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WO1996008508A1 - Nouvel antibiotique peptidique humain (fall-39) et son utilisation - Google Patents

Nouvel antibiotique peptidique humain (fall-39) et son utilisation Download PDF

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Publication number
WO1996008508A1
WO1996008508A1 PCT/SE1995/001030 SE9501030W WO9608508A1 WO 1996008508 A1 WO1996008508 A1 WO 1996008508A1 SE 9501030 W SE9501030 W SE 9501030W WO 9608508 A1 WO9608508 A1 WO 9608508A1
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WO
WIPO (PCT)
Prior art keywords
fall
polypeptide
pharmaceutical composition
peptide
pharmaceutically acceptable
Prior art date
Application number
PCT/SE1995/001030
Other languages
English (en)
Inventor
Hans G. Boman
Birgitta Agerberth
Gudmundur H. Gudmundsson
Hans Gunne
Original Assignee
Boman Hans G
Birgitta Agerberth
Gudmundsson Gudmundur H
Hans Gunne
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 Boman Hans G, Birgitta Agerberth, Gudmundsson Gudmundur H, Hans Gunne filed Critical Boman Hans G
Priority to AU35368/95A priority Critical patent/AU3536895A/en
Publication of WO1996008508A1 publication Critical patent/WO1996008508A1/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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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 a new human polypeptide (FALL-39), its therapeutic use, pharmaceutical compositions containing same as an active ingredient, a cDNA sequence, and a method for inhibiting microbial growth.
  • the peptides most studied are the cecropins and the magainins in the first group and the defensin family in the fourth group.
  • the animal peptide antibiotics can be divided into two groups: Those which like the defensins accumulate in the granule of phagocytes and those which are delivered into body fluids or epithelial layers. Peptides which have evolved to kill engulfed microbes inside phagocytic vac ⁇ oles can in released form be cytotoxic to the host and this is the case for the defensins
  • Animal peptide antiobiotics differ from the "classical" antibiotics in several respects (Boman, H.G. (1995) Annu. Rev. Immunol . 13 , in press; Boman, H.G. (1994) in Antimicrobial Peptides . Ciba Symposium No. 186 , pp 1-4, ed. Goode, J. (John Wiley & Sons Ltd. Chichester, P019 1UD, UK)).
  • the animal peptides are all gene encoded and they are made as preproproteins which are processed to the mature peptide by defined pathways. The actual processing steps have been studied for cecropins (Boman, H.G., Boman, I.A., Andreu, D., Li, Z.-q., Merrifield, R.B.,
  • the present invention has for its main object to provide new polypeptides of biological activity but different from classical antibiotics, such as penicillins and tetracyclins.
  • Another object of the invention is to provide new human polypeptides having antimicrobial activity against bacteria without being cytotoxic.
  • Yet another object of the invention is to provide pharmaceutical compositions containig such polypeptides as active ingredients.
  • Still another object of the invention is to provide a method for inhibiting microbial growth in animals including man.
  • a further object of the invention is to provide the clone enabling expression of a precursor protein for the new polypeptides.
  • polypeptide comprising the following amino acid sequence:
  • polypeptide comprising the following amino sequence:
  • the new polypeptides according to the invention find therapeutic applications, such as used as antimicrobial agents, such as antibacterial agents.
  • the invention also includes pharmaceutical compositions containing as active ingredients one or more of the polypeptides according to the invention in an effective amount together with a pharmaceuticallly acceptable carrier or diluent.
  • Said amount is suitably antimicrobially active, such as antibacterially active.
  • the carrier or diluent is preferably adapted for oral, intravenuous, intramuscular or subcutaneous administration.
  • the invention includes a method for inhibiting microbial growth in animals, such as mammals including man, said method comprising the step of administering to an animal subject to a disorder caused by microbial attack a polypeptide as defined above, a functional derivative or a pharmaceutically acceptable salt thereof, or a composition as defined above in an inhibitory amount.
  • Such method can be directed to intestinal use constituted by oral administration of a composition as defined above in a slow release form.
  • the method can also be directed to administration by injection of such a composition in an injectable dose form.
  • compositions of this invention are pharmaceutically acceptable salts of the polypeptides of this invention.
  • Such salts are formed by methods well known to skilled artisans.
  • base salts of the polypeptides can be prepared according to conventional methods.
  • polypeptide is used said term is intended to include both functional derivatives and pharmaceutically acceptable salts of the polypeptides.
  • the active polypeptide according to the present invention can be formulated for use in human or veterinary medicine for therapeutic or prophylactic use.
  • the active preparations are normally administered orally, rectally or parenterally, such as by injection in the form of a pharmaceutical preparation or composition comprising the active constituents in combination with a pharmaceutically acceptable carrier which may be solid, semi-solid or liquid, or contained in a capsule, such as when orally admi bred.
  • a pharmaceutically acceptable carrier which may be solid, semi-solid or liquid, or contained in a capsule, such as when orally admi bred.
  • the administration may also take the form of to pical application.
  • examples of pharmaceutical preparations there may be mentioned tablets, drops, solutions and suppositories.
  • the active constituent constitutes the minor part of the preparation, such as from about 0.1 to about 50% thereof based on weight.
  • the polypeptide of the invention can be mixed with a solid, pulverulent or other carrier, for example lactose, saccharose, sorbitol, mannitol, starch, such as potatoe starch, corn starch, millopectine, cellulose derivative or gelatine, and may also include lubricants, such as magnesium or calcium stearate, or polyethylene glycol waxes compressed to the formation of tablets or bodies for dragees.
  • a solid, pulverulent or other carrier for example lactose, saccharose, sorbitol, mannitol, starch, such as potatoe starch, corn starch, millopectine, cellulose derivative or gelatine
  • lubricants such as magnesium or calcium stearate, or polyethylene glycol waxes compressed to the formation of tablets or bodies for dragees.
  • the dose units may also be presented in a coated form of enteric type.
  • Liquid preparations for oral application or for injection can be made in the form of elexirs, syrups or suspensions, for example solutions containing from 0.1 to 20% by weight of active substance, sugar and a mixture of ethanol, water, glycerol, propyleneglycol and possibly other additives of a conventional nature.
  • An important aspect of the present invention is the use of the polypeptides of the invention in products for the treatment of urinary tract infections and sexually transmitted diseases, such as disorders created by clamydia, gonococchi and HIV virus.
  • Such treatment can be performed by using a solution of the antimicrobially active polypeptide in lavage of invaded organs, cavities or tubes, such as the urinary tract, the bladder, urethral organs and passages, renal organs and associated passages, male genital organs and associated passages, etc.
  • the dose by which the active constituent is administered may vary within wide limits and is dependent on different factors, such as the seriousness of the disorder, the age and the weight of the patient and can be adjusted individually.
  • Figure 1 shows the cDNA sequence for prepro-FALL-39 with translation of the open reading frame
  • Figure 2 illustrates two Northern blot analyses with RNA from a leucemic child, a healthy child and a child with leucemia in remission in comparison with mRNA from human bone marrow;
  • Figure 3 shows an Edmundson wheel plot for residues E13-V34 in the sequence of FALL-39;
  • Figure 4 shows CD spectra of a water solution of synthetic FALL-39 before and after adding of medium E; and Figure 5 shows an inhibition zone assay for FALL-39 on E.coli.
  • a liquid lysate of a human bone marrow ⁇ gtll cDNA library (Clontech) was used to isolate template DNA by
  • RNA was isolated by an RNA separator kit
  • the completed 39-residue peptide was cleaved from the resin with liquid hydrogen fluoride:anisole:methyl sulfide (10:1:1) for 60 min at 0°C.
  • the cleavage product was washed with ether in order to remove the scavengers and then extracted into 30% acetic acid and lyophilized.
  • the peptide was purified by HPLC on a Vydac C18 column, utilizing a linear gradient of 80% acetonitrile (20-75% for 30 min) in 0.1% trifluoroacetic acid.
  • the molecular mass was analysed by a time-of-flight mass spectrometer (Biolon 20).
  • the CD spectra were recorded with a J-710 spectropolarimeter (JASCO, Japan) at the BioScience Center of Pharmacia.
  • FALL-39 is indicated by a dashed underlining. The regions towards which the three successive primers were directed are indicated as a full line for the corresponding sequence and dotted lines for the two complementary sequences.
  • the cathelin sequence starts with bases 101-115 , translated to QVLSY . * is a stop signal.
  • a comparison with the genomic DNA sequence for PR-39 shows that both the signal sequences and the propart are partly conserved.
  • the C-terminal ends of the cathelin parts differ in the six last residues: in prepro-FALL-39 the end is a typical dibasic cleavage site (KR, residue 130-131) instead of an elastase site (SV) in prepro-PR-39.
  • Fig. 2 (a) Overexposure of the film in Fig. 2 (a) showed a faint signal from peripheral blood leukocyte RNA.
  • a filter preloaded with human mRNA from heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas gave no signals.
  • FALL-39 was synthesized with residues 132-170 of the precursor structure.
  • FIG. 3 shows an Edmundson wheel plot for residues E13-V34 in the sequence for FALL- 39.
  • the dotted line divides the helix into a hydrophilic and a hydrophobic part.
  • the start of the helix is at the top of the wheel.
  • a plot of residues F7-V34 does not give a perfect amphipathic helix.
  • the synthetic peptide was investigated for its helix content by CD spectroscopy.
  • Fig. 4 shows CD spectra of a water solution of synthetic FALL-39 (86 ⁇ M) and the same solution after adding 2% of 50 times concentrated Medium E. Values below 190 nm are believed to be artifacts from the solvents. About 50% of helix formation was induced in the presence of 30% trifluoroethanol.
  • Fig. 5 shows the inhibition zones produced by FALL-39 on E.coli D21 accor ding to Hultmark (Hultmark, D., Engstr ⁇ m, A., Andersson, K., Steiner, H., Bennich, H. and Boman, H.G. (1983) EMBO J. 2, 571-576).
  • the bacteria were grown in thin agarose plates with LB medium supplemented with basal medium E (MedE).
  • the zone area should normally be a function of the log of the amounts of peptide applied to each well as shown in the upper curve. In the absence of MedE there is a concentration dependence only for the three highest amounts of peptide.
  • the graph also shows the zones obtained with our standard assay conditions.
  • FALL-39 does not seem to be cytotoxic for human cells.
  • the present invention relates to the provision of novel human antibacterial peptides which are free of cystein.
  • the sequence of the clone for prepro-FALL-39 indicates that the molecule belongs to the family of cathelin like precursors.
  • FALL-39 residues 13-34, inclusive are capable of forming a perfect amphiphatic helix (cf. Fig. 4).
  • Medium E is a basal salt medium designed specially for E.coli (Vogel, H.J. and D.M. Bonner (1956) J. Bio . Chem. 218 , 97-106).

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne de nouveaux polypeptides comprenant la séquence aminoacide suivante: E K I G K E F K R I V O R I K D F L R N L V. Elle concerne également leurs dérivés fonctionnels et leurs sels pharmaceutiquement acceptables; une composition pharmaceutique contenant ces polypeptides comme principes actifs; un procédé pour inhiber la prolifération microbienne chez l'animal et l'homme; et une séquence d'ADNc capable d'exprimer une protéine précurseur de ce polypeptide.
PCT/SE1995/001030 1994-09-13 1995-09-12 Nouvel antibiotique peptidique humain (fall-39) et son utilisation WO1996008508A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU35368/95A AU3536895A (en) 1994-09-13 1995-09-12 A new human peptide antibiotic (fall-39) and its use

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE9403055-8 1994-09-13
SE9403055A SE9403055L (sv) 1994-09-13 1994-09-13 Nytt humant peptidantibiotikum (fall-39) och dess användning

Publications (1)

Publication Number Publication Date
WO1996008508A1 true WO1996008508A1 (fr) 1996-03-21

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PCT/SE1995/001030 WO1996008508A1 (fr) 1994-09-13 1995-09-12 Nouvel antibiotique peptidique humain (fall-39) et son utilisation

Country Status (3)

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AU (1) AU3536895A (fr)
SE (1) SE9403055L (fr)
WO (1) WO1996008508A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002060468A3 (fr) * 2001-03-30 2003-01-23 Univ Iowa Res Found Nouvelles activites antivirales de defensines theta de primates et mammaliennes
WO2002095076A3 (fr) * 2001-05-23 2003-09-25 Toyoda Chuo Kenkyusho Kk Polypeptides modifies presentant une resistance a la protease et/ou une sensibilite a la protease
WO2003039443A3 (fr) * 2001-11-05 2004-08-19 Deutsches Krebsforsch Nouveaux marqueurs genetiques pour leucemies
US7173007B1 (en) * 2003-04-02 2007-02-06 The Regents Of The University Of California Therapy for microbial infections
US7718618B2 (en) 2003-10-21 2010-05-18 The Regents Of The University Of California Human cathelicidin antimicrobial peptides
US7777000B2 (en) 2003-03-06 2010-08-17 The Regents Of The University Of California Anti-viral activity of cathelicidin peptides
US7776823B2 (en) 2003-10-21 2010-08-17 The Regents Of The University Of California Human cathelicidin antimicrobial peptides
US8012933B2 (en) 2003-01-29 2011-09-06 Lipopeptide Ab Use of the cathelicidin LL-37 and derivatives therof for wound healing

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4674511A (en) * 1979-04-30 1987-06-23 American Hospital Supply Corporation Medical electrode
US5063932A (en) * 1989-10-03 1991-11-12 Mieczyslaw Mirowski Controlled discharge defibrillation electrode
US5095916A (en) * 1985-06-20 1992-03-17 Medtronic, Inc. Cardioversion and defibrillation lead system
US5114424A (en) * 1989-09-07 1992-05-19 Siemens Aktiengesellschaft Multipart planar electrode for an hf-surgery device

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4674511A (en) * 1979-04-30 1987-06-23 American Hospital Supply Corporation Medical electrode
US5095916A (en) * 1985-06-20 1992-03-17 Medtronic, Inc. Cardioversion and defibrillation lead system
US5114424A (en) * 1989-09-07 1992-05-19 Siemens Aktiengesellschaft Multipart planar electrode for an hf-surgery device
US5063932A (en) * 1989-10-03 1991-11-12 Mieczyslaw Mirowski Controlled discharge defibrillation electrode

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002060468A3 (fr) * 2001-03-30 2003-01-23 Univ Iowa Res Found Nouvelles activites antivirales de defensines theta de primates et mammaliennes
WO2002095076A3 (fr) * 2001-05-23 2003-09-25 Toyoda Chuo Kenkyusho Kk Polypeptides modifies presentant une resistance a la protease et/ou une sensibilite a la protease
WO2003039443A3 (fr) * 2001-11-05 2004-08-19 Deutsches Krebsforsch Nouveaux marqueurs genetiques pour leucemies
US8012933B2 (en) 2003-01-29 2011-09-06 Lipopeptide Ab Use of the cathelicidin LL-37 and derivatives therof for wound healing
US8506994B2 (en) 2003-01-29 2013-08-13 Lipopeptide Ab Use of the cathelicidin LL-37 and derivatives thereof for wound healing
US8936807B2 (en) 2003-01-29 2015-01-20 Lipopeptide Ab Use of the cathelicidin LL-37 and derivatives thereof for wound healing
US9125875B2 (en) 2003-01-29 2015-09-08 Lipopeptide Ab Use of the cathelicidin LL-37 and derivatives thereof for wound healing
US7777000B2 (en) 2003-03-06 2010-08-17 The Regents Of The University Of California Anti-viral activity of cathelicidin peptides
US7173007B1 (en) * 2003-04-02 2007-02-06 The Regents Of The University Of California Therapy for microbial infections
US7718618B2 (en) 2003-10-21 2010-05-18 The Regents Of The University Of California Human cathelicidin antimicrobial peptides
US7776823B2 (en) 2003-10-21 2010-08-17 The Regents Of The University Of California Human cathelicidin antimicrobial peptides

Also Published As

Publication number Publication date
AU3536895A (en) 1996-03-29
SE9403055L (sv) 1996-03-14
SE9403055D0 (sv) 1994-09-13

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