肥
満細胞脱顆粒抑制剤に関するものである。DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to chymase and tributase inhibitors, particularly mast cell degranulation inhibitors.
(従来の技術)
アレルギー性鼻炎、アレルギー性結膜炎、喘、=、a麻
疹、食物性アレルギー等の、rgE(免疫グロブリンE
)の関与するアレルギー性炎症において1組織中の肥満
細胞(mast cell)が重要な役;(Aを担って
いることは既に明らかにされている(例えば「感染・ア
レルギー・免疫病学j 。(Prior art) rgE (immunoglobulin E
It has already been clarified that mast cells in one tissue play an important role in allergic inflammation associated with
1978年6月111医学書院発行参照)。即ち、細胞
表面に結合したIgHに対して抗原が結合することによ
り、細胞内顆粒が放出され、その中に含まれるヒスタミ
ンや5RS−A等が炎症を成立させると考えられている
。(Refer to June 1978, published by Igaku Shoin, 111). That is, it is thought that intracellular granules are released by antigen binding to IgH bound to the cell surface, and histamine, 5RS-A, etc. contained therein cause inflammation.
一方、勝沼らは、肥満細胞70粒中にキブーゼ(Ghy
mase)が存在し、これが肥満細胞の脱顆粒に必須で
あると共に、遊蕩した該酵素がIgGを分解し好中球遊
走因子を作ることを明らかにした(「生化学」第57巻
1076頁(1985)) 、更に勝沼らは、キマーゼ
に対する抗体及びキマーゼ阻害作用のあるキモスタチン
(C1lBOstaLin)により脱顆粒が抑制される
ことを明らかにしたが、アプロチニン(aprotin
in)やα1−アンチキモトリプシン(α1−anti
chymotrypsin)などの分子Q8000以ト
の蛋白性キマーゼ阻害剤では、その抑制は認め難かった
(”Biochemistr71nt、、”10.pp
、883〜871(1985))[発明が解決しようと
する問題点コ
木発明者等は更に研究を進める中で、肥満細胞顆粒中に
はキマーゼ以外にも存在するトリプターゼ(Trypt
ase)が、プロトロンビア (Prothra −m
bin)をトロンビン(Thrombin)に活性化さ
せ得ること、及び該活性化による血液凝固系を介する炎
症反応への関与の可能性が重要であるとの知見を得て、
これと前記キマーゼに関する実験事実から、肥満細胞顆
粒中の二種のプロテアーゼ、即ち、キマーゼ及びトリプ
ターゼに対する阻害が、脱顆粒の抑制、ひいてはアレル
ギー反応の抑制に有意義であろうとの認識を抱くに至っ
たが、現在までのところ、両プロテアーゼに対し強力な
作用を持つ、殊にキマーゼに対し強い活性を示す高分子
性阻害物質は見出されていない。On the other hand, Katsunuma et al. found that quibuze (Ghy) was present in 70 mast cells.
It was revealed that this enzyme exists and is essential for mast cell degranulation, and that the free enzyme degrades IgG and creates neutrophil migration factor (Seikagaku, Vol. 57, p. 1076). Furthermore, Katsunuma et al. revealed that degranulation was suppressed by antibodies against chymase and chymostatin (C1lBOstaLin), which has a chymase inhibitory effect, but aprotinin (aprotinin)
in) and α1-antichymotrypsin (α1-anti
In protein chymase inhibitors with molecular Q8000 or higher, such as chymotrypsin, the inhibition was hardly observed ("Biochemistr71nt," 10.pp.
, 883-871 (1985)) [Problems to be Solved by the Invention] During further research, the inventors discovered that tryptase (Tryptase), which exists in mast cell granules in addition to chymase,
Ase) is prothrombia (Prothra-m
Obtaining the knowledge that it is possible to activate thrombin (bin) into thrombin, and that the possibility of involvement in inflammatory reactions via the blood coagulation system due to this activation is important,
Based on this and the experimental facts regarding chymase mentioned above, we have come to realize that inhibition of two types of proteases in mast cell granules, namely chymase and tryptase, may be meaningful in suppressing degranulation and, in turn, suppressing allergic reactions. However, to date, no polymeric inhibitor has been found that exhibits strong activity against both proteases, particularly against chymase.
しかし、仮にかかる高分子性50害物質を発見すること
ができれば、他の低分子性インヒビターにおけるが如き
中線拡散による標的肥満細胞以外の細胞への無差別な細
胞内侵入は生じ難いものと想像されるので、副作用の恐
れのない安全な医薬品開発への展望が開けるものと推測
される。However, if such a high-molecular-weight inhibitor were to be discovered, it would be difficult to indiscriminately invade cells other than the target mast cells through midline diffusion, as is the case with other low-molecular-weight inhibitors. It is assumed that this will open up prospects for the development of safe drugs without the fear of side effects.
[発明完成の経過]
そこで本発明者らは、従来から種々の高分子性プロテア
ーゼインヒビターの存在が知られなが゛らも、基体的な
生理学的挙動については不IJであった植物性プロテア
ーゼインヒビターに探究の手を進めたところ、ここに大
豆由来のポウマン−八−り(BowIlan Birk
)型トリプシンインヒビターが両酵素に対しwJ著な阻
害作用を有すること、及びこのものが肥満細胞顆粒から
のIgE誘発によるヒスタミンの放(11を効果的に抑
制することを発見した。[Process of Completion of the Invention] Therefore, the present inventors have developed a plant protease inhibitor, which has not been known to have basic physiological behavior, although the existence of various polymeric protease inhibitors has been known for some time. As I continued my research, I discovered BowIlan Birk, which is derived from soybeans.
)-type trypsin inhibitor has a significant inhibitory effect on both enzymes, and it has been found that it effectively suppresses IgE-induced histamine release (11) from mast cell granules.
[問題点を解決するための手段]
未発11は以上の知見を基礎とするものであって、その
要旨は大豆由来のボウマン−バーク(Bowman B
irk)型トリプシンインヒビターを有効成分とする肥
満細胞脱顆粒抑制剤に存する。[Means for solving the problem] Unexploited No. 11 is based on the above knowledge, and its gist is that soybean-derived Bowman-Bark (Bowman B)
irk) type trypsin inhibitor as an active ingredient.
ここに、発明の主体である大豆由来のボウマン−バーク
型トリプシンインヒビクー(以下BBIと略す)は、約
6千〜8千の分子量を有し、他のタイプのトリプシンイ
ンヒビターである分子量約2万のクニッッ(Kunit
z) p )リプンンインヒビターと共に大豆の水溶性
画分中に存在している。Here, the soybean-derived Bowman-Birk trypsin inhibitor (hereinafter abbreviated as BBI), which is the main subject of the invention, has a molecular weight of about 6,000 to 8,000, while other types of trypsin inhibitors have a molecular weight of about 2. Kunit
z) p) Present in the water-soluble fraction of soybean together with lipunin inhibitor.
因に、これら両種インヒビターの基本的性質はrMeL
hod in EnzymologyJ 19巻853
頁(1970)及び「クン白質研究の新しい視点(化学
研究を中心として)」共立出版刊(1982)に記載さ
れ、かつそれらの構造も既に判明しているので(Eur
、J、Bio−ches、 、32.417;J、Bi
ochem、 、74,8S7 (1973) )それ
の化学的及び生化学的方法による合成や修飾も今日では
可能である。従って、ここにいうBBIは、大豆から原
始的に得られた天然ボウマン−バーク型トリプシンイン
ヒビターのみならず、その活性フラグメント若しくは修
飾物又はそれらの化学的又は生化学合成物を包含する概
念である。Incidentally, the basic properties of these two types of inhibitors are rMeL
hod in EnzymologyJ Volume 19 853
(1970) and “New Perspectives on White Matter Research (Focusing on Chemical Research)” published by Kyoritsu Shuppan (1982), and their structures have already been determined (Eur.
,J,Bio-ches, ,32.417;J,Bi
ochem, 74, 8S7 (1973)) Its synthesis and modification by chemical and biochemical methods is also possible today. Therefore, BBI as used herein is a concept that encompasses not only the natural Bowman-Birk type trypsin inhibitor originally obtained from soybean, but also its active fragments or modifications, or chemical or biochemical compounds thereof.
ところで、天然BBIの調製法としては既に種々の方法
が知られており1.に発りJではどの方法で作られたも
のでもよいが、一般的には、大豆、脱脂大豆、大豆ホエ
ーなどを出発原料として、これを水性媒質又は極性有機
溶剤(例えば低級アルコール類若しくは低級)偕肋族ケ
トン類又はジオキサン“や)による抽出、膜分離、等電
点沈澱、塩析等による濃縮、分画によって先ず粗精製物
の状態にまで予備的に精製した後、この組成物を、更に
ゲル鑓過、イオン交換又は吸着などの精製手段を施すこ
とにより、ポリアクリルアミドゲルディスク電気泳動で
単一のパターンを示す標品にまで純化することが可能で
ある。しかし実用的には、活性さえI)]らかであれば
粗製品で充分である。なお、活性のアッセイには、公知
の方法、例えばクニッツ(Kunitz)のカゼイン消
化法又は合成基質(例えばN−Benzoylargi
nine−p−’n1toroanilide )を利
用することができる。なお?ロー性及び純度の検定には
、SDS含有ポリアクリルアミドゲル電気泳動法又は高
速液体クロマトグラフィーを利用するのがよい。By the way, various methods are already known for preparing natural BBI. It can be made by any method, but in general, soybeans, defatted soybeans, soybean whey, etc. are used as starting materials, and this is mixed in an aqueous medium or a polar organic solvent (e.g., lower alcohols or lower). After first preliminarily purifying the composition to a crudely purified state by extraction with a group of ketones or dioxane, membrane separation, isoelectric precipitation, concentration by salting out, and fractionation, this composition is Furthermore, by applying purification methods such as gel filtration, ion exchange, or adsorption, it is possible to purify the specimen to a specimen that shows a single pattern in polyacrylamide gel disk electrophoresis. A crude product is sufficient as long as it is clear.The activity can be assayed using known methods such as the Kunitz casein digestion method or synthetic substrates (e.g. N-Benzoylargi).
nine-p-'n1toroanilide) can be used. In addition? SDS-containing polyacrylamide gel electrophoresis or high-performance liquid chromatography may be used to test for purity and purity.
[作用]
大豆由来のBBIは、肥満細胞より精製したキマーゼ及
びトリプターゼを強力に阻Iヒする。この阻害機作は、
BBIの分子鎖中のキモトリプシン系及びトリプシン系
酵素の活性中心に対する特異的結合作用によるものと理
解される。[Effect] BBI derived from soybean strongly inhibits chymase and tryptase purified from mast cells. This inhibitory mechanism is
It is understood that this is due to the specific binding action to the active centers of chymotrypsin and trypsin enzymes in the molecular chain of BBI.
更に大豆由来のBBIは、肥満細胞からの脱顆粒を抑制
する。この作用は、上の両酵素に対する阻害作用の結果
と考えられるが、BBIの分子量(約8,0QQ)から
考えて、この間、細胞膜との界面において伺等かの取り
込み機作が存在することは確実であろう。Furthermore, soybean-derived BBI inhibits degranulation from mast cells. This effect is thought to be the result of an inhibitory effect on both of the enzymes mentioned above, but considering the molecular weight of BBI (approximately 8.0 QQ), it is unlikely that some uptake mechanism exists at the interface with the cell membrane during this time. It would be certain.
とまれ、上のBBIでは、その高分子性から他の低分子
性インヒビターにおれるが如さ単純拡散による被標的細
胞以外の他種細胞中への侵入は生じないと考えられ、事
実、標的肥満細胞以外の細胞への影響は現在のところ発
見されていない。However, due to its high molecular nature, the BBI mentioned above is not considered to invade into cells of other species other than the target cell by simple diffusion, as is the case with other low-molecular-weight inhibitors, and in fact, it is believed that the above BBI does not invade into cells of other species other than the target cell by simple diffusion, as it does with other low-molecular-weight inhibitors. No effects on cells other than cells have been discovered so far.
以上のように、BBIは肥満細胞に作用してその脱顆粒
を抑制するため、アレルギー性疾患に対する有力な薬剤
としての効用が嘱望される。As described above, since BBI acts on mast cells and suppresses their degranulation, it is expected to be effective as a powerful drug for allergic diseases.
[実施例]
以下、実施例及び参考例により発明をより具体的に説明
するが、例示は当然説明用のものであって、発明思想の
限定を意味するものではない。[Examples] Hereinafter, the invention will be described in more detail with reference to Examples and Reference Examples, but the examples are of course for illustration only and do not mean a limitation on the idea of the invention.
参考例1(BBIの調製例)
低変性脱脂大豆から分離大豆蛋白を製造する過程で得ら
れる大豆ホエーを濃縮し、このC線動(粗蛋白資金75
.5%)1容に対し05容のアセトンを加えて約1時間
攪拌後、遠心分路して得られた旧情に対し、更に1,5
容のアセトンを加えて約1時間攪拌し、生じた沈kj、
画分を木に対して透析した。この透析液に’150量の
0.5 M燐酸ナトリウム緩衝液(p)17.Q)を加
えてPH7,0に調節後、DEAE−セルロースイオン
交換カラムに通して、該樹脂に吸71させた0次いで、
カラムを0〜0.4 Mの直線食塩濃度勾配で溶出し、
溶出液をアラクシ3ンコレクターにより分画し、BBI
に富む両分とクニッツ型トリプシンインヒビターに富む
両分を得た。Reference Example 1 (Preparation example of BBI) Soybean whey obtained in the process of producing isolated soybean protein from low-denatured defatted soybeans was concentrated, and this C linear activity (crude protein fund 75
.. 5%) Add 0.5 volumes of acetone to 1 volume and stir for about 1 hour, then centrifuge and separate.
of acetone and stirred for about 1 hour, the resulting precipitate kj,
The fractions were dialyzed against wood. To this dialysate was added 150 volumes of 0.5 M sodium phosphate buffer (p) 17. After adjusting the pH to 7.0 by adding Q), it was passed through a DEAE-cellulose ion exchange column and absorbed into the resin.
The column was eluted with a linear salt gradient from 0 to 0.4 M;
The eluate was fractionated using an aracin collector, and BBI
Two fractions were obtained, one enriched with Kunitz-type trypsin inhibitor and the other enriched with Kunitz-type trypsin inhibitor.
BBIに富む両分は、e錦、透析後、そのpHを4.0
に調製し、CMセルロースイオン交換カラムに通し、吸
着したBBIを0〜015Mの食塩濃度勾配にて溶出し
、溶出物を凍結乾燥して精製BBIを得た。またクニッ
ツ型の両分も、等電沈鍛後、凍結乾燥し、精製インヒビ
ターを得た。各標品の純度は、SO8含有ポリアクリル
アミドゲル電気泳動及び高速液体クロマトグラフィー(
ゲル鑓過法)にて測定したところ両者ともに蛋白質中9
5%以上であった。また、BBIの比活性は。The BBI-rich fraction is e-nishiki, and after dialysis, its pH is adjusted to 4.0.
was prepared, passed through a CM cellulose ion exchange column, and the adsorbed BBI was eluted with a 0-015M salt concentration gradient, and the eluate was lyophilized to obtain purified BBI. Both parts of the Kunitz mold were also freeze-dried after isoelectric precipitation to obtain purified inhibitors. The purity of each sample was determined by SO8-containing polyacrylamide gel electrophoresis and high performance liquid chromatography (
When measured by gel filtration method, both proteins contained 9.
It was 5% or more. Also, the specific activity of BBI is.
Sigma社製トリプシン「タイプ−XIJ(7500
〜9000 BAEE unit/ mgの活性蛋白)
及び合成基質(N−Benzyl−DL−Argini
ne−p−nitroanil ide:BAPA)を
用いた測定で、5〜6 unit/■g蛋白であった(
同条件で2unitのBAPAを氷解するトリプシンの
活性を50%阻害するとき阻害活性をfun己とした。Sigma trypsin “Type-XIJ (7500
~9000 BAEE units/mg of active protein)
and synthetic substrate (N-Benzyl-DL-Argini
When measured using ne-p-nitroanil ide: BAPA), it was 5 to 6 units/g protein (
The inhibitory activity was defined as fun when the activity of trypsin to thaw 2 units of BAPA under the same conditions was inhibited by 50%.
)。).
参考例2(BBIによるプロテアーゼの阻害)ラット舌
より精製したキマーゼ及びラット腹腔内肥満細胞より精
製したトリプターゼに対する阻害能は、」−記BBI及
びクニッツ型インヒビターを加え、キマーゼの場合、S
ue−Leu−Val−Tyr−MCA(Sue =サ
クシニル、MCA = 4−メチルクマリル−7−アミ
ド)を基質とし、p)18.5において測定し、またト
リブターゼの場合は、Boc−Phe−Se r−Ar
g−MGA (Boc=第三級ブチロキシカルボニル
)をA(質としてpH7,5において測定した。BBI
及びりこツツ型インヒビターの水溶液又はキモスタチy
(C:bymosLaLin : (財)蛋白質研
究!2劫会)のジメチルスルホキシド溶液をキマーゼ又
はトリプターゼに添加し、25℃で5分間保温した後、
基質溶液を加えた。酵素反応精製物は蛍光光度計にて測
定し、各511害剤の50%阻害率を求めた。結果を下
表1として示す。Reference Example 2 (Inhibition of protease by BBI) The inhibitory ability for chymase purified from rat tongue and tryptase purified from rat intraperitoneal mast cells was determined by adding BBI and Kunitz-type inhibitor, and in the case of chymase, S
ue-Leu-Val-Tyr-MCA (Sue = succinyl, MCA = 4-methylcoumaryl-7-amide) as substrate, measured at p) 18.5, and in the case of tributase, Boc-Phe-Ser- Ar
g-MGA (Boc=tertiary butyroxycarbonyl) was measured at pH 7.5 as A (quality).BBI
Aqueous solution of and Norikotsutu type inhibitor or Kymostachiy
(C:bymosLaLin: (Foundation) Protein Research! Nikokai) dimethyl sulfoxide solution was added to chymase or tryptase, and after incubating at 25°C for 5 minutes,
Substrate solution was added. The enzyme reaction purified product was measured using a fluorometer to determine the 50% inhibition rate of each 511 harmful agent. The results are shown in Table 1 below.
表1
上表1から明らかな様に、クニッツ型インヒビターのキ
マーゼに対する阻害能は強くなく、またキモスタチンの
トリプターゼに対する阻害部も弱いものであった。BB
Iのみがキマーゼ及びトリプターゼの双方に対し強い3
11害能を示した。これは、従来の各種インヒビターも
見られない特徴である・
実施例(BBIによる脱顆粒の抑制)
雄性ウィスタ一種ラットに、N 1ppost ran
gy 1usbracilienSisの幼虫を皮下接
種し、 IgE抗体値の上昇したラットの腹腔から肥満
細胞を純度95〜99%の純度で調製した。Table 1 As is clear from Table 1 above, the inhibitory ability of Kunitz-type inhibitors against chymase was not strong, and the inhibitory ability of chymostatin against tryptase was also weak. BB
Only I is strong against both chymase and tryptase3
11 showed harmful effects. This is a characteristic that is not seen in conventional inhibitors.Example (Suppression of degranulation by BBI)
Mast cells with a purity of 95 to 99% were prepared from the peritoneal cavities of rats with elevated IgE antibody levels after subcutaneous inoculation of gy1usbraciliensis larvae.
脱顆粒の測定は遊離ヒスタミン酸を指標として行った。Degranulation was measured using free histamic acid as an indicator.
即ち、11当たり4 X 105個の肥満細胞を01%
牛血清アルブミン含有タイロード液中に浮遊させ、BB
I又は他のインヒビターを加えて0〜60分間3分間3
侃
の抗うッ) IgE抗体を加え、37℃10分間内にお
けるヒスタミンの遊離量を液体クロマトグラフィーによ
り定量した.インヒビター無添加の場合の対照を100
%とした結果をr表2に示す。i.e. 4 x 105 mast cells per 11%
Suspended in Tyrode's solution containing bovine serum albumin, BB
I or other inhibitors for 0 to 60 minutes 3
An IgE antibody was added, and the amount of histamine released within 10 minutes at 37°C was determined by liquid chromatography. The control without inhibitor was 100.
The results expressed as % are shown in Table 2.
(以下余白)
表2
上表から明らかなように、低分子インヒビターであるキ
モスタチン及びロイペプチンはヒスタミンの遊離を抑制
する効果を示し、特に前者の効果は顕著であった.一方
,高分子インヒビターであるα1−抗ギモトリプシン及
びアプロチニンは、自体キマーゼ抑制効果有するに拘ら
ず、ヒスタミンの遊離を抑制する効果がなく、殊に後者
は却ってヒスタミンのisを促進した。(Leaving space below) Table 2 As is clear from the above table, the low molecular weight inhibitors chymostatin and leupeptin showed the effect of suppressing histamine release, and the effect of the former was particularly remarkable. On the other hand, the macromolecular inhibitors α1-antigimotrypsin and aprotinin have no effect of inhibiting histamine release, although they themselves have a chymase inhibitory effect, and the latter in particular promoted histamine IS.
以上に対し、BBIは高分子インヒビターであるにも拘
らず、ヒスタミンの遊離を効果的に抑制する効果を示し
た。In contrast, although BBI is a polymeric inhibitor, it showed the effect of effectively suppressing the release of histamine.
[発明の効果〕
以上,説明した通り、大豆由来のBBIは、肥満細胞中
のキマーゼ及びトリプターゼを強く阻害すると共に、該
細胞からの脱顆粒を抑制するので、炎症その他1種々の
アレルギー症状への適応が期待される。[Effects of the Invention] As explained above, soybean-derived BBI strongly inhibits chymase and tryptase in mast cells and suppresses degranulation from these cells, so it is effective against inflammation and other allergic symptoms. Adaptation is expected.
Claims (1)
irk)型トリプシンインヒビターを有効成分とする肥
満細胞脱顆粒抑制剤。(1) Bowman-Bark (Bowman B) derived from soybean
A mast cell degranulation inhibitor containing a trypsin inhibitor (irk) type trypsin inhibitor as an active ingredient.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP61196347A JPS6351335A (en) | 1986-08-20 | 1986-08-20 | Degranulation inhibitor for mast cell |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP61196347A JPS6351335A (en) | 1986-08-20 | 1986-08-20 | Degranulation inhibitor for mast cell |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS6351335A true JPS6351335A (en) | 1988-03-04 |
JPH0586933B2 JPH0586933B2 (en) | 1993-12-14 |
Family
ID=16356325
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP61196347A Granted JPS6351335A (en) | 1986-08-20 | 1986-08-20 | Degranulation inhibitor for mast cell |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS6351335A (en) |
Cited By (12)
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---|---|---|---|---|
WO1995003333A3 (en) * | 1993-07-26 | 1995-03-23 | Ucp Gen Pharma Ag | Tryptase inhibitor |
US5567425A (en) * | 1993-11-30 | 1996-10-22 | Lxr Biotechnology Inc. | Compositions which inhibit apoptosis, methods of purifying the compositions and uses thereof |
US5620888A (en) * | 1994-12-29 | 1997-04-15 | Lxr Biotechnology, Inc. | Cell strains for use in identifying potentially therapeutically effective agents |
US5637486A (en) * | 1993-04-30 | 1997-06-10 | Lxr Biotechnology Inc. | Methods of identifying potentially therapeutically effective agents and cell strains for use therein |
EP0841933A1 (en) * | 1995-07-25 | 1998-05-20 | The Trustees Of The University Of Pennsylvania | Bowman-birk inhibitor compositions for treatment of imflammatory disease |
US6004579A (en) * | 1995-09-14 | 1999-12-21 | Lxr Biotechnology, Inc. | Compositions which inhibit apoptosis, methods of making the compositions and uses thereof |
WO2001062293A1 (en) * | 2000-02-22 | 2001-08-30 | Suntory Limited | Preventive or therapeutic drugs for various eosinophilia-related diseases containing chymase inhibitors as the active ingredient |
US6949528B1 (en) | 1998-03-18 | 2005-09-27 | Goddard John G | Compositions containing lysophosphatidic acids which inhibit apoptosis and uses thereof |
US6949529B2 (en) | 1997-03-19 | 2005-09-27 | Sky High, Llc | Compositions containing lysophosphotidic acids which inhibit apoptosis and uses thereof |
WO2009060915A1 (en) * | 2007-11-06 | 2009-05-14 | San-Ei Gen F.F.I., Inc. | Salivary secretion promoter |
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-
1986
- 1986-08-20 JP JP61196347A patent/JPS6351335A/en active Granted
Cited By (29)
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US5637486A (en) * | 1993-04-30 | 1997-06-10 | Lxr Biotechnology Inc. | Methods of identifying potentially therapeutically effective agents and cell strains for use therein |
US5681703A (en) * | 1993-04-30 | 1997-10-28 | Lxr Biotechnology Inc. | Methods of identifying potentially therapeutically effective agents and cell strains for use therein |
WO1995003333A3 (en) * | 1993-07-26 | 1995-03-23 | Ucp Gen Pharma Ag | Tryptase inhibitor |
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US5624672A (en) * | 1993-11-30 | 1997-04-29 | Lxr Biotechnology Inc. | Compositions which inhibit apoptosis, methods of purifying the compositions and uses thereof |
US5567425A (en) * | 1993-11-30 | 1996-10-22 | Lxr Biotechnology Inc. | Compositions which inhibit apoptosis, methods of purifying the compositions and uses thereof |
US5635186A (en) * | 1993-11-30 | 1997-06-03 | Lxr Biotechnology Inc. | Compositions which inhibit apoptosis, methods of purifying the compositions and uses thereof |
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US6306398B1 (en) | 1993-11-30 | 2001-10-23 | Lxr Biotechnology, Inc. | Compositions which inhibit apoptosis, methods of purifying the compositions and uses thereof |
US5759548A (en) * | 1993-11-30 | 1998-06-02 | Lxr Biotechnology Inc. | Compositions which inhibit apoptosis, methods of purifying the compositions and uses thereof |
US5635187A (en) * | 1993-11-30 | 1997-06-03 | Lxr Biotechnology Inc. | Compositions which inhibit apoptosis, methods of purifying the compositions and uses thereof |
US5620888A (en) * | 1994-12-29 | 1997-04-15 | Lxr Biotechnology, Inc. | Cell strains for use in identifying potentially therapeutically effective agents |
EP0841933A4 (en) * | 1995-07-25 | 1998-08-05 | Univ Pennsylvania | Bowman-birk inhibitor compositions for treatment of imflammatory disease |
EP0841933A1 (en) * | 1995-07-25 | 1998-05-20 | The Trustees Of The University Of Pennsylvania | Bowman-birk inhibitor compositions for treatment of imflammatory disease |
US6004579A (en) * | 1995-09-14 | 1999-12-21 | Lxr Biotechnology, Inc. | Compositions which inhibit apoptosis, methods of making the compositions and uses thereof |
US6949529B2 (en) | 1997-03-19 | 2005-09-27 | Sky High, Llc | Compositions containing lysophosphotidic acids which inhibit apoptosis and uses thereof |
US7259273B1 (en) | 1998-03-18 | 2007-08-21 | Goddard John G | Compositions containing lysophosphotidic acids which inhabit apoptosis and uses thereof |
US6949528B1 (en) | 1998-03-18 | 2005-09-27 | Goddard John G | Compositions containing lysophosphatidic acids which inhibit apoptosis and uses thereof |
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WO2017150724A1 (en) * | 2016-03-03 | 2017-09-08 | 学校法人藤田学園 | Soybean allergy antigen |
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---|---|
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