AU8988998A

AU8988998A - Linear peptides derived from antibiotic peptides, preparation and use for vectoring active substances

Info

Publication number: AU8988998A
Application number: AU89889/98A
Authority: AU
Inventors: Bernard Calas; Alain Chavanieu; Gerard Grassy; Michel Kaczorek
Original assignee: Synt EM SA
Current assignee: Synt EM SA
Priority date: 1997-08-12
Filing date: 1998-08-06
Publication date: 1999-03-01
Anticipated expiration: 2018-08-06
Also published as: FR2767323A1; CA2298932A1; IL134293A0; EP1003771A1; AU754617B2; JP2001512739A; WO1999007728A2; FR2767323B1; WO1999007728A3

Description

1 LINEAR PEPTIDES DERIVED FROM ANTIBIOTIC PEPTIDES, PREPARATION AND USE FOR VECTORING ACTIVE SUBSTANCES The invention concerns linear peptides derived from antibiotic peptides and their use for vectoring active substances. More particularly, the subject of the invention is new compounds formed from a linear 5 derivative of an antibiotic peptide coupled to at least one active substance, and the preparation of these compounds and compositions containing them. In addition to their immunity system responsible for specific defence mechanisms against infectious 10 agents, vertebrates have numerous peptides with anti microbial activity (Nicolas P. et al., 1995, Annual Rev. Microbiol. 49, 277-304) . These peptides only exist in invertebrates having a short lifetime and a high renewal rate, in whom a memory immunity system, long in forming 15 and developing appropriate response, is ill-adapted. The anti-microbial peptides of vertebrates, irrespective of their origin, lower or higher vertebrates, myeloid or non-myeloid tissue, have a certain number of properties in common : 20 - high basicity due to the presence of numerous arginines and lysines, - the ability to form amphipathic structures. By amphipathic structure is meant structures in which the hydrophobic residues are separated in space 25 from hydrophilic residues, - a very wide activity spectrum. They are able to rapidly destroy bacteria (Gram+ and Gram-), fungi, a few protozoa, membrane viruses and even some cancer cell lines. R 1RA4 2 According to their structure, antibiotic peptides can be divided into three major families : - amphipathic a-helical antibiotic peptides: cecropins and maganins (Maloy W.L. et al., 1995, 5 BioPolymer 37, 105-122), - P-stranded antibiotic peptides linked by disulphide bonds defensins (Lehrer R.I. et al., 1991, Cell 64:229-230 ; Lehrer R.I. et al., 1993, Ann. Rev. Immunol. 11:105-128), protegrins (Kokryakov V.N. et al., 10 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), - antibiotic peptides, having destructured chains with many angles due to the presence of multiple 15 prolines : bactenecins and PR39 (Frank R.W. et al., 1991, Eur. J. Biochem. 202, 849-854). Despite the diversity of their sequences, most antibiotic peptides act by direct lysis of the membrane of pathogenic cells. Their basicity promotes their 20 interaction with negatively charged phospholipids, and being amphipathic they are subsequently able to incorporate themselves into the membrane in which they aggregate to form pores through which the cell loses its substance. It is generally accepted that their 25 preferential selectivity for prokaryote cells is due to the special composition of their membranes which contain more anionic phospholipids than those of eukaryotes. Also, the plasma membranes of mammalian cells all contain cholesterol whose role is to modulate their fluidity, 30 which could hinder the incorporation of antibiotic peptides. However, the specificity of the latter for 3 micro-organisms is low, meaning that they show strong cytotoxicity which limits their use. The presence of antibiotic peptides in vertebrates, and more particularly in mammalians, raises 5 numerous queries. Immunologists assume that the compounds having non-specific anti-microbial activity found in invertebrates constitute an ancestral means of defence which later developed leading to much more complex memory systems. What is the advantage therefore, in mammalians 10 for example, of having preserved some peptides with antibiotic activity ? It is supposed that these small molecules that are always present in biological fluids, or sequestered in some lymphocyte structures, could form a first line of defence while awaiting the secretion of 15 specific antibodies (Nicolas P. et al., 1995, Annual Rev. Microbiol. 49, 277-304). They could also, within the macrophages, take part in the destruction of plasma membranes of pathogenic organisms. Regardless of their exact role, antibiotic 20 peptides are of considerable interest owing to their wide spectrum of activity and the difficulty encountered by micro-organisms to set up inactivation strategies. On this account very numerous research studies have been conducted to endeavour to find new molecules and to 25 obtain better performing analogues than the parent peptides. It is possible that in the future these antibiotic peptides are called upon to replace the antibiotics derived from bacteria or fungi. For example, PCT international patent applications published under 30 numbers W095/03325, W096/37508 and W097/02287 describe a new class of antibiotic peptides called "protegrins", isolated from porcine leukocytes or even prepared by 4 chemical synthesis or genetic engineering and having antibacterial, antiviral and antifungal activities. At the present time, P-stranded antibiotic peptides linked by disulphide bonds (defensins, 5 protegrins, tachyplesins) are a particular subject of research on account of their powerful anti-microbial activity (bacteria, some viruses, fungi and parasites). Within this family, protegrins and tachyplesins are certainly the most promising molecules given the 10 simplicity of their structure and the relative ease with which they can be synthesised. The name protegrins denotes a group of five peptides called PG-1, PG-2, PG-3, PG-4 and PG-5 whose sequences are given below, closely resembling and 15 isolated from porcine leukocytes (V.N. Kokryakov et al., FEBS lett. 327, 231-236) : PG-1 : RGGRLCYCRRRFCVCVGR-NH 2 PG-2 : RGGRLCYCRRRFCICV..-NH 2 PG-3 : RGGGLCYCRRRFCVCVGR-NH 2 20 PG-4 : RGGRLCYCRGWICFCVGR-NH 2 PG-5 : RGGRLCYCRPRFCVCVGR-NH 2 Tachyplesins (Tamura H. et al., 1993, Chem. Pharm. Bul. Tokyo 41, 978-980) denoted T1, T2 and T3 and polyphemusins (Muta T., 1994, CIBA Found. Sym. 186, 160 25 174) denoted P1 and P2 whose sequences are given below, are homologous peptides isolated from the hemolymph of two crabs Tachyplesus tridentatus for Tachyplesins T1, T2 and T3, and Limmulus polyphemus for Polyphemusins P1 and P2. 30 P1 RRWCFRVCYRGFCYRKCR-NH 2 5 P2 : RRWCFRVCYKGFCYRKCR-NH 2 Ti : KWCFRVCYRGICYRRCR-NH 2 T2 : RWCFRVCYRGICYRKCR-NH 2 T3 : KWCFRVCYRGICYKRCR-NH 2 5 Protegrins, tachyplesins and polyphemusins contain a high proportion of base residues (lysines and arginines) and have four cysteines which form two parallel disulphide bonds. These three families of peptides also show homologies with some defensins in 10 particular with the human defensin NP-1 (Kokryakov V.N. et al., 1993, Febs Let. 327, 231-236). Tachyplesins and protegrins have a closely resembling three-dimensional structure. It is an anti parallel P strand stabilised by the two disulphide bonds. 15 These bonds play an important role in the antibacterial activity of protegrins and tachyplesins. Their removal, either by protecting the SH groups with acetamidomethyls, or by replacing the cysteines with alanines or glycines, leads to obtaining analogues virtually devoid of in vivo 20 activity (Lehrer R.I. et al., 1996, Eur. J. Biochem. 240:352-357). As previously indicated, protegrins and tachyplesins have substantial lysis activity on prokaryote cells. Research work conducted by the 25 Applicant on the cytotoxicity of these peptides on cultured mammalian cells, have shown that, prior to the death of the cells, there are non-negligible quantities of protegrins and tachyplesins in the cytoplasm of said cells. It was considered that the presence of peptides in 30 the cytoplasm could be the outcome of transport via pores, but these pores are only permeable to ions and small molecules and their diameter is too small to give AL/ I-Il, 6 passageway to antibiotic peptides. It would seem that protegrins and tachyplesins, in addition to perforating the plasma membrane, are able to pass through it. The cytotoxicity and antimicrobial activity of 5 protegrins and tachyplesins are known to derive from their ability to aggregate inside the membrane to form multimeric channels (Mangoni M. et al., 1996, Febs Let. 383, 93-98). The Applicant therefore considered that this aggregation might be connected with the tertiary structure 10 of these antibiotic peptides, which comprise several cysteine residues, and linear derivatives of protegrins and tachyplesins in which the cysteines are replaced by various natural amino acids have been prepared. These peptides were coupled, at their N-terminal end, to a 15 fluorescent molecule or to biotin and the distribution of these markers inside the cell was observed under confocal microscopy. In this way, it was found that these peptides are non-toxic and have no lytic activity but are, on the other 20 hand, able to pass rapidly through the membranes of mammalian cells via a passive mechanism. These linear derivatives of antibiotic peptides therefore constitute a new, non-toxic, system for vectoring active substances. 25 By vectoring system is meant, according to the invention, a process capable of conveying said active substance to a target, such as for example : - to cause an active substance to pass through the cell membrane and to allow the distribution of said 30 substance in the cytoplasm and/or in the nuclear compartment.

LU

7 - to bring an active substance to a particular organ, for example to cause this active substance to pass through the blood-brain barrier, - to force this active substance to interact 5 specifically with a given cell type, erythrocytes for example. The subject of the present invention is therefore peptides derived from antibiotic peptides or analogues thereof, characterised in that they are devoid of a 10 disulphide bond. By analogue of antibiotic peptides is meant a peptide whose amino acid sequence has been modified without causing any modification in the antibiotic properties of said peptide. 15 The absence of a disulphide bond in the peptides of the, invention, may be obtained by any means known to those skilled in the art, for example by : - removing, or replacing with other amino acids, the cysteine residues of the antibiotic peptide 20 sequence, - blocking the -SH groups of the cysteine residues such that they do not form a disulphide bond, provided, evidently, that the peptide obtained has vectoring properties that are not toxic for the 25 previously described cells. These modifications may be conducted during the preparation of the peptides of the invention, more particularly by chemical synthesis or the expression of a gene coding for said peptide, or directly on an 30 antibiotic peptide through the action of chemical agents 8 enabling the opening and blocking of the -SH groups of the cysteine residues. The above modifications advantageously concern all the cysteine residues of the antibiotic peptide, but 5 should the presence of a single cysteine residue not allow the formation of a disulphide bond, the peptides of the invention may contain a single cysteine. Natural antibiotic peptides generally have 4 or 6 cysteine residues able to form two or three disulphide bonds, 10 therefore in the peptides of the invention only one of these cysteines can be maintained and the three or five others are modified or blocked. The antibiotic peptides from which the peptides of the invention are derived may be defensins, 15 protegrins, tachyplesins or their analogues whose antibiotic properties are imparted to them by their tertiary structure resulting from the presence of disulphide bonds. Linear peptides of the invention meet one of the 20 following formulas : BXXBXXXXBBBXXXXXXB (I) BBXXXBXXXBXXXXBBXB (II) which may also be represented by the following single formula (III) : 25 B(XB)X(XB)X(XB)XX(XB)B(XB)XXX(XB) (XB)XB in which : - the B groups, identical or different, represent an amino acid residue whose side chain carries a base group, and 30 - the X groups, identical or different, represent an aliphatic or aromatic amino acid residue, RA4- 9 or are made up of a sequence of at least 5, preferably at least 7, successive amino acids of either of formulas (I) or (II), if this sequence has vectoping properties that are non-toxic for the previously 5 described cells. B and X may or may not be natural amiho acids, including D-amino acids. As an example the following denotations of B and X may be cited : 10 - B is chosen from among arginine, lysine, diaminoacetic acid, diaminobutyric acid, diaminoproprionic acid, ornithine. - X is chosen from among glycine, alanine, valine, norleucine, isoleucine, leucine, cysteine, 15 cysteineAcm, penicillamine, methionine, serine, threonine, asparagine, glutamine, phenylalanine, histidine, tryptophan, tyrosine, proline, Abu, carboxylic amino-l cyclohexane acid, Aib, carboxylic 2-aminotetraline, 4 bromophenylalanine, tert-Leucine, 4-chlorophenylalanine, 20 P-cyclohexylalanine, 3,4-dichlorophenylalanine,- 4 fluorophenylalanine, homoleucine, P-homoleucine, homophenylalanine, 4-methylphenylalanine, 1 naphthylalanine, 2-naphthylalanine, 4-nitrophenylalanine, 3-nitrotyrosine, norvaline, phenylglycine, 3 25 pyridylalanine, [2-thienyl alanine. The invention also concerns peptide derivatives having the formula (I) or (II) such as said peptides in retro form, or moieties of peptides having the formula (I) or (II) made up of five, preferably seven, successive 30 amino acids of either formula (I) or (II).

10 Among the peptides of the invention, special mention can be made of those meeting the following formulas :

RXXRXUXURRRXUXUXXR-NH

2 (V) 5 RRXUXRXUXRXXUXRRUR-NH2 (VI) in which : - U represents serine or threonine - R represents arginine, and - the X groups, identical or different, 10 represent an amino acid which may or may not be natural (including D-amino acids), either aliphatic or aromatic, such as glycine, alanine, valine, norleucine, isoleucine, leucine, cysteine, cysteineAc, penicillamine, methionine, serine, threonine, asparagine, glutamine, phenylalanine, 15 histidine, tryptophan, tyrosine, proline, Abu, carboxylic amino-1-cyclohexane acid, Aib, carboxylic 2 aminotetraline, 4-bromophenylalanine, tert-Leucine, 4 chlorophenylalanine, P-cyclohexylalanine, 3,4 dichlorophenylalanine, 4-fluorophenylalanine, 20 homoleucine, p-homoleucine, homophenylalanine, 4 methylphenylalanine, 1-naphthylalanine, 2 naphthylalanine, 4-nitrophenylalanine, 3-nitrotyrosine, norvaline, phenylglycine, 3-pyridylalanine, [2 thienyl]alanine. 25 Among the peptides of formulas (I) and (II) or their derivatives, the invention specifically considers those derived from protegrins and tachyplesins referred to in tables I and II below. 47 11 Table I : Protegrin derivatives Code Sequence Modification SM1738 RGGRLSYSRRRFSVSVGR Head of series SM1736 rggrlsysrrrfsvsvgr Aa of D form of SM1738 SM1727 RGVSVSFRRRSYSLRGGR Retro form of SM1738 SM1739 EGGELSYSEEEFSVSVGE Reversed charge (R -> E) SM2187 RGGRLAYRLLRFAIRVGR Increased amphipathicity SM2188 OGGOXXBOXXOBXXXOXG Increased hydrophobicity SM2189 RAARLGYRXXRFGZRVGR Increased amphipathicity SM2194 YRRRFSVSVR C-terminal end of SM2193 SM2195 RRLSYSRRRF N-terminal end of SM2193 SM2193 RRLSYSRRRFSVSVR Reduced flexibility (G deletion) SM2196 RGGRLSYSRRRFSTSTGR Inhibition dimerisation Table II : Tachyplesin derivatives Code Sequence Modification SM1726 KWSFRVSYRGISYRRSR Head of series SM2307 RWSFRVSYRGISYRRSR K -+ R mutation SM2392 rwsfrvsyrgisyrrsr Aa of D form (of SM2307) SM2309 kwsfrvsyrgisyrrsr Aa of D form (of SM1726) SM2310 RSRRYSIGRYSVRFSWK Retro form SM2190 OBXBOXXBOGXOBXXOX Increased hydrophobicity SM2191 KWAFRVAYRGIRYLLRL Increased amphipathicity SM2192 KYAWRVAHRGIRWLLRX Increased amphipathicity In the sequences of tables I and II above, B 5 represents Naphthylalanine, 0 represents Ornithine, X represents Norleucine and Z represents Norvaline. The invention also concerns the use of the above peptides for vectoring one or more active substances both for therapeutic and for diagnostic applications. As 10 active substance, the invention gives particular 12 consideration to proteins or moieties of proteins, such as polypeptides or peptides, antibodies or parts of antibodies, nucleic acids and oligonucleotides or ribozymes, or even, obviously, active chemical molecules 5 for the treatment or prevention of human or animal pathologies, such as for example, but not restricted to, anti-tumorals, antivirals, anti-inflammatories, agents preventing the degradation of organs and/or tissues, etc... 10 In the field of diagnostics, the active substance may be a radioactive marker, a stain marker, or any other means or substance able to reveal a metabolism or a pathology. A further purpose of the invention is therefore 15 compounds of formula (IV) as follows and compositions containing them in k' j - Z) M(IV) in which : - A represents a linear peptide derived from an 20 antibiotic peptide in accordance with the invention, - Z represents an active substance, such as defined above, - Y represents a signal agent, - n is 0 or more, advantageously 0 or 1, 25 - m is 1 or more, preferably up to 10, advantageously up to 5. Therefore, the above formula (IV) compounds are formed from a peptide of the invention coupled with one or more active substances, identical or different, 30 represented by the (Z) group in formula (IV), and optionally one or more signal agents, represented by the -1 R 4 13 (Y) group in formula (IV), having an addressing role for the compound of formula (IV) towards a cell type, a site or cell compartment or a given tissue. More particularly, the signal agent (Y) is an oligopeptide or a protein, 5 such as a signal peptide, a nuclear localising signal, an antibody moiety, or a chemical molecule ligand or anti ligand of a receptor. In a special embodiment of the compounds of formula (IV), group (Y) is fixed to group (Z). 10 This coupling, symbolised by the horizontal lines in formula (IV), may be conducted by any acceptable linking means, taking into consideration the chemical nature, the size and number of groups (Z) and (Y) in the compounds of formula (IV), such as covalent, hydrophobic 15 or ionic bonds, which may or may not be cleaved in physiological media. Coupling may be conducted at any site of peptide (A), at which functional groups such as OH, -SH, -COOH, '-NH2 are naturally present or have been inserted. 20 The invention gives consideration to the fixation of several (Z) groups to one and the same site of peptide (A) either directly, if this site comprises several functional groups as is the case for a C- or N-terminal lysine, or indirectly via an intermediate group carrying 25 several reaction groups enabling the fixation of several (Z) groups. The preferred coupling positions for the active substance are at the N-terminal and C-terminal ends or at the primary amino groups carried by the side chains of 30 the lysines of peptide (A). If the C-terminal end of peptide (A) is used to attach active substance (Z), the N-terminal end is available for optional coupling to a signal agent (Y) enabling the compound of the invention 14 to be addressed either towards the nucleus, or towards a given tissue type. For example, if the C-terminal end of a linear peptide of the invention is used to couple an active 5 substance made up of a fluorescent marker, or biotin, or a medicinal molecule such as doxorubicin, the covalent peptide-drug complex distributes itself after administration within the cytoplasm of the target cell. It is possible to bring this complex into the nuclear 10 compartment by using the N-terminal end of the peptide to couple a short basic sequence, for example of around 7 amino acids, corresponding to a nuclear localising signal. Under these conditions, the biotin or doxorubicin are found in the cell nucleus. 15 In the same way, it is possible to vector a drug towards a given cell type, by using the N-terminal end of the linear peptide of the invention coupled at its C terminal end to a medicinal agent, to add a peptide sequence able to specifically recognise a determinant 20 present on the surface of cell type. Synthetic pentadecapeptide aM2 for example (Swolapenko G.B. et al., 1995, The Lancet 346, 1662-65) a moiety of a monoclonal antibody, directed against an antigen expressed by breast cancer cells (Tumour Associated Antigen Polymorphic 25 Epithelial Mucin) maintains good affinity for these cells. It is therefore possible, by associating ciM2 with a linear peptide-medicinal agent complex, to bring this group preferably towards the cells which express the antigen characteristic related.to breast cancer. 30 The compounds of formula (IV) may be prepared by chemical synthesis or by using molecular biology techniques.

-T.

15 For chemical syntheses, commercially available equipment can be used allowing the incorporation of non natural amino acids, such as D enantiomers and residues with side chains of different hydrophobicity and size to 5 those of their natural homologues. At the time of synthesis it is evidently possible to conduct a wide range of modifications, for example to insert a lipid (prenyl or myristyl) on the N-terminal so as to be able to anchor the peptide of the invention and hence the 10 formula (IV) compound to a lipid membrane such as that of a liposome made up of positively charged lipids. It is also possible to replace one or more peptide bonds (-CO NH-) by equivalent structures such as -CO-N(CH 3 )-, -CH 2 CH 2 -, -CO-CH 2 -, or to interpose groups such as -CH 2 -, 15 -NH-, -0-. It is also possible to obtain formula (IV) compounds, or part thereof having a protein nature, from an encoding nucleic acid sequence. A further purpose of the invention is a nucleic acid molecule comprising or 20 made up of a nucleic sequence coding for a linear peptide derived from an antibiotic peptide. More particularly, the invention concerns a nucleic acid molecule comprising at least one sequence coding for a formula (IV) compound or part thereof having a protein nature. These nucleic 25 acid sequences may be DNAs or RNAs and be associated with control sequences and/or inserted in vectors. The vector used is chosen in relation to the host to which it will be transferred ; it may be any vector such as a plasmid. These nucleic acids and vectors are useful for producing 30 the linear peptides and formula (IV) compounds, or part of the latter having a protein nature, in a host cell. The preparation of these vectors and the production or expression in a host of linear peptides or formula (IV) compounds may be conducted using molecular biology and 16 genetic engineering techniques well known to those skilled in the art. By way of example, said method for producing a peptide of the invention consists of : 5 - transferring a nucleic acid molecule or a vector containing said molecule into a host cell, - culturing said host cell under conditions enabling the production of the peptide, - isolating, by any appropriate means, the 10 peptides of the invention. The host cell used in this type of method may be chosen from among prokaryotes or eukaryotes, in particular from among bacteria, yeasts, mammalian, plant or insect cells. The invention therefore also concerns 15 transformed cells expressing the linear peptides or formula (IV) compounds or part of the latter having a protein nature. The invention also relates to - pharmaceutical compositions comprising as 20 active ingredient at least one formula (IV) compound optionally associated with an acceptable* vehicle or carrier, - diagnostic agents containing at least one formula (IV) compound. 25 Other characteristics and advantages of the invention will become apparent in the following description concerning the preparation of formula (IV) compounds and the research work which led to revealing the vectoring properties of the linear peptides of the 30 invention derived from antibiotic peptides. -0,~ 17 Example 1 : Fixing biotin and doxorubicin onto linear analogues of antibiotic peptides. 1) Preparation of linear peptides The three peptides with the sequences given below 5 were synthesised :

RGGRLXYXRRRFXVXVGR-NH

2

RRWXFRVXYRGFXYRKXR-NH

2

KWXFRVXYRGIXYRRXR-NH

2 in which X represents the serine, threonine or 10 alanine residues. These peptides are respectively derived from the sequences of Protegrin PG-l having the formula:

RGGRLCYCRRRFCVCVGR-NH

2 of Tachyplesin 1 having the formula 15 KWCFRVCYRGICYRRCR-NH 2 of Polyphemusin having the formula

KWXFRVXYRGIXYRRXR-NH

2 These three peptides may be prepared indifferently either from BOC chemistry or from FMOC 20 chemistry using conventional synthesis methods in solid or homogeneous phase. 2) Fixing biotin onto linear peptides The peptide is synthesised in solid phase and, after incorporation of the N-terminal arginine, 5 25 aminopentanoic acid is added. The FMOC or BOC N-terminal is removed, and on the peptide still adhering to the resin, the N-hydroxy succimido biotin ester is caused to react in dimethylformamide. After 15 hours' reaction at room temperature, the biotinylated peptide is cut from 18 the carrier through the action of trifluoroacetic acid or hydrofluoric acid following well-established protocols in peptide chemistry. The peptide is then purified by high pressure liquid chromatography. 5 3) Fixing doxorubicin onto a linear peptide To fix doxorubicin, solid phase synthesis is made of the peptide having the formula

RGGRLXYXRRRFXVXVGR-NH

2 After cleaving from the purification substrate, 10 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 diisopropylcarbodiimide and 1-hydroxybenzotriazole mixture. After two hours' reaction 15 at room temperature, the doxorubicin is added and the mixture is stirred for 12 hours at 0*C. The peptide doxorubicin unit is then purified by high pressure liquid chromatography. Example 2 : Ability of the linear peptides of the 20 invention to pass through cell membranes. 1) Cell models The ability of the peptides to pass through the membranes was tested on various cell types (MCF7, MCF7R, HL60, HL60R, HeLa). 25 The cells are cultured on RPMI 1640 (Gibco) to which is added 10 % (v/v) fetal calf serum, 2mM glutamine and 2mM pencillin/streptomycin at 37 0 C. 30 000 cells are seeded in Lab Tek chambers and cultured for 1 day. 2) Treatment with linear peptides-biotin 30 prepared according to example 1 (2)

STF

19 The cells are incubated in Opti-Mem (Gibco) for one hour before being treated for variable time periods with biotin-labelled peptides. The latter are obtained in accordance with 5 example 1 (2) by treating 1 equivalent of linear peptide with 2 equivalents of N-hydroxysuccinimide biotin ester, then purified by high pressure liquid chromatography. The cells are then fixed with a 3.7 % solution of paraformaldehyde for 5 minutes at 250C, then rinsed three 10 times with PBS. They are then permeabilised with 0.1 % Triton (1 min. room temperature). After three rinsings in PBS the cells are incubated 10 min with 200 pl TexRed antibodies diluted to 300 th and rinsed three times in PBS. The slides are finally mounted with a Mowiol-Dabco 15 solution and observed under an Axiophot photomicroscope. 3) Treatment with linear peptides-doxorubicin prepared in accordance with example 1 (3) The cells are incubated for 15 minutes, then rinsed with PBS and the doxorubicin present in the cell 20 is determined by chromatography. 4) Results a) Among the peptides studied, those which pass the most easily through the membranes are those with the following formulas : 25 RXXRXUXURRRXUXUXXR-NH 2 (V)

RRXUXRXUXRXXUXRRUR-NH

2 (VI) in which - U represents serine or threonine, - R represents arginine, and S TS 20 - the X groups, identical or different, represent an amino acid which may or may not be natural (including D-amino acids), either aliphatic or aromatic, such as glycine, alanine, valine, norleucine, isoleucine, 5 leucine, cysteine, cysteineAcm, penicillamine, methionine, serine, threonine, asparagine, glutamine, phenylalanine, histidine, tryptophan, tyrosine, proline, Abu, carboxylic amino-1-cyclohexane acid, Aib, carboxylic 2 aminotetraline, 4-bromophenylalanine, tert-Leucine, 4 10 chlorophenylalanine, p-cyclohexylalanine, 3,4 dichlorophenylalanine, 4-fluorophenylalanine, homoleucine, p-homoleucine, homophenylalanine, 4 methylphenylalanine, 1-naphthylalanine, 2 naphthylalanine, 4-nitrophenylalanine, 3-nitrotyrosine, 15 norvaline, phenylglycine, 3-pyridylalanine, [2 thienyl] alanine. b) The results of the experiments conducted with doxorubicin show a significant increase in the plasma and nuclear concentration of doxorubicin when the latter is 20 coupled with the linear peptide of the invention compared with the use of doxorubicin alone. c) The experiments with biotin were conducted more especially on MCF7 cells treated at different times with a complex of biotin and a peptide of the invention 25 having the formula : biotin-RGGRLSYSRRRFSVSVGR-NH2 This work was photographed (not shown) - Control in which the cell was treated with biotin alone, 30 - Treatment of the cell for 2 minutes with a complex biotin-linear peptide of the invention, 7rrs 21 - Treatment of the cell for 30 minutes with a complex: biotin-linear peptide of the invention. It can be seen in these photographs that biotin alone does not enter the cell and accumulates weakly 5 around the cell. Conversely, with the complex of the invention, it can be seen that the biotin is rapidly led by the linear peptide of the invention inside the cell in which it is present in the cytoplasm and cell nucleus. Example 3 : Internalisation ability of the linear 10 peptides of the invention Linear peptides of the invention derived from Protegrins and Tachyplesins were tested on different cell lines for the purposes of assessing their respective internalisation. 15 1) Experimental conditions The cells were seeded at approximately 104 cells per dish, 24 h before the addition of biotinylated peptides. On the day of the experiment confluence was 60 80%. The biotinylated peptides are incubated with the 20 cells at a concentration of 10 iM for 15 minutes at 370C in an atmosphere of 95% humidity and 5% C02 in an OptiMem medium. The cells are washed three times with PBS at room temperature and are then fixed with formalin (3.7% formaldehyde in PBS, 10 min at room temperature). They 25 are then washed in PBS and permeabilised for 15 min with PBS-TritonX-100. Development is made with streptavidin Texas-Red for 15 min away from light and the cells are then slide mounted. They are observed under a fluorescence microscope and compared with a positive 30 control (Ap43-58), well described in the literature, and with a negative control. The cell nuclei were Hoechst stained.

22 2) Cell lines All the lines tested are of human origin and were commercially obtained from ATCC. - Non-tumoral lines : MRC5 (lung fibroblast), 5 HuVeC (endothelial, umbilical cord) - Tumoral lines : HT29 (colon carcinoma), HepG2 (hepatoblastoma), A172 (glioblastoma), HMCB (melanoma). The cells are cultured at 37*C in an atmosphere of 95% humidity and 5% C02. The culture medium is the one 10 recommended by ATCC. 3) Tested peptides The two series of tested peptides are those given in tables I and II. 4) Results 15 The internalisation results are shown in tables III and IV below. The peptides penetrate the cells with different degrees of internalisation. Some (such as SM1739 and SM2190) are not internalised whereas others (such as SM2307, SM2187 etc...) penetrate with good 20 efficacy. We also observed that some peptides enter into a given cell type more than in others. SM2196 for example has better internalisation in tumoral cells (HepG2, A172 and HT29) than in non-tumoral cells (MRC5 and HuVeC) . Conversely, the SM1738 peptide has greater penetration 25 in non-tumoral lines than in tumoral lines. These results suggest the existence of cell tropism. Generally it would appear that the retro form of the heads of series does not significantly modify internalisation. Increased hydrophobicity has a negative 30 effect for both families of tested peptides. It is therefore advisable to avoid increasing hydrophobia. On 23 the other hand, an increase in amphipathicity seems to have a positive effect at least for the Protegrin family. Table III : Protegrin derivatives HepG2 A172 HMCB HuVeC MRC5 HT29 Internalisation SM1738 + + + +++ +++ + Reference SM1727 0 ++ ++ +++ + + No significant effect SM1736 ++ + +++ ++++ ++++ + No significant effect SM1739 0 + + 0 0 0 Negative effect SM2187 +++ +++ ++++ +++ ++++ +++ Positive effect SM2189 +++ ++ +++ ++ ++++ ++ Positive effect SM2188 0 0 0 ++ 0 0 Negative effect SM2193 ++ ++ +++ ++ 0 0 Negative effect SM2194 0 + +++ + + 0 Negative effect SM2195 ++++ 0 +++ + + ++++ Contradictory SM2196 ++++ ++++ ++ + + ++++ Tropism 5 Fluorescence microscopy photographs of internalisation are shown in figures 1 and 2. In the A172 and HT29 lines, the SM1738 peptide, shown as an example, appears to be mainly localised in the cytoplasm and in a perinuclear zone. For the HuVec line, the peptide is 10 mainly localised in the cytoplasm. The left column corresponds to nucleus staining with Hoechst. Table IV : Tachyplesin derivatives HepG2 A172 HMCB HuVeC MRC5 HT29 Internalisation SM1726 +++ + +++++ +++ +++ +++ Reference SM2310 ND ++ ++++ +++ ++ +++ No effect SM2309 ND ++++ ++ ++ ++++ ++++ ND SM2191 ++ ++ ++ ND +++ +++ No effect SM2192 + +++ ++++ +++ ++++ ++ No effect 24 SM2190 0 0 0 0 0 0 Negative effect SM2307 ND +++++ +++++ ++++ ++++ +++++ Positive effect SM2392 ND +++ ++++ ++ +++ ++++ No effect ND = not determined The internalisation photographs are shown in appended figures 3 and 4. For the 3 cell lines shown (A172, HT29, HuVeC) the biotinylated peptide is localised 5 in the cytoplasm in diffuse manner and also distinctly labels the nucleolus. The left column corresponds to nucleus staining with Hoechst. Example 4 : Internalisation of vectored doxo rubicin 10 The cells are seeded to approximately 104 cells per dish 24 h before the addition of the products. On the day of the experiment confluence is 60-80%. The free doxorubicin or the doxorubicin coupled to the SM1738 vector are incubated with the MCF7 cells at a 15 concentration of 10 pM for 60 minutes at 37 0 C in an atmosphere of 95% humidity and 5% C02 in the culture medium. The subcell localisation of doxorubicin, naturally fluorescent, was determined by confocal microscopy. The results are given in appended figure 5. 20 The localisation is partly cytoplasmic and partly nuclear. The nucleus in this case is labelled in diffuse manner. In the peptide sequences listed below, the amino acids are represented by their one-letter code, but they 25 may also be represented by their three-letter code according to the following nomenclature : A Ala alanine 25 C Cys cysteine D Asp aspartic acid E Glu glutamic acid F Phe phenylalanine 5 G Gly glycine H His histidine I Ile isoleucine K Lys lysine L Leu leucine 10 M Met methionine N Asn asparagine P Pro proline Q Gln glutamine R Arg arginine 15 S Ser serine T Thr threonine V Val valine W Trp tryptophan Y Tyr tyrosine I77

Claims

1. Peptide derived from an antibiotic peptide or an analogue therr f, characterised in that it is devoid of a disulphide bond. 5

2. Peptide derived from an antibiotic peptide or an analogue thereof, characterised in that all the cysteine residues, optionally except one, are removed, replaced by another amino acid residue or blocked at their SH group 10 level.

3. Linear peptide according to any one of claims 1 to 2, characterised in that it meets one of the following formulas : 15 BXXBXXXXBBBXXXXXXB (I) BBXXXBXXXBXXXXBBXB (II) in which : - the B groups, identical or different, represent an amino acid residue whose side chain carries 20 a base group, and - groups X, identical or different, represent an aliphatic or aromatic amino acid residue, or in that it is made up of a succession of at least 5, preferably at least 7, successive amino acids 25 of either of formulas (I) or (II).

4. Linear peptide according to claim 3, characterised in that the B groups are chosen from among arginine, lysine, diaminoacetic acid, diaminobutyric 30 acid, diaminoproprionic acid, ornithine. 27

5. Linear peptide according to one of claims 3 to 4, characterised in that the X groups are chosen from among glycine, alanine, valine, norleucine, isoleucine, Am leucine, cysteine, cysteinem, penicillamine, methionine, 5 serine, threonine, asparagine, glutamine, phenylalanine, histidine, tryptophan, tyrosine, proline, Abu, carboxylic amino-1-cyclohexane acid, Aib, carboxylic 2 aminotetraline, 4-bromophenylalanine, tert-Leucine, 4 chlorophenylalanine, p-cyclohexylalanine, 3,4 10 dichlorophenylalanine, 4-fluorophenylalanine, homoleucine, $-homoleucine, homophenylalanine, 4 methylphenylalanine, 1-naphthylalanine, 2 naphthylalanine, 4-nitrophenylalanine, 3-nitrotyrosine, norvaline, phenylglycine, 3-pyridylalanine, [2 15 thienyl]alanine.

6. Linear peptide according to any one of claims 1 to 5, characterised in that it meets one of the following formulas 20 RXXRXUXURRRXUXUXXR-NH 2 (V) RRXUXRXUXRXXUXRRUR-NH 2 (VI) in which - U represents serine or threonine, - R represents arginine, and 25 - the X groups, identical or different, represent an amino acid which may or may not be natural, including D-amino acids, either aliphatic or aromatic, such as glycine, alanine, valine, norleucine, isoleucine, leucine, cysteine, cysteine , penicillamine, methionine, 30 serine, threonine, asparagine, glutamine, phenylalanine, histidine, tryptophan, tyrosine, proline, Abu, carboxylic 28 amino-1-cyclohexane acid, Aib, carboxylic 2 aminotetraline, 4-bromophenylalanine, tert-Leucine, 4 chlorophenylalanine, $-cyclohexylalanine, 3,4 dichlorophenylalanine, 4-fluorophenylalanine, 5 homoleucine, $-homoleucine, homophenylalanine, 4 methylphenylalanine, 1-naphthylalanine, 2 naphthylalanine, 4-nitrophenylalanine, 3-nitrotyrosine, norvaline, phenylglycine, 3-pyridylalanine, [2 thienyl]alanine. 10

7. Linear peptide according to any one of claims 1 to 6, having the following sequences RGGRLSYSRRRFSVSVGR, RGVSVSFRRRSYSLRGGR, 15 EGGELSYSEEEFSVSVGE, RGGRLAYRLLRFAIRVGR, OGGOXXBOXXOBXXXOXG, RAARLGYRXXRFGZRVGR, YRRRFSVSVR, 20 RRLSYSRRRF, RRLSYSRRRFSVSVR, RGGRLSYSRRRFSTSTGR, in which B represents Napthylalanine, 0 represents Ornithine, X represents Norleucine and Z 25 represents Norvaline.

8. Linear peptide according to any one of claims 1 to 6, having the following sequences : 29 KWSFRVSYRGISYRRSR, RWSFRVSYRGISYRRSR, RSRRYSIGRYSVRFSWK, OBXBOXXBOGXOBXXOX, 5 KWAFRVAYRGIRYLLRL, KYAWRVAHRGIRWLLRX in which B represents Napthylalanine, 0 represents Ornithine, X represents Norleucine and Z represents Norvaline. 10

9. Use of an antibiotic peptide or an analogue thereof, devoid of a disulphide bond, to vector active substances in an organism. 15

10. Use of a peptide according to any one of claims 1 to 8 to vector active substances in an organism.

11. Compound with the following formula (IV) in X./ - Z)M(IV) n m 20 in which : - A represents a linear peptide derived from an antibiotic peptide or from an analogue thereof, - Z represents an active substance - Y represents a signal agent 25 - n is 0 or more, advantageously 0 or 1, - m is 1 or more, preferably up to 10, advantageously up to 5. 30

12. Compound of formula (IV) in which A is defined as in any one of claims 1, to 8.

13. Compound according to one of claims 11 or 12, characterised in that the coupling between the linear 5 peptide (A) and group (Z) or groups (Z) and (Y) is made by one or more covalent, hydrophobic or ionic bonds.

14. Compound according to any one of claims 11 to 13, characterised in that at least one of the active 10 substances (Z) is attached by a covalent bond either to the N-terminal or C-terminal ends or to the primary amino groups, carried by the side chains of the lysines, of linear peptide (A).

15 15. Compound according to any one of claims 11 to 14, characterised in that at least one signal agent (Y), if present, is attached by a covalent bond to the N-terminal end of linear peptide (A). 20

16. Pharmaceutical composition, characterised in that as active ingredient it comprises at least one compound of formula (IV) according to any one of claims 11 to 15.

17. Diagnostic agent made up of at least one compound 25 of formula (IV) according to any one of claims 11 to 15.