JPH0137400B2 - - Google Patents
Info
- Publication number
- JPH0137400B2 JPH0137400B2 JP60111321A JP11132185A JPH0137400B2 JP H0137400 B2 JPH0137400 B2 JP H0137400B2 JP 60111321 A JP60111321 A JP 60111321A JP 11132185 A JP11132185 A JP 11132185A JP H0137400 B2 JPH0137400 B2 JP H0137400B2
- Authority
- JP
- Japan
- Prior art keywords
- resin
- amino
- alanine
- cells
- protected
- 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
Links
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- 125000001176 L-lysyl group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C([H])([H])C([H])([H])C([H])([H])C(N([H])[H])([H])[H] 0.000 claims 1
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- 206010020983 Hypogammaglobulinaemia Diseases 0.000 description 1
- 229930064664 L-arginine Natural products 0.000 description 1
- 235000014852 L-arginine Nutrition 0.000 description 1
- 239000004395 L-leucine Substances 0.000 description 1
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- IZPVWNSAVUQBGP-CIUDSAMLSA-N Leu-Ser-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O IZPVWNSAVUQBGP-CIUDSAMLSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 201000002481 Myositis Diseases 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
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- 241001494479 Pecora Species 0.000 description 1
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- 239000012507 Sephadex™ Substances 0.000 description 1
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- BTWMICVCQLKKNR-DCAQKATOSA-N Val-Leu-Ser Chemical compound CC(C)[C@H]([NH3+])C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C([O-])=O BTWMICVCQLKKNR-DCAQKATOSA-N 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Chemical compound CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
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- 125000003545 alkoxy group Chemical group 0.000 description 1
- 230000029936 alkylation Effects 0.000 description 1
- 238000005804 alkylation reaction Methods 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 238000005576 amination reaction Methods 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- 210000001557 animal structure Anatomy 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 150000001540 azides Chemical class 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000000984 branchial region Anatomy 0.000 description 1
- 210000001669 bursa of fabricius Anatomy 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 150000001735 carboxylic acids Chemical class 0.000 description 1
- UOCJDOLVGGIYIQ-PBFPGSCMSA-N cefatrizine Chemical group S([C@@H]1[C@@H](C(N1C=1C(O)=O)=O)NC(=O)[C@H](N)C=2C=CC(O)=CC=2)CC=1CSC=1C=NNN=1 UOCJDOLVGGIYIQ-PBFPGSCMSA-N 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 235000013985 cinnamic acid Nutrition 0.000 description 1
- 229930016911 cinnamic acid Natural products 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 125000000753 cycloalkyl group Chemical group 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 125000006286 dichlorobenzyl group Chemical group 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 125000000031 ethylamino group Chemical group [H]C([H])([H])C([H])([H])N([H])[*] 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 1
- 239000001530 fumaric acid Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 150000002367 halogens Chemical group 0.000 description 1
- 125000003187 heptyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- 229960002885 histidine Drugs 0.000 description 1
- 150000002431 hydrogen Chemical class 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 229960003136 leucine Drugs 0.000 description 1
- 108010083708 leucyl-aspartyl-valine Proteins 0.000 description 1
- 230000021633 leukocyte mediated immunity Effects 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 230000001926 lymphatic effect Effects 0.000 description 1
- 210000004324 lymphatic system Anatomy 0.000 description 1
- 210000003738 lymphoid progenitor cell Anatomy 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 239000011976 maleic acid Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 1
- 150000004702 methyl esters Chemical class 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- WBYWAXJHAXSJNI-UHFFFAOYSA-N methyl p-hydroxycinnamate Natural products OC(=O)C=CC1=CC=CC=C1 WBYWAXJHAXSJNI-UHFFFAOYSA-N 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 206010028417 myasthenia gravis Diseases 0.000 description 1
- 230000001538 myasthenic effect Effects 0.000 description 1
- TXOUDPQJASQYBZ-UHFFFAOYSA-N n,n'-dihexylmethanediimine Chemical compound CCCCCCN=C=NCCCCCC TXOUDPQJASQYBZ-UHFFFAOYSA-N 0.000 description 1
- PSZYNBSKGUBXEH-UHFFFAOYSA-N naphthalene-1-sulfonic acid Chemical compound C1=CC=C2C(S(=O)(=O)O)=CC=CC2=C1 PSZYNBSKGUBXEH-UHFFFAOYSA-N 0.000 description 1
- 230000002232 neuromuscular Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 125000000612 phthaloyl group Chemical group C(C=1C(C(=O)*)=CC=CC1)(=O)* 0.000 description 1
- 229920003053 polystyrene-divinylbenzene Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- 125000001501 propionyl group Chemical group O=C([*])C([H])([H])C([H])([H])[H] 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- GAPYKZAARZMMGP-UHFFFAOYSA-N pyridin-1-ium;acetate Chemical compound CC(O)=O.C1=CC=NC=C1 GAPYKZAARZMMGP-UHFFFAOYSA-N 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000003134 recirculating effect Effects 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229960001153 serine Drugs 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 229950000244 sulfanilic acid Drugs 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 229960002898 threonine Drugs 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 239000000724 thymus hormone Substances 0.000 description 1
- 125000003944 tolyl group Chemical group 0.000 description 1
- 125000002088 tosyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1C([H])([H])[H])S(*)(=O)=O 0.000 description 1
- 201000008827 tuberculosis Diseases 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 229960004295 valine Drugs 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 125000005023 xylyl group Chemical group 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/57581—Thymosin; Related peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Description
【発明の詳細な説明】
本発明は新規なポリペプチド類、新規なポリペ
プチド類の製造方法、及び前記ポリペプチド類の
用途に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel polypeptides, methods for producing novel polypeptides, and uses of said polypeptides.
多くのポリペプチド類が動物の各種器管から単
離されていることはよく知られている。しかしな
がら、約10年前までは生まれた人間の体重の約
0.8％をしめる器官である胸線については殆んど
知るところがなく、僅かに神経筋遮断物質が胸線
に存在しているという仮設が提出されていたにす
ぎない。胸線の機能に関する強い関心と初期の推
論及び実験にも拘らず、最近まで胸線の機能に関
しては殆んど分つていなかつた。しかしながら、
現在は胸線は上皮（内分泌）成分とリンパ（免疫
学的）成分とを有する複器官であり従つて胸線は
身体の免疫機能に関与していることが分つた。即
ち、胸線は第３鰓弓に由来する上皮ストロマ及び
造血組織の幹細胞に由来するリンパ球からなる複
器であることは公知である（Goldstein等の
「The Human Thymus」，Heinemann，
London，1969年参照）、リンパ球は胸線内部で分
化されてＴ細胞と言われる成熟した胸線由来細胞
として胸線から離れ、血液、リンパ、脾臓、リン
パ節へと循環する。胸線内部での幹細胞分化の誘
導は胸線の上皮細胞の分泌物を媒介として起るよ
うに思われるが、生物試験に伴う種々の困難によ
り存在すると考えられるホルモン類の完全な単離
及び構造の特定ができなかつたというのがこれま
での実情であつた。 It is well known that many polypeptides have been isolated from various animal organs. However, until about 10 years ago, about the weight of a human being born at birth
Little is known about the thoracic gland, an organ that accounts for 0.8% of the population, and only a hypothesis has been proposed that a neuromuscular blocking substance exists in the thoracic gland. Despite intense interest and early speculation and experimentation regarding the function of the thoracic gland, until recently very little was known about its function. however,
It is now known that the thoracic gland is a complex organ having an epithelial (endocrine) component and a lymphatic (immunological) component, and that the thoracic gland is therefore involved in the immune function of the body. That is, it is known that the thymus is a complex organ consisting of an epithelial stroma derived from the third branchial arch and lymphocytes derived from stem cells of hematopoietic tissue (Goldstein et al., "The Human Thymus", Heinemann, et al.
London, 1969), lymphocytes differentiate within the thymus and leave the thymus as mature thymus-derived cells called T cells, circulating to the blood, lymph, spleen, and lymph nodes. Induction of stem cell differentiation within the thymus appears to occur mediated by secretions of thymus epithelial cells, but due to various difficulties associated with biological testing, complete isolation and structure of the hormones believed to be present have not been achieved. The reality has been that it has not been possible to identify the
胸線は身体の免疫特性に関連があることが分つ
てきたので胸線から単離された物質に多大の関心
が向けられてきた、近年、牛の胸線に存在する物
質に関する科学的研究に基づく比較的大部の論文
集が発表されている。現に本出願人も上記分野の
研究に関する多数の論文を発表してきた。関連す
る刊行物としては、例えば、「The Lancet」，
1968年７月20日号、p119−122，「Triangle」，
Vol，11，No.１，p.7−14，1972，「Annals of
the New York Academy Immunology」，
Vol.4，No.２，p.181−189，1969，［Nature」，
Vol.247.p.11−14，1974，「Proceedings of the
National Academy of Science，USA」，
Vol.71，p.1474−1478，1974，「Cell」，Vol.5，
p.361−365及び367−370，1975「Lancet」，Vol.2，
p.256−259，1975，「Proceedings of the
National
Academy of Sciences USA」，Vol.72，p.11
−15，1975，「Biochemistry」，Vol.14，p.2214−
2218，1974，「Nature」，Vol.255.p.423−
424.1975，がある。 In recent years, scientific research into the substances present in the thoracic gland of cattle has attracted much attention as substances isolated from the thoracic gland have been found to be associated with the body's immune properties. A relatively large collection of papers based on this has been published. In fact, the present applicant has published numerous papers related to research in the above field. Related publications include "The Lancet",
July 20, 1968 issue, p119-122, "Triangle",
Vol. 11, No. 1, p. 7-14, 1972, “Annals of
the New York Academy Immunology”
Vol.4, No.2, p.181-189, 1969, [Nature]
Vol.247.p.11-14, 1974, “Proceedings of the
National Academy of Science, USA”
Vol.71, p.1474-1478, 1974, "Cell", Vol.5,
p.361-365 and 367-370, 1975 "Lancet", Vol.2,
p.256-259, 1975, “Proceedings of the
National Academy of Sciences USA”, Vol.72, p.11
−15, 1975, “Biochemistry”, Vol.14, p.2214−
2218, 1974, “Nature”, Vol.255.p.423−
424.1975, there is.
Goldstein及びManganaroによる「Annals of
the New York Academy of Sciences」，
Vol.183，p.230−240，1971の論文には、動物の
筋無力症神経筋障害を引起す胸線ポリペプチドの
存在が開示されている。この神経筋障害は人間の
病気である重症性筋無力症に似たものである。更
に上記論文には、牛の胸線に含まれる上記に示し
たと別のポリペプチド類により２つの効果が生じ
ることが示されている。これらのポリペプチド類
の１つはチモトキシン（Thymotoxin）と名付け
られているもので、これは筋炎を引起すと思われ
ている。このポリペプチドは単離されていない
が、分子量約7000であると思われ、強陽電荷を有
しかつPH8.0でCM−セフアデツクス（CM−
Sephadex）に保持されることが前記論文に示さ
れている。 “Annals of
the New York Academy of Sciences”
Vol. 183, p. 230-240, 1971, discloses the existence of a thoracic polypeptide that causes myasthenic neuromuscular disorder in animals. This neuromuscular disorder is similar to the human disease myasthenia gravis. Furthermore, the above article shows that other polypeptides contained in the bovine thorax produce two effects. One of these polypeptides, named Thymotoxin, is thought to cause myositis. Although this polypeptide has not been isolated, it is believed to have a molecular weight of approximately 7000, has a strong positive charge, and has a pH of 8.0.
Sephadex).
「Nature」，247，11.1975年１月４日号には、
チミン（Thymin）及びチミン（Thymin
）と同定された物質が記載されている。これら
の物質は牛の胸線から単離された新ポリペプチド
類であり、種々の治療分野に特定の用途を有する
ことが見出された。過去、牛の胸線から単離され
た他の物質に対しても同様の各称が用いられてい
るので、チミン及び物質は現在チモポイエチ
ン（Thymopoietin）及びチモポイエチン
（Thymopoietin）とそれぞれ呼称されている。
これらの物質及びその製造法は1975年８月22日付
で米国に出願された米国特許出願第606843号明細
書に記載されている。 “Nature”, 247 , 11. In the January 4, 1975 issue,
Thymin and Thymin
) are listed. These substances are new polypeptides isolated from bovine thorax and have been found to have specific applications in various therapeutic areas. Thymine and the substance are now referred to as Thymopoietin and Thymopoietin, respectively, as similar names have been used in the past for other substances isolated from the thorax of cattle.
These materials and methods of making them are described in US Patent Application No. 606,843, filed August 22, 1975.
米国特許第4002602号明細書には、遍在性造免
疫性ポリペプチド（Ubiquitous Immunopoietic
Polypeptide）として記載されている長鎖ポリペ
プチド類が開示されている。このポリペプチドは
後にユビキチン（Ubiquitin）と名付けられたが、
74−アミノ酸ポリペプチドであり試験管中ナノグ
ラム単位の濃度で骨髄又は脾臓に存在する先駆物
質からＴ細胞及びＢ細胞の免疫細胞の分化を誘導
する能力を特徴とするものである。従つて、上記
ポリペプチドは胸線又は免疫欠陥又は欠失等の治
療分野に有効である。 Ubiquitous Immunopoietic Polypeptide (Ubiquitous Immunopoietic Polypeptide)
Long chain polypeptides are disclosed. This polypeptide was later named ubiquitin,
It is a 74-amino acid polypeptide characterized by its ability to induce T-cell and B-cell immune cell differentiation from precursors present in the bone marrow or spleen at nanogram concentrations in vitro. Accordingly, the above polypeptides are useful in therapeutic areas such as thymus or immune defects or deficiencies.
1977年１月11日付で発行された米国特許第
4002740号は明細書には、合成トリデカペプチド
組成物が開示されている。これらのトリデカペプ
チド組成物は補体受容体であるＢ−リンパ球では
なくＴ−リンパ球の分化を誘導することができ
る。従つて、このポリペプチドは上述米国特許出
願第606843号明細書に於いてチモポイエチレンと
して単離された長鎖ポリペプチド類の特性の内多
くのものをその特性として示した。 U.S. Patent No. 1, issued January 11, 1977.
No. 4002740 discloses synthetic tridecapeptide compositions. These tridecapeptide compositions are capable of inducing differentiation of T-lymphocytes rather than B-lymphocytes, which are complement receptors. Thus, this polypeptide exhibits many of the properties of the long chain polypeptides isolated as thymopoiethylene in the above-mentioned US patent application Ser. No. 6,06,843.
本発明は一定の活性部構造単位を有する合成５
−アミノ酸ポリペプチドを製造する方法であり、
このポリペプチドは上述刊行物及び米国特許第
4002602号明細書に於いて遍在性造免疫性ポリペ
プチド（UBIP）として単離された長鎖ポリペプ
チドの持つ多くの特性をその特性として示すこと
が分つた。 The present invention provides synthetic 5
- A method for producing an amino acid polypeptide,
This polypeptide has been described in the above-mentioned publications and in U.S. Pat.
It was found that the long chain polypeptide isolated as ubiquitous immunogenic polypeptide (UBIP) in the specification of No. 4002602 exhibits many properties as its properties.
従つて、本発明の目的は生物学的に重要である
新規なポリペプチド類の製法を提供することであ
る。 Therefore, it is an object of the present invention to provide a method for producing novel biologically important polypeptides.
本発明の他の目的はナノグラム単位の濃度でＢ
−先駆体細胞とＴ−先駆体細胞とを分化でき、従
つて人間及び動物の免疫系に非常に有効である新
規なポリペプチド類の製法を提供することであ
る。 Another object of the present invention is to provide B in nanogram concentrations.
- To provide a method for the preparation of novel polypeptides which are capable of differentiating precursor cells and T-precursor cells and are therefore highly effective for the human and animal immune system.
以下、本発明を更に詳しく説明する。 The present invention will be explained in more detail below.
本発明の方法による新規なポリペプチド類は活
性部位又は成分として下記構造単位を有するもの
である。 The novel polypeptides according to the method of the present invention have the following structural unit as an active site or component.
−Ｘ−Ｙ−Ｚ−GLN−LYS−
上式に於いて、ＸはTYR，ＹはASN，Ｚは
ILEを表わす。 -X-Y-Z-GLN-LYS- In the above formula, X is TYR, Y is ASN, and Z is
Represents ILE.
更に、本発明においては前記ポリペプチドの製
造過程で形成される新規なポリペプチド−樹脂中
間体が生ずるものであり、この中間体は下記式で
表わされる。 Furthermore, in the present invention, a novel polypeptide-resin intermediate is formed during the production process of the polypeptide, and this intermediate is represented by the following formula.
上記式に於いて、Ｘ，Ｙ及びＺは前述の通りで
あり、R1，R2及びR3は、必要により、それぞれ
示されている各アミノ酸に結合している保護基で
あり、樹脂は反応支持体として作用する固相高分
子物質である。本発明は固担ペプチド合成法によ
るポリペプチドの製法である。 In the above formula, X, Y and Z are as described above, R 1 , R 2 and R 3 are protecting groups bonded to each of the indicated amino acids, if necessary, and the resin is It is a solid phase polymeric material that acts as a reaction support. The present invention is a method for producing polypeptides using a solid-supported peptide synthesis method.
上述の如く、本発明は種々の分野に治療効果を
有する新規なポリペプチド類、ポリペプチド類の
製造過程で生じる種々の中間体、治療用組成物、
前記ポリペプチド類を利用する用途、及びポリペ
プチド類の製造方法に関するものである。 As mentioned above, the present invention relates to novel polypeptides having therapeutic effects in various fields, various intermediates generated in the process of producing polypeptides, therapeutic compositions,
The present invention relates to uses of the polypeptides and methods for producing the polypeptides.
本発明の参考例によれば、活性部位として下記
アミノ酸構造単位を有するポリペプチド類を得る
こともできる。 According to a reference example of the present invention, polypeptides having the following amino acid structural unit as an active site can also be obtained.
Ｘ−Ｙ−Ｚ−GLN−LYS
上式に於いて、ＸはTYR、ＹはASN、及びＺ
はILEであることが好ましい。 X-Y-Z-GLN-LYS In the above formula, X is TYR, Y is ASN, and Z
is preferably an ILE.
更に、活性部位として上記構造体を含むもので
あつて下記一般式で表わされるペンタポリペプチ
ドが提供されている。 Furthermore, a pentapolypeptide containing the above structure as an active site and represented by the following general formula is provided.
上式で、Ｘ，Ｙ及びＺは上述の通りであり、Ｒ
及びR′はそれぞれペンタペプチド構造体に結合
している置換体であり、これらの置換体はアミノ
酸の基本形活性構造体の生化学的活性に実質上影
響を与えないものである。式（）の意味すると
ころは、これらの２つの末満アミノ酸にペンタペ
プチド分子の生化学的活性に実質的な影響を及ぼ
さない官能基又は誘導体（即ち、Ｒ及びR′）を
結合させることによりこのペンタペプチド類の末
端アミノ酸を改質することができるということで
ある。従つて、末端アミノ酸基及びカルボン酸基
は或る種のポリペプチド類の場合と同様にペタペ
プチドの生化学的活性付与に関して必須のもので
ないということである。 In the above formula, X, Y and Z are as described above, and R
and R' are each a substituent attached to the pentapeptide structure, and these substituents do not substantially affect the biochemical activity of the basic active structure of the amino acid. What is meant by formula () is that by attaching to these two terminal amino acids functional groups or derivatives (i.e., R and R′) that do not substantially affect the biochemical activity of the pentapeptide molecule. This means that the terminal amino acids of these pentapeptides can be modified. Therefore, the terminal amino acid group and carboxylic acid group are not essential for imparting biochemical activity to the petapeptide, as is the case with certain polypeptides.
これらの官能基の置換としては遊離アミノ基の
アシル化及び遊離カルボン酸基のアミド化のよう
な通常の置換、及び他のアミノ酸類の置換が含ま
れる。これらの観点から見る時、本発明のペンタ
ペプチド類は特異なものであると思われる。何故
なら、本発明のペンタペプチド類は前記ペプチド
構造単位をその一部として含む反鎖天然ペプチド
類と同じ生化学活性を示すからである。それ故、
ペプチド分子の生化学的活性は分子の立体化学的
特徴、即ち分子の特定な折りたたみ構造に起因し
ていると思われる。ポリペプチドの結合は剛直で
なく柔軟でありかつシート状、らせん状等の形状
で存在している。その結果、分子全体としては柔
軟となり一定の状態で折り重なつている。本発明
者は本発明のペンタペプチドは長鎖の天然ポリペ
プチドと同様な折り重なり構造を有しそれ故同様
の生化学的特性を示すことを見出した。この様な
訳で、実質上生化学的活性に影響を与えない置換
基であるか又はペンタペプチドの分子の天然重畳
構造を阻害しない置換基であればペンタペプチド
は種々の官能基で置換することができる。 Substitutions of these functional groups include conventional substitutions such as acylation of free amino groups and amidation of free carboxylic acid groups, as well as substitutions of other amino acids. When viewed from these points of view, the pentapeptides of the present invention appear to be unique. This is because the pentapeptides of the present invention exhibit the same biochemical activity as the anti-chain natural peptides containing the peptide structural unit as a part thereof. Therefore,
The biochemical activity of peptide molecules is believed to be due to the stereochemical characteristics of the molecule, ie, the specific folded structure of the molecule. Polypeptide bonds are not rigid but flexible, and exist in sheet-like, spiral-like, and other forms. As a result, the molecule as a whole becomes flexible and folds in a certain way. The inventors have found that the pentapeptides of the invention have a folded structure similar to long-chain natural polypeptides and therefore exhibit similar biochemical properties. For this reason, pentapeptides can be substituted with various functional groups as long as the substituents do not substantially affect the biochemical activity or do not interfere with the natural folded structure of the pentapeptide molecule. I can do it.
ペンタペプチド分子の生化学的特性及び天然重
畳構造の保持する能力は次の事実から明らかであ
る。即ち、本発明のペンタペプチド構造体は米国
特許第4002602号に於いてユビキチン
（Ubiquitin）として開示された天然の74−アミノ
酸ペプチドと同じ生化学特性を示すという事実で
ある。この長鎖のポリペプチドの分子の内部に於
いて上記特許に記載されている他のアミノ酸と結
合しているという形ではあるが本発明のペンタペ
プチドを同定することができる。しかしながら、
本発明のペンタペプチドとユビキチンの生化学活
性は同一でありかつ末端アミノ酸が置換されてい
るアミノ酸類及びペプチド鎖は基本形ペンタペプ
チド構造体の生化学特性に影響を及ぼさない故に
本発明のペンタペプチドが活性部位であるという
ことを上記特許は直接立証しているのである。 The biochemical properties of the pentapeptide molecule and its ability to preserve the natural folded structure are evident from the following facts. That is, the pentapeptide structure of the present invention exhibits the same biochemical properties as the natural 74-amino acid peptide disclosed as Ubiquitin in US Pat. No. 4,002,602. The pentapeptide of the present invention can be identified in the interior of the molecule of this long polypeptide chain, albeit in a form that is linked to other amino acids described in the above-mentioned patents. however,
The biochemical activities of the pentapeptide of the present invention and ubiquitin are the same, and the amino acids and peptide chains in which the terminal amino acids are substituted do not affect the biochemical properties of the basic pentapeptide structure. The above patent directly proves that it is the active site.
このことから、式のＲ及びR′としては基本
形活性構造体の生化学的活性に実質上影響を与え
ないかぎりいかなる置換体でも使用できることが
理解されるであろう。Ｒ及びR′は例えば下記置
換基のいずれでもよいと推定される。 From this, it will be understood that any substitution can be used for R and R' in the formula as long as it does not substantially affect the biochemical activity of the basic active structure. It is presumed that R and R' may be, for example, any of the following substituents.
Ｒ R′
水素 OH
C1−C7アルキル NH2
C5−C12アリール NHR7
C6−C20アルカリール Ｎ（R7）2
C6−C20アルアルキル OR7
C2−C7アルカノイル
C1−C7アルケニル
C1−C7アルキニル
ここで、R7はC1−C7のアルキル、C2−C7のア
ルケニル、C2−C7のアルキニル、C6−C20のアリ
ール，C6−C20のアルカリール又はC6−C20のアル
アルキルを示す。 R R'Hydrogen OH C 1 -C 7 alkyl NH 2 C 5 -C 12 aryl NHR 7 C 6 -C 20 alkaryl N(R 7 ) 2 C 6 -C 20 aralkyl OR 7 C 2 -C 7 alkanoyl C 1 - C7 alkenyl C1 - C7 alkynyl where R7 is C1 - C7 alkyl, C2 - C7 alkenyl, C2 - C7 alkynyl, C6 - C20 aryl, C It represents 6 - C20 alkaryl or C6 - C20 aralkyl.
Ｒ及びR′は又中性アミノ酸基又は炭素原子数
１−20のポリペプチド鎖の残基であつてもよい。
これらの例を下記に示す。 R and R' may also be neutral amino acid groups or residues of a polypeptide chain containing 1 to 20 carbon atoms.
Examples of these are shown below.
Ｒ R′
ASP GLU
SER SER
LEU THR
SER−ASP LEU
LEU−SER−ASP HIS
LEU−ASP VAL
LEU−SER ARG
GLU−SER
GLU−THR
GLU−SER−LEU
GLU−SER−THR
GLU−SER−THR−LEU
GLU−SER−THR−LEU−HIS
GLU−SER−THR−LEU−HIS−
LEU
GLU−SER−THR−LEU−HIS−
LEU−VAL
GLU−SER−THR−LEU−HIS−
LEU−VAL−LEU
GLU−SER−THR−LEU−HIS−
LEU−VAL−LEU−ARG
GLU−SER−THR−LEU−HIS−
LEU−VAL−LEU−ARG−LEU
GLU−SER−THR−LEU−HIS−
LEU−VAL−LEU−ARG−LEU−
ARG
本発明の他の参考例によれば、下記式を有する
新規なペンタペプチドをも得ることができる。 R R′ ASP GLU SER SER LEU THR SER−ASP LEU LEU−SER−ASP HIS LEU−ASP VAL LEU−SER ARG GLU−SER GLU−THR GLU−SER−LEU GLU−SER−THR GLU−SER−THR−LEU GLU−SER−THR−LEU−HIS GLU−SER−THR−LEU−HIS−
LEU GLU−SER−THR−LEU−HIS−
LEU−VAL GLU−SER−THR−LEU−HIS−
LEU−VAL−LEU GLU−SER−THR−LEU−HIS−
LEU−VAL−LEU−ARG GLU−SER−THR−LEU−HIS−
LEU−VAL−LEU−ARG−LEU GLU−SER−THR−LEU−HIS−
LEU−VAL−LEU−ARG−LEU−
ARG According to another reference example of the present invention, a novel pentapeptide having the following formula can also be obtained.
上式で、Ｒは水素、メチル、エチル等のC1−
C7のアルキル、フエニル等のC5−C12のアリー
ル、アセチル、プロピオニル等のC1−C7のアル
カノイル、R′はOH，NH2，NHR7又はＮ（R7）2
を示す。最も好ましいポリペプチド類はＲが水
素、R′がOHであるペプチド類である。 In the above formula, R is C 1 − such as hydrogen, methyl, ethyl, etc.
C7 alkyl, C5 - C12 aryl such as phenyl, C1 - C7 alkanoyl such as acetyl, propionyl, R' is OH, NH2 , NHR7 or N( R7 ) 2
shows. The most preferred polypeptides are those in which R is hydrogen and R' is OH.
ペンタペプチドと塩を形成し得る酸としては塩
酸、臭化水素酸、過塩素酸、硝酸、チオシアン
酸、硫酸、燐酸等の無機酸と蟻酸、酢酸、プロピ
オン酸、グリコール酸、乳酸、ピルビン酸、蓚
酸、マロン酸、コハク酸、マレイン酸、フマール
酸、アントラニル酸、桂皮酸、ナフタリンスルホ
ン酸、スルフアニル酸等の有機酸とを挙げること
ができる。 Acids that can form salts with pentapeptides include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, as well as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, Examples include organic acids such as oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalenesulfonic acid, and sulfanilic acid.
上記構造体に於いてペプチドのアミノ酸成分は
便宜上略号で示したが、これらの略号は下記アミ
ノ酸を示す。 In the above structure, the amino acid components of the peptide are indicated by abbreviations for convenience, and these abbreviations indicate the following amino acids.
アミノ酸 略号
Ｌ−チロシン TYR
Ｌ−アスパラギン ASN
Ｌ−アスパラギン ASP
Ｌ−イソロイシン ILE
Ｌ−セリン SER
Ｌ−グルタミン GLN
Ｌ−ロイシン LEU
Ｌ−リシン LYS
Ｌ−グルタミン酸 GLU
Ｌ−スレオニン THR
Ｌ−ヒスチジン HIS
Ｌ−バリン VAL
Ｌ−アルギニン ARG
Ｌ−アラニン ALA
本発明のポリペプチド類は５−アミノ酸ペプチ
ド類であり、このペプチドは米国特許第4002602
号に開示されている如く牛の胸線から単離された
74−アミノ酸ポリペプチドであるユビキチン
（UBIP）に似た特性を示すことが分つた。本発
明のペプチド類はナノグラム単位の濃度でＢ先駆
体細胞と同時にＴ先駆体細胞の選択的分化を誘導
することができる点にその特徴がある。本発明の
ポリペプチド類は米国特許第4002602号に開示さ
れている長鎖ポリペプチド類と同様に試験管中で
免疫細胞先駆体細胞を分化させることが分つた。
即ち、本発明のポリペプチド類は例えナノグラム
単位の濃度でも胸線分化抗原TL及びTHY−１
（θ）の獲得により測定されるようなＴ先駆体細
胞とＢ細胞の識別マーカー（marker）である補
体受容体の獲得により測定されるようなＢ先駆体
細胞との分化を誘導することが分つた。 Amino acid abbreviations L-tyrosine TYR L-asparagine ASN L-asparagine ASP L-isoleucine ILE L-serine SER L-glutamine GLN L-leucine LEU L-lysine LYS L-glutamic acid GLU L-threonine THR L-histidine HIS L-valine VAL L-Arginine ARG L-Alanine ALA The polypeptides of the present invention are 5-amino acid peptides, which are disclosed in US Pat.
isolated from the thorax of cattle as disclosed in No.
It was found that it exhibits properties similar to ubiquitin (UBIP), a 74-amino acid polypeptide. The peptides of the present invention are characterized in that they can induce selective differentiation of T precursor cells as well as B precursor cells at nanogram concentrations. It has been found that the polypeptides of the present invention, like the long polypeptides disclosed in US Pat. No. 4,002,602, differentiate immune cell precursor cells in vitro.
That is, the polypeptides of the present invention, even at nanogram concentrations, can inhibit thymus differentiation antigens TL and THY-1.
inducing differentiation between T precursor cells, as measured by the acquisition of (θ), and B precursor cells, as measured by the acquisition of complement receptors, which are the distinguishing markers for B cells. Divided.
本発明のポリペプチド類の生化学的分化特性の
重要性を理解するには、免疫に関する胸線の機能
は広義に言えばＴ細胞と言われる胸線に由来する
細胞即ちリンパ球を生成することであるというこ
とができることを銘記すべきである。Ｔ細胞は再
循環小リンパ球貯槽の大部分を形成している。Ｔ
細胞は免疫学的特異性を有しかつ実行器細胞とし
て（同種移植反応等の）細胞介在による免疫応答
に直接関与している。しかしながら、Ｔ細胞は液
性抗体を分泌することはない。これらの抗体は胸
線とは無関係に骨髄に直接由来する細胞により分
泌される。これらの骨髄に由来する細胞はＢ細胞
と言われる。しかしながら、多くの抗原とは対称
的に、Ｂ細胞が抗体を生成するには適当に反応性
のあるＴ細胞の存在が必要である。Ｔ細胞とＢ細
胞の協働によるこの生成過程の機構は未だ完全に
は理解されていない。 To understand the importance of the biochemical differentiation characteristics of the polypeptides of the present invention, it is important to understand that the function of the thymus in relation to immunity is, broadly speaking, to produce cells derived from the thymus called T cells, namely lymphocytes. It should be noted that it can be said that T cells form the majority of the recirculating small lymphocyte reservoir. T
The cells have immunological specificity and are directly involved in cell-mediated immune responses (such as allograft reactions) as executive cells. However, T cells do not secrete humoral antibodies. These antibodies are secreted by cells derived directly from the bone marrow, independent of the thymus. These bone marrow-derived cells are called B cells. However, in contrast to many antigens, the presence of suitably reactive T cells is required for B cells to produce antibodies. The mechanism of this generation process through cooperation between T cells and B cells is still not completely understood.
上述のことから、作用効果という観点に於いて
胸線は細胞免疫の発現及び多くの液性抗体反応に
対して必要なものでありかつ造血性幹細胞を分化
してＴ細胞を胸線内で誘導することにより上記発
現及び反応系に胸線は影響を与えているというこ
とができる。この誘導的影響は胸線の上皮細胞の
分泌物、即ち胸線ホルモン類を媒介として行われ
る。 From the above-mentioned point of view, the thymus is necessary for the expression of cellular immunity and many humoral antibody reactions, and it is necessary to differentiate hematopoietic stem cells and induce T cells within the thymus. Therefore, it can be said that the thymus has an influence on the above expression and reaction system. This inductive effect is mediated by the secretions of the epithelial cells of the thymus, ie, thymus hormones.
更に、胸線及びリンパ球細胞系の作用及び体内
でのリンパ球の循環を理解するには、幹細胞は骨
髄に生じて血液に運ばれて胸線に達するというこ
とを挙げておく必要がある。胸線の内部に於い
て、幹細胞は免疫学的に有能なＴ細胞に分化し、
Ｔ細胞は血流に移行しＢ細胞と共に種々の組織、
リンパ管系び血流間を循環する。 Furthermore, in order to understand the action of the thymus and lymphocyte cell lineage and the circulation of lymphocytes in the body, it is necessary to mention that stem cells arise in the bone marrow and are carried in the blood to reach the thymus. Inside the thymus, stem cells differentiate into immunologically competent T cells,
T cells move into the bloodstream and together with B cells enter various tissues,
Circulate through the lymphatic system and bloodstream.
抗体を分泌する身体細胞も又造血幹細胞から発
生するものであるが身体細胞の分化は胸線により
決まるものではない。従つて、身体細胞は骨髄由
来細胞即ちＢ細胞と名付けられている。鳥類に於
いては、身体細胞は胸線に類似するフアブリキウ
ス嚢という器官で分化される。哺乳類では、フア
ブリキウス嚢に相当する器官は見出されておらず
Ｂ細胞は骨髄内で分化すると考えられている。こ
の分化を行わせる生理的物質は全く分つていな
い。 Somatic cells that secrete antibodies also originate from hematopoietic stem cells, but the differentiation of somatic cells is not determined by the thymus. Therefore, the body cells are named bone marrow-derived cells or B cells. In birds, somatic cells are differentiated in an organ called the capsule of Fabricius, which resembles the thorax. In mammals, no organ corresponding to the bursa of Fabricius has been found, and B cells are thought to differentiate within the bone marrow. The physiological substances that cause this differentiation are completely unknown.
上述の如く、本発明のポリペプチド類は人間及
び動物の治療に有効である。本発明のポリペプチ
ド類は造血組織から生じる造リンパ球幹細胞を分
化して身体の免疫応答に関与することができる胸
線由来細胞即ちＴ細胞に誘導することができかつ
Ｂ細胞の分化をも誘導できるので、ポリペプチド
類は多くの治療上の用途を有するものであると思
われる。第１に、本発明のポリペプチド化合物は
胸線の機能の内或る種の機能を遂行できるので
種々の胸線機能分野及び免疫分野に適用できる。
用途の主分野は先天性胸線欠如の状態であるダイ
ジヨージ（Di Deorge）症候群の治療である。本
発明のポリペプチド類の注入によりこの欠陥又は
欠失に打勝つことができる。他の適用例としては
身体のＢ細胞分化性推定ホルモンの欠如に依る無
ガンマグロブリン血症の治療である。これもポリ
ペプチド類の注入により治療することができる。
低濃度で用いても非常に活性があるポリペプチド
類の生化学的特性の故に、ポリペプチド類は細胞
免疫及び液性免疫の治療的刺激を増大又は促進
し、従つて細菌性又はマイコプラズマ伝染病、結
核、癩病、急性及び慢性ビールス性伝染病等の生
物体中にある慢性伝染性病気の治療に有効であり
身体の総合的免疫を助成するという点に於いて有
効である。更に、ポリペプチド化合物は細胞又は
液性免疫が問題である分野、特に前述ダイジヨー
ジ症候群に於ける如く免疫に欠損がある分野に有
効であると思われる。又、ポリペプチド類の前述
特性の故に、ポリペプチド類は試験管中でのＴ細
胞の表面抗原の発育に有効であり、機能的能力を
発現させて細胞分裂物質及び抗原に応答させるこ
とに有効であり、かつ細胞を協働させてＢ細胞の
能力を高めて種々の抗体を生成させることができ
る。更に、ポリペプチド類は補体の表面受容体の
発育により測定される如くＢ細胞を試験管中で発
育させることができる。本発明のポリペプチド類
は又ユビキチン応答性リンパ球の無制限の増植を
禁止するにも有効である。 As mentioned above, the polypeptides of the invention are useful in human and animal therapy. The polypeptides of the present invention can induce lymphoid stem cells originating from hematopoietic tissues to differentiate into thymus-derived cells, ie, T cells, which can participate in the body's immune response, and also induce the differentiation of B cells. As such, polypeptides are likely to have many therapeutic uses. First, the polypeptide compound of the present invention can perform certain functions of the thymus, and thus can be applied to various thymus functions and immunological fields.
The main field of use is the treatment of Di Deorge syndrome, a condition of congenital absence of the thymus. This defect or deletion can be overcome by injection of polypeptides of the invention. Another application is the treatment of agammaglobulinemia due to the body's lack of B-cell differentiation presumptive hormones. This can also be treated by injection of polypeptides.
Because of the biochemical properties of polypeptides that are highly active even when used at low concentrations, polypeptides can increase or promote therapeutic stimulation of cellular and humoral immunity, thus preventing bacterial or mycoplasmal infections. It is effective in the treatment of chronic infectious diseases in living organisms such as tuberculosis, leprosy, acute and chronic viral infectious diseases, and is effective in supporting the overall immunity of the body. Furthermore, the polypeptide compounds are believed to be useful in fields where cellular or humoral immunity is a problem, particularly in fields where there is a deficiency in immunity, such as in the aforementioned Daiji-Yoji syndrome. Also, because of the aforementioned properties of polypeptides, they are effective in developing surface antigens of T cells in vitro and in developing functional capacity to respond to cell division substances and antigens. , and can enhance the ability of B cells to produce various antibodies by cooperating with cells. Additionally, the polypeptides are capable of in vitro development of B cells as measured by the development of surface receptors for complement. The polypeptides of the invention are also effective in inhibiting the uncontrolled expansion of ubiquitin-responsive lymphocytes.
Ｔ細胞の特性を用いて細胞を回復させることが
できるという生物体中での性質又Ｂ細胞の特性を
用いて細胞を回復させることができるという生物
体中でのポリペプチドの性質もポリペプチドの重
要な特性の１つである。 The property of polypeptides in living organisms to be able to use the properties of T cells to recover cells and the property of polypeptides in living organisms to be able to use the properties of B cells to recover cells is also a property of polypeptides. This is one of the important characteristics.
本発明のポリペプチド類の更に重要な性質はポ
リペプチド類は非常に低濃度で非常に活性である
ということである。即ち、ポリペプチド類は10n
ｇ／ml以上の濃度で活性であり、約0.05−1μｇ／
mlの濃度で活性は最大となる。このポリペプチド
の担体としてはこの様な目的に対して通常用いら
れる公知の担体はいずれも使用可能であり、例え
ば通常の塩水溶液を使用できる。この場合牛の血
清アルブミン等のタンパク質希釈剤を含ませて上
記低濃度で用いられるポリペプチドがガラス器具
に吸着損失することのないようにすることが好ま
しい。本発明のポリペプチド類は体重１Kgに対し
て約0.1mg以上の範囲で活性である。ダイジヨー
ジ症侯群の治療には、ポリペプチド類は体重１Kg
当り約1.0−10mg投与すればよい。一般には、上
記投与量はこれまで挙げた他の症状又は病気の処
置に対しても有効である。 A further important property of the polypeptides of the invention is that they are highly active at very low concentrations. That is, polypeptides are 10n
It is active at concentrations above 0.05-1 μg/ml.
The activity is maximum at a concentration of ml. As a carrier for this polypeptide, any known carrier commonly used for such purposes can be used, for example, a common salt aqueous solution can be used. In this case, it is preferable to include a protein diluent such as bovine serum albumin to prevent the polypeptide used at the low concentration from being adsorbed and lost to the glassware. The polypeptides of the present invention are active in a range of about 0.1 mg or more per 1 kg of body weight. Polypeptides are recommended for the treatment of Daijiyogi syndrome, with a body weight of 1 kg.
Approximately 1.0-10 mg per dose may be administered. Generally, the above dosages will also be effective for treating other conditions or diseases mentioned above.
本発明のポリペプチド類は「Journal Of
American Chemical Society」85，p.2149−
2154，1963、に記載されているようなメリフイー
ルドの概念と同様の概念を用いて製造された。即
ち、数種の保護形アミノ酸を段階的に固形樹脂粒
に共有結合で結合されている生長ペプチド鎖に付
加することにより合成を行つた。この方法では、
薬剤及び副生物は濾過及び中間体の再結晶により
除去された。この方法を一般的に言えば、ペプチ
ド鎖の第１のアミノ酸を固体高分子に共有結合で
結合させ、引続きアミノ酸を一度に１種類づつ階
段的に付加して所望の構造単位を構成する方法で
ある。最後に、ペプチドを固形支持体から分離し
かつ保護基も除く。この方法によれば生長するペ
プチド鎖は完全に不溶性の固体粒子に結合してい
るので濾過洗浄して薬剤及び副生物を除くには都
合がよい。 The polypeptides of the present invention are described in "Journal Of
American Chemical Society” 85, p.2149−
2154, 1963, using a concept similar to that of Merrifield. That is, the synthesis was carried out by stepwise addition of several protected amino acids to a growing peptide chain covalently bonded to solid resin granules. in this way,
Drugs and byproducts were removed by filtration and recrystallization of the intermediate. Generally speaking, this method involves covalently bonding the first amino acid of a peptide chain to a solid polymer, followed by stepwise addition of amino acids one at a time to form the desired structural unit. be. Finally, the peptide is separated from the solid support and the protecting groups are also removed. According to this method, the growing peptide chain is bound to completely insoluble solid particles, making it convenient to filter and wash to remove drugs and by-products.
アミノ酸類は適当なポリマーに結合させること
ができるがこの場合前記ポリマーは使用溶媒に不
溶性でなければならず又濾過を容易に行わせるこ
とができる物理的に安定な形状でなければならな
い。又、ポリマーは第１の保護形アミノ酸を共有
結合で確実に結合できる官能基を含むものでなけ
ればならない。種々のポリマーが使用可能であり
例えばセルローズ、ポリビニルアルコール、ポリ
メタアクレート、スルホン化ポリスチレンを挙げ
ることができるが、本発明で行つた合成ではスチ
レン及びジビニルベンゼンのクロルメチル化供重
合体が用いられた。 The amino acids can be attached to a suitable polymer, which must be insoluble in the solvent used and must be in a physically stable form to permit easy filtration. The polymer must also contain a functional group that can reliably bind the first protected amino acid through a covalent bond. Although a variety of polymers can be used, including cellulose, polyvinyl alcohol, polymethacrylate, and sulfonated polystyrene, the synthesis carried out in the present invention used a chloromethylated copolymer of styrene and divinylbenzene. .
活性ではあるが反応には関与すべきでないアミ
ノ酸の各種官能基はポリペプチドの場合通常使用
される公知の保護基により合成反応の間中保護し
ておいた。チロシン及びリシンの官能基は、最終
ポリペプチド生成物に悪影響を与えずに段階的付
加反応の完了時に除くことができる保護基により
保護された。カツプリングが完了したかどうかを
陽性蛍光の指示により決めるためにフルオレスア
ミン（Fluorescamine）を使用するという点
（Felix et alによるAnalyt.Biochem.，52，377，
1973参照）に於いてポリペプチドの合成は固体合
成法の改良法により行われた。カツプリングの完
了が指示されない場合には、α−アミノ保護解除
を行う前に同一の保護形アミノ酸を用いてカツプ
リングを繰返した。 Various functional groups of the amino acids, which are active but should not participate in the reaction, were protected throughout the synthesis reaction with known protecting groups commonly used in the case of polypeptides. The tyrosine and lysine functional groups were protected with protecting groups that could be removed upon completion of the stepwise addition reaction without adversely affecting the final polypeptide product. The use of Fluorescamine to determine whether coupling is complete by positive fluorescence indication (Felix et al., Analyt.Biochem., 52, 377,
(1973), the synthesis of polypeptides was carried out by an improved solid-state synthesis method. If coupling was not indicated to be complete, coupling was repeated using the same protected amino acid before α-amino deprotection.
合成の一般的方法としては先づアミノ基を保護
されたＬ−リシンをアミンを含む無水アルコール
中で樹脂に対してエステル化した。結合したアミ
ノ酸樹脂を濾過し、アルコール、次いで水で洗浄
し、乾燥した。次いでリシンアミノ酸のα−アミ
ノ基の保護基（例えばｔ−BOC、即ちｔ−ブチ
ルオキシカルボニル）を他の保護基に影響を与え
ることなく除去した。遊離アミノ基を有する結合
アミノ酸樹脂を保護形Ｌ−グルタミン酸、好まし
くはアルフア−ｔ−BOC−Ｌ−グルタミン、と
反応させてＬ−グルタミンを結合させた。更に、
保護形Ｌ−イソロイシン，Ｌ−アスパラギン及び
Ｌ−チロシンを用いて反応を繰返し完全な分子を
合成した。上記反応は下記の如く行つた。 As a general method of synthesis, L-lysine with a protected amino group is first esterified with a resin in an amine-containing absolute alcohol. The bound amino acid resin was filtered, washed with alcohol, then water, and dried. The protecting group for the α-amino group of the lysine amino acid (e.g. t-BOC, ie t-butyloxycarbonyl) was then removed without affecting other protecting groups. The bound amino acid resin having free amino groups was reacted with a protected form of L-glutamic acid, preferably alpha-t-BOC-L-glutamine, to bind L-glutamine. Furthermore,
The complete molecule was synthesized by repeating the reaction using protected forms of L-isoleucine, L-asparagine and L-tyrosine. The above reaction was carried out as follows.
樹脂
↓ α−BOC−Lys−COOH
α−BOC−Lys−樹脂
↓ α−アミノ保護基を除く
H2N−Lys−樹脂
↓ α−BOC−Ｌ−Gln
α−BOC−Gln−Lys−樹脂
↓ α−アミノ保護基を除く
H2N−Gln−Lys−樹脂
↓ α−BOC−Ile−COOH
α−BOC−Ile−Gln−Lys−樹脂
↓ α−アミノ保護基を除く
H2N−Ile−Gln−樹脂は
↓ α−BOC−Asn−COOH
α−BOC−Asn−Ile−Gln−Lys−樹脂
↓ α−アミノ保護基を除く
H2N−Asn−Ile−Gln−Iys−樹脂
↓ α−R3−Tyr−COOH
α−R3−Tyr−Asn−Ile−Gln−Lys−樹脂
↓ 全ての保護基を除く
H2N−Tyr−Asn−Ile−Gln−Lys−COOH
上記一連の反応に於いて、R1及びR2は各アミ
ノ酸の反応性側鎖の保護基であり、これらの保護
基はα−アミノ基の保護基を除いて次の反応を行
う時何ら影響を受けたり除かれたりしない、又α
−R3はα−アミノ基の保護基である。上記中間
体ペンタペプチド樹脂の場合、R1はＯ−２，６
−ジクロルベンジル等の保護基、R2はε−２−
クロルベンジルオキシカルボニル、R3はｔ−ブ
チルオキシカルボニルであることが好ましい。樹
脂は本発明の方法に有効であるものであれば前述
樹脂のいずれでもよい。上記一連の反応に於い
て、ALA，をTYR，ASN又はILEと代えて反応
を行つても同様にペプチド類を合成できる。 Resin ↓ α-BOC-Lys-COOH α-BOC-Lys- resin ↓ H 2 N-Lys- resin excluding α-amino protecting group ↓ α-BOC-L-Gln α-BOC-Gln-Lys- resin ↓ α −H 2 N−Gln−Lys− resin excluding the amino protecting group ↓ α−BOC−Ile−COOH α−BOC−Ile−Gln−Lys− resin ↓ H 2 N−Ile−Gln− excluding the α−amino protecting group The resin is ↓ α-BOC-Asn-COOH α-BOC-Asn-Ile-Gln-Lys- resin ↓ H 2 N-Asn-Ile-Gln-Iys- resin without α-amino protecting group ↓ α-R 3 − Tyr−COOH α−R 3 −Tyr−Asn−Ile−Gln−Lys− resin ↓ Remove all protecting groups H 2 N−Tyr−Asn−Ile−Gln−Lys−COOH In the above series of reactions, R 1 and R2 are protecting groups for the reactive side chains of each amino acid, and these protecting groups are not affected or removed in the next reaction except for the protecting group for the α-amino group, or α
-R 3 is a protecting group for α-amino group. In the case of the above intermediate pentapeptide resin, R 1 is O-2,6
-Protecting group such as dichlorobenzyl, R 2 is ε-2-
Chlorbenzyloxycarbonyl, R 3 is preferably t-butyloxycarbonyl. The resin may be any of the aforementioned resins as long as it is effective in the method of the present invention. In the above series of reactions, peptides can be synthesized in the same way even if ALA is replaced with TYR, ASN, or ILE.
最終中間体が得られた後、ペプチド−樹脂を分
割してR1，R2及びR3保護基と樹脂を除く。保護
基は無水フツ化水素で処理する等の通常の手段で
行い、その結果得られる遊離ペプチドを回収す
る。 After the final intermediate is obtained, the peptide-resin is split to remove the R 1 , R 2 and R 3 protecting groups and the resin. Protecting groups are achieved by conventional means such as treatment with anhydrous hydrogen fluoride, and the resulting free peptide is recovered.
本発明のポリペプチド類を製造するには、メリ
フイールド法に於ける如く不溶性ポリマーを利用
することが好ましいが、勿論他の方法で製造する
ことも出来る。例えば、Ｎ−メチル−ベンズヒド
リルアミン樹脂あるいはベンズヒドリルアミン樹
脂等の別の固体支持体を使用することもでき、こ
れらの樹脂を使用することが有利な場合もある。
これらの樹脂を用いる場合には、Ｃ−末端アミノ
酸を直接樹脂に付着させ、得られたペプチドは
HF中で樹脂基体から分離させてＣ−末端アミド
類を得ることができる。Ｎ−メチル−ベンズヒド
リルアミン樹脂を利用する方法はモナハン
（Monahan）等により「Biochemical and
Biophysical Research Communications」48，
1100〜1105（1972）に記載されている。又ベンズ
ヒドリルアミンを使用する方法はジエイ・リビエ
ール（J.Rivier）等によりJournal of Medicinal
Chemistry，1973，Vol.16，545−549，に記載さ
れている。 In order to produce the polypeptides of the present invention, it is preferable to utilize insoluble polymers, such as in the Merrifield method, but it is of course possible to produce them by other methods. Other solid supports can also be used, for example N-methyl-benzhydrylamine resins or benzhydrylamine resins, and it may be advantageous to use these resins.
When using these resins, the C-terminal amino acid is attached directly to the resin, and the resulting peptide is
C-terminal amides can be obtained by separation from the resin substrate in HF. A method using N-methyl-benzhydrylamine resin was described by Monahan et al. in ``Biochemical and
Biophysical Research Communications” 48 ,
1100-1105 (1972). The method of using benzhydrylamine was also described in the Journal of Medicinal by J. Rivier et al.
Chemistry, 1973, Vol. 16, 545-549.
本発明の基本形ペンタペプチドの種々の誘導体
を公知の方法で製造することができる。この様な
誘導体を製造するには、一連の反応を阻害する可
能性のあるペプチドの官能基を保護しておき、所
望の生成物を得るようにすることが必要であろ
う。例えば、アスパラギン酸のα−カルボン酸基
あるいはリシンのα−アミノ基は、誘導体を製造
する時には保護しておく必要がある。 Various derivatives of the basic pentapeptide of the present invention can be produced by known methods. To prepare such derivatives, it may be necessary to protect functional groups on the peptide that may inhibit a series of reactions to yield the desired product. For example, the α-carboxylic acid group of aspartic acid or the α-amino group of lysine must be protected when producing derivatives.
上述の如く、本発明の方法を行うにはアミノ基
を保護又は封鎖することが必要でありこれにより
反応をコントロールし所望の生成物を得る。有効
に使用し得る適当なアミノ保護基とは強塩基性ア
ミノ基を保護するための塩を形成できるもの、又
はベンジルオキシカルボニル及びｔ−ブチルオキ
シカルボニル等のウレタン保護置換基がある。分
子のカルボキシル基端で反応するアミノ酸のα−
アミノ基を保護するためにはｔ−ブチルオキシカ
ルボニルＬ（tBOC）又はｔ−アミルオキシカル
ボニル（AOC）を用いることが好ましい。これ
はこれらのBOC及びAOC保護基は反応後次の工
程（この工程で斯るα−アミノ基自身が反応す
る）に先立つて酸（例えばトリフルオル酢酸）の
比較的温和な作用で容易に除くことができる。こ
の酸処理は他の反応性側鎖を保護するための基に
は別に何の影響も与えない。従つて、次の反応に
対してα−アミノ基を保護し、かつペプチド分子
に特に影響を与えない条件下で後に除くことがで
きるすべての物質との反応によりα−アミノ基を
保護することができるということである。斯る物
質の例としてはアミノ基をアシル化し得る有機カ
ルボン酸の誘導体を挙げることができる。 As mentioned above, carrying out the process of the invention requires protecting or capping the amino group, thereby controlling the reaction and obtaining the desired product. Suitable amino protecting groups that can be effectively used include those capable of forming salts for protecting strongly basic amino groups, or urethane protecting substituents such as benzyloxycarbonyl and t-butyloxycarbonyl. α- of amino acids that react at the carboxyl end of the molecule
In order to protect the amino group, it is preferable to use t-butyloxycarbonyl L (tBOC) or t-amyloxycarbonyl (AOC). This is because these BOC and AOC protecting groups can be easily removed by the relatively mild action of an acid (e.g. trifluoroacetic acid) after the reaction and prior to the next step (in which the α-amino group itself reacts). I can do it. This acid treatment has no particular effect on the groups used to protect other reactive side chains. Therefore, it is possible to protect the α-amino group against subsequent reactions and by reaction with any substance that can be subsequently removed under conditions that do not particularly affect the peptide molecule. It means that it can be done. Examples of such substances include derivatives of organic carboxylic acids capable of acylating amino groups.
一般に、アミノ基は下記式で示される基を含む
化合物との反応により保護することが出来る。 Generally, an amino group can be protected by reaction with a compound containing a group represented by the following formula.
上式で、Ｒはアミノ基が次のカツプリング反応
に関与することを防止し、かつ分子を破壊するこ
となく除去できる基であればよい。このような基
Ｒとしては飽和又は不飽和直鎖又は分岐鎖アルキ
ルで、好ましくは炭素数１−10のもの、アリー
ル、好ましくは炭素数６−15のもの、シクロアル
キル、好ましくは炭素数５−８のもの、アルアル
キル、好ましくは炭素数７−18のもの、アルカリ
ール、好ましくは炭素数７−18のもの、又はイソ
ニコチニル等の複素環式基がある。 In the above formula, R may be any group as long as it prevents the amino group from participating in the next coupling reaction and can be removed without destroying the molecule. Such radicals R include saturated or unsaturated straight-chain or branched alkyl, preferably with 1 to 10 carbon atoms, aryl, preferably with 6 to 15 carbon atoms, cycloalkyl, preferably with 5 to 15 carbon atoms. 8, aralkyl, preferably having 7 to 18 carbon atoms, alkaryl, preferably having 7 to 18 carbon atoms, or heterocyclic groups such as isonicotinyl.
アリール、アルアルキル及びアルカリールの各
基は炭素数１−約４の１種又はそれ以上のアルキ
ルにより更に置換されていてもよい。Ｒとして好
ましい基はｔ−ブチル、ｔ−アミル、フエニル，
トリル、キシリル、及びベンジルの各基である。
特に好ましい特定のアミノ保護基としてはベンジ
ルオキシカルボニル、フエニル環が１つ又はそれ
以上のCl又はBr等のハロゲンで置換されている
置換ベンジルオキシカルボニル、ニトロ、メトキ
シ等の低級アルコキシ、低級アルキル、ｔ−ブチ
ルオキシカルボニル、ｔ−アミルオキシカルボニ
ル、シクロヘキシルオキシカルボニル、ビニルオ
キシカルボニル、アダマンチルオキシカルボニ
ル、ビフエニルイソプロポキシカニボニル等であ
る。使用可能な他の保護基としてはイソニコチニ
ルオキシカルボニル、フタロイル、ｐ−トリルス
ルホニル、ホルミル等である。 The aryl, aralkyl and alkaryl groups may be further substituted with one or more alkyl groups having from 1 to about 4 carbon atoms. Preferred groups as R are t-butyl, t-amyl, phenyl,
These are tolyl, xylyl, and benzyl groups.
Particularly preferred specific amino protecting groups include benzyloxycarbonyl, substituted benzyloxycarbonyl in which the phenyl ring is substituted with one or more halogens such as Cl or Br, lower alkoxy such as nitro, methoxy, lower alkyl, t -butyloxycarbonyl, t-amyloxycarbonyl, cyclohexyloxycarbonyl, vinyloxycarbonyl, adamantyloxycarbonyl, biphenylisopropoxycanibonyl, and the like. Other protecting groups that can be used include isonicotinyloxycarbonyl, phthaloyl, p-tolylsulfonyl, formyl, and the like.
本発明の方法を行うには、ペプチドは遊離α−
アミノ基を保護形α−アミノ基を含む化合物と反
応させることで得られる。反応即ちカツプリング
をさせるには、反応を受ける化合物のカルボキシ
ル基を活性化させて、カルボキシル基をペプチド
鎖に結合している遊離アミノ基と反応させる。活
性化するには、カルボキシル基をエステル、無水
物、アジド、酸クロライド等の反応性基に変えれ
ばよい。 To carry out the method of the invention, the peptide is free α-
It is obtained by reacting an amino group with a compound containing a protected α-amino group. To effect the reaction, or coupling, the carboxyl group of the compound undergoing the reaction is activated, causing the carboxyl group to react with a free amino group attached to the peptide chain. Activation can be achieved by changing the carboxyl group to a reactive group such as ester, anhydride, azide, or acid chloride.
アミノ酸成分はアミノ基とカルボキシル基の両
者を含んでいるが、これらの反応中通常一方の基
が反応に関与し他方は保護されている。カツプリ
ングに先立つて、反応したペプチドのα−又は末
端位のアミノ基に付いている保護基は、他の保護
基、例えばリシン分子のε−アミノ基に付いてい
る基に実質的に影響を与えない条件下で除去され
る。この除去するに当つての好ましい方法は酸に
よる温和な加水分解であり、例えば室温でのトリ
フルオル酢酸との反応にる加水分解である。 The amino acid component contains both an amino group and a carboxyl group, and during these reactions, usually one group participates in the reaction and the other is protected. Prior to coupling, the protecting group attached to the alpha or terminal amino group of the reacted peptide substantially affects other protecting groups, such as the group attached to the epsilon amino group of the lysine molecule. removed under no conditions. The preferred method for this removal is mild hydrolysis with an acid, such as reaction with trifluoroacetic acid at room temperature.
上述一連の工程により下記式で示されるペンタ
ペプチドが生成する。 Through the series of steps described above, a pentapeptide represented by the following formula is produced.
このペンタペプチドも又本発明のポリペプチド
の基本形活性構造単位を含んでいる。式の置換
ペンタペプチドは勿論TYR及びLYSの末端アミ
ノ酸基は上述の如く更に置換されていてもよい
が、この置換ペンタペプチドは上記基本形ペンタ
ペプチドを適当な薬剤と反応させて所望の誘導体
を得ることができる。この種のアシル化、エステ
ル化、アミド化等の反応は周知の反応である。更
に、他のアミノ酸類、即ち基本形ペンタペプチド
分子の生化学的活性に影響を与えないアミノ酸基
をペプチド鎖に付加してもよい。この付加反応は
ペンタペプチドの合成と同じ連続反応で行うこと
ができる。 This pentapeptide also contains the basic active structural unit of the polypeptide of the invention. Of course, the terminal amino acid groups of TYR and LYS in the substituted pentapeptide of the formula may be further substituted as described above, but this substituted pentapeptide can be prepared by reacting the above basic pentapeptide with an appropriate drug to obtain the desired derivative. I can do it. These types of reactions such as acylation, esterification, amidation, etc. are well known reactions. Additionally, other amino acids, ie, amino acid groups that do not affect the biochemical activity of the basic pentapeptide molecule, may be added to the peptide chain. This addition reaction can be carried out in the same continuous reaction as the synthesis of pentapeptide.
以下、本発明を実施例で具体的に説明するが、
本発明はこれらの実施例に限定されるものでな
い。実施例及び本明細書本文に於いては、特に指
示がない場合は部は重量部である。 Hereinafter, the present invention will be specifically explained with examples.
The present invention is not limited to these examples. In the examples and the text of this specification, parts are parts by weight unless otherwise specified.
〔実施例 １〕
本発明のポリペプチドを合成するために、下記
物質を購入した。[Example 1] In order to synthesize the polypeptide of the present invention, the following materials were purchased.
α−BOC−Ｌ−グルタミン−Ｏ−ニトロフエ
ニル−エステル
α−BOC−ε−２−クロル−ベンジルオキシ
カルボニル−Ｌ−リシン
α−BOC−Ｌ−アスパラギン
α−BOC−Ｌ−イソロイシン
α−BOC−Ｏ−２，６−ジクロルベンジル−
Ｌ−チロシン
上記薬剤に於いて、BOCはｔ−ブチルオキシ
カルボニルを示す。アミノ酸の逐次反応に際して
用いた試薬、ジヘキシルカーボジイミド、フルオ
レスアミン（Fluorescamine）及び樹脂も市販品
を購入した。 α-BOC-L-glutamine-O-nitrophenyl-ester α-BOC-ε-2-chloro-benzyloxycarbonyl-L-lysine α-BOC-L-asparagine α-BOC-L-isoleucine α-BOC-O- 2,6-dichlorobenzyl-
L-Tyrosine In the above drug, BOC represents t-butyloxycarbonyl. The reagents used in the sequential reactions of amino acids, dihexyl carbodiimide, Fluorescamine, and resin were also purchased from commercial sources.
使用した樹脂は粒径200−400メツシユのポリス
チレンジビニルベンゼン樹脂であり、１％のジビ
ニルベンゼンと樹脂１ｇ当り0.75ミリモルのクロ
ライドを含有していた。 The resin used was a polystyrene divinylbenzene resin with a particle size of 200-400 mesh, containing 1% divinylbenzene and 0.75 mmol chloride/g resin.
ポリペプチドを合成するに当り、２ミリモルの
α−BOC−ε−２−クロルベンジルオキシカル
ボニル−Ｌ−リシンを１ミリモルのトリエチルア
ミンを含む無水アルコール中80℃で24時間処理し
て２ミリモルのクロルメチル化樹脂にエステル化
した。得られた結合アミノ酸樹脂を濾過し、無水
アルコールで洗浄しかつ乾燥した。次いで、残り
のα−BOCアミノ酸を同様にしてペプチド−樹
脂の保護解除されたα−アミノ基に順次結合さ
せ、直接結合しているα−BOC−Ｌ−グルタミ
ン−Ｏ−ニトロフエニルエステル以外は当量のジ
シクロヘキシルカーボジイミドを用いて本発明の
ポリペプチドを合成した。各カツプリング反応
後、少量の樹脂をフルオレスアミンで試験し、も
し陽性螢光となつた場合にはカツプリングは不完
全であるとして同一の保護形アミノ酸を用いて反
応を繰返した。数回カツプリング反応を行つた結
果、中間体ペンタペプチド−樹脂が得られた。 To synthesize the polypeptide, 2 mmol of α-BOC-ε-2-chlorobenzyloxycarbonyl-L-lysine was treated in absolute alcohol containing 1 mmol of triethylamine at 80°C for 24 hours to chloromethylate 2 mmol. Esterified into resin. The resulting bound amino acid resin was filtered, washed with absolute alcohol and dried. The remaining α-BOC amino acids were then sequentially attached to the deprotected α-amino groups of the peptide-resin, except for the directly attached α-BOC-L-glutamine-O-nitrophenyl ester. Polypeptides of the invention were synthesized using an equivalent amount of dicyclohexylcarbodiimide. After each coupling reaction, a small amount of the resin was tested with fluorescein amine, and if positive fluorescence the coupling was considered incomplete and the reaction was repeated with the same protected amino acid. After several coupling reactions, an intermediate pentapeptide-resin was obtained.
このペンタペプチド−樹脂の分割と保護基の除
去を０℃60分間無水フツ化水素を用いてケルフ分
割装置（Peninsula Lab.Inc.製）で行い、この場
合スカベンジヤーとしてペプチド−樹脂１ｇに対
して1.2mlのアニソールを用いた。ペプチド混合
物を凍結真空乾燥し、無水エーテルで洗浄した。
このペプチドを1N酢酸中ｐ−６ビオ−ゲル
（Bio−Gel）上に担持してクロマトグラフ分析に
付した。得られたポリペプチドは純度94％で下記
構造を有することが分つた。 The resolution of this pentapeptide-resin and the removal of the protective groups were carried out using anhydrous hydrogen fluoride for 60 minutes at 0°C using a Kelf resolution apparatus (manufactured by Peninsula Lab. Inc.). 1.2 ml of anisole was used. The peptide mixture was lyophilized in vacuo and washed with anhydrous ether.
This peptide was loaded on p-6 Bio-Gel in 1N acetic acid and subjected to chromatographic analysis. It was found that the obtained polypeptide had a purity of 94% and had the following structure.
〔薬理試験例〕
上記ポリペプチドの活性及び特性を調べるため
に、健康な生後５〜６週間のnu／nuマウスの両
性について試験を行つた。マウスはBALB／ｃ
背地（Thy−1.2表面抗原を示す胸線細胞）で飼
育されかつ通常の条件下に維持された。抗血清に
間しては、抗Thy−1.2血清をThy−１同族マウ
スで調整した。 [Pharmacological Test Example] In order to investigate the activity and properties of the above polypeptide, tests were conducted on healthy 5-6 week old nu/nu mice of both sexes. The mouse is BALB/c
The mice were reared on the lining (thymocytes displaying Thy-1.2 surface antigen) and maintained under normal conditions. Among antisera, anti-Thy-1.2 serum was prepared in Thy-1 homologous mice.
Thy−1+T細胞又はCR+B細胞の分化の試験管
中誘導を行うため、試験管中で前胸線細胞
（Prothymocyte）からの胸線細胞の分化誘導を
コムロ及びボイス（Komuro＆Boyse）により記
載された方法（Lancet，１，740，1973）に準じ
て行つた。この方法ではThy−1.2の獲得をＴ細
胞分化の目安として利用している。一方、試験管
中でのCR-B細胞先駆体からのCR+B細胞への分
化の誘導も胸線細胞の場合と同様の条件下で行つ
たが、この場合分析の基準としてうさぎの抗体及
び非分解性の補体を多少凝集する程度の量塗被さ
れた羊の赤血球を結合するCR+B細胞の能力を用
いた。不連続な牛の血清アルブミン勾配上で分別
された健康なnu／nuマウスの脾臓細胞群を両先
駆体（Thy−1-及びCR-）源として用いた。これ
は前記細胞群は殆んど又は全くThy−1+細胞を含
まずしかもCR+細胞の数は少しであるという理由
による。 In order to induce in vitro differentiation of Thy-1 + T cells or CR + B cells, Komuro & Boyse described the induction of differentiation of thymocytes from prothymocytes in vitro. It was carried out according to the method described in (Lancet, 1, 740, 1973). This method uses the acquisition of Thy-1.2 as a measure of T cell differentiation. On the other hand, induction of differentiation of CR - B cell precursors into CR + B cells in vitro was performed under similar conditions as for thymocytes, but in this case rabbit antibodies and The ability of CR + B cells to bind sheep red blood cells coated with a moderately agglutinating amount of non-degradable complement was used. A population of healthy nu/nu mouse spleen cells sorted on a discontinuous bovine serum albumin gradient was used as a source for both precursors (Thy-1 - and CR - ). This is because the cell population contains little or no Thy-1 + cells and only a small number of CR + cells.
この結果、このポリペプチドはＴリンパ球及び
補体受容体（CR+）Ｂリンパ球の分化を誘導する
という点に於いてユビキチンと同様の作用選択性
を示した。このペンタペプチドは10nｇ−1μｇ／
mlの濃度範囲でThy−1+T細胞の分化を誘導しか
つ10nｇ−1μｇ／mlの濃度範囲でCR+B細胞の分
化も誘導した。 As a result, this polypeptide showed the same selectivity of action as ubiquitin in inducing the differentiation of T lymphocytes and complement receptor (CR + ) B lymphocytes. This pentapeptide is 10ng-1μg/
It induced differentiation of Thy-1 + T cells in the concentration range of 10 ng-1 μg/ml and also induced differentiation of CR + B cells in the concentration range of 10 ng-1 μg/ml.
〔参考例 ３〕
実施例１と同様にして合成し、未だ樹脂に結合
しているペンタペプチド類をジクロルメタン中で
トリフルオル酢酸で処理してチロシンのｔ−
BOC保護基を除いた。得られたペプチドを次い
で無水酢酸でアシル化し、更に樹脂基板から分離
させかつ保護基をすべてHFを用いて取り除き下
記式のアシル化誘導体を得た。[Reference Example 3] Synthesized in the same manner as in Example 1, the pentapeptides still bound to the resin were treated with trifluoroacetic acid in dichloromethane to release the t-
BOC protecting group was removed. The obtained peptide was then acylated with acetic anhydride, further separated from the resin substrate, and all protecting groups were removed using HF to obtain an acylated derivative of the following formula.
〔参考例 ４〕
実施例１のペンタペプチド類のアミノ化誘導体
を得るために、基板としてベンズヒドリルアミン
を用いて前述J.Rivier等の方法により保護された
ペンタペプチドを合成した。次いで弗化水素を利
用して樹脂基板に付着している保護ペンタペプチ
ドを開裂分離して下記式のアミノ化誘導体を得
た。 [Reference Example 4] In order to obtain an aminated derivative of the pentapeptide of Example 1, a protected pentapeptide was synthesized by the method of J. Rivier et al. described above using benzhydrylamine as a substrate. Next, the protected pentapeptide attached to the resin substrate was cleaved and separated using hydrogen fluoride to obtain an aminated derivative of the following formula.
上記誘導体を同定するために、薄層クロマトグ
ラフ法及び電気泳動法を用い下記結果を得た。 In order to identify the above derivative, the following results were obtained using thin layer chromatography and electrophoresis.
薄層クロマトグラフ法
試料：30μｇ
シリカゲル（ブリンクマンガラス板、５×20
cm、厚さ0.25mm）
Ｒ：ｎ−ブタノール：ピリジン
：酢酸（HOAc）：水30：15：３：12
Ｒ：酢酸エチル（EtoAc）：ピリジン
：酢酸：水５：５：１：３
Ｒ：酢酸エチル：ｎ−ブタノール：酢酸
：水１：１：１：１
スプレー試薬：パウリ試薬、ニンヒドリン
及び2
化合物の種類
Ac−Tyr−Asn−Ile−Gln−Lys
Tyr Asn−Ile−Gln−Lys−NH2
薄層クロマトグラフ法
Ｒ Ｒ Ｒ
0.47 0.87 0.54
0.48 0.87 0.37
電気泳動法
陰極へ移動したペプチド
−1.35cm
−6.60cm
電気泳動法
ワツトマン３mm厚濾紙（11.5cm×56.5mm）
試料：100μｇ
PH5.6、ピリジン−アセテート緩衝溶液、
100V、１時間
スプレー試薬：パウリ試薬及びニンヒドリン
参考例３のアセチル化ペンタペプチドを６％ト
ウミー（Twmoey）溶液中1μｇ／mlの濃度で用
いた時、実施例１の基本形ペンタペプチドに匹敵
する最大及び最小活性度を示した。Thin layer chromatography Sample: 30 μg silica gel (Brinkmann glass plate, 5 x 20
cm, thickness 0.25 mm) R: n-butanol: pyridine: acetic acid (HOAc): water 30:15:3:12 R: ethyl acetate (EtoAc): pyridine: acetic acid: water 5:5:1:3 R: Ethyl acetate: n-butanol: acetic acid: water 1:1:1:1 Spray reagent: Pauli reagent, ninhydrin and 2 types of compounds Ac-Tyr-Asn-Ile-Gln-Lys Tyr Asn-Ile-Gln-Lys-NH 2 Thin layer chromatography R R R 0.47 0.87 0.54 0.48 0.87 0.37 Electrophoresis Peptide transferred to cathode -1.35cm -6.60cm Electrophoresis Watzmann 3mm thick filter paper (11.5cm x 56.5mm) Sample: 100μg PH5.6, pyridine-acetate buffer solution,
100V, 1 hour Spray reagents: Pauli's reagent and ninhydrin When the acetylated pentapeptide of Reference Example 3 was used at a concentration of 1 μg/ml in 6% Twmoey solution, the maximum and showed minimal activity.
参考例４のアミド化ペンタペプチドは、６％ト
ウミー溶液中1μｇ／mlの濃度で用いた時実施例
１の基本形ペンタペプチドに匹敵する活性を示し
た。 The amidated pentapeptide of Reference Example 4 exhibited activity comparable to the basic pentapeptide of Example 1 when used at a concentration of 1 μg/ml in 6% Toumy's solution.
〔参考例 ５〕
実施例１の方法に従い合成し、樹脂に付着して
いる状態の基本形ペンタペプチドをエステル交換
条件下メタノール中でナトリウムメトキシドでエ
ステル交換して樹脂から開裂分離させた。保護基
をすべて除去して下記式を有するステル化生成物
を得た。[Reference Example 5] The basic pentapeptide synthesized according to the method of Example 1 and attached to the resin was cleaved and separated from the resin by transesterification with sodium methoxide in methanol under transesterification conditions. All protecting groups were removed to obtain a stellated product having the following formula:
〔参考例 ７〕
実施例１と同様にして合成したペプチドのジエ
チルアミン誘導体を参考例５のメチルエステル化
合物から製造した。即ち、参考例５のメチルエス
テルをジメチルホルムアミド中でジエチルアミン
と反応させて、下記式のジアミノ置換アミドを得
た。 [Reference Example 7] A diethylamine derivative of a peptide synthesized in the same manner as in Example 1 was produced from the methyl ester compound of Reference Example 5. That is, the methyl ester of Reference Example 5 was reacted with diethylamine in dimethylformamide to obtain a diamino-substituted amide of the following formula.
このジエチルアミン誘導体は、実施例１の樹脂
基板に付着している基本形ペンタペプチドをジエ
チルアミンと反応させて樹脂から前記ペンタペプ
チドを開裂分離させる反応からも同様に得ること
ができる。樹脂から除去しかつ保護基を除いて得
られる生成物はジエチルアミド誘導体である。 This diethylamine derivative can be similarly obtained by reacting the basic pentapeptide attached to the resin substrate of Example 1 with diethylamine to cleave and separate the pentapeptide from the resin. The product obtained upon removal from the resin and removal of the protecting groups is a diethylamide derivative.
〔参考例 ８〕
本参考例では、実施例１の基本形ペンタペプチ
ドを臭化エチルと反応させてＮ−エチルチロシン
誘導体を得た。この反応を行うにあたつて、リシ
ンのα−アミノ基をε−２−クロルベンジルオキ
シカルボニル基で保護し、TYRの末端アミノ基
をジクロルメタン中でトリフルオル酢酸と反応さ
せて遊離させた。この保護中間体をアルキル化条
件下で化学量論量の臭化エチルと反応させた。
LYSα−アミノ酸から上記保護基を除去して下記
式を有する置換ポリペプチドを得た。[Reference Example 8] In this Reference Example, the basic pentapeptide of Example 1 was reacted with ethyl bromide to obtain an N-ethyltyrosine derivative. In carrying out this reaction, the α-amino group of lysine was protected with an ε-2-chlorobenzyloxycarbonyl group, and the terminal amino group of TYR was liberated by reaction with trifluoroacetic acid in dichloromethane. This protected intermediate was reacted with a stoichiometric amount of ethyl bromide under alkylation conditions.
The above protecting group was removed from the LYSα-amino acid to obtain a substituted polypeptide having the following formula.
〔参考例 ９〕
本参考例では、参考例８のエチルアミノポリペ
プチドのアミノ化誘導体を得た。即ち、基本形ペ
ンタペプチドを樹脂基板に結合させたまま参考例
８の反応を行い、基板から分離させずにＮ−エチ
ル誘導体を得た。次いで、樹脂基板に付着してい
るこの中間体生成物をジメチルホルムアミド溶媒
中無水アンモニアで処理し樹脂から分離させ下記
式のアミドポリペプチドを得た。 [Reference Example 9] In this Reference Example, an aminated derivative of the ethylamino polypeptide of Reference Example 8 was obtained. That is, the reaction of Reference Example 8 was carried out while the basic pentapeptide was bound to the resin substrate, and the N-ethyl derivative was obtained without separating it from the substrate. This intermediate product adhering to the resin substrate was then treated with anhydrous ammonia in a dimethylformamide solvent to separate it from the resin, yielding an amide polypeptide of the following formula.
〔参考例 11〕
本参考例では、参考例３と同様にして合成し、
樹脂基板に付着しているままのアシル化ペンタペ
プチドをアミノ化反応条件下で無水アンモニアと
反応させてこの化合物を遊離させ、保護基を除去
して下記式のポリペプチドを得た。 [Reference Example 11] In this reference example, synthesis was performed in the same manner as in Reference Example 3,
The acylated pentapeptide still attached to the resin substrate was reacted with anhydrous ammonia under amination reaction conditions to liberate this compound, and the protecting group was removed to yield a polypeptide of the following formula.
以上、本発明は活性部位として前述の特異なア
ミノ酸構造単位を有する新規なポリペプチドを提
供するものである。このポリペプチドは、胸線分
化抗原TL及びTHY−１（θ）の獲得により測定
されるようなＴ−先駆体細胞とＢ細胞の識別の目
安である補体の受容体の獲得により測定されるよ
うなＢ−先駆体細胞とを分化することができる。
従つて、ペプチドは胸線機能及び免疫分野、例え
ば先天性胸線欠損の治療、に有効である。このペ
プチドは非常に低濃度で活性である。 As described above, the present invention provides a novel polypeptide having the aforementioned unique amino acid structural unit as an active site. This polypeptide is measured by the acquisition of complement receptors, which is a measure of differentiation between T-precursor cells and B cells, as measured by the acquisition of thymus differentiation antigens TL and THY-1 (θ). B-precursor cells such as B-progenitor cells can be differentiated.
The peptides are therefore useful in the field of thymic function and immunity, for example in the treatment of congenital thymic defects. This peptide is active at very low concentrations.
本発明はいくつかの好ましい実施態様について
説明してきたが、これらの態様に本発明が限定さ
れるものでない。 Although the invention has been described in terms of several preferred embodiments, the invention is not limited to these embodiments.
Claims (1)
性高分子樹脂にエステル化して共有結合で結合
し、α−アミノ保護基をＬ−リシン部から除いて
α−アミノ保護形Ｌ−グルタミンと反応させてＬ
−グルタミンをＬ−リシン−樹脂と結合させ、Ｌ
−グルタミン部からα−アミノ保護基を除いてα
−アミノ保護形Ｌ−イソロイシン又はＬ−アラニ
ンと反応させてＬ−イソロイシン又はＬ−アラニ
ンをＬ−グルタミン−Ｌ−リシン−樹脂に結合さ
せ、α−アミノ保護基を除いてα−アミノ保護形
Ｌ−アスパラギン又はＬ−アラニンと反応させて
Ｌ−アスパラギン又はＬ−アラニンをＬ−イソロ
イシン又はＬ−アラニン−Ｌ−グルタミン−Ｌ−
リシン−樹脂に結合させ、α−アミノ保護基を除
いてα−アミノ保護形Ｌ−チロシン又はＬ−アラ
ニンと反応させてＬ−チロシン又はＬ−アラニン
をＬ−アスパラギン又はＬ−アラニン−Ｌ−イソ
ロイシン又はＬ−アラニン−Ｌ−グルタミン−Ｌ
−リシン−樹脂に結合させ、 下記式を有するペプチド中間体であつて、 上式で、R1，R2及びR3はアミノ酸の保護基で
あるペプチド中間体を形成し、 全てのアミノ保護基を前記ペプチド中間体から
除き、ペプチドを高分子樹脂から分離して下記
式、 で表わせるペンタペプチド化合物の製造方法。 ２ R1はＯ−２，６−ジクロルベンジル，R2は
ε−２−クロルベンジルオキシカルボニル、及び
R3はｔ−ブチルオキシカルボニルであることを
特徴とする特許請求の範囲第１項に記載のペンタ
ペプチド化合物の製造方法。 ３ 反応アミノ酸類の反応性側鎖は反応期間中保
護されていることを特徴とする特許請求の範囲第
１項に記載の方法。 ４ 高分子樹脂はセルロース、ポリビニルアルコ
ール、ポリメタアクリレート、スルホン化ポリス
チレン及びスチレンとジビニルベンゼンとのクロ
ルメチル共重合体からなる群より選ばれる樹脂で
あることを特徴とする特許請求の範囲第１項に記
載の方法。[Scope of Claims] 1 L-lysine whose amino group is protected is esterified to an insoluble polymer resin and bonded with a covalent bond, and the α-amino protecting group is removed from the L-lysine moiety to form the α-amino protected form. By reacting with L-glutamine, L
- Combine glutamine with L-lysine-resin,
-Remove the α-amino protecting group from the glutamine moiety to α
- React with amino-protected L-isoleucine or L-alanine to bind L-isoleucine or L-alanine to L-glutamine-L-lysine-resin, remove the α-amino protecting group, and remove α-amino protected L-isoleucine or L-alanine. - React with asparagine or L-alanine to convert L-isoleucine or L-alanine-L-glutamine-L-
lysine-resin, the α-amino protecting group is removed and the α-amino protected form of L-tyrosine or L-alanine is reacted to convert L-tyrosine or L-alanine into L-asparagine or L-alanine-L-isoleucine. or L-alanine-L-glutamine-L
- lysine - a peptide intermediate bound to a resin and having the following formula, In the above formula, R 1 , R 2 and R 3 form a peptide intermediate which is a protecting group for amino acids, and all amino protecting groups are removed from the peptide intermediate and the peptide is separated from the polymer resin to form the following formula: , A method for producing a pentapeptide compound expressed as 2 R 1 is O-2,6-dichlorobenzyl, R 2 is ε-2-chlorobenzyloxycarbonyl, and
The method for producing a pentapeptide compound according to claim 1, wherein R 3 is t-butyloxycarbonyl. 3. The method according to claim 1, wherein the reactive side chains of the reactive amino acids are protected during the reaction. 4. Claim 1, wherein the polymeric resin is a resin selected from the group consisting of cellulose, polyvinyl alcohol, polymethacrylate, sulfonated polystyrene, and a chloromethyl copolymer of styrene and divinylbenzene. Method described.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US85177877A | 1977-11-15 | 1977-11-15 | |
| US851778 | 1977-11-15 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6150999A JPS6150999A (en) | 1986-03-13 |
| JPH0137400B2 true JPH0137400B2 (en) | 1989-08-07 |
Family
ID=25311658
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1956878A Granted JPS5476522A (en) | 1977-11-15 | 1978-02-22 | Novel pentapeptides and manufacture |
| JP60111321A Granted JPS6150999A (en) | 1977-11-15 | 1985-05-22 | Manufacture of pentapeptide composition and intermediate polypeptide |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1956878A Granted JPS5476522A (en) | 1977-11-15 | 1978-02-22 | Novel pentapeptides and manufacture |
Country Status (7)
| Country | Link |
|---|---|
| JP (2) | JPS5476522A (en) |
| BE (1) | BE864122A (en) |
| CA (1) | CA1105923A (en) |
| CH (1) | CH640217A5 (en) |
| DE (1) | DE2804567C2 (en) |
| FR (1) | FR2408581A1 (en) |
| SE (1) | SE446866B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4215111A (en) * | 1979-03-14 | 1980-07-29 | Ortho Pharmaceutical Corporation | Peptides having ubiquitin-like activity |
| US4215112A (en) * | 1979-03-14 | 1980-07-29 | Ortho Pharmaceutical Corporation | Tripeptides and methods |
| DE2938420A1 (en) * | 1979-09-22 | 1981-04-09 | Hoechst Ag, 6000 Frankfurt | NEW PEPTIDES AND METHOD FOR THEIR PRODUCTION |
| US4426324A (en) * | 1979-09-28 | 1984-01-17 | Hoffmann-La Roche Inc. | Immunopotentiating peptides |
| JPH0528542U (en) * | 1991-04-03 | 1993-04-16 | 品川白煉瓦株式会社 | Long nozzle for continuous casting |
| US8148141B2 (en) | 2001-05-07 | 2012-04-03 | Hipep Laboratories | Peptide-immobilized substrate and method for measuring target protein |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3864481A (en) * | 1972-12-14 | 1975-02-04 | St Lukes Hospital | Anti disease producing synthetic material for the prevention suppression and diagnosis of multiple sclerosis and method of treatment therefor |
| US4002602A (en) * | 1974-03-11 | 1977-01-11 | Gideon Goldstein | Ubiquitous immunopoietic polypeptide (UBIP) and methods |
| US4113858A (en) * | 1975-01-20 | 1978-09-12 | St. Luke's Hospital | Novel compounds, compositions and methods of their use |
-
1978
- 1978-01-27 SE SE7801031A patent/SE446866B/en not_active IP Right Cessation
- 1978-02-03 DE DE2804567A patent/DE2804567C2/en not_active Expired
- 1978-02-08 CA CA296,484A patent/CA1105923A/en not_active Expired
- 1978-02-17 FR FR7804598A patent/FR2408581A1/en active Granted
- 1978-02-20 BE BE185307A patent/BE864122A/en not_active IP Right Cessation
- 1978-02-22 JP JP1956878A patent/JPS5476522A/en active Granted
- 1978-02-24 CH CH206378A patent/CH640217A5/en not_active IP Right Cessation
-
1985
- 1985-05-22 JP JP60111321A patent/JPS6150999A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| SE446866B (en) | 1986-10-13 |
| FR2408581B1 (en) | 1983-07-22 |
| CA1105923A (en) | 1981-07-28 |
| DE2804567A1 (en) | 1979-05-17 |
| CH640217A5 (en) | 1983-12-30 |
| JPS5476522A (en) | 1979-06-19 |
| JPS6150999A (en) | 1986-03-13 |
| JPS6149320B2 (en) | 1986-10-29 |
| DE2804567C2 (en) | 1986-01-16 |
| BE864122A (en) | 1978-08-21 |
| SE7801031L (en) | 1979-05-16 |
| FR2408581A1 (en) | 1979-06-08 |
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