AU754617B2

AU754617B2 - Linear peptides derived from antibiotic peptides, preparation and use for vectoring active substances

Info

Publication number: AU754617B2
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: 2002-11-21
Anticipated expiration: 2018-08-06
Also published as: WO1999007728A3; AU8988998A; FR2767323A1; CA2298932A1; JP2001512739A; FR2767323B1; WO1999007728A2; EP1003771A1; IL134293A0

Description

I II 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 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 agents, vertebrates have numerous peptides with antimicrobial 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 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 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 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.

I I I 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, 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., 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 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 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 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, which could hinder the incorporation of antibiotic peptides. However, the specificity of the latter for iii 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 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 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 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 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 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 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 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, 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 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 isolated from porcine leukocytes Kokryakov et al., FEBS lett. 327, 231-236) PG-1 RGGRLCYCRRRFCVCVGR-NH 2 PG-2 RGGRLCYCRRRFCICV..-NH 2 PG-3 RGGGLCYCRRRFCVCVGR-NH 2 PG-4 RGGRLCYCRGWICFCVGR-NH 2

RGGRLCYCRPRFCVCVGR-NH

2 Tachyplesins (Tamura H. et al., 1993, Chem.

Pharm. Bul. Tokyo 41, 978-980) denoted Tl, T2 and T3 and polyphemusins (Muta 1994, CIBA Found. Sym. 186, 160- 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.

P1 RRWCFRVCYRGFCYRKCR-NH 2 'i 1 P2 RRWCFRVCYKGFCYRKCR-NH 2 T1 KWCFRVCYRGICYRRCR-NH 2 T2 RWCFRVCYRGICYRKCR-NH 2 T3 KWCFRVCYRGICYKRCR-NH 2 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 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 antiparallel p strand stabilised by the two disulphide bonds.

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 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 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 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

I

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 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 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 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 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.

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 substance in the cytoplasm and/or in the nuclear compartment.

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 specifically with a given cell type, erythrocytes for example.

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

The present invention therefore provides peptides derived from antibiotic peptides or analogues thereof, 25 wherein said peptides are devoid of a disulphide bond.

The present invention provides an isolated peptide derived from an antibiotic peptide or an analogue thereof wherein said peptide is devoid of disulphide bonds, and wherein said peptide has one of the following 30 formulae

RGGRLSYSRRRFSVSVGR,

RGVSVSFRRRSYSLRGGR,

S* EGGELSYSEEEFSVSVGE,

RGGRLAYRLLRFAIRVGR,

OGGOXXBOXXOBXXXOXG,

-RA RAARLGYRXXRFGZRVGR,

SYRRRFSVSVR,

H:\RBell\Keep\89889.98 .doc 06/09/02 7a

RRLSYSRRRF,

RRLSYSRRRFSVSVR,

RGGRLSYSRRRFSTSTGR,

in which B represents Napthylalanine, O represents Ornithine, X represents Norleucine and Z represents Norvaline.

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.

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 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 S: previously described cells.

These modifications may be conducted during the preparation of the peptides of the invention, more 25 particularly by chemical synthesis or the expression of a gene coding for said peptide, or directly on an antibiotic peptide through the action of chemical agents *o H:\RBell\Keep\89889.98 .doc 06/09/02 ml 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 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, 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, 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 following formulas BXXBXXXXBBBXXXXXXB (I) BBXXXBXXXBXXXXBBXB

(II)

which may also be represented by the following single formula (III) B(XB)X(XB)B)X(XB)XX(XB)B( (XB)XXX(XB) B) in which the B groups, identical or different, represent an amino acid residue whose side chain carries a base group, and the X groups, identical or different, represent an aliphatic or aromatic amino acid residue, I, or are made up of a sequence of at least preferably at least 7, successive amino acids of either of formulas or if this sequence has vectoring properties that are non-toxic for the previously described cells.

B and X may be natural or non-naturally occuring amino acids, including D-amino acids.

As an example the following denotations of B and X may be cited 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, cysteine^, penicillamine, methionine, serine, threonine, asparagine, glutamine, phenylalanine, histidine, tryptophan, tyrosine, proline, Abu, carboxylic amino-1cyclohexane acid, Aib, carboxylic 2-aminotetraline, 4bromophenylalanine, tert-Leucine, 4-chlorophenylalanine, 20 P-cyclohexylalanine, 3,4-dichlorophenylalanine, 4fluorophenylalanine, homoleucine, P-homoleucine, homophenylalanine, 4-methylphenylalanine, 1naphthylalanine, 2-naphthylalanine, 4-nitrophenylalanine, 3-nitrotyrosine, norvaline, phenylglycine, 3- S 25 pyridylalanine, [2-thienyl]alanine.

The invention also concerns peptide derivatives having the formula or (II) such as said peptides in retro form, or moieties of peptides having the formula or (II) made up of five, preferably seven, successive amino acids of either formula or (II).

H:\RBell\Keep\89889.98 .doc 06/09/02 I. f Among the peptides of the invention, special mention can be made of those meeting the following formulas

RXXRXUXURRRXUXUXXR-NH

2

(V)

RRXUXRXUXRXXUXRRUR-NH

2

(VI)

in which U represents serine or threonine R represents arginine, and 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 Am penicillamine, methionine, serine, threonine, asparagine, glutamine, phenylalanine, histidine, tryptophan, tyrosine, proline, Abu, carboxylic amino-l-cyclohexane acid, Aib, carboxylic 2aminotetraline, 4-bromophenylalanine, tert-Leucine, 4chlorophenylalanine, P-cyclohexylalanine, 3,4dichlorophenylalanine, 4-fluorophenylalanine, homoleucine, P-homoleucine, homophenylalanine, 4methylphenylalanine, l-naphthylalanine, 2naphthylalanine, 4-nitrophenylalanine, 3-nitrotyrosine, norvaline, phenylglycine, 3-pyridylalanine, [2thienyl]alanine.

Among the peptides of formulas and (II) or their derivatives, the invention specifically considers those derived from protegrins and tachyplesins referred to in tables I and II below.

I 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 represents Naphthylalanine, tables I and II above, B O 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 active substance, the invention gives particular 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 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...

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 compounds of formula (IV) as follows and compositions containing them

(Z)

m

(IV)

in which A represents a linear peptide derived from an 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 i, m is 1 or more, preferably up to advantageously up to Therefore, the above formula (IV) compounds are formed from a peptide of the invention coupled with one or more active substances, identical or different, represented by the group in formula and optionally one or more signal agents, represented by the 13 group in formula 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 is an oligopeptide or a protein, such as a signal peptide, a nuclear localising signal, an antibody moiety, or a chemical molecule ligand or antiligand of a receptor.

In a special embodiment of the compounds of formula group is coupled to group This coupling, symbolised by the horizontal lines in formula may be conducted by any acceptable linking means, taking into consideration the chemical nature, the size and number of groups and in the compounds of formula such as covalent, hydrophobic or ionic bonds, which may or may not be cleaved in physiological media. Coupling may be conducted at any site of peptide at which functional groups such as -OH, SH, -COOH, -NH2 are naturally present or have been inserted.

The invention gives consideration to the coupling of several groups to one and the same site of peptide 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 coupling of several 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 the 30 lysines of peptide If the C-terminal end of peptide is used to attach active substance the N-terminal end is available for optional coupling to a signal agent enabling the compound of the invention H:\RBell\Keep\89889.98 .doc 06/09/02 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 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 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.

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 Cterminal end to a medicinal agent, to add a peptide sequence able to specifically recognise a determinant present on the surface of cell type. Synthetic pentadecapeptide cM2 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 Epithelial Mucin) maintains good affinity for these cells. It is therefore possible, by associating aM2 with a linear peptide-medicinal agent complex, to bring this group preferably towards the cells which express the antigen characteristic related to breast cancer.

The compounds of formula (IV) may be prepared by chemical synthesis or by using molecular biology techniques.

For chemical syntheses, commercially available equipment can be used allowing the incorporation of nonnatural amino acids, such as D enantiomers and residues with side chains of different hydrophobicity and size to 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 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 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 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 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 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 ,1I 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 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 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 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 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.

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 invention derived from antibiotic peptides.

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 were synthesised

RGGRLXYXRRRFXVXVGR-NH

2

RRWXFRVXYRGFXYRKXR-NH

2

KWXFRVXYRGIXYRRXR-NH

2 in which X represents the serine, threonine or alanine residues.

These peptides are respectively derived from the sequences of Protegrin PG-1 having the formula

RGGRLCYCRRRFCVCVGR-NH

2 of Tachyplesin 1 having the formula

KWCFRVCYRGICYRRCR-NH

2 of Polyphemusin having the formula

KWXFRVXYRGIXYRRXR-NH

2 These three peptides may be prepared either by BOC chemistry or by FMOC chemistry using conventional synthesis methods in solid or homogeneous phase.

2) Fixing biotin onto linear peptides The peptide is synthesised in solid phase and, I S. after incorporation of the N-terminal arginine, aminopentanoic acid is added. The FMOC or BOC N-terminal 25 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 S* H:\RBell\Keep\89889.98 .doc 06/09/02 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.

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, 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 Nterminal is activated by the diisopropylcarbodiimide and l-hydroxybenzotriazole mixture. After two hours' reaction at room temperature, the doxorubicin is added and the mixture is stirred for 12 hours at 0 C. The peptidedoxorubicin unit is then purified by high pressure liquid chromatography.

Example 2 Ability of the linear peptides of the 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, HL60R, HeLa).

The cells are cultured on RPMI 1640 (Gibco) to which is added 10 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 prepared according to example 1 (2) 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 example 1 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 25 0 C, then rinsed three 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 J1 TexRed antibodies diluted to 3 00 th and rinsed three times in PBS. The slides are finally mounted with a Mowiol-Dabco 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 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

RXXRXUXURRRXUXUXXR-NH

2

(V)

RRXUXRXUXRXXUXRRUR-NH

2

(VI)

in which S U represents serine or threonine, S R represents arginine, and 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, cysteineAcm, penicillamine, methionine, serine, threonine, asparagine, glutamine, phenylalanine, histidine, tryptophan, tyrosine, proline, Abu, carboxylic amino-l-cyclohexane acid, Aib, carboxylic 2aminotetraline, 4-bromophenylalanine, tert-Leucine, 4chlorophenylalanine, p-cyclohexylalanine, 3,4dichlorophenylalanine, 4-fluorophenylalanine, homoleucine, p-homoleucine, homophenylalanine, 4methylphenylalanine, 1-naphthylalanine, 2naphthylalanine, 4-nitrophenylalanine, 3-nitrotyrosine, norvaline, phenylglycine, 3-pyridylalanine, [2thienyl]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 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 having the formula biotin-RGGRLSYSRRRFSVSVGR-NH 2 This work was photographed (not shown) Control in which the cell was treated with biotin alone, Treatment of the cell for 2 minutes with a complex biotin-linear peptide of the invention, f K J f t 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 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 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.

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 The biotinylated peptides are incubated with the cells at a concentration of 10 pM for 15 minutes at 37 0

C

in an atmosphere of 95% humidity and 5% CO2 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 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 control (Ap43-58), well described in the literature, and with a negative control.

The cell nuclei were Hoechst stained.

f 22 2) Cell lines All the lines tested are of human origin and were commercially obtained from ATCC.

Non-tumoral lines MRC5 (lung fibroblast), HuVeC (endothelial, umbilical cord) Tumoral lines HT29 (colon carcinoma), HepG2 (hepatoblastoma), A172 (glioblastoma), HMCB (melanoma).

The cells are cultured at 37 0 C in an atmosphere of 95% humidity and 5% C02. The culture medium is the one recommended by ATCC.

3) Tested peptides The two series of tested peptides are those given in tables I and II.

4) Results 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 penetrate with good 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 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 effect for both families of tested peptides. It is therefore advisable to avoid increasing hydrophobia. On I V 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 Fluorescence microscopy photographs 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 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 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 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 doxorubicin 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 concentration of 10 [tM for 60 minutes at 37°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 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 may also be represented by their three-letter code according to the following nomenclature A Ala alanine C Cys cysteine D Asp aspartic acid E Glu giutarnic acid F Phe phenylalanine Gly glycine H His histidine I Ile isoleucine K Lys lysine L Leu leucine M Met methionine N Asn asparagine P Pro praline Q Gin giutamine R Arg arginine S Ser serine T Thr threonine V Val vaiine W Trp tryptophan Y Tyr tyrosine EDITORIAL NOTE FOR 89889/98 THE FOLLOWING SEQUENCE LISTING NUMBERED PAGES 34 40 IS PART OF THE

DESCRIPTION

THE CLAIMS FOLLOW ON PAGE 26 34 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME SYNT:EM S. A. et al.

STREET Parc Scientifique Georges Besse TOWN :Nimes COUNTRY France ZIP CODE :30000 (ii) TITLE OF THE INVENTION :Linear peptides derived from antibiotic peptides, preparation and use for vectoring actives substances (iii) SEQUENCE NUMBER 16 (iv) COMPUTER READABLE

FORM

SUPPORT TYPE :Diskette COMPUTER Macintosh OPERATING SYSTEM :Macintosh SOFTWARE Patentln Release Version ff1.30 (OEB) PRESENT APPLICATION

DATA

APPLICATION NUMBER :PCT 98/01757 (vI) PRIOR APPLICATION

DATA:

0:00(A) APPLICATION NUMBER :97/10297 FILING DATE 12/08/97 0 so 0 *0 0 *0 0 0 INFORMATION ON SEQ ID NO :1 000 0 0 0 *0 0 SEQUENCE

CHARACTERISTICS

0.0 LENGHT 18 TYPE :amino acids FORM :linear (ii) MOLECULE TYPE :peptide s0 (ix) CHARACTERISTICS 0 0 0(A) NAME/KEY :Linear peptide derived from 0 antibiotic peptide 060 0(xii) DESCRIPTION DE LA SEQUENCE: SEQ ID NO :1 00 Arg Gly Gly Arg Leu Ser Tyr Ser Arg Arg Arg Phe Se~r Val Ser Val 0 10 0-00. Gy Arg INFORMATION ON SEQ ID NO :2 36 (ix) CHARACTERISTICS NAME/1(EY Linear peptide derived from antibiotic peptide (xii) DESCRIPTION DE LA SEQUENCE: SEQ ID NO :4 Arg Gly Gly Arg Leui Ala Tyr Arg Leu Leu Jkrg Phe Ala Ile Arg Val 1 5 10 Gly iArg INFORMATION ON SEQ ID NO 1 SEQUENCE CHARACTERISTIC

S

LENCHT 18 TYPE :amino acids FORM :linear (ii) MOLECULE TYPE peptide (ix) CHARACTERISTICS NAME/K'EY Linear peptide derivE antibiotic peptide (xii) DESCRIPTION DE LA SEQUENCE: SEQ ID NO Gly Gly Xaa Xaa. Xaa Xaa xaa. Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xza 10 ,1 ~d f rom *.0 0 e as* 0g Xaa Xaa. Gly INFORMATION ON SEQ ID NO :16 SEQUENCE

CHARACTERISTICS

LENGET 18 TYPE amino acids FORM linear (ii) MOLECUL.E TYPE :peptide (ix) CHARACTERISTICS NAM~EIKEY Linear peptide derived from antibiotic peptide (xii) DESCRIPTION DE LA SEQUENCE: SEQ ID NO :6 Arg Ala Ala Arg Leu~ Gly Tyr Arq Xaa Arg ene Gly Xaa Arg Val 10 Gly Arg 37 IN1FORMAATION ON SEQ ID NO t7 SEQUENCE CIIAIACTERtSTICS LENGHT TYPE amino acids (1D) FORM :liflealr (ii) MOLECULE TYPE :peptide (ix) CHARACTERISTICS NAME/KEY Linear peptide derived f rom antibiotic peptide (xii) DESCRIPTION DE L 1 A SEQUENCE: SEQ ID NO 7 Lyr Arg Arg Arg Phe Ser Val Ser Val Arg INFORMATION ON SEQ ID NO 8 SEQUENCE

CHARACTERISTIC.$

LENGIIT TYPE amino acids FORM linear (ii) MOLECULE TYPE ;peptide (ix) CHARACTERISTICS NAME/KEY Linear peptidle derived f ran antibiotic peptide (xii) DESCRIPTION DE ItA SEQUENCE: SEQ ID NO ;8 Arg Arg Leu Ser Tyr Ser Arg Arg Arg Phe 1 5 S 0O I I.

S

(1O)INFORM1ATION ON SEQ ID:NO 9 SEQUENCE

CHARACTERISTICS

LENGHT TYPE amin6 acids FORM line~.r (ii) MOLECULE TYPE eptide (ix) CHARACTERISTICS NAME/KEY LiJnear peptide derived from antibiotic peptlde (xii) DESCRIPTION DE LA SEQUENCE: SEQ ID NO 9 Arg Arg Leu Ser Tyr Ser Arg Arg Ar4 Phe Ser Val Ser Val Arg 10 is 38 (11) INFORMATION ON'SEQ ID NO SEQUENCE

CHAACTERISTICS

LENGHT :18 TYPE amino acid's FORM :linear (ii) MOLECULE TYPE :peptide (ix) CHARACTERISTICS NAME/KEY Linear peptide derived from antibiotic peptide (xii) DESCRIPTION DE LA SEQUENCE: SEQ ID NO Arq Gly Gly Arq Leu Ser Tyr Ser Arg Arg Arg ?~he Ser Thr Set Thr 10 Gly Arg 99 9 9 9.

99 9 9.

.9 *9 9 9.

9*99 9* 9 99 99 9 9.

(12) INFORMATION ON SEQ ID NO 1 SEQUENCE

CHARACTERISTICS

LENGHT :17 TYPE amino acids FORM :linear (ii) MOLECULJE TYPE :peptide (ix) CHARACTERISTICS i(A) NAME/KEY Linear peptide derived f rom antibiotic peptide (xii) DESCRIPTION DE LA SEQUENCE: SEQ ID NO 1 Lys Trp Ser Phe Arg Val Ser Tyr Arg Gly Ile Ser Tyr Arg Arq Ser 510 9. 9 9 9.

9 6 9 @9 Is 9 ~9 9999 I I. 99 I. 9 99999 9 (13) INFORKATION.ON SEQ ID NO 4 12 SEQUENCE

CHARACTERISTICS

LENGHT 17 TYPE :amino acids FORM linear (ii) MOLECULE TYPE :peptide (ix) CHARACTERISTICS NAME/KEY Linear peptide derived from antibiotic peptide 39 (xii) DESCRIPTION DE LA SEQUENCE: SEQ ID NO 12 Artg Trp Ser Phe Arg Val Ser Tyr Arg Gly Ille Ser Tyr Arg Arg Ser 10 Rrg (14) INFORMATION ON SEQ ID) NO :13 SEQUENCE

CHARACTERISTICS

LENGRT :17 TYPE amino acids FORM linear (ii) MOLECULE-TYPE ;peptide (ix) CHARACTERISTIC'S NAI4E/KEY Linear peptide derived antibiotic peptide (xii) DESCRIPTION DE LA SEQUENCE: SEQ ID NO :13 Ser Arg Arg Tyr Ser Ile Gly 7 rq Tyr SGr Val Arg Phe Ser Trp 5 10 f rom Axg 1

S.

S S

S.

5* 0

S.

5 0

S.

*OOS

50 S. 5 5* S S @5 S. S S

OS..

0 S 5.

S S S. 0505 @005 0 S 0 '056@.

5 Lys (15) INFORMATION ON SEQ ID NO 14 SEQUENCE

CHARACTERISTICS

LENGET 17 (B3) TYPE amiuno acidB FORM :linear (ii) MOLECUL~E TYPE :peptide (ix) CHARACTERISTICS NAME/KEY :Linear peptide derived from antibiotic peptide (xii) DESCRIPTION DE LA SEQUENCE: SEQ ID NO :14 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa. Xaa Xaa Xas Xaa Xaa 1 5 10 is Xaa (16) INFORMATION, ON SEQ, ID NO 40 SEQUENCE CHARACTERISTIC8 LENGUT :17 TYPE amino acids FORM :linear (ii) MOLECULE TYPE :peptid6 (ix) CHARACTERISTICS NANE/KEY Linear peptide derived from antibiotic peptide (Xii) DESCRIPTION DE LA SEQUENCE: SEQ ID NO Lys Trp Ala Phe Arg Val A-1a Tyr O~xq Gly I'le Arg Tyr Leu Levi Arg 1 3.0 Leu (17) INFORMATION ON SEQ. ID NO 16 SEQUENCE

CHARACTERISTICS

LENGET 17 TYPE :amino acids FORM :linear (ii) MOLECULE TYPE I:peptide (ix) CHARACTERISTICS NAM'E/KEY Linear peptide derived fromn antibiotic peptide (xii) DESCRIPTION DE LA SEOUENCE; SEQ ID NO :16 Lys Tyr Ala Trp Atg Val Ala HisjArg Gly Tle Arg Trp Levi Leu Arg 10 Xaa

Claims

1. An isolated peptide derived from an antibiotic peptide or an analogue thereof wherein said peptide is devoid of disulphide bonds, and wherein said peptide has one of the following formulae RGGRLSYSRRRFSVSVGR, RGVSVS FRRRSYSLRGGR, EGGELSYSEEEFSVSVGE, RGGRL 1 AYRLLRFAI RVGR, OGGOXXBOXXOBXXXOXG, PAARLGYRXXRFGZRVGR, YRRRFSVSVR, RRLSYSRRRF, RRLSYSRRRFSVSVR, RGGRLSYSRRRFSTSTGR, *0 00. represents represents in which B represents Napthylalanine, 0 Ornithine, X represents Norleucine and Z Norvaline.

2. An isolated peptide derived from an antibiotic peptide or an analogue thereof wherein said peptide is devoid of disulphide bonds and wherein said peptide has one of the following formulae KWSFRVSYRGI SYRRSR, S T 1-1RWSFRVSYRGISYRRSR, H:\KarraR\Keep\speci\Biotech\89889-98.Response to second ORdoc 13/05/02 27 RSRRYSIGRYSVRFSWK, OBXBOXXBOGXOBXXOX, KWAFRVAYRGIRYLLRL, KYAWRVAHRGIRWLLRX in which B represents Napthylalanine, 0 represents Ornithine, X represents Norleucine and Z represents Norvaline.

3. Use of an P-stranded antibiotic peptide or an analogue thereof, wherein said peptide or analogue is devoid of disulphide bonds as a result of the removal, the replacement by another amino acid or the blocking of the cysteine residues at their SH group level of all the cysteine residues, to vector active substances in an 15 organism.

4. Use according to claim 3, wherein said peptide has one of the following formulas BXXBXXXXBBBXXXXXXB (I) BBXXXBXXXBXXXXBBXB (II) in which .i* the B groups are the same or different, and represent an amino acid residue whose side chain carries a basic group, and 25 the X groups are the same or different, and represent an aliphatic or aromatic amino acid residue.

H:\KarraR\Keep\speci\Biotech\89889-98.Response to second OR.doc 13/05/02 28 Use according to claim 3 or claim 4, wherein said peptide has one of the following formulae BXXBXXXXBBBXXXXXXB (I) BBXXXBXXXBXXXXBBXB (I I) in which: -the B groups are chosen from among arginine, lysine, diaminoacetic acid, diaminobutyric acid, diaminoproprionic acid, ornithine; the X groups are chosen from among glycine, alanine, valine, norleucine, isoleucine, leucine, cysteine, cysteine Ac, 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, P-cyclohexylalanine, 3,4- dichlorophenylalanine, 4-f luorophenylalanine, hornoleucine, 1-homoleucine, homophenylalanine, 4-methylphenylalanine, 1 -naphthylalanine, 2 -naphthylalanine, 4- nitrophenylalanine, 3-nitrotyrosine, norvaline, phenylglycine, 3-pyridylalanine, and [2-thienyl] alanine.

6. Use according to claim 3, wherein said peptide has one of the following formulae RXXRXUXURRRXUXUXXR -NH 2 (V) RRXUXRXUXRXXUXRRUR -NH 2 (VI) in which: U represents serine or threonine, ZT~N.- R represents arginine, and H:\KarraR\Keep\speci\Biotech\89889-98.Response to second ORdoc 13/05/02 29 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 Acm 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- chlorophenylalanine, P-cyclohexylalanine, 3,4- dichlorophenylalanine, 4-fluorophenylalanine, homoleucine, P-homoleucine, homophenylalanine, 4-methylphenylalanine, 1-naphthylalanine, 2-naphthylalanine, 4- nitrophenylalanine, 3-nitrotyrosine, norvaline, phenylglycine, 3-pyridylalanine, and [2-thienyl]alanine.

7. Use of a peptide according to claim 1 or claim 2 to vector active substances in an organism.

8. A compound with the following formula (IV) (IV) in which A represents a linear peptide derived from 25 a P-stranded antibiotic peptide or an analogue thereof, wherein said peptide or analogue is devoid of disulphide bonds as a result of the removal, the replacement by another amino acid or the blocking of the cysteine residues at their SH group level of all the cysteine residues, HI\KarraR\Keep\speci\Biotech\89889-98.Response to second OR.doc 13/05/02 30 Z represents an active substance Y represents a signal agent n is 0 or more, advantageously 0 or 1, m is 1 or more, preferably up to advantageously up to 5, and each of the horizontal lines represents a covalent or ionic bond.

9. A compound according to claim 8, wherein said linear peptide has one of the following formulae BXXBXXXXBBBXXXXXXB (I) BBXXXBXXXBXXXXBBXB (II) in which the B groups are the same or different, and represent an amino acid residue whose side chain carries a basic group, and the X groups are the same or different, and represent an aliphatic or aromatic amino acid residue.

A compound according to claim 8 or claim 9, wherein said linear peptide has one of the following formulae BXXBXXXXBBBXXXXXXB (I) BBXXXBXXXBXXXXBBXB (II) in which y the B groups are chosen from among arginine, lysine, diaminoacetic acid, diaminobutyric acid, diaminoproprionic acid, and ornithine; H:\RBell\Keep\89889.98 .doc 06/09/02 31 the X groups are chosen from among glycine, alanine, valine, norleucine, isoleucine, leucine, Acm cysteine, cysteine 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- chiorophenylalanine, 1-cyclohexylalanine, 3,4- dichiorophenylalanine, 4-fluorophenylalanine, homoleucine, P-homoleucine, homophenylalanine, 4-methyiphenylalanine, l-naphthylalanine, 2-naphthylalanine, 4- nitrophenylalanine, 3-nitrotyrosine, norvaline, phenyiglycine, 3-pyridylalanine, and [2-thienyllalanine.

11. A compound according to claim 8, wherein said linear peptide has one of the following formulae RXXRXUXURRRXUXUXXR NH2 (V) RRXUXRXUXRXXUXRRUR-NH 2 (VI) in which U represents serine or threonine, R represents arginine, and the X groups are the same or different, and represent a natural or non-naturally occurring amino acid including D-amino acids, either aliphatic or aromatic, such as glycine, alanine, valine, norleucine, isoleucine, leucine, cysteine, cysteine penicillamine, methionine, serine, threonine, asparagine, glutamine, phenylalanine, histidine, tryptophan, tyrosine, proline, Abu, carboxylic amino-l-cyclohexane acid, Aib, carboxylic 2- h:\KarraR\Keep\speci\Biotech\89889-98.Response to second OR.doc 13/05/02 32 aminotetraline, 4-bromophenylalanine, tert-Leucine, 4- chlorophenylalanine, 0-cyclohexylalanine, 3,4- dichlorophenylalanine, 4-fluorophenylalanine, homoleucine, P-homoleucine, homophenylalanine, 4-methylphenylalanine, 1-naphthylalanine, 2-naphthylalanine, 4- nitrophenylalanine, 3-nitrotyrosine, norvaline, phenylglycine, 3-pyridylalanine, or [2-thienyl]alanine.

12. A compound according to any one of claims 8 to 11, wherein said linear peptide is a peptide according to claim 1 or claim 2.

13. A compound according to any one of claims 8 to 12, wherein the coupling between the linear peptide (A) and group or groups and is made by one or more covalent or ionic bonds.

14. A compound according to any one of claims 8 to 13, wherein at least one of the active substances 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

15. A compound according to any one of claims 8 to 14, wherein at least one signal agent if present, is attached by a covalent bond to the N-terminal end of linear peptide

16. A pharmaceutical composition, which comprises as active ingredient at least one compound of formula (IV) according to any one of claims 8 to H:\RBell\Keep\89889.98 .doc 06/09/02 33

17. A diagnostic agent comprising at least one compound of formula (IV) according to any one of claims 8 to

18. An isolated peptide according to claim 1 or claim 2, substantially as herein described with reference to any of the examples or figures.

19. Use according to claim 3, substantially as herein described with reference to any of the examples or figures. A compound according to claim 8, substantially as herein described with reference to any of the examples or 15 figures. *Dated this 13 day of May 2002 SYNT:EM By their Patent Attorneys GRIFFITH HACK DtFellows Institutey of Patent and

20 By Trade Marktent Attorneystralia 4* H:\KarraR\Keep\speci\Biotech\9889-98.Response to second OR.doc 13/05/02