JP2712611B2 - Toxoplasma growth inhibitor - Google Patents
Toxoplasma growth inhibitorInfo
- Publication number
- JP2712611B2 JP2712611B2 JP1224677A JP22467789A JP2712611B2 JP 2712611 B2 JP2712611 B2 JP 2712611B2 JP 1224677 A JP1224677 A JP 1224677A JP 22467789 A JP22467789 A JP 22467789A JP 2712611 B2 JP2712611 B2 JP 2712611B2
- Authority
- JP
- Japan
- Prior art keywords
- glu
- toxoplasma
- gly
- peptide
- cells
- 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.)
- Expired - Fee Related
Links
- 241000223996 Toxoplasma Species 0.000 title claims description 32
- 239000003966 growth inhibitor Substances 0.000 title claims description 5
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 36
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 5
- SOEATRRYCIPEHA-BQBZGAKWSA-N Gly-Glu-Glu Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O SOEATRRYCIPEHA-BQBZGAKWSA-N 0.000 claims description 3
- 108010002311 N-glycylglutamic acid Proteins 0.000 claims description 3
- 125000001433 C-terminal amino-acid group Chemical group 0.000 claims description 2
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 description 19
- 230000000694 effects Effects 0.000 description 17
- 230000012010 growth Effects 0.000 description 15
- 230000002401 inhibitory effect Effects 0.000 description 10
- 238000000034 method Methods 0.000 description 9
- 241001465754 Metazoa Species 0.000 description 8
- 210000002540 macrophage Anatomy 0.000 description 7
- 239000011347 resin Substances 0.000 description 7
- 229920005989 resin Polymers 0.000 description 7
- 229940079593 drug Drugs 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 102000004196 processed proteins & peptides Human genes 0.000 description 6
- 239000000243 solution Substances 0.000 description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 239000012981 Hank's balanced salt solution Substances 0.000 description 4
- 230000000975 bioactive effect Effects 0.000 description 4
- 239000006059 cover glass Substances 0.000 description 4
- 244000045947 parasite Species 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 230000001938 anti-toxoplasmal effect Effects 0.000 description 3
- 108010029907 obioactin Proteins 0.000 description 3
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 2
- KRHYYFGTRYWZRS-UHFFFAOYSA-N Fluorane Chemical compound F KRHYYFGTRYWZRS-UHFFFAOYSA-N 0.000 description 2
- -1 amide compound Chemical class 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 229910000040 hydrogen fluoride Inorganic materials 0.000 description 2
- 230000003308 immunostimulating effect Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 125000000896 monocarboxylic acid group Chemical group 0.000 description 2
- 210000003024 peritoneal macrophage Anatomy 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 125000006239 protecting group Chemical group 0.000 description 2
- 239000002356 single layer Substances 0.000 description 2
- 238000010532 solid phase synthesis reaction Methods 0.000 description 2
- AQTUACKQXJNHFQ-LURJTMIESA-N (2s)-2-[(2-methylpropan-2-yl)oxycarbonylamino]pentanedioic acid Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)CCC(O)=O AQTUACKQXJNHFQ-LURJTMIESA-N 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 238000002738 Giemsa staining Methods 0.000 description 1
- BUZMZDDKFCSKOT-CIUDSAMLSA-N Glu-Glu-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O BUZMZDDKFCSKOT-CIUDSAMLSA-N 0.000 description 1
- IEFJWDNGDZAYNZ-BYPYZUCNSA-N Gly-Glu Chemical compound NCC(=O)N[C@H](C(O)=O)CCC(O)=O IEFJWDNGDZAYNZ-BYPYZUCNSA-N 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 206010053759 Growth retardation Diseases 0.000 description 1
- 108700021154 Metallothionein 3 Proteins 0.000 description 1
- 102100028708 Metallothionein-3 Human genes 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 201000005485 Toxoplasmosis Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 208000037919 acquired disease Diseases 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- 230000009435 amidation Effects 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000005915 ammonolysis reaction Methods 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000003855 balanced salt solution Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000034303 cell budding Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 201000002491 encephalomyelitis Diseases 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 210000000416 exudates and transudate Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000008125 glucin Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 1
- 230000000521 hyperimmunizing effect Effects 0.000 description 1
- 229960001438 immunostimulant agent Drugs 0.000 description 1
- 239000003022 immunostimulating agent Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 210000003200 peritoneal cavity Anatomy 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002953 preparative HPLC Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000003812 trophozoite Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Description
【発明の詳細な説明】 （産業上の利用分野） 本発明は、トキソプラズマ増殖抑制作用を有する生理
活性ペプチドを含有するトキソプラズマ増殖抑制剤に関
するものである。Description: TECHNICAL FIELD The present invention relates to a toxoplasma growth inhibitor containing a physiologically active peptide having a toxoplasma growth inhibitory action.
（発明の背景） トキソプラズマ（Toxoplasma）は広く世界各地域の哺
乳類、鳥類等のほとんどあらゆる細胞内に寄生しうる原
虫の一種で、ヒトにおける脳髄膜炎をはじめ各種の先天
性、後天性障害を引き起こし、また家畜の病原体として
も恐れられている。このトキソプラズマは健康な宿主の
マクロファージに摂取されても、一般微生物と異なり、
マクロファージの殺機能から逃避し、内部出芽による分
裂増殖を繰り返す。Mammalian BACKGROUND OF THE INVENTION Toxoplasma (Toxoplasma) is widely over the world, a type of parasite that can infest almost any intracellular avian or the like, including encephalomyelitis in humans various congenital causes acquired disorders It is also feared as a livestock pathogen. Unlike normal microorganisms, this toxoplasma is ingested by healthy host macrophages,
Escape from the killing function of macrophages and repeat division and proliferation by internal budding.
一方、トキソプラズマ過免疫動物では、マクロファー
ジ内での虫体の増殖抑制、虫体への殺作用が観察されて
おり、その血清中には該動物の正常細胞内のトキソプラ
ズマ増殖を抑制する液性因子（リンホカイン）が存在す
ることが知られている（文献1;以下説明中に参照される
文献は本明細書末尾に参考文献として列挙する）。On the other hand, in toxoplasma hyperimmunized animals, growth suppression of the parasite in macrophages and killing of the parasite have been observed, and a humoral factor that suppresses the growth of toxoplasma in normal cells of the animal is contained in the serum. (Lymphokine) is known to exist (Reference 1; references referred to in the description below are listed as references at the end of the specification).
またトキソプラズマ過免疫動物の脾臓細胞をトキソプ
ラズマ原虫溶解抗原（Toxoplasma lysateantigen;TLE）
の存在下で培養した培養上清中にも、同種細胞内でのト
キソプラズマ増殖を抑制するリンホカインが存在するこ
とがわかっている（文献2,3）。In addition, the spleen cells of a Toxoplasma hyperimmune animal are treated with Toxoplasma lysateantigen (TLE).
It has been found that there is also a lymphokine that suppresses the growth of Toxoplasma in allogeneic cells even in the culture supernatant cultured in the presence of E. coli (References 2 and 3).
このリンホカインはＴリンパ球産生物質と推察される
分子量３〜４万の糖蛋白で、Toxo-GIF（Toxoplasma Gro
wth Inhibitory Factor;トキソプラズマ増殖抑制因子）
と呼ばれている（文献2,3）。このToxo-GIFはマクロフ
ァージ内での増殖のみならず他の体細胞でのトキソプラ
ズマ増殖をも抑制する。しかし宿主と同じ動物種の細胞
内における原虫の増殖を阻止するのみで、異種の動物で
の増殖は阻止できない（文献3,4）。このような種族特
異性があるため、Toxo-GIFはヒトや宿主以外の動物のト
キソプラズマ感染症の予防，治療に用いることができな
かった。This lymphokine is a glycoprotein with a molecular weight of 30,000 to 40,000, which is presumed to be a T-lymphocyte-producing substance, and Toxo-GIF ( Toxoplasma Gro
wth Inhibitory Factor)
(References 2 and 3). This Toxo-GIF suppresses not only the growth in macrophages but also the growth of Toxoplasma in other somatic cells. However, it can only prevent the growth of protozoa in cells of the same animal species as the host, but cannot prevent the growth in heterologous animals (References 3 and 4). Due to such race specificity, Toxo-GIF could not be used to prevent or treat toxoplasmosis in animals other than humans and hosts.
このような状況下で本発明者らが見いだしたトキソプ
ラズマ免疫動物血清の加水分解による低分子量ポリペチ
ド物質は種族特異性のないトキソプラズマ増殖抑制物質
として画期的なものであった（文献5,6,7）。このトキ
ソプラズマ免疫動物血清由来加水分解物はオビオアクチ
ン（Obioactin）とよばれている（文献８）。このオビ
オアクチンは分子量3,000〜5,000のポリペプチド物質で
あり同種細胞だけでなく、異種細胞内におけるトキソプ
ラズマ増殖を抑制する（文献９）。さらにオビオクアチ
ンはトキソプラズマ以外の他の原虫や細菌、ウィルス等
に対する抗微生物活性、さらには抗腫瘍活性をも有する
もので、免疫賦活剤としての用途を開くものであった
（文献5,6,7,17）。Under such circumstances, the low molecular weight polypeptide substance obtained by hydrolysis of the serum of a Toxoplasma immunized animal found by the present inventors was a revolutionary substance as a Toxoplasma growth inhibitor having no race specificity (Refs. 5, 6, 7). The hydrolyzate derived from the serum of an animal immunized with Toxoplasma is called Obioactin (Reference 8). This obioactin is a polypeptide substance having a molecular weight of 3,000 to 5,000, and suppresses the proliferation of Toxoplasma in not only the same kind of cells but also the different kinds of cells (Reference 9). Furthermore, obioquatin also has antimicrobial activity against other protozoa, bacteria, viruses, etc. other than toxoplasma, and also has antitumor activity, and has opened up its use as an immunostimulant (References 5, 6, 7, 17).
発明者はさらに、このオビオアクチンを精製してその
活性中心を明らかにし、その知見から Gly-Glu-Glu-Glu-Glu-Glu など一連の新たな抗トキソプラズマペプチドを合成した
（文献18;特開平1-175996）。The inventors further purified this obioactin to reveal its active center, and synthesized a series of new anti-toxoplasma peptides such as Gly-Glu-Glu-Glu-Glu-Glu based on the findings (Reference 18; -175996).
今回、発明者はこれら新規生理活性ペプチドの類縁体
を探索する過程で、より短かいペプチドでもトキソプラ
ズマ増殖抑制作用を有する新たな生理活性ペプチドを見
出した。これは、より活性中心に迫った抗トキソプラズ
マペプチドをより簡単なステップで低コスで大量生産が
できることを示す。またペプチドの溶解性、安定性を改
善する目的で種々の修飾を試みたところ、アミド化した
ペプチドにトキソプラズマ増殖抑制作用を有するものを
見出し、より有用な抗トキソプラズマペプチドの存在を
明らかにした。本発明はこのような知見に基き完成され
たものである。In the process of searching for analogs of these novel bioactive peptides, the present inventors have found a new bioactive peptide having a toxoplasma growth inhibitory action even with a shorter peptide. This indicates that the anti-toxoplasma peptide closer to the active center can be mass-produced with simpler steps and at lower cost. In addition, various modifications were made to improve the solubility and stability of the peptide. As a result, an amidated peptide having an inhibitory action on toxoplasma growth was found, and the existence of a more useful anti-toxoplasma peptide was revealed. The present invention has been completed based on such findings.
（発明の目的） すなわち本発明は、より活性中心に迫ったもので、か
つ低コストで大量生産ができるトキソプラズマ増殖抑制
作用を有する新規生理活性ペプチドを提供することを目
的とする。(Purpose of the Invention) That is, an object of the present invention is to provide a novel bioactive peptide having a toxoplasma growth inhibitory action which is closer to the active center and can be mass-produced at low cost.
また本発明は、溶解性安定性に優れ、薬剤としての有
用性に優れた新規生理活性ペプチドを提供することを目
的とする。Another object of the present invention is to provide a novel bioactive peptide having excellent solubility stability and excellent utility as a drug.
（発明の構成） 本発明のこの目的は、下記アミノ酸配列のペプチドの
内少なくとも一つのペプチドを含有する、トキソプラズ
マ増殖抑制剤。(Constitution of the Invention) The object of the present invention is a toxoplasma growth inhibitor comprising at least one peptide having the following amino acid sequence:
Gly-Glu-Glu-Glu Gly-Glu-Glu Gly-Glu Gly-Glu-Glu-Glu-Glu-Glu-NH2 により達成される。なお左末端のアミノ酸はＮ末端アミ
ノ酸であり、右末端のアミノ酸はＣ末端のアミノ酸であ
る。従って第４列のペプチドのＣ末端側のアミノ酸（‐
Glu-NH2）は、α−COOH基がアミド化されていることを
示す。It is accomplished by Gly-Glu-Glu-Glu Gly -Glu-Glu Gly-Glu Gly-Glu-Glu-Glu-Glu-Glu-NH 2. The left terminal amino acid is the N-terminal amino acid, and the right terminal amino acid is the C-terminal amino acid. Therefore, the amino acid at the C-terminal side of the peptide in the fourth row (-
Glu-NH 2 ) indicates that the α-COOH group is amidated.
これらは固相法や液相法などの従来より公知の方法に
より合成できる（文献15,16）。These can be synthesized by a conventionally known method such as a solid phase method or a liquid phase method (References 15, 16).
アミド化ペプチドの場合には、以下のような方法によ
って合成できる。In the case of an amidated peptide, it can be synthesized by the following method.
固相法による合成では、パラメチルベンズヒドリルア
ミン樹脂を用いて骨格となる基本ペプチドを合成し、そ
の後フッ化水素処理して樹脂から脱離させると共にアミ
ド体を生成する。In the synthesis by the solid-phase method, a basic peptide serving as a skeleton is synthesized using paramethylbenzhydrylamine resin, and then treated with hydrogen fluoride to be eliminated from the resin and to generate an amide compound.
液相法の場合は、NH2基を予め保護基でマスクしてか
らCOOH基にＮ−ヒドロキシスクシンイミドを導入し、そ
の後、アンモノリシスさせてCOOH基をアミド化する。こ
のアミド化Gluに、Ｚ（OMe）‐Glu（OBzl）及びＺ（OM
e）‐Glyを順次活性カップリング法で反応させ、基本骨
格を伸長することができる。In the case of the liquid phase method, the NH 2 group is masked with a protecting group in advance, N-hydroxysuccinimide is introduced into the COOH group, and then the COOH group is amidated by ammonolysis. Z (OMe) -Glu (OBzl) and Z (OM
e) -Gly can be sequentially reacted by the active coupling method to extend the basic skeleton.
（実施例） Toxo-GIF活性測定方法 Toxo-GIF活性の測定は本発明者らの方法で行なった
（文献11,12）。(Example) Toxo-GIF activity measurement method Toxo-GIF activity was measured by the present inventors' methods (References 11 and 12).
成熟BALB/C系雌マウス腹腔内に滅菌0.1％グリコーゲ
ン加生理食塩水1mlを注入し、５日後に冷ハンクス液（H
anks' balanced salt solution;HBSS）にて腹腔内を洗
浄し腹腔内滲出細胞（peritoneal exudate cell）を採
取した。遠心操作（250G,5分）により２回洗浄後、細胞
を１×106cells/mlの割合で10％熱非働化子牛血清（C
S）添加199培養液（Tc-199）に浮遊させた。この細胞浮
遊液を1mlずつ径15mmの円形カバーグラスを落とし込ん
だ培養シャーレ（Multi-dish-tray,FB-16-24-TC,米国Li
nblo Chemical社製）内に分注し、５％炭酸ガス培養器
内で培養した。混入した赤血球やリンパ球系細胞を除去
するために、各培養シャーレを２時間ごとに３回、HBSS
で静かに洗浄後、一夜炭酸ガス培養器内で培養してカバ
ーグラス上にマクロファージのモノレーヤを形成させ
た。1 ml of sterile 0.1% glycogen-added saline was injected into the abdominal cavity of a mature BALB / C female mouse, and 5 days later, a cold Hanks solution (H
The peritoneal cavity was washed with anks' balanced salt solution (HBSS), and peritoneal exudate cells were collected. After washing twice by centrifugation (250 G, 5 minutes), the cells were washed at a rate of 1 × 10 6 cells / ml in 10% heat-inactivated calf serum (C
S) The cells were suspended in an added 199 culture solution (Tc-199). A culture Petri dish (multi-dish-tray, FB-16-24-TC, Li
nblo Chemical Co.) and cultured in a 5% carbon dioxide incubator. In order to remove contaminating red blood cells and lymphoid cells, each culture dish was washed 3 times every 2 hours with HBSS.
Gently, and cultured overnight in a carbon dioxide incubator to form a macrophage monolayer on a cover glass.
このモノレーヤをTc-199培養液で再び洗浄した後、ト
キソプラズマRH株栄養型虫体をマクロファージ１×106
個当たり１×105個となるように添加・感染させた。細
胞内に穿入しなかった遊離虫体は感染１時間後にHBSSで
洗浄除去した。After the monolayer was washed again with the Tc-199 culture solution, the trophozoites of the Toxoplasma RH strain were transformed into macrophages 1 × 10 6
The cells were added and infected at 1 × 10 5 cells. The free parasites that did not penetrate into the cells were washed away with HBSS one hour after infection.
このカバーグラスに各試料を含む培養液（マクロファ
ージ１×106個当たり1ml）を添加し、48時間培養した
後、May-Giemsa染色して細胞内のトキソプラズマ数を算
定した。カバーグラス上の一定視野での細胞100個当た
りについて、含有虫体数が０個である細胞の数、含有虫
体数が１個から５個である細胞の数、含有虫体数が６個
以上である細胞の数をそれぞれ数え百分率を求める。同
様な操作をカバーグラスの他の４ケ所の視野でも行な
い、合計５ケ所での値からそれぞれの平均値と標準偏差
を求めてトキソプラズマ含有細胞出現率とした。A culture solution containing each sample (1 ml per 1 × 10 6 macrophages) was added to the cover glass, cultured for 48 hours, and then subjected to May-Giemsa staining to calculate the number of intracellular toxoplasma. Per 100 cells in a fixed field of view on a cover glass, the number of cells containing 0 worms, the number of cells containing 1 to 5 worms, and 6 worms The above number of cells is counted and the percentage is determined. The same operation was performed for the other four fields of view of the cover glass, and the average value and standard deviation were calculated from the values at a total of five points to obtain the appearance rate of the Toxoplasma-containing cells.
また活性の別の表現方法として、試料添加によるトキ
ソプラズマ（Tp）含有細胞（百分率）の減少の割合から
Toxo-GIF活性を百分率で表示した。Another expression of the activity is based on the ratio of decrease in the percentage of toxoplasma (Tp) -containing cells due to sample addition.
Toxo-GIF activity was expressed as a percentage.
試料は各粉末試料を、3mlの10％のCS-Tc199培地に溶
解し、0.45μｍ孔の膜フィルタで濾過滅菌したものを用
いた。 Samples were prepared by dissolving each powder sample in 3 ml of 10% CS-Tc199 medium and filtering and sterilizing with a 0.45 μm pore membrane filter.
合成ペプチドのToxo-GIF活性 以下のオリゴペプチドを合成しToxo-GIF活性を測定し
た。Toxo-GIF activity of synthetic peptide The following oligopeptides were synthesized and Toxo-GIF activity was measured.
Gly-Glu-Glu-Glu-Glu-Glu （Glycil-penta-Glutaminate;1Gly＋5Glu） Gly-Glu-Glu-Glu-Glu （Glycil-tetra-Glutaminate;1Gly＋4Glu） Gly-Glu-Glu-Glu （Glycil-tri-Glutaminate;1Gly＋3Glu） Gly-Glu-Glu （Glycil-di-Glutaminate;1Gly＋2Glu） Gly-Glu （Glycil-mono-Glutaminate;1Gly＋1Glu） これらの合成ペプチドを0.7mMの濃度で培養液（10％
‐CS-Tc199）に溶解して、マウス腹腔マクロファージを
用いてToxo-GIF活性を測定した。Gly-Glu-Glu-Glu-Glu-Glu (Glycil-penta-Glutaminate; 1Gly + 5Glu) Gly-Glu-Glu-Glu-Glu (Glycil-tetra-Glutaminate; 1Gly + 4Glu) Gly-Glu-Glu-Glu (Glycil-tri- Glutaminate; 1Gly + 3Glu) Gly-Glu-Glu (Glycil-di-Glutaminate; 1Gly + 2Glu) Gly-Glu (Glycil-mono-Glutaminate; 1Gly + 1Glu) These synthetic peptides are cultured at a concentration of 0.7 mM in a culture solution (10%
-CS-Tc199), and Toxo-GIF activity was measured using mouse peritoneal macrophages.
結果を下記の第１表に示す。 The results are shown in Table 1 below.
第１表に示されるように各ペプチドのToxo-GIF活性値
は、1Gly＋4Gluでは−2.5％、1Gly＋3Gluでは14.5％、1
Gly＋2Gluでは65.0％、1Gly＋1Gluでは84.1％であり、1
Gly＋4Gluはトキソプラズマ増殖抑制活性が見られない
ものの、より短かい各ペプチド1Gly＋3Glu、1Gly＋2Glu
及び1Gly＋1Gluでは短くなるにつれて強くなるトキソプ
ラズマ増殖抑制活性が認められた。 As shown in Table 1, the Toxo-GIF activity value of each peptide was -2.5% for 1Gly + 4Glu, 14.5% for 1Gly + 3Glu, 1
65.0% for Gly + 2Glu, 84.1% for 1Gly + 1Glu, 1
Gly + 4Glu has no Toxoplasma growth inhibitory activity, but each shorter peptide 1Gly + 3Glu, 1Gly + 2Glu
In addition, 1Gly + 1Glu showed an inhibitory activity on toxoplasma growth which became stronger as the length became shorter.
また最も活性の高い1Gly＋1Gluについて、前回の出願
においてToxo-GIFの活性の存在を明らかにした1Gly＋5G
luとの比較を行なった。In addition, regarding the most active 1Gly + 1Glu, the existence of Toxo-GIF activity was clarified in the previous application.
Compared with lu.
第２表に示すように、Gly＋1Gluは1Gly＋5Gluと同程
度のToxo-GIF活性を示し、1Gly＋5Gluと同様のトキソプ
ラズマ増殖抑制作用や免疫賦活作用を有する薬剤として
の用途が期待できることがわかった。なお試料濃度0.7m
Mを重量濃度に換算すると、1Gly＋5Gluでは0.50mg/ml、
1Gly＋1Gluでは0.14mg/mlとなり、1Gly＋1Gluは重量当
りで1Gly＋5Gluの約1/3の量で同程度の抑制効果が得ら
れることを示している。このことは薬剤として投与する
際により少ない投与量で良いことを示している。 As shown in Table 2, Gly + 1Glu exhibited the same level of Toxo-GIF activity as 1Gly + 5Glu, indicating that it could be expected to be used as a drug having a Toxoplasma growth inhibitory action and an immunostimulatory action similar to 1Gly + 5Glu. Sample concentration 0.7m
When M is converted to a weight concentration, 0.50 mg / ml for 1Gly + 5Glu,
1Gly + 1Glu is 0.14 mg / ml, which indicates that 1Gly + 1Glu can achieve the same level of inhibitory effect with about 1/3 of 1Gly + 5Glu by weight. This indicates that a smaller dose is required for administration as a drug.
アミド化ペプチドのToxo-GIF活性 公知の固相法によりアミド化ペプチド、Gly-Glu-Glu-
Glu-Glu-Glu-NH2を合成した。すなわち、パラメチルベ
ンズヒドリルアミン樹脂（導入量1.5mモル，アプライド
バイオシステム社製）にBoc-Glu（OcHex）ｔ−ブトキシ
カルボニル−グルタミン酸）及び、Boc-Gly（ｔ−ブト
キシカルボニル−グルシン）をDCC-HOBT法（ジシクロヘ
キシルカルボジイミド−ヒドロキシベンゾトリアゾロー
ル法）にて順次カップリングさせ、保護ペプチド樹脂を
得た。この樹脂を乾燥した後、０℃で１時間フッ化水素
処理して保護基を除去すると共に、樹脂から脱離させ
た。得られたペプチドは大量分取用高速液体クロマトグ
ラフィーで精製し高純度の精製物を得た。Toxo-GIF activity of amidated peptide Amidated peptide, Gly-Glu-Glu-
Glu-Glu-Glu-NH 2 was synthesized. That is, Boc-Glu (OcHex) t-butoxycarbonyl-glutamic acid) and Boc-Gly (t-butoxycarbonyl-glucin) were added to paramethylbenzhydrylamine resin (introduced amount: 1.5 mmol, manufactured by Applied Biosystems) in DCC. Coupling was successively performed by the -HOBT method (dicyclohexylcarbodiimide-hydroxybenzotriazolol method) to obtain a protected peptide resin. After the resin was dried, the resin was treated with hydrogen fluoride at 0 ° C. for 1 hour to remove the protecting group and to be eliminated from the resin. The obtained peptide was purified by high performance preparative high performance liquid chromatography to obtain a highly purified product.
このアミド化ペプチド、Gly-Glu-Glu-Glu-Glu-Glu-NH
2を0.7mM（0.50mM）の濃度で培養液（10％‐CS-Tc199）
に溶解し、BALB/マウス（８週齢）の腹腔マクロファー
ジを用いてToxo-GIF活性を測定した。This amidated peptide, Gly-Glu-Glu-Glu-Glu-Glu-NH
2 at a concentration of 0.7 mM (0.50 mM) in culture solution (10% -CS-Tc199)
And Toxo-GIF activity was measured using peritoneal macrophages of BALB / mouse (8 weeks old).
第３表に示すようにアミド化ペプチドもトキソプラズ
マ増殖抑制活性を有し、しかもその活性はアミド化され
る前のGly-Glu-Glu-Glu-Glu-Gluと同程度かむしろ高い
ものであった。As shown in Table 3, the amidated peptide also had the activity of inhibiting the growth of toxoplasma, and its activity was comparable or higher than that of Gly-Glu-Glu-Glu-Glu-Glu before amidation. .
そこで、アミド化ペプチドの濃度依存製を調べた。第
４表に示すように0.05mg/ml以上の濃度でToxo-GIF活性
を示し、0.5mg/mlの濃度で活性はほぼ飽和していた。1.
0mg/ml濃度では細胞の凝縮と剥離を示す細胞障害像が認
められた。この結果は非アミド化ペプチドGly-Glu-Glu-
Glu-Glu-Gluと同様であった（文献18）。Therefore, the concentration-dependent product of the amidated peptide was examined. As shown in Table 4, Toxo-GIF activity was exhibited at a concentration of 0.05 mg / ml or more, and the activity was almost saturated at a concentration of 0.5 mg / ml. 1.
At a concentration of 0 mg / ml, a cytotoxic image indicating cell condensation and detachment was observed. This result indicates that the non-amidated peptide Gly-Glu-Glu-
It was similar to Glu-Glu-Glu (Reference 18).
このように溶解性と安定性に優れると考えられるアミ
ド化ペプチド、Gly-Glu-Glu-Glu-Glu-Glu-NH2も非アミ
ド化ペプチドと同様に薬剤として使用できると考えられ
る。Thus solubility and amidated peptide is considered to be excellent in stability, believed Gly-Glu-Glu-Glu- Glu-Glu-NH 2 can be used as well as non-amidated peptide as a drug.
（発明の効果） 以上のように本発明の生理活性ペプチドは、トキソプ
ラズマ増殖抑制作用を有する薬剤として使用でき、従来
のものよりもより活性中心に迫ったもので、かつ低コス
トで大量生産ができる。 (Effect of the Invention) As described above, the physiologically active peptide of the present invention can be used as a drug having an inhibitory action on toxoplasma growth, is closer to the active center than conventional ones, and can be mass-produced at low cost. .
また、アミド化ペプチドは溶解性・安定性に優れ、薬
剤としての有用性に優れる。Further, the amidated peptide is excellent in solubility and stability, and is excellent in utility as a drug.
参考文献 1.Sethi,K.K.et al.J.Immunol.131,1151-1558,（1975） 2.Shirahata,T.,Shimizu,K.＆ Suzuki,N.Z.Parasitenk
d.49,11-23（1976） 3.Nagasawa,H.et al.Immunobiol.156,307-319（1980） 4.Matsumoto,Y.et al.Zbl.Bakt.Hyg.A 250,383-391（19
81） 5.特開昭57-142922号 6.特開昭57-144983号 7.米国特許第4482543号 8.Suzuki,N.et al.Zbl.Bakt.Hyg.I.Abt.Orig.A 250,356
-366（1984） 9.Suuki,N.et al.Zbl.Bakt.Hyg.I.Abt.Orig.A 256,367-
380（1984） 10.Igarashi,I.et al.Zbl.Bakt.Hyg.I.Abt.Orig.A 244,
472-482（1979） 11.Nagasawa,H.et al.Jpn.J.Vet.Sci.43 307-319（198
1） 12.Sakurai,H.,et al.Jpn.J.Trop.Med.Hyg.10,183-195
（1982） 13.Benson R.J.,et al.Proc.Nat.Acad.Sci.,USA.72,619
-622（1975） 14.Bohlen,P.「Method in Enzymoloy」91,17-26（198
3） 15.Kent.S.B.,et al.U.Ragnarsen編「Peptides 1984」
（Almqvist and Wiksell,Stockholm,Sweden,1984）p18
5. 16.Kent.S.B.H.et al.N.Izumiya編「Peptide Chemisitr
y」（Protein Reseach Foundation,B.H.Osaka,Japan,19
85）p217. 17.Osaki,H.et al.Zbl.Bakt.Hyg.I.Abt.Orig.A 256,328
-334（1984） 18.特開平1-175996号References 1. Sethi, KKet al. J. Immunol. 131 , 1151-1558, (1975) 2. Shirahata, T., Shimizu, K. & Suzuki, NZ Parasitenk
d. 49 , 11-23 (1976) 3. Nagasawa, H. et al. Immunobiol. 156 , 307-319 (1980) 4. Matsumoto, Y. et al. Zbl. Bakt. Hyg. A 250 , 383-391 (19
81) 5. JP-A-57-142922 6. JP-A-57-144983 7. U.S. Pat. No. 4,482,543 8. Suzuki, N. et al. Zbl. Bakt. Hyg. I. Abt. Orig.A 250 , 356
-366 (1984) 9.Suuki, N.et al.Zbl.Bakt.Hyg.I.Abt.Orig.A 256 , 367-
380 (1984) 10.Igarashi, I.et al.Zbl.Bakt.Hyg.I.Abt.Orig.A 244 ,
472-482 (1979) 11.Nagasawa, H. et al. Jpn. J. Vet. Sci. 43 307-319 (198
1) 12.Sakurai, H., et al.Jpn.J.Trop.Med.Hyg. 10 , 183-195
(1982) 13.Benson RJ, et al. Proc. Nat. Acad. Sci., USA. 72 , 619
-622 (1975) 14.Bohlen, P. "Method in Enzymoloy" 91 , 17-26 (198
3) 15. Kent. SB, et al. U. Ragnarsen, edited by Peptides 1984.
(Almqvist and Wiksell, Stockholm, Sweden, 1984) p18
5. 16.Kent.SBHet al.N.Izumiya ed., Peptide Chemisitr
y ”(Protein Reseach Foundation, BHOsaka, Japan, 19
85) p217. 17.Osaki, H.et al.Zbl.Bakt.Hyg.I.Abt.Orig.A 256, 328
-334 (1984) 18.JP-A-1-175996
Claims (1)
も一つのペプチドを含有する、トキソプラズマ増殖抑制
剤。 Gly-Glu-Glu-Glu Gly-Glu-Glu Gly-Glu Gly-Glu-Glu-Glu-Glu-Glu-NH2 （左末端はＮ末端アミノ酸、右末端はＣ末端アミノ酸で
ある。最下行のＣ末端のアミノ酸（‐Glu-NH2）はα−C
OOH基がアミド化されていることを示す）A toxoplasma growth inhibitor comprising at least one peptide having the following amino acid sequence: Gly-Glu-Glu-Glu Gly -Glu-Glu Gly-Glu Gly-Glu-Glu-Glu-Glu-Glu-NH 2 ( left end N-terminal amino acid, the right end is the C-terminal amino acids. Bottom row C The terminal amino acid (-Glu-NH 2 ) is α-C
OOH group is amidated.)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1224677A JP2712611B2 (en) | 1989-09-01 | 1989-09-01 | Toxoplasma growth inhibitor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1224677A JP2712611B2 (en) | 1989-09-01 | 1989-09-01 | Toxoplasma growth inhibitor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0390099A JPH0390099A (en) | 1991-04-16 |
| JP2712611B2 true JP2712611B2 (en) | 1998-02-16 |
Family
ID=16817492
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1224677A Expired - Fee Related JP2712611B2 (en) | 1989-09-01 | 1989-09-01 | Toxoplasma growth inhibitor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2712611B2 (en) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5859953A (en) * | 1981-10-02 | 1983-04-09 | Toyo Jozo Co Ltd | Novel peptide |
-
1989
- 1989-09-01 JP JP1224677A patent/JP2712611B2/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| Ｂｕｌｌ．Ｃｈｅｍ．Ｓｏｃ．Ｊｐｎ．，Ｖｏｌ．５１，Ｎｏ．１２ （１９７８） Ｐ．３５２２−Ｐ．３５２６ |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0390099A (en) | 1991-04-16 |
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