JPH04230400A - Polypeptide derivative having vip-like activity and its use - Google Patents
Polypeptide derivative having vip-like activity and its useInfo
- Publication number
- JPH04230400A JPH04230400A JP2408425A JP40842590A JPH04230400A JP H04230400 A JPH04230400 A JP H04230400A JP 2408425 A JP2408425 A JP 2408425A JP 40842590 A JP40842590 A JP 40842590A JP H04230400 A JPH04230400 A JP H04230400A
- Authority
- JP
- Japan
- Prior art keywords
- vip
- activity
- fermentation method
- polypeptide
- peptide
- 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
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 32
- 230000000694 effects Effects 0.000 title claims abstract description 23
- 229920001184 polypeptide Polymers 0.000 title claims description 21
- 102000004196 processed proteins & peptides Human genes 0.000 title claims description 21
- UKAUYVFTDYCKQA-UHFFFAOYSA-N -2-Amino-4-hydroxybutanoic acid Natural products OC(=O)C(N)CCO UKAUYVFTDYCKQA-UHFFFAOYSA-N 0.000 claims abstract description 18
- UKAUYVFTDYCKQA-VKHMYHEASA-N L-homoserine Chemical compound OC(=O)[C@@H](N)CCO UKAUYVFTDYCKQA-VKHMYHEASA-N 0.000 claims abstract description 10
- 239000003814 drug Substances 0.000 claims abstract description 8
- 201000001881 impotence Diseases 0.000 claims abstract description 6
- 229940124630 bronchodilator Drugs 0.000 claims abstract description 5
- 229940124597 therapeutic agent Drugs 0.000 claims abstract description 4
- 125000000539 amino acid group Chemical group 0.000 claims description 5
- QJPWUUJVYOJNMH-VKHMYHEASA-N L-homoserine lactone Chemical compound N[C@H]1CCOC1=O QJPWUUJVYOJNMH-VKHMYHEASA-N 0.000 claims description 3
- STGQSBKUYSPPIG-CIUDSAMLSA-N His-Ser-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CN=CN1 STGQSBKUYSPPIG-CIUDSAMLSA-N 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 19
- 239000000126 substance Substances 0.000 abstract description 9
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 abstract description 6
- 150000001413 amino acids Chemical class 0.000 abstract description 6
- 238000000855 fermentation Methods 0.000 abstract description 6
- 230000004151 fermentation Effects 0.000 abstract description 6
- 229930182817 methionine Natural products 0.000 abstract description 6
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 abstract description 5
- 229940079593 drug Drugs 0.000 abstract description 4
- 239000000813 peptide hormone Substances 0.000 abstract description 3
- 150000001408 amides Chemical class 0.000 abstract description 2
- 108010003205 Vasoactive Intestinal Peptide Proteins 0.000 abstract 5
- 102400000015 Vasoactive intestinal peptide Human genes 0.000 abstract 5
- 235000019454 L-leucine Nutrition 0.000 abstract 2
- 239000004395 L-leucine Substances 0.000 abstract 2
- 229940024606 amino acid Drugs 0.000 abstract 2
- 210000004899 c-terminal region Anatomy 0.000 abstract 2
- 229960003136 leucine Drugs 0.000 abstract 2
- 102000015731 Peptide Hormones Human genes 0.000 abstract 1
- 108010038988 Peptide Hormones Proteins 0.000 abstract 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 abstract 1
- 238000004519 manufacturing process Methods 0.000 description 5
- 230000001766 physiological effect Effects 0.000 description 5
- 241000700199 Cavia porcellus Species 0.000 description 4
- 230000009435 amidation Effects 0.000 description 4
- 238000007112 amidation reaction Methods 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 229960002101 secretin Drugs 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 206010006482 Bronchospasm Diseases 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- 108010086019 Secretin Proteins 0.000 description 2
- 102100037505 Secretin Human genes 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 208000006673 asthma Diseases 0.000 description 2
- 210000000621 bronchi Anatomy 0.000 description 2
- 230000007885 bronchoconstriction Effects 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 150000002596 lactones Chemical group 0.000 description 2
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
- 230000002040 relaxant effect Effects 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- OWMZNFCDEHGFEP-NFBCVYDUSA-N secretin human Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(N)=O)[C@@H](C)O)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)C1=CC=CC=C1 OWMZNFCDEHGFEP-NFBCVYDUSA-N 0.000 description 2
- 210000002460 smooth muscle Anatomy 0.000 description 2
- MTJGVAJYTOXFJH-UHFFFAOYSA-N 3-aminonaphthalene-1,5-disulfonic acid Chemical compound C1=CC=C(S(O)(=O)=O)C2=CC(N)=CC(S(O)(=O)=O)=C21 MTJGVAJYTOXFJH-UHFFFAOYSA-N 0.000 description 1
- LVRVABPNVHYXRT-BQWXUCBYSA-N 52906-92-0 Chemical compound C([C@H](N)C(=O)N[C@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(O)=O)C(C)C)C1=CC=CC=C1 LVRVABPNVHYXRT-BQWXUCBYSA-N 0.000 description 1
- 102000051325 Glucagon Human genes 0.000 description 1
- 108060003199 Glucagon Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- LRQKBLKVPFOOQJ-YFKPBYRVSA-N L-norleucine Chemical compound CCCC[C@H]([NH3+])C([O-])=O LRQKBLKVPFOOQJ-YFKPBYRVSA-N 0.000 description 1
- 101800002372 Motilin Proteins 0.000 description 1
- 102000002419 Motilin Human genes 0.000 description 1
- 108010047386 Pituitary Hormones Proteins 0.000 description 1
- 102000006877 Pituitary Hormones Human genes 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 210000000941 bile Anatomy 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000004531 blood pressure lowering effect Effects 0.000 description 1
- 230000003523 bronchorelaxing effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 210000005226 corpus cavernosum Anatomy 0.000 description 1
- 210000001198 duodenum Anatomy 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000027119 gastric acid secretion Effects 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- MASNOZXLGMXCHN-ZLPAWPGGSA-N glucagon Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 MASNOZXLGMXCHN-ZLPAWPGGSA-N 0.000 description 1
- 229960004666 glucagon Drugs 0.000 description 1
- 230000004116 glycogenolysis Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
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- ITMCEJHCFYSIIV-UHFFFAOYSA-N triflic acid Chemical compound OS(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【０００１】0001
【産業上の利用分野】本発明は、ＶＩＰ様活性を有する
新規なポリペプチド誘導体及びその用途に係わる。本発
明物質は医薬品成分として殊に喘息及びインポテンツの
治療に用いることができる。TECHNICAL FIELD The present invention relates to novel polypeptide derivatives having VIP-like activity and uses thereof. The substances according to the invention can be used as pharmaceutical ingredients, in particular for the treatment of asthma and impotence.
【０００２】0002
【従来技術】ＶＩＰ（Ｖａｓｏａｃｔｉｖｅ Ｉｎｔ
ｅｓｔｉｎａｌ Ｐｏｌｙｐｅｐｔｉｄｅ）は、Ｓａ
ｉｄ ＆ Ｍｕｔｔにより１９７０年にブタ十二指
腸よりセクレチンを精製抽出する段階の副分画から精製
された、２８個のアミノ酸からなるペプチドホルモンで
ある。１９７４年にアミノ酸の一次構造が明らかになり
、セクレチン、グルカゴン等と類似していることからグ
ルカゴン−セクレチンファミリ−に属するペプチドホル
モンであるとされるに至っている。ＶＩＰは消化管以外
に神経系にも広く存在が認められており、その生理作用
も多彩で、強い血管拡張・血圧降下作用、平滑筋弛緩作
用、腸液分泌促進、膵液・胆汁分泌促進、胃酸分泌抑制
、グリコ−ゲン分解、種々の下垂体ホルモン分泌促進や
生殖器の血流調節、気管支拡張作用等を有することが明
らかになっている。[Prior art] VIP (Vasoactive Int.
estinal Polypeptide) is Sa
It is a peptide hormone consisting of 28 amino acids that was purified from a sub-fraction during the purification and extraction of secretin from pig duodenum in 1970 by ID & Mutt. The primary structure of amino acids was clarified in 1974, and because it is similar to secretin, glucagon, etc., it has come to be considered a peptide hormone belonging to the glucagon-secretin family. In addition to the gastrointestinal tract, VIP is widely recognized to exist in the nervous system, and its physiological effects are diverse, including strong vasodilation and blood pressure lowering effects, smooth muscle relaxing effects, promotion of intestinal fluid secretion, promotion of pancreatic juice and bile secretion, and gastric acid secretion. It has been revealed that it suppresses glycogenolysis, promotes secretion of various pituitary hormones, regulates blood flow in the reproductive organs, and dilates the bronchodilator.
【０００３】これらの種々多様な薬理作用の中でも、Ｖ
ＩＰは気管支並びに海面体小柱の平滑筋弛緩作用を利用
して喘息やインポテンツの治療に用いる薬物として期待
されている。このＶＩＰの構造的な特徴として、Ｃ末端
がアミド化されておりこれが生理活性の発現に重要な役
割を果たしているとされてきた。このため大腸菌を用い
てポリペプチドを作製すると、Ｃ末端がアミド型のペプ
チドを作製するためには、発現したペプチドを精製しＣ
末端アミド化酵素等の特殊な技術を用いなければならな
かった。しかしながら、この反応は、収率も悪く酵素も
高価で工業化には適していなかった。一方、本発明者等
はＣ末端がアミド化されているモチリン等においてはＣ
末端がホモセリンの形でも十分活性を有することを発見
し、これを廉価に大量生産する方法を見いだした（特願
昭６４−２８６号）。Among these various pharmacological actions, V
IP is expected to be used as a drug for the treatment of asthma and impotence by utilizing its relaxing effect on the smooth muscles of the bronchi and trabeculae of the corpus cavernosum. A structural feature of this VIP is that the C-terminus is amidated, which has been considered to play an important role in the expression of physiological activity. Therefore, when a polypeptide is produced using E. coli, in order to produce a peptide with an amide type at the C-terminus, the expressed peptide must be purified and the C-terminus must be purified.
Special techniques such as terminal amidation enzymes had to be used. However, this reaction was not suitable for industrialization because the yield was poor and the enzyme was expensive. On the other hand, the present inventors found that in motilin, etc., in which the C-terminus is amidated, C
It was discovered that the homoserine terminal has sufficient activity, and a method for mass-producing it at low cost was discovered (Japanese Patent Application No. 64-286).
【０００４】更にＶＩＰの１７位はメチオニンであるが
、これをロイシン又はノルロイシンに変換したアミノ酸
構造を有するポリペプチドもＶＩＰと同様な生理活性を
示すことが報告されており（特開昭６２−２４６５９５
号）、１７位のメチオニンは活性に及ぼす影響はほとん
どないものと考えられている。Furthermore, although the 17th position of VIP is methionine, it has been reported that a polypeptide having an amino acid structure in which methionine is converted to leucine or norleucine also exhibits the same physiological activity as VIP (Japanese Unexamined Patent Publication No. 246595/1983).
No.), methionine at position 17 is thought to have almost no effect on activity.
【０００５】[0005]
【発明が解決しようとする課題及び発明の目的】慣用技
術によれば、ＶＩＰを得るには合成法または、生物試料
からの抽出に依らざるを得ず、安価で大量な精製は極め
て困難であった。また、バイオテクノロジ−による技術
を用いてもＣ末端のアミド化または塩基性アミノ酸の付
加が活性に重要であると言われているために工業的に有
利なＶＩＰの製法をもたらすには至らなかった。[Problems to be Solved by the Invention and Objectives of the Invention] According to conventional technology, VIP must be obtained by synthesis or extraction from biological samples, and it is extremely difficult to purify it at low cost and in large quantities. Ta. Furthermore, even using biotechnology techniques, it has not been possible to create an industrially advantageous method for producing VIP because it is said that amidation at the C-terminus or addition of basic amino acids is important for activity. .
【０００６】本発明は従来技術におけるこのような困難
をふまえてなされたものであり、ブロムシアンによる切
断によってＣ末端にホモセリンを有するＶＩＰ様ポリペ
プチドを作成し、アミド化処理等の煩雑な操作を行わな
くてもＶＩＰと同等以上の活性を有するポリペプチドを
提供することにある。本発明の附随的な、但し重要な目
的は非常に効率的にしかも廉価に提供できる上記のＶＩ
Ｐ様活性を有するポリペプチドを有効成分とする医薬品
、殊に気管支拡張剤およびインポテンツ治療剤を提供す
ることにある。[0006] The present invention was made in view of these difficulties in the prior art, and involves creating a VIP-like polypeptide having homoserine at the C-terminus by cleavage with bromcyanide, and then performing complicated operations such as amidation treatment. The objective is to provide a polypeptide that has an activity equivalent to or higher than that of VIP even without the use of VIP. Ancillary but important object of the present invention is to provide the above-mentioned VI in a highly efficient and inexpensive manner.
The object of the present invention is to provide a pharmaceutical agent, in particular a bronchodilator and a therapeutic agent for impotence, containing a polypeptide having P-like activity as an active ingredient.
【０００７】[0007]
【課題を解決し、目的を達成する手段及び作用】本発明
者等は鋭意検討を重ねた結果、発酵法又は合成法により
得られ、且つ１７位がメチオニン（Ｍｅｔ）以外のアミ
ノ酸残基であるポリペプチドに関して、ブロムシアンに
より切断した後に、得られるポリペプチドはＣ末端がホ
モセリン（ラクトンも含む）になっているポリペプチド
自体が、もとのＶＩＰと同等又はそれ以上の活性を有す
ることを見いだし、これによって本発明を基本的には完
成するに至った。
従って、本発明は、一般式（１）
Ｈｉｓ−Ｓｅｒ−Ａｓｐ−Ａｌａ−Ｖａｌ−Ｐｈｅ−Ｔ
ｈｒ−Ａｓｐ−Ａｓｎ−Ｔｙｒ−Ｔｈｒ−Ａｒｇ−Ｌｅ
ｕ−Ａｒｇ−Ｌｙｓ−Ｇｌｎ− Ｘ −Ａｌａ−Ｖ
ａｌ−Ｌｙｓ−Ｌｙｓ−Ｔｙｒ−Ｌｅｕ−Ａｓｎ−Ｓｅ
ｒ−Ｉｌｅ−Ｌｅｕ−Ａｓｎ− Ｙ
［式中 ＸはＭｅｔ以外のアミノ酸残基を意味し、Ｙ
はホモセリン（ホモセリンラクトンも含む、Ｈｓｅで示
される）を意味する。］によって示されるＶＩＰ様活性
を有する新規ポリペプチドにて解決されるとともに、本
発明による既述の主目的が達成される。[Means and effects for solving the problem and achieving the object] As a result of intensive studies, the present inventors found that the amino acid residue is obtained by a fermentation method or a synthesis method, and the 17th position is an amino acid residue other than methionine (Met). Regarding polypeptides, we found that after cleavage with bromcyanide, the resulting polypeptide itself has homoserine (including lactone) at the C-terminus, and has an activity equal to or greater than that of the original VIP, As a result, the present invention has been basically completed. Therefore, the present invention provides general formula (1) His-Ser-Asp-Ala-Val-Phe-T
hr-Asp-Asn-Tyr-Thr-Arg-Le
u-Arg-Lys-Gln-X-Ala-V
al-Lys-Lys-Tyr-Leu-Asn-Se
r-Ile-Leu-Asn-Y [wherein X means an amino acid residue other than Met, Y
means homoserine (also includes homoserine lactone, denoted by Hse). ] The above-mentioned main objectives of the present invention are achieved with a novel polypeptide having VIP-like activity.
【０００８】[0008]
【実施例】次に、製造例及び薬理活性試験例に関して本
発明をさらに詳細に説明する。尚、以下においてはＶＩ
Ｐの１７位がロイシンに変換されたＶＩＰ様ポリペプチ
ドの合成法に関して説明するが醗酵法を用いた場合、メ
チオニン以外のアミノ酸残基であれば、同様にして他の
ＶＩＰ様ポリペプチドを製造し得ることに留意されたい
。EXAMPLES Next, the present invention will be explained in more detail with reference to production examples and pharmacological activity test examples. In addition, in the following, VI
We will explain the method for synthesizing a VIP-like polypeptide in which position 17 of P is converted to leucine, but if fermentation is used, other VIP-like polypeptides can be produced in the same way as long as the amino acid residue is other than methionine. Note that you get
【０００９】製造例
（１）ＶＩＰ様生理活性ポリペプチドの合成Ｌ−ロイシ
ン−１７−ＶＩＰ−Ｈｓｅの合成はペプチド合成機で合
成終了後、通常の脱保護基、脱樹脂によってＣ末端にホ
モセリン（Ｈｓｅ）を与えるＢｏｃ−Ｈｓｅ（Ｂｚｌ）
−４−（ｏｘｙｍｅｔｈｙｌ）ｐｈｅｎｙｌａｃｅｔｉ
ｃ ａｃｉｄを化学合成し、市販のＡｍｉｎｏｍｅｔ
ｈｙｌ樹脂を用いてアプライドバイオシステムズ社のペ
プチドシンセサイザー４３１Ａにて行った。合成したＬ
−ロイシン−１７−ＶＩＰ−Ｈｓｅの配列を以下に示す
。Ｈｉｓ−Ｓｅｒ−Ａｓｐ−Ａｌａ−Ｖａｌ−Ｐｈｅ−
Ｔｈｒ−Ａｓｐ−Ａｓｎ−Ｔｙｒ−Ｔｈｒ−Ａｒｇ−Ｌ
ｅｕ−Ａｒｇ−Ｌｙｓ−Ｇｌｎ−Ｌｅｕ−Ａｌａ−Ｖａ
ｌ−Ｌｙｓ−Ｌｙｓ−Ｔｙｒ−Ｌｅｕ−Ａｓｎ−Ｓｅｒ
−Ｉｌｅ−Ｌｅｕ−Ａｓｎ−Ｈｓｅ合成したサンプルは
トリフルオロメタンスルホン酸法にて脱保護基、脱樹脂
を行ない、さらに０．５Ｍアンモニアで氷中３０分処理
した後、ウォーターズ社製のマイクロボンダスフェアの
Ｃ−１８カラム（１９ｍｍ×１５ｃｍ）を用いてＨＰＬ
Ｃにて以下の条件下で精製した。
溶出液：０．１％トリフルオロ酢酸中１５％から５０％
のアセトニトリルの直線勾配、３５分間流速：７．０ｍ
ｌ／ｍｉｎ
ＨＰＬＣによるメインピーク部分を回収して凍結乾燥す
る。以上の過程でペプチドサンプルのＣ末端は、ホモセ
リンまたはホモセリンラクトンとなっている。その一部
をアプライドバイオシステムズ社のペプチドシークエン
サーにて調べたところ、サンプルはＣ末端のホモセリン
を含めて正しく化学合成されていることが確認された。Production Example (1) Synthesis of VIP-like physiologically active polypeptide After the synthesis of L-leucine-17-VIP-Hse is completed using a peptide synthesizer, homoserine ( Boc-Hse(Bzl) giving Hse)
-4-(oxymethyl)phenylaceti
C acid was chemically synthesized and commercially available Aminomet
This was carried out using Hyl resin on an Applied Biosystems Peptide Synthesizer 431A. Synthesized L
-The sequence of Leucine-17-VIP-Hse is shown below. His-Ser-Asp-Ala-Val-Phe-
Thr-Asp-Asn-Tyr-Thr-Arg-L
eu-Arg-Lys-Gln-Leu-Ala-Va
l-Lys-Lys-Tyr-Leu-Asn-Ser
-Ile-Leu-Asn-Hse The synthesized sample was deprotected and resin removed using the trifluoromethanesulfonic acid method, and then treated with 0.5M ammonia in ice for 30 minutes. HPL using C-18 column (19 mm x 15 cm)
It was purified at C under the following conditions. Eluent: 15% to 50% in 0.1% trifluoroacetic acid
Linear gradient of acetonitrile, 35 minutes Flow rate: 7.0 m
The main peak portion obtained by l/min HPLC is collected and freeze-dried. Through the above process, the C-terminus of the peptide sample becomes homoserine or homoserine lactone. When a part of the sample was examined using an Applied Biosystems peptide sequencer, it was confirmed that the sample was chemically synthesized correctly, including homoserine at the C-terminus.
【００１０】生理活性試験例（気管支収縮抑制活性の測
定）上記製造例で得られたＶＩＰアナログを被験物質と
して、（株）ペプチド研究所から購入したＶＩＰを対照
物質とし、モルモット気管支を用いるマグヌス法［”Ｐ
ｅｐｔｉｄｅｓ”第６巻、第５９７ー６０１頁（１９８
５年）］に従い気管支収縮抑制活性を測定した結果は第
１図に示される通りであった。この図から明らかなよう
に、本発明により得られたＬ−ロイシン−１７−ＶＩＰ
−Ｈｓｅが示す気管支弛緩作用はもとのＶＩＰ以上のレ
ベルであることが確認された。また第２図の実際の気管
支弛緩作用を示すチャートからももとのＶＩＰより強い
気管支弛緩作用を示すことがわかる。Physiological activity test example (measurement of bronchoconstriction inhibitory activity) Magnus method using guinea pig bronchi, using the VIP analog obtained in the above production example as the test substance and VIP purchased from Peptide Institute Co., Ltd. as the control substance. [”P
eptides” Volume 6, pp. 597-601 (198
The results of measuring the bronchoconstriction inhibitory activity according to the method (5 years)] are as shown in Figure 1. As is clear from this figure, L-leucine-17-VIP obtained by the present invention
- It was confirmed that the bronchial relaxation effect exhibited by Hse was at a level higher than that of the original VIP. Moreover, from the chart showing the actual bronchial relaxation effect in FIG. 2, it can be seen that the bronchial relaxation effect is stronger than that of the original VIP.
【００１１】製剤例
本発明物質１ｍｇを含有するようにバイアルに無菌的に
分配し、凍結乾燥等により水分を除去して密封する。使
用時は注射用生理食塩水等を添加し、溶解して用いる。
尚、本発明物質の安定化剤としてヒト血清アルブミン等
を用いることができる。Formulation Example The substance of the present invention is aseptically dispensed into vials containing 1 mg, moisture is removed by lyophilization, etc., and the vials are sealed. When using, add physiological saline for injection and dissolve. Incidentally, human serum albumin or the like can be used as a stabilizer for the substance of the present invention.
【００１２】0012
【発明の効果】本発明によるポリペプチド誘導体は、Ｖ
ＩＰと同程度以上の生理活性を示す。本発明によるＣ末
端にホモセリン（ラクトンも含む）を有すＶＩＰ様活性
を有するポリペプチド（ＶＩＰアナログ）は、本発明者
が開発した方法（特願平１−２１６０３４）を応用して
大腸菌等により容易にしかも非常に廉価に作成できる。
従って本発明による上記のポリペプチド調製に際しては
従来行なわれていた収率の悪く、高価であったアミド化
処理法等に比して製造コストが著しく低減し、収率も向
上し、結果として生産効率が高まり、ＶＩＰ様生理活性
物質の廉価な且つ大量の供給が可能となる。Effect of the invention The polypeptide derivative according to the present invention has V
Shows physiological activity comparable to or higher than IP. The polypeptide (VIP analog) having VIP-like activity having homoserine (including lactone) at the C-terminus according to the present invention can be obtained by using E. coli etc. by applying the method developed by the present inventor (Japanese Patent Application No. 1-216034). It can be produced easily and at a very low cost. Therefore, when preparing the above-mentioned polypeptide according to the present invention, the production cost is significantly reduced and the yield is improved compared to the conventional amidation treatment method, which had a poor yield and was expensive. Efficiency is increased, and VIP-like physiologically active substances can be supplied at low cost and in large quantities.
【図１】は本発明によるＶＩＰ様活性を有するポリペプ
チドを被験物質とし、（株）ペプチド研究所から購入し
た純ＶＩＰを対照物質とし、これら物質が示すモルモッ
ト気管支弛緩作用をマグヌス法によって測定した結果を
示すグラフである。[Figure 1] The polypeptide having VIP-like activity according to the present invention was used as a test substance, and pure VIP purchased from Peptide Institute Co., Ltd. was used as a control substance, and the guinea pig bronchorelaxant effect of these substances was measured by the Magnus method. It is a graph showing the results.
【図２】はモルモット気管支弛緩作用をマグヌス法によ
って測定したときの実際のチャートを示す図である。
（被検薬物１０−７モル）FIG. 2 is a diagram showing an actual chart when the guinea pig bronchial relaxation effect was measured by the Magnus method. (Test drug 10-7 mol)
【図３】はモルモット気管支弛緩作用をマグヌス法によ
って測定したときの実際のチャートを示す図である。
（被検薬物１０−８モル）FIG. 3 is a diagram showing an actual chart when the guinea pig bronchial relaxation effect was measured by the Magnus method. (Test drug 10-8 mol)
Claims (3)
−Ａｌａ−Ｖａｌ−Ｐｈｅ−Ｔｈｒ−Ａｓｐ−Ａｓｎ−
Ｔｙｒ−Ｔｈｒ−Ａｒｇ−Ｌｅｕ−Ａｒｇ−Ｌｙｓ−Ｇ
ｌｎ− Ｘ −Ａｌａ−Ｖａｌ−Ｌｙｓ−Ｌｙｓ−
Ｔｙｒ−Ｌｅｕ−Ａｓｎ−Ｓｅｒ−Ｉｌｅ−Ｌｅｕ−Ａ
ｓｎ− Ｙ ［式中ＸはＭｅｔ以外のアミノ酸残基を意味し、Ｙはホ
モセリン（ホモセリンラクトンも含む、「Ｈｓｅ」と略
す）］によって示され、且つＶＩＰ様活性を有している
ことを特徴とするポリペプチド。[Claim 1] Formula (I) His-Ser-Asp
-Ala-Val-Phe-Thr-Asp-Asn-
Tyr-Thr-Arg-Leu-Arg-Lys-G
ln-X-Ala-Val-Lys-Lys-
Tyr-Leu-Asn-Ser-Ile-Leu-A
sn- Y [in the formula, X means an amino acid residue other than Met, Y is homoserine (also includes homoserine lactone, abbreviated as "Hse")], and is characterized by having VIP-like activity polypeptide.
とする気管支拡張剤。2. A bronchodilator containing the polypeptide according to claim 1 as a main component.
とするインポテンツ治療剤。3. A therapeutic agent for impotence comprising the polypeptide according to claim 1 as a main component.
Priority Applications (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2408425A JPH04230400A (en) | 1990-12-27 | 1990-12-27 | Polypeptide derivative having vip-like activity and its use |
| EP91109490A EP0463450B1 (en) | 1990-06-26 | 1991-06-10 | Vasoactive intestinal polypeptide analogues and use thereof |
| AT91109490T ATE101619T1 (en) | 1990-06-26 | 1991-06-10 | VIP ANALOGS AND THEIR USE. |
| ES91109490T ES2063406T3 (en) | 1990-06-26 | 1991-06-10 | PROCEDURE FOR THE PREPARATION OF VASOACTIVE INTESTINAL POLYPEPTIDE ANALOGS. |
| DE69101187T DE69101187T2 (en) | 1990-06-26 | 1991-06-10 | VIP analogues and their use. |
| DK91109490.2T DK0463450T3 (en) | 1990-06-26 | 1991-06-10 | Vasoactive intestinal polypeptide analogs and their utilization |
| US08/201,092 US5428015A (en) | 1990-06-26 | 1994-02-24 | Vasoactive intestinal polypeptide analogues and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2408425A JPH04230400A (en) | 1990-12-27 | 1990-12-27 | Polypeptide derivative having vip-like activity and its use |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH04230400A true JPH04230400A (en) | 1992-08-19 |
Family
ID=18517880
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2408425A Pending JPH04230400A (en) | 1990-06-26 | 1990-12-27 | Polypeptide derivative having vip-like activity and its use |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH04230400A (en) |
-
1990
- 1990-12-27 JP JP2408425A patent/JPH04230400A/en active Pending
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