EP1680136A1

EP1680136A1 - Peptides having, for example, an antiangiogenic activity and applications thereof in therapeutics

Info

Publication number: EP1680136A1
Application number: EP04787446A
Authority: EP
Inventors: Etienne Jacotot; Annie Borgne-Sanchez; Sylvie Dupont; Dominique Rebouillat
Original assignee: Theraptosis SA
Current assignee: Theraptosis SA
Priority date: 2003-09-25
Filing date: 2004-09-24
Publication date: 2006-07-19
Also published as: US20080171697A1; FR2860236B1; FR2860236A1; WO2005030238A1; JP2007506713A; IL174547A0

Abstract

The invention relates to cyclised peptides corresponding to sequence SEQ ID N°1: X1X2RGDX3FGX4X5LLFIHFX6IGSX7HSX8IX9, wherein: the letters without any numerical index correspond to amino acids defined by the single-letter international code; X1 is G or GG having an amino-terminal end which may or may not be free; X2 is either a C, in which case X2 = X4, the two Cs being connected by a disulphide bridge, or X2 is capable of forming a lactam bridge with X4, either X2 or X4 being an amino acid bearing an acid group, such as A or D, and the other bearing an amino function such as Q, N; X3 is either an M pattern or a norleucine pattern; X5 is one or several di-, tri-, or tetra-peptide patterns comprising G or a combination of G and S, or X5 is a C pattern having a side-chain which serves as a covalent linkage point with a 3-nitro-2-pyridinesulphenyl group which is located at the N-terminal end of the next amino acid (L); X6 is either an R pattern or a K pattern; X7 is either an R pattern or a K pattern; X8 is either an R pattern or a K pattern; and X9 is an aliphatic amino acid (such as G or A) having an amide C-terminal end. The inventive peptides can be used as active ingredients in medicaments, for example, for the treatment of pathologies linked to hypervascularisation.

Description

"PEPTIDES WITH ESPECIALLY ANTI-ANGIOGENIC ACTIVITY AND THEIR THERAPEUTIC APPLICATIONS"

The invention relates to peptides having in particular an anti-angiogenic activity and to their applications in therapy. The inventors' research into therapeutically active peptides led them to develop constructs which have proved to be of great interest with regard to their anti-angiogenic properties. The invention therefore relates to such peptides and the use of their therapeutic properties to develop drugs. It thus relates to pharmaceutical compositions containing these peptides as active principle. It also relates to the use of these peptides to manufacture drugs with anti-angiogenic effect, for the treatment of pathologies linked to hypervascularization. The peptides according to the invention are characterized in that they are cyclized peptides, corresponding to the sequence SEQ ID No. 1: XιX ₂ RGDX ₃ FGX4X5LLFIHFX ₅ IGSX ₇ HSX ₈ IX9 in which: the letters without numerical index correspond amino acids defined by the international one-letter code. Xi is either a G or a GG whose amino-terminal end is free, alkylated, acylated, in particular acetylated, or comprises a labeling group such as the biotinyl group, X ₂ is either a C, in which case X ₂ = X ₄ , the 2 Cs being then united by a disulfide bridge, or X ₂ is capable of forming a lactam bridge with X ₄ , one of X ₂ or X ₄ being an amino acid carrying an acid group, such as A or D , the other carrying an amino function, like Q, N. X ₃ is either a motif M or a nor-Leucine motif X is either a motif or a succession of two di-, tri- or tetra-peptide motifs composed of G or of a combination of G and S, such as GG, GGG, GGGG, GGS, GGGS, GGSGGS, or even X ₅ is a C motif whose side chain (thiol function) serves as a covalent bonding point with a 3-nitro-2-pyridinesulfenyl group (Npys; Drijfhout et al ., 1988 Int J Peptide protein Res, 32: 161-166) located on the N-terminal end of the following amino acid (L), X ₆ is either an R motif or a K motif - X ₇ is either an R motif, either a KX ₈ motif is either an R motif, or a K Xg motif is an aliphatic amino acid (such as G, or A) whose C-terminal end is amidated. These peptides contain from 25 to 35 amino acids. A peptide of this type responds to the sequence SEQ ID No. 2: GG * CRGDMFG * CGGLLFIHFRIGSRHSRIG (^ indicates a disulfide bridge connecting the two motifs C). Other peptides are as defined above and have an alkyl group at their N-terminal end. In still other peptides, one or more amino acids are replaced by their dextrorotatory form ( _D aa). Other peptides according to the invention correspond to SEQ ID No. 1 above, but have one or more peptide bonds to form bioisosteres. Examples include the reduction of an amide bridge to -CH ₂ NH-, or a retro-inverso reaction, as defined by Goodman and Ro (1995, in Burger's Medicinal Chemistry. Fifth ed vol.l pages 803- 861 , edited by ME olff). As variants of the peptide of sequence SEQ ID No. 2 exhibiting the RGD motif by a disulfide bridge between two cysteines, mention will be made of the peptides of sequence SEQ ID No. 3 to 10: SEQ ID N ° 3: GG * CRGDMFG * CGGLLRIHFRIGSRHSRIG

SEQ ID N ° 4: GG * CRGDMFG * CGG-LFIHFRIGSRHSRIG

SEQ ID N ° 5: GG * CRGDMFG * CGGSLFIHFRIGSRHSRIG

SEQ ID N ° 6: GG * CRGDMFG * CGGLLFIHFKIGSRHSRIG

SEQ ID N ° 7: GG * CRGDMFG * CGGLLFIHF ^N RIGSRHSRIG ( ^N R representing an N-alkylarginine motif)

SEQ ID N ° 8: GG * CRGDMFG * CGGLLSRHFRIGSRHSRIG

SEQ ID N ° 9: GG * CRGDMFG * CGGLLSIHFRIGSRHSRIG

SEQ ID N ° 10: GG * CRGDMFG * CGGLLFRHFRIGSRHSRIG

Other peptides of the invention include a sequence SEQ ID No. 11: XRGDMF-GX 'exposing the RGD motif by a lactam bridge between the amino acids X (X) -CO-NH- (X'), X and X 'being amino acids such that one carries an acid group, and the other carries an amino. Preferred peptides of this group correspond to the sequences SEQ ID No. 12 to SEQ ID No. 23:

SEQ ID N ° 12: GGXRGDMFGX 'GGLLFIHFRIGCRHSRIG

SEQ ID N ° 13: GGXRGDMFGX 'GGLLFIFFRIGCRFSRIG

SEQ ID N ° 14: GGXRGDMFGX 'GGLLFIHFRIGSRHSRIG

SEQ ID N ° 15: GGXRGDMFGX 'GGLLRIHFRIGSRHSRIG

SEQ ID N ° 16: GGXRGDMFGX 'GG-LFIHFRIGSRHSRIG

SEQ ID N ° 17: GGXRGDMFGX 'GGSLFIHFRIGSRHSRIG

SEQ ID N ° 18: GGXRGDMFGX 'GGLLFIHFKIGSRHSRIG

SEQ ID N ° 19: GGXRGDMFGX 'GGLLFIHFRIGSRHSRIG ( ^N R representing an N-alkylarginine motif)

SEQ ID N ° 20: GGXRGDMFGX 'GGLLSRHFRIGSRHSRIG

SEQ ID N ° 21: GGXRGDMFGX 'GGLLSIHFRIGSRHSRIG

SEQ ID N ° 22: GGXRGDMFGX 'GGLLFRHFRIGSRHSRIG

SEQ ID N ° 23: GGXRGDMFGX 'GGLLFIHFRIGSRHSRIG Said sequences can be modified, that is to say correspond to the native peptide but contain one or more different acids, chemically modified, as long as these modifications do not affect the desired function. These include the replacement of Met by nor-Leu, Arg by N-alkyl Arg, which in particular allows the construction to be stabilized. These modifications also include an acyl group, in particular N-terminal acetyl. The peptides of the invention are further characterized in that they induce apoptosis of human endothelial cells expressing the αVβ3 receptors. They are also advantageously characterized in that they undergo endocytosis by human endothelial cells expressing the αVβ3 receptors, are localized in the itochondrial compartment and exert a mitochondriotoxic effect. When a peptide as developed above, or as defined above, is brought into contact with endothelial cells, a specific recognition of the Vβ3 integrins is observed on the surface of the endothelial cells, which allows endocytosis chimeric peptide. Once internalized, the peptide is temporarily located in the lysosomes, as shown in confocal microscopy and is gradually distributed in the mitochondrial compartment. It will be noted that the specificity of the peptides of the invention results from the addition of the mitochondrio-toxic part to ligands of the integrins to exert a toxicity by the way of the mitochondrio-toxicity, the ligands of the integrins being present for purposes of targeting and not themselves having angiostatic activity. As illustrated by the examples given below, the treatment of primary human endothelial cells with doses of peptides of the order of a micromolar leads to a dissipation of the mitochondrial transmembrane potential (Δψm), to the release of mitochondrial cytochrome c, to l 'exposure of phosphatidyl-serine and condensation of nuclear chromatin. These peptide constructions have the advantage of an absence of toxicity on negative Vβ3 cells. The invention therefore also aims to take advantage of these properties to selectively induce PMM and apoptosis of angiogenic endothelial cells in the context of therapeutic strategies, in particular anti-cancer, treatment of polyarthritis, diabetic retinopathy. The pharmaceutical compositions according to the invention are characterized in that they contain a therapeutically effective amount of at least one peptide, as defined above, in combination with a phar aceutically acceptable vehicle. These compositions are advantageously in the dosage forms suitable for their administration by injectable route. Mention will in particular be made of injectable solutions intended for administration by the intravenous route. The invention further relates to the use of peptide constructs as defined above for manufacturing anti-angiogenic drugs for the treatment of pathologies due to hypervascularization. Mention will in particular be made of the treatment of solid tumors such as pulmonary tumors, adenomas, melanomas, cancers of the prostate, breast, colon, pancreas, osteosarcomas. The invention also applies to the treatment of diabetic retinopathies and polyarthritis. The dosages of the administration forms and the treatments will be determined by a person skilled in the art depending on the pathology to be treated and the condition of the patient. Other characteristics and advantages of the invention will be given in the examples which follow relating to the construction SEQ ID No. 2 (hereafter TEAM-VP) SEQ ID No. 2: GG * CRGDMFG * C-GG-LLFIHFRIGSRHSRIG -amide with or without biotin, "*" indicating cyclization by formation of a disulfide bridge. Reference will be made to FIGS. 1 to 3, which represent, respectively, FIG. 1, the analysis of cytotoxicity on endothelial cells, FIG. 2, the recognition of the CycRGD motif by the integrins αVβ3, FIG. 3, the effects of TEAM-VP on isolated itochondria and HUVEC cells.

Examples: Analysis of the cytotoxicity of TEAM-VP on endothelial cells a. The HUVEC cells were incubated for 24 h with 5-30 μM of peptide CycRGD, LLFIHFRIGSRHSRIG-amide (C4) or TEAM-VP, then labeled with 7-AAD and analyzed by flow cytometry (Figure la). b. The HUVEC cells were incubated for 24, 48, 72 and 96 h with 15 μM of TEAM-VP and then labeled with 7-AAD and analyzed by flow cytometry (Figure lb).

Recognition of the CycRGD motif by the αVβ3 integrins a. Cell binding analysis: HUVEC cells were incubated for 45 min at room temperature with the following peptides: GGCRGDMFGCGG-amide (linear RGD), GG * CRADMFG * CGG-amide (CycRAD) and GG * CRGDMFG * CGG-amide ( CycRGD) (0.5 to 2 μM) marked FITC and analyzed by flow cytometry (Figure 2a). b. Hunt for the CycRGD peptide by itself: The HUVEC cells were pre-incubated or not for 30 min at room temperature with 200 μM of unlabeled CycRGD peptide before addition of FITC-CycRGD peptide (10 μM) for 45 min and analyzed by flow cytometry (Figure 2b). vs. Competition for integral sites:

The HUVEC cells were pre-incubated or not for 30 min at room temperature with 25 μM of CycRGD, CycRAD, GRGDS and GRGES peptide before addition of FITC-CycRGD peptide (0.5 μM), then analyzed by flow cytometry (Figure 2c). d. Correlation between expression of integrins, binding and toxicity of peptides: The cells HUVEC, HMVECd, MCF-7, MDA, HeLa, HT-29, Jurkat, CEM and PBMC were labeled with the antibodies directed against the integrins αVβ3 and αVβ5 and analyzed by flow cytometry. The binding of the CycRGD peptide and the induction of apoptosis by TEAM-VP on the different cell types were measured (Figure 2d).

+ Study of the peptide entry process:

The FITC-CycRGD and TEAM-VP (FITC) peptides enter the HUVECs and co-locate with the dextran beads (5 hours of co-treatment). The entry of TEAM-VP and dextran beads is inhibited by the treatment of sodium azide + deoxyglucose, indicating entry of the peptide by endocytosis. No entry of the FITC-CycRAD peptide was observed in the HUVECs, nor was there any entry of the FITC-CycRGD peptide in the HeLa. + TEAM-VP intracellular routing:

In the confocal, HUVECs treated with TEAM-VP are observed for 8, 24 and 32 hours.

TEAM-VP revealed with Streptavidin-Texas Red co-distributes with lysosomes (anti-Lamp2-FITC) at 8 h of treatment and seems to leave these organelles at 24 h. TEAM-VP revealed with Streptavidin-FITC partially co-distributes with the mitochondria (anti-VDAC) at 24h and totally at 32h. We does not observe co-distribution with the Golgi apparatus (anti-Golgin) throughout the treatment.

Effects of TEAM-VP on isolated mitochondria and HUVEC cells a. Effect on isolated mitochondria: Induction of mitochondrial swelling:

The isolated mitochondria were incubated with the peptide CycRGD or TEAM-VP in the presence or not of bongkrekic acid (BA, 50 μM), cyclosporin A (CsA, 10 μM), and DIDS (8 μM). Induction of the mitochondrial membrane potential drop: The isolated mitochondria were incubated with 1 μM of TEAM-VP or its controls (C1, C2, C3), labeled with JC-1 and analyzed by flow cytometry (FIG. 3a),

SEQ ID N ° 24 Cl = GG * CRADMFG * CGGLLFlHFRIGSRHSRIGamide SEQ ID N ° 25 Cl = GG * CRGDMFG * CGGLLFIHFAIGSRHSAIGamide SEQ ID N ° 26 C3 = RKKRRQRRRGGLLFIHFRIGSRHSRIGamide

b. Release of cytochrome c:

The isolated mitochondria were incubated with Alamethicin (5 ug / ml) or TEAM-VP (10 μM) and the supernatant analyzed in Western Blot with an anti-cytochrome c (Figure 3b). vs. Analysis of nuclear apoptosis:

HUVEC cells treated with TEAM-VP (15-40 μM) in the presence or absence of a caspase inhibitor for 8, 16, 24 and 48 h were labeled with Hoechst and observed with an inverted microscope. The percentages of cells with intact nuclei, stage I or stage II are reported (Figure 3c). d. Induction of the mitochondrial membrane potential drop and exposure of phosphatidyl serines in cellula:

The HUVEC cells were incubated for 24 hours with 15 μM of TEAM-VP peptide, CycRGD and C4 then labeled with JC-1 or with an anti-PARP or Annexin-V-FITC and analyzed by flow cytometry (Figure 3d).

+ Cytochrome C in cellula release:

Release of cytochrome c observed under microscopy after 24 hours of treatment with TEAM-VP (10 μM) and double labeling of fixed cells with an anti-cytochrome c and an anti-VDAC.

+ Loss of mitochondrial membrane potential in cellula:

Fall in potential observed under the microscope after labeling with

JC-1 of the HUVEC cells treated for 16-24 hours with 15 μM of TEAM-VP peptide.

Animal models:

The animal models used to demonstrate the efficacy of the products correspond to those conventionally used (see in particular Kisher et al., 2001 Cancer Research 61: 7669-7674,

Galaup et al., 2003 Mol Therapy 7: 731-740).

Claims

1. Peptides having in particular an anti-angiogenic activity, characterized in that they are cyclized peptides, corresponding to the sequence SEQ ID No 1: XιX ₂ RGDX ₃ FGX ₄ X ₅ LLFIHFX ₆ IGSX ₇ HSX ₈ IXg in which: letters without a numerical index correspond to the amino acids defined by the international one-letter code. Xi is either a G or a GG whose amino-terminal end is free, alkylated, acylated, in particular acetylated, or comprises a labeling group such as the biotinyl group, X ₂ is either a C, in which case X ₂ = X ₄ , the 2 Cs being then united by a disulfide bridge, or X ₂ is capable of forming a lactam bridge with X ₄ , one of X ₂ or X ₄ being an amino acid carrying an acid group, such as A or D , the other carrying an amino function, such as Q, N. X ₃ is either a motif M or a nor-Leucine motif X ₅ is either a motif or a succession of two di-, tri- or tetra-peptide motifs composed of G or a combination of G and S, such as GG, GGG, GGGG, GGS, GGGS, GGSGGS, or even X ₅ is a C motif whose side chain (thiol function) serves as a covalent bonding point with a 3-nitro-2-pyridinesulfenyl group located on the N-terminal end of the next amino acid (L). X ₆ is either an R motif, or a KX ₇ motif is either an R motif, or a KX ₈ motif is either an R motif, or a K motif Xg is an aliphatic amino acid (such as G, or A) whose C-terminal end is amidated.

2. Peptide according to claim 1, characterized in that it responds to the sequence SEQ ID No. 2: GG * CRGDMFG * CGGLLFIHFRIGSRHSRIG (* indicates a disulfide bridge connecting the two C motifs).

3. Peptides according to claim 1 or 2, characterized in that they are modified with respect to the native peptide and in particular have an alkyl group at their N-terminal end and / or in that one or more amino acids are replaced by their dextrorotatory form ( _D aa) and / or in that they comprise one or more peptide bonds to form bioisosteres, for example the reduction of an amide bridge into -CH ₂ NH-, or a retro-inverso reaction.

4. Peptides according to claim 2, in which the RGD motif is exposed by a disulfide bridge between two cysteines, in particular the peptides of sequences SEQ ID N ° 3 to 10:

SEQ ID N ° 3: GG * CRGDMFG * CGGLLRIHFRIGSRHSRIG

SEQ ID N ° 4 GG * CRGDMFG * CGG-LFIHFRIGSRHSRIG SEQ ID N ° 5 GG * CRGDMFG * CGGSLFIHFRIGSRHSRIG SEQ ID N ° 6 GG * CRGDMFG * CGGLLFIHFKIGSRHSRIG SEQ ID N ° 7 TR representing an N-alkylarginine motif)

SEQ ID N ° 8: GG * CRGDMFG * CGGLLSRHFRIGSRHSRIG SEQ ID N ° 9: GG * CRGDMFG * CGGLLSIHFRIGSRHSRIG SEQ ID N ° 10: GG * CRGDMFG * CGGLLFRHFRIGSRHSRIG 5. Peptides according to claim 1, characterized in that they contain a sequence

SEQ ID No. 11: XRGDMFGX 'exposing the RGD motif by a lactam bridge between the amino acids X (X) -CO-NH- (X'), X and X 'being amino acids such that one carries a group acid, and the other carries an amino. 6. Peptides according to claim 5, characterized in that they correspond to the sequences SEQ ID No. 12 to SEQ ID No. 23: SEQ ID NO: 12 GGXRGDMFGX 'GGLLFIHFRIGCRHSRIG SEQ ID NO: 13 GGXRGDMFGX' GGLLFIFFRIGCRFSRIG SEQ ID NO: 14 GGXRGDMFGX 'GGLLFIHFRIGSRHSRIG SEQ ID NO: 15 GGXRGDMFGX' GGLLRIHFRIGSRHSRIG SEQ ID NO: 16 GGXRGDMFGX GG-LFIHFRIGSRHSRIG SEQ ID NO: 17 GGXRGDMFGX 'GGSLFIHFRIGSRHSRIG SEQ ID N ° 18 GGXRGDMFGX 'GGLLFIHFKIGSRHSRIG SEQ ID N ° 19 GGXRGDMFGX' GGLLFIHF ^N RIGSRHSRIG ( ^N R representing an N-alkylarginine motif)

SEQ ID N ° 20 GGXRGDMFGX 'GGLLSRHFRIGSRHSRIG SEQ ID N ° 21 GGXRGDMFGX' GGLLSIHFRIGSRHSRIG SEQ ID N ° 22 GGXRGDMFGX 'GGLLFRHFRIGSRHSRIG SEQ ID N ° 23 GGXIGGGFRGIFRGFRGFGRGFRGFGRGFRGFRGFRGFRGFRGGFRGFGRGFGRGFRGFGRGFGRGFGRGFGGFRGFRGFGGFRGFGFRGFGFRGGFGFRGGFGFRGGFGFRGFGFRGGFGFRGFGFRGFGFRGGFGFRGFGFGGFGGFGGFGGFGGFGGFGGFGGFGGFGGFGGFGGFGGFGGFGGFGG

6, characterized that they induce apoptosis of human endothelial cells expressing the αVβ3 receptors. 8. Peptides according to any one of claims 1 to

7, characterized in that they undergo endocytosis by human endothelial cells expressing the αVβ3 receptors, are located in the mitochondrial compartment and exert a mitochondriotoxic effect. 9. Pharmaceutical compositions, characterized in that they contain a therapeutically effective amount of at least one peptide as defined in any one of claims 1 to 8, in association with a pharmaceutically acceptable vehicle. 10. Pharmaceutical compositions according to claim 9, characterized in that they are in dosage forms suitable for their administration by injectable route, in particular in the form of injectable solutions intended for administration by intravenous route. 11. Use of peptides according to any one of claims 1 to 8, for manufacturing anti-drugs angiogenic drugs for the treatment of pathologies due to hypervascularisation. 12. Use according to claim 11, for the manufacture of medicaments for the treatment of solid tumors such as lung tumors, adenomas, melanomas, cancers of the prostate, breast, colon, pancreas, osteosarcomas, treatment of diabetic retinopathies and polyarthritis.