WO 2012/066376 Al 24 May 20 12 (24.05.2012) W P O P C T

(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2012/066376 Al 24 May 20 12 (24.05.2012) W P O P C T

(51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C07K 14/47 (2006.01) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, (21) International Application Number: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, PCT/IB20 10/003 158 DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, (22) International Filing Date: HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, 18 November 20 10 (18.11.20 10) KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, (25) Filing Language: English OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, SE, SG, (26) Publication Language: English SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (71) Applicants (for all designated States except US): CENTRE NATIONAL DE LA RECHERCHE SCIEN- (84) Designated States (unless otherwise indicated, for every TIFIQUE - CNRS - [FR/FR]; 3, rue Michel Ange, F- kind of regional protection available): ARIPO (BW, GH, 7501 6 Paris (FR). UNIVERSITE DE MONTPELLIER 2 GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, - SCIENCES ET TECHNIQUES [FR/FR]; Place Eugene ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, Bataillon, F-34095 MONTPELLIER CEDEX (FR). TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, (72) Inventors; and MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, (75) Inventors/Applicants (for US only): BARRERE, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, Stephanie [FR/FR]; 5, rue des Mattes, F-34230 Vendemi- ML, MR, NE, SN, TD, TG). an (FR). NARGEOT, Joel [FR/FR]; 9, rue de Nazareth, Bat. Gl, F-34090 Montpellier (FR). LEBLEU, Bernard Declarations under Rule 4.17 : [FR/FR]; 140, rue Pioch de Boutonnet, Bat. Al, F-34090 — of inventorship (Rule 4.17(iv)) Montpellier (FR). BOISGUERIN, Prisca [FR/FR]; 8, rue Published: la Preneuse, F-30240 Le Grau-du-Roi (FR). PIOT, Chris- tophe [FR/FR]; 172 avenue de Peyre Grosse, F-34980 — with international search report (Art. 21(3)) Saint Clement de Riviere (FR). — with sequence listing part of description (Rule 5.2(a)) (74) Agents: POPPING, Barbara et al; Cabinet Plasseraud, 52, rue de la Victoire, F-75440 Paris Cedex 09 (FR).

© (54) Title: INHIBITORS OF APOPTOSIS AND USES THEREOF (57) Abstract: The invention relates to fragments of the Daxx and FADD proteins inhibiting cell apoptosis mediated by the Fas re - ceptor. The invention also relates to conjugates comprising said fragments, and to the medical applications of said fragments and conjugates. INHIBITORS OF APOPTOSIS AND USES THEREOF

FIELD OF THE INVENTION The invention relates to inhibitors of apoptosis and to their uses, in particular medical treatments.

INTRODUCTION Coronary heart disease is the leading cause of death worldwide, and 3.8 million men and 3.4 million women die of the disease each year. As the population grows older and comorbidities (e.g., obesity and metabolic syndrome) become more prevalent, as in recent years, the enormous public health burden caused by ischemic heart disease is likely to increase even further (reviewed in Yellon et al, N Engl J Med 2007; 357: 1121-1 135). Myocardial infarction is the leading cause of death in both Europe and the United states. Myocardial infarction (MI) or acute myocardial infarction (AMI) - commonly known as a heart attack - is the interruption of blood supply to part of the heart, causing cell death. This is most commonly due to occlusion (blockage) of a coronary artery. AMI remains a frequent (more than 1.5 million new cases per year in the United States) and disabling (leading to heart failure) disease. Infarct size is a major determinant of myocardial functional recovery and mortality after AMI. Currently, the most effective way to limit infarct size is to reperfuse the jeopardized myocardium as soon as possible with the use of coronary angioplasty or thrombolysis and to prevent reocclusion of the coronary artery with the use of antiplatelet therapy. Reperfusion, or restoration of blood flow to the ischemic myocardium, is achieved with thrombolytic therapy that dissolves the thrombus or through dilatation of the occluded artery by percutaneous coronary angioplasty. Reperfusion is necessary for the salvage of myocardial cells and cardiac function in general; however, reperfusion initiates a cascade of events that leads to 'reperfusion injury' (RI). This also occurs following recovery from cardioplegic arrest of the heart during bypass surgery. RI is characterized by arrhythmias, endothelial dysfunction leading to the no-reflow phenomenon, myocardial stunning (reversible loss of myocardial contractility) and, ultimately, cell death. Lethal reperfusion injury culminates in apoptotic death of cardiac cells that were viable immediately before myocardial reperfusion. The participation of a highly- regulated form of cell death during myocardial ischemia/reperfusion may lead to novel therapeutic interventions in the reperfusion phase. However, the apoptosis signalling pathways functional during myocardial ischemia/reperfusion have not yet been fully delineated in vivo. Finding new treatments for inhibiting apoptosis, and in particular for treating myocardial infarction and reperfusion injury, thus constitute a real challenge to protect cardiac function and to save lives.

SUMMARY OF THE INVENTION The invention is based on the finding that it is possible to decrease apoptosis of cardiac cells after myocardial infarction by inhibiting the Fas signalling pathway. The Fas Receptor upon binding to the FasL (Fas Ligand) trimerizes and induces apoptosis through a cytoplasmic domain called DD (Death Domain) that interacts with signalling adaptors like FAF-1 (Fas-Associated Factor- 1), FADD (Fas- Associated Death Domain), Daxx (Death-Domain Associated protein), FAP-1, FLASH (FLICE-associated huge) and RIP (Receptor-Interacting Protein). Daxx and FADD bind independently to Fas and activate distinct apoptotic pathways. Daxx can enhance Fas-mediated apoptosis by activating the INK kinase cascade, culminating in the phosphorylation and activation of transcription factors such as c- Jun. In contrast, FADD triggers, through a cascade of caspases signalling, the release of mitochondrial pro-apoptotic factors like CytoC (Cytochrome-C) and SMAC (Second Mitochondria-derived Activator of Caspases) also called Diablo. The inventors have shown that the inhibition of the interaction of Fas Receptor with Daxx and FADD leads to a strong decrease of the apoptosis of cardiac cells after myocardial infarction. The invention thus relates to peptides consisting of : - a fragment of at most 130 amino acids of an amino acid sequence having at least 80% identity with SEQ ID NO:l, wherein said fragment comprises the amino acid sequence as shown in SEQ ID NO:6, or - a fragment at most 130 amino acids of an amino acid sequence having at least 80% identity with SEQ ID NO:8, wherein said fragment comprises the amino acid sequence as shown in SEQ ID NO: 12, or derivatives thereof, wherein said peptides or derivatives thereof are capable of inhibiting cell apoptosis. The invention also relates to conjugates comprising a peptide or a derivative thereof according to the invention linked to a Cell Penetrating Peptide. The invention also relates to the peptides or derivatives thereof according to the invention, or to the conjugates according to the invention, for use in a method for treatment of the human or animal body. The invention still relates to the peptides or derivatives thereof according to the invention, or to the conjugates according to the invention, for use in a method for inhibiting cell apoptosis in the human or animal body. The invention also relates to the peptides or derivatives thereof according to the invention, or to the conjugates according to the invention, for use in a method for treatment of acute myocardial infarction (AMI), cerebral infarction, organ transplantations, cardiac interventions (extra-corporally circulation and temporary vessel occlusion), or acute circulation perturbations (state of shock), in the human or animal body. The invention further relates to the peptides or derivatives thereof according to the invention, or to the conjugates according to the invention, for use in a method for treatment of ischemia, in particular cardiac ischemia, ischemic colitis, mesenteric ischemia, brain ischemia, limb ischemia or skin ischemia, in the human or animal body. The invention also relates to the peptides or derivatives thereof according to the invention, or to the conjugates according to the invention, for use in a method for treatment of reperfusion injury in the human or animal body.

DEFINITIONS As used herein, the percentage of sequence identity refers to comparisons among amino acid sequences, and is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the amino acid sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical amino acid residue occurs in both sequences or an amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. ScL USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al, eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)). Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al, 1990, J. Mol. Biol. 215: 403-410 and Altschul et al, 1977, Nucleic Acids Res. 3389-3402, respectively. In the context of the invention, the term "treating" or "treatment", means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies.

As used herein, "subject" refers to a human or animal that may benefit from the administration of a fragment according to the invention. By a "therapeutically effective amount" according to the invention, it is meant a sufficient amount to treat the disease, at a reasonable benefit/risk ratio applicable to any medical treatment.

DETAILED DESCRIPTION OF THE INVENTION Peptides inhibiting cell apoptosis The invention relates to peptides consisting of : - a fragment of at most 130 amino acids of an amino acid sequence having at least 80% identity with SEQ ID NO:l, wherein said fragment comprises the amino acid sequence as shown in SEQ ID NO:6, or - a fragment at most 130 amino acids of an amino acid sequence having at least 80% identity with SEQ ID NO:8, wherein said fragment comprises the amino acid sequence as shown in SEQ ID NO: 12, or derivatives thereof, wherein said peptides or derivatives thereof are capable of inhibiting cell apoptosis.

In a particular embodiment, the invention relates to peptides consisting of a fragment of at most 130 amino acids of an amino acid sequence having at least 80% identity with SEQ ID NO:l, wherein said fragment comprises the amino acid sequence as shown in SEQ ID NO:7, or a derivatives thereof, wherein said peptides or derivatives thereof are capable of inhibiting cell apoptosis.

In another particular embodiment, the invention relates to peptides consisting of a fragment of at most 130 amino acids of an amino acid sequence having at least 80% identity with SEQ ID NO:l, wherein said fragment comprises the amino acid sequence as shown in SEQ ID NO:5, or a derivatives thereof, wherein said peptides or derivatives thereof are capable of inhibiting cell apoptosis.

In still another particular embodiment, the invention relates to peptides consisting of a fragment at most 130 amino acids of an amino acid sequence having at least 80% identity with SEQ ID NO: 8, wherein said fragment comprises the amino acid sequence as shown in SEQ ID NO:9, or a derivatives thereof, wherein said peptides or derivatives thereof are capable of inhibiting cell apoptosis.

Indeed, the inventors have shown that peptides comprising the particular amino acid sequences SEQ ID NO:5 ("DaxxP"), SEQ ID NO:6 ("DaxxP15") or SEQ ID NO:7 ("DaxxP14"), which are fragments of the Daxx protein (SEQ ID NO:l); and peptides comprising the particular amino acid sequence SEQ ID NO: 12 ("FADDp") or SEQ ID NO:9 ("FADDp 15"), which are fragments of the FADD protein (SEQ ID NO:8), interact with the Fas receptor and thereby are capable of decreasing cell apoptosis.

The peptides according to the invention are fragments of an amino acid sequence having at least 80% identity with SEQ ID NO:l or SEQ ID NO:8. In particular embodiments, the peptides according to the invention are fragments of an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:l or SEQ ID NO:8.

The peptides or derivatives thereof according to the invention are able to inhibit the cell apoptosis, in particular cardiomyocyte apoptosis. This ability of the peptides or derivatives thereof according to the invention to inhibit cell apoptosis can be measured by any suitable method known by the skilled person, typically with a cell apoptosis detection kit. An example of a kit suitable for measuring cell apoptosis is the Cell Death Detection ELISAPLUS® kit (Cat. No. 11 774 425 001, Roche Applied Science).

The peptides according to the invention consist of fragments of at most 130 amino acids. In particular embodiments, the peptides according to the invention consist of fragments of at most 120, 110, 100, 90, 80, 70, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19,

18, 17, 16, 15, 14, 13, 12, 11, 10, 9 amino acids. When the peptides according to the invention comprises the amino acid sequence of SEQ ID NO:6, said peptides comprise at least 14 amino acids. When the peptides according to the invention comprises the amino acid sequence of

SEQ ID NO:7, said peptides comprise at least 15 amino acids. When the peptides according to the invention comprises the amino acid sequence of SEQ ID NO:5, said peptides comprise at least 16 amino acids. When the peptides according to the invention comprises the amino acid sequence of SEQ ID NO: 12, said peptides comprise at least 9 amino acids.

In a particular embodiment, the peptide according to the invention consists of SEQ ID NO:6. In a particular embodiment, the peptide according to the invention consists of SEQ ID NO:7. In a particular embodiment, the peptide according to the invention consists of SEQ ID NO:5. In a particular embodiment, the peptide according to the invention consists of SEQ ID NO:12. In a particular embodiment, the peptide according to the invention consists of SEQ ID NO:9.

Derivatives of thepeptides according to the invention The invention also relates to the derivatives of the peptides according to the invention, retaining the activity of inhibiting cell apoptosis. The derivatives typically consist of peptides according to the invention which are chemically or biologically modified. Examples of derivatives are peptides according to the invention wherein: - at least one amino acid of the peptide has been substituted, inserted or deleted, and/or - at least one amino acid of the peptide is chemically altered or derivatized. Such "chemically altered or derivatized" amino acids include, for example, naturally occurring amino acid derivatives, for example 4-hydroxyproline for proline, 5- hydroxylysine for lysine, homoserine for serine, ornithine for lysine, and the like. Other "chemically altered or derivatized" amino acids include, e.g., a label, such as fluorescein, tetramethylrhodamine or cyanine dye Cy5.5; or one or more post- translational modifications such as acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation, sulfatation, glycosylation or lipidation. Indeed, certain chemical modifications, in particular N-terminal glycosylation, have been shown to increase the stability of peptides in human serum {Powell et al, Pharma Res 1993:10:1268-1273). "Chemically altered or derivatized" amino acids also include those with increased membrane permeability obtained by N- myristoylation (Brand, et al, Am J Physiol Cell Physiol 1996; 270:C1362-C1369). It is understood that a derivative of a peptide according to the invention may contain more than one of the "chemically altered or derivatized" amino acids. Other derivatives of the peptides according to the invention are peptidomimetics of said peptides. Peptidomimetics refer to a synthetic chemical compound, which has substantially the same structural and/or functional characteristics of the peptides according to the invention. The mimetic can be entirely composed of synthetic, non- natural amino acid analogs, or can be a chimeric molecule including one or more natural amino acids and one or more non-natural amino acid analogs. The mimetic can also incorporate any number of natural amino acid conservative substitutions that do not destroy the mimetic 's activity. Routine testing can be used to determine whether a mimetic has the requisite activity, using Test A according to the invention. The phrase "substantially the same", when used in reference to a mimetic or peptidomimetic, means that the mimetic or the peptidomimetic has one or more activities or functions of the referenced molecule, e.g., inhibition of cell apoptosis. The techniques for developing peptidomimetics are conventional. For example, peptide bonds can be replaced by non-peptide bonds or non-natural amino acids that allow the peptidomimetic to adopt a similar structure, and therefore biological activity, to the original peptide. Further modifications can also be made by replacing chemical groups of the amino acids with other chemical groups of similar structure. The development of peptidomimetics can be aided by determining the tertiary structure of the original fragment/peptide, either free or bound to the intracellular region of the Fas receptor, by NMR spectroscopy, crystallography and/or computer- aided molecular modeling. Once a potential peptidomimetic compound is identified, it may be synthesized and its capability of inhibiting cell apoptosis can be assayed. Peptidomimetics can contain any combination of non-natural structural components, which are typically from three structural groups: residue linkage groups other than the natural amine bond ("peptide bond") linkages; non-natural residues in place of naturally occurring amino acid residues; residues which induce secondary structural mimicry (e.g. beta turn, gamma turn, beta sheet, alpha helix conformation); or other changes which confer resistance to proteolysis. For example, lysine mimetics can be generated by reacting lysinyl with succinic or other carboxylic acid anhydrides. Lysine and other alpha-amino-containing residue mimetics can also be generated by reaction with imidoesters, such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transamidase- catalyzed reactions with glyoxylate. One or more residues can also be replaced by an amino acid (or peptidomimetic residue) of the opposite chirality. Thus, any amino acid naturally occurring in the L- configuration (which can also be referred to as R or S, depending upon the structure of the chemical entity) can be replaced with the same amino acid or a mimetic, but of the opposite chirality, referred to as the D-amino acid, but which can additionally be referred to as the R- or S-form. As will be appreciated by one skilled in the art, the peptidomimetics of the present invention can also include one or more of the modifications described herein for the "chemically altered or derivatized" amino acids, e.g., a label, or one or more post- translational modifications. The peptides, derivatives and peptidomimetics can be produced and isolated using any method known in the art. Peptides can be synthesized, whole or in part, using usual chemical methods. Techniques for generating peptide and peptidomimetic libraries are well known, and include, for example, multipin, tea bag, split-couple-mix techniques and SPOT synthesis.

Conjugates The invention also relates to conjugates comprising a peptide or derivative thereof according to the invention, linked to a Cell Penetrating Peptide or CPP. Indeed, to facilitate the uptake of the peptides or derivatives thereof according to the invention across cell membranes, such as the plasma membrane and/or the nuclear membrane of a cell, the inventors have shown that it is very useful to conjugate those peptides or derivatives thereof with a "cellpenetrating peptides" (CPP). CPP are well known peptides which can be conjugated to cargos to facilitate transport of the cargos across the membranes. CPP are for instance well described by Lebleu B. et ah, Advanced Drug Delivery Reviews 60 (2008) 517-529 and by Said Hassane F. et a , Cell. Mol. Life Sci. (2010) 67:715-726. Any CPP can be used to improve the cytoplasmic delivery of fragments or derivatives thereof according to the invention. Examples of CPP which can be conjugated with the fragments or derivatives thereof according to the invention are the following: Name Sequence Tat GRKKRPvQRPvRPPQ

RXPv RXRRXRRXRRXR Bpep RXRRBRRXRRBRXB Pip2b RXRRXRRXPJHILFQNrRMKWHK wherein:

- X = aminohexyl, β-alanyl, p-aminobenzoyl, isonipecotyl, or 4-aminobutyryl - B = betaAlanine - small letter = D-amino acid (D-amino acids may be replaced by L-amino acids). A CPP typically has two or more cationic amino acids with hydrophobic amino acids or spacer groups separating some of the cationic amino acids. For example, the cationic amino acid is Arginine (R). Still typically, a CPP generally has at least 3 or 4 Arginine residues. In some embodiments the CPP contains 5, 6 or more Arginine residues. It is also known that the presence of aminohexyl and betaAlanine in the CPP sequence is advantageous in that it helps minimize the immunogenicity of the peptide. The CPP is typically linked to the N-terminal or C-terminal end of the peptide or derivative thereof according to the invention, preferably to the C-terminal. Chemical linkage may be performed via a disulphide bond, thioether or thiol-maleimide linkage. In a particular embodiment, the peptide or derivative thereof according to the invention is linked to the CPP through a linker. Any type of linker can be used by the skilled person, provided that said linker allows chemical linkage of the peptide or derivative thereof to the CPP. A range of linkers are possible, including amino acid sequences having a C-terminal Cysteine residue that permits formation of a disulphide, thioether or thiol-maleimide linkage. Other ways of linking the peptide or derivative thereof according to the invention to the CPP include use of a C-terminal aldehyde to form an oxime. Still another type of linkers use click chemistry. Examples of linkers are amino acid or amino acid sequences chosen from the group comprising: C, BC, XC, GC, BBCC, BXCC, XBC, X, XX, B, BB, BX or XB, wherein: - X = aminohexyl, β-alanyl, p-aminobenzoyl, isonipecotyl, or 4-aminobutyryl - B = betaAlanine.

Applications The invention also relates to the peptides or derivatives thereof according to the invention, or to the conjugates according to the invention, for use in a method for treatment of the human or animal body.

More particularly, the invention concerns the peptides, derivatives thereof and conjugates according to the invention, for use in a method for inhibiting cell apoptosis in the human or animal body. The invention also relates to methods for inhibiting cell apoptosis in a subject in need thereof, said methods comprising the step of administering to said subject an effective amount of a peptide, a derivative thereof and/or a conjugate according to the invention. Said "effective amount" is an amount sufficient for inhibiting apoptosis in cells of said subject.

The invention also relates to the peptides or derivatives thereof according to the invention, or to the conjugates according to the invention, for use in a method for treatment of acute myocardial infarction (AMI), cerebral infarction, organ transplantations, cardiac interventions (extra-corporally circulation and temporary vessel occlusion), or acute circulation perturbations (state of shock), in the human or animal body. The invention also relates to methods for treatment of acute myocardial infarction (AMI), cerebral infarction, organ transplantations, cardiac interventions (extra- corporally circulation and temporary vessel occlusion), or acute circulation perturbations (state of shock), in a subject in need thereof, said methods comprising the step of administering, to said subject, a therapeutically effective amount of a peptide, a derivative thereof and/or a conjugate according to the invention.

The invention also relates to the peptides or derivatives thereof according to the invention, or to the conjugates according to the invention, for use in a method for treatment of ischemia, in particular cardiac ischemia, ischemic colitis, mesenteric ischemia, brain ischemia, limb ischemia or skin ischemia, in the human or animal body. The invention also relates to methods for treatment of ischemia, in particular cardiac ischemia, ischemic colitis, mesenteric ischemia, brain ischemia, limb ischemia or skin ischemia, in a subject in need thereof, said methods comprising the step of administering, to said subject, a therapeutically effective amount of a peptide, a derivative thereof and/or a conjugate according to the invention.

The invention also relates to the peptides or derivatives thereof according to the invention, or to the conjugates according to the invention, for use in a method for treatment of reperfusion injury, in the human or animal body. The invention also relates to methods for treatment of reperfusion injury, in a subject in need thereof, said methods comprising the step of administering, to said subject, a therapeutically effective amount of a peptide, a derivative thereof and/or a conjugate according to the invention.

In the methods for treatment according to the invention, the peptides, the derivatives thereof or the conjugates according to the invention can be combined with other agents employed in the treatment of apoptosis, ischemia, and/or reperfusion injury. In particular, combined treatments with agents targeting the intrinsic pathway of apoptosis, i.e. the mitochondrial pathway, are of great interest. In one embodiment, the methods for treatment according to the invention also comprises the step of administering cyclosporine A and/or the BH4 peptide to said human or animal body. Indeed, cyclosporine A was shown to inhibit the mitochondrial PTP opening, decreases infarct size both in patients and in animal models of AMI (Gomez et al, Cardiovasc Res. 2009 ;83(2):226-33; Piot et al, N Engl J Med. 2008;359(5):473-81; Mewton N et al, J Am Coll Cardiol. 2010 Mar 23;55(12): 1200-5). BH4 derived from the antiapoptotic Bcl-xl protein has been reported to be efficacious in decreasing apoptosis during ischemia-reperfusion when administrated as a conjugate of Tat protein at the time of reperfusion (Ono et al, Eur J Cardiothorac Surg. 2005;27(1):1 17-21; Donnini S et al, Cell Cycle 2009; 8(8): 1271-8).

In the methods for treatment according to the invention, all the compounds (peptides, derivatives, conjugates, combined products) may be formulated for administration by a number of routes, including but not limited to, intravenous, parenteral, intra-arterial, intramuscular, oral and nasal. The products may be formulated in fluid or solid form. Fluid formulations may be formulated for administration by injection to a selected region of the human or animal body. All the compounds may be administered before and during ischemia, before, at the time and after reperfusion.

Further aspects and advantages of this invention will be disclosed in the following figures and examples, which should be regarded as illustrative and not limiting the scope of this application.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Determination of the Daxx epitope by SPOT synthesis. (A) The amino

acid sequence of Daxx was dissected in overlapping peptide arrays (pepscan; 15 mer peptides with a shift of 3 amino acids) and analysed in enzyme-linked blot. In the black square is represented the epitope sequences given below with the spot numbers and the signal intensities (BLU). Incubation conditions: His-tagged intracellular region of Fas receptor [10 µg/ml]; antibodies: anti-His-(mouse) (Sigma H1029; 1:6,000) / anti-mouse-HRP (Calbiochem 401207; 1:2,000), exposure time: 1 min. (B) Alignment of the found peptide with the Daxx-DN (highlighted) sequence used in [Roubille et al, Circulation, 2007;1 16:2709-2717]. Our DaxxP correspond to the N- terminal region of the Daxx-DN sequence. Figure 2 : Analysing of the Daxx epitope length by SPOT synthesis. (A) The length of Daxx-2 11 and Daxx-2009 peptide sequences were analysed shortening the given sequences by the N-terminus, the C-terminus and in both directions Incubation conditions: His-tagged intracellular region of Fas receptor [10 µg/ml]; antibodies: anti-His-(mouse) (Sigma H1029; 1:6,000) / anti-mouse-HRP (Calbiochem 401207; 1:2,000), exposure time: 1 min. (B) Out of the Daxx-21 1 and Daxx-209 sequence, the in red mention portion revealed the highest signal intensities (BLU) corresponding to spot number 6 and 27, respectively. Figure 3 : Anti-apoptotic effect of DaxxP in vitro. A. Workflow for the detection of apoptosis using the Cell Death detection ELISA PLUS kit {Roche Diagnostics). Cardiomyocytes : 40,000 cells/wells were seeded in a gelatin-coated 24-well plate and grown overnight. The next day, the cells were incubated with 10 µΜ STS or 10 µΜ STS + 1 µΜ peptide (in OptiMEM) for 6 h. Thereafter, solutions were removed, replaced by complete medium and further incubated for 48 h. After the reperfusion phase, the cells were lysed and DNA fragmentation was detected according to manufacturer's instructions. B - Quantification of DNA fragmentation in cardiomyocytes: Data were normalized to 100% STS. Data shown are the means ± SEM of duplicates of four independent experiments. Figure 4 : Anti-apoptotic effect of DaxxP in other cell types. Quantification of DNA fragmentation in murine cardiomyocytes, NG118-15 (as model for neuronal cells) and C2C12 (as model for muscle cells). Cardiomyocytes : 40,000 cells/wells were seeded in a gelatin-coated 24-well plate and grown overnight. C2C12: 20,000 cells/wells were seeded in a 24-well plate and grown overnight. The next day, the cells were incubated with 1 µΜ STS or 1 µΜ STS + 1 µΜ peptide (in OptiMEM) for 1.5 hour. NG1 18-15 : 15,000 cells/well were seeded in a polyO-coated 24-well plate and grow over night. The next day, the cells were incubated with 500 nM STS + 1 µΜ peptide (in OptiMEM) for 1 h. Data were normalized to 100% STS. Data shown are the means ± SEM of duplicates of four independent experiments. At the moment, we have measured Tat-DaxxP13 only in cardiomyocytes. Figure 5: Determination of FADDplS and its anti-apoptotic effect in cardiomyocytes. (A) The protein sequences of FADD were dissected in overlapping peptide arrays (pepscan; 15 mer peptides with a shift of 3 amino acids) and analysed in enzyme-linked blot. In the black square is represented the epitope sequences given below with the spot numbers and the signal intensities (BLU). Incubation conditions: His-tagged intracellular region of Fas receptor [10 µg/ml]; antibodies: anti-His- (mouse) (Sigma H1029; 1:6,000) / anti-mouse-HRP (Calbiochem 401207; 1:2,000), exposure time: 1 min. (B) Out of the FADD-1 1 sequence, the in red mention portion revealed the highest signal intensities (BLU) corresponding to spot number 24 and to the sequence of FADDp. (C) Quantification of DNA fragmentation reduction by Tat- FADDp in cardiomyocytes: Data were normalized to 100% STS. Data shown are the means ± SEM of duplicates of four independent experiments. (For cell culture condition, see Figure legend 3). Figure 6: Experimental protocol. C57B16 mice underwent a surgical protocol of myocardial ischemia-reperfusion (IR). The black box represents the period of ischemia. Infarct size or cell death measurements were performed at the end of

surgery for each protocol (indicated by †). . 40 minutes of Ischemia, 60 minutes

of Reperfusion. IR24 : 40 minutes Ischemia and 24 hours Reperfusion. Figure 7: Cardioprotective effects of Tat-DaxxP (lmg/kg - IV) in mice subjected

to IR.60'. Infarct size (in % of area at risk) and internucleosomal DNA fragmentation determined by ELISA were quantified in mice subjected to IR o' and treated by control saline, Tat or Tat-DaxxP (IV injection 5 minutes before reperfusion). Means ±

SEM were plotted for (A): Area at risk/LV mass (left ventricle), (B): Infarct size (in % of area at risk) and (C): (I/NI ratio) corresponding to the ratio of soluble nucleosome in the ischemic versus the non-ischemic portion of LV tissues. Statistical analysis was done using One-way ANOVA with Neuman-Keuls post test for multiple comparisons (GraphPad Prism software). P<0.05, P<0.01 and P<0.001 versus Tat-DaxxP were noted *, **, *** respectively. P=ns (not significant) for P>0.05. Figure 8: Cardioprotective effects of Tat-DaxxP (lmg/kg-IV) in mice subjected

to IR 24h- Infarct size (in % of area at risk) and internucleosomal DNA fragmentation

determined by ELISA were quantified in mice subjected to IR 24H and treated by control saline, Tat or Tat-DaxxP (r injection 5 minutes before reperfusion). Means ±

SEM were plotted for (A): Area at risk/LV mass (left ventricle), (B): Infarct size (in % of area at risk) and (C): (I/NI ratio) corresponding to the ratio of soluble nucleosome in the ischemic versus the non-ischemic portion of LV tissues. Note that results did not reach significance due to a lack of power (preliminary results). Statistical analysis was done using One-way ANOVA with Neuman-Keuls post test for multiple comparisons (GraphPad Prism software). P<0.05, P<0.01 and P<0.001 versus Tat-DaxxP were noted *, **, *** respectively. P=ns (not significant) for P>0.05.

EXAMPLES Synthesis of membrane-bound inverted peptide arrays The peptides were synthesized on N-modified CAPE-membranes [S. Bhargava, et al, Mol. Recognit. 2002, 15, 145.] and prepared by a MultiPep SPOT-robot (INTAVIS Bioanalytical Instruments AG, Cologne, Germany). Array design was performed with the aid of the in-house software LISA 1.71. The synthesis started with spot definition by a standard protocol [R. Frank, Tetrahedron 1992, 48, 9217.], followed by the coupling of a solution of Fmoc-cysteine-(Trt)-Opfp (0.3M) in N-methylpyrolidone

(NMP) and Fmoc-alanine-Opfp (double coupling, 15 min reaction each). After Fmoc cleavage with piperidine in DMF (20 %), 4-hydroxymethylphenoxyacetic acid (HMPA) dissolved in dimethylformamid (DMF, 0.6M solution) and activated with EEDQ (1.1 equiv) was added, and samples were directly spotted on the membrane

(4x coupling, 15 min reaction each). The membrane was acetylated with acetic anhydride in DMF (2%), washed with DMF (5x3 min), ethanol (2x3 min), and diethyl ether (2x3 min), and finally air dried. Solutions of Fmoc-amino acid-OH (0.4M) activated with Ι , -carbonyldiimidazole (CDI, 3 equiv) in DMF were spotted on the

membrane (4x coupling, 15 min reaction time each). Proline, tyrosine, and glutamine were activated with l,l'-carbonyldi(l,2,4-triazole) (CDT). The Fmoc group was removed from the spots, and the sequences of the peptides were completed by the standard SPOT synthesis protocol [R. Frank, Tetrahedron 1992, 48, 9217] followed by a N-terminal tag with β-alanine. For standard SPOT synthesis Fmoc-aa-Opfp were used with the following side chain protection: E-, D-(OtBu); C,- S-, T-, Y-(tBu); K-, W-(Boc); N-, Q-, H-(Trt); R-(Pbf). For thioether cyclization all peptides were N-acylated with bromoacetic acid 2,4-

dinitrophenyl ester in DMF (1M), double coupling, 15 min reaction time each. The membrane was washed with DMF (3x3 min) and dichlormethane (DCM, 3x3 min) and dried. To enable cyclization the trityl side-chain protecting group of the

cysteine was cleaved with trifluoroacetic acid (TFA, 7%), H20 (2%) in DCM (1x5

min) followed by TFA (7%), TIBS (3 %), H20 (2%) in DCM. The membrane was washed with DCM (3x3 min), DMF (2x3 min), and DMF (2x3 min). The peptides were cyclized overnight by treatment with 25 % aqueous C S2CO3/DMF (1:1). The

membrane was washed with DMF (2x3 min), H20 (2x3 min), ethanol (2x3 min), and diethyl ether (2x3 min) and air-dried. Hydrolysis and side-chain deprotection were achieved through one treatment with

TFA (60 %), TIBS (3 %), and H20 (2%) in DCM for 2.5 h without shaking, followed by washing steps (DCM 3x3 min, DMF 3x3 min, ethanol 3x3 min, diethyl ether 2x3

min), followed by TFA (90 %), TIBS (3 %), and H20 (2%) in DCM for 30 min without shaking. The membrane was washed with DCM (3x3 min), DMF (3x3 min),

ethanol (2x3 min), phosphate buffer (pH 7.4, 0.1M, 2x3 min), H20 (2x3 min), ethanol (2x3 min), and diethyl ether (2x3 min) and air dried. Design and delivery of fragments according to the invention The primary sequence of the Daxx protein (SEQ ID NO: 1) is dissected in overlapping

15 mer peptides (3 amino acids shift) and all peptides (243 peptides) are synthesized on cellulose membrane by SPOT synthesis as described above. The peptide library is incubated with the His-tagged intra-cellular region of the Fas receptor (Sigma). The interaction between Fas and the peptides is determined with a sandwich of anti- His(mouse)/anti-mouse-HRP and the signals are revealed using a Lumilmager (Roche) as shown in Figure 1A. Spot no. 2 11 (SEQ ID NO:2) has the higher signal intensity and the spots no. 209-210 (SEQ ID NO:3 and SEQ ID NO:4) include the minimal epitope sequence (KSRKEKKQT). Nevertheless, the sequence length analysis of the 15mer sequences

KSRKEKKQTGSGPLG (spot 2 11, SEQ ID NO:2) and SGPPCKKSRKEKKQT (spot 209, SEQ ID NO:3) revealed that it is not possible to shorter the sequences arbitrarily

to the minimal epitope found in Figure 1. Using the SPOT synthesis as mentioned above, we have analysed the influence of the peptide length by shortening the given sequences by the N-terminus, the C-terminus and in both directions (Figure 2A). The peptides sequences with the highest signal intensities are shown in Figure 2B. We have then done a merged combination of the highest signal intensity of the Daxx- 2 11 and the Daxx-209 which correspond to a 16mer peptide (KKSRKEKKQTGSGPLG), called the Daxx peptide or DaxxP (SEQ ID NO:5). DaxxP has an important in vitro and in vivo anti-apoptotic activity. Taken together, these results suggest that it is highly credible that the peptide DaxxP could be shortened by one or tow amino acids at the N-terminus. These peptides are called DaxxP14 (SRKEKKQTGSGPLG; SED ID NO:6) and DaxxP15 (KSRKEKKQTGSGPLG; SED ID NO:7) and should keep an important anti- apoptotic activity. On the other side, it is also possible to elongate the peptide by the C-terminus due to the fact that the dominant negative protein (Daxx-DN) [Roubille et al., Circulation, 2007;1 16:2709-2717] correspond to the Daxx-21 1 peptide till the end of the Daxx protein (Figure IB). Applications in AMI DaxxP peptides conjugated to CPPs have been assayed for their anti-apoptotic activity in primary cardiomyocytes and for their ability to reduce infarct size in a mouse surgical I/R model after systemic administration. In vitro evaluation In a first step, we have quantified the cellular uptake of the peptides in Table 1 in primary cell culture of mouse cardiomyocytes using flow cytometry measurements (FACS - with CF-labeled peptides) and controlled the absence of any cytotoxicity.

amino-hexanoic acid; B = -alanine, all peptides are C-terminally amidated. For FACS measurements all peptides are labeled with carboxyfluorescein). nc = negative control corresponding to spot number 4 1 of Figure 1. scr = scrambled version of DaxxP; mDaxxP is the mouse homolog of human DaxxP. Additionally, we have cross-validated the potential interaction of the fragments with the intracellular region of the Fas receptor by measuring the binding affinities (Kd) using the surface plasmon resonance (SPR) technique (Biacore Life Science, Sweden). Binding affinities of the fragment alone and in conjugation with the CPPs are summarized in Table 2.

Table 2 : Measurement of the binding affinities (Kd, in µΜ ) of the used constructs. All experiments are performed on a Biacore instrument. For each condition, the mean value of three independent experiments and the corresponding standard deviation are plotted.

Thereafter, we have evaluated the capacity of these peptides to inhibit apoptosis induced by staurosporine. The apoptosis was determined using the Cell Death detection ELISA PLUS kit (Roche) measuring the DNA fragmentation. In most publications, the anti-apoptotic peptides are administrated 3-4 h prior apoptosis induction, which is far away from a clinical application. For that reason, we used a protocol administrating the peptides together with the staurosporine. Figure 3 clearly show a reduction of DNA fragmentation of 44% using Tat-DaxxP and of 55% using Pip2b-DaxxP, respectively. This is not shown with the CPP alone and with the negative control Tat-ncDaxxP. The enrichment factor is calculated as suggested by the suppliers (DNA fragmentation of treated cells/DNA fragmentation of un-treated cells). To better compare the results, the DNA fragmentation (described by the enrichment factor) is related to the staurosporine treated cells (=100%). We have also analyzed the anti-apoptotic effects of the peptides in murine NG1 18-15 (model for neuronal cells) and in murine C2C12 (model for muscle cells) (Figure 4).

We could observe the highest reduction of DNA fragmentation of 66%> using Bpep- DaxxP in NG1 18-15 and of 21% using Tat-DaxxP in C2C12. This clearly reveals the importance of the optimal CPP choice for the appropriated application or biological context. Furthermore, we have determined the binding epitope of FADD protein (SEQ ID NO: 8) in the same way as for the Daxx epitope (details see above). As shown in

Figure 5A, Spot no. 11, corresponding to the peptide FADDpl5 (VGKRKLERVQSGLDL; SEQ ID NO:9), has the higher signal intensity and the

spots no. 10 and 12 (SEQ ID NO: 10 and SEQ ID NO: 11) share a minimal epitope sequence with FADDpl5. Using a peptide library dissecting the length, we could determine the minimal fragment of FADDpl5, which correspond to FADDp (SEQ ID NO: 12; KRKLERVQS), Figure 5B. In cardiomyocytes, the decrease in apoptosis was 29% (Figure 5C) using the conjugates of Tat-FADDp. In vivo evaluation In a second step, we have evaluated the cardioprotective effects of DaxxP in an in vivo model of myocardial ischemia-reperfusion. Acute myocardial ischemia and reperfusion were performed in C57B16 mice subjected to a surgical model of reversible coronary occlusion. Male mice (22-28 g) were anesthetized and ventilated via a tracheal intubation using a Harvard rodent respirator. Body temperature was maintained between 36.8°C and 37.0°C via a thermo-regulated surgical table. The chest was opened by a left lateral thoracotomy and a reversible coronary artery snare occluder was placed around the left coronary artery. Mice have been randomly allocated to 2 different surgical protocols of myocardial ischemia-reperfusion (Figure 6). At the end of reperfusion, the artery was re-occluded and phtalocyanine blue dye was injected into the left ventricle cavity and allowed to perfuse the non-ischemic portions of the myocardium. To determine the effect of DaxxP on myocardial infarct size, peptides were administered intravenously (caudal vein) 5 minutes before reperfusion during the surgical protocol of ischemia-reperfusion. The control groups were treated with physiological serum (vehicle) and Tat or Pip2b peptide (for CPP alone). The dose of 1 g/g was chosen ^molar range). At the end of reperfusion, mice were re-anaesthetized, the coronary ligature definitively tighten, the blue dye injected and the harvested left ventricles were dedicated to infarct size (TTC method, Schwartz ER et al Journal of Thrombosis and Thrombolysis. 2000; 10 (2): 181-187.) or DNA fragmentation measurements (Cell death ELISA kit, Roche) in order to investigate the cardioprotective effects against ischemia-reperfusion injuries. Results are shown in Figure 7. When Tat-DaxxP (lmg/kg) was injected in vivo intravenously, infarct size was decreased by 56,2 % versus control (p<0,05) and 53,4 % versus Tat peptide alone (p<0,01) (area at risk was comparable among groups; p=ns - Figure 7A). This cardioprotection was correlated to a drastic decrease in specific DNA fragmentation, a hallmark of apoptosis, in left ventricles from Tat-DaxxP injected mice versus control or Tat-injected mice (see Figure 7B). The cardioprotective effects of Tat- DaxxP were maintained when the reperfusion duration was prolonged from 1 hour to 24 hours (see Figure 8).

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. CLAIMS

1. A peptide consisting of : - a fragment of at most 130 amino acids of an amino acid sequence having at least 80% identity with SEQ ID NO:l, wherein said fragment comprises the amino acid sequence as shown in SEQ ID NO:6, or - a fragment at most 130 amino acids of an amino acid sequence having at least 80% identity with SEQ ID NO:8, wherein said fragment comprises the amino acid sequence as shown in SEQ ID NO: 12, or a derivative thereof, wherein said peptide or derivative thereof is capable of inhibiting cell apoptosis.

2. The peptide according to claim 1, wherein said peptide consists of a fragment of at most 130 amino acids of an amino acid sequence having at least 80% identity with SEQ ID NO:l, and wherein said fragment comprises the amino acid sequence as shown in SEQ ID NO:7 or SEQ ID NO:5.

3. The peptide according to claim 1, wherein said peptide consists of a fragment of at most 130 amino acids of an amino acid sequence having at least 80% identity with SEQ ID NO: 8, and wherein said fragment comprises the amino acid sequence as shown in SEQ ID NO:9.

4. The peptide according to any one of claims 1-3, wherein said fragment of at most 130 amino acids is a fragment of at most 100 amino acids, particularly of at most 75 amino acids, more particularly of at most 50 amino acids, most particularly of at most 25 amino acids.

5. The peptide according to any one of claims 1-4, wherein said peptide consists of an amino acid sequence as shown in SEQ ID NO:6, SEQ ID NO: 12, SEQ ID NO:7, SEQ ID NO:5 or SEQ ID NO:9. 6. The peptide according to any one of claims 1-5, wherein said derivative is a peptide wherein: - at least one amino acid of the peptide has been substituted, inserted or deleted, and/or - at least one amino acid of the peptide is chemically altered or derivatized.

7. The peptide according to any one of claims 1-5, wherein said derivative is a peptidomimetic of said fragment.

8. A conjugate comprising a peptide or a derivative thereof as defined in any one of claims 1-7 linked to a Cell Penetrating Peptide.

9. The conjugate according to claim 8, wherein said peptide or said derivative thereof is linked to the Cell Penetrating Peptide through a linker.

10. The conjugate according to claim 8 or 9, wherein said Cell Penetrating Peptide is selected from the group comprising Tat, RXR, Bpep and Pip2b.

11. The peptide or derivative thereof according to any one of claim 1-7, or the conjugate according to any one of claims 8-10, for use in a method for treatment of the human or animal body.

12. The peptide or derivative thereof according to any one of claim 1-7, or the conjugate according to any one of claims 8-10, for use in a method for inhibiting cell apoptosis in the human or animal body.

13. The peptide or derivative thereof according to any one of claim 1-7, or the conjugate according to any one of claims 8-10, for use in a method for treatment of acute myocardial infarction (AMI), cerebral infarction, organ transplantations, cardiac interventions (extra-corporally circulation and temporary vessel occlusion), or acute circulation perturbations (state of shock), in the human or animal body. 14. The peptide or derivative thereof according to any one of claim 1-7, or the conjugate according to any one of claims 8-10, for use in a method for treatment of ischemia, in particular cardiac ischemia, ischemic colitis, mesenteric ischemia, brain ischemia, limb ischemia or skin ischemia, in the human or animal body.

15. The peptide or derivative thereof according to any one of claim 1-7, or the conjugate according to any one of claims 8-10, for use in a method for treatment of reperfusion injury in the human or animal body.

16. The peptide, the derivative thereof or the conjugate according to any one of claims 11-15, wherein said method also comprises the step of administering cyclosporine A and/or BH4 to said human or animal body.

International application No PCT/IB201Q/0O3158

A . CLASSIFICATION O F SUBJECT MATTER INV. C07K14/47 ADD.

According to International Patent Classification (IPC) or to both national classification and IPC

B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols C07K

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practical, search terms used)

EPO-Internal , BIOSIS, EMBASE, WPI Data, Sequence Search

C . DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

WO 98/34946 Al (MASSACHUSETTS INST 1 ,2,4, TECHNOLOGY [US] ) 6-16 13 August 1998 (1998-08-13) c l aims 26-28,35,36; f i gures 6A, 8B exampl es 3,7

CHEN JIN-ZHONG ET AL: "DAXX i nteracts 1 ,2,4, wi t h phage Phi C31 integrase and i nhi bi t s 6-16 recombination" , NUCLEIC ACIDS RESEARCH, vol . 34, no. 21, December 2006 (2006-12) , pages 6298-6304, XP002634400, ISSN : 0305-1048 abstract

/ -

Further documents are listed in the continuation of Box C . See patent family annex.

* Special categories of cited documents : "T" later document published after the international filing date or priority date and not in conflict with the application but "A" document defining the general state of the art which is not cited to understand the principle or theory underlying the considered to be of particular relevance invention "E" earlier document but published on or after the international "X" document of particular relevance; the claimed invention filing date cannot be considered novel or cannot be considered to "L" document which may throw doubts on priority claim(s) or involve an inventive step when the document is taken alone which is cited to establish the publication date of another " document of particular relevance; the claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the "O" document referring to a n oral disclosure, use, exhibition or document is combined with one or more other such docu¬ other means ments, such combination being obvious to a person skilled in the art. "P" document published prior to the international filing date but later than the priority date claimed "&" document member of the same patent family

Date of the actual completion of the international search Date of mailing of the international search report

28 Apri l 2011 17/08/2011

Name and mailing address of the ISA/ Authorized officer European Patent Office, P.B. 5818 Patentlaan 2 NL - 2280 HV Rijswijk Tel. (+31-70) 340-2040, Fax: (+31-70) 340-3016 Gri esinger, Iri na International application No PCT/IB201Q/0O3158

C(Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

DATABASE Uni Prot [Onl i ne] 1 ,2,4,6

20 March 2007 (2007-03-20) , "SubName: Ful =Death-associated protei n 6 ; Fl ags : Fragment; " , XP002634401, retri eved from EBI accessi on no. UNI PR0T:A2V6V1 Database accession no. A2V6V1 the whole document

KR 2005 0107007 A (DONGBU HANNONG CHEMICAL 1 ,2,4, CO LTD [KR] ) 11 November 2005 (2005-11-11) 6-16 abstract; claims 3,4

R0UBI LLE FRANCOIS ET AL: "Myocardial 11-16 expression of a dominant-negative form of Daxx decreases infarct si ze and attenuates apoptosi s i n an i n v i vo mouse model of i schemia/reperfusi on i njury" , CI RCULATION, vol . 116, no. 23 , December 2007 (2007-12) , pages 2709-2717, XP002634402 , ISSN : 0009-7322

JUNG ET AL: "Subcel l ular local i zation of 11-16 Daxx determines i t s opposi ng functions i n i schemi c cel l death" , FEBS LETTERS, ELSEVI ER, AMSTERDAM, NL, vol . 581 , no. 5 , 24 February 2007 (2007-02-24) , pages 843-852, XP005916836, ISSN : 0014-5793, D0I : D0I : 1Θ. 1 16/ . FEBSLET . 2ΘΘ7 .Gl . 055 abstract International application No Information on patent family members PCT/IB201Q/0O3158

Patent document Publication Patent family Publication cited in search report date member(s) date

WO 9834946 A l 13-08-1998 NONE

KR 20050107007 A 11-11-2005 NONE International Application No. PCTY Ι Β2Θ10/ G03158

FURTHER INFORMATION CONTINUED FROM PCT/ISA/ 210

Thi s International Searching Authori t y found mul tiple (groups of) i nventions i n thi s international appl i cation , as fol l ows :

1. claims: 2(completely) 1, 4-16(partial ly)

relate t o fragments of at most 130 amino acids of Daxx according t o SEQ I D NO: 1 such as the fragments according t o SEQ I D NOs: 5-7, the corresponding conjugates and medi cal uses .

2 . claims: 3(completely) 1, 4-16(partial ly)

relate t o fragments of at most 130 amino acids of FADD according t o SEQ I D NO: 8 such as the fragments according t o SEQ I D NOs: 9-12 , the correspondi ng conjugates and medi cal uses . International application No. PCT/IB201Q/003158 INTERNATIONAL SEARCH REPORT

Box No. II Observations where certain claims were found unsearchable (Continuation of item 2 of first sheet)

This international search report has not been established in respect of certain claims under Article (2)(a) for the following reasons:

□ Claims Nos.: because they relate to subject matter not required to be searched by this Authority, namely:

□ Claims Nos.: because they relate to parts of the international application that do not comply with the prescribed requirements to such an extent that no meaningful international search can be carried out, specifically:

□ Claims Nos.: because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a).

Box No. Ill Observations where unity of invention is lacking (Continuation of item 3 of first sheet)

This International Searching Authority found multiple inventions in this international application, as follows:

see addi tional sheet

1. 1 As all required additional search fees were timely paid by the applicant, this international search report covers all searchable ' claims.

2 . I I As all searchable claims could be searched without effort justifying an additional fees, this Authority did not invite payment of additional fees.

As only some of the required additional search fees were timely paid by the applicant, this international search report covers ' ' only those claims for which fees were paid, specifically claims Nos. :

I I No required additional search fees were timely paid by the applicant. Consequently, this international search report is restricted to the invention first mentioned in the claims; it is covered by claims Nos.: (compl etely) ; 1, 4-16(partial ly)

Remark on Protest The additional search fees were accompanied by the applicant's protest and, where applicable, the ' ' payment of a protest fee. The additional search fees were accompanied by the applicant's protest but the applicable protest ' ' fee was not paid within the time limit specified in the invitation.

I INo protest accompanied the payment of additional search fees.

Form PCT/ISA/21 0 (continuation of first sheet (2)) (April 2005)