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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
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• • A—HUMAN NECESSITIES
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  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
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  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
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  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4747—Apoptosis related proteins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/82—Translation products from oncogenes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the invention relates to the use of peptides isolated to induce cell death by targeting the mitochondria and their biological applications.
• the mitochondrion performs essential functions in the control of apoptosis in vertebrates.
• the mitochondrial pathway of apoptosis is defined by a major event: the permeabilization of the outer mitochondrial membrane, which leads to the release into the cytoplasm of toxic proteins (such as cytochrome c) normally contained in the intermembrane space.
• toxic proteins such as cytochrome c
• Bcl-2 family proteins The proteins of this family, which are either pro-apoptotic (like Bax and Bid) or anti-apoptotic (like Bcl-2 and Bcl-xL), form homo- and heterodimers, all of these protein-protein interactions. participating in the regulation of apoptosis.
• Proteins homologous to Bcl-2 share a globular structure (reminiscent of certain bacterial membrane-forming bacterial toxins) with between 6 and 9 amphipathic alpha-helices.
• a supersecondary (“helix-elbow-helix”) motif consisting of two anti-parallel "hairpin” alpha helices (alpha5- alpha6 in Bax), able to insert into the membranes biological and to form pores.
• the 3D structure of Bax is given on the Figurel.
• Each helix of the motif has a size of about 15-20 amino acids (order of magnitude for an alpha helix capable of crossing a lipid bilayer), separated by a bend formed of 3-4 residues.
• the membrane insertion regions of the Bcl-2 family proteins have been evolutionarily selected to integrate specifically into the mitochondrial membranes of animal cells. It is important to test experimentally if these regions, apart from the global context of the proteins which contain them, are capable of reaching the mitochondria, of interfering with their structure (such as for example their membranes) and / or their function (for example their transmembrane potential) and cause a cell death. To date, the ability of peptides derived from this membrane insertion motif to trigger an apoptotic process by acting on mitochondria alone is not supported by any experimental evidence. Such peptides could represent an alternative to the artificial cationic peptides used to trigger cell death (Ellerby et al., Nat Med, 1999), which are not very active and therefore not conducive to industrial development.
• the work of the inventors has focused on the study of peptides from these regions to determine whether they alone can target mitochondrial membranes and cause cell death by apoptosis.
• poropeptides from the Greek word Poros ⁇ : hole or sea passage, figuratively: an effective way around a difficulty
• a poropeptide designating a peptide having the ability to form pores in membranes lipids or destabilize biological membranes such as mitochondrial membranes.
• Poropeptides designed from the membrane insertion regions of proteins of the Bcl-2 family, have thus been shown to be capable of inducing the apoptosis of harmful cells, for example tumor cells.
• the purpose of the invention is therefore the use of peptides whose sequence derives from the membrane insertion regions of the Bcl-2 family proteins to induce cell death by targeting the mitochondria. It also aims to provide new peptide molecules developed from these regions.
• the invention also aims to provide, with these different molecules, high efficiency means to restore an apoptosis process in cancer cells while minimizing the maximum impact on surrounding healthy cells.
• This targeting step may for example be based on the vectorization of peptides by coupling to a domiciliation molecule ensuring their selective targeting at the tumor site, sparing healthy tissue. Since all living cells in the body (healthy or pathological) have mitochondria, poropeptides inducing the breakdown of mitochondrial permeability can be used to induce the death of many cell types. With advances in therapeutic targeting techniques, it may be possible to selectively eliminate certain cell populations that are harmful to the body.
• the invention thus relates to the use of membrane insertion helix derivatives of Bcl-2 family proteins to induce cell death by targeting the mitochondria, characterized in that they are peptides corresponding to the a5 regions. and / or a6 of the Bax protein or equivalent regions present in the other proteins of the Bcl-2 family.
• the invention further relates to derivatives, variants, mutants or fragments of the aforementioned peptides.
• Synthetic peptides derived from those defined above comprise, for example, means allowing cell penetration or recognition of a particular cell type.
• Advantageous means correspond to a transduction motif allowing their internalization in the targeted cell. It is in particular a polyarginine unit, advantageously comprising 8 arginine residues. Other modifications correspond to deletions and / or substitutions of one or more amino acids. Thus, one or more Cysteine residues of said regions may be replaced by Serine or Glycine motifs.
• the invention also relates to conjugates that contain a peptide derived from those defined above and an addressing molecule, such as recognizing molecules. the endothelium, to target a particular tissue or cell type, such as certain tumor cells or angiogenic endothelial cells.
• the invention also relates to functional equivalents of the peptides defined above, such as peptides comprising modifications resulting from post-translational processes such as glycosylation or chemical modifications such as coupling with lipids, sugars, nucleotide sequences or d amino acids since these modifications do not modify the proapoptotic activity of said peptides according to the tests given in the experimental part below.
• Functional equivalents also include peptides of which one or more amino acids are D-conforming amino acids.
• the invention also encompasses retro-peptides and retro-inverso-peptides.
• the invention is directed in particular to the peptides derived from regions a5 and a6, for example, the peptide of sequence SEQ ID No. 1: NH 2 -NWGRVVALFYFASKLVLKALSTKVPELIR-COOH.
• these "mitochondrial-toxic” compounds are able to trigger apoptosis by destabilizing the mitochondrial membranes, especially the outer mitochondrial membrane.
• they induce the release of multiple toxic molecules contained in the inter-membrane space of mitochondria, in particular cytochrome c.
• the invention relates to the use of the peptides defined above as medicaments.
• compositions of the invention are characterized in that they comprise a therapeutically effective amount of at least one peptide as defined above in association with a pharmaceutically acceptable vehicle.
• the present invention also relates to a pharmaceutical composition
• a pharmaceutical composition comprising as active ingredient at least one peptide as defined previously associated in said composition with one or more pharmaceutically acceptable carriers, diluents or excipients.
• compositions according to the invention can be carried out according to the techniques known to those skilled in the art by any of the accepted modes of administration for the therapeutic agents. These methods include systemic, topical, oral or central administration, for example by surgery or intraocular administration. We can also mention the subcutaneous implantation of biodegradable implants.
• the dosage for administration of the peptides according to the invention is chosen according to many factors including the type, species, age, weight, sex and medical condition of the subject; the severity of the condition to be treated; the route of administration; the state of renal and hepatic function of the subject and the nature of the particular compound, or salt, used.
• the normally experienced person skilled in the art will determine and prescribe the effective amount to prevent, hinder or halt the progress of the medical condition to be treated.
• the above compounds are particularly advantageous for initiating an apoptotic process after internalization in tumor cells or neovascular tumor-irrigating cells.
• the invention also relates to the use of the aforementioned peptides as mitochondrial targeting molecules for delivering a compound or polypeptide to the mitochondrial membranes to induce apoptosis.
• poropeptides are generally useful in situations with increased neovascularization, such as rheumatoid arthritis or obesity. Indeed, sustained angiogenesis characterizes severe forms of arthritis and the maintenance of adipose mass during obesity. Pathological angiogenesis is also found in psoriasis and some forms of diabetes. According to another advantageous embodiment of said poropeptides, they are used to counter certain other types of harmful cells, such as immune cells that recognize the self and cause autoimmune diseases.
• the poropeptides as defined above have all the characteristics of antimicrobial peptides: amphipathic, cationic character, destabilization of lipid membranes.
• the present invention therefore also relates to the use of the peptides as defined above for the preparation of a molecule with antibiotic properties capable of killing resistant bacteria.
• therapeutic peptides are generally metabolized and eliminated more easily than conventional drugs, giving the patient a better chance of healing while minimizing the complications associated with standard therapies.
• the poropeptides can be used, according to the invention, as adjuvants in addition to conventional drugs, thus reducing the doses and side effects of the latter.
• the invention relates to cosmetic compositions containing in their active principle at least one peptide as defined above in combination with a vehicle acceptable in cosmetology.
• the peptides of the invention have stabilizing properties for these compositions.
• the invention is further directed to agronomic or agri-food compositions containing in their active ingredient at least one peptide as defined above, in combination with a vehicle acceptable in agronomy and in the food field.
• Figures 1 to 11 represent, respectively, - Figure 1, A) the "channel" domain of colicin A; B) the 3D structure of Bax; C) the ⁇ -helical hairpin motif of Bcl-2 family proteins (alpha5-alpha6 in Bax);
• FIG. 2 constructs coding for fusions between GFP (green fluorescent protein) and the various regions of anchoring or membrane insertion of the pro-apoptotic protein Bax;
• FIG. 6 the apoptosis rate of wild-type embryonic fibroblasts (MEFs) or deficient in Bax and Bak (MEF-DKO);
• FIG. 7 the mitochondrial transmembrane potential of cells expressing GFP or GFP fused with the peptides used according to the invention.
• FIGS. 10 and 11 the effect of microinjection of peptide fragments of Bax on viability and embryonic (Fig. 9) and cellular morphology (Fig. 10);
• FIG. 12 the proapoptotic effect of a poropeptide of the invention.
• Plasmid constructs comprising inserts corresponding to the nucleic acids encoding one or more Bax regions fused to GFP: amino acids 1-192 (whole Bax protein: ⁇ ! - ⁇ 2- ⁇ 3- ⁇ 4- ⁇ 5- ⁇ 6- ⁇ 6'- ⁇ 7- ⁇ 8 - ⁇ 9), 20-37 (a1), 106-134 (a5), 130-150 (a6, 102-150 (a5-a6, 102-192 (a5-a6-a6'-a7-a8-a9 or a5 -a9, 169-192 (a9.
• the nucleic acids coding for the various anchoring or membrane insertion regions present on the pro-apoptotic protein Bax were cloned into the pEGFP-C1 plasmid.
• the inserted sequences are given in Figure 2.
• a construct (GFP-KLAK) encoding a GFP-KLAKLAKKLAKLAK polypeptide was also made.
• Artificial peptides enriched with KLA sequences are known to target the negatively charged bacterial membrane and exhibit antibiotic activity.
• FIG. upper half corresponds to the Western Blot analysis, using an anti-GFP antibody, of HT 1080 fibrosarcoma cell lysates transfected transiently by the above plasmid constructs; the middle part gives the immunoblot obtained using an antibody recognizing the apoptotic cleavage fragment of PARP; the lower part gives the immunoblot obtained using an antibody recognizing the activated form of caspase-3.
• the different constructs were transfected into human cells cultured in vitro (HT-1080 and SK-MEL-28), and the subcellular localization and cytotoxicity induced by GFP-fused Bax membrane insertion fragments was determined.
• FIG 4 shows the subcellular localization of the complete GFP protein relative to that of inserts composed of Bax-fused membrane insertion fragments.
• MEF-DKO cells were co-transfected with the pEGFP-C1 plasmids encoding the various Bax inserts and the MitoDsRed vector. It is observed that peptides corresponding to or comprising the membrane insertion helices of Bax and / or the TM domain are sufficient to address GFP (in green) to mitochondrial membranes (marked in red with the MitoDsRed protein, comprising the mitochondrial targeting CoxVIII). Similar results were obtained using an antibody directed against the mitochondrial protein Hsp70 (bottom inserts).
• Figure 5 shows the cellular toxicity induced by the expression of the different constructs.
• Figure 5A shows the death rate (apoptosis / necrosis) of HT-1080 cells expressing GFP alone or in fusion with different membrane insertion fragments of the Bax death protein. The cells were treated (+) or not (-) with the general caspase inhibitor zVAD.fmk ( ⁇ ). Cell death was quantified 24 h after transfection by counting the number of green cells (expressing GFP) under an epifluorescence microscope.
• the type of cell death was determined by membrane permeability to propidium iodide (necrosis) and nuclear morphology (condensed or fragmented nuclei) after staining with Hoechst 33342 (apoptosis). Green cells negative for propidium iodide and having a pyknotic nucleus were counted as being in apoptosis. The results represent the mean values (with standard deviations) of three experiments.
• the green cells (expressing the construction) presenting a pycnotic nucleus are apoptosis, those stained in red by propidium iodide are in necrosis.
• apoptotic cell death is demonstrated by Annexin-V labeling, which specifically recognizes phosphatidylserines extemalized by apoptotic cells (Fig. 5B).
• Annexin-V labeling specifically recognizes phosphatidylserines extemalized by apoptotic cells (Fig. 5B).
• cell death was quantified using the Annexin V-Cyanine 3 assay by 24h flow cytometry after transient cell transfection with the plasmids of interest.
• the GFP-Bax-a5 and GFP-Bax-a6 constructs are more effective at inducing apoptosis than the GFP-KLA construct.
• FIG. 6 shows that the toxic proteins encoded by the inserts do not act via the endogenous Bax and Bak proapoptic proteins, since murine fibroblasts deficient in Bax and Bak (MEF-DKO) are not resistant to stress. induced by their expression (see also Fig.3).
• the graph (FIG. 6A) corresponds to kinetics of cell death (quantified by flow cytometry using the Annexin V-Cy3 assay) of wild-type MEF versus MEF-DKO murine fibroblasts. It is observed that the fibroblasts deficient for Bax and Bak are as sensitive to the induction of apoptosis by the expression of the GFP-Bax-alpha5 protein as to wild murine fibroblasts.
• FIG. 7 shows that cells (HT-1080) expressing the polypeptides comprising the Bax cc5 and / or cc6 helices undergo a significant fall in their mitochondrial transmembrane potential ( ⁇ ), unlike the cells expressing GFP alone, suggesting the implication of a mitochondria-dependent pathway in the induction of apoptosis.
• FIG. 7A The histograms (FIG. 7B) show the average values of three flow cell cytometry quantification experiments (expressing GFP) with Mitotracker incorporation defects (ie a weak ⁇ ).
• the results presented show that the regions of the pro-apoptic protein Bax corresponding to amino acids 106-104 and 130-150 are capable of targeting mitochondrial membranes and of inducing apoptotic cell suicide. These regions are alpha helix structured within the complete Bax protein (Suzuki et al., 2000, Cell, 103, 645-654) and have an amphipatic character favorable to the destabilization of biological membranes.
• Poropeptide of sequence SEQ ID No. 1 It is an amphipatic peptide (see FIG. 8), longer than the a5 helix deduced from the 3D structure of the Bax protein in solution (Suzuki et al., 2000). ). It contains a serine residue incorporated in position 126 in place of the natural cysteine residue in order to limit the formation of disulfide bridges. ⁇ Characterization of its mode of action by incubation with isolated mitochondria.
• the peptide is incubated with the mitochondria for 5, 15, 30 or 60 min, and the mitochondrial release of cytochrome c in the supernatant (SN) is measured by Western Blot.
• the mitochondrial fractions (Mito) are calibrated using an antibody directed against mitochondrial proteins HSP70 or ATPase (subunit 6).
• the poropeptide-Bax (106-134)
• the cytochrome c is released from the inter-membrane space of the mitochondria at 10 ⁇ and in 5 min of incubation. No release is observed when an artificial antimicrobial peptide (KLAK) of sequence SEQ ID No. 2 KLAKLAKKLAKLAK is used.
• poropeptide-Bax [106-134], of sequence SEQ ID No. 1 has a strong ability to induce the permeabilization of mitochondrial membranes.
• Poropeptide-Bax [106-134] was microinjected in vivo.
• the zebrafîsh (Danio rerio) model was used because (i) the apoptosis machinery is conserved between human and fish cells; (ii) the egg corresponds to an integrated cellular system compared to isolated mitochondria; (ii) zebrafish eggs are easily injectable routinely.
• A Zebrafis eggs were microinjected at stage 1-2 cell (s) with increasing concentrations of poropeptide-Bax [106-134], Bax-BH3 peptide or with ultracure water Cmock ' ).
• a concentration of 100 ⁇ l inside the microinjection capillary corresponds to approximately 6 ⁇ 10 12 peptide molecules per egg.
• the histograms represent the percentage of mortality 24 h after fertilization and the percentage of surviving embryos with severe malformations. The data presented are representative of 2 independent experiments giving the same results.
• FIG. 1 A Photomicrographs showing SK-MEL-28 human melanoma cells injected with fluorescent tracer Dextran-FITC alone Cmock ') or mixed with Poropeptide-Bax [106-134]. The cells were photographed 12 h after microinjection. The micro-injected cells with the poropeptide-Bax [106-134] exhibit morphological manifestations characteristic of apoptosis (cytoplasmic budding, retraction and cell rounding). Magnification x 800.
• FIG. 1 A Photomicrographs showing SK-MEL-28 human melanoma cells injected with fluorescent tracer Dextran-FITC alone Cmock ') or mixed with Poropeptide-Bax [106-134]. The cells were photographed 12 h after microinjection. The micro-injected cells with the poropeptide-Bax [106-134] exhibit morphological manifestations characteristic of apoptosis (cytoplasmic budding, retraction and cell rounding). Magnification x 800.
• micro-injection of poropeptide-Bax [106-134] induces a substantial cell death, whereas micro-injected cells by Bax-BH3 or KLAK have no significant effect.
• Micro-injected cells control peptide (R8) SEQ ID NO: 3 sequence RRRRRRRR (a polyarginine intracellular transduction pattern) have viability rates comparable to the control.
• poropeptide-Bax [106-134] exerts dose-dependent cytotoxic effects after introduction into zebrafis eggs or tumor cells cultured in vitro, and is more effective in inducing cell death than in a pro-apoptotic peptide derived from the same protein (Bax-BH3) or other peptides known to be active at the membrane level (KLAK and R8).
• a pro-apoptotic peptide derived from the same protein Bax-BH3
• KLAK and R8 peptides known to be active at the membrane level
• the R8-Scr-FITC peptide is a "scramble" sequence of the sequence present in the R8-Bax [106-134] -FITC peptide, that is to say a control sequence in which the amino acids (their order in the sequence) were mixed.
• D Death rate (apoptosis / necrosis) of HeLa cells treated with poropeptide R8-Bax [106-134].
• the type of cell death was determined 24 h after transfection by observation of cell permeability to propidium iodide (necrosis) or nuclear morphology after staining of DNA with Hoechst 33342 (apoptosis). Green cells (expressing GFP) were visualized by epifluorescence microscopy. About 300 cells were counted by experiment. The results are an average of three independent experiments ( ⁇ standard deviation is also shown).
• the two peptides are rapidly internalized (in less than one hour) by HeLa cells in vitro culture (FIG. 12A).
• the poropeptide R8-Bax [106-134] -FITC inhibits HeLa cell viability in a dose-dependent and incubation-time manner (Fig. 12B).
• a pan-caspase inhibitor zVAD.fmk added in vitro, significantly inhibited R8-Bax [106-134] -FITC-induced cell death (Fig.
• FIG. 12C Apoptosis rate of wild-type fibroblastic cells (MEFs) or deficient in death proteins Bax and Bak (MEF DKO) treated with FITC-R8-Bax poropeptide [106- 134] or FITC-R8-control peptide Bax [Scr].
• Apoptosis was quantified using the Annexin V-Cyanine 3 test by flow cytometry 6h or 24h after treatment, in the presence or absence of the caspase inhibitor zVAD.fmk. The results correspond to the percentage of apoptotic cells having internalized the fluorescent peptide in each condition.

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