WO1999007728A2 - Linear peptides derived from antibiotic peptides, preparation and use for vectoring active substances - Google Patents

Linear peptides derived from antibiotic peptides, preparation and use for vectoring active substances Download PDF

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Publication number
WO1999007728A2
WO1999007728A2 PCT/FR1998/001757 FR9801757W WO9907728A2 WO 1999007728 A2 WO1999007728 A2 WO 1999007728A2 FR 9801757 W FR9801757 W FR 9801757W WO 9907728 A2 WO9907728 A2 WO 9907728A2
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Prior art keywords
peptides
peptide
groups
antibiotic
linear peptide
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PCT/FR1998/001757
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French (fr)
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WO1999007728A3 (en
Inventor
Bernard Calas
Gérard Grassy
Alain Chavanieu
Michel Kaczorek
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Synt:Em (S.A.)
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Priority to CA002298932A priority Critical patent/CA2298932A1/en
Priority to EP98941556A priority patent/EP1003771A1/en
Priority to IL13429398A priority patent/IL134293A0/en
Priority to AU89889/98A priority patent/AU754617B2/en
Priority to JP2000506230A priority patent/JP2001512739A/en
Publication of WO1999007728A2 publication Critical patent/WO1999007728A2/en
Publication of WO1999007728A3 publication Critical patent/WO1999007728A3/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A61K47/6897—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies
    • A61K47/6898—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies using avidin- or biotin-conjugated antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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 linear peptides derived from antibiotic peptides and their use for vectorizing active substances. More particularly, the subject of the invention is new compounds formed from a linear derivative of an antibiotic peptide linked to at least one active substance, as well as the preparation of these compounds and the compositions containing them.
  • Antimicrobial peptides from vertebrates whatever their origin, lower or higher vertebrates, myeloid or non-myeloid tissues, have a number of common properties:
  • amphipathic structure is understood to mean structures in which the hydrophobic residues are spatially separated from the hydrophilic residues.
  • antibiotic peptides can be classified into three main families:
  • antibiotic peptides with destructured chains containing numerous bends linked to the presence of multiple prolines bactenecins and PR39 (Frank, R. W. et al., 1991, Eur. J. Biochem. 202, 849-
  • antibiotic peptides act by direct lysis of the membrane of pathogenic cells. Their basic nature facilitates their interaction with negatively charged phospholipids, and their amphipatic nature then allows them to be incorporated into the membrane where they aggregate to form pores through which the cell loses its substance. It is generally accepted that their preferential selectivity for prokaryotic cells is due to the particular composition of their membranes which contain more anionic phospholipids than those of eukaryotes. In addition, the plasma membranes of mammalian cells all contain cholesterol, the role of which is to modulate its fluidity. and which could hinder the incorporation of antibiotic peptides. However, the specificity of the latter for microorganisms is low so that they have a high cytotoxicity which limits their use.
  • antibiotic peptides are of considerable interest because of their broad spectrum of action and the difficulty that microorganisms have in implementing inactivation strategies. As a result, a great deal of research is undertaken to try to find new molecules and to obtain analogues which are more efficient than the parent peptides. In the future, these antibiotic peptides may be called upon to replace antibiotics from bacteria or fungi.
  • the PCT international patent applications published under the numbers O95 / 03325, WO96 / 37508 and WO97 / 02287 describe a new class of antibiotic peptides, designated "protectins", isolated from pig leukocytes or else prepared by chemical synthesis or by genetic engineering and exhibiting antibacterial, antiviral and antifungal activities.
  • Protinins are used to designate a set of five peptides designated PG-1, PG-2, PG-3, PG-4 and PG-5, the sequences of which are given below, closely related and isolated from pig leukocytes ( VN Kokryakov & col. FEBS lett. 327, 231-236):
  • Proteins, tachyplines and polyphemusins contain a high proportion of basic residues (lysines and arginines) and have four cysteines which form two parallel disulfide bridges. These three families of peptides also exhibit homologies with certain defensins and in particular with human defensin NP-1 (Kokryakov, V. N. et al., 1993, Febs Let. 327, 231-236).
  • Tachyplines and protectins have a similar three-dimensional structure. It is an antiparallel ⁇ sheet stabilized by the two disulfide bridges. These bridges play an important role in the antibacterial activity of protectins and tachyplesins. Their removal, either by protecting the SH groups with acetamidomethyls, or by replacing the cysteines with alanines or glycines, leads to analogs practically devoid of activity in vivo (Lehrer, RI et al., 1996, Eur. J. Biochem. 240: 352-357).
  • the protectins and the tachyplesins have an important lytic activity on the prokaryotic cells.
  • the research carried out by the Applicant on the cytotoxicity of these peptides on cultured mammalian cells has made it possible to demonstrate, before the death of the cells, non-negligible amounts of protectins and tachyplesins in the cytoplasm of said cells. . It has been envisaged that the presence of peptides in the cytoplasm could result from transport through pores, but these pores are only permeable to ions and small molecules and their diameter is too small to allow the passage of peptides. antibiotics. It would seem that the proteines and tachyplines, in addition to puncturing the plasma membrane, are able to cross it.
  • vectorization system a process capable of transporting said active substance to a target, such as for example:
  • the subject of the present invention is therefore peptides derived from antibiotic peptides or analogs thereof, characterized in that they are devoid of the disulfide bridge.
  • analog Pept ides antibiotic, a peptide whose amino acid sequence has been modi f ied without this n 'enta viable modif ication in the antibiotic properties of said peptide.
  • the above modifications advantageously relate to all the cysteine residues of the antibiotic peptide, but as soon as the presence of a single cysteine residue does not allow the formation of a disulfide bridge, the peptides of the invention can contain a single cysteine.
  • Natural antibiotic peptides generally have 4 or 6 cysteine residues capable of forming two or three disulfide bridges, also in the peptides of the invention, only one of these cysteines can be maintained and the other three or five are modified or blocked .
  • the antibiotic peptides from which the peptides of the invention are derived may be defensins, protectins, tachyplesins or their analogs, the antibiotic properties of which are imparted to them by their tertiary structure resulting from the presence of disulfide bridges.
  • Linear peptides according to the invention correspond to one of the following formulas:
  • BBXXXBXXXBXXXXBBXB (II) which can also be represented by the following unique formula (III): B (XB) X (XB) X (XB) XX (XB) B (XB) XXX (XB) (XB) XB in which:
  • - groups B identical or different, represent an amino acid residue whose side chain carries a basic group
  • - groups X identical or different, represent an aliphatic or aromatic amino acid residue, or consist of '' a sequence of at least
  • B and X can be natural amino acids or not, including amino acids of configuration D.
  • B and X can be cited: - B is chosen from arginine, lysine, diaminoacetic acid, diaminobutyric acid, 1 diaminopropionic acid, 1 ornithine.
  • - X is chosen from glycine, alanine, valine, norleucine, isoleucine, leucine, cysteine, cysteine, penicillamine, methionine, serine, threonine, 1 asparagine, glutamine, phenylalanine, histidine, tryptophan, tyrosine, proline, Abu, amino-1-cyclohexane carboxylic acid, Aib, 2-aminotetraline carboxylic, 4-bromophenylalanine, tert-Leucine, 4-chlorophenylalanine, ⁇ -cyclohexylalanine, 3, 4-dichlorophenylalanine, 4-fluorophenylalanine, 1 homoleucine, ⁇ -homoleucine,
  • the invention also relates to derivatives of the peptides of formulas (I) or (II) such as said peptides in retro form, or fragments of the peptides of formulas (I) or (II) consisting of five and preferably seven successive amino acids of one of the formulas (I) or (II).
  • derivatives of the peptides of formulas (I) or (II) such as said peptides in retro form, or fragments of the peptides of formulas (I) or (II) consisting of five and preferably seven successive amino acids of one of the formulas (I) or (II).
  • groups X represent a natural or unnatural amino acid (including amino acids of configuration D) aliphatic or aromatic, such as glycine, alanine, valine, norleucine, isoleucine, leucine, cysteine , the
  • ⁇ rjrn cysteine penic il lamine, methionine, serine, threonine, asparagine, glutamine, phenylalanine, histidine, tryptophan, tyrosine, proline, 1 'bu, amino-1 acid -carboxylic cyclohexane, Aib, carboxylic 2-aminotetralin, 4-bromophenylalanine, tert-Leucine, 4-chlorophenylalanine, ⁇ -cyclohexylalanine, 3,4-dichlorophenylalanine, 4-f luorophenylalanine, 1 'homo leuc , ⁇ -homo leucine, 1 homophenylalanine,
  • B represents Napthylalanine
  • O represents Ornithine
  • X represents Norleucine
  • Z represents Norvaline.
  • the invention also relates to the use of the above peptides for the vectorization of one or more active substances both for therapeutic and diagnostic applications.
  • active substance the invention envisages in particular proteins or protein fragments, such as polypeptides or peptides, antibodies or part of antibodies, nucleic acids and oligonucleotides or ribozymes, or, of course, active chemical molecules for the treatment or prevention of human or animal pathologies, such as, for example and without limitation, anti-tumor agents , antivirals, anti-inflammatory agents, agents preventing the degradation of organs and / or tissues, etc.
  • the active substance can be a radioactive marker, a colored marker, or any other means or substance capable of revealing a metabolism or a pathology.
  • the invention therefore also relates to compounds of formula (IV) below, as well as the compositions containing them:
  • - Y represents a signal agent
  • - n is 0 and or more, and advantageously 0 or 1
  • - m is 1 or more, and preferably up to 10 advantageously up to 5.
  • the compounds of formula (IV) above are formed from a peptide of the invention coupled to one or more active substances, identical or different, represented by the group (Z) in the formula (IV), and optionally one or more signal agents, represented by the group (Y) in formula (IV), having a role of addressing the compound of formula
  • the signal agent (Y) is an oligopeptide or a protein, such as a signal peptide, a nuclear localization signal, an antibody fragment, or a chemical molecule ligand or anti-ligand of a receptor.
  • the group (Y) is attached to the group (Z).
  • the coupling symbolized by the horizontal lines in the formula (IV), can be carried out by any acceptable means of connection taking into account the chemical nature, the bulk and the number of groups (Z) and (Y) in the compounds of formula (IV), such as covalent, hydrophobic or ionic bonds, cleavable or non-cleavable in physiological media.
  • the coupling can be carried out at any site of the peptide (A), in which functional groups such as -OH, -SH, -COOH, -NH2 are naturally present or have been introduced.
  • the invention also envisages the attachment of several groups (Z) to the same site of the peptide (A), either directly, if this site comprises several functional groups, as in the case of a C- or N-terminal lysine, either indirectly via an intermediate group carrying several reaction groups making it possible to fix therein several groups (Z).
  • the preferred coupling positions for the active substance are at the N-terminal or C-terminal ends or else at the level of the primary amino groups carried by the side chains of the lysines of peptide (A).
  • the N-terminal end of the peptide (A) is used to hook the active substance (Z)
  • the N-terminal end is available for possible coupling to a signal agent (Y) allowing the addressing the compound of the invention either towards the nucleus, or even towards a particular tissue type.
  • the covalent peptide-drug complex after administration is distributed in the cytoplasm of the target cell. It is possible to bring this complex into the nuclear compartment by coupling to the N-terminal end of the peptide a short basic sequence, for example of about 7 amino acids, corresponding to a nuclear localization signal. Under these conditions, biotin or doxorubicin is found in the nucleus of the cell.
  • the pentadecapeptide 0CM2 (Swolapenko, GB et al., 1995, The Lancet 346, 1662-65) synthetic, fragment of a monoclonal antibody, directed against an antigen expressed by breast cancer cells (Tumor Associated Antigen Polymorphic Epithelial Mucin), retains a good affinity for these cells. It is therefore possible, by combining aM2 with a linear peptide-drug set, to bring this set preferentially towards the cells which express the antigenic characteristic linked to breast cancer.
  • the compounds of formula (IV) can be prepared by chemical synthesis or by using molecular biology techniques. It is possible to use for chemical synthesis commercial devices making it possible to incorporate non-natural amino acids, such as the D enantiomers and residues having side chains having hydrophobicities and bulkings different from those of their natural counterparts. During the synthesis, it is obviously possible to make a wide range of modifications, for example introducing a lipid (prenyl or myristyl) onto the N-terminal so as to be able to anchor the peptide of the invention and therefore the compound of formula (IV) to a lipid membrane such as that of a liposome consisting of positively charged lipids.
  • a lipid prenyl or myristyl
  • the present invention also relates to a nucleic acid molecule comprising or consisting of a nucleic sequence coding for a linear peptide derived from antibiotic peptide. More particularly the invention relates to a nucleic acid molecule comprising at least one sequence coding for a compound of formula (IV) or part of that of a protein nature.
  • These nucleic acid sequences can be DNA or RNA and be associated with control sequences and / or be inserted into vectors. The vector used is chosen according to the host to which it will be transferred; it can be any vector such as a plasmid.
  • nucleic acids and vectors are useful for producing linear peptides and compounds of formula (IV) or part of them of a protein nature in a cellular host.
  • such a process for producing a peptide according to the invention consists in: transferring a nucleic acid molecule or a vector containing said molecule to a cellular host,
  • the cell host used in this type of process can be chosen from prokaryotes or eukaryotes and in particular from bacteria, yeasts, mammalian, plant or insect cells.
  • the invention therefore also relates to the transformed cells expressing the linear peptides or the compounds of formula (IV) or part of these of a protein nature.
  • the invention also relates to: pharmaceutical compositions comprising as active principle at least one compound of formula (IV) optionally associated with an acceptable vehicle or support.
  • Example 1 Binding of biotin and doxorubic ine on linear analogues of antibiotic peptides.
  • RGGRLCYCRRRFCVCVGR-NH2 tachyplesin 1 of formula: KWCFRVCYRGICYRRCR-NH2, polyphemusine of formula:
  • These three peptides can be prepared either from a BOC chemistry or from a FMOC chemistry, by conventional methods of synthesis in solid or homogeneous phase.
  • the peptide After cleavage of the purification support, the peptide is treated with glutaric anhydride in the presence of triethylamine. The peptide is then purified and the -COOH group carried by the glutaryl at the N-terminal is activated by the mixture of diisopropylcarbodiimide and 1-hydroxybenzotriazole. After two hours of reaction at room temperature, doxorubicin is added and the mixture is stirred for 12 hours at 0 ° C. The whole pept ide-doxorubic ine is then purified by high pressure liquid chromatography.
  • Example 2 Ability of linear peptides of the invention to pass cell membranes.
  • the ability of peptides to pass membranes has been tested on various cell types (MCF7, MCF7R, HL60, HL60R, HeLa).
  • the cells are cultured on RPMI 1640
  • the cells are incubated in Opti-Mem (Gibco) for one hour before being treated for variable times with the peptides labeled with biotin.
  • Example 1 The latter are obtained in accordance with Example 1 (2) by treating 1 equivalent of linear peptide with 2 equivalents of N-hydroxysuccinimide ester of biotin, then purified by high pressure liquid chromatography.
  • the cells are then fixed with a 3.7% paraformaldehyde solution for 5 minutes at
  • the cells are incubated for 15 minutes, then rinsed with PBS and then the doxorubicin present in the cell is determined by chromatography.
  • - U represents serine or threonine
  • - R represents arginine
  • groups X represent a natural or unnatural amino acid (including amino acids of configuration D) aliphatic or aromatic, such as glycine, alanine, valine, norleucine, isoleucine, leucine, cysteine , the
  • .A.cm cysteine penicillamine, methionine, serine, threonine, asparagine, glutamine, phenylalanine, histidine, tryptophan, tyrosine, proline, 1 'Abu, amino acid 1-cyclohexane carboxylic, Aib, 2-aminotetralin carboxylic, 4-bromophenylalanine, tert-Leucine, 4-chlorophenylalanine, ⁇ -cyclohexylalanine, 3,4-dichlorophenylalanine, 4 -fluorophenylalanine, 1 'homoleucine ⁇ -homoleucine, 1 homophenylalanine,
  • biotin alone does not enter the cell and accumulates weakly around it.
  • biotin is rapidly entrained by the linear peptide of the invention inside the cell where it is present in the cytoplasm and in the nucleus of the cell .
  • Example 3 Capacity for internalization of the linear peptides of the invention.
  • Linear peptides of the invention derived from Proteins and Tachyplesins were tested on different cell lines, with the aim of evaluating their respective internalization.
  • the cells are seeded at approximately 10 cells per well, 24 h before the addition of the biotinylated peptides. They are then at 60-80% confluence on the day of the experiment.
  • the biotinylated peptides are incubated with the cells at a concentration of 10 ⁇ M, for 15 minutes, at 37 ° C. in an atmosphere at 95% humidity and 5% CO 2 in an OptiMem medium.
  • the cells are washed three times with PBS at room temperature and then are fixed with formalin (3.7% formaldehyde in PBS, 10 min at room temperature). They are then washed with PBS and permeabilized for 15 min with PBS-TritonX-100.
  • the cell nuclei were stained with Hoechst.
  • Non-tumor lines MRC5 (lung fibroblast), HuVeC (endothelial, umbilical cord).
  • Tumor lines HT29 (colon carcinoma), HepG2 (hepatoblastoma), A172 (glioblastoma), HMCB (elanoma).
  • the cells are cultured at 37 ° C. in an atmosphere at 95% humidity and 5% CO 2.
  • the culture medium is that recommended by the ATCC.
  • FIGS. 1 and 2 The fluorescence microscopy photos of the internalization are presented in FIGS. 1 and 2.
  • the peptide SM 1738 presented by way of example, appears to be located mainly in the cytoplasm and in a perinuclear zone.
  • the peptide has a localization mainly cytoplasmic.
  • the left column corresponds to the coloring of the nucleus by Hoechst.
  • the internal i sat ion photos are shown in Figures 3 and 4 in the appendix.
  • the biotinylated peptide is localized in the cytoplasm in a diffuse fashion and also clearly marks nuc leole.
  • the left column corresponds to the coloring of the nucleus by Hoechst.
  • the cells are seeded at approximately 10 cells per well, 24 h before the addition of the products. They are then at 60-80% confluence on the day of the experiment.
  • Free doxorubicin on the one hand, or doxorubicin coupled to the vector SM1738 on the other hand are incubated with MCF7 cells at a concentration of 10 ⁇ M, for 60 minutes, at 37 ° C. in an atmosphere at 95% humidity. and 5% CO 2 in the culture medium.
  • the subcellular localization of the naturally fluorescent doxorubicin was determined by confocal microscopy. The results are presented in Figure 5 in the appendix. Localization is partly cytoplasmic and partly nuclear. The nucleus is then diffusedly marked.
  • amino acids are represented by their one-letter code, but they can also be represented by their three-letter code according to the nomenclature below.

Abstract

The invention concerns peptides derived from antibiotic peptides or analogues thereof, characterised in that they are devoid of sulphide bond. The invention also concerns the use of these linear peptides for vectoring chemical substances and chemical compounds formed by said peptides coupled with at least an active substance. The invention further concerns the preparation of said peptides and compositions containing them.

Description

PEPTIDES LINEAIRES DERIVES DE PEPTIDES ANTIBIOTIQUES, LEUR PRÉPARATION ET LEUR UTILISATION POUR VECTORISER DES SUBSTANCES ACTIVES. LINEAR PEPTIDES DERIVED FROM ANTIBIOTIC PEPTIDES, THEIR PREPARATION AND THEIR USE FOR VECTORIZING ACTIVE SUBSTANCES.
La présente invention concerne des peptides linéaires dérivés de peptides antibiotiques et leur utilisation pour vectoriser des substances actives. Plus particulièrement l'invention a pour objet de nouveaux composés formés d'un dérivé linéaire d'un peptide antibiotique lié à au moins une substance active, ainsi que la préparation de ces composés et les compositions les contenant .The present invention relates to linear peptides derived from antibiotic peptides and their use for vectorizing active substances. More particularly, the subject of the invention is new compounds formed from a linear derivative of an antibiotic peptide linked to at least one active substance, as well as the preparation of these compounds and the compositions containing them.
A côté de leur système immunitaire responsable de mécanismes spécifiques de défense contre les agents infectieux, les vertébrés possèdent de nombreux peptides à activité antimicrobienne (Nicolas, P. et al., 1995, Annual Rev. Microbiol . 49, 277-304). Ces peptides sont seulement présents chez les invertébrés à courte durée de vie et à taux de renouvellement élevé, chez lesquels un système immunitaire à mémoire, long à s'établir et à développer une réponse appropriée, serait inadapté.Besides their immune system responsible for specific defense mechanisms against infectious agents, vertebrates have numerous peptides with antimicrobial activity (Nicolas, P. et al., 1995, Annual Rev. Microbiol. 49, 277-304). These peptides are only present in short-lived, high-turnover invertebrates, in which a long-established immune system with memory and in developing an appropriate response would be inadequate.
Les peptides antimicrobiens des vertébrés, quelque soit leur origine, vertébrés inférieurs ou supérieurs, tissus myéloïdes ou non myéloïdes, possèdent un certain nombre de propriétés communes :Antimicrobial peptides from vertebrates, whatever their origin, lower or higher vertebrates, myeloid or non-myeloid tissues, have a number of common properties:
- Une forte basicité due à la présence de nombreuses arginines et lysines. - La capacité de former des structures amphipatiques . On entend par structure amphipatique des structures dans lesquelles les résidus hydrophobes sont spatialement séparés des résidus hydrophiles.- Strong basicity due to the presence of many arginines and lysines. - The ability to form amphipathic structures. The term “amphipatic structure” is understood to mean structures in which the hydrophobic residues are spatially separated from the hydrophilic residues.
- Un très large spectre d'activité. Ils sont capables de détruire rapidement des bactéries (Gram+ et- A very broad spectrum of activity. They are able to quickly destroy bacteria (Gram + and
Gram- ) , des champignons, quelques protozoaires, des virus à membrane et même certaines lignées de cellules cancéreuses .Gram-), fungi, some protozoa, membrane viruses and even certain cancer cell lines.
Selon leur structure, les peptides antibiotiques peuvent être classés en trois grandes familles :According to their structure, antibiotic peptides can be classified into three main families:
- Les peptides antibiotiques à hélices amphipatiques : cécropines et maganines (Maloy, W. L. et al., 1995, BioPolymer 37, 105-122) .- Antibiotic peptides with amphipatic helices: cecropins and maganins (Maloy, W. L. et al., 1995, BioPolymer 37, 105-122).
- Les peptides antibiotiques à feuillets β réunis par des ponts disulfures : défensines (Lehrer, R.- Antibiotic peptides with β sheets united by disulfide bridges: defensins (Lehrer, R.
I. et al., 1991, Cell 64:229-230 ; Lehrer, R. I. et al., 1993, Ann . Rev . Immunol . 11:105-128) , protégrines (Kokryakov, V. N. et al. , 1993, FEBS 337:231-236) , tachyplésines (Nakamura, T. et al., 1988, J. Biol. Chem. 263:16709-16713 ; Miyata, T et al., 1989, J. Biochem.I. et al., 1991, Cell 64: 229-230; Lehrer, R. I. et al., 1993, Ann. Rev. Immunol. 11: 105-128), protectins (Kokryakov, VN et al., 1993, FEBS 337: 231-236), tachyplesins (Nakamura, T. et al., 1988, J. Biol. Chem. 263: 16709-16713; Miyata, T et al., 1989, J. Biochem.
106:663-668) . les peptides antibiotiques à chaînes déstructurées contenant de nombreux coudes liés à la présence de multiples prolines : bacténécines et PR39 (Frank, R. W. et al., 1991, Eur . J. Biochem. 202, 849-106: 663-668). antibiotic peptides with destructured chains containing numerous bends linked to the presence of multiple prolines: bactenecins and PR39 (Frank, R. W. et al., 1991, Eur. J. Biochem. 202, 849-
854) .854).
Malgré la diversité de leurs séquences, la plupart des peptides antibiotiques agissent par lyse directe de la membrane des cellules pathogènes. Leur nature basique facilite leur interaction avec les phospholipides chargés négativement, et leur caractère amphipatique leur permet ensuite de s'incorporer dans la membrane où ils s'agrègent pour former des pores par lesquels la cellule perd sa substance. Il est généralement admis que leur sélectivité préférentielle pour les cellules procaryotes, est due à la composition particulière de leurs membranes qui contiennent davantage de phospholipides anioniques que celles d ' eucaryotes . De plus, les membranes plasmiques de cellules de mammifères contiennent toutes du cholestérol, dont le rôle est d'en moduler la fluidité et qui pourrait gêner l'incorporation des peptides antibiotiques. Toutefois, la spécificité de ces derniers pour les microorganismes est faible si bien qu'ils présentent une forte cytotoxicité ce qui en limite l'utilisation.Despite the diversity of their sequences, most antibiotic peptides act by direct lysis of the membrane of pathogenic cells. Their basic nature facilitates their interaction with negatively charged phospholipids, and their amphipatic nature then allows them to be incorporated into the membrane where they aggregate to form pores through which the cell loses its substance. It is generally accepted that their preferential selectivity for prokaryotic cells is due to the particular composition of their membranes which contain more anionic phospholipids than those of eukaryotes. In addition, the plasma membranes of mammalian cells all contain cholesterol, the role of which is to modulate its fluidity. and which could hinder the incorporation of antibiotic peptides. However, the specificity of the latter for microorganisms is low so that they have a high cytotoxicity which limits their use.
La présence de peptides antibiotiques chez les vertébrés et plus particulièrement chez les mammifères soulève de nombreuses questions. Les immunologistes supposent que les composés à activité antimicrobienne non-spécifique que l'on rencontre au niveau des invertébrés constituent un moyen ancestral de défense qui a ensuite évolué pour conduire aux systèmes à mémoire beaucoup plus complexes . Quel est donc l'intérêt pour les mammifères, par exemple, d'avoir conservé certains peptides à activité antibiotique ? On admet que ces petites molécules toujours présentes dans les fluides biologiques, ou encore séquestrées dans certaines structures lymphocytaires , pourraient constituer une première ligne de défense en attendant que les anticorps spécifiques soient sécrétés (Nicolas, P. et al., 1995, Annual Rev. Microbiol . 49, 277-304). Ils pourraient également participer au sein des macrophages, à la destruction des membranes plasmiques des organismes pathogènes. Quelque soit leur rôle exact, les peptides antibiotiques ont un intérêt considérable du fait de leur large spectre d'action et de la difficulté que les microorganismes ont à mettre en place des stratégies d' inactivation. De ce fait de très nombreuses recherches sont entreprises pour essayer de trouver de nouvelles molécules et d'obtenir des analogues plus performant que les peptides parents. Il se peut que dans l'avenir, ces peptides antibiotiques soient appelés à remplacer les antibiotiques issus de bactéries ou de champignons . Ainsi les demandes de brevet internationales PCT publiées sous les numéros O95/03325, WO96/37508 et WO97/02287 décrivent une nouvelle classe de peptides antibiotiques, désignés "protégrines " , isolés de leucocytes de porcs ou encore préparés par synthèse chimique ou par génie génétique et présentant des activités antibactériennes, antivirales et antifongiques .The presence of antibiotic peptides in vertebrates and more particularly in mammals raises many questions. Immunologists assume that the compounds with non-specific antimicrobial activity found in invertebrates constitute an ancestral defense which has then evolved to lead to much more complex memory systems. So what is the advantage for mammals, for example, of having preserved certain peptides with antibiotic activity? We admit that these small molecules always present in biological fluids, or even sequestered in certain lymphocyte structures, could constitute a first line of defense while waiting for specific antibodies to be secreted (Nicolas, P. et al., 1995, Annual Rev. Microbiol. 49, 277-304). They could also participate within macrophages, in the destruction of the plasma membranes of pathogenic organisms. Whatever their exact role, antibiotic peptides are of considerable interest because of their broad spectrum of action and the difficulty that microorganisms have in implementing inactivation strategies. As a result, a great deal of research is undertaken to try to find new molecules and to obtain analogues which are more efficient than the parent peptides. In the future, these antibiotic peptides may be called upon to replace antibiotics from bacteria or fungi. Thus, the PCT international patent applications published under the numbers O95 / 03325, WO96 / 37508 and WO97 / 02287 describe a new class of antibiotic peptides, designated "protectins", isolated from pig leukocytes or else prepared by chemical synthesis or by genetic engineering and exhibiting antibacterial, antiviral and antifungal activities.
Actuellement, les peptides antibiotiques à feuillets β réunis par des ponts disulfures (défensines, protégrines, tachyplésines ) sont particulièrement étudiés étant donné leur puissante activité antimicrobienne (bactéries, certains virus, champignons et parasites) . Dans cette famille, les protégrines et les tachyplésines sont certainement les molécules les plus prometteuses étant donné la simplicité de leur structure et la facilité relative de leur synthèse.Currently, β-sheet antibiotic peptides joined by disulfide bridges (defensins, protectins, tachyplines) are particularly studied given their powerful antimicrobial activity (bacteria, certain viruses, fungi and parasites). In this family, protectins and tachyplines are certainly the most promising molecules given the simplicity of their structure and the relative ease of their synthesis.
On désigne sous le nom de protégrines un ensemble de cinq peptides désignés PG-1, PG-2, PG-3, PG-4 et PG-5 dont les séquences sont données ci-dessous, étroitement apparentés et isolés de leucocytes de porc (V.N. Kokryakov & col. FEBS lett . 327, 231-236) :Protinins are used to designate a set of five peptides designated PG-1, PG-2, PG-3, PG-4 and PG-5, the sequences of which are given below, closely related and isolated from pig leukocytes ( VN Kokryakov & col. FEBS lett. 327, 231-236):
PG-1 RGGRLCYCRRRFCVCVGR-NH2 PG-2 RGGRLCYCRRRFCICV.. -NH2 PG-3 RGGGLCYCRRRFCVCVGR-NH2 PG-4 RGGRLCYCRGWICFCVGR-NH2 PG-5 RGGRLCYCRPRFCVCVGR-NH2 Les tachyplésines (Tamura, H. et al., 1993, Chem. Pharm. Bul. Tokyo 41, 978-980), désignées Tl , T2 et T3 et les polyphémusines (Muta, T., 1994, CIBA Found. Sym. 186, 160-174), désignées PI et P2 , dont les séquences sont données ci-dessous, sont des peptides homologues isolés de l' hémolymphe de deux crabes, Tachyplesus tridentatus pour les tachyplésines Tl , T2 et T3 et Limmulus polyphemus pour les polyphémusines Pi et P2. PI : RRWCFRVCYRGFCYRKCR-NH2PG-1 RGGRLCYCRRRFCVCVGR-NH2 PG-2 RGGRLCYCRRRFCICV .. -NH2 PG-3 RGGGLCYCRRRFCVCVGR-NH2 PG-4 RGGRLCYCRGWICFCVGR-NH2 PG-5, RGGRLCYCRPRFCVC. . Tokyo 41, 978-980), designated Tl, T2 and T3 and the polyphemusines (Muta, T., 1994, CIBA Found. Sym. 186, 160-174), designated PI and P2, the sequences of which are given below. below, are homologous peptides isolated from the hemolymph of two crabs, Tachyplesus tridentatus for the tachyplesins Tl, T2 and T3 and Limmulus polyphemus for the polyphemusins Pi and P2. PI: RRWCFRVCYRGFCYRKCR-NH2
P2 : RRWCFRVCYKGFCYRKCR-NH2 Tl : KWCFRVCYRGICYRRCR-NH2P2: RRWCFRVCYKGFCYRKCR-NH2 Tl: KWCFRVCYRGICYRRCR-NH2
T2 : RWCFRVCYRGICYRKCR-NH2T2: RWCFRVCYRGICYRKCR-NH2
T3 : K CFRVCYRGICYKRCR-NH2T3: K CFRVCYRGICYKRCR-NH2
Protégrines, tachyplésines et polyphémusines contiennent une forte proportion de résidus basiques (lysines et arginines) et possèdent quatre cystéines qui forment deux ponts disulfures parallèles. Ces trois familles de peptides présentent également des homologies avec certaines défensines et en particulier avec la défensine humaine NP-1 (Kokryakov, V. N. et al., 1993, Febs Let. 327, 231-236).Proteins, tachyplines and polyphemusins contain a high proportion of basic residues (lysines and arginines) and have four cysteines which form two parallel disulfide bridges. These three families of peptides also exhibit homologies with certain defensins and in particular with human defensin NP-1 (Kokryakov, V. N. et al., 1993, Febs Let. 327, 231-236).
Tachyplésines et protégrines possèdent une structure tridimensionnelle voisine. Il s'agit d'un feuillet β antiparalléle stabilisé par les deux ponts disulfures. Ces ponts jouent un rôle important dans l'activité antibactérienne des protégrines et des tachyplésines. Leur suppression, soit en protégeant les groupements SH par des acétamidométhyles , soit en remplaçant les cystéines par des alanines ou des glycines, conduit à des analogues pratiquement dénués d'activité in vivo (Lehrer, R. I. et al., 1996, Eur . J. Biochem. 240:352-357).Tachyplines and protectins have a similar three-dimensional structure. It is an antiparallel β sheet stabilized by the two disulfide bridges. These bridges play an important role in the antibacterial activity of protectins and tachyplesins. Their removal, either by protecting the SH groups with acetamidomethyls, or by replacing the cysteines with alanines or glycines, leads to analogs practically devoid of activity in vivo (Lehrer, RI et al., 1996, Eur. J. Biochem. 240: 352-357).
Comme indiqué précédemment, les protégrines et les tachyplésines ont une importante activité lytique sur les cellules procaryotes. Les travaux de recherche réalisés par la Demanderesse sur la cytotoxicité de ces peptides sur des cellules de mammifère en culture, ont permis de mettre en évidence, avant la mort des cellules, des quantités non-négligeables de protégrines et de tachyplésines dans le cytoplasme desdites cellules. Il a été envisagé que la présence des peptides dans le cytoplasme pouvait résulter d'un transport par le biais de pores, mais ces pores ne sont perméables qu'aux ions et aux petites molécules et leur diamètre est trop petit pour permettre le passage des peptides antibiotiques. Il semblerait que les protégrines et tachyplésines, en plus de perforer la membrane plasmique, soient capables de la traverser.As indicated previously, the protectins and the tachyplesins have an important lytic activity on the prokaryotic cells. The research carried out by the Applicant on the cytotoxicity of these peptides on cultured mammalian cells has made it possible to demonstrate, before the death of the cells, non-negligible amounts of protectins and tachyplesins in the cytoplasm of said cells. . It has been envisaged that the presence of peptides in the cytoplasm could result from transport through pores, but these pores are only permeable to ions and small molecules and their diameter is too small to allow the passage of peptides. antibiotics. It would seem that the proteines and tachyplines, in addition to puncturing the plasma membrane, are able to cross it.
Il est connu que la cytotoxicité et l'activité antimicrobienne des protégrines et des tachyplésines sont dus à leur capacité de s'agréger à 1 ' intérieur de la membrane pour former des canaux multimèriques (Mangoni, M. et al . , 1996, Febs Let. 383, 93-98) . La Demanderesse a alors envisagé que cette agrégation soit reliée à la structure tertiaire de ces peptides antibiotiques qui comportent plusieurs résidus cystéines, et des dérivés linéaires des protégrines et des tachyplésines dans lesquels les cystéines sont remplacées par divers acides aminés naturels, ont été synthétisés. Ces peptides ont été couplés, par leur extrémité N-terminale, à une molécule fluorescente ou à la biotine et la répartition de ces marqueurs à l'intérieur de la cellule a été observée par microscopie confocale.It is known that the cytotoxicity and the antimicrobial activity of protectins and tachyplesins are due to their capacity to aggregate inside the membrane to form multimeric channels (Mangoni, M. et al., 1996, Febs Let 383, 93-98). The Applicant then envisaged that this aggregation be linked to the tertiary structure of these antibiotic peptides which contain several cysteine residues, and linear derivatives of the protectins and tachyplines in which the cysteines are replaced by various natural amino acids, have been synthesized. These peptides were coupled, by their N-terminal end, to a fluorescent molecule or to biotin and the distribution of these markers inside the cell was observed by confocal microscopy.
Il a ainsi maintenant été trouvé que ces peptides sont non-toxiques et sans activité lytique mais sont par contre capables de traverser rapidement les membranes des cellules de mammifères par un mécanisme passif .It has thus now been found that these peptides are non-toxic and without lytic activity but are on the other hand capable of rapidly crossing the membranes of mammalian cells by a passive mechanism.
Ces dér ivé s l inéaire s de s pept ide s antibiotiques constituent donc un nouveau système de vectorisation de substances actives qui est non toxique .These linear derivatives of antibiotic peptides therefore constitute a new vectorization system for active substances which is non-toxic.
Par système de vectorisation , on entend selon l ' invention , un processus capable de transporter ladite substance active j usqu ' à une cible , comme par exemple :By vectorization system is meant according to the invention, a process capable of transporting said active substance to a target, such as for example:
- de faire traverser la membrane cellulaire à une substance active et de permettre la distribution de cel le -c i dans le cytoplasme et / ou dans le compartiment nucléaire , - d ' amener une substance active au niveau d ' un organe particulier , par exemple de faire franchir à cette substance active la barrière hémato-encéphalique ,- to make an active substance cross the cell membrane and allow the distribution of this in the cytoplasm and / or in the nuclear compartment, to bring an active substance to a particular organ, for example to make this active substance cross the blood-brain barrier,
- de f orcer cette subs tance ac tive à interagir spécifiquement avec un type cellulaire donné , comme par exemple les hématies .- to force this active substance to interact specifically with a given cell type, such as red blood cells.
La présente invention a donc pour obj et des peptides dérivés des peptides antibiotiques ou d ' analogues de ceux-ci , caractérisés en ce qu ' ils sont dépourvus de pont disulfure .The subject of the present invention is therefore peptides derived from antibiotic peptides or analogs thereof, characterized in that they are devoid of the disulfide bridge.
On entend par analogue de pept ides antibiotiques , un peptide dont la séquence en acides aminés ' a été modi f iée sans que cela n ' entaîne de modif ication dans les propriétés antibiotiques dudit peptide .The term analog Pept ides antibiotic, a peptide whose amino acid sequence has been modi f ied without this n 'entaîne modif ication in the antibiotic properties of said peptide.
L'absence de pont disulfure dans les peptides de 1 ' invention peut être obtenue par tout moyen connu de l'homme du métier, comme par exemple : en supprimant ou en remplaçant par d'autres acides aminés les résidus de cystéine de la séquence du peptide antibiotique,The absence of disulfide bridge in the peptides of the invention can be obtained by any means known to those skilled in the art, such as for example: by deleting or replacing with other amino acids the cysteine residues of the sequence of the antibiotic peptide,
- en bloquant les groupes -SH des résidus cystéines de façon à ce qu'ils ne forment pas de pont disufure, dès lors bien entendu que le peptide obtenu présente les propriétés de vectorisation sans toxicité pour les cellules décrites précédemment. Ces modifications peuvent être réalisées lors de la préparation des peptides de l'invention, plus particulièrement par synthèse chimique ou par expression d'un gène codant pour ledit peptide, ou directement sur un peptide antibiotique par action d'agents chimiques permettant d'ouvrir et de bloquer les groupes -SH des résidus de cystéine. Les modifications ci-dessus concernent avantageusement tous les résidus de cystéines du peptide antibiotique, mais dès lors que la présence d'un unique résidu de cystéine ne permet pas la formation de pont disulfure, les peptides de l'invention peuvent contenir une seule cystéine. Les peptides antibiotiques naturels présentent généralement 4 ou 6 résidus de cystéine capables de former deux ou trois ponts disulfures, aussi dans les peptides de l'invention, l'une seulement de ces cystéines peut être maintenue et les trois ou cinq autres sont modifiées ou bloquées.- By blocking the -SH groups of the cysteine residues so that they do not form a disufure bridge, therefore of course that the peptide obtained has the vectoring properties without toxicity for the cells described above. These modifications can be carried out during the preparation of the peptides of the invention, more particularly by chemical synthesis or by expression of a gene coding for said peptide, or directly on an antibiotic peptide by the action of chemical agents making it possible to open and to block the -SH groups of cysteine residues. The above modifications advantageously relate to all the cysteine residues of the antibiotic peptide, but as soon as the presence of a single cysteine residue does not allow the formation of a disulfide bridge, the peptides of the invention can contain a single cysteine. Natural antibiotic peptides generally have 4 or 6 cysteine residues capable of forming two or three disulfide bridges, also in the peptides of the invention, only one of these cysteines can be maintained and the other three or five are modified or blocked .
Les peptides antibiotiques dont dérivent les peptides de l'invention peuvent être des défensines, des protégrines, des tachyplésines ou leurs analogues, dont les propriétés antibiotiques leur sont conférées par leur structure tertiaire résultant de la présence de ponts disulfures.The antibiotic peptides from which the peptides of the invention are derived may be defensins, protectins, tachyplesins or their analogs, the antibiotic properties of which are imparted to them by their tertiary structure resulting from the presence of disulfide bridges.
Des peptides linéaire selon 1 ' invention répondent à 1 ' une des formules suivantes :Linear peptides according to the invention correspond to one of the following formulas:
BXXBXXXXBBBXXXXXXB (I)BXXBXXXXBBBXXXXXXB (I)
BBXXXBXXXBXXXXBBXB (II) qui peuvent être aussi représentées par la formule unique (III) suivante : B(XB)X(XB)X(XB)XX(XB)B(XB)XXX(XB) (XB)XB dans lesquelles :BBXXXBXXXBXXXXBBXB (II) which can also be represented by the following unique formula (III): B (XB) X (XB) X (XB) XX (XB) B (XB) XXX (XB) (XB) XB in which:
- les groupes B, identiques ou différents, représentent un résidus d'acide aminé dont la chaîne latérale porte un groupement basique, et - les groupes X, identiques ou différents, représentent un résidus d'acide aminé aliphatique ou aromatique, ou sont constitués d'une séquence d'au moins- groups B, identical or different, represent an amino acid residue whose side chain carries a basic group, and - groups X, identical or different, represent an aliphatic or aromatic amino acid residue, or consist of '' a sequence of at least
5 et de préférence d'au moins 7 acides aminés successifs de l'une des formules (I) ou (II), dès lors que cette séquence présente les propriétés de vectorisation sans toxicité pour les cellules décrites précédemment.5 and preferably at least 7 successive amino acids of one of the formulas (I) or (II), since this sequence presents the vectorization properties without toxicity to the cells described above.
B et X peuvent être des acides aminés naturels ou non, y compris des acides aminés de configuration D.B and X can be natural amino acids or not, including amino acids of configuration D.
On peut citer comme exemple, les significations de B et X suivantes : - B est choisi parmi l'arginine, la lysine, l'acide diaminoacétique , l'acide diaminobutyrique , 1 ' acide diaminopropionique , 1 ' ornithine .As an example, the following meanings of B and X can be cited: - B is chosen from arginine, lysine, diaminoacetic acid, diaminobutyric acid, 1 diaminopropionic acid, 1 ornithine.
- X est choisi parmi la glycine, l'alanine, la valine, la norleucine, 1 ' isoleucine, la leucine, la cystéine, la cystéine , la penicillamine , la methionine, la serine, la threonine, 1 ' asparagine, la glutamine, la phénylalanine , l'histidine, le tryptophane, la tyrosine, la proline, l'Abu, l'acide amino-1-cyclohexane carboxylique , l'Aib, la 2- aminotétraline carboxylique, la 4-bromophénylalanine, tert-Leucine , la 4-chlorophénylalanine , la β- cyclohexylalanine, la 3 , 4-dichlorophénylalanine, la 4- f luorophénylalanine, 1 ' homoleucine, la β-homoleucine,- X is chosen from glycine, alanine, valine, norleucine, isoleucine, leucine, cysteine, cysteine, penicillamine, methionine, serine, threonine, 1 asparagine, glutamine, phenylalanine, histidine, tryptophan, tyrosine, proline, Abu, amino-1-cyclohexane carboxylic acid, Aib, 2-aminotetraline carboxylic, 4-bromophenylalanine, tert-Leucine, 4-chlorophenylalanine, β-cyclohexylalanine, 3, 4-dichlorophenylalanine, 4-fluorophenylalanine, 1 homoleucine, β-homoleucine,
1 ' homophénylalanine , la 4-méthylphénylalanine, la 1- naphtylalanine , la 2 -naphtylalanine , la 4- nitrophénylalanine, la 3 -ni tro tyrosine, la norvaline, la phénylglycine , la 3 -pyr idylalanine , la [2- thiényl] alanine .1 homophenylalanine, 4-methylphenylalanine, 1-naphthylalanine, 2-naphthylalanine, 4-nitrophenylalanine, 3-ni tro tyrosine, norvaline, phenylglycine, 3-pyr idylalanine, [2-thienyl] alan .
L'invention concerne aussi des dérivés des peptides de formules (I) ou (II) comme lesdits peptides sous forme rétro, ou des fragments des peptides de formules (I) ou (II) constitués de cinq et de préférence sept acides aminés successifs de l'une des formules (I) ou (II) . Parmi les peptides de l'invention, on peut citer plus particulièrement ceux répondant aux formules suivantes :The invention also relates to derivatives of the peptides of formulas (I) or (II) such as said peptides in retro form, or fragments of the peptides of formulas (I) or (II) consisting of five and preferably seven successive amino acids of one of the formulas (I) or (II). Among the peptides of the invention, there may be mentioned more particularly those corresponding to the following formulas:
RXXRXUXURRRXUXUXXR-NH2 (V) RRXUXRXUXRXXUXRRUR-NH2 (VI) dans lesquellesRXXRXUXURRRXUXUXXR-NH2 (V) RRXUXRXUXRXXUXRRUR-NH2 (VI) in which
- U représente la serine ou la threonine,- U represents serine or threonine,
- R représente l'arginine, et- R represents arginine, and
- les groupes X identiques ou différents représentent un acide aminés naturel ou non (y compris acides aminés de configuration D) aliphatiques ou aromatiques, comme la glycine, l'alanine, la valine, la norleucine, 1 ' isoleucine, la leucine, la cystéine, la- identical or different groups X represent a natural or unnatural amino acid (including amino acids of configuration D) aliphatic or aromatic, such as glycine, alanine, valine, norleucine, isoleucine, leucine, cysteine , the
Δrjrn cystéine , la penic il lamine, la methionine, la serine, la threonine, 1 ' asparagine , la glutamine, la phénylalanine, l'histidine, le tryptophane, la tyrosine, la proline, 1 ' bu , l'acide amino-1-cyclohexane carboxylique, l'Aib, la 2-aminotétraline carboxylique, la 4 -bromophénylalanine , tert-Leucine , la 4- chlorophénylalanine, la β-cyclohexylalanine, la 3,4- dichlorophénylalanine , la 4-f luorophénylalanine , 1 ' homo leucine, la β-homo leucine, 1 'homophénylalanine, laΔrjrn cysteine, penic il lamine, methionine, serine, threonine, asparagine, glutamine, phenylalanine, histidine, tryptophan, tyrosine, proline, 1 'bu, amino-1 acid -carboxylic cyclohexane, Aib, carboxylic 2-aminotetralin, 4-bromophenylalanine, tert-Leucine, 4-chlorophenylalanine, β-cyclohexylalanine, 3,4-dichlorophenylalanine, 4-f luorophenylalanine, 1 'homo leuc , β-homo leucine, 1 homophenylalanine,
4-méthylphénylalanine , la 1-naphtylalanine , la 2- naphtylalanine , la 4-ni trophénylalanine , la 3- nitro tyrosine, la norvaline, la phénylglycine, la 3- pyridylalanine, la [2-thiényl] alanine .4-methylphenylalanine, 1-naphthylalanine, 2-naphthylalanine, 4-ni trophenylalanine, 3-nitro tyrosine, norvaline, phenylglycine, 3-pyridylalanine, [2-thienyl] alanine.
Parmi les peptides de formules (I) et (II) ou leurs dérivés, l'invention envisage à titre spécifique ceux dérivés de protégrines et de tachyplésines rapportés dans les tableaux I et II ci- dessous . Tableau I : dérivés de protégrinesAmong the peptides of formulas (I) and (II) or their derivatives, the invention specifically contemplates those derived from protectins and tachyplines reported in Tables I and II below. Table I: derivatives of protectins
Figure imgf000013_0001
Figure imgf000013_0001
Tableau II : dérivés de tachyplésinesTable II: derivatives of tachyplesins
Figure imgf000013_0002
Figure imgf000013_0002
Dans les séquences des tableaux I et II ci- dessus, B représente la Napthylalanine, O représente l'Ornithine, X représente la Norleucine et Z représente la Norvaline.In the sequences of Tables I and II above, B represents Napthylalanine, O represents Ornithine, X represents Norleucine and Z represents Norvaline.
L'invention concerne aussi l'utilisation des peptides ci-dessus pour la vectorisation d'une ou plusieurs substances actives tant pour des applications thérapeutiques que de diagnostic. A titre de substance active, l'invention envisage notamment des protéines ou fragments de protéines, comme des polypeptides ou peptides, des anticorps ou partie d'anticorps, des acides nucléiques et oligonucléotides ou des ribozymes, ou encore, bien entenu des molécules chimiques actives pour le traitement ou la prévention de pathologies humaines ou animales, comme par exemple et de manière non limitative des antitumoraux, des antiviraux, des agents anti-inflammatoires, des agents empêchant la dégradation d'organes et/ou de tissus, etc..The invention also relates to the use of the above peptides for the vectorization of one or more active substances both for therapeutic and diagnostic applications. As an active substance, the invention envisages in particular proteins or protein fragments, such as polypeptides or peptides, antibodies or part of antibodies, nucleic acids and oligonucleotides or ribozymes, or, of course, active chemical molecules for the treatment or prevention of human or animal pathologies, such as, for example and without limitation, anti-tumor agents , antivirals, anti-inflammatory agents, agents preventing the degradation of organs and / or tissues, etc.
Dans le domaine du diagnostic, la substance active peut être un marqueur radioactif, un marqueur coloré, ou tout autre moyen ou substance capable de révéler un métabolisme ou une pathologie.In the diagnostic field, the active substance can be a radioactive marker, a colored marker, or any other means or substance capable of revealing a metabolism or a pathology.
L'invention a donc également pour objet des composés de formule (IV) suivante, ainsi que les compositions les contenant :The invention therefore also relates to compounds of formula (IV) below, as well as the compositions containing them:
(Y=- A.) (-Z)m (Y = - A.) (-Z) m
11 ιv dans laquelle - A représente un peptide linéaire dérivé d'un peptide antibiotique conforme à l'invention, 11 ιv in which - A represents a linear peptide derived from an antibiotic peptide according to the invention,
- Z représente une substance active, comme défini ci-dessus,- Z represents an active substance, as defined above,
- Y représente un agent signal, - n est 0 et ou plus, et avantageusement 0 ou 1,- Y represents a signal agent, - n is 0 and or more, and advantageously 0 or 1,
- m est 1 ou plus, et de préférence jusqu'à 10 avantageusement jusqu'à 5.- m is 1 or more, and preferably up to 10 advantageously up to 5.
Ainsi, les composés de formule (IV) ci- dessus sont formés à partir d'un peptide de l'invention couplé à une ou plusieurs substances actives, identiques ou différentes, représentées par le groupe (Z) dans la formule (IV), et éventuellement un ou plusieurs agents de signal, représentés par le groupe (Y) dans la formule (IV), ayant un rôle d'adressage du composé de formuleThus, the compounds of formula (IV) above are formed from a peptide of the invention coupled to one or more active substances, identical or different, represented by the group (Z) in the formula (IV), and optionally one or more signal agents, represented by the group (Y) in formula (IV), having a role of addressing the compound of formula
(IV) vers un type cellulaire, un site ou compartiment de la cellule ou un tissus particulier. Plus particulièrement, l'agent signal (Y) est un oligopeptide ou une protéine, comme un peptide signal, un signal de localisation nucléaire, un fragment d'anticorps, ou une molécule chimique ligand ou anti-ligand d'un récepteur.(IV) to a cell type, a site or compartment of the particular cell or tissue. More particularly, the signal agent (Y) is an oligopeptide or a protein, such as a signal peptide, a nuclear localization signal, an antibody fragment, or a chemical molecule ligand or anti-ligand of a receptor.
Dans une forme toute particulière de réalisation des composés de formule (IV) , le groupe (Y) est fixé au groupe (Z) .In a very particular embodiment of the compounds of formula (IV), the group (Y) is attached to the group (Z).
Le couplage, symbolisé par les traits horizontaux dans la formule (IV), peut être réalisé par tout moyen de liaison acceptable compte tenu de la nature chimique, de l'encombrement et du nombre de groupes (Z) et (Y) dans les composés de formule (IV), comme des liaisons covalentes, hydrophobes ou ioniques, clivables ou non-clivables dans les milieux physiologiques. Le couplage peut être effectué en n'importe quel site du peptide (A), dans lequel des groupements fonctionels tels que -OH, -SH, -COOH, -NH2 sont naturellement présents ou ont été introduits. L'invention envisage aussi la fixation de plusieurs groupes (Z) sur un même site du peptide (A), soit directement, si ce site comporte plusieurs groupements fonctionnels, comme dans le cas d'une lysine C- ou N-terminale, soit indirectement via un groupe intermédiaire portant plusieurs groupements réactionnels permettant d'y fixer plusieurs groupes (Z) .The coupling, symbolized by the horizontal lines in the formula (IV), can be carried out by any acceptable means of connection taking into account the chemical nature, the bulk and the number of groups (Z) and (Y) in the compounds of formula (IV), such as covalent, hydrophobic or ionic bonds, cleavable or non-cleavable in physiological media. The coupling can be carried out at any site of the peptide (A), in which functional groups such as -OH, -SH, -COOH, -NH2 are naturally present or have been introduced. The invention also envisages the attachment of several groups (Z) to the same site of the peptide (A), either directly, if this site comprises several functional groups, as in the case of a C- or N-terminal lysine, either indirectly via an intermediate group carrying several reaction groups making it possible to fix therein several groups (Z).
Les positions de couplage préférées pour la substance active sont au niveau des extrémités N- terminale ou C-terminale ou bien au niveau des groupements aminés primaires portés par les chaînes latérales des lysines du peptide (A) . Dans le cas où l'on utilise l'extrémité C-terminale du peptide (A) pour accrocher la substance active (Z), l'extrémité N- terminale est disponible pour le couplage éventuel à un agent signal (Y) permettant l'adressage du composé de l'invention soit vers le noyau, soit encore vers un type tissulaire particulier.The preferred coupling positions for the active substance are at the N-terminal or C-terminal ends or else at the level of the primary amino groups carried by the side chains of the lysines of peptide (A). In the case where the C-terminal end of the peptide (A) is used to hook the active substance (Z), the N-terminal end is available for possible coupling to a signal agent (Y) allowing the addressing the compound of the invention either towards the nucleus, or even towards a particular tissue type.
En effet, par exemple dans le cas du couplage à l'extrémité C-terminale d'un peptide linéaire de l'invention, d'une substance active constituée par un marqueur fluorescent, ou de la biotine, ou encore d'une molécule médicamenteuse telle que la doxorubicine, le complexe covalent peptide-drogue après administration se répartit dans le cytoplasme de la cellule cible. Il est possible d'amener ce complexe dans le compartiment nucléaire en couplant à l'extrémité N-terminale du peptide une courte séquence basique, par exemple d'environ 7 acides aminés, correspondant à un signal de localisation nucléaire. Dans ces conditions, la biotine ou la doxorubicine se retrouvent dans le noyau de la cellule.Indeed, for example in the case of the coupling at the C-terminal end of a linear peptide of the invention, of an active substance constituted by a fluorescent marker, or of biotin, or also of a drug molecule such as doxorubicin, the covalent peptide-drug complex after administration is distributed in the cytoplasm of the target cell. It is possible to bring this complex into the nuclear compartment by coupling to the N-terminal end of the peptide a short basic sequence, for example of about 7 amino acids, corresponding to a nuclear localization signal. Under these conditions, biotin or doxorubicin is found in the nucleus of the cell.
De la même manière, il est possible de vectoriser une drogue vers un type cellulaire donné, en ajoutant à l'extrémité N-terminale du peptide linéaire de l'invention couplé à son extrémité C-terminale à un médicament, une séquence peptidique capable de reconnaître spécifiquement un déterminant présent à la surface de type cellulaire. Ainsi, le pentadecapeptide 0CM2 (Swolapenko, G. B. et al., 1995, The Lancet 346, 1662-65) synthétique, fragment d'un anticorps monoclonal, dirigé contre un antigène exprimé par les cellules de cancer du sein (Tumour Associated Antigen Polymorphic Epithelial Mucin) , conserve une bonne affinité pour ces cellules. Il est donc possible en associant aM2 à un ensemble peptide linéaire-médicament, d'amener cet ensemble préférentiellement vers les cellules qui expriment la caractéristique antigénique liée au cancer mammaire.Likewise, it is possible to vectorize a drug towards a given cell type, by adding to the N-terminal end of the linear peptide of the invention coupled to its C-terminal end to a drug, a peptide sequence capable of specifically recognize a determinant present on the cell-like surface. Thus, the pentadecapeptide 0CM2 (Swolapenko, GB et al., 1995, The Lancet 346, 1662-65) synthetic, fragment of a monoclonal antibody, directed against an antigen expressed by breast cancer cells (Tumor Associated Antigen Polymorphic Epithelial Mucin), retains a good affinity for these cells. It is therefore possible, by combining aM2 with a linear peptide-drug set, to bring this set preferentially towards the cells which express the antigenic characteristic linked to breast cancer.
Les composés de formule (IV) peuvent être préparés par synthèse chimique ou en utilisant des techniques de biologie moléculaire. On peut utiliser pour les synthèses chimiques des appareils commerciaux permettant d'incorporer des acides aminés non-naturels, tels que les énantiomères D et des résidus ayant des chaînes latérales ayant des hydrophobicités et des encombrements différents de ceux de leurs homologues naturels. Au cours de la synthèse, il est évidemment possible de réaliser un large éventail de modifications, par exemple introduire sur le N-terminal un lipide (prenyl ou myristyl) de façon à pouvoir ancrer le peptide de l'invention et donc le composé de formule (IV) à une membrane lipidique telle que celle d'un liposome constitué de lipides chargés positivement. Il est également possible de remplacer une ou plusieurs liaisons peptidiques (-CO-NH-) par des structures équivalentes comme -CO-N(CH3)-, -CH2-CH -, -CO-CH2-, ou bien d' intercaller des groupes comme -CH2-, -NH- , -O- .The compounds of formula (IV) can be prepared by chemical synthesis or by using molecular biology techniques. It is possible to use for chemical synthesis commercial devices making it possible to incorporate non-natural amino acids, such as the D enantiomers and residues having side chains having hydrophobicities and bulkings different from those of their natural counterparts. During the synthesis, it is obviously possible to make a wide range of modifications, for example introducing a lipid (prenyl or myristyl) onto the N-terminal so as to be able to anchor the peptide of the invention and therefore the compound of formula (IV) to a lipid membrane such as that of a liposome consisting of positively charged lipids. It is also possible to replace one or more peptide bonds (-CO-NH-) with equivalent structures such as -CO-N (CH 3 ) -, -CH 2 -CH -, -CO-CH 2 -, or d '' intercalate groups like -CH 2 -, -NH-, -O-.
On peut également obtenir les composés de formule (IV) ou partie de ceux-ci de nature proteique à partir d'une séquence d'acide nucléique codant pour celui-ci. La présente invention a aussi pour objet une molécule d'acide nucléique comprenant ou constituée par une séquence nucléique codant pour un peptide linéaire dérivé de peptide antibiotique. Plus particulièrement l'invention concerne une molécule d'acide nucléique comprenant au moins une séquence codant pour un composé de formule (IV) ou une partie de celui de nature proteique. Ces séquences d'acides nucléiques peuvent être des ADN ou ARN et être associées à des séquences de contrôle et/ou être insérées dans des vecteurs. Le vecteur utilisé est choisi en fonction de l'hôte dans lequel il sera transféré; il peut s'agir de tout vecteur comme un plasmide. Ces acides nucléiques et vecteurs sont utiles pour produire les peptides linéaires et les composés de formule (IV) ou partie de ceux-ci de nature proteique dans un hôte cellulaire. La préparation de ces vecteurs ainsi que la production ou l'expression dans un hôte des peptides linéaires ou des composés de formuleIt is also possible to obtain the compounds of formula (IV) or part of these of a protein nature from a nucleic acid sequence coding for it. The present invention also relates to a nucleic acid molecule comprising or consisting of a nucleic sequence coding for a linear peptide derived from antibiotic peptide. More particularly the invention relates to a nucleic acid molecule comprising at least one sequence coding for a compound of formula (IV) or part of that of a protein nature. These nucleic acid sequences can be DNA or RNA and be associated with control sequences and / or be inserted into vectors. The vector used is chosen according to the host to which it will be transferred; it can be any vector such as a plasmid. These nucleic acids and vectors are useful for producing linear peptides and compounds of formula (IV) or part of them of a protein nature in a cellular host. The preparation of these vectors as well as the production or expression in a host of linear peptides or compounds of formula
(IV) peuvent être réalisées par les techniques de biologie moléculaire et de génie génétique bien connues de l'homme du métier.(IV) can be carried out by molecular biology and genetic engineering techniques well known to those skilled in the art.
A titre d'exemple, un tel procédé de production d'un peptide selon l'invention consiste : à transférer une molécule d'acide nucléique ou un vecteur contenant ladite molécule dans un hôte cellulaire,By way of example, such a process for producing a peptide according to the invention consists in: transferring a nucleic acid molecule or a vector containing said molecule to a cellular host,
- à cultiver ledit hôte cellulaire dans des conditions permettant la production du peptide,- cultivating said cell host under conditions allowing the production of the peptide,
- à isoler, par tous moyens appropriés les peptides de l'invention. L'hôte cellulaire mis en oeuvre dans ce type de procédé peut être choisi parmi les procaryotes ou les eucaryotes et notamment parmi les bactéries, les levures, les cellules de mammifères, de plantes ou d'insectes. L'invention concerne donc aussi les cellules transformées exprimant les peptides linéaires ou les composés de formule (IV) ou partie de ceux-ci de nature proteique.- to isolate, by any appropriate means, the peptides of the invention. The cell host used in this type of process can be chosen from prokaryotes or eukaryotes and in particular from bacteria, yeasts, mammalian, plant or insect cells. The invention therefore also relates to the transformed cells expressing the linear peptides or the compounds of formula (IV) or part of these of a protein nature.
L'invention se rapporte aussi : aux compositions pharmaceutiques comprenant comme principe actif au moins un composé de formule (IV) éventuellement associé à un véhicule ou support acceptable.The invention also relates to: pharmaceutical compositions comprising as active principle at least one compound of formula (IV) optionally associated with an acceptable vehicle or support.
- aux agents de diagnostic constitués d'au moins un composé de formule (IV) .- diagnostic agents consisting of at least one compound of formula (IV).
D'autres caractéristiques et avantages de l'invention apparaîtront dans la description qui suit se rapportant à la préparation de composés de formule (IV) ainsi qu'aux travaux de recherche ayant mené à la mise en évidence des propriétés de vectorisation des peptides l i néa i re s de l ' i nven t i on dé r ivé s de pep t i de s antibiotiques .Other characteristics and advantages of the invention will appear in the following description relating to the preparation of compounds of formula (IV) as well as to research work which has led to the demonstration of vectorization properties of peptides li nei i re s of the inven ti on r rov ies of pep ti of s antibiotics.
Exemple 1 : Fixation de la biotine et de la doxorubic ine sur des analogues l inéaires de peptides antibiotiques .Example 1: Binding of biotin and doxorubic ine on linear analogues of antibiotic peptides.
1 ) Préparation des peptides linéaires .1) Preparation of linear peptides.
Les trois peptides de séquences ci-dessous ont été synthétisés :The three peptides of sequences below have been synthesized:
RGGRLXYXRRRFXVXVGR-NH2RGGRLXYXRRRFXVXVGR-NH2
RRXFRVXYRGFXYRKXR-NH2RRXFRVXYRGFXYRKXR-NH2
K XFRVXYRGIXYRRXR-NH2 dans lesquels X représente les résidus serine, threonine ou alanine.K XFRVXYRGIXYRRXR-NH2 in which X represents the serine, threonine or alanine residues.
Ces peptides dérivent repectivement des séquences de le protégrine PG-1 de formule :These peptides are respectively derived from the PG-1 protectin sequences of formula:
RGGRLCYCRRRFCVCVGR-NH2 , de la tachyplésine 1 de formule : KWCFRVCYRGICYRRCR-NH2 , de la polyphemusine de formule :RGGRLCYCRRRFCVCVGR-NH2, tachyplesin 1 of formula: KWCFRVCYRGICYRRCR-NH2, polyphemusine of formula:
KWXFRVXYRGIXYRRXR-NH2.KWXFRVXYRGIXYRRXR-NH2.
Ces trois peptides peuvent être préparés indifféremment soit à partir d'une chimie BOC, soit à partir d'une chimie FMOC, par des procédés classiques de synthèse en phase solide ou homogène.These three peptides can be prepared either from a BOC chemistry or from a FMOC chemistry, by conventional methods of synthesis in solid or homogeneous phase.
2) Fixation de la biotine sur les peptides linéaires. Le peptide est synthétisé en phase solide et après incorporation de 1 ' arginine N-terminale on ajoute l'acide 5-aminopentanoique . Le Fmoc ou le Boc N- terminal est enlevé et on fait réagir sur le peptide toujours accroché à la résine le N-hydroxy succimido ester de la biotine dans le diméthylformamide . Après 15 heures de réaction à température ambiante, le peptide biotinilé est coupé du support par action de l'acide trifluoroacétique ou de l'acide fluorohydrique selon des protocoles bien établis dans la chimie des peptides. Le peptide est ensuite purifié par chromatographie liquide à haute pression.2) Fixation of biotin on linear peptides. The peptide is synthesized in the solid phase and after incorporation of the N-terminal arginine, 5-aminopentanoic acid is added. The N-terminal Fmoc or Boc is removed and the N-hydroxy succimido ester of biotin in dimethylformamide is reacted on the peptide still attached to the resin. After 15 hours of reaction at room temperature, the peptide biotinile is cut from the support by the action of trifluoroacetic acid or hydrofluoric acid according to protocols well established in peptide chemistry. The peptide is then purified by high pressure liquid chromatography.
3 ) Fixation de la doxorubicine sur un peptide linéaire3) Fixation of doxorubicin on a linear peptide
Pour fixer la doxorubicine, on synthétise en phase solide le peptide de formule :To fix doxorubicin, the peptide of formula is synthesized in solid phase:
RGGRLXYXRRRFXVXVGR-NH2RGGRLXYXRRRFXVXVGR-NH2
Après clivage du support de purification, le peptide est traité par l'anhydre glutarique en présence de triéthylamine . Le peptide est alors purifié et le groupement -COOH porté par le glutaryl en N-terminal est activé par le mélange diisopropylcarbodiimide et 1- hydroxybenzotriazole . Après deux heures de réaction à température ambiante, de la doxorubicine est ajoutée et le mélange est agité pendant 12 heures à 0°C. L'ensemble pept ide-doxorubic ine est alors purifié par chromatographie liquide à haute pression.After cleavage of the purification support, the peptide is treated with glutaric anhydride in the presence of triethylamine. The peptide is then purified and the -COOH group carried by the glutaryl at the N-terminal is activated by the mixture of diisopropylcarbodiimide and 1-hydroxybenzotriazole. After two hours of reaction at room temperature, doxorubicin is added and the mixture is stirred for 12 hours at 0 ° C. The whole pept ide-doxorubic ine is then purified by high pressure liquid chromatography.
Exemple 2 : Capacité des peptides linéaires de l'invention à passer les membranes de cellules.Example 2: Ability of linear peptides of the invention to pass cell membranes.
1) Modèles cellulaires .1) Cellular models.
La capacité des peptides à passer les membranes a été testée sur divers types cellulaires (MCF7, MCF7R, HL60, HL60R, HeLa) . Les cellules sont cultivées sur RPMI 1640The ability of peptides to pass membranes has been tested on various cell types (MCF7, MCF7R, HL60, HL60R, HeLa). The cells are cultured on RPMI 1640
(Gibco) auquel on ajoute 10% (v/v) de sérum veau foetal, 2mM glutamine and 2mM pénicilline/streptomycine, a 37 °C. 30 000 cellules sont ensemencées dans des chambres Lab Tek et cultivées pendant 1 jour. 2 ) Traitement par les peptides linéaires- biotine préparés conformément à l'exemple 1 (2).(Gibco) to which 10% (v / v) of fetal calf serum, 2mM glutamine and 2mM penicillin / streptomycin are added, at 37 ° C. 30,000 cells are seeded in Lab Tek chambers and cultured for 1 day. 2) Treatment with linear peptides-biotin prepared in accordance with Example 1 (2).
Les cellules sont incubées dans de l'Opti- Mem (Gibco) pendant une heure avant d'être traitées pendant des temps variables avec les peptides marqués à la biotine.The cells are incubated in Opti-Mem (Gibco) for one hour before being treated for variable times with the peptides labeled with biotin.
Ces derniers sont obtenus conformément à l'exemple 1(2) en traitant 1 équivalent de peptide linéaire par 2 équivalents d'ester de N- hydroxysuccinimide de la biotine, puis purifié par chromatographie liquide à haute pression.The latter are obtained in accordance with Example 1 (2) by treating 1 equivalent of linear peptide with 2 equivalents of N-hydroxysuccinimide ester of biotin, then purified by high pressure liquid chromatography.
Les cellules sont ensuite fixées avec une solution à 3.7% de paraformaldéhyde pendant 5 minutes àThe cells are then fixed with a 3.7% paraformaldehyde solution for 5 minutes at
25°C, puis rincées trois fois avec du PBS. Elles sont ensuite perméabilisées par du Triton 0.1% (1 min, température ambiante). Après trois rinçages au PBS, les cellules sont incubées 10 min avec 200 μl d'anticorps TexRed dilué au 300eme et rincées trois fois au PBS. Les lames sont enfin montées avec une solution Mowiol-Dabco et observées au photomicroscope Axiophot .25 ° C, then rinsed three times with PBS. They are then permeabilized with 0.1% Triton (1 min, room temperature). After three rinses with PBS, the cells are incubated for 10 min with 200 μl of TexRed antibody diluted to 300th and rinsed three times with PBS. The slides are finally mounted with a Mowiol-Dabco solution and observed with the Axiophot photomicroscope.
3 ) Traitement par les peptides linéaires- doxorubicine préparés conformément à l'exemple 1 (3).3) Treatment with linear peptides- doxorubicin prepared in accordance with Example 1 (3).
Les cellules sont incubées pendant 15 minutes, puis rincées avec du PBS et ensuite la doxorubicine présente dans la cellule est dosée par chromatographie .The cells are incubated for 15 minutes, then rinsed with PBS and then the doxorubicin present in the cell is determined by chromatography.
4) Résultats. a) Parmi les peptides étudiés, ceux qui passent le plus facilement les membranes sont ceux répondant aux formules suivantes :4) Results. a) Among the peptides studied, those which pass the membranes most easily are those corresponding to the following formulas:
RXXRXUXURRRXUXUXXR-NH2 (V)RXXRXUXURRRXUXUXXR-NH2 (V)
RRXUXRXUXRXXUXRRUR-NH2 (VI ) dans lesquellesRRXUXRXUXRXXUXRRUR-NH2 (VI) in which
- U représente la serine ou la threonine, - R représente l'arginine, et- U represents serine or threonine, - R represents arginine, and
- les groupes X identiques ou différents représentent un acide aminés naturel ou non (y compris acides aminés de configuration D) aliphatiques ou aromatiques, comme la glycine, l'alanine, la valine, la norleucine, 1 ' isoleucine, la leucine, la cystéine, la- identical or different groups X represent a natural or unnatural amino acid (including amino acids of configuration D) aliphatic or aromatic, such as glycine, alanine, valine, norleucine, isoleucine, leucine, cysteine , the
.A.cm cystéine , la penicillamine, la methionine, la serine, la threonine, 1 ' asparagine , la glutamine, la phénylalanine , l'histidine, le tryptophane, la tyrosine, la proline, 1 ' Abu , l'acide amino-1-cyclohexane carboxylique, l'Aib, la 2-aminotétraline carboxylique, la 4-bromophénylalanine , tert-Leucine , la 4- chlorophénylalanine, la β-cyclohexylalanine, la 3,4- dichlorophénylalanine , la 4 -fluorophénylalanine , 1 'homoleucine, la β-homoleucine, 1 'homophénylalanine, la.A.cm cysteine, penicillamine, methionine, serine, threonine, asparagine, glutamine, phenylalanine, histidine, tryptophan, tyrosine, proline, 1 'Abu, amino acid 1-cyclohexane carboxylic, Aib, 2-aminotetralin carboxylic, 4-bromophenylalanine, tert-Leucine, 4-chlorophenylalanine, β-cyclohexylalanine, 3,4-dichlorophenylalanine, 4 -fluorophenylalanine, 1 'homoleucine β-homoleucine, 1 homophenylalanine,
4-méthylphénylalanine, la 1-naphtylalanine , la 2- naphtylalanine , la 4-nitrophénylalanine , la 3- nitrotyrosine, la norvaline, la phénylglycine, la 3- pyridylalanine, la [2-thiényl] alanine. b) Les résultats des expériences menées avec la doxorucine montrent une augmentation significative de la concentration plasmique et nucléaire en doxorubicine lorsque celle-ci est couplée au peptide linéaire de l'invention par rapport à l'utilisation de doxorubicine seule. c) Les expériences avec la biotine ont été effectuées plus particulièrement sur des cellules MCF7 traitées à différents temps par un complexe biotine- peptide de 1 ' invention de formule : biotine-RGGRLSYSRRRFSVSVGR-NH24-methylphenylalanine, 1-naphthylalanine, 2-naphthylalanine, 4-nitrophenylalanine, 3-nitrotyrosine, norvaline, phenylglycine, 3-pyridylalanine, [2-thienyl] alanine. b) The results of the experiments carried out with doxorucin show a significant increase in the plasma and nuclear concentration in doxorubicin when this is coupled to the linear peptide of the invention compared to the use of doxorubicin alone. c) The experiments with biotin were carried out more particularly on MCF7 cells treated at different times with a biotin-peptide complex of the invention of formula: biotin-RGGRLSYSRRRFSVSVGR-NH2
Ces travaux ont donné lieu à des clichés (non représentés) :These works gave rise to photographs (not shown):
- Contrôle dans lequel la cellule a été traitée avec la biotine seule. - Traitement de la cellule pendant 2 minutes avec un complexe biot ine-pept ide linéaire de 1 ' invention.- Control in which the cell has been treated with biotin alone. - Treatment of the cell for 2 minutes with a linear ine-pept ide biot complex of the invention.
Traitement de la cellule pendant 30 minutes avec un complexe biotine-peptide linéaire de 1 ' invention.Treatment of the cell for 30 minutes with a linear biotin-peptide complex of the invention.
On observe dans ces clichés que la biotine seule ne rentre pas dans la cellule et s'accumule faiblement autour de celle-ci. A l'inverse, avec le complexe de l'invention, on constate que la biotine est entraînée rapidement par le peptide linéaire de 1 ' invention à 1 ' intérieur de la cellule où elle est présente dans le cytoplasme et dans le noyau de la cellule .We observe in these photos that biotin alone does not enter the cell and accumulates weakly around it. Conversely, with the complex of the invention, it can be seen that biotin is rapidly entrained by the linear peptide of the invention inside the cell where it is present in the cytoplasm and in the nucleus of the cell .
Exemple 3 : Capacité d ' internalisation des peptides linéaires de l'invention.Example 3: Capacity for internalization of the linear peptides of the invention.
Des peptides linéaires de l'invention dérivés de Protégrines et Tachyplésines ont été testés sur différentes lignées cellulaires, dans le but d'évaluer leur internalisation respective.Linear peptides of the invention derived from Proteins and Tachyplesins were tested on different cell lines, with the aim of evaluating their respective internalization.
1) Conditions Expérimentales. Les cellules sont ensemencées à environ 10 cellules par puits, 24 h avant l'addition des peptides biotinylés. Elles sont alors à 60-80% de confluence le jour de l'expérience. Les peptides biotinylés sont incubés avec les cellules à la concentration de lOμM, pendant 15 minutes, à 37°C dans une atmosphère à 95% d'humidité et 5% de C02 dans un milieu OptiMem, . Les cellules sont lavées trois fois avec du PBS à température ambiante puis sont fixées à la formaline (3,7% de formaldéhyde dans PBS, 10 min à température ambiante) . Elles sont ensuite lavées avec du PBS et perméabilisées pendant 15 min avec du PBS-TritonX-100. La révélation est faite avec de la streptavidine-Texas- Red pendant 15 min à l'abri de la lumière et les cellules sont ensuite montées entre lame et lamelle. Elles sont observées en microscopie à fluorescence, et comparées à un témoin positif (Ap43-58), bien décrit dans la littérature, et à un témoin négatif.1) Experimental Conditions. The cells are seeded at approximately 10 cells per well, 24 h before the addition of the biotinylated peptides. They are then at 60-80% confluence on the day of the experiment. The biotinylated peptides are incubated with the cells at a concentration of 10 μM, for 15 minutes, at 37 ° C. in an atmosphere at 95% humidity and 5% CO 2 in an OptiMem medium. The cells are washed three times with PBS at room temperature and then are fixed with formalin (3.7% formaldehyde in PBS, 10 min at room temperature). They are then washed with PBS and permeabilized for 15 min with PBS-TritonX-100. The revelation is made with streptavidin-Texas-Red for 15 min protected from light and the cells are then mounted between slide and coverslip. They are observed under fluorescence microscopy, and compared to a positive control (Ap43-58), well described in the literature, and to a negative control.
Les noyaux des cellules ont été colorés avec du Hoechst.The cell nuclei were stained with Hoechst.
2) Lignées cellulaires.2) Cell lines.
Toutes les lignées testées sont d'origine humaine et ont été obtenues commercialement auprès de 1 ' ATCC .All the lines tested are of human origin and have been obtained commercially from ATCC.
Lignées non tumorales : MRC5 (fibroblaste de poumon), HuVeC (endothéliale, cordon ombilical) .Non-tumor lines: MRC5 (lung fibroblast), HuVeC (endothelial, umbilical cord).
Lignées tumorales : HT29 (carcinome du colon) , HepG2 (hépatoblastome) , A172 (glioblastome) , HMCB ( élanome) . Les cellules sont cultivées à 37°C dans une atmosphère à 95% d'humidité et 5% de C02 • Le milieu de culture est celui recommandé par l'ATCC.Tumor lines: HT29 (colon carcinoma), HepG2 (hepatoblastoma), A172 (glioblastoma), HMCB (elanoma). The cells are cultured at 37 ° C. in an atmosphere at 95% humidity and 5% CO 2. The culture medium is that recommended by the ATCC.
3 ) Peptides testés . Les deux séries de peptides tes tées sont celles des tableaux I et II .3) Peptides tested. The two sets of peptides tested are those of Tables I and II.
4 ) Résultats .4) Results.
Les résultats d' internalisation sont montrés dans les tableaux III et IV ci-dessous. Les peptides pénètrent dans les cellules avec différents degrés d' internalisation. Certains (tels que SM1739 et SM2190) ne sont pas internalisés alors que d'autres (tels que SM2307, SM2187, etc...) pénètrent avec une bonne efficacité. Nous avons aussi observé que certains peptides pénètrent beaucoup plus dans un type cellulaire que dans d'autres. Par exemple, le SM2196 a une meilleure internalisation dans les cellules tumorales (HepG2, A172, et HT29) que dans les cellules non tumorales (MRC5 et HuVeC) . A l'inverse, le peptide SM1738 pénètre beaucoup plus dans les lignées non tumorales que dans les lignées tumorales. Ces résultats suggèrent qu'il y aurait un tropisme cellulaire.The internalization results are shown in Tables III and IV below. Peptides enter cells with varying degrees of internalization. Some (such as SM1739 and SM2190) are not internalized while others (such as SM2307, SM2187, etc ...) penetrate with good efficiency. We also observed that certain peptides penetrate much more in a cell type than in others. For example, SM2196 has better internalization in tumor cells (HepG2, A172, and HT29) than in non-tumor cells (MRC5 and HuVeC). Conversely, the SM1738 peptide penetrates much more into non-tumor lines than into tumor lines. These results suggest that there is a cellular tropism.
De manière générale, il apparaît que la forme rétro des têtes de série ne modifie pas de façon significative 1 ' internalisation . L'augmentation de 1' hydrophobie a un effet négatif, pour les deux familles de peptides testées. Il convient donc éviter d'augmenter 1 ' hydrophobie . En revanche, une augmentation de 1 ' amphipathie semble avoir un effet positif, au moins pour la famille Protégrine.In general, it appears that the retro shape of the heads of series does not significantly modify internalization. The increase in hydrophobicity has a negative effect for the two families of peptides tested. It is therefore advisable to avoid increasing hydrophobicity. On the other hand, an increase in amphipathy seems to have a positive effect, at least for the Protégrine family.
Tableau III : dérivés de protégrinesTable III: derivatives of protectins
Figure imgf000025_0001
Figure imgf000025_0001
Les photos de microscopie à fluorescence de 1 ' internalisation sont présentées aux figures 1 et 2. Dans les lignées A172 et HT29, le peptide SM 1738, présenté à titre d'exemple, apparaît localisé majoritairement dans le cytoplasme et dans une zone périnucléaire . Dans le cas de la lignée HuVec , le peptide a une localisation majoritairement cytoplasmique . La colonne de gauche correspond à la coloration du noyau par Hoechst .The fluorescence microscopy photos of the internalization are presented in FIGS. 1 and 2. In the lines A172 and HT29, the peptide SM 1738, presented by way of example, appears to be located mainly in the cytoplasm and in a perinuclear zone. In the case of the HuVec line, the peptide has a localization mainly cytoplasmic. The left column corresponds to the coloring of the nucleus by Hoechst.
Tableau II : dérivés de tachyplésinesTable II: derivatives of tachyplesins
Figure imgf000026_0001
nd : non déterminé
Figure imgf000026_0001
na: not determined
Les photos de 1 ' internal i sat ion sont présentées aux f igures 3 et 4 en annexe . Pour les 3 lignées cellulaires présentées (A172 , HT29 , HuVeC ) , le peptide biotinylé est localisé dans le cytoplasme de façon dif fuse et marque également de façon nette le nuc léole . La colonne de gauche correspond à la coloration du noyau par Hoechst .The internal i sat ion photos are shown in Figures 3 and 4 in the appendix. For the 3 cell lines presented (A172, HT29, HuVeC), the biotinylated peptide is localized in the cytoplasm in a diffuse fashion and also clearly marks nuc leole. The left column corresponds to the coloring of the nucleus by Hoechst.
Exemple Internal i sat ion de l a doxorubicine vectorisée ,Example Internal i sat ion of vectorized doxorubicin,
Les cellules sont ensemencées à environ 10 cellules par puits, 24 h avant l'addition des produits. Elles sont alors à 60-80% de confluence le jour de l'expérience. La doxorubicine libre d'une part, ou la doxorubicine couplée au vecteur SM1738 d'autre part, sont incubés avec les cellules MCF7 à la concentration de 10 μM, pendant 60 minutes, à 37°C dans une atmosphère à 95% d'humidité et 5% de C02 dans le milieu de culture. La localisation subcellulaire de la doxorubicine, naturellement fluorescente, a été déterminée par microscopie confocale . Les résultats sont présentés à la figure 5 en annexe. La localisation est pour partie cytoplasmique et pour partie nucléaire. Le noyau est alors marqué de façon diffuse.The cells are seeded at approximately 10 cells per well, 24 h before the addition of the products. They are then at 60-80% confluence on the day of the experiment. Free doxorubicin on the one hand, or doxorubicin coupled to the vector SM1738 on the other hand, are incubated with MCF7 cells at a concentration of 10 μM, for 60 minutes, at 37 ° C. in an atmosphere at 95% humidity. and 5% CO 2 in the culture medium. The subcellular localization of the naturally fluorescent doxorubicin was determined by confocal microscopy. The results are presented in Figure 5 in the appendix. Localization is partly cytoplasmic and partly nuclear. The nucleus is then diffusedly marked.
Dans les séquences peptidiques rapportées ci-dessus, les acides aminés sont représentés par leur code à une lettre, mais ils peuvent être aussi représentés par leur code à trois lettres selon la nomenclature ci-dessous.In the peptide sequences reported above, the amino acids are represented by their one-letter code, but they can also be represented by their three-letter code according to the nomenclature below.
A Ala alanineTo Ala alanine
C Cys cystéineC Cysteine
D Asp acide aspartiqueD Asp aspartic acid
E Glu acide glutamiqueE Glu glutamic acid
F Phe phénylalanineF Phe phenylalanine
G Gly glycine H H H Hiiss histidineG Gly glycine H H H Hiiss histidine
I Ile isoleucineI isoleucine island
K Lys lysineK lysine lysine
L Leu leucineL Leu leucine
M Met methionine N N A Assnn asparagineM Met methionine N N A Assnn asparagine
P Pro prolineP Pro proline
Q Gin glutamineQ Gin glutamine
R Arg arginineR Arg arginine
S Ser serine T T T Thhrr threonineS Ser serine T T T Thhrr threonine
V Val valineV Val valine
W Trp tryptophaneW Trp tryptophan
Y Tyr tyrosine Y Tyr tyrosine

Claims

REVENDICATIONS
1) Peptide dérivé d'un peptide antibiotique ou d'un analogue de celui-ci, caractérisé en ce qu'il est dépourvu de pont disulfure.1) Peptide derived from an antibiotic peptide or an analog thereof, characterized in that it has no disulfide bridge.
2) Peptide dérivé d'un peptide antibiotique ou d'un analogue de celui-ci, caractérisé en ce que tous les résidus cystéines, éventuellement sauf un, sont supprimés, remplacés par un autre résidu d'acide aminé ou bloqués au niveau de leur groupe SH.2) Peptide derived from an antibiotic peptide or an analogue thereof, characterized in that all the cysteine residues, optionally except one, are deleted, replaced by another amino acid residue or blocked at their level. SH group.
3) Peptide linéaire selon l'une quelconque des revendications 1 à 2, caractérisé en ce qu'il répond à l'une des formules suivantes :3) Linear peptide according to any one of claims 1 to 2, characterized in that it corresponds to one of the following formulas:
BXXBXXXXBBBXXXXXXB ( I )BXXBXXXXBBBXXXXXXB (I)
BBXXXBXXXBXXXXBBXB (II) dans lesquelles :BBXXXBXXXBXXXXBBXB (II) in which:
- les groupes B, identiques ou différents, représentent un résidu d'acide aminé dont la chaîne latérale porte un groupement basique, etthe groups B, which are identical or different, represent an amino acid residue whose side chain carries a basic group, and
- les groupes X, identiques ou différents, représentent un résidu d'acide aminé aliphatique ou aromatique, ou en ce qu'il est constitué d'une succession d'au moins 5, et de préférence d'au moins 7 acides aminés successifs de l'une des formules (I) ou (II) •the X groups, identical or different, represent an aliphatic or aromatic amino acid residue, or in that it consists of a succession of at least 5, and preferably at least 7 successive amino acids of one of the formulas (I) or (II) •
4) Peptide linéaire selon la revendication4) Linear peptide according to claim
3, caractérisé en ce que les groupes B sont choisis parmi l' arginine, la lysine, l'acide diaminoacétique, l'acide diaminobutyrique, l'acide diaminopropionique, 1 ' ornithine . 5) Peptide linéaire selon l'une des revendications 3 à 4, caractérisé en ce que les groupes X sont choisis parmi la glycine, l' alanine, la valine, la norleucine, 1 ' isoleucine, la leucine, la cystéine, la3, characterized in that the groups B are chosen from arginine, lysine, diaminoacetic acid, diaminobutyric acid, diaminopropionic acid, ornithine. 5) Linear peptide according to one of claims 3 to 4, characterized in that the groups X are chosen from glycine, alanine, valine, norleucine, 1 isoleucine, leucine, cysteine,
_Α.cm cystéine , la penicillamine, la methionine, la serine, la threonine, 1 ' asparagine , la glutamine, la phénylalanine, 1 'histidine, le tryptophane, la tyrosine, la proline, 1 ' Abu , l'acide amino-1-cyclohexane carboxylique, l'Aib, la 2-aminotétraline carboxylique, la 4-bromophénylalanine , tert-Leucine , la 4- chlorophénylalanine, la β-cyclohexylalanine, la 3,4- dichlorophénylalanine , la 4- f luorophénylalanine , l 'homo leucine, la β-homoleucine, 1 ' homophénylalanine , la_Α.cm cysteine, penicillamine, methionine, serine, threonine, asparagine, glutamine, phenylalanine, histidine, tryptophan, tyrosine, proline, 1 'Abu, amino-1 acid -carboxylic cyclohexane, Aib, carboxylic 2-aminotetralin, 4-bromophenylalanine, tert-Leucine, 4-chlorophenylalanine, β-cyclohexylalanine, 3,4-dichlorophenylalanine, 4-fluorophenylalanine, homo leucine , β-homoleucine, 1 homophenylalanine,
4-méthylphénylalanine , la 1-naphtylalanine , la 2- naphtylalanine, la 4-ni trophénylalanine , la 3- nitrotyrosine, la norvaline, la phénylglycine, la 3- pyridylalanine, la [2-thiényl] alanine .4-methylphenylalanine, 1-naphthylalanine, 2-naphthylalanine, 4-ni trophenylalanine, 3-nitrotyrosine, norvaline, phenylglycine, 3-pyridylalanine, [2-thienyl] alanine.
6) Peptide linéaire selon l'une quelconque des revendications 1 à 5, caractérisé en ce qu'il répond à 1 ' une des formules suivantes :6) Linear peptide according to any one of claims 1 to 5, characterized in that it corresponds to one of the following formulas:
RXXRXUXURRRXUXUXXR-NH2 (V)RXXRXUXURRRXUXUXXR-NH2 (V)
RRXUXRXUXRXXUXRRUR-NH2 (VI) dans lesquelles - U représente la serine ou la threonine,RRXUXRXUXRXXUXRRUR-NH2 (VI) in which - U represents serine or threonine,
- R représente l' arginine, et- R represents arginine, and
- les groupes X identiques ou différents représentent un acide aminés naturel ou non, y compris acides aminés de configuration D, aliphatiques ou aromatiques, comme la glycine, l' alanine, la valine, la norleucine, 1 ' isoleucine, la leucine, la cystéine, la cystéine , la penicillamine, la methionine, la serine, la threonine, 1 ' asparagine , la glutamine, la phénylalanine, l' histidine, le tryptophane, la tyrosine, la proline, 1 ' Abu , l'acide amino- 1-cyclohexane carboxylique, l'Aib, la 2-aminotétraline carboxylique, la 4 -bromophénylalanine , tert-Leucine , la 4- chlorophénylalanine , la β-cyclohexylalanine, la 3,4- dichlorophénylalanine , la 4- fluorophénylalanine , 1 'homoleucine, la β-homoleucine, 1 ' homophénylalanine, la 4-méthylphénylalanine , la 1-naphtylalanine , la 2- naphtylalanine , la 4-nitrophénylalanine , la 3- nitrotyrosine, la norvaline, la phénylglycine, la 3- pyridylalanine, la [2-thiényl] alanine .- identical or different groups X represent a natural or unnatural amino acid, including amino acids of configuration D, aliphatic or aromatic, such as glycine, alanine, valine, norleucine, isoleucine, leucine, cysteine , cysteine, penicillamine, methionine, serine, threonine, asparagine, glutamine, phenylalanine, histidine, tryptophan, tyrosine, proline, 1 'Abu, amino acid 1- cyclohexane carboxylic, Aib, 2-aminotetralin carboxylic, 4-bromophenylalanine, tert-Leucine, 4-chlorophenylalanine, β-cyclohexylalanine, 3,4- dichlorophenylalanine, 4-fluorophenylalanine, 1 homoleucine, β-homoleucine, 1 'homophenylalanine, 4-methylphenyl 1-naphthylalanine, 2-naphthylalanine, 4-nitrophenylalanine, 3-nitrotyrosine, norvaline, phenylglycine, 3-pyridylalanine, [2-thienyl] alanine.
7) Peptide linéaire selon l'une quelconque des revendications 1 à 6 , de séquences suivantes :7) Linear peptide according to any one of claims 1 to 6, of the following sequences:
RGGRLSYSRRRFSVSVGR,RGGRLSYSRRRFSVSVGR,
RGVSVSFRRRSYSLRGGR,RGVSVSFRRRSYSLRGGR,
EGGELSYSEEEFSVSVGE, RGGRLAYRLLRFAIRVGR,EGGELSYSEEEFSVSVGE, RGGRLAYRLLRFAIRVGR,
OGGOXXBOXXOBXXXOXG,OGGOXXBOXXOBXXXOXG,
RAARLGYRXXRFGZRVGR,RAARLGYRXXRFGZRVGR,
YRRRFSVSVR,YRRRFSVSVR,
RRLSYSRRRF, RRLSYSRRRFSVSVR,RRLSYSRRRF, RRLSYSRRRFSVSVR,
RGGRLSYSRRRFSTSTGR, où B représente la Napthylalanine, O représente l'Ornithine, X représente la Norleucine et Z représente la Norvaline.RGGRLSYSRRRFSTSTGR, where B represents Napthylalanine, O represents Ornithine, X represents Norleucine and Z represents Norvaline.
8) Peptide linéaire selon l'une quelconque des revendications 1 à 6 , de séquences suivantes :8) Linear peptide according to any one of claims 1 to 6, of the following sequences:
K SFRVSYRGISYRRSR,K SFRVSYRGISYRRSR,
RWSFRVSYRGISYRRSR, RSRRYSIGRYSVRFSWK,RWSFRVSYRGISYRRSR, RSRRYSIGRYSVRFSWK,
OBXBOXXBOGXOBXXOX,OBXBOXXBOGXOBXXOX,
KWAFRVAYRGIRYLLR ,KWAFRVAYRGIRYLLR,
KYAWRVAHRGIRWLLRX où B représente la Napthylalanine, O représente l'Ornithine, X représente la Norleucine et Z représente la Norvaline. 9 ) Utilisation d ' un peptide antibiotique ou d ' un analogue de celui-ci , dépourvu de pont disulfure , pour vec tori ser des substances ac t ives dans un organisme .KYAWRVAHRGIRWLLRX where B represents Napthylalanine, O represents Ornithine, X represents Norleucine and Z represents Norvaline. 9) Use of an antibiotic peptide or an analog thereof, devoid of a disulfide bridge, for the delivery of active substances in an organism.
10) Utilisation d'un peptide selon l'une quelconque des revendications 1 à 8, pour vectoriser des substances actives dans un organisme.10) Use of a peptide according to any one of claims 1 to 8, to vectorize active substances in an organism.
11) Composé de formule (IV) suivante :11) Compound of formula (IV) below:
(Y-r-ή 11—A-) (-Z)m1LL ( IV) dans laquelle(Yr- ή 11 — A-) (-Z) m 1LL (IV) in which
- A représente un peptide linéaire dérivé d'un peptide antibiotique ou d'un analogue de celui-ci,- A represents a linear peptide derived from an antibiotic peptide or an analog thereof,
- Z représente une substance active,- Z represents an active substance,
- Y représente un agent signal,- Y represents a signal agent,
- n est 0 et ou plus, et avantageusement 0 ou 1, - m est 1 ou plus, et de préférence jusqu'à- n is 0 or more, and advantageously 0 or 1, - m is 1 or more, and preferably up to
10, avantageusement jusqu'à 5.10, advantageously up to 5.
12) Composé de formule (IV) dans laquelle A est définie comme dans l'une quelconque des revendications 1 à 8.12) Compound of formula (IV) in which A is defined as in any one of claims 1 to 8.
13) Composé selon l'une des revendications 11 ou 12, caractérisé en ce que le couplage entre le peptide linéaire (A) et le groupe (Z) ou les groupes (Z) et (Y) est réalisé par une ou plusieurs liaisons covalentes , hydrophobes ou ioniques .13) Compound according to one of claims 11 or 12, characterized in that the coupling between the linear peptide (A) and the group (Z) or the groups (Z) and (Y) is carried out by one or more covalent bonds , hydrophobic or ionic.
14) Composé selon l'une quelconque des revendications 11 à 13, caractérisé en ce que l'une au moins des substances actives (Z) est fixée par une liaison covalente soit aux extrémités N-terminale ou C- terminale, soit au niveau des groupements aminés primaires portés par les chaînes latérales des lysines, du peptide linéaire (A) .14) Compound according to any one of claims 11 to 13, characterized in that at least one of the active substances (Z) is fixed by a covalent bond either at the N-terminal or C- ends terminal, ie at the level of the primary amino groups carried by the side chains of the lysines, of the linear peptide (A).
15) Composé selon l'une quelconque des revendications 11 à 14, caractérisé en ce qu'au moins un agent signal (Y), s'il est présent, est fixé par une liaison covalente à l'extrémité N-terminale du peptide linéaire (A) .15) Compound according to any one of claims 11 to 14, characterized in that at least one signal agent (Y), if present, is attached by a covalent bond to the N-terminal end of the linear peptide (AT) .
16) Composition pharmaceutique caractérisé en ce qu'elle comprend comme principe actif au moins un composé de formule (IV) selon l'une quelconque des revendications 11 à 15.16) Pharmaceutical composition characterized in that it comprises as active principle at least one compound of formula (IV) according to any one of claims 11 to 15.
17) Un agent de diagnostic constitué d'au moins un composé de formule (IV) selon l'une quelconque des revendications 11 à 15. 17) A diagnostic agent consisting of at least one compound of formula (IV) according to any one of claims 11 to 15.
PCT/FR1998/001757 1997-08-12 1998-08-06 Linear peptides derived from antibiotic peptides, preparation and use for vectoring active substances WO1999007728A2 (en)

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FR2767323A1 (en) 1999-02-19
CA2298932A1 (en) 1999-02-18
EP1003771A1 (en) 2000-05-31
WO1999007728A3 (en) 1999-06-24
FR2767323B1 (en) 2001-01-05
AU754617B2 (en) 2002-11-21

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