WO1996017872A1 - Intermediaire pour la production d'un peptide surfactant - Google Patents

Intermediaire pour la production d'un peptide surfactant Download PDF

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Publication number
WO1996017872A1
WO1996017872A1 PCT/JP1995/001114 JP9501114W WO9617872A1 WO 1996017872 A1 WO1996017872 A1 WO 1996017872A1 JP 9501114 W JP9501114 W JP 9501114W WO 9617872 A1 WO9617872 A1 WO 9617872A1
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WO
WIPO (PCT)
Prior art keywords
peptide
leu
val
surfactant
arg
Prior art date
Application number
PCT/JP1995/001114
Other languages
English (en)
Japanese (ja)
Inventor
Tsunetomo Takei
Eiji Ohtsubo
Original Assignee
Tokyo Tanabe Company Limited
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Tokyo Tanabe Company Limited filed Critical Tokyo Tanabe Company Limited
Priority to AU25768/95A priority Critical patent/AU2576895A/en
Publication of WO1996017872A1 publication Critical patent/WO1996017872A1/fr

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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
    • C07K14/785—Alveolar surfactant peptides; Pulmonary surfactant peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to peptide derivatives. More specifically, the present invention relates to a peptide derivative which is an intermediate for producing a synthetic peptide useful as an active ingredient for treating respiratory distress syndrome.
  • Respiratory distress syndrome is a disease in which the surface activity of the alveolar surface is reduced due to lack of pulmonary surfactant and the alveoli collapse, resulting in severe respiratory distress. Is high. It is known that pulmonary surfactant preparations are effective for respiratory distress syndrome in newborns. In recent years, as apoproteins specific to mammalian lung surfactant, lipophilic surfactant topoprotein A and surfactant topoprotein D, and hydrophobic surfactant topoprotein B (hereinafter referred to as “SP-B”).
  • SP-B hydrophobic surfactant topoprotein B
  • SP-c surfactant Apoprotein C
  • Human lung-derived SP-C [SEQ ID NO: 1] is an apoprotein with 35 amino acids, a rich N-terminal amino acid, Val-rich, and extremely strong hydrophobicity.
  • the isolated SP-C also consists of 34 to 35 amino acids, and the amino acid sequence at the N-terminal varies depending on the animal species, but has very high homology to humans.
  • hydrophilic peptide portion having a specific sequence which is a partial structure of SP-C on the N-terminal side, and a hydrophobic peptide portion mainly composed of Leu and / or Nle on the C-terminal side.
  • Synthetic peptides containing amino acid sequences can be easily isolated and purified, can be manufactured in large quantities, can be formic acid, trifluoroacetic acid (TFA), trifluoroethanol, Dissolves well in dimethyl sulfoxide (DMSO), form-of-cloth, mixed form-of-form-methanol, methanol, ethylenechlorohydrin or tetrahydrofuran.
  • Xaa is absent or represents Cys or Ser
  • Xbb represents His or Asn
  • Xcc represents Leu or lie
  • W represents a hydrophobic peptide moiety.
  • the present invention is used as an intermediate for the production of the above-mentioned synthetic peptide (hereinafter referred to as “surfactant peptide”) which exhibits a strong surface activity by being combined with a lipid mixture. It is a peptide derivative in which the N-terminal and functional side chain of a hydrophilic peptide represented by the following specific sequence are protected (hereinafter, referred to as “the peptide derivative of the present invention”).
  • Xaa-Pro-Val-Xbb-Xcc-Lys-Arg (Xaa is absent or represents Cys or Ser, Xbb represents His or Asn, and Xcc represents Leu or lie.)
  • the peptide derivative of the present invention is a peptide derivative capable of easily producing surfactant peptide by condensing with a previously prepared hydrophobic peptide moiety.
  • the protecting group for the N-terminus and the functional side chain of the peptide derivative of the present invention is not particularly limited as long as it is a protecting group used in usual peptide synthesis.
  • the protecting groups of 9-fluorenylmethyloxycarbonyl (Fmoc), 2-chlorobenzyloxycarbonyl (2-CLZ) or t-butyloxycarbonyl (Boc) are the protective groups of Lys. Boc, carbobenzoxy (Z) or tosyl (Tos) groups as bases, Trt, Fmoc, Boc, Dnp, Bom, Bzl or Tos groups as His protecting groups, and Arg protecting groups as Mtr, Pmc, Mts or Tos groups.
  • peptide derivative of the present invention include Fmoc-Pro-Val-His (, rt) -Leu-Lys (, Boc) -Argi > tr), Fmoc-Pro-Val-Asn-Leu-Lys ( (Boc) -Arg (Mtr), Fmoc-Pro-Val-Asn-Ile-Lys (Boc) -Arg (Mtr) or Fmoc-Cys (Acm)-Pro-Val-His (Trt)-Leu-Ly s (Boc )-Arg (Mtr).
  • Surfactant peptide produced from the peptide derivative of the present invention and a hydrophobic peptide portion is mixed with choline phosphoglyceride, acidic phospholipid and fatty acid as a lipid mixture.
  • a pulmonary surfactant useful as a therapeutic agent for respiratory distress syndrome can be produced.
  • the molecular weight of the synthesized peptides was measured by the fast atom bombardment method (FABMS).
  • FABMS fast atom bombardment method
  • the mass spectrometer used was JMS-S102A (produced by Honden Electronics Co., Ltd.), and the ion source used was a cesium gun (lOKeV).
  • the title peptide derivative B was synthesized and purified by a peptide synthesizer system 9050 (manufactured by Millipore) in the same manner as in Example 1.
  • the amino acid was sequentially extended in the N-terminal direction on the resin, and H-Nle- (Nle-Nle-0-resin) was synthesized by a multipeptide solid-phase synthesis system.
  • the peptide derivative A of Example 1 was added to N-hydroxyquinobenzotriazole and N, N'-diisopropylcarpoide. The mixture was added and shaken for 8 hours, and the condensation reaction was performed twice. The confirmation of the condensation reaction was carried out by a force test using the ninhydrin method.
  • a 20% solution of pyridin-DMF was added to deprotect the F-moc protecting group at the N-terminal. It was washed six times with methanol and dried under reduced pressure. While stirring the dried peptide-0-resin (100 mg) under ice cooling, m-cresol (0.2 ml), 1,2-ethanedithiol (0.5 ml), thioanisole (1 2 ml), TFA (7.5 ml) and trimethylsilyl bromide (4 ml), and the mixture was stirred under ice-cooling for 120 minutes to obtain a functional side chain.
  • the peptide was cut out from the resin together with the deprotection, and filtered through a glass filter (G3).
  • the filtrate was concentrated under reduced pressure to about 5 ml by an evaporator, and getyl ether was added to precipitate the peptide.
  • the peptide precipitate was taken out using a glass filter (G3), washed with getyl ether five times, dried under reduced pressure, and further purified by HPLC to obtain a surfactant peptide of [SEQ ID NO: 4].
  • C was prepared.
  • H-Leu- (Leu) prepared in the same manner as in Reference Example 1. -Leu-0-resin is cut out of the resin and purified to H-Leu- (Leu) ,. -Leu-OH was obtained.
  • the precipitated dicyclourea was removed by filtration, and water was added to the filtrate.
  • the product was extracted with a black hole form, washed sequentially with a 10% aqueous sodium hydrogen carbonate solution, a 0.1N aqueous hydrochloric acid solution and distilled water, and the solvent was distilled off under reduced pressure. Then, after recrystallization from ethanol / water (4: 1), the crystals were dried under reduced pressure to obtain 3.58 g of a condensate (yield: 80%).
  • the surfactant peptides obtained in Reference Examples 1 and 2 were treated with 12N hydrochloric acid-TFA containing 5% (v / v) phenol [2: 1 (V / V)] at 150 ° C under vacuum. After acid hydrolysis for 2, 4, 6, 12, 24, 48 and 72 hours to remove the acid, the hydrolysis products were analyzed by Shimadzu Amino Acid Automatic Analysis System (LC-9A). In the hydrolysis for 1 to 72 hours, the amino acid value showing higher recovery was adopted, and the amino acid composition value was calculated. The value almost coincided with the calculated value.
  • LC-9A Shimadzu Amino Acid Automatic Analysis System
  • 1,2-dino-noremitoyl glycerol (3) -phosphocholin (21 Omg), 1-noreno-tomi-one 2-oleoyl-1 sn-glycero (3) -phospho-1L-serine (90.Omg) ) And palmitic acid (33.Omg) were dissolved in a chloroform solution (4: 1 (VZV)) (100 ml), and surfactant peptide C of SEQ ID NO: 4 (11 Omg) was dissolved in TFA (0. 5 ml). These solutions were mixed and evaporated to dryness under reduced pressure.
  • the resulting residue The suspension was suspended in a water-ethanol mixture [9: 1 (V / V)] (110 ml) at 45 ° C for 25 minutes. This suspension was frozen at 55 ° C and dried at 100-120 Hg vacuum for 28 hours to give 348.7 mg of a white powder of lung surfactant.
  • the pulmonary surfactant prepared from the surfactant peptide and the lipid mixture has a good suspension property and a strong surface activity, and thus is useful as a therapeutic agent for respiratory distress syndrome.
  • Val Val Val Val Val Leu Leu Val Val Val lie Val Gly Ala Leu Leu Met
  • Leu Arg lie Pro Cys Cys Pro Val Asn Leu Lys Arg Leu Leu Val Val

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Toxicology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pulmonology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un dérivé peptidique présentant la séquence Xaa-Pro-Val-Xbb-Xcc-Lys-Arg (dans laquelle Xaa est absent ou représente Cys ou Ser; Xbb représente His ou Asn; et Xcc représente Leu ou Ile). Ce dérivé est capable de produire efficacement et facilement un peptide hautement soluble dans le méthanol, etc, et le peptide surfactant obtenu peut être facilement combiné à un mélange lipidique et convient donc à la préparation d'un surfactant pulmonaire. Ce surfactant, qui présente une bonne suspensibilité et une puissante activité de surface, est donc utile en tant que remède contre le syndrome de détresse respiratoire.,
PCT/JP1995/001114 1994-12-07 1995-06-06 Intermediaire pour la production d'un peptide surfactant WO1996017872A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU25768/95A AU2576895A (en) 1994-12-07 1995-06-06 Intermediate for producing surfactant peptide

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP6/303397 1994-12-07
JP30339794 1994-12-07

Publications (1)

Publication Number Publication Date
WO1996017872A1 true WO1996017872A1 (fr) 1996-06-13

Family

ID=17920539

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP1995/001114 WO1996017872A1 (fr) 1994-12-07 1995-06-06 Intermediaire pour la production d'un peptide surfactant

Country Status (2)

Country Link
AU (1) AU2576895A (fr)
WO (1) WO1996017872A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05294996A (ja) * 1992-04-17 1993-11-09 Tokyo Tanabe Co Ltd 合成ペプチド、それを含有する肺サーファクタント及び呼吸窮迫症候群治療剤
JPH069428A (ja) * 1992-06-24 1994-01-18 Tokyo Tanabe Co Ltd 気道部ウィルス疾患の予防及び治療剤
WO1994025480A1 (fr) * 1993-04-30 1994-11-10 Tokyo Tanabe Company Limited Procede pour purifier un polypeptide hydrophobe
WO1995015980A1 (fr) * 1993-12-08 1995-06-15 Tokyo Tanabe Company Limited Nouveau peptide synthetique, tensioactif pulmonaire contenant ledit peptide et remede contre le syndrome de souffrance respiratoire

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05294996A (ja) * 1992-04-17 1993-11-09 Tokyo Tanabe Co Ltd 合成ペプチド、それを含有する肺サーファクタント及び呼吸窮迫症候群治療剤
JPH069428A (ja) * 1992-06-24 1994-01-18 Tokyo Tanabe Co Ltd 気道部ウィルス疾患の予防及び治療剤
WO1994025480A1 (fr) * 1993-04-30 1994-11-10 Tokyo Tanabe Company Limited Procede pour purifier un polypeptide hydrophobe
WO1995015980A1 (fr) * 1993-12-08 1995-06-15 Tokyo Tanabe Company Limited Nouveau peptide synthetique, tensioactif pulmonaire contenant ledit peptide et remede contre le syndrome de souffrance respiratoire

Also Published As

Publication number Publication date
AU2576895A (en) 1996-06-26

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