WO2008040978A1

WO2008040978A1 - N-capped peptides with np-1 antagonist activity

Info

Publication number: WO2008040978A1
Application number: PCT/GB2007/003765
Authority: WO
Inventors: Haiyan Jia; Ian Zachary; Michelle Tickner; Rehan Aqil; Shaheda Shaikh; Chris Chapman; Ashley Jarvis; David Selwood
Original assignee: Ark Therapeutics Ltd.
Priority date: 2006-10-04
Filing date: 2007-10-04
Publication date: 2008-04-10
Also published as: WO2008040978A8; GB0619609D0

Abstract

The present invention is a peptide derivative having formula (I) or a N-terminal fragment thereof that substantially retains NP-1 antagonist activity, in cyclic form, wherein each X1 is an amino acid residue having a side chain which includes, or has been modified to include, a functional group capable of forming a bond with another X1 group; X2 is an arginine, alanine or proline residue or a direct bond; and R1 is an alkyl group containing at least two carbon atoms, an alkenyl, aryl, alkylaryl, cycloalkyl, heterocyclic or heteroaromatic group; a chelating group capable of chelating an imaging isotope; or a linking group for attachment to or incorporation into an imaging nanoparticle. The compounds of the invention may be useful for stimulating nerve repair, treating neurodegeneration, immune system modulation and treating cancer and other neovascular diseases.

Description

N-CAPPED PEPTIDES WITH NP-1 ANTAGONIST ACTIVITY

Field of the Invention

This invention relates to N-capped derivatives of peptides which are fragments of VEGF (vascular endothelial growth factor) and which have activity of potential benefit in therapy. Background of the Invention

VEGF-A is a secreted polypeptide which is essential for formation of the vascular system in embryogenesis and plays a major role in angiogenesis in a variety of disease states. VEGF expression is upregulated by hypoxia and several cytokines in diverse cell types, and elicits multiple biological activities in vivo and in vitro, including the differentiation, proliferation, migration and survival of endothelial cells, increased vascular permeability, monocyte migration, and increased endothelial production of the vasodilatory factors NO and prostacyclin.

Human VEGF-A exists in multiple isoforms, of 121 , 145, 165, 189 and 206 amino acids, generated by alternative mRNA splicing, of which VEGF_12I, VEGF₁₄₅ and VEGF₁₆₅ are known to be secreted and biologically active. Two distinct protein tyrosine kinase receptors for VEGF have been identified, i.e. FIM (VEGFR1) and KDR/Flk-1 (VEGFR2). KDR/flk-1 is thought to be the receptor which primarily mediates the mitogenic effects of VEGF in endothelial cells and angiogenesis in vivσ, the function of FIt-I in endothelial cells is unknown. A non- tyrosine kinase transmembrane protein, neuropilin-1 (NP-1), has been identified as an additional receptor for VEGF, which specifically binds VEGF_16S- VEGF promotes the survival of tumour cells expressing NP-1, including some breast carcinoma cells (Bachelder et al, Cancer Res 61 : 5736-5740, 2001). The role of NP-1 in mediating biological effects of VEGF is still largely unknown.

NP-1 is a receptor for a family of molecules called semaphorins or collapsins which play a key role in the guidance of neuronal axons during mammalian development. In particular, NP-1 is known to mediate the growth cone-collapsing and chemorepulsive activity of semaphorin-3A. NP-1 is expressed on immune system cells and may mediate immunosuppression by binding semaphorin-3A (Lepelletier, Eur. J. Immunol., 36(7) 1782-93). The immunosuppressive role of semaphorin-3A on T-cell proliferation is mediated by inhibition of actin cytoskeleton reorganization (Romeo, Adv. Exp. Med. Biol. 2002, 515, 49-54). Soker et al, J. Biol. Chem. 271(10): 5761-5767 (1996), discloses that a GST fusion protein containing the 44 amino acids encoded by VEGF exon 7 binds to NP-1.

Soker et al, J. Biol. Chem. 272(10): 31582-31588 (1997), discloses that a region of exon 7 is necessary for inhibition of VEGF binding to HUVECs. The shortest active peptide is SEQ ID NO. 1 which is

CSCKNTDSRCKARQLELNERTCRC i.e. VEGF (137-160), or amino acids 22-44 of exon 7 and amino acid 1 of exon 8. The terminal cysteine residue (C¹³⁷ in VEGF) is apparently essential for activity and the molecule's 3D structure. It is suggested that there may be intradisulfide bonding within the VEGF monomer. 3D structures, including the disulfide connectivity, of the C-terminal residues (109-165) of VEGF, are disclosed by Fairbrother et al., Structure 6, 637-48, and by Stauffer et al., J. Biomol. NMR 23, 57-61. WO03/082918 discloses VEGF peptides, which have NP-1 antagonist activity, but wherein the N-terminal Cys residue of SEQ ID NO. 1 is not present. An active peptide has SEQ ID NO. 2 which is

SCKNTDSRCKARQLELNERTCRCDKPRR and is named EG3287. Further studies of EG3287 can be found in Cheng et al., J. Biol. Chem.

2004, 279(29) 30654-61. This publication describes how anti-chemorepulsive effects of VEGF and placental growth factor-2 in dorsal root ganglion neurons are mediated via neuropilin-1 and cyclooxygenase-derived prostanoid production. Jia et al., J. Biol. Chem. 2006, 12, 281(19) 13493-502, describes how characterization of EG3287 reveals the importance of the region of vascular endothelial growth factor encoded by exon 8 for NP-1 binding and the role of NP-1 in KDR signalling. Summary of the Invention

The present invention is based on the discovery that, by capping the amino-terminal nitrogen of EG3287 (or EG3287 analogues) with certain acyl groups, NP-1 antagonist activity is improved.

According to a first aspect of the present invention, novel peptide derivatives are of formula I

(I)

or a N-terminal fragment thereof that substantially retains NP-1 antagonist activity, in cyclic form, wherein each X₁ is an amino acid residue having a side chain which includes, or has been modified to include, a functional group capable of forming a bond with another X₁ group;

X₂ is an arginine, alanine or proline residue or a direct bond; and R₁ is an alkyl group containing at least two carbon atoms, an alkenyl, aryl, alkylaryl, cycloalkyl, heterocyclic or heteroaromatic group; a chelating group capable of chelating an imaging isotope; or a linking group for attachment to or incorporation into an imaging nanoparticle.

According to a second aspect, peptide derivatives of the invention, where R₁ is a chelating or linking group, may be useful as diagnostic agents in medical imaging. Especially for this purpose, the peptides may comprise the imaging isotope. According to a third aspect, peptide derivatives of the invention are useful as therapeutic agents. They may be useful for stimulating nerve repair, for treating neurodegeneration, cancer or other neovascular diseases. They may also be used in the treatment of diseases where modulation of the immune system is required, for example, following transplant surgery. Yet other conditions that may be treated using a compound of the invention include skin diseases such as psoriasis, diseases requiring immunomodulation, angiogenesis in the eye, diabetes, macular degeneration, glaucoma and heart failure. Description of the Preferred Embodiments

It will be appreciated that the peptide derivatives of the invention contain an asymmetrically substituted carbon atom. The presence of this asymmetric centre in a compound of formula (I) can give rise to stereoisomers, and in each case the invention is to be understood to extend to all such stereoisomers, including enantiomers and diastereomers, and mixtures including racemic and non-racemic mixtures thereof. As used in this specification, alone or in combination, the term "alkyl" refers to a straight or branched chain alkyl moiety containing between two and twenty carbon atoms and includes, for example, ethyl, propyl, isopropyl, butyl, te/t-butyl, pentyl, octyl, stearyl, and the like.

The term "alkenyl" refers to a straight or branched chain alkenyl moiety containing up to twenty carbon atoms and includes for example, propenyl, heptadecenyl, butenyl, and the like. The term "aryl" means phenyl, biphenyl or naphthyl group.

The term "cycloalkyl" refers to a saturated alicyclic moiety and includes for example, benzofused cyclopentyl and cyclohexyl and the like.

The term "heterocyclic" refers to a saturated heterocyclic moiety containing one or more heteroatoms selected from N, O and S, and includes, for example, benzofused pyrrolidinyl, tetrahydrofuranyl, piperidinyl, dioxolane and the like.

The term "heteroaromatic" refers to an aromatic ring system in which at least one atom is selected from O, N and S and includes, for example, benzofused furanyl, thiophenyl, pyridyl, indolyl, pyridazinyl, piperazinyl, pyrimidinyl and the like.

The term "N-terminal fragment", as used herein, refers to any fragment of a peptide of the invention, which retains the terminal NC(O)R group. Preferably,

X₂ is present. Peptide derivatives of the invention may include modifications of the given sequence. Such modifications are well known to those skilled in the art. lsosteric replacements include Abu for Cys (this may be desirable where the peptide should retain an even number of Cys residues for cyclisation), Phe for

Tyr and different alkyl/aryl substituents. The shifting of substituents within an amino acid residue, from a C atom to a N atom, to produce peptoids having greater resistance to proteolysis, and other modifications, are known and are included within the scope of this invention.

Each X₁ group is any amino acid residue having a side chain including a functional group capable of forming a bond with another X₁ group. The functional group may be inherent in the amino acid, for example the -SH group in a cysteine residue. Alternatively, the side chain may be modified to include such a functional group. One example of this is modifying 2,3-diaminopropionic acid to contain a 3-NHC(O)CH₂CI group. This group is capable of forming a thioether bond with the -SH group in a cysteine residue. In an embodiment of the invention, R₁ includes a chelating group capable of chelating an imaging isotope. An imaging isotope is an isotope capable of enhancing medical images in techniques such as medical magnetic resonance imaging. Examples of such isotopes are gadolinium and iron. The peptide derivative may additionally include the imaging isotope. In another embodiment, the R₁ group includes a linking group for attachment to or incorporation into an imaging nanoparticle or liposome. In this case where Ri is an alkyl moiety, it is preferred that R₁ contains more than three carbon atoms, more preferably more than four carbon atoms, yet more preferably more than five carbon atoms and, most preferably, more than six carbon atoms. Longer chains of carbon atoms, for example, stearyl or cis-9-heptadecene, are preferable for incorporation into nanoparticles or liposomes.

The activity of peptides of the invention means that they may be useful in the treatment of diseases in which NP-1 may have a significant role in pathology. For therapeutic use, peptides of the invention may be formulated and administered by procedures, and using components, known to those of ordinary skill in the art. The appropriate dosage of the peptide may be chosen by the skilled person having regard to the usual factors such as the condition of the subject to be treated, the potency of the compound, the route of administration etc. Suitable routes of administration include oral, intravenous, intraperitoneal, intramuscular, intranasal and subcutaneous.

A NP-1 antagonist may compete with semaphorin-3A for binding to NP-1, and thereby antagonise inhibitory effects of semaphorin-3A on axonal outgrowth and migration in nerve cells. Potential applications of this are in promoting neurite outgrowth, in stimulating nerve repair or treating neurodegeneration. Further, an NP-1 antagonist may promote the survival of semaphorin-3A responsive neurones, an effect that would confirm or enhance its utility in the applications given above, and may extend these applications, e.g. to treating neuronal death caused by episodes of ischaemia as in stroke and some eye diseases. NP-1 plays an important role in angiogenesis and may be essential for

VEGF-induced angiogenesis in cancer, eye disease, rheumatoid arthritis and other diseases. Therefore, NP-1 antagonists may have applications in the inhibition of VEGF-dependent angiogenesis in disease.

NP-1 antagonists may also play a role in immunosuppression. Therefore, it may be useful to give a peptide of the invention before, during or after a transplant.

In addition, a NP-1 antagonist may compete with VEGF for binding to NP- 1 in tumour cells and promote cell death in NP-1 -expressing tumour cells. Potential applications of this are in anti-cancer therapy. Peptides of the invention may be synthesised by known methods.

Examples are given below. In particular, a linear peptide may be produced by automated peptide synthesis, followed by cyclisation. Known cyclisation procedures include those described by Tarn et al, JACS 113:6657-62 (1991). Other cyclisations, e.g. Mitsunobu, thioether formation, or olefin metathesis ring closure, may also be used. Cyclisation is also possible using amides, to produce a double amide bicyclic system. This method may offer the advantage of the greater stability of the cyclic products in the bloodstream.

In a preferred embodiment, a peptide of the invention is bicyclic. It may be cyclised by the formation of disulfide bridges across Cys residues, or it may be cyclised by the condensation of amide bonds. Alternatively, thioether bridges may be formed between, for example, -SH on one residue and -CH₂CI on another residue.

The following Methods 1 to 3 illustrate the synthesis of N-capped peptides of the invention. Method 1

(ι) I₂, TFA/H₂O

(M) LC-MS purification

.1 . N .- -SS--CC-K-N-T-D-S-R-C-K-A-R-Q-L-E-L-N-E-R-T-C-R-C-D-K-P-R-R-OH

W = ^Wan9 [p-alkoxybenzyl alcohol] linker Trt = triphenylmethyl; Acm = acetamidom ethyl Method 2

R = φ = Wang [p-alkoxybenzyl alcohol] linker Trt = triphenylmethyl; Acm = acetamidomethyl

X = Dpr (modified as C(O)CH₂CI)

Method 3

Acm Acm

I I

H₂N-S-C-K-N-T-D-S-R-C-K-A-R-Q-L-E-L-N-E-R-T-C-R-C-D-K-P-A-R

Trt TrI

I HOBt, DIC, RCO₂H DMF

Acm Acm

N-S-C-K-N-T-D-S-R-C-K-A-R-Q-L-E— L-N-E-R-T-C-R-C-D-K-P-A-R

Trt Trt

0) I₂, TFA/H₂O

(n) LC-MS purification

JL. N-S- C-K- N— T— D-S- R- 1C-K-A- R-Q- L— E— L— N-E- R— T— C-R-C-D-K— P-A— R-OH

= Wang [p-alkoxybenzyl alcohol] linker Trt = triphenylmethyl; Acm = acetamidomethyl

The following illustrates the invention further.

Abbreviations

Acm, acetamidomethyl; Aib, 2-aminoisobutyric acid; Ala or A, alanine; Arg or R, Arginine; Asn or N, asparagine; Asp or D₁ aspartic acid; Cys or C, cysteine; GIn or Q, glutamine; GIu or E, glutamic acid; Leu or L, leucine; Lys or K, lysine; Pro or P, proline; Ser or S, serine; Thr or T₁ threonine; Tyr or Y, tyrosine; Fmoc, 9-fluorenylmethoxy-carbonyl; Pbf, 2,2,4,6,7- pentamethyldihydrobenzofuran-5-sulfonyl; Boc, terf-butoxy carbonyl; Trt, trityl; tBu or tert-Bu, tert-butyl; HBTU, 2-(1H-benzotriazol-1-yl)-1 ,1,3,3- tetramethyluronium hexafluorophosphate; HOBt, 1 -hydroxybenzotriazole; HPLC, high performance liquid chromatography; LC-MS, liquid chromatography mass spectrometry; Mtt, methyltrityl; TLC, thin-layer chromatography. General Method for resin-bound linear peptide synthesis

Linear peptides were synthesised by an automated single solid phase approach (Applied Biosystems 433A peptide synthesiser) using the Fmoc- Arg(Pbf)-p-alkoxybenzyl alcohol resin (0.59 mmol/g loading). Amino acids were attached by Fmoc strategy on a 0.25 mmol scale using Fastmoc™ chemistry with a single coupling time of 21 min. The resin and the amino acid derivatives, Fmoc-Aib-OH, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc- ASp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gln(Trt)-OH, FmOc-GIu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc- Ser(tBu)-OH, Fmoc-Thr(tBu)-OH and Fmoc-Tyr(tBu)-OH, were purchased from Bachem AG, Bubendorf, Switzerland. Each amino acid was added sequentially to the growing peptide chain from the C- to the N-termini applying 0.45M HBTU/HOBt in N,N-dimethylformamide (Applied Biosystems, Warrington, UK) and N,N-diisopropylethylamine (Fisher Chemicals, Loughborough, UK) as coupling reagents. Removal of the N-Fmoc protecting group was carried out with 20% piperidine (Romil, Cambridge, UK) in N-methylpyrrolidone (M56 Chemicals, Runcorn, UK) followed by sequential washes with N- methylpyrrolidone. The coupling reagent, HBTU/HOBt is 3.6-fold excess (0.9 mmol) and all amino acid derivatives 4-fold excess (1.0 mmol). All solvents used were of HPLC-grade quality.

Procedure for the solid-phase coupling of N-deprotected resin-bound peptide with capping acids

N-deprotected peptidyl resin (250 mg) was swollen with N, N- dimethylformamide (2 mL), for 30 minutes, at room temperature, after which time the solvent was removed. 2-(1H-benzotriazole-1yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (3 eq), 1 -hydroxybenzotriazole (3 eq) and the capping acid (3 eq) were added to the resin in N,N-dimethylformamide (2 mL). N, N- diisopropylethylamine (9 eq) was added to the reaction mixture and gently agitated for 150 minutes. The reagents were removed from the capped peptidyl resin using nitrogen and the resin was washed with N,N-dimethylformamide, dichloromethane, methanol and dichloromethane and dried in vacuo. Monitoring of the capping was conducted using the Kaiser test. Peptide cleavage

The peptides were cleaved from the resin with simultaneous deprotection using 95% trifluoroacetic acid at room temperature, for 2 h, in the presence of 1.25% triisopropyl silane, 1.25% water and 2.5% ethane dithiol. The filtrate was collected, the resin was washed with dichloromethane and the solutions combined and agitated for a further 60 minutes. The peptide was precipitated by adding the cleavage solution, dropwise, to cold (-78⁰C) diethyl ether and the resulting suspension was maintained at this temperature for a further 15 minutes. The peptide was then pelleted by centrifugation at 3000 rpm for 5 minutes (4⁰C). The diethyl ether was decanted off, and the process was repeated twice more. The resulting peptide pellet was thoroughly dried under vacuum for 30 minutes, dissolved in trifluoroacetic acid/acetonitrile/water and lyophilized overnight (-54⁰C, 0.08 mbar). Characterization of crude peptides

Crude N-capped peptides were characterised by reverse-phase LC-MS using the following methods:

Method A (Shimadzu QP-8000): Analytical C-18 column, Thermo betabasic, 100 x 4.6 mm, 5 μM, AB gradient of 5-95% for B, over 30 minutes, at a flow rate of 1 mL/minute, where eluent A was 0.1% trifluoroacetic acid/water and eluent B was 0.1% trifluoroacetic acid in 60% acetonitrile/water.

Method B (Waters ZQ): Analytical C-18 column, Thermo betabasic, 100 x 4.6 mm, 5 μM, AB gradient of 5-95% for B, over 25 minutes, at a flow rate of 1 mL/minute, where eluent A was 0.1% formic acid/water and eluent B was 0.1% formic acid/acetonitrile.

Production of bicyclic peptides

The crude linear precursors were dissolved in the minimum trifluoroacetic acid and diluted to 2 L/0.25 mmol with water. The first disulfide bridge was formed between unprotected Cys residues using potassium hexacyanoferrate (III). The peptide solution was adjusted to pH 7.5 with aqueous ammonium hydroxide. To this solution, 0.01 M potassium hexacyanoferrate (III) was added dropwise to excess, until a slight yellow colour remained. The completion of the reaction was confirmed by LC-MS sampling after acidification. The pH of the solution was adjusted to 4 using 50% aqueous acetic acid. The crude reaction mixture was stirred with Bio-Rex 70 weak cation-exchange resin (BioRad, CA) overnight and packed into a glass column. After thorough washing with water, the peptide was eluted using 50% aqueous acetic acid and detected by thin layer TLC using ninhydrin. Ninhydrin positive fractions were pooled and lyophilized (-54⁰C, 0.08 mbar). The crude monocyclic peptide was then subjected to iodine-deprotection/oxidation between Cys(Acm) protected residues to provide the second disulfide bridge. A solution of the peptide (5 mg/ml) in 10% aqueous trifluoroacetic acid was mixed vigorously with 8 equivalents of iodine and the resulting suspension stirred for, typically, 1.5 h. LC-MS sampling was used to determine reaction completion.

Purification and characterization of the N-capped bicyclic peptides The reaction mixture was diluted x 2 with eluent A, filtered through a 0.45 mm disposable filter and purified directly via (mass-directed) preparative reverse-phase LC-MS using the following methods:

Method C (Shimadzu QP-8000): Preparative C-18 column, Thermo betabasic, 100 x 21.2 mm, 5 μM, AB gradient of 5-95% for B, over 40 minutes, at a flow rate of 20 mL/minute, where eluent A was 0.1% trifluoroacetic acid/water and eluent B was 0.1% trifluoroacetic acid in 60% acetonitrile/water. Method D (Waters ZQ): Preparative C-18 column, Thermo betabasic,

100 x 21.2 mm, 5 μM, AB gradient of 5-95% for B, over 30 minutes, at a flow rate of 1 mL/minute, where eluent A was 0.1% formic acid/water and eluent B was 0.1 % formic acid/acetonitrile.

The relevant fractions were collected, evaporated, lyophilized (-54⁰C, 0.08 mbar) and stored at 4⁰C. Confirmation of the structure was performed by analytical reverse-phase LC-MS using a related method (A and B) to the preparative method.

Purified using Method C.

EG00086[batch A]: Off-white solid, 3.9 mg, HPLC (Method A) R, 17.69 min.; purity > 75%; MS m/z- 874 [M + 4] <⁺. Purified using Method D. EG00086[batch B]: White solid, 18.6 mg, HPLC (Method B) R, 10.71 min.; purity > 90%; MS m/z- 873.6 [M + 4J⁴⁺.

Purified using Method C. EG00087[batch A]: Off-white solid, 1.4 mg, HPLC (Method A) R, 10.97-11.51 min.; purity > 75%; MS m/z- 884 [M + 4] **. Purified using Method D.

EG00087[batch B]: White solid, 18.5 mg, HPLC (Method B) R_t 10.12 min.; purity

> 95%; MS m/z- 884.5 [M + 4] **.

Purified using Method C.

EG00088: Off-white solid, 4.8 mg, HPLC (Method A) R, 16.17 min.; purity >

75%; MS m/z- 885 [M + 4] **.

Purified using Method C.

EG00089: Off-white solid, 1.2 mg, HPLC (Method A) R, 11.90 min.; purity >

75%; MS m/z- 887 [M + 4] *^*.

Purified using Method C.

EG00090: Off-white solid, 1.3 mg, HPLC (Method A) R, 12.07 min.; purity >

75%; MS m/z- 894 [M + 4] **.

Purified using Method D. EG00187: White solid, 9.3 mg, HPLC (Method B) R, 4.0 min.; purity > 95%; MS m/z- 852.3 [M + 4] **.

Purified using Method D.

EG00188: White solid, 2.2 mg, HPLC (Method B) R, 3.9 min.; purity > 95%; MS m/z- 862.6 [M + 4] **.

The following compounds were synthesised by Bachem (UK) Ltd.

H-SCKNTDSRCKARQLELNERTCRCDKPR-OH i 1

EG00206: White solid, 20 mg, HPLC (Method B) R, 1.66 min.; purity > 95%; MS m/z- 642.7 [M + 5]⁵⁺

EG00254: White solid, 100 mg, HPLC (Method B) R, 4.32 min.; purity > 95%; MS m/z- 667.9 [M + 5J⁵⁺.

Described below are experimental methods for various binding and cell- adhesion studies, which were carried out on peptide derivatives of the invention. Results of these studies are given below. Cell culture

Porcine aortic endothelial cells expressing neuropilin-1 (PAE/NP-1) were cultured in Ham's F12 medium containing 10% fetal bovine serum (FBS) and 25 μg/ml hygromycin B. PAE cells expressing KDR (PAE/KDR) were grown in Ham's F12 medium containing 10% FBS and 250 μg/ml Gentamicin G418. Human carcinoma cell lines (DU145, A549 and ACHN) were grown in RPMI 1640 medium containing 10% FBS and L-glutamine. 1²⁵I-VEGF-Ai₆₅ binding

Confluent cells in 24-well plates were washed twice with phosphate- buffered saline (PBS). At 4°C various concentrations of peptides diluted in binding medium (Dulbecco's modified Eagle's medium, 25 mM HEPES pH 7.3 containing 0.1% BSA) were added, followed by addition of 0.1 nM of ¹²⁵I-VEGF- Ai₆₅ (1200-1800 Ci/mmol, GE Healthcare). After 2 h of incubation at 4°C, the medium was aspirated and washed 4 times with cold PBS. The cells were lysed with 0.25 M NaOH, 0.5% SDS solution, and the bound radioactivity of the lysates was measured in a gamma counter. Non-specific binding was determined in the presence of 100-fold excess unlabelled VEGF-A₁₆S. Cell matrix adhesion

Cell adhesion to extracellular matrix proteins (basement membrane protein complex, laminin I, collagen IV, fibronectin or vitronectin) was measured by the lnnocyte ECM cell adhesion assay (Calbiochem). Cells were detached with a non-enzyme cell dissociation solution (Sigma), washed and resuspended in RPMI 1640 medium. Cells with or without peptide treatment were seeded at 3x10⁴ cells per matrix-coated well of 96-well plates. After 1.5 h incubation, cells were washed with PBS. The attached cells were labeled with the green fluorescent dye calcein-AM and measured using a fluorescence plate reader at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. Results

Table 1 shows that both EG00086 and EG00087 bind to cells expressing NP-1 receptors. No binding is observed to cells expressing KDR receptors. Table 1

Table 2 shows that both EG00086 and EG00087 are as potent as the parental peptide EG3287 in the inhibition of ¹²⁵I-VEGF binding to DU 145, A549 and ACHN cells Table 2

Table 3 shows that both EG00086 and EG00087 are more effective than EG3287, at concentrations of 10 μM, in the inhibition adhesion of DU 145 and ACHN cancer cells to extracellular matrix proteins. Table 3

Table 4 shows biological results for three compounds embodying the present invention. The IC_5O of EG3287 is 2.8 μM. Table 4

Claims

1. A peptide derivative having formula I

X₂ is an arginine, alanine or proline residue or a direct bond; and

R₁ is an alkyl group containing at least two carbon atoms, an alkenyl, aryl, alkylaryl, cycloalkyl, heterocyclic or heteroaromatic group; a chelating group capable of chelating an imaging isotope; or a linking group for attachment to or incorporation into an imaging nanoparticle.

2. A peptide derivative according to claim 1 , wherein each X₁ is independently a cysteine, aspartate, glutamate, lysine, diaminobutyric acid, allylglycine or homoallylglycine residue, or -NHCH(CH₂NHCOCH₂CI)COO-.

3. A peptide derivative according to claim 1 or claim 2, wherein X₂ is an arginine or alanine residue and R₁ is an alkyl, aryl or alkylaryl group.

4. A peptide derivative according to claim 3, wherein X₂ is an arginine residue.

5. A peptide derivative according to any preceding claim, wherein R₁ is heptyl, -CH₂-naphthyl, naphthyl-OH, biphenyl or CH₂CH(Ph)₂.

6. A peptide derivative according to claim 5, wherein X₂ is a direct bond and R₁ is heptyl.

7. A peptide derivative according to any preceding claim, which comprises SEQ ID NO. 2.

8. A peptide derivative according to any preceding claim, in bicyclic form.

9. A peptide derivative according to any preceding claim, for use in therapy.

10. A peptide derivative according to any preceding claim, for stimulating nerve repair.

11. A peptide derivative according to any of claims 1 to 9, for use in the treatment of neurodegeneration.

12. A peptide derivative according to any of claims 1 to 9, for use in immune system modulation.

13. A peptide derivative according to any of claims 1 to 9, for use in the treatment of cancer.

14. A method of stimulating nerve repair, using a peptide derivative according to any of claims 1 to 8.

15. A method of treating neurodegeneration, using a peptide derivative according to any of claims 1 to 8.

16. A method of immune system modulation, using a peptide derivative according to any of claims 1 to 8.

17. A method of treating cancer, using a peptide derivative according to any of claims 1 to 8.

18. A peptide derivative according to any of claims 1 to 8, wherein Ri is a chelating or linking group and which additionally comprises the imaging isotope.

19. A peptide derivative according to claim 18, for diagnostic use.