JPH03271300A - New peptide - Google Patents
New peptideInfo
- Publication number
- JPH03271300A JPH03271300A JP2070358A JP7035890A JPH03271300A JP H03271300 A JPH03271300 A JP H03271300A JP 2070358 A JP2070358 A JP 2070358A JP 7035890 A JP7035890 A JP 7035890A JP H03271300 A JPH03271300 A JP H03271300A
- Authority
- JP
- Japan
- Prior art keywords
- peptide
- acid
- amino acid
- solid phase
- amino
- 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.)
- Pending
Links
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- OXNIZHLAWKMVMX-UHFFFAOYSA-N picric acid Chemical compound OC1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-N 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002953 preparative HPLC Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- XEYBRNLFEZDVAW-UHFFFAOYSA-N prostaglandin E2 Natural products CCCCCC(O)C=CC1C(O)CC(=O)C1CC=CCCCC(O)=O XEYBRNLFEZDVAW-UHFFFAOYSA-N 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000001698 pyrogenic effect Effects 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
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- WPLOVIFNBMNBPD-ATHMIXSHSA-N subtilin Chemical compound CC1SCC(NC2=O)C(=O)NC(CC(N)=O)C(=O)NC(C(=O)NC(CCCCN)C(=O)NC(C(C)CC)C(=O)NC(=C)C(=O)NC(CCCCN)C(O)=O)CSC(C)C2NC(=O)C(CC(C)C)NC(=O)C1NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C1NC(=O)C(=C/C)/NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)CNC(=O)C(NC(=O)C(NC(=O)C2NC(=O)CNC(=O)C3CCCN3C(=O)C(NC(=O)C3NC(=O)C(CC(C)C)NC(=O)C(=C)NC(=O)C(CCC(O)=O)NC(=O)C(NC(=O)C(CCCCN)NC(=O)C(N)CC=4C5=CC=CC=C5NC=4)CSC3)C(C)SC2)C(C)C)C(C)SC1)CC1=CC=CC=C1 WPLOVIFNBMNBPD-ATHMIXSHSA-N 0.000 description 1
- 239000001384 succinic acid Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- TUNFSRHWOTWDNC-HKGQFRNVSA-N tetradecanoic acid Chemical compound CCCCCCCCCCCCC[14C](O)=O TUNFSRHWOTWDNC-HKGQFRNVSA-N 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 102000003390 tumor necrosis factor Human genes 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 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)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、ヒトインターロイキン−１活性を有する新規
なペプチド、そのエステル、そのアミド、そのアシル化
体及びこれらの塩に関する。DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a novel peptide having human interleukin-1 activity, its ester, its amide, its acylated product, and salts thereof.
（発明の背景）
生体内に異物２例えば細菌やウィルスの侵入が起こった
場合には、侵入局所での炎症反応が惹き起こされ、さら
には免疫応答による生体防御反応が誘導される。これら
一連の生体反応に関与する物質として、マクロファージ
よシ産生される因子が注目され、その生物学的作用の違
いよシ、リンパ球活性化因子、Ｂ細胞活性化因子、ある
いは内因性発熱物質等と呼ばれていたが、互いに物理化
学的性質が類似しているので。(Background of the Invention) When a foreign substance 2, such as a bacteria or virus, invades a living body, an inflammatory reaction is triggered at the site of the invasion, and furthermore, a biological defense reaction by an immune response is induced. Factors produced by macrophages have attracted attention as substances involved in a series of these biological reactions, and their biological effects differ, such as lymphocyte activating factors, B cell activating factors, and endogenous pyrogens. They were called because they have similar physicochemical properties.
インターロイキン−１（ＩＬ−１）として統一し呼称さ
れた。It was unified and named as interleukin-1 (IL-1).
ＩＬ−１は、当初単球、マクロファージ、あるいは単球
性白血病株から産生されるとされたが。IL-1 was initially thought to be produced by monocytes, macrophages, or monocytic leukemia strains.
その後皮膚ケラチノサイト、脳の星状細胞、グリア細胞
、腎糸球体メサンギウム細胞、眼の角膜細胞等、多くの
組織由来の細胞から産生されることが明らかになるにつ
れ、その多彩な役割が注目されてきた。Later, as it became clear that it is produced by cells derived from many tissues, such as skin keratinocytes, brain astrocytes, glial cells, renal glomerular mesangial cells, and eye corneal cells, its diverse roles have attracted attention. Ta.
ＩＬ−１およびその前駆体をコードする２種類の遺伝子
がクローニングされ、その全アミノ酸配列も決定された
（ＩＬ−１αとＩＬ−１β） ［Ｎａｔｕｒｅ３１２、
４５８（１９８４）、 Ｐｒｏｃ、 Ｎａｔｌ、 Ａｃ
ａｄ、Ｓｃｉ、 ＵＳＡ。Two genes encoding IL-1 and its precursors have been cloned, and their entire amino acid sequences have been determined (IL-1α and IL-1β) [Nature 312,
458 (1984), Proc, Natl, Ac.
ad, Sci, USA.
８１、７９０７（１９８４）、 Ｎａｔｕｒｅ、 ３１
５．６４１ （１９８５）ｒＮｕｃｌｅｉｃ Ａｃ１ｄ
Ｒｅａ、、１３．５８６９（１９８５）コ。81, 7907 (1984), Nature, 31
5.641 (1985) rNucleic Ac1d
Rea, 13.5869 (1985).
現在では遺伝子組み換え法により大量に、かつ均一７’
ｆＩＬ−１が得られるようになり、ＩＬ−１の多様な生
物学的作用が認められてきた。その主な作用として、腫
瘍増殖の抑制や完全退縮が認められること［Ｊ、 Ｉｍ
ｍｕｎｏｌ、、１３６．３０９８（１９８６）。Currently, through genetic recombination, 7' can be produced uniformly in large quantities.
fIL-1 has become available, and various biological actions of IL-1 have been recognized. Its main effects include suppression of tumor growth and complete regression [J, Im
Munol, 136.3098 (1986).
Ｊｐｎ、 Ｊ、 Ｃ１１ｎ、 Ｉｍｍｕｎｏｌ、、 ９
．３３０（１９８６）コ、放射線障害防止作用［Ｊ、
Ｉｍｍｕｎｏｌ、 １３６．２４８３（１９８６）］。Jpn, J, C11n, Immunol, 9
．． 330 (1986), Prevention of radiation damage [J,
Immunol, 136.2483 (1986)].
日和見感染に対する防御作用［Ｉｎｆｅｃｔ−Ｉｍｍｕ
ｎ、、５５＋１４３６（１９８７）］が期待されている
ほか、ＩＬ−１は内因性発熱物質であること、また結合
組織系細胞、即ち溝膜細胞や線維芽細胞からのプロスタ
グランジンＥ２ （Ｐ Ｇ Ｅ２ ）の産生亢進作用を
有していることが報告されている。筺た最近では。Protective effect against opportunistic infections [Infect-Immu
In addition, IL-1 is expected to be an endogenous pyrogen, and prostaglandin E2 (P G It has been reported that it has the effect of enhancing the production of E2). Recently,
ＩＬ−１は線維芽細胞の増殖を促進することから動脈硬
化の増悪に関与するとの報告や、β細胞の疾病の治療に
対して傷害的に働くこともあり。IL-1 has been reported to be involved in exacerbation of arteriosclerosis because it promotes the proliferation of fibroblasts, and it may also have a detrimental effect on the treatment of β-cell diseases.
ＩＬ−１の医薬としての利用を複雑にしている。This complicates the medicinal use of IL-1.
最近いくつかのＩＬ−１α誘導体（特開昭６２−１８５
０９７ ）や、ＩＬ−１β誘導体（特開昭６３−１５２
３９８ ）、 ＩＬ−１の部分合成ペプチド（特開昭６
２−１８５０９５ ）が報告され、ＩＬ−１の作用の抽
出や改善が試みられている。壕だ本発明者らは、既に作
成したＩＬ−１β誘導体が天然のヒトＩＬ−１βと比較
して改良されたＩＬ−１活性を有し、医薬品として有用
に使用できることを知り。Recently, some IL-1α derivatives (JP-A-62-185
097) and IL-1β derivatives (JP-A-63-152)
398), partially synthetic peptide of IL-1 (Japanese Unexamined Patent Publication No. 6
2-185095) has been reported, and attempts have been made to extract and improve the effects of IL-1. However, the present inventors found that the IL-1β derivatives that had already been prepared had improved IL-1 activity compared to natural human IL-1β, and could be usefully used as pharmaceuticals.
特許出願（特願昭６３−２７２０１９ ） している。A patent application (Japanese Patent Application No. 63-272019) has been filed.
（解決手段）
本発明者らは、かかる技術水準下に種々研究した結果、
下記式（Ｉ）で示される文献未載のデカペプチド、その
エステル、そのアミド、そのアシル化体やこれらの塩が
、ＩＬ−１活性を有することをつきとめ２本発明を完成
させるに至った。(Solution Means) As a result of various researches based on this technical level, the present inventors found that
The inventors have found that a decapeptide represented by the following formula (I), which has not been described in any literature, its ester, its amide, its acylated product, and its salt have IL-1 activity, and have completed the present invention.
Ａｌａ−Ｌｅｕ−Ｇｌｙ−Ｌｅｕ−Ｌｙｓ−Ｇｌｕ−Ｌ
ｙｓ−Ａｓｎ−Ｌｅｕ−Ｔｙｒ （Ｉ）すなわち２本発
明は、上記式（１）で示されるペプチド、そのエステル
、そのアミド、そのアシル化体又はこれらの塩を発明の
構成とする。Ala-Leu-Gly-Leu-Lys-Glu-L
ys-Asn-Leu-Tyr (I), that is, 2 The present invention comprises a peptide represented by the above formula (1), an ester thereof, an amide thereof, an acylated product thereof, or a salt thereof.
本発明の目的は、上記の新規化合物の提供にある。The object of the present invention is to provide the above-mentioned novel compounds.
本発明の他の目的は、下記の記載及び実施例により明ら
かにされる。Other objects of the invention will become apparent from the following description and examples.
本発明者らはペプチド合成に先立ち、ヒ）ＩＬ−１タン
パク質の活性部位の同定を行った。ヒトＩＬ−１βタン
パク質は結晶化され、Ｘ線解析によりその立体構造が明
らかにされている［ＥＭＢＯ。Prior to peptide synthesis, the present inventors identified the active site of IL-1 protein. Human IL-1β protein has been crystallized and its three-dimensional structure has been revealed by X-ray analysis [EMBO.
Ｊ、、７．３３９（１９８８）コ。これによると、ヒト
ＩＬ−１βは、１２本のβ−シートを構成するアミノ酸
と、それを連絡するαループを構成するアミノ酸の部分
からなり、２本の逆平行なβ−シートの組が１辺を成す
四面体構造を呈している。さらには、ＩＬ−１βの活性
部分として想定されているアミノ酸配列［Ｊ、 Ｉｍｍ
ｕｎｏｌ、、 １３７．３２０１（１９８６）］が、４
番目のβ−シートの後半から５番目のβ−シートの前半
部分を構成している［蛋白質・核酸・酵素、 ３３．１
７９３（１９８８）コことが示唆されている。J, 7.339 (1988). According to this, human IL-1β consists of amino acids constituting 12 β-sheets and amino acids constituting an α-loop connecting them, and a set of two antiparallel β-sheets forms one It has a tetrahedral structure with edges. Furthermore, the amino acid sequence postulated as the active part of IL-1β [J, Imm
unol, 137.3201 (1986)], but 4
It constitutes the first half of the fifth β-sheet from the second half of the second β-sheet [Proteins/Nucleic Acids/Enzymes, 33.1
793 (1988).
また、ＩＬ−１のアミノ酸配列は、ヒト、ウシ。Furthermore, the amino acid sequence of IL-1 is human and bovine.
マウス、ウサギらでよく保存されておジ２種特異性が低
いことが知られている。さらに、タンパク質の活性部位
は、比較的親水性の高い領域に多く見いだされることが
知られている。It is well conserved in mice and rabbits, and is known to have low bispecies specificity. Furthermore, it is known that many active sites of proteins are found in relatively highly hydrophilic regions.
本発明者らはこれらの点に着目し、特によく種間で保存
されており、比較的親水性の高い領域である。５番目と
６番目のβ−シートを連絡し、かつ四面体の頂点の一つ
を構成するαループ部分に相当するペプチド（Ｐ ｒｏ
ｅ、 Ｎａｔｌ、 Ａｃａｄ、 Ｓ ｃｉ。The present inventors focused on these points, and found that this region is particularly well conserved among species and has relatively high hydrophilicity. A peptide (Pro
e, Natl, Acad, Sci.
ＵＳＡ、 ８１．７９０７（１９８４）の文献に示され
たＩＬ−１βのアミノ酸配列において１７５位から１８
４位）を合成したものである。USA, 81.7907 (1984) in the amino acid sequence of IL-1β, from position 175 to position 18.
4)).
（化合物） 以下に９本発明化合物につき詳述する。(Compound) Nine compounds of the present invention will be described in detail below.
式（Ｉ）のペプチドのエステルには、Ｃ末端のカルボキ
シル基及びＧｌｕ （グルタミン酸残基）のγ−カルボ
キシル基の一方又は双方と、アルコール成分とが結合し
て形成するエステルが金管れ、このようなエステル形成
アルコール成分としてＦｉ、 メタノール、エタノー
ル、プロパノールウインプロパツール、ブタノール、イ
ンブタノール、 ｔｅｒｔ−ブタノールなどの炭素数
１乃至６個の直鎖又は分岐状の低級アルコールや、ベン
ジル基などのアラルキル基が挙げられる。The ester of the peptide of formula (I) includes an ester formed by combining an alcohol component with one or both of the carboxyl group at the C-terminus and the γ-carboxyl group of Glu (glutamic acid residue), and such Examples of ester-forming alcohol components include Fi, straight-chain or branched lower alcohols having 1 to 6 carbon atoms such as methanol, ethanol, propanolwinpropertool, butanol, imbutanol, and tert-butanol, and aralkyl groups such as benzyl groups. can be mentioned.
また９式（Ｉ）のペプチドのアミドとしては、Ｃ末端の
カルボキシル基及びＧｌｕ （グルタミン酸残基）のγ
−カルボキシル基の一方又は双方がカルバモイル基であ
る化合物が挙げられる。The amide of the peptide of formula 9 (I) includes the C-terminal carboxyl group and the γ of Glu (glutamic acid residue).
Examples include compounds in which one or both of the -carboxyl groups are carbamoyl groups.
筐た２式（Ｉ）のペプチドのアシル化体としては。As an acylated form of the peptide of formula (I),
Ｎ末端のアミノ基及びＬｙｓ （リジン残基）のε−ア
ミノ基のいくつか、捷たはすべてと、有機酸成分とが結
合して形成するアシル化体が含すれ、このような有機酸
成分としては、ギ酸、酢酸、ミリスチン酸、コハク酸、
マレイン酸、グルクロン酸などが挙げられる。This includes acylated products formed by bonding some, all, or all of the amino group at the N-terminus and the ε-amino group of Lys (lysine residue) with an organic acid component; Examples include formic acid, acetic acid, myristic acid, succinic acid,
Examples include maleic acid and glucuronic acid.
本発明のペプチド、そのエステル、そのアミド、そのア
シル化体は、酸付加塩を形成する場合がある。かかる塩
としては、塩酸、臭化水素酸、ヨウ化水素酸、硫酸、硝
酸、リン酸等の鉱酸、ギ酸、酢酸、炭酸、トリフルオロ
酢酸、プロピオン酸、シュウ酸、マロン酸、コハク酸。The peptide of the present invention, its ester, its amide, and its acylated product may form acid addition salts. Such salts include mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, carbonic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid.
フマール酸°、マレイン酸、 乳酸、 ＩＪンゴ酸、
酒石酸、メタンスルホン酸、エタンスルホン酸。fumaric acid, maleic acid, lactic acid, IJ malic acid,
Tartaric acid, methanesulfonic acid, ethanesulfonic acid.
ピクリン酸などの有機酸との酸付加塩が挙げられる。Examples include acid addition salts with organic acids such as picric acid.
また９本発明のペプチド、そのエステル、そのアミド、
そのアシル化体は、塩基との塩を形成する場合がある。In addition, 9 peptides of the present invention, esters thereof, amides thereof,
The acylated product may form a salt with a base.
そのような塩としてはカリウム、ナトリウムなどのアル
カリ金属、カルシウム、マグネシウムなどのアルカリ土
類金属、アルミニウムなどの■価の金属との塩、アンモ
ニウム塩。Such salts include salts with alkali metals such as potassium and sodium, alkaline earth metals such as calcium and magnesium, salts with valent metals such as aluminum, and ammonium salts.
々どが挙げられる。There are many examples.
本発明化合物はデカベグチド又はその誘導体であるから
少々くとも１０個の不斉炭素原子を含み、異性体が存在
する。本発明にはこれら異性れる。従って９本発明化合
物を構成するアミノ酸残基はＬ体のアミノ酸残基に限定
されるものではなく、Ｄ体、ＤＬ体であってもよい。中
でも２本発明化合物の好適なＩＬ−１活性剤としては天
然型のものあるいは１０個のアミノ酸残基の一部がＤ型
となったものが挙げられる。Since the compound of the present invention is decabegutide or a derivative thereof, it contains at least 10 asymmetric carbon atoms and has isomers. The present invention includes these isomers. Therefore, the amino acid residues constituting the compound of the present invention are not limited to L-configuration amino acid residues, but may be D-configuration or DL-configuration. Among these, preferable IL-1 activators of the two compounds of the present invention include natural types and those in which some of the 10 amino acid residues are D-type.
なお９本発明ペプチド（Ｉ）を示すアミノ酸残基の記号
は、当分野で慣用されているアミノ酸の三文字略記法に
基づいて表わしたものであり。Note that the symbols for amino acid residues representing the peptide (I) of the present invention are expressed based on the three-letter abbreviation for amino acids commonly used in the art.
Ａｌａ ：アラニン、Ｌｅｕ：ｏイシン、ｃｔｙニゲリ
シン。Ala: alanine, Leu: oisine, cty nigericin.
Ｌｙｓ ：リジン、Ｇｌｕ：グルタミン酸、Ａｓｎ：ア
スパラギン。Lys: lysine, Glu: glutamic acid, Asn: asparagine.
Ｔｙｒ ：チロシン のそれぞれの残基を示している。Tyr: Tyrosine The respective residues are shown.
〈製法〉
本発明ペプチド、そのエステル、そのアミドそのアシル
化体、又はこれらの塩は、ペプチド合成法の一つである
各種保護基、カップリング試薬などを駆使して行うカッ
プリング法、Ｃ端活性化法、Ｎ端活性化法などの液相法
を用いて合成することはもちろん可能であるが簡便に生
産及び精製が可能な固相法［Ｍｅｒｒｉｆｉｅｌｄ ［
Ｊ、Ａｍ。<Production method> The peptide of the present invention, its ester, its amide, its acylated product, or a salt thereof can be produced by a coupling method using various protecting groups, coupling reagents, etc., which is one of the peptide synthesis methods, and a C-terminus method. It is of course possible to synthesize using liquid phase methods such as activation method and N-terminal activation method, but solid phase method [Merrifield [
J.Am.
Ｃｈｅｍ、 Ｓｏｃ、、 ８５．２１８５ （１９６３
） ］によって初めて紹介され、その後改良が加えられ
ている方法］を適用して合成するのが有利である。固相
法によってペプチドを合成するに当っては、優れたペプ
チド自動合成機９例えばアプライド・バイオシステムズ
社のペプチド自動合成機４３０ Ａなどが市販されてお
り、このような装置の標準的運転プログラムに従って行
なえばよい。なお２本発明ペプチドの製法として、現在
市販品装置の適用のみに限定されるべきでないことはい
うまでもない。Chem, Soc, 85.2185 (1963
It is advantageous to synthesize by applying the method first introduced by ) and improved since then. When synthesizing peptides by the solid phase method, excellent automatic peptide synthesizers9 such as the Applied Biosystems automatic peptide synthesizer 430A are commercially available, and the standard operating program for such devices is followed. Just do it. It goes without saying that the method for producing the peptide of the present invention should not be limited to the application of currently commercially available equipment.
エステルは９本発明ペプチドあるいはそのＣ端活性化体
に前記アルコールを縮合剤、酸、塩基触媒の存在下又は
非存在下に作用させる常法により、アミドは１本発明ペ
プチドあるいはそのＣ端活性化体にアンモニア又はその
塩を作用させる常法によりそれぞれ合成される。また。Esters can be obtained by a conventional method in which the above-mentioned alcohol is applied to the peptide of the present invention or its C-terminal activated product in the presence or absence of a condensing agent, an acid, or a base catalyst; Each is synthesized by a conventional method in which ammonia or its salt is applied to the body. Also.
ペプチド自動合成の際に初発担体として、ベンズヒドリ
ルアミンを結合させた固相担体を用いれば固相担体より
遊離させたときにＣ末端のアミドとして得られる。さら
に、ペプチド自動合成の最終段階で、有機酸成分活性化
体を作用させる常法により、Ｎ末端アミノ基及びＬｙｓ
のε−アミノ基のいくつか、またはすべてがアシル化さ
れた誘導体を合成することができる。If a solid phase support to which benzhydrylamine is bound is used as an initial support during automatic peptide synthesis, the amide can be obtained as a C-terminal amide when released from the solid phase support. Furthermore, in the final stage of automatic peptide synthesis, the N-terminal amino group and Lys
It is possible to synthesize derivatives in which some or all of the ε-amino groups are acylated.
これらの合成ペプチドは、さらに精製度を高めるために
９分取用逆相高性能液体クロマトグラフィー（ＨＰＬＣ
）によって精製するのが有利である。These synthetic peptides were subjected to 9-preparative reverse-phase high-performance liquid chromatography (HPLC) to further increase the degree of purification.
).
ｋお、この精製法適用によりペプチド自動台て単離する
ことができる。＋
なお、純度は分析用逆相ＨＰＬＣによって確立した。By applying this purification method, peptides can be isolated using an automated system. + Note that purity was established by analytical reverse phase HPLC.
また９合成ペプチドの純度、安定性を維持するためには
、凍結乾燥するのが好適である。なお９本発明ペプチド
を製造する方法として、このペプチド（Ｉ）をコードす
るＤＮＡを用いて遺伝子工学的手法により生産させるこ
とによって行うことも可能である。Furthermore, in order to maintain the purity and stability of the 9 synthetic peptide, it is preferable to freeze-dry it. Note that the peptide of the present invention can also be produced by genetic engineering using DNA encoding this peptide (I).
（発明の効果） 本発明の新規ペプチド（Ｉ）やそのエステル。(Effect of the invention) The novel peptide (I) of the present invention and its ester.
そのアミド、そのアシル化体及びこれらの塩（以下ペプ
チド類という）は、ＩＬ−１βに認められるのと同様の
薬理作用を有しているが、幾つかの作用については選択
性があり、その作用が改善されている。す々わち１本発
明の新規ペプチド類は、リンパ球活性化作用、インター
ロイキン−２（ＩＬ−２）の産生亢進作用、抗体産生の
増強やコロニー刺激因子（Ｃ８Ｆ）亢進作用等免疫系細
胞に対する作用の増大１発熱作用の減少やＰＧＥ、産生
先進作用の減少などで改良されたＩＬ−１活性を示すこ
とができる。Its amides, its acylated forms, and their salts (hereinafter referred to as peptides) have pharmacological actions similar to those observed in IL-1β, but some actions are selective; The effect is improved. The novel peptides of the present invention have effects on immune system cells such as lymphocyte activation, interleukin-2 (IL-2) production enhancement, antibody production enhancement, and colony stimulating factor (C8F) enhancement effects. Improved IL-1 activity can be demonstrated by increasing the effect on IL-1, reducing the pyrogenic effect, and reducing the PGE and advanced effect on production.
さらに２本発明の新規ペプチド類は、 ＩＬ−１と同
様マクロファージ、血管内皮細胞や線維芽細胞などから
のＣ８Ｆ産生を促進し［Ｊ、 Ｃ１１ｎ。Furthermore, the two novel peptides of the present invention, like IL-1, promote C8F production from macrophages, vascular endothelial cells, fibroblasts, etc. [J, C11n.
Ｉｎｖｅｓｔ、、８１，９２ （１９８８）； Ｂｌｏ
ｏｄ、６８，１３１６（１９８６）コかつ放射線障害防
止作用を有していることから。Invest, 81, 92 (1988); Blo
od, 68, 1316 (1986) and has an action to prevent radiation damage.
化学療法剤治療や放射線治療後の顆粒球減少症や放射線
障害防止に有用であり、また９日和見感染に対する抗感
染剤、抗炎症剤としても有用である。It is useful for preventing granulocytopenia and radiation damage after chemotherapeutic treatment and radiotherapy, and is also useful as an anti-infective and anti-inflammatory agent for opportunistic infections.
更に９本発明の新規ペプチド類は、他の生理活性物質、
化学療法剤や免疫増強剤と併用して用いることによりそ
の治療効果を高めることができる。例えば、ＩＬ−２，
腫瘍壊死因子（ＴＮＦα、 ＴＮＦβ）ヤインターフェ
ロンーガンマ（ＩＦＮγ）等と併用することにより、免
疫増強作用や抗腫瘍作用に於て、相加並びに相乗効果が
認められる。化学療法剤や免疫増強剤等と併用すること
により、その抗腫瘍効果を増強し、かつ副作用を低減せ
しめることができる。また、５−ＦＵによる造血障害に
対して、Ｇ−Ｃ８Ｆとの併用により相乗的に回復作用を
果たすことや肺臓中のコロニー形成能の増強作用を有す
ることが期待できる［Ｊ、 ＩｍｍｕｎｏＬ、１４０．
３８３０（１９８８）］。Furthermore, the novel peptides of the present invention may contain other physiologically active substances,
The therapeutic effect can be enhanced by using it in combination with chemotherapeutic agents and immune enhancers. For example, IL-2,
When used in combination with tumor necrosis factors (TNFα, TNFβ), interferon-gamma (IFNγ), etc., additive and synergistic effects are observed in immune-enhancing effects and anti-tumor effects. By using it in combination with chemotherapeutic agents, immune enhancers, etc., its antitumor effect can be enhanced and side effects can be reduced. In addition, it is expected to have a synergistic recovery effect on hematopoietic disorders caused by 5-FU when used in combination with G-C8F, and an enhancement effect on colonization ability in the lungs [J, ImmunoL, 140.
3830 (1988)].
（実施例）
以下に実施例を掲記し本発明を更に詳細に説明する。な
お、実施例は本発明を限定するものではない。(Example) The present invention will be described in further detail by referring to Examples below. Note that the examples do not limit the present invention.
実施例１
ペプチド合成及び精製
ペプチドの合成はアプライド・バイオシステムズ社のペ
プチド自動合成機４３０Ａ型を用イテ。Example 1 Peptide synthesis and purification Peptide synthesis was performed using an automatic peptide synthesizer model 430A manufactured by Applied Biosystems.
同社市販の試薬、溶媒類を使用し、同機の標準的な運転
プログラムに従って行った。即ち、α−ｔ−フチルオキ
シカルボニループロモペンジルオキシカルボニルーＬ−
チロシン（０，５ミリモル）が結合したポリスチレン固
相担体を、 ５０％トリフルオロ酢酸−塩化メチレンで
処理し、ｔ−ブチルオキシカルボニル基を除去し、Ｌ−
チロシンのα−アミノ基を遊離させた。これに４当量の
α−ｔ−ブチルオキシカルボニル−Ｌ−ロイシンを２当
量のジシクロへキシルカルボジイミドで活性化し、生成
したＬ−ロイシンの活性エステルを縮合させた。これら
の工程はプログラムに従ってチロシンが合成された。以
下、トリフルオロ酢酸によるｔ−ブチルオキシカルボニ
ルの脱離と、ジシクロへキシルカルボジイミドを活性化
剤とするアミノ酸の縮合をＬ−アスノくラギン。The test was carried out using reagents and solvents commercially available from the company, and according to the standard operating program for the machine. That is, α-t-phthyloxycarbonyl-promopenzyloxycarbonyl-L-
A polystyrene solid support bound to tyrosine (0.5 mmol) was treated with 50% trifluoroacetic acid-methylene chloride to remove the t-butyloxycarbonyl group and
The α-amino group of tyrosine was liberated. To this, 4 equivalents of α-t-butyloxycarbonyl-L-leucine was activated with 2 equivalents of dicyclohexylcarbodiimide, and the resulting active ester of L-leucine was condensed. Tyrosine was synthesized according to the program in these steps. Hereinafter, the elimination of t-butyloxycarbonyl with trifluoroacetic acid and the condensation of amino acids using dicyclohexylcarbodiimide as an activator will be described as L-asuno-ragin.
Ｌ−リジン、Ｌ−グルタミン酸、Ｌ−リジン。L-lysine, L-glutamic acid, L-lysine.
Ｌ−ロイシン、Ｌ−グリシン、Ｌ−ロイシン。L-leucine, L-glycine, L-leucine.
Ｌ−アラニンで自動的にくり返しく但し、Ｎ−末端をｔ
−Ｂｏｃで保護したものを用いているが。Automatically repeats with L-alanine, but the N-terminus is t
-I use one protected with Boc.
リジンｅ−ア□ノ基についてはｐ−クロロベンジルオキ
シカルボニル基で、グルタミン酸のγ−カルボキシル基
についてはベンジル基で保護したものを用いた）、固相
担体上にデカペプチドを合成した。ただし、Ｌ−アスノ
くラギンの活性には４当量のジシクロへキシルカルボジ
イミドと４当量のＮ−ヒドロキシベンゾトリアゾールを
用いた。樹脂から合成ペプチドの脱離は、 ４３０Ａ
ユーザーズマニユアルに記述される操作によりトリフル
オロメタンスルホン酸で処理することによって達成され
、凍結乾燥後４２５■の粗製ペプチドを得た。得られた
粗製ペプチドは逆相カラムを用いた分取用ＨＰＬＣで精
製した。即ち、粗製ペプチドを５０％アセトニトリル水
溶液に溶解し、逆相カラム（センシュー科学。The lysine e-ano group was protected with a p-chlorobenzyloxycarbonyl group, and the γ-carboxyl group of glutamic acid was protected with a benzyl group), and a decapeptide was synthesized on a solid support. However, 4 equivalents of dicyclohexylcarbodiimide and 4 equivalents of N-hydroxybenzotriazole were used for the activity of L-asunochragin. Desorption of synthetic peptides from resin is performed using 430A
This was achieved by treatment with trifluoromethanesulfonic acid according to the procedure described in the user's manual, yielding 425 μ of crude peptide after lyophilization. The obtained crude peptide was purified by preparative HPLC using a reverse phase column. That is, the crude peptide was dissolved in a 50% acetonitrile aqueous solution, and a reverse phase column (Senshu Scientific Co., Ltd.) was used.
ＯＤＳ −Ｈ−５２５１，ｔ ２０ ｘ ２５０ｍｍ）
にかげて、Ａ液（０，１％トリフルオロ酢酸）５％とＢ
液（０，１％トリフルオロ酢酸−７０％アセトニトリル
）９５％との混液からＡ液４０％とＢ液６０％との混液
までのグラジェントで溶出した。ペプチドを含むフラク
ション（島津５ＰＤ−６Ａ装置による２１０μｍの吸収
を分析して同一性を確認した）を集め。ODS-H-5251, t 20 x 250mm)
However, 5% of solution A (0.1% trifluoroacetic acid) and B
Elution was performed using a gradient from a mixture of 95% of solution (0.1% trifluoroacetic acid-70% acetonitrile) to a mixture of 40% of solution A and 60% of solution B. Fractions containing the peptide (identity was confirmed by analyzing absorption at 210 μm using a Shimadzu 5PD-6A instrument) were collected.
減圧下で乾固し、 Ａｌｔ−Ｌｅｕ−Ｇｌｙ−Ｌｙｓ−
Ｇｌｕ−’Ｌｙｓ−Ａｓｎ−Ｌｅｕ−Ｔｙｒのトリフル
オロ酢酸塩として白色の粉末を得た。Dry under reduced pressure to obtain Alt-Leu-Gly-Lys-
A white powder was obtained as the trifluoroacetate of Glu-'Lys-Asn-Leu-Tyr.
得られたペプチドは５０％アセトニトリル水溶液に溶解
し９分析用逆相カラム（センシュー科学、 ０ＤＳ−
Ｈ−１２５１，ｒ４．６Ｘ２５０ｍｍ） にかげ。The obtained peptide was dissolved in a 50% acetonitrile aqueous solution and applied to a 9 analytical reverse phase column (Senshu Kagaku, 0DS-
H-1251, r4.6X250mm) Shadow.
流速０．７ ｍ１７分、Ａ液１００％からＢ液１００％
まで３５分間のグラジェント溶出によるＨＰＬＣによる
単一性を確認した（保持時間約２１分に単一ピークが認
められた）。得られたペプチドを１５０℃。Flow rate 0.7 m 17 minutes, 100% liquid A to 100% liquid B
Uniformity was confirmed by HPLC using gradient elution for up to 35 minutes (a single peak was observed at a retention time of about 21 minutes). The obtained peptide was heated to 150°C.
１時間で６Ｎ塩酸（１％フェノールを含む）によって加
水分解した後、アミノ酸分析によって同定した。分析に
よって得られた残基の値を第１表に示す。After hydrolysis with 6N hydrochloric acid (containing 1% phenol) for 1 hour, it was identified by amino acid analysis. The residue values obtained by the analysis are shown in Table 1.
第 １ 表
実施例 ２
リンパ球活性化因子（ＬＡＦ）活性の測定ＬＡＦ活性「
Ｍｉｚｅｌ、 Ｓ、 Ｂ、 ｅｔ ａｌ、 Ｊ、 Ｉ
ＩｍｍｕｎａＬ、 １２０ 。Table 1 Example 2 Measurement of lymphocyte activating factor (LAF) activity LAF activity
Mizel, S, B, et al, J, I
ImmunaL, 120.
１４９７ （１９７８）］は、マイトジェンによるマウ
ス周線細胞***作用を促進させる生物活性であり、これ
によって実施例１で得られたペプチド（Ｉ）のトリフル
オロ酢酸塩の活性を測定した。1497 (1978)] is a biological activity that promotes the mitogenic action of mouse periclinal cells by mitogens, and the activity of the trifluoroacetate of peptide (I) obtained in Example 1 was measured using this activity.
析液の５０μｌを９６穴平底型マルチプレートに入れ、
それぞれに４−６週令の雄のＣ３Ｈ／ ＨｅＪマウスよ
り採取した脚線細胞（ｌ Ｘ １０７ｃｅｌｌｓ／ｍｔ
）とコンカナバリンＡ（０，６μｇ／ｍ７）を含む細胞
浮遊液を。Put 50 μl of the analysis solution into a 96-well flat-bottom multiplate,
Leg line cells (l x 107 cells/mt) collected from 4-6 week old male C3H/HeJ mice were used for each.
) and a cell suspension containing concanavalin A (0.6 μg/m7).
１００μを加え、５％二酸化炭素を含む３７℃の培養器
内で、４８時間培養した。ついで３Ｈ−チミジンを１．
８５ ｋＢｑ ／２０μｌ／穴加え、さらに１８時間培
養後、セルノ・−ペスタを用いて、細胞をグラスファイ
バーフィルター上に集め、液体シンチレイションカウン
ターでその細胞内に取り込まれた３Ｈ−チミジン量を計
測することにより、脚線細胞の増殖を測定した。100μ was added and cultured for 48 hours in an incubator at 37°C containing 5% carbon dioxide. Then 3H-thymidine was added to 1.
After adding 85 kBq/20 μl/well and culturing for 18 hours, cells were collected on a glass fiber filter using Cerno-Pesta, and the amount of 3H-thymidine incorporated into the cells was measured using a liquid scintillation counter. The proliferation of leg line cells was measured by this method.
第１図は実施例１で得られたペプチド（Ｉ）のトリフル
オロ酢酸塩の脚線細胞の増殖におよぼす影響を示すグラ
フである。縦軸には胸線細胞４゜
に取り込まれた３Ｈ−チミジンの量（ｃｐｍ）として。FIG. 1 is a graph showing the effect of the trifluoroacetate of peptide (I) obtained in Example 1 on the proliferation of leg line cells. The vertical axis shows the amount (cpm) of 3H-thymidine incorporated into thymus cells.
横軸にはこの合成ペプチドの濃度をμｇ／ｍｌとして示
した。合成ペプチドによる３Ｈ−チミジンの正味の取り
込み量で示した。第１図から明らかなように９本発明合
成ペプチド（■）トリフルオロ酢酸塩がマウス胸腺細胞
***活性を有することが判る。The concentration of this synthetic peptide is shown in μg/ml on the horizontal axis. It is shown as the net amount of 3H-thymidine uptake by the synthetic peptide. As is clear from FIG. 1, 9 synthetic peptides of the present invention (■) trifluoroacetate have mouse thymocyte division activity.
第１図は本発明の実施例１で得られたペプチド（Ｉ）の
トリフルオロ酢酸塩が周線細胞の増殖におよぼす影響を
示す図である。FIG. 1 is a diagram showing the influence of the trifluoroacetate of peptide (I) obtained in Example 1 of the present invention on proliferation of pericytes.
Claims (1)
ｙｓ−Ａｓｎ−Ｌｅｕ−Ｔｙｒで表わされるペプチド、
そのエステル、そのアミド、そのアシル化体、又はこれ
らの塩。[Claims] Formula Ala-Leu-Gly-Leu-Lys-Glu-L
A peptide represented by ys-Asn-Leu-Tyr,
Its ester, its amide, its acylated product, or a salt thereof.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2070358A JPH03271300A (en) | 1990-03-19 | 1990-03-19 | New peptide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2070358A JPH03271300A (en) | 1990-03-19 | 1990-03-19 | New peptide |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH03271300A true JPH03271300A (en) | 1991-12-03 |
Family
ID=13429131
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2070358A Pending JPH03271300A (en) | 1990-03-19 | 1990-03-19 | New peptide |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH03271300A (en) |
-
1990
- 1990-03-19 JP JP2070358A patent/JPH03271300A/en active Pending
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