JPH03197493A - Novel peptide - Google Patents
Novel peptideInfo
- Publication number
- JPH03197493A JPH03197493A JP1339080A JP33908089A JPH03197493A JP H03197493 A JPH03197493 A JP H03197493A JP 1339080 A JP1339080 A JP 1339080A JP 33908089 A JP33908089 A JP 33908089A JP H03197493 A JPH03197493 A JP H03197493A
- Authority
- JP
- Japan
- Prior art keywords
- peptide
- present
- ala
- hplc
- synthesis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 66
- 239000002220 antihypertensive agent Substances 0.000 claims abstract description 6
- 239000004480 active ingredient Substances 0.000 claims description 5
- 230000015572 biosynthetic process Effects 0.000 abstract description 15
- 238000003786 synthesis reaction Methods 0.000 abstract description 15
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 abstract description 7
- 229940030600 antihypertensive agent Drugs 0.000 abstract description 4
- -1 butyloxycarbonyl- protected amino Chemical group 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 2
- 238000002360 preparation method Methods 0.000 abstract description 2
- 239000011347 resin Substances 0.000 abstract description 2
- 229920005989 resin Polymers 0.000 abstract description 2
- 150000001875 compounds Chemical class 0.000 abstract 1
- 230000008030 elimination Effects 0.000 abstract 1
- 238000003379 elimination reaction Methods 0.000 abstract 1
- 238000002955 isolation Methods 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- 125000006239 protecting group Chemical group 0.000 abstract 1
- LMBFAGIMSUYTBN-MPZNNTNKSA-N teixobactin Chemical compound C([C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H](CCC(N)=O)C(=O)N[C@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H]1C(N[C@@H](C)C(=O)N[C@@H](C[C@@H]2NC(=N)NC2)C(=O)N[C@H](C(=O)O[C@H]1C)[C@@H](C)CC)=O)NC)C1=CC=CC=C1 LMBFAGIMSUYTBN-MPZNNTNKSA-N 0.000 abstract 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 19
- 238000010828 elution Methods 0.000 description 14
- 125000003275 alpha amino acid group Chemical group 0.000 description 12
- 108090000623 proteins and genes Proteins 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 238000002347 injection Methods 0.000 description 9
- 239000007924 injection Substances 0.000 description 9
- 238000003556 assay Methods 0.000 description 8
- 230000002401 inhibitory effect Effects 0.000 description 7
- 239000012071 phase Substances 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 108010010803 Gelatin Proteins 0.000 description 5
- 229920002472 Starch Polymers 0.000 description 5
- 229920000159 gelatin Polymers 0.000 description 5
- 239000008273 gelatin Substances 0.000 description 5
- 235000019322 gelatine Nutrition 0.000 description 5
- 235000011852 gelatine desserts Nutrition 0.000 description 5
- 239000008107 starch Substances 0.000 description 5
- 235000019698 starch Nutrition 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 230000004531 blood pressure lowering effect Effects 0.000 description 4
- 239000001913 cellulose Substances 0.000 description 4
- 229920002678 cellulose Polymers 0.000 description 4
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 241000251468 Actinopterygii Species 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 102400000344 Angiotensin-1 Human genes 0.000 description 2
- 101800000734 Angiotensin-1 Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- QIAFMBKCNZACKA-UHFFFAOYSA-N N-benzoylglycine Chemical compound OC(=O)CNC(=O)C1=CC=CC=C1 QIAFMBKCNZACKA-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 235000019359 magnesium stearate Nutrition 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000010647 peptide synthesis reaction Methods 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 238000004007 reversed phase HPLC Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000003826 tablet Substances 0.000 description 2
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003276 anti-hypertensive effect Effects 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000012059 conventional drug carrier Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000008057 potassium phosphate buffer Substances 0.000 description 1
- 238000013094 purity test Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000005556 structure-activity relationship Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000011287 therapeutic dose Methods 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
Description
【発明の詳細な説明】
［産業上の利用分野コ
本発明は新規なペプチド、更に詳細には優れた血圧降下
作用を有し、医薬品として有用なペプチド及びその用途
に関する。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel peptide, and more particularly to a peptide that has an excellent antihypertensive effect and is useful as a pharmaceutical, and uses thereof.
［従来の技術及び発明が解決しようとする課題］従来、
魚類の組織中から有効成分を抽出採取することが行われ
ており、そのうちのあるものはすでに医薬品等として実
用に供されている。しかも、まだ未発見の生理活性物質
が存在する可能性が秘められていることから、本発明者
は、魚類の組織中の成分について種々研究をしてきたと
ころ、魚類組織中から抽出単離したオクタペプチドが強
い血圧降下作用を有することを見出し、先に特許出願し
た（特願昭６３−１４３２５８号）。[Prior art and problems to be solved by the invention] Conventionally,
Active ingredients are extracted and collected from the tissues of fish, and some of them are already in practical use as pharmaceuticals. Moreover, since there is a hidden possibility that there are still undiscovered physiologically active substances, the present inventor has conducted various studies on the components in fish tissues, and found that octase isolated from fish tissues. They discovered that the peptide had a strong blood pressure lowering effect and filed a patent application (Japanese Patent Application No. 143258/1983).
しかし、該オクタペプチドよりもさらに優れた血圧降下
作用を示す物質の開発が望まれていた。However, there has been a desire to develop a substance that exhibits a blood pressure lowering effect that is even better than the octapeptide.
［課題を解決するための手段］
そこで、本発明者は該オクタペプチドの各種誘導体を合
成し、アンジオテンシン１転換酵素（ＡＣＥ）阻害活性
を指標として血圧降下作用との構造活性相関につき検討
してきたところ、特定のへキサペプチドが強いＡＣＥ阻
害活性を有し、血圧降下剤として有用であることを見出
し、本発明を完成した。[Means for Solving the Problems] Therefore, the present inventors have synthesized various derivatives of the octapeptide and have investigated the structure-activity relationship with blood pressure lowering effect using angiotensin 1 converting enzyme (ACE) inhibitory activity as an index. discovered that a specific hexapeptide has strong ACE inhibitory activity and is useful as an antihypertensive agent, and completed the present invention.
すなわち、本発明は次式（１）
％式％（）
［式中、Ｒ１はＰｒｏ、Ａｌａ、 Ｈｉｓ、 Ｇｉｎ、
アセチル−Ｇ Ｉｎ。That is, the present invention is based on the following formula (1) % formula % () [wherein R1 is Pro, Ala, His, Gin,
Acetyl-G In.
Ｐｈｅ、 Ａｒｇ、アセチル−ＩｌｅまたはＭｅｔを示
し、Ｒ２はＩｌｅまたはＶａｌを示し、ＲａはＡｌａ、
ＬｙｓまたはＡｓｐを示す］
で表わされるヘキサペプチド及びこれを有効成分とする
血圧降下剤を提供するものである。Phe, Arg, acetyl-He or Met, R2 represents He or Val, Ra is Ala,
Lys or Asp] The present invention provides a hexapeptide represented by the following formula and an antihypertensive agent containing the hexapeptide as an active ingredient.
本発明のペプチド（１）は、通常のペプチド合成法に従
って、構成アミノ酸をペプチド結合させることにより製
造することができる。The peptide (1) of the present invention can be produced by peptide bonding the constituent amino acids according to a conventional peptide synthesis method.
ペプチド合成法としては、液相法または固相法のいずれ
の合成法を用いてもよく、また市販のペプチド合成機を
用いることもできる。ペプチド（１）は、通常ペプチド
分離に用いられる逆相カラムクロマトグラフィーにより
精製単離できる。As the peptide synthesis method, either a liquid phase method or a solid phase method may be used, and a commercially available peptide synthesizer may also be used. Peptide (1) can be purified and isolated by reverse-phase column chromatography, which is commonly used for peptide separation.
［作用］
本発明のペプチド（１）の血圧降下作用をＣｕｓｈｍａ
ｎｎ ＆ Ｃｈｅｕｎｇの方法（Ｂｉｏｃｈｅｍ、 Ｐ
ｈａｒｍａｃｏｌ、。[Action] The blood pressure lowering effect of the peptide (1) of the present invention can be demonstrated by Cushma.
nn &Cheung's method (Biochem, P
harmacol,.
２０、１６３７（１９７１））を用いてアンジオテンシ
ン１転換酵素（ＡＣＥ）阻害活性として測定した。20, 1637 (1971)) as angiotensin 1 converting enzyme (ACE) inhibitory activity.
ＡＣＥ １〜１０ｍＵ、基質旧ｐ−Ｈｉｓ−Ｌｅｕ （
ペプチド研究新製） ２．５ｍＭ、リン酸カリウム緩衝
液（ｐＨ８、３）１００ｍＭ及び食塩３００ｍＭからな
る溶液に、試料の所定量を加え、この混合物（０，２５
ｍ１＋）を３７°Ｃで３０分間インキュベートした後、
ＩＮ塩酸０．２５１ｎｆｌを加えて酵素反応を停止した
。酢酸エチル１．５ｍ１ｌを加え、１０秒間強く撹拌し
、生成した馬尿酸を抽出した。ACE 1-10 mU, substrate old p-His-Leu (
A predetermined amount of the sample was added to a solution consisting of 2.5mM (Peptide Research Shinsei), 100mM of potassium phosphate buffer (pH 8,3), and 300mM of sodium chloride, and the mixture (0,25
m1+) at 37°C for 30 min.
The enzyme reaction was stopped by adding 0.251 nfl of IN hydrochloric acid. 1.5 ml of ethyl acetate was added and stirred strongly for 10 seconds to extract the produced hippuric acid.
酢酸エチル層１ｍｌから溶媒を留去し、残渣を蒸留水１
ｍｌに溶解し、２２８ｎｍの吸光度を測定した（ＯＤｓ
）。The solvent was distilled off from 1 ml of ethyl acetate layer, and the residue was dissolved in distilled water.
ml and measured the absorbance at 228 nm (ODs
).
インキュベート前に、あらかじめＩＮ塩酸を加えたもの
をブランクとしく ０Ｄｓｂα）、吸光度の差（ＯＤｓ
−ＯＤｓＭ ）を求めた。また試料を添加しなかったも
のをコントロール群（ＯＤｃ）とし、同様にＯＤｃ −
０Ｄｃｂｌｌを求めた。ＡＣＥ阻害活性は次の式によっ
て算出した。Before incubation, add IN hydrochloric acid in advance as a blank (0Dsbα), and measure the difference in absorbance (ODs
-ODsM) was determined. In addition, the group to which no sample was added was defined as a control group (ODc), and similarly ODc −
0Dcbll was determined. ACE inhibitory activity was calculated using the following formula.
０ＤＳ−〇ＤＳｂｒｌ）ｘｌｏ。0DS-〇DSbrl)xlo.
ＡＣＥ阻害活性（％）−（１０Ｄｃ−ＯＤｃＭ結果を第
１表に示す。ACE inhibitory activity (%)-(10Dc-ODcM results are shown in Table 1.
以下余白
第１表
また、本発明のペプチド（１−Ａ）〜（Ｉ−Ｌ）は、そ
の有効投与量の過剰量である５０ｍｇ／ｋｇをラットに
静脈内投与した場合においても、異常が認められなかっ
た。Table 1 in the margin below: Furthermore, when the peptides (1-A) to (IL) of the present invention were intravenously administered to rats at 50 mg/kg, which is an excess of their effective dose, no abnormalities were observed. I couldn't.
叙上の如く、本発明の（１）式で表わされるペプチドは
、いずれも優れたＡＣＥ阻害活性を示し、血圧降下剤と
して有用である。As mentioned above, all of the peptides represented by formula (1) of the present invention exhibit excellent ACE inhibitory activity and are useful as antihypertensive agents.
その用法としては静脈内投与または経口投与か好ましい
。As for its usage, intravenous administration or oral administration is preferred.
使用可能な剤形としては、注射剤またはカプセル剤、錠
剤、粉末剤、顆粒剤等の経口投与剤がある。Usable dosage forms include injections or oral preparations such as capsules, tablets, powders, and granules.
本発明のペプチド（１）の投与量は、患者の症状の程度
、投与方法等によりその最適量はかなり異なるが、成人
１日当りの治療量は０．１〜１０００ ｍｇである。The optimal dose of the peptide (1) of the present invention varies considerably depending on the severity of the patient's symptoms, administration method, etc., but the therapeutic dose for an adult is 0.1 to 1000 mg per day.
また、本発明のペプチド（１）は、慣用の任意の製薬用
担体、基剤または賦形剤と共に慣用の方法にて医薬用製
剤とすることができる。経口投与剤としては脂溶性のカ
プセル剤、錠剤、粉末剤また −
は顆粒剤として、注射剤としては水溶性注射剤または凍
結乾燥粉末剤として使用するのが好ましい。Moreover, the peptide (1) of the present invention can be made into a pharmaceutical preparation by a conventional method together with any conventional pharmaceutical carrier, base, or excipient. For oral administration, it is preferably used as a lipophilic capsule, tablet, powder, or granule, and for injection, it is preferably used as a water-soluble injection or lyophilized powder.
賦形剤としては慣用の物質が使用可能であるが、ヒト血
清アルブミン、ショ糖、ゼラチン、デンプン、またはポ
リエチレングリコール等を使用することが好ましい。Although conventional substances can be used as excipients, it is preferable to use human serum albumin, sucrose, gelatin, starch, polyethylene glycol, or the like.
［実施例］
以下、実施例を挙げて本発明を更に説明するが、本発明
はこれら実施例に限定されるものではない。[Examples] Hereinafter, the present invention will be further explained with reference to Examples, but the present invention is not limited to these Examples.
実施例１ ペプチド（Ｉ −Ａ）の合成全自動ペプチド
シンセサイザー モデル４３０−Ａ（アブライトバイオ
システムズ社製）により、ブチロキシカルボニル保護ア
ミノ酸（Ｂｏｃ−アミノ酸）を用いたＭｅｒｒｉｆｉｅ
ｌｄ法にて合成した後、６０％トリフルオロ酢酸（ＴＦ
Ａ）／ＣＨ２Ｃｌ１２を用いて脱保護基及びＨＦガスに
よる樹脂からの脱離を行なった。Example 1 Synthesis of Peptide (I-A)Merrifie synthesis using a butyroxycarbonyl-protected amino acid (Boc-amino acid) using a fully automatic peptide synthesizer model 430-A (manufactured by Abrite Biosystems)
After synthesis using the ld method, 60% trifluoroacetic acid (TF
A) /CH2Cl12 was used for deprotection and removal from the resin using HF gas.
得られたペプチドを、センシューＯＤＳ ２１５１）１
（センシュー科学■製）カラムを用い、０１％ＴＦＡ→
７０％アセトニトリル１０１％ＴＦＡの直線濃度勾配（
６０分）、流速１ 、０ｙｎｌＪ ／ｍｔｎ、検出ＵＶ
２８０ｎｍの溶出条件で逆相ＨＰＬＣに付し、本発明の
ペプチド（Ｉ−Ａ）を精製した。The obtained peptide was added to Senshu ODS 2151)1
Using a column (manufactured by Senshu Kagaku ■), 01% TFA→
Linear concentration gradient of 70% acetonitrile 101% TFA (
60 minutes), flow rate 1, 0ynlJ/mtn, detection UV
The peptide (IA) of the present invention was purified by reverse phase HPLC under the elution condition of 280 nm.
得られたペプチドはＨＰＬＣにて純度を検定したが、第
１図に示す通り、単一成分であることが確認された。The purity of the obtained peptide was tested by HPLC, and as shown in FIG. 1, it was confirmed that it was a single component.
また、気相プロテインシークエンサー モデル４７７Ａ
（アプライドバイオシステムズ社製）にてアミノ酸配
列が次式であることを確認した。In addition, gas phase protein sequencer model 477A
(manufactured by Applied Biosystems), the amino acid sequence was confirmed to be the following formula.
Ｐｒｏ−Ｔｈｒ−Ｈｉｓ−１１ｅ−Ａｌａ−Ｔｒｐ
（Ｉ −Ａ ）実施例２ ペプチド（１−Ｂ）の合
成
実施例１と同様にして、本発明ペプチド（１−Ｂ）を合
成し、ＨＰＬＣで純度検定（第２図）を行い、気相プロ
ティンシークエンサーモデル４７７Ａでアミノ酸配列が
次式であることを確認した。Pro-Thr-His-11e-Ala-Trp
(I-A) Example 2 Synthesis of Peptide (1-B) Peptide (1-B) of the present invention was synthesized in the same manner as in Example 1, and purity assay (Fig. 2) was performed by HPLC. The amino acid sequence was confirmed to be of the following formula using a protein sequencer model 477A.
Ｐｒｏ−Ｔｈｒ−■１ｓ−Ｖａｌ−Ａｌａ−Ｔｒｐ
（Ｉ −Ｂ ）実施例３ ペプチド（Ｉ−Ｃ）の合
成
実施例１と同様にして、本発明ペプチド（ＩＣ）を合成
した。Pro-Thr-■1s-Val-Ala-Trp
(I-B) Example 3 Synthesis of Peptide (IC) The peptide (IC) of the present invention was synthesized in the same manner as in Example 1.
得られたペプチドを、ＹＭＣ−Ｐａｃｋ Ｒ−ＯＤＳ−
５（山村化学（株）製）カラムを用い、０１％ＴＦＡ−
７０％アセトニトリル１０１％ＴＦＡの直線濃度勾配（
２５分）、流速１．０ｍｆｌ／ｍｉｎ、検出ＵＶ２１０
ｎｍの溶出条件で逆相ＨＰＬＣに付し、本発明のペプチ
ド（Ｉ−Ｃ）を精製した。The obtained peptide was transferred to YMC-Pack R-ODS-
5 (manufactured by Yamamura Kagaku Co., Ltd.) using 01% TFA-
Linear concentration gradient of 70% acetonitrile 101% TFA (
25 minutes), flow rate 1.0 mfl/min, detection UV 210
The peptide (IC) of the present invention was purified by reverse phase HPLC under the elution conditions of nm.
得られたペプチドはＨＰＬＣにて純度を検定したが、第
３図に示す通り、単一成分であることが確認された。The purity of the obtained peptide was tested by HPLC, and as shown in FIG. 3, it was confirmed that it was a single component.
また、気相プロテインシークエンサー モデル４７７Ａ
でアミノ酸配列が次式であることを確認した。In addition, gas phase protein sequencer model 477A
The amino acid sequence was confirmed to be the following formula.
Ｐｒｏ−Ｔｈｒ−Ｈｉｓ−１１ｅ−Ｌｙｓ−Ｔｒｐ
（ｒ −Ｃ）実施例４ ペプチド（Ｉ ＝Ｄ）の合
成実施例１と同様にして、本発明ペプチド（Ｉ−Ｄ）を
合成し、ＨＰＬＣで純度検定（第４図）を行い、気相プ
ロティンシークエンサーモデル４７７Ａでアミノ酸配列
が次式であることを確認した。Pro-Thr-His-11e-Lys-Trp
(r-C) Example 4 Synthesis of peptide (I = D) The peptide (I-D) of the present invention was synthesized in the same manner as in Example 1, and the purity assay (Fig. 4) was performed by HPLC. The amino acid sequence was confirmed to be of the following formula using a protein sequencer model 477A.
Ａｌａ−Ｔｈｒ−Ｈｉｓ−１１ｅ−Ａｌａ−Ｔｒｐ
（１−Ｄ ）実施例５ ペプチド（Ｉ−Ｅ）の合成
実施例３と同様にして、本発明ペプチド（ＩＥ）を合成
し、ＨＰＬＣで純度検定（第５図）を行い、気相プロテ
ィンシークエンサーモデル４７７Ａでアミノ酸配列が次
式であることを確認した。Ala-Thr-His-11e-Ala-Trp
(1-D) Example 5 Synthesis of Peptide (IE) The peptide (IE) of the present invention was synthesized in the same manner as in Example 3, and the purity assay was performed by HPLC (Fig. 5). It was confirmed that the amino acid sequence of model 477A was as follows.
Ｈｉｓ−Ｔｈｒ−Ｈｉｓ−１１ｅ−Ａｌａ−Ｔｒｐ
（Ｉ −Ｅ ）実施例６ ペプチド（１−Ｆ）の合
成
実施例１と同様にして、本発明ペプチド（ＩＦ）を合成
し、［（ＰＬＣで純度検定（第６図）を行い、気相プロ
ティンシークエンサーモデル４７７Ａでアミノ酸配列が
次式であることを確認した。His-Thr-His-11e-Ala-Trp
(I-E) Example 6 Synthesis of Peptide (1-F) The peptide (IF) of the present invention was synthesized in the same manner as in Example 1, and the purity assay (FIG. 6) was performed using PLC. The amino acid sequence was confirmed to be of the following formula using a protein sequencer model 477A.
Ｇｉｎ−Ｔｈｒ−Ｈｉｓ−１１ｅ−Ａｌａ−Ｔｒｐ
（Ｉ −Ｆ ）実施例７ ペプチド（ｔ−Ｇ）の合
成
実施例１と同様にして、本発明ペプチド（１−Ｇ）を合
成し、ＨＰＬＣで純度検定（第７図）を行い、気相プロ
ティンシークエンサーモデル４７７Ａでアミノ酸配列が
次式であることを確認した。Gin-Thr-His-11e-Ala-Trp
(I-F) Example 7 Synthesis of peptide (t-G) The peptide (1-G) of the present invention was synthesized in the same manner as in Example 1, and the purity assay (Fig. 7) was performed by HPLC. The amino acid sequence was confirmed to be of the following formula using a protein sequencer model 477A.
Ａｃ−Ｇｌｎ−Ｔｈｒ−Ｈｉｓ−１１ｅ−Ａｌａ−Ｔｒ
ｐ （Ｉ −Ｇ ）実施例８ ペプチド（ｒ−Ｈ）
の合成
実施例３と同様にして、本発明ペプチド（ＩＨ）を合成
し、ＨＰＬＣで純度検定（第８図）を行い、気相プロテ
ィンシークエンサーモデル４７７Ａでアミノ酸配列が次
式であることを確認した。Ac-Gln-Thr-His-11e-Ala-Tr
p (I-G) Example 8 Peptide (r-H)
The peptide (IH) of the present invention was synthesized in the same manner as in Synthesis Example 3, and the purity assay was performed by HPLC (Figure 8), and the amino acid sequence was confirmed to be the following formula using a gas phase protein sequencer model 477A. .
Ｐｈｅ−Ｔｈｒ−Ｉｔｓ−１１ｅ−Ａｌａ−Ｔｒｐ
（１−Ｈ）実施例９ ペプチド（１−１）の合成
９−
０
実施例１と同様にして、本発明ペプチド（Ｉ■）を合成
し、ＨＰＬＣで純度検定（第９図）を行い、気相プロテ
ィンシークエンサーモデル４７７Ａでアミノ酸配列が次
式であることを確認した。Phe-Thr-Its-11e-Ala-Trp
(1-H) Example 9 Synthesis of peptide (1-1) 9-0 The peptide (I) of the present invention was synthesized in the same manner as in Example 1, and the purity assay (Fig. 9) was performed by HPLC. The amino acid sequence was confirmed to be the following formula using a gas phase protein sequencer model 477A.
Ａｒｇ−Ｔｈｒ−Ｈｉｓ−１１ｅ−Ａｌａ−Ｔｒｐ
（Ｉ−１）実施例１０ ペプチド（Ｉ−Ｊ）の合
成実施例１と同様にして、本発明ペプチド（ＩＪ）を合
成し、ＨＰＬＣで純度検定（第１０図）を行い、気相プ
ロティンシークエンサーモデル４７７Ａでアミノ酸配列
が次式であることを確認した。Arg-Thr-His-11e-Ala-Trp
(I-1) Example 10 Synthesis of Peptide (I-J) The peptide (IJ) of the present invention was synthesized in the same manner as in Example 1, and the purity assay was performed by HPLC (Fig. 10). It was confirmed that the amino acid sequence of model 477A was as follows.
Ａｃ−１１ｅ−Ｔｈｒ−Ｈｉｓ−１１ｅ−Ａｌａ−Ｔｒ
ｐ （Ｉ −Ｊ ）実施例１１ ペプチド（１−
Ｋ）の合成実施例１と同様にして、本発明ペプチド（■
Ｋ）を合成し、ＨＰＬＣで純度検定（第１１図）を行い
、気相プロティンシークエンサーモデル４７７Ａでアミ
ノ酸配列が次式であることを確認した。Ac-11e-Thr-His-11e-Ala-Tr
p (I-J) Example 11 Peptide (1-
Synthesis of the peptide of the present invention (■
K) was synthesized, a purity test was performed by HPLC (Fig. 11), and the amino acid sequence was confirmed to be the following formula using a gas phase protein sequencer model 477A.
Ｍｅｔ−Ｔｈｒ−Ｈｉｓ−１１ｅ−Ａｌａ−Ｔｒｐ
（Ｉ −Ｋ ）実施例１２ ペプチド（１−Ｌ）
の合成実施例１と同様にして、本発明ペプチド（■Ｌ）
を合成し、ＨＰＬＣで純度検定（第１２図）を行い、気
相プロティンシークエンサーモデル４７７Ａでアミノ酸
配列が次式であることを確認した。Met-Thr-His-11e-Ala-Trp
(I-K) Example 12 Peptide (1-L)
The peptide of the present invention (■L) was prepared in the same manner as in Synthesis Example 1.
was synthesized, the purity was tested by HPLC (Fig. 12), and the amino acid sequence was confirmed to be the following formula using a gas phase protein sequencer model 477A.
Ｐｒｏ−Ｔｈｒ−Ｈｉｓ−１１ｅ−Ａｓｐ−Ｔｒｐ
（Ｉ−Ｌ ）実施例１３ 水溶性注射剤
実施例１で得られたペプチド（Ｉ ’−Ａ ）１００ｍ
ｇを、注射用蒸留水にて調製した０１４Ｍ塩化ナトリウ
ム含有００１Ｍリン酸緩衝液（ｐＨ７，０） １００ｒ
ｒｆｌに溶解した。水液をメンブランフィルタ−を使用
して無菌的に濾過し、濾液をガラス容器に１ｍｌずつ充
填して密封し、水溶性注射剤とした。Pro-Thr-His-11e-Asp-Trp
(IL) Example 13 Water-soluble injection Peptide (I'-A) obtained in Example 1 100m
g, 100r of 001M phosphate buffer containing 014M sodium chloride (pH 7,0) prepared with distilled water for injection
Dissolved in rfl. The aqueous solution was aseptically filtered using a membrane filter, and each 1 ml of the filtrate was filled into a glass container and sealed to obtain a water-soluble injection.
実施例１４ 水溶性注射剤
実施例８で得られたペプチド（Ｉ −Ｈ）１００ｍｇを
、注射用蒸留水にて調製した０、１．４Ｍ塩化ナトリウ
ム含有００１Ｍリン酸緩衝液（ｐＨ７，０） １００雄
に溶解した。水液をメンブランフィルタ−を使用して無
菌的に濾過し、濾液をガラス容器に１ ｒｒａずつ充填
して密封し、水溶性注射剤とした。Example 14 Water-soluble injection 100 mg of the peptide (I-H) obtained in Example 8 was prepared in distilled water for injection in 001M phosphate buffer containing 0 and 1.4M sodium chloride (pH 7.0) 100 Dissolved in males. The aqueous solution was aseptically filtered using a membrane filter, and the filtrate was filled into glass containers of 1 rra each and sealed to obtain a water-soluble injection.
実施例１５ 錠剤
実施例３で得られたペプチド（■−Ｃ）２０ｇデンプン
１００ｇゼラチン
結晶セルロース
５ｇ
０ｇ
計
１９０ｇ上記成分から本発明のペプチド（Ｉ −Ｃ）２
０ｍｇを含む錠剤１０００個を得た。Example 15 Tablet Peptide (■-C) obtained in Example 3 20g Starch 100g Gelatin Crystalline cellulose 5g 0g Total
190g Peptide (I-C)2 of the present invention from the above ingredients
1000 tablets containing 0 mg were obtained.
ペプチド（１−Ｃ）及びデンプンをゼラチン水溶液と混
合した後乾燥し、粉砕して細がい粉末とした。これに結
晶セルロース、次いでステアリン酸マグネシウムを混和
した後、錠剤機にかけて有効成分２０■を含む錠剤１０
００個を得た。Peptide (1-C) and starch were mixed with an aqueous gelatin solution, dried, and ground into fine powder. After mixing this with crystalline cellulose and then magnesium stearate, it was passed through a tablet machine into 10 tablets containing 20 cm of the active ingredient.
Obtained 00 pieces.
実施例１６ 錠剤
実施例５で得られたペプチド（Ｉ−Ｅ） ２０ｇデン
プン １００ｇゼラチン
１５ｇ結晶セルロース
５０ｅ計
１９０ｇ上記成分
から本発明のペプチド（Ｉ−Ｅ）２０■を含む錠剤１０
００個を得た。Example 16 Tablet Peptide (I-E) obtained in Example 5 20g starch 100g gelatin 15g crystalline cellulose 50e total
190g 10 tablets containing 20■ of the peptide (I-E) of the present invention from the above ingredients
Obtained 00 pieces.
ペプチド（Ｉ−Ｅ）及びデンプンをゼラチン水溶液と混
合した後乾燥し、粉砕して細かい粉末とした。これに結
晶セルロース、次いでステアリン酸マグネシウムを混和
した後、錠剤機にかけて有効成分２０ｍｇを含む錠剤１
０００個を得た。Peptide (I-E) and starch were mixed with an aqueous gelatin solution, dried and ground into a fine powder. After mixing this with crystalline cellulose and then magnesium stearate, it was passed through a tablet machine to form a tablet containing 20 mg of the active ingredient.
Obtained 000 pieces.
［発明の効果］
本発明のへキサペプチドは優れたＡＣＥ阻害活性を有し
、血圧降下剤として有用である。[Effects of the Invention] The hexapeptide of the present invention has excellent ACE inhibitory activity and is useful as a hypotensive agent.
第１図は、本発明のペプチド（Ｉ −Ａ）をＨＰＬＣに
付したときの溶出パターンを、第２図は、本発明のペプ
チド（Ｉ−Ｂ）をＨＰＬＣに付したときの溶出パターン
を、第３図は、本発明のペプチド（Ｉ−Ｃ）をＨＰＬＣ
に付したときの溶出パターンを、第４図は、本発明のペ
プチド（１−Ｄ）をＨＰＬＣに付したときの溶出パター
ンを、第５図は、本発明のペプチド（Ｉ−Ｅ）をＨＰＬ
Ｃに付したときの溶出パターンを、第６図は、本発明の
ペプチド（Ｉ−Ｆ）をＨＰＬＣに付したときの溶出パタ
ーンを、第７図は、本発明のペプチド（１−Ｇ）をＨＰ
ＬＣに付したときの溶出パター３
４−
ンを、第８図は、本発明のペプチド（１−Ｈ）をＨＰＬ
Ｃに付したときの溶出パターンを、第９図は、本発明の
ペプチド（Ｉ−１）を肝ＬＣに付したときの溶出パター
ンを、第１０図は、本発明のペプチド（ＩＪ）をＨＰＬ
Ｃに付したときの溶出パターンを、第１１図は、本発明
のペプチド（１−Ｋ）を肝ＬＣに付したときの溶出パタ
ーンを、第１２図は、本発明のペプチド（■−Ｌ）をＩ
（ＰＬＣに付したときの溶出パターンを示す図面である
。
以 上Figure 1 shows the elution pattern when the peptide (I-A) of the present invention was subjected to HPLC, and Figure 2 shows the elution pattern when the peptide (I-B) of the present invention was subjected to HPLC. Figure 3 shows the HPLC analysis of the peptide (I-C) of the present invention.
Figure 4 shows the elution pattern when the peptide (1-D) of the present invention was subjected to HPLC, and Figure 5 shows the elution pattern when the peptide (I-E) of the present invention was subjected to HPLC.
Figure 6 shows the elution pattern when the peptide of the present invention (IF) was subjected to HPLC, and Figure 7 shows the elution pattern when the peptide of the present invention (1-G) was subjected to HPLC. HP
Figure 8 shows the elution pattern when the peptide (1-H) of the present invention was subjected to LC.
Figure 9 shows the elution pattern when the peptide (I-1) of the present invention was subjected to liver LC, and Figure 10 shows the elution pattern when the peptide (IJ) of the present invention was subjected to HPLC.
FIG. 11 shows the elution pattern when the peptide of the present invention (1-K) was subjected to liver LC, and FIG. 12 shows the elution pattern when the peptide of the present invention (1-K) was subjected to liver LC. I
(This is a drawing showing the elution pattern when subjected to PLC.
Claims (2)
I ）［式中、Ｒ＾１はＰｒｏ、Ａｌａ、Ｈｉｓ、Ｇｌｎ
、アセチル−Ｇｌｎ、Ｐｈｅ、Ａｒｇ、アセチル−Ｉｌ
ｅまたはＭｅｔを示し、Ｒ＾２はＩｌｅまたはＶａｌを
示し、Ｒ＾３はＡｌａ、ＬｙｓまたはＡｓｐを示す］ で表わされるペプチド。(1) The following formula (I) R^1-Thr-His-R^2-R^3-Trp(
I) [where R^1 is Pro, Ala, His, Gln
, acetyl-Gln, Phe, Arg, acetyl-Il
e or Met, R^2 represents He or Val, and R^3 represents Ala, Lys or Asp].
下剤。(2) A hypotensive agent containing the peptide according to claim 1 as an active ingredient.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1339080A JP2627672B2 (en) | 1989-04-04 | 1989-12-27 | New peptide |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1-85276 | 1989-04-04 | ||
| JP8527689 | 1989-04-04 | ||
| JP1339080A JP2627672B2 (en) | 1989-04-04 | 1989-12-27 | New peptide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03197493A true JPH03197493A (en) | 1991-08-28 |
| JP2627672B2 JP2627672B2 (en) | 1997-07-09 |
Family
ID=26426291
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1339080A Expired - Fee Related JP2627672B2 (en) | 1989-04-04 | 1989-12-27 | New peptide |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2627672B2 (en) |
-
1989
- 1989-12-27 JP JP1339080A patent/JP2627672B2/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| BIOCHEM.BIOPHYS.RES.COMMUN.=1988 * |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2627672B2 (en) | 1997-07-09 |
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