WO2008040986A1 - Courts antagonistes peptidiques de la neuropiline-1 et leur utilisation - Google Patents

Courts antagonistes peptidiques de la neuropiline-1 et leur utilisation Download PDF

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Publication number
WO2008040986A1
WO2008040986A1 PCT/GB2007/003775 GB2007003775W WO2008040986A1 WO 2008040986 A1 WO2008040986 A1 WO 2008040986A1 GB 2007003775 W GB2007003775 W GB 2007003775W WO 2008040986 A1 WO2008040986 A1 WO 2008040986A1
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WO
WIPO (PCT)
Prior art keywords
hplc
peptide
purity
white solid
fmoc
Prior art date
Application number
PCT/GB2007/003775
Other languages
English (en)
Inventor
Haiyan Jia
Azadeh Bagherzadeh
Ian Zachary
Michelle Tickner
Chris Chapman
Ashley Jarvis
David Selwood
Original Assignee
Ark Therapeutics Ltd.
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 Ark Therapeutics Ltd. filed Critical Ark Therapeutics Ltd.
Publication of WO2008040986A1 publication Critical patent/WO2008040986A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1019—Tetrapeptides with the first amino acid being basic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/02—Non-specific cardiovascular stimulants, e.g. drugs for syncope, antihypotensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06086—Dipeptides with the first amino acid being basic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

  • This invention relates to short peptides which have NP-1 antagonist activity and which are of potential benefit in therapy.
  • NP-1 non-tyrosine kinase transmembrane protein
  • semaphorins or collapsins which play a key role in the guidance of neuronal axons during mammalian development.
  • NP-1 is known to mediate the growth cone-collapsing and chemorepulsive activity of semaphorin 3A.
  • NP-1 may have a significant role in pathology.
  • Such conditions include stroke, ischaemic eye disease, cancer and rheumatoid arthritis.
  • a novel peptide of the invention has up to 7 amino acids and comprises the sequence K-X 1 -Xa-R, wherein Xi is P or A, and wherein X 2 is any amino acid or COCONH.
  • Compounds of the invention may be useful as therapeutic agents. They may be useful for stimulating nerve repair, for treating neurodegeneration, or for treating cancer. They may also be used in the treatment of diseases where modulation of the immune system is required, for example, following transplant surgery. Yet other conditions that may be treated using a compound of the invention include skin diseases such as psoriasis, diseases requiring immunomodulation, angiogenesis in the eye, diabetes, macular degeneration, glaucoma and heart failure. Description of the Preferred Embodiments It will be appreciated that the compounds according to the invention contain an asymmetrically substituted carbon atom.
  • peptide of the invention can give rise to stereoisomers, and in each case the invention is to be understood to extend to all such stereoisomers, including enantiomers and diastereomers, and mixtures including racemic and non-racemic mixtures thereof.
  • Peptides of the invention may be synthesised by known methods. A general method is described herein.
  • peptides of the invention may include modifications of the given sequence. Such modifications are well known to those skilled in the art. lsosteric replacements include Abu for Cys (this may be desirable where the peptide should retain an even number of Cys residues for cyclisation), Phe for Tyr and different alkyl/aryl substituents. The shifting of substituents within an amino acid residue, from a C atom to an N atom, to produce peptoids having greater resistance to proteolysis, and other modifications, are known and are included within the scope of this invention. Peptides of the invention may also include N modifications, such as N-alkylation or N-acetylation. Other terminal modifications will also be known to those skilled in the art. Such modified peptides may act as prodrugs and/or have modified immunogenecity.
  • the activity of the compounds of the invention means that they may be useful in the treatment of diseases in which NP-1 may have a significant role in pathology.
  • compounds of the invention may be formulated and administered by procedures, and using components, known to those of ordinary skill in the art.
  • the appropriate dosage of the compound may be chosen by the skilled person having regard to the usual factors such as the condition of the subject to be treated, the potency of the compound, the route of administration etc. Suitable routes of administration include oral, intravenous, intramuscular, intraperitoneal, intranasal and subcutaneous.
  • a NP-1 antagonist may compete with semaphorin 3A for binding to NP-1 , and thereby antagonise inhibitory effects of semaphorin 3A on axonal outgrowth and migration in nerve cells. Potential applications of this are in promoting neurite outgrowth, in stimulating nerve repair or treating neurodegeneration. Further, an NP-1 antagonist may promote the survival of semaphorin 3A- responsive neurones, an effect that would confirm or enhance its utility in the applications given above, and may extend these applications, e.g. to treating neuronal death caused by episodes of ischaemia as in stroke and some eye diseases.
  • NP-1 plays an important role in angiogenesis and may be essential for VEGF-induced angiogenesis in cancer, eye disease, rheumatoid arthritis and other diseases. Therefore, NP-1 antagonists may have applications in the inhibition of VEGF-dependent angiogenesis in disease. NP-1 antagonists may also play a role in immunosupression. Therefore, it may be useful to give a compound of the invention before, during or after a transplant.
  • a NP-1 antagonist may compete with VEGF for binding to NP- 1 in tumour cells and promote cell death in NP-1 -expressing tumour cells. Potential applications of this are in anti-cancer therapy.
  • the following Examples illustrate the invention. Experimental Detail Abbreviations Abu, aminobutyric acid; Aib, 2-aminoisobutyric acid; Ala or A, alanine;
  • Arg or R Arginine; asparagine; Asp or D, aspartic acid; GIn or Q, glutamine; GIu or E, glutamic acid; Leu or L, leucine; Lys or K, lysine; Phe of F, phenylalanine; Pro or P, proline; Ser or S, serine; Thr or T, threonine; Tyr or Y, tyrosine; Boc, tert-butoxy carbonyl; Fmoc, 9-fluorenylmethoxy-carbonyl; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; Trt, trityl; tBu, tert-butyl; HPLC, high performance liquid chromatography; LC-MS, liquid chromatography mass spectrometry; TLC, thin-layer chromatography.
  • Peptides were purified by reverse-phase HPLC using a preparative C-18 column. The relevant fractions were collected, evaporated, lyophilized (-54 0 C, 0.08 mbar) and stored at 4 0 C.
  • H-DKAAR-OH EG00105 White solid, 22 mg, HPLC R t 9.20 min.; purity > 95%; MS m/z - 560.3
  • H-KPFR-OH EG00119 White solid, 30 mg, HPLC R t 11.43 min.; purity > 95%; MS m/z -
  • H-TKPRR-OH EG00208 White solid, 20 mg, HPLC R 1 8.49 min.; purity > 95%; MS m/z- 657.9
  • Porcine aortic endothelial cells expressing neuropilin-1 were cultured in Ham's F12 medium containing 10% fetal bovine serum (FBS) and 25 ⁇ g/ml hygromycin B.
  • PAE cells expressing KDR were grown in
  • Human carcinoma cell lines (DU145, A549 and ACHN) were grown in RPMI
  • 125 I-VEGF-A 165 binding Confluent cells in 24-well plates were washed twice with phosphate- buffered saline (PBS). At 4°C various concentrations of peptides or peptidomimetics diluted in binding medium (Dulbecco's modified Eagle's medium, 25 mM HEPES pH 7.3 containing 0.1% BSA) were added, followed by addition of 0.1 nM of 125 I-VEGF-A 165 (1200-1800 Ci/mmol, GE Healthcare). After 2 h of incubation at 4°C, the medium was aspirated and washed 4 times with cold PBS. The cells were lysed with 0.25 M NaOH, 0.5% SDS solution, and the bound radioactivity of the lysates was measured in a ⁇ counter. Non-specific binding was determined in the presence of 100-fold excess unlabelled VEGF-
  • Cell adhesion to extracellular matrix proteins was measured by the lnnocyte ECM cell adhesion assay (Calbiochem).
  • Cells were detached with a non-enzyme cell dissociation solution (Sigma), washed and resuspended in RPMI 1640 medium.
  • Cells with or without peptide or peptidomimetic treatment were seeded at 3 x 10 4 cells per matrix-coated well of 96-well plates. After 1.5 h incubation, cells were washed with PBS. The attached cells were labelled with the green fluorescent dye caicein-AM and measured using a fluorescence plate reader at an excitation wavelength of 485 nm and an emission wavelength of 520 nm.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne un peptide comportant jusqu'à 7 acides aminés et comprenant la séquence K-X1-X2-R, dans laquelle X1 est P ou A et dans laquelle X2 est n'importe quel acide aminé ou COCONH. Les peptides de l'invention peuvent être utilisés pour traiter le cancer et la neurodégénérescence et pour stimuler la réparation des nerfs.
PCT/GB2007/003775 2006-10-04 2007-10-04 Courts antagonistes peptidiques de la neuropiline-1 et leur utilisation WO2008040986A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB0619610.9A GB0619610D0 (en) 2006-10-04 2006-10-04 Compounds and their use
GB0619610.9 2006-10-04

Publications (1)

Publication Number Publication Date
WO2008040986A1 true WO2008040986A1 (fr) 2008-04-10

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2007/003775 WO2008040986A1 (fr) 2006-10-04 2007-10-04 Courts antagonistes peptidiques de la neuropiline-1 et leur utilisation

Country Status (2)

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GB (1) GB0619610D0 (fr)
WO (1) WO2008040986A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060153775A1 (en) * 2000-06-02 2006-07-13 Bracco International B.V. Compounds for targeting endothelial cells, compositions containing the same and methods for their use
WO2006130504A2 (fr) * 2005-05-27 2006-12-07 Advanced Cell Technology, Inc. Methodes destinees a l'identification de ligands pour des cellules souches et cellules derivees de celles-ci

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060153775A1 (en) * 2000-06-02 2006-07-13 Bracco International B.V. Compounds for targeting endothelial cells, compositions containing the same and methods for their use
WO2006130504A2 (fr) * 2005-05-27 2006-12-07 Advanced Cell Technology, Inc. Methodes destinees a l'identification de ligands pour des cellules souches et cellules derivees de celles-ci

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
GERDIN B ET AL: "Structural requirements for microvascular permeability-increasing ability of peptides. Studies on analogues of a fibrinogen pentapeptide fragment.", BIOCHIMICA ET BIOPHYSICA ACTA 9 JUN 1983, vol. 757, no. 3, 9 June 1983 (1983-06-09), pages 366 - 370, XP002463280, ISSN: 0006-3002 *
GERDIN B ET AL: "STRUCTURAL SIMILARITIES BETWEEN SYNTHETIC ANALOGS TO A VASOACTIVE PEPTIDE DERIVED FROM HUMAN FIBRINOGEN AND OTHER BIOLOGICALLY ACTIVE PEPTIDES", PROGRESS IN FIBRINOLYSIS, CHURCHILL LIVINGSTONE, GB, 1981, pages 1 - 3, XP008078625, ISSN: 0262-0790 *
LEUNG-TACK J ET AL: "Inhibition of phagocyte functions by a synthetic peptide Lys-Pro-Pro-Arg (postin)", PROTIDES OF THE BIOLOGICAL FLUIDS, PERGAMON PRESS, OXFORD, GB, vol. 34, 1986, pages 205 - 208, XP008087147, ISSN: 0079-7065 *
NICOLAIDES E D ET AL: "ANTI-WRITHING ACTIVITY OF SOME PEPTIDES RELATED TO NEUROTENSIN AND TUFSIN", INTERNATIONAL JOURNAL OF PEPTIDE AND PROTEIN RESEARCH, MUNKSGAARD, COPENHAGNE, DK, vol. 25, no. 4, 1 April 1985 (1985-04-01), pages 435 - 441, XP000577690, ISSN: 0367-8377 *
VON WRONSKI MATHEW A ET AL: "Tuftsin binds neuropilin-1 through a sequence similar to that encoded by exon 8 of vascular endothelial growth factor.", THE JOURNAL OF BIOLOGICAL CHEMISTRY 3 MAR 2006, vol. 281, no. 9, 3 March 2006 (2006-03-03), pages 5702 - 5710, XP002463279, ISSN: 0021-9258 *
WILLIAMS G ET AL: "A complementary peptide approach applied to the design of novel semaphorin/neuropilin antagonists", JOURNAL OF NEUROCHEMISTRY, NEW YORK, NY, US, vol. 92, no. 5, March 2005 (2005-03-01), pages 1180 - 1190, XP002363727, ISSN: 0022-3042 *

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Publication number Publication date
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