Agonist agents of CD47 inducing programmed cell death and their use in the treatments of diseases associated with defects in programmed cell death

Abstract

The present invention relates to cyclic peptides mimetics of the C-terminal binding domain of TSP-1. The present invention also relates to the use of these cyclic peptides as agonists of CD47 and their ability to trigger programmed cell death (PCD). The present invention further relate to a pharmaceutical composition for use in the treatment of diseases associated with defects in PCD such as cancers and immunological disorders (including chronic inflammation) and comprising at least one cyclic peptide according to the invention.

Claims

1. An isolated cyclic peptide of general formula (Ia): ##STR00049## or a pharmacologically acceptable salt thereof, wherein: Z.sub.1 is nothing or an heterochiral sequence D-Pro-L-Pro or any sequence of two amino acids or analogs of amino acid able to mimic said heterochiral sequence or mimic a beta turn; B.sub.2 represents the peptidic sequence X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X.sub.10 (SEQ ID NO: 2) and comprises between 6 and 10 amino acids, wherein: X.sub.1 refers to nothing or serine; X.sub.2 refers to nothing, arginine, or N-methyl arginine; X.sub.3 refers to phenylalanine; X.sub.4 refers to tyrosine; X.sub.5 refers to valine; X.sub.6 refers to valine; X.sub.7 refers to methionine, lysine, or norleucine; X.sub.8 refers to tryptophan; X.sub.9 refers to nothing or lysine; X.sub.10 refers to nothing or glutamine; Z.sub.2 is nothing or an heterochiral sequence D-Pro-L-Pro or any sequence of two amino acids or analogs of amino acid able to mimic said heterochiral sequence or mimic a beta turn; B.sub.3 is a peptidic sequence of between 6 and 10 amino acids comprising the following sequence: -X.sub.19-X.sub.14-X.sub.15-X.sub.20-X.sub.21-X.sub.16-X.sub.22-X.sub.23-X.sub.17-X.sub.18- (SEQ ID NO: 36) wherein: X.sub.14 is nothing, glycine, alanine, or serine; X.sub.15 is isoleucine or leucine; X.sub.16 is lysine or alanine; X.sub.17 is nothing, asparagine, alanine, glutamine or lysine; X.sub.18 is nothing, serine, or glycine; X.sub.19 is nothing or serine; X.sub.20 is serine, alanine or leucine; X.sub.21 is valine or alanine; X.sub.22 is valine; and X.sub.23 is valine or alanine; wherein said isolated cyclic peptide comprises an even number of amino acids and wherein said isolated cyclic peptide comprises between 8 and 26 amino acids.

2. A pharmaceutical composition comprising the isolated cyclic peptide of claim 1 and a pharmaceutically acceptable carrier.

3. The isolated cyclic peptide according to claim 1, wherein both Z.sub.1 and Z.sub.2 are nothing.

4. The isolated cyclic peptide according to claim 1, wherein Z.sub.1 or Z.sub.2 is D-Pro-L-Pro.

5. A method of treating a disease associated with defects in PCD in a subject in need thereof comprising administering a therapeutically effective amount of the isolated cyclic peptide of claim 1 to the subject.

6. The method of claim 5, wherein the disease is cancer selected from the group consisting of adrenal cortical cancer, anal cancer, bile duct cancer, multiple myeloma, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, Castleman disease, cervical cancer, colorectal cancer, endometrial cancer, esophagus cancer, gallbladder cancer, gastrointestinal carcinoid tumors, Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, liver cancer, lung cancer, mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, melanoma, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, vaginal cancer, vulvar cancer, and uterine cancer.

7. The method of claim 5, wherein the disease is an immunological disorder.

8. The method of claim 5, wherein the disease is chronic inflammation.

9. The isolated cyclic peptide according to the claim 1 wherein said peptide is selected from a group consisting of PKTD1 (SEQ ID NO: 9), PKTD10 (SEQ ID NO: 17), PKTD10-1 (SEQ ID NO: 18), PKTD10-3 (SEQ ID NO: 20), PKTD10-4 (SEQ ID NO: 21), PKTD10-5 (SEQ ID NO: 22), PKTD10-7 (SEQ ID NO: 24), PKTD10-8 (SEQ ID NO: 25), PKTD12 (SEQ ID NO: 29), PKTD18 (SEQ ID NO: 35), PKD8 (SEQ ID NO: 41), PKD9 (SEQ ID NO: 43), PKD10 (SEQ ID NO: 44), PKTD10-RNMe (SEQ ID NO: 54), PKTD10-X-RNMe (SEQ ID NO: 55), PKTD11-RNMe (SEQ ID NO: 60).

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIGS. 1 and 2. Two pathways closely connected leading to different types of PCD showing that PCD is a complex network and highly regulated process.

(2) FIG. 3. A. TSP-1 is a multidomain protein, each domain involved in biological function: the heparin-binding domain (HBD), the von Willebrand C domain (vWCD), three type 1 properdin repeats, three type 2 EGFlike repeats, seven type 3 calcium-binding repeats and the C-terminal CBD. Some of the domains interact with extracellular matrix components or membrane receptors (arrows). Key amino acid sequences responsible for TSP-1 ligation to integrins (RGD) and CD47 (RFYVVMWK) (SEQ ID NO: 3) are indicated. Additional illustrated sequences are set forth as SEQ ID NOS: 65 and 62. 3B. This figure shows the CD47 and 4N1 interaction. The open conformation of the TSP-1 C-terminal domain (MRMS=2 Å) was used for protein-protein docking experiments against a homology model of the extracellular part of the CD47receptor. Two putative TSP-1:CD47 interaction regions [(1) and (2), respectively] were proposed by molecular modeling (see Floquet et al, 2008)..sup.xlii.

(3) FIG. 4. CD47 is used as a target to eliminate cancer cells. Therapeutic targeting of CD47 using monoclonal antibodies (mAb) can induce the elimination of cancer cells through multiple mechanisms. 1) Through phagocytic uptake of tumor cells by macrophages: by inhibiting the CD47-SIRPα interaction with a blocking anti-CD47 mAb, a blocking anti-SIRPα mAb, or a recombinant SIRPα protein (shown as a bivalent Fc-fusion protein). 2) Anti-CD47 antibodies can stimulate an anti-tumor adaptive immune response leading to the phagocytic uptake of tumor cells by DCs and subsequent antigen presentation to CD4 and CD8 T cells. 3) By NK cell-mediated ADCC and CDC induction and tumor cell elimination: an anti-CD47 antibody can eliminate tumor cells through antibody Fc-dependent mechanisms. 4) Function blocking of CD47 may also promote tumor reduction by blocking several of its actions in tumor cells. 5) Finally, CD47 stimulation can induce direct cell death induction. Modified from.sup.xliii.

(4) FIG. 5 and FIG. 6. Binding curve measured by MST. The measurement method is based on the directed movement of molecules along a temperature gradient, an effect termed “thermophoresis”. A local temperature difference ΔT leads to a local change in molecule concentration (depletion or enrichment), quantified by the Soret coefficient S.sub.T: c.sub.hot/c.sub.cold=exp(−S.sub.TΔT). Jurkat or Mec-1 cell membrane preparations are labeled using the Nanotemper NT-647 labeling kit as described elsewhere.xliv The labeled preparation is eluted with PBS and stored at 4° C. A stock solution of each peptide is prepared in DMSO (5 mM) and then diluted with PBS. For the peptides evaluated by MST, we have kept the concentration of the NT.115-labeled membrane constant, while the concentration of the ligand (peptide) was varied. After a short incubation the samples were loaded into MST premium glass capillaries and the MST analysis was performed using the Monolith NT.115-pico. FIG. 5: MST curve observed for PKT16 [(D)Lys-(N-Me)Arg-Phe-Tyr-Val-Val-Nle-Trp-Lys-(D)Lys] (SEQ ID NO: 66) a linear peptide derived from the C-terminal binding domain of TSP-1. The Kd=1600 nM. FIG. 6A to 6H: MST curves observed for PKTD1, PKTD10, PKTD10-1, PKTD10-3, PKTD10-4, PKTD10-5, PKTD10-8 [see structures and sequences in Table I] cyclic peptide analogues of the C-terminal binding domain of TSP-1. The Kd (1.9 and 49 nM respectively). The Kd ratio between PKT16 and PKTD1 or PKTD10 for example highlights the fact that these cyclic analogues (i.e. PKTD1 or PKTD10) are much more efficient in CD47 ligation. Such affinities of the linear peptide analogues develop to date have never been reached.

(5) FIG. 7A to 7F. Those Figures show results of the effects of several cyclic peptides of the invention (PKTD1, PKTD7, PKTD10, PKTD11, PKTD16, PKD10 and PKD10-FF) in comparison with a linear analog (PKT16) on the viability of tumor cells that were evaluated on 5 cell lines (MCF-7, human breast cancer cells; HCT-116, human colon cancer cells; BxPC3, human pancreas cancer cells; A549 and human lung cancer cells) by cytotoxic assay and by counting directly the number of cells. Noticeably, PKT16, the linear analogue is not efficient at 100 microM on these cancer cell lines whereas the cyclic analogues designed to mimic a hairpin involving the beta strands 6 and 7, or 7 and 8, of the C-terminal binding domain of TSP-1 (such as PKTD1, PKTD7, PKTD9, PKTD10, PKTD10-X-NMe, PKTD11, PKTD11-NMe, PKTD18, PKD8 and PKD10 for example among others) are efficient in inducing cell death on these cancer cell lines. Of importance, the simple cyclisation of the beta strand number 7 (4N1 binding epitope of TSP-1) is not sufficient to improve the peptide efficiency since the cyclisation of the 4N1 sequence (peptide PKC1) led to a cyclic analogue that has no potency in inducing cell death.

DETAILED DESCRIPTION OF THE INVENTION

(6) Isolated Cyclic Peptides

(7) The present invention relates to an isolated cyclic peptide of general formula (I):

(8) ##STR00001##
or a pharmacologically acceptable salt or a biologically active derivative thereof, wherein:

(9) B.sub.1 is nothing or a peptidic sequence comprising between 6 and 10 amino acids derived from the beta-strand No 6 of TSP-1 of sequence YAGFVF (SEQ ID NO: 1);

(10) Z.sub.1 is nothing or an heterochiral sequence D-Pro-L-Pro (also designated p-P, p being a D-proline and P a L-proline) or any sequence of two amino acids or analogs of amino acid able to mimic said heterochiral sequence or mimic a beta turn, example of amino acids or analogs of amino acid of said sequence are nipecotic acid, isonipecotic acid, piperidine carboxylic acid, silaproline, thioproline and any other substituted derivative thereof (fluoro, methyl, bromo etc), pseudo proline, substituted proline, N-methyl amino acids, cyclopropyl amino acids (see Karoyan et al. Target in heterocyclic system, 2004 and Karoyan et al. ChemBioChem (2011), 12(7), 1039-1042 and Larregola et al. Journal of Peptide Science (2011), 17(9), 632-643) or biaryl amino acids templates; with the proviso that Z.sub.1 is nothing if B.sub.1 is nothing; in a preferred embodiment, Z.sub.1 is D-Pro-L-Pro;

(11) B.sub.2 represents the peptidic sequence X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X.sub.10 (SEQ ID NO: 2) is derived from the beta-strand No 7 of TSP-1 (of sequence RFYVVMWK, SEQ ID NO: 3) wherein: X.sub.1 refers to nothing or serine or any amino acid with similar properties such as glycine or alanine or threonine; X.sub.2 refers to nothing or arginine or any amino acid with similar properties such as homoarginine, lysine, ornithine, phenylalanine, naphtylalanine, N-methyl arginine or homophenylalanine or any other ring substituted analogues in ortho, meta or para position; for example for arginine, derivatives include any other side chain involving a guanido function and/or one or more than one amine function; X.sub.3 refers to phenylalanine or any amino acid with similar properties including naphtylalanine, homophenylalanine or any other ring substituted analogues in ortho, meta or para position such as para-fluoro-phenylalanine, para-amino-phenylalanine or para-nitro-phenylalanine; tyrosine or any amino acid with aromatic side chains; X.sub.4 refers to tyrosine or any amino acid with aromatic side chains, phenylalanine or any amino acid with similar properties including naphtylalanine, homophenylalanine or any other ring substituted analogues in ortho, meta or para position such as para-fluoro-phenylalanine, para-amino-phenylalanine or para-nitro-phenylalanine; X.sub.5 refers to valine or any amino acid with similar properties including leucine, isoleucine, terleucine, methionine; X.sub.6 refers to valine or any amino acid with similar properties including leucine, isoleucine, terleucine, methionine; X.sub.7 refers to methionine or lysine or any amino acid with similar properties including valine, methionine, norleucine, leucine or isoleucine or terleucine; X.sub.8 refers to tryptophan, tyrosine, phenylalanine, naphthyl-alanine, para-fluoro-phenylalanine, para-amino-phenylalanine, para-nitro-phenylalanine, D-prolino-tryptophane or D-prolino-homotryptophane; X.sub.9 refers to nothing or lysine or any amino acid with similar properties including arginine, homoarginine, ornithine, phenylalanine, naphtylalanine, N-methyl arginine or homophenylalanine or any other ring substituted analogues in ortho, meta or para position or histidine; X.sub.10 refers to nothing or glutamine or alanine or any amino acid with similar properties including asparagine;

(12) Z.sub.2 is nothing or an heterochiral sequence D-Pro-L-Pro (also designated p-P) or any sequence of two amino acids or analogs of amino acid able to mimic said heterochiral sequence or mimic a beta turn, example of amino acids or analogs of amino acid of said sequence are nipecotic acid, isonipecotic acid, piperidine carboxylic acid, silaproline, thioproline and any other substituted derivative thereof (fluoro, methyl, bromo etc), pseudo proline, substituted proline, N-methyl amino acids, cyclopropyl amino acids (Karoyan et al. Target in heterocyclic system, 2004 and Karoyan et al. ChemBioChem (2011), 12(7), 1039-1042 and Larregola et al. Journal of Peptide Science (2011), 17(9), 632-643) or biaryl amino acids templates; with the proviso that Z.sub.2 is nothing if B.sub.3 is nothing; in a preferred embodiment, Z.sub.2 is D-Pro-L-Pro;

(13) B.sub.n represents B.sub.2 or B.sub.3;

(14) B.sub.3 is nothing or a peptidic sequence comprising between 6 and 10 amino acids derived from the beta-strand No 8 of TSP-1 (of sequence GLSVKVVNS, SEQ ID NO: 4); with the proviso that if B.sub.1 is a peptidic sequence comprising between 6 and 10 amino acids residues derived from the beta-strand No 6 of TSP-1 then B.sub.n is nothing and if B.sub.n is B.sub.2 or B.sub.3 (that is to say a peptidic sequence) then B.sub.1 is nothing; and wherein said isolated cyclic peptide comprises between 8 and 26 amino acids, preferably between 14 and 22 amino acids; according to an other embodiment, isolated cyclic peptide comprises between 18 and 22 amino acids.

(15) Except when explicitly mentioned, all amino acids are indifferently of the (D) or (L) configuration.

(16) The present invention thus encompasses cyclic peptides of formula B.sub.1-Z.sub.1-B.sub.2, B.sub.1-B.sub.2, B.sub.2-Z.sub.2-B.sub.3, B.sub.2-B.sub.3, B.sub.2-B.sub.2 (each B.sub.2 being identical or different), B.sub.2-Z.sub.2-B.sub.2 (each B.sub.2 being identical or different) and B.sub.2.

(17) In an embodiment, B.sub.1 comprises the following sequence: YAGFVFG (SEQ ID NO: 5).

(18) In another embodiment, B.sub.1 comprises the following sequence: -X.sub.11-Y-A-G-F-V-F-G-X.sub.12-X.sub.13- (SEQ ID NO: 6) wherein:

(19) X.sub.11 is nothing or aspartic acid or any amino acid with similar properties including glutamic acid;

(20) X.sub.12 is nothing or tyrosine or any amino acid with aromatic side chains and

(21) X.sub.13 is nothing or serine or any amino acid with similar properties including glycine.

(22) In an embodiment, B.sub.3 comprises the following sequence: -X.sub.19-X.sub.14-X.sub.15-X.sub.20-X.sub.21-X.sub.16-X.sub.22-X.sub.23-X.sub.17-X.sub.18- (SEQ ID NO: 36); preferably, B.sub.3 comprises the following sequence -X.sub.19-X.sub.14-X.sub.15-S-V-X.sub.16-V-V-X.sub.17-X.sub.18- wherein:

(23) X.sub.14 is nothing or glycine or alanine or any amino acid with similar properties including serine;

(24) X.sub.15 is isoleucine or leucine or alanine or any amino acid with similar properties including terleucine, valine, methionine;

(25) X.sub.16 is lysine or alanine or any amino acid with similar properties including arginine, homoarginine, lysine, ornithine, phenylalanine, naphtylalanine, N-methyl arginine or homophenylalanine or any other ring substituted analogues (ortho, meta, para), histidine, or methionine or any amino acid with similar properties including valine, leucine, isoleucine, terleucine;
X.sub.17 is nothing or asparagine or alanine or any amino acid with similar properties including glutamine or lysine or any amino acid with similar properties including arginine, homoarginine, lysine, ornithine, phenylalanine, naphtylalanine, N-methyl arginine or homophenylalanine or any other ring substituted analogues (ortho, meta, para), histidine;
X.sub.18 is nothing, serine or glycine or any amino acid with similar properties;
X.sub.19 is nothing or serine or alanine or any amino acid with similar properties;
X.sub.20 is serine or alanine or any amino acid with similar properties including leucine, isoleucine, terleucine;
X.sub.21 is valine or alanine or any amino acid with similar properties including leucine, isoleucine, terleucine;
X.sub.22 is valine or alanine or any amino acid with similar properties including leucine, isoleucine, terleucine; and
X.sub.23 is valine or alanine or any amino acid with similar properties including leucine, isoleucine, terleucine.

(26) The isolated cyclic peptide of general formula (I) of the invention yet comprises at least parts of the beta-sheet No 7, of the beta-sheets No 7 and 8 or at least parts of the beta-sheets No 6 and 7 of the C-terminal domain of the TSP-1 but cannot be the entire sequence of the C-terminal domain of the TSP-1 (as described by Kosfeld M D, Frazier W A (1993) Identification of a new cell adhesion motif in two homologous peptides from the COOH-terminal cell binding domain of human thrombospondin. J Biol Chem 268: 8806-8814), because this domain has not the same biologic activity as cyclic peptides of the invention.

(27) According to a specific embodiment, the present invention relates to an isolated cyclic peptide of general formula (Ia):

(28) ##STR00002##
or a pharmacologically acceptable salt or a biologically active derivative thereof, wherein:

(29) Z.sub.1 is nothing or an heterochiral sequence D-Pro-L-Pro (also designated p-P, p being a D-proline and P a L-proline) or any sequence of two amino acids or analogs of amino acid able to mimic said heterochiral sequence or mimic a beta turn, example of amino acids or analogs of amino acid of said sequence are nipecotic acid, isonipecotic acid, piperidine carboxylic acid, silaproline, thioproline and any other substituted derivative thereof (fluoro, methyl, bromo etc), pseudo proline, substituted proline, N-methyl amino acids, cyclopropyl amino acids (see Karoyan et al. Target in heterocyclic system, 2004 and Karoyan et al. ChemBioChem (2011), 12(7), 1039-1042 and Larregola et al. Journal of Peptide Science (2011), 17(9), 632-643) or biaryl amino acids templates; in a preferred embodiment, Z.sub.1 is D-Pro-L-Pro;

(30) B.sub.2 represents the peptidic sequence X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X.sub.10 (SEQ ID NO: 2) derived from the beta-strand No 7 of TSP-1 (of sequence RFYVVMWK, SEQ ID NO: 3) and comprises between 6 and 10 amino acids, wherein: X.sub.1 refers to nothing or serine or any amino acid with similar properties such as glycine or alanine or threonine; X.sub.2 refers to nothing or arginine or any amino acid with similar properties such as homoarginine, lysine, ornithine, phenylalanine, naphtylalanine, N-methyl arginine (RNMe) or homophenylalanine or any other ring substituted analogues in ortho, meta or para position; for example for arginine, derivatives include any other side chain involving a guanido function and/or one or more than one amine function; X.sub.3 refers to phenylalanine or any amino acid with similar properties including naphtylalanine, homophenylalanine or any other ring substituted analogues in ortho, meta or para position such as para-fluoro-phenylalanine, para-amino-phenylalanine or para-nitro-phenylalanine; tyrosine or any amino acid with aromatic side chains; X.sub.4 refers to tyrosine or any amino acid with aromatic side chains, phenylalanine or any amino acid with similar properties including naphtylalanine, homophenylalanine or any other ring substituted analogues in ortho, meta or para position such as para-fluoro-phenylalanine, para-amino-phenylalanine or para-nitro-phenylalanine; X.sub.5 refers to valine or any amino acid with similar properties including leucine, isoleucine, terleucine, methionine; X.sub.6 refers to valine or any amino acid with similar properties including leucine, isoleucine, terleucine, methionine; X.sub.7 refers to methionine or lysine or any amino acid with similar properties including valine, methionine, norleucine, leucine or isoleucine or terleucine; X.sub.8 refers to tryptophan, tyrosine, phenylalanine, naphthyl-alanine, para-fluoro-phenylalanine, para-amino-phenylalanine, para-nitro-phenylalanine, D-prolino-tryptophane or D-prolino-homotryptophane; X.sub.9 refers to nothing or lysine or any amino acid with similar properties including arginine, homoarginine, ornithine, phenylalanine, naphtylalanine, N-methyl arginine or homophenylalanine or any other ring substituted analogues in ortho, meta or para position or histidine; X.sub.10 refers to nothing or glutamine or any amino acid with similar properties including asparagine; X.sub.10 may also refers to alanine;
preferably, if X.sub.2 is nothing then X.sub.1 is nothing and/or if X.sub.9 is nothing then X.sub.10 is nothing; preferably, B.sub.2 comprises at least the 6 amino acids -X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-; more preferably, B.sub.2 comprises at least the peptidic fragment -F-Y-V-V-M-W- (SEQ ID NO: 37);

(31) Z.sub.2 is nothing or an heterochiral sequence D-Pro-L-Pro (also designated p-P, p being a D-proline and P a L-proline) or any sequence of two amino acids or analogs of amino acid able to mimic said heterochiral sequence or mimic a beta turn, example of amino acids or analogs of amino acid of said sequence are nipecotic acid, isonipecotic acid, piperidine carboxylic acid, silaproline, thioproline and any other substituted derivative thereof (fluoro, methyl, bromo etc), pseudo proline, substituted proline, N-methyl amino acids, cyclopropyl amino acids (see Karoyan et al. Target in heterocyclic system, 2004 and Karoyan et al. ChemBioChem (2011), 12(7), 1039-1042 and Larregola et al. Journal of Peptide Science (2011), 17(9), 632-643) or biaryl amino acids templates; in a preferred embodiment, Z.sub.1 is D-Pro-L-Pro;

(32) B.sub.n represents B.sub.2 or B.sub.3;

(33) B.sub.3 is a peptidic sequence comprising between 6 and 10 amino acids derived from the beta-strand No 8 of TSP-1 (of sequence GLSVKVVNS, SEQ ID NO: 4); B.sub.3 comprises the following sequence: -X.sub.19-X.sub.14-X.sub.15-X.sub.20-X.sub.21-X.sub.16-X.sub.22-X.sub.23-X.sub.17-X.sub.18- (SEQ ID NO: 36); preferably, B.sub.3 comprises the following sequence: -X.sub.19-X.sub.14-X.sub.15-S-V-X.sub.16-V-V-X.sub.17-X.sub.18- wherein:

(34) X.sub.14 is nothing or glycine or alanine or any amino acid with similar properties including serine;

(35) X.sub.15 is isoleucine or leucine or alanine or any amino acid with similar properties including terleucine, valine, methionine;

(36) X.sub.16 is lysine or alanine or any amino acid with similar properties including arginine, homoarginine, homolysine, ornithine, phenylalanine, naphtylalanine, N-methyl arginine (RNMe) or homophenylalanine or any other ring substituted analogues (ortho, meta, para), histidine, or methionine or any amino acid with similar properties including valine, leucine, isoleucine, terleucine;

(37) X.sub.17 is nothing, asparagine or alanine or any amino acid with similar properties including glutamine or lysine or any amino acid with similar properties including arginine, homoarginine, homolysine, ornithine, phenylalanine, naphtylalanine, N-methyl arginine (RNMe) or homophenylalanine or any other ring substituted analogues (ortho, meta, para), histidine;

(38) X.sub.18 is nothing, serine or glycine or any amino acid with similar properties;

(39) X.sub.19 is nothing, serine or alanine or any amino acid with similar properties;

(40) X.sub.20 is serine or alanine or any amino acid with similar properties including leucine, isoleucine, terleucine;

(41) X.sub.21 is valine or alanine or any amino acid with similar properties including leucine, isoleucine, terleucine;

(42) X.sub.22 is valine or alanine or any amino acid with similar properties including leucine, isoleucine, terleucine; and

(43) X.sub.23 is valine or alanine or any amino acid with similar properties including leucine, isoleucine, terleucine;

(44) preferably, if X.sub.14 is nothing then X.sub.19 is nothing and/or if X.sub.17 is nothing then X.sub.18 is nothing; preferably B.sub.3 comprises at least the 6 amino acids -X.sub.15-S-V-X.sub.16-V-V-; more preferably, B.sub.3 comprises at least the peptidic fragment -L-S-V-K-V-V (SEQ ID NO: 38);

(45) wherein said isolated cyclic peptide comprises an even number of aminoacids (that is to say B.sub.2 and B.sub.n have the same number of amino acids and both consist in a fragment of 6, 7, 8, 9 or 10 amino acids) and wherein said isolated cyclic peptide comprises between 8 and 26 amino acids, preferably between 12 and 22 amino acids; more preferably, isolated cyclic peptides of the invention consist in 12, 14, 16, 18, 20 or 22 amino acids.

(46) The present invention thus encompasses cyclic peptides of formula B.sub.2-B.sub.3, Z.sub.1-B.sub.2-B.sub.3, B.sub.2-Z.sub.2-B.sub.3, B.sub.2-B.sub.2 (each B.sub.2 being identical or different) and B.sub.2-Z.sub.2-B.sub.2 (each B.sub.2 being identical or different).

(47) In a preferred embodiment, B.sub.2 and B.sub.3 are arranged so that X.sub.5 of B.sub.2 faces X.sub.16 of B.sub.3 and X.sub.8 of B.sub.2 faces X.sub.15 of B.sub.3 as illustrated below:

(48) ##STR00003##

(49) According to a particular embodiment, both Z.sub.1 and Z.sub.2 can be nothing; if Z.sub.1 consists in two amino acids then Z.sub.2 is nothing and if Z.sub.2 consists in two amino acids then Z.sub.1 is nothing.

(50) Solutions of the cyclic peptides of the invention as free base or pharmacologically acceptable salts can be prepared.

(51) The peptides thereof according to the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

(52) Examples of isolated cyclic peptide according to the present invention are as described in Table I:

(53) TABLE-US-00001 TABLE I PEP- STRUCTURE/ FORMULA TIDES AMINO: ACID SEQUENCE (linear representation) AND MW PKPH12 (SEQ ID NO: 39)   —F—Y—V—V—M—W—L—S—V—K—V—V— PKPH12P (SEQ ID NO: 40) F—Y—V—V—M—P.sub.Z.sup.3hW—L—S—V—K—V—V— P.sub.Z.sup.3hW being D-prolino-homo-tryptophane (see Karoyan and Chassaing, Tetrahedron Letters, 1997, p84) PKD8 (SEQ ID NO: 41) Chemical Formula: C.sub.86H.sub.124F.sub.3N.sub.16O.sub.17S Molecular Weight: 1743.09 —F—Y—V—V—M—W—p—P—L—S—V—K—V—V— PKD8FF (SEQ ID NO: 42) Chemical Formula: C.sub.86H.sub.124F.sub.3N.sub.16O.sub.16S Molecular Weight: 1727.09 —F—F—V—V—M—W—p—P—L—S—V—K—V—V— PKD9 (SEQ ID NO: 43) Chemical Formula: C.sub.87H.sub.126F.sub.3N.sub.16O.sub.17 Molecular Weight: 1725.06 —F—Y—V—V—X—W—p—P—L—S—V—K—V—V— X = NLe PKD10 (SEQ ID NO: 44) Chemical Formula: C.sub.89H.sub.127F.sub.6N.sub.17O.sub.18 Molecular Weight: 1837.09 —F—Y—V—V—K—W—p—P—L—S—V—K—V—V— PKD10FF (SEQ ID NO: 45) 0 Chemical Formula: C.sub.89H.sub.127F.sub.6N.sub.17O.sub.17 Molecular Weight: 1821.09 —F—F—V—V—K—W—p—P—L—S—V—K—V—V— PKTDi4 (SEQ ID NO: 46) Chemical Formula: C.sub.86H.sub.124F.sub.3N.sub.16O.sub.17S Exact Mass: 1741.90 Molecular Weight: 1743.09 —p—P—F—Y—V—V—M—W—L—S—V—K—V—V— PKD11 (SEQ ID NO: 47) Chemical Formula: C.sub.98H.sub.142F.sub.6N.sub.22O.sub.21S Exact Mass: 2109.03 Molecular Weight: 2110.40 —R—F—Y—V—V—M—W—p—P—L—S—V—K—V—V—N— PKD11RNMe (SEQ ID NO: 48) Chemical Formula: C.sub.99H.sub.144F.sub.6N.sub.22O.sub.21S Exact Mass: 2123.05 Molecular Weight: 2124.42 —R*—F—Y—V—V—M—W—p—P—L—S—V—K—V—V—N— R* = NMethylarginine PKD12 (SEQ ID NO: 49) Chemical Formula: C.sub.102H.sub.148F.sub.9N.sub.22O.sub.21S Exact Mass: 2220.08 Molecular Weight: 2221.48 —R—F—Y—V—V—M—W—p—P—I—S—V—K—V—V—K— PKD12RNMe (SEQ ID NO: 50) Chemical Formula: C.sub.103H.sub.150F.sub.9N.sub.22O.sub.21S Exact Mass: 2234.09 Molecular Weight: 2235.51 —R*—F—Y—V—V—M—W—p—P—I—S—V—K—V—V—K— R* = NMethylarginine PKTDi3 (SEQ ID NO: 51) Chemical Formula: C.sub.96H.sub.139F.sub.6N.sub.19O.sub.20S Exact Mass: 2024.01 Molecular Weight: 2025.33 —p—P—F—Y—V—V—M—W—K—G—L—S—V—K—V—V— PKTDi5 (SEQ ID NO: 52) Chemical Formula: C.sub.98H.sub.142F.sub.6N.sub.22O.sub.21S Exact Mass: 2109.03 Molecular Weight: 2110.40 —p—P—R—F—Y—V—V—M—W—L—S—V—K—V—V—N— PKTD7 (SEQ ID NO: 14)   —R—F—Y—V—V—M—W—K—p—P—G—I—S—V—K—V—V—N— Chemical Formula: C.sub.102H.sub.160N.sub.25O.sub.21S.sub.3+ Molecular Weight: 2104.61 PKTD14 (SEQ ID NO: 31) Chemical Formula: C.sub.105H.sub.154F.sub.6N.sub.24O.sub.23S.sub.2 Exact Mass: 2297.10 Molecular Weight: 2298.64 —R—F—Y—V—V—M—W—K—p—P—G—L—S—V—M—V—V—N— PKTD15 (SEQ ID NO: 32) 0 Chemical Formula: C.sub.108H.sub.157F.sub.9N.sub.25O.sub.24S Exact Mass: 2391.14 Molecular Weight: 2392.64 —R—F—Y—V—V—M—W—K—p—P—G—L—S—V—K—V—V—N— PKTD17 (SEQ ID NO: 34) Chemical Formula: C.sub.130H.sub.172F.sub.12N.sub.26O.sub.24S.sub.2 Exact Mass: 2801.23 Molecular Weight: 2803.08 —R—F—Y—V—V—M—W—K—p—P—R—F—Y—V—V—M—W—K— PKTDi2 (SEQ ID NO: 53) Chemical Formula: C.sub.108H.sub.157F.sub.9N.sub.25O.sub.24S Exact Mass: 2391.14 Molecular Weight: 2392.64 —p—P—R—F—Y—V—V—M—W—K—G—L—S—V—K—V—V—N— PKTD1 (SEQ ID NO: 9) Chemical Formula: C.sub.118H.sub.173F.sub.12N.sub.27O.sub.28S Exact Mass: 2676.25 Molecular Weight: 2677.88 —S—R—F—Y—V—V—M—W—K—p—P—G—I—S—V—K—V—V—K—S— PKTD3 (SEQ ID NO: 10) Chemical Formula: C.sub.116H.sub.169F.sub.12N.sub.27O.sub.28S Exact Mass: 2648.22 Molecular Weight: 2649.83 —S—R—F—Y—V—V—M—W—K—p—P—G—I—S—A—K—V—V—K—S— PKTD4 —D—Y—A—G—F—V—F—G—Y—p—P—R—F—Y—V—V—M—W—K—Q— (SEQ ID NO: 11) PKTD5 —Y—A—G—F—V—F—G—Y—p—P—R—F—Y—V—V—M—W—K— (SEQ ID NO: 12) PKTD6 (SEQ ID NO: 13) Chemical Formula: C.sub.114H.sub.167F.sub.9N.sub.27O.sub.28S Exact Mass: 2565.21 Molecular Weight: 2566.80 —S—R—F—Y—V—V—M—W—K—p—P—G—I—S—V—K—V—V—N—S— PKTD8 —D—Y—A—G—F—V—F—G—Y—Q—p—P—S—R—F—Y—V—V—M—W—K—Q— (SEQ ID NO: 15) PKTD9 (SEQ ID NO: 16) Chemical Formula: C.sub.126H.sub.162F.sub.6N.sub.26O.sub.27S Molecular Weight: 2618.89 —Y—A—G—F—V—F—G—Y—Q—p—P—S—R—F—Y—V—V—M—W—K— PKTD10 (SEQ ID NO: 17) Chemical Formula: C.sub.114H.sub.167F.sub.9N.sub.27O.sub.28S Molecular Weight: 2566.80 —S—R—F—Y—V—V—M—W—K—p—P—G—L—S—V—K—V—V—N—S— PKTD10-1 (SEQ ID NO: 18)   —S—R—F—Y—V—V—M—W—K—p—P—A—L—S—V—K—V—V—N—S— Chemical Formula: C.sub.111H.sub.179N.sub.27O.sub.24S.sub.4+ Molecular Weight: 2307.88 PKTD10-2 (SEQ ID NO: 19)   —S—R—F—Y—V—V—M—W—K—p—P—G—A—S—V—K—V—V—N—S— Chemical Formula: C.sub.107H.sub.171N.sub.27O.sub.24S.sub.4+ Molecular Weight: 2251.77 PKTD10-3 (SEQ ID NO: 20) 0   —S—R—F—Y—V—V—M—W—K—p—P—G—L—A—V—K—V—V—N—S— Chemical Formula: C.sub.110H.sub.177N.sub.27O.sub.23S.sub.4+ Molecular Weight: 2277.85 PKTD10-4 (SEQ ID NO: 21)   —S—R—F—Y—V—V—M—W—K—p—P—G—L—S—A—K—V—V—N—S— Chemical Formula: C.sub.108H.sub.173N.sub.27O.sub.24S.sub.4+ Molecular Weight: 2265.79 PKTD10-5 (SEQ ID NO: 22)   —S—R—F—Y—V—V—M—W—K—p—P—G—L—S—V—A—V—V—N—S— Chemical Formula: C.sub.107H.sub.169N.sub.26O.sub.24S.sub.3+ Molecular Weight: 2235.75 PKTD10-6 (SEQ ID NO: 23)   —S—R—F—Y—V—V—M—W—K—p—P—G—L—S—V—K—A—V—N—S— Chemical Formula: C.sub.108H.sub.173N.sub.27O.sub.24S.sub.4+ Molecular Weight: 2265.79 PKTD10-7 (SEQ ID NO: 24)   —S—R—F—Y—V—V—M—W—K—p—P—G—L—S—V—K—V—A—N—S— Chemical Formula: C.sub.108H.sub.173N.sub.27O.sub.24S.sub.4+ Molecular Weight: 2265.79 PKTD10-8 (SEQ ID NO: 25)   —S—R—F—Y—V—V—M—W—K—p—P—G—L—S—V—K—V—V—A—S— Chemical Formula: C.sub.107H.sub.169N.sub.26O.sub.24S.sub.3+ Molecular Weight: 2235.75 PKTD10-9 (SEQ ID NO: 26)   —S—R—F—Y—V—V—M—W—K—p—P—G—L—S—V—K—V—V—N—A— Chemical Formula: C.sub.110H.sub.177N.sub.27O.sub.23S.sub.4+ Molecular Weight: 2277.85 PKTD10- RNMe (SEQ ID NO: 54) Chemical Formula: C.sub.115H.sub.169F.sub.9N.sub.27O.sub.28S Molecular Weight: 2580.82 —S—R*—F—Y—V—V—M—W—K—p—P—G—L—S—V—K—V—V—N—S— R* = RNMe PKTD10-X- RNMe (SEQ ID NO: 55) Chemical Formula: C.sub.116H.sub.171F.sub.9N.sub.27O.sub.28 Molecular Weight: 2562.79 —S—R*—F—Y—V—V—X—W—K—p—P—G—L—S—V—K—V—V—N—S— R* = RNMe and X = NLe PKTD10-3- X-RNMe (SEQ ID NO: 56) Chemical Formula: C.sub.116H.sub.171F.sub.9N.sub.27O.sub.27 Molecular Weight: 2546.79 —S—R*—F—Y—V—V—X—W—K—p—P—G—L—A—V—K—V—V—N—S— R* = RNMe and X = NLe PKTD12 (SEQ ID NO: 29) 0   —R—F—Y—V—V—M—W—K—Q—p—P—S—G—L—S—V—K—V—V—N— Chemical Formula: C.sub.110H.sub.177N.sub.27O.sub.24S.sub.4+ Molecular Weight: 2293.85 PKTD16 —S—R—F—Y—V—V—M—W—K—p—P—S—R—F—Y—V—V—M—W—K— (SEQ ID NO: 33) PKTD18 (SEQ ID NO: 35) Chemical Formula: C.sub.113H.sub.165F.sub.9N.sub.27O.sub.27S Molecular Weight: 2536.77 —S—R—F—Y—V—V—M—W—K—p—P—G—L—S—V—K—V—V—N—G— PKTDi1 (SEQ ID NO: 57) Chemical Formula: C.sub.109H.sub.158F.sub.9N.sub.26O.sub.27S Exact Mass: 2466.14 Molecular Weight: 2467.66 —p—P—S—R—F—Y—V—V—M—W—K—G—L—S—V—K—V—V—N—S— PKTD11 (SEQ ID NO: 28)   —S—R—F—Y—V—V—M—W—K—Q—p—P—S—G—L—S—V—K—V—V—N—S— Chemical Formula: C.sub.115H.sub.180N.sub.29O.sub.30S.sub.3+ Molecular Weight: 2480.94 PKTD11Q (SEQ ID NO: 58) Chemical Formula: C.sub.120H.sub.177F.sub.9N.sub.29O.sub.31S Molecular Weight: 2724.95 —S—R—F—Y—V—V—M—W—K—A—p—P—S—G—L—S—V—K—V—V—N—S— PKTD11S (SEQ ID NO: 59) Chemical Formula: C.sub.122H.sub.179F.sub.9N.sub.29O.sub.32S Molecular Weight: 2766.99 —S—R—F—Y—V—V—M—W—K—Q—p—P—A—G—L—S—V—K—V—V—N—S— PKTD11- RNMe (SEQ ID NO: 60) Chemical Formula: C.sub.123H.sub.181F.sub.9N.sub.29O.sub.33S Molecular Weight: 2797.02 —S—R*—F—Y—V—V—M—W—K—Q—p—P—S—G—L—S—V—K—V—V—N—S— R* = RNMe PKTD11-X- RNMe (SEQ ID NO: 61) Chemical Formula: C.sub.124H.sub.183F.sub.9N.sub.29O.sub.33 Molecular Weight: 2778.98 —S—R*—F—Y—V—V—X—W—K—Q—p—P—S—G—L—S—V—K—V—V—N—S— R* = RNMe and X = NLe
Synthesis of the Cyclic Peptides of the Invention

(54) The cyclic peptides of the invention are prepared as described in the experimental part.

(55) Briefly, cyclic peptides of the invention are synthesized using a mixed solid/solution phase procedure leading to a linear peptide that is then cyclized. Cyclization include S—S bridges, thioether bridges, C—C bonds, C—N, ester bonds, carbon-heteroatom bonds, O—O, N—N, cyclization using scaffolds . . . .

(56) Biologically Active Derivatives of the Cyclic Peptides of the Invention

(57) As used herein, the term “biologically active derivatives” include the functional variants of the peptide to which it refers. More particularly, in the context of the invention, the derivative designates “biologically active derivative of the cyclic peptide of general formula (I)” are variants retaining the biological activity and the specificity of the parent peptide. Thus, in the context of the invention, said “biologically active derivatives” have are agonist of CD47 and able trigger PCD and to treat diseases associated with defects in PCD such as cancer and immunological disorders.

(58) Preferably, the ability to trigger PCD and the antiproliferative effect of one biologically active derivative of a given cyclic peptide of general formula (I) has to be of at least about 70%, preferably between 80 and 90%, more preferably between 90 and 99% and even more preferably 100% of the antiproliferative effect, in particular to inhibit cell proliferation, of said given cyclic peptide of general formula (I) as assessed in vitro by conventional proliferation techniques.

(59) Also, the biologically active derivatives have preferably the same specificity as the cyclic peptides of general formula (I) toward cell proliferation as assessed in vitro by conventional cellular experiments.

(60) Said biologically active derivative can be either an allelic variant of the peptide, or a peptidomimetic variant of the peptide.

(61) An “allelic variant of the peptide” has the same amino acid sequence as one cyclic peptide of general formula (I), except that one or more amino acids have been replaced by other amino acids or suppressed, the final peptide retaining the biological activity and specificity of the parent cyclic peptide of general formula (I). Preferably, such allelic variant has at least 50%, preferably 70%, preferably 80%, more preferably 90% and even more preferably 95% of identity as compared with the parent cyclic peptide of general formula (I).

(62) As used herein, “percentage of identity” between two amino acid sequences denotes the percentage of amino acids residues that are identical between the two sequences to be compared, obtained after the best alignment (optimum alignment), this percentage being purely statistical and the differences between the two sequences being distributed randomly and along their entire length. Sequence comparisons between two amino acid sequences can be performed for example with the BLAST program available on the website ncbi.nlm.nih.gov/gorf/b12.html the parameters used being those given by default (in particular for the parameters “open gap penalty”:5 and “extension gap penalty”:2, the matrix selected being for example the “BLOSUM 62” matrix as suggested by the program, the percentage identity between the two sequences to be compared being calculated directly by the program).

(63) Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid or another.

(64) The biologically active derivative of the cyclic peptide of general formula (I) can also be a peptidomimetic variant, which is an organic molecule that mimics some properties of the parent peptide, including at least one or more properties of interest that preferably is its biological activity.

(65) Preferred peptidomimetics are obtained by structural modification of cyclic peptides according to the invention, preferably using unnatural amino acids, D amino acid instead of L amino acids, conformational restraints, isosteric replacement or other modifications.

(66) Other preferred modifications include, without limitation, those in which one or more amide bond is replaced by a non-amide bond, and/or one or more amino acid side chain is replaced by a different chemical moiety, or one or more side chain is protected by a protecting group, and/or double bonds and/or cyclization and/or stereo specificity is introduced into the amino chain to increase rigidity and/or binding affinity.

(67) Still other preferred modifications include those intended to enhance resistance to enzymatic degradation, improvement in the bioavailability, and more generally in the pharmacokinetic properties, compared to the parent cyclic peptide of general formula (I).

(68) Examples of such peptidomimetics include, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Chemical derivatives also include peptides that contain one or more naturally-occurring amino acid derivatives of the twenty standard amino acids. For examples: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine. The term “conservative substitution” also includes the use of non-natural amino acids aimed to control and stabilize peptides or proteins secondary structures. These non-natural amino acids are chemically modified amino acids such as prolinoamino acids, beta-amino acids, N-methylamino acids, cyclopropylamino acids, alpha,alpha-substituted amino acids as describe here below. These non-natural amino acids may include also fluorinated, chlorinated, brominated- or iodinated modified amino acids.

(69) Other examples of modifications include conjugation for example with lipids or carbohydrate.

(70) All of these variations are well known in the art. Thus, given the peptide sequences of the cyclic peptide of general formula (I), those skilled in the art are enabled to design and produce peptidomimetics having biological characteristics similar to or superior to such peptides.

(71) Preferred peptidomimetic variants of the cyclic peptide of general formula (I) retain at least the biological activity and specificity of said cyclic peptide of general formula (I).

(72) The biologically active derivatives of the cyclic peptides of general formula (I) can be conveniently synthesized using art recognized techniques.

(73) Therapeutic Use of the Cyclic Peptide and/or the Biologically Active Derivative Thereof of the Invention

(74) The present invention provides an isolated cyclic peptide of general formula (I) or a biologically active derivative thereof for its use as medicine.

(75) The present invention also provides an isolated cyclic peptide of general formula (I) or a biologically active derivative thereof for use as agonist agent for triggering programmed cell death (PCD), as agent that activates CD47 and as agonist agent of CD47. More specifically, an isolated cyclic peptide of general formula (I) or a biologically active derivative thereof is useful for the treatment of diseases involving interaction of TSP1 and CD47, in particular for the treatment of diseases associated with defects in PCD; example of diseases involving apoptosis are described by Favoloro et al. (Role of apoptosis in disease, AGING, May 2012, vol. 4, No 5, pp. 330-349), importance of cell death in disease is also described by R A Knight and G Melino (Cell death in disease: from 2010 onwards, Cell Death and Disease (2011) 2, e202; doi:10.1038/cddis.2011.89); carcinogenesis is also associated with inflammation that is a defensive process against tissue injury, example of TSP-1 role in the modulation of the inflammatory process and its resolution are given by Zenaida Lopez-Dee et al. (“Thrombospondin-1: Multiple Path to inflammation, Mediators of Inflammation, Volume 2011, Article ID 296069, 10 pages doi:10.1155/2011/296069). Once this self-protective strategy is initiated, an effective resolution of the process is crucial to avoid major and unnecessary tissue damage. If the underlying event inducing inflammation is not addressed and homeostasis is not restored, the inflammation process become chronic and lead to angiogenesis, carcinogenesis and diseases associated with immunological disorders (see Favoloro et al.) including chronic inflammation (See Lopez-Dee et al.), such as for example, multiple sclerosis, Crohn disease, psoriasis, ulcerative colitis, arthritis and asthma.

(76) In a particular embodiment, isolated cyclic peptide of general formula (I) or a biologically active derivative thereof may be useful in the treatment of a cancer selected form the group consisting of adrenal cortical cancer, anal cancer, bile duct cancer, multiple myeloma, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, Castleman disease, cervical cancer, colorectal cancer, endometrial cancer, esophagus cancer, gallbladder cancer, gastrointestinal carcinoid tumors, Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, liver cancer, lung cancer, mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, melanoma, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, vaginal cancer, vulvar cancer, and uterine cancer.

(77) In another particular embodiment, isolated cyclic peptide of general formula (I) or a biologically active derivative thereof may be useful in the treatment of diseases associated with chronic inflammation, including immunological diseases, selected from the group consisting of multiple sclerosis, Crohn disease, psoriasis, ulcerative colitis, arthritis and asthma.

(78) In another embodiment, the present invention relates to a method of therapeutically treating cancer and diseases associated with chronic inflammation by administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising at least an isolated cyclic peptide of general formula (I) or a biologically active derivative thereof.

(79) Pharmaceutical Composition Comprising a Cyclic Peptide and/or a Biologically Active Derivative Thereof of the Invention

(80) The present invention also relates to pharmaceutical composition comprising an isolated cyclic peptide of general formula (I) or a biologically active derivative thereof and a pharmaceutically acceptable carrier.

(81) In a particular embodiment of the invention, the isolated cyclic peptide of general formula (I) to be incorporated in the pharmaceutical composition of the invention is selected in group consisting of PKTD1 (SEQ ID NO: 9), PKTD3 (SEQ ID NO: 10), PKTD4 (SEQ ID NO: 11), PKTD5 (SEQ ID NO: 12), PKTD6 (SEQ ID NO: 13), PKTD7 (SEQ ID NO: 14), PKTD8 (SEQ ID NO: 15), PKTD9 (SEQ ID NO: 16), PKTD10 (SEQ ID NO: 17), PKTD10-1 (SEQ ID NO: 18), PKTD10-2 (SEQ ID NO: 19), PKTD10-3 (SEQ ID NO: 20), PKTD10-4 (SEQ ID NO: 21), PKTD10-5 (SEQ ID NO: 22), PKTD10-6 (SEQ ID NO: 23), PKTD10-7 (SEQ ID NO: 24), PKTD10-8 (SEQ ID NO: 25), PKTD10-9 (SEQ ID NO: 26), PKTD11 (SEQ ID NO: 27), PKTD12 (SEQ ID NO: 29), PKTD14 (SEQ ID NO: 31), PKTD15 (SEQ ID NO: 32), PKTD16 (SEQ ID NO: 33), PKTD17 (SEQ ID NO: 34) and PKTD18 (SEQ ID NO: 35), PKPH12 (SEQ ID NO: 39), PKPH12P (SEQ ID NO: 40), PKD8 (SEQ ID NO: 41), PKD8FF (SEQ ID NO: 42), PKD9 (SEQ ID NO: 43), PKD10 (SEQ ID NO: 44), PKD10FF (SEQ ID NO: 45), PKTDi4 (SEQ ID NO: 46), PKD11 (SEQ ID NO: 47), PKD11RNMe (SEQ ID NO: 48), PKD12 (SEQ ID NO: 49), PKD12RNMe (SEQ ID NO: 50), PKTDi3 (SEQ ID NO: 51), PKTDi5 (SEQ ID NO: 52), PKTDi2 (SEQ ID NO: 53), PKTD10-RNMe (SEQ ID NO: 54), PKTD10-X-RNMe (SEQ ID NO: 55), PKTD10-3-X-RNMe (SEQ ID NO: 56), PKTDi1 (SEQ ID NO: 57), PKTD11Q (SEQ ID NO: 58), PKTD11S (SEQ ID NO: 59), PKTD11-RNMe (SEQ ID NO: 60), PKTD11-X-RNMe (SEQ ID NO: 61) or a pharmacologically acceptable salt thereof or a biologically active derivative thereof.

(82) For the purpose of the invention, suitable pharmaceutically acceptable carriers include, but are not limited to: water, salt solutions (e.g., NaCl), alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, and polyvinyl pyrolidone, lipids such as but not limited to: phospholipids, sphinglipids, glycerol-fatty acid esters . . . .

(83) The pharmaceutical composition of the invention can be sterilized and if desired, mixed with auxiliary agents, e. g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds. The pharmaceutical composition of the invention, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

(84) The pharmaceutical composition of the invention can be a liquid solution, suspension, emulsion, tablet including sterile lyophilized formulation, pill, capsule, sustained release formulation, or powder. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrolidone, sodium saccharine, cellulose, magnesium carbonate, etc. Some appropriate precise formulations are described, for example, in Remington, The Science and Practice of Pharmacy, 19th edition, 1995, Mack Publishing Company.

(85) The pharmaceutical composition of the invention can be formulated in accordance with the routine procedures as a composition adapted for intravenous administration to an individual. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer or a sterile lyophilized formulation to be reconstituted prior injection, such injection can be intravenous, intramuscular, subcutaneous, intrathecal, such pharmaceutical composition can also be inhaled through nasal and/or pulmonary delivery. In a preferred embodiment the pharmaceutical composition of the invention is a liquid composition that is dedicated to be administered by injection, and for example, by intratumoral injection. Said intratumoral injection can be obtained for example by using stereotactic neurosurgery. This administration can be performed prior to or after a surgical operation intended to remove the tumor. In the first case, the composition enables to inhibit the growth of the tumor and avoid dissemination of the tumor cells and the occurrence of dramatic symptoms on the subject; in the second case, the composition can be used to destroy all the tumor cells that have not be removed during the surgical operation.

(86) The effective dose of an isolated cyclic peptide of general formula (I) varies in function of numerous parameters such as, for example, the chosen administration method, the weight, age, sex, and the sensitivity of the individual to be treated. Consequently, the optimal dose must be determined individually, in function of the relevant parameters, by a medical specialist. In order to predict the expected active doses in human from the first animal studies presented hereunder, one can also use the fc.sub.2 and C.sub.T values as described by Rocchetti et al (2007).

(87) The following examples describe the high specificity and therapeutic efficiency of the cyclic peptides of general formula (I). They are however not limitative, in particular concerning the nature of amino acid sequence of the invention, and the experimental conditions to use it.

EXAMPLES

(88) 1. Synthesis and Characterization of the Cyclic Peptides of the Invention

(89) General Methods:

(90) All commercial chemicals and solvents were reagent grade and were used without further purification unless otherwise specified. All reactions except those in aqueous media were carried out with the use of standard techniques for the exclusion of moisture. All reactions were performed under argon or nitrogen in oven-dried glassware using anhydrous solvents and standard syringe techniques. Protected amino acid derivatives, HATU, HBTU, HFIP, pseudoproline dipeptides and 2-CTC resin were purchased from Iris Biotech (Marktredwitz, Germany) DIPEA, NMP, piperidine solution, DMF, IPA, TFA, PyBOP were obtained from Sigma-Aldrich. Preloaded 2-CTC resins, Dmb-amino acid, PyOxim, and Oxyma Pure were from Merck Novabiochem.

(91) Solid-phase peptide syntheses were performed in polypropylene Torviq syringes fitted with a polyethylene porous disc at the bottom and closed with an appropriate piston. Solvent and soluble reagents were removed through back and forth movements. Removal of the Fmoc group was carried out with piperidine/DMF (20%, v/v) (1×1 min, 1×10 min). Washings between deprotection, coupling, and final deprotection steps were carried out with NMP (3×1 min), IPA (3×1 min) and NMP (3×1 min). Peptide synthesis transformations and washes were performed at 20° C. Supported coupling reactions were monitored by classical Kaiser test (directly prepared solution kit from Sigma-Aldrich). Compounds molecular weights were calculated using ChemBioDraw® Ultra 12. All final products were of >95% purity unless otherwise indicated (determined by analytical reverse phase LCMS). Analytical data are given in Table II.

(92) Five methods were conducted for LC-MS analysis:

(93) Method A: analytical HPLC was conducted on a X-Select CSH C18 XP column (2.5 μm 30×4.6 mm id) eluting with 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B), using the following elution gradient 0-3.20 minutes: 5% to 100% B, 3.20-4 minutes 100% B, at a flow rate of 1.8 ml/minute at 40° C. The mass spectra (MS) were recorded on a Waters ZQ mass spectrometer using electrospray positive ionisation [ES+ to give MH.sup.+ molecular ions] or electrospray negative ionisation [ES.sup.− to give (M-H).sup.− molecular ions] modes. The cone voltage was 20V.
Method B: analytical HPLC was conducted on a X-Select CSH C18 XP column (2.5 μm 30×4.6 mm id) eluting with 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B), using the following elution gradient 0-10 minutes: 40% to 100% B, 10-11 minutes 100% B, at a flow rate of 1.8 ml/minute at 40° C. The mass spectra (MS) were recorded on a Waters ZQ mass spectrometer using electrospray positive ionisation [ES.sup.+ to give MH.sup.+ molecular ions] or electrospray negative ionisation [ES− to give (M-H)− molecular ions] modes. The cone voltage was 20V.
Method C: analytical HPLC was conducted on a X-Select CSH C18 XP column (2.5 μm 30×4.6 mm id) eluting with 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B), using the following elution gradient 0-3.20 minutes: 0% to 50% B, 3.20-4 minutes 100% B, at a flow rate of 1.8 ml/minute at 40° C. The mass spectra (MS) were recorded on a Waters ZQ mass spectrometer using electrospray positive ionisation [ES+ to give MH+ molecular ions] or electrospray negative ionisation [ES− to give (M-H)− molecular ions] modes. The cone voltage was 20V.
Method D: analytical HPLC was conducted on a X-Select CSH C18 XP column (2.5 μm 30×4.6 mm id) eluting with 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B), using the following elution gradient 0-6 minutes: 0% to 50% B, 6-7 minutes 100% B, at a flow rate of 1.8 ml/minute at 40° C. The mass spectra (MS) were recorded on a Waters ZQ mass spectrometer using electrospray positive ionisation [ES+ to give MH+ molecular ions] or electrospray negative ionisation [ES− to give (M-H)− molecular ions] modes. The cone voltage was 20V.
Method E: Analytical HPLC was conducted on a X-Select CSH C18 XP column (2.5 μm 30×4.6 mm id) eluting with 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B), using the following elution gradient 0-3 minutes: 5% to 100% B, 3-4 minutes 100% B, at a flow rate of 1.8 ml/minute at 40° C. The high resolution mass spectra (MS) were recorded on a Waters LCT mass spectrometer using electrospray positive ionisation [ES+ve to give MH+ molecular ions] or electrospray negative ionisation [ES−ve to give (M-H)− molecular ions] modes.

(94) Purifications were performed by reverse phase HPLC on either a Waters semi-preparative HPLC-system connected to a Breeze software or a Dionex semi-preparative HPLC-system connected to a Chromeleon softwares, using a C18 semi-preparative columns from AIT using as eluent A, H.sub.2O containing 0.1% of TFA and as eluent B, CH.sub.3CN containing 0.1% of TFA, at a flow rate of 5 mL/min. UV detection was done at 220 nm and at 280 nm. Purification gradients were chosen to get a ramp of approximately 1% solution B per minute in the interest area.

(95) Cyclic Peptide Synthesis:

(96) Cyclic peptides were synthesized using a mixed solid/solution phase procedure. A typical supported synthesis is reported as described earlier (see Peptidomimetic Antibiotics Target Outer-Membrane Biogenesis in Pseudomonas aeruginosa, Nityakalyani Srinivas et al. published 19 Feb. 2010, Science 327, 1010 (2010)). 2-Chlorotritylchloride resin was previously swelled in anhydrous CH.sub.2Cl.sub.2 for 2 h. Fmoc-Aa-OH (0.32 mmol) was coupled to 2-CTC resin (400 mg, loading=1.6 mmol/g) in the presence of diisopropyethylamine (DIPEA, 4 eq.) in CH.sub.2Cl.sub.2 (4 mL). The unreacted sites on the resin were capped by washing with a mixture of CH.sub.2Cl.sub.2/MeOH/DIPEA (7:2:1) followed by MeOH. After removal of the Fmoc-group using 20% piperidine in N,N-dimethylformamide (DMF), chain elongation was performed with standard Fmoc-protected amino acids (Bachem, Switzerland), using 20% piperidine/DMF for Fmoc deprotection, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/1-hydroxybenzotriazole (HBTU/HOBt) for activation, DIPEA as base and N-methyl-2-pyrrolidinone (NMP) as solvent. When assembly of the linear peptide chain was complete, additional MeOH wash was carried for final washing step (1×1 min, 1×15 min) in order to shrink the resin.

(97) Linear peptides were cleaved from the resin by 2 times treatment with HFIP/CH.sub.2Cl.sub.2 cocktail (1:4, v/v) for 15 min each. The reaction mixture is filtered and the resin is sequentially rinsed with CH.sub.2Cl.sub.2 and MeOH. The filtrates are pooled and the solvents were subsequently evaporated under reduced pressure. At the end, the crude linear peptide was precipitated 3 times using dry-ice cold EtO.sub.2 and recovered after centrifugations (3×5 min, 7800 rpm) and drying (under nitrogen flow). The latter system is especially suitable for the cleavage of fully protected fragments to be cyclized in solution, as it eliminates the use of a carboxylic acid in the cleavage step.

(98) For cyclization, the resulting linear peptide (1 eq.) was dissolved in DMF (1 mg/mL concentration), PyBOP (2 eq.), and HOBt (2 eq.) were added to the solution. The pH was adjusted to 8 by adding DIPEA (1% v/v) and the mixture was stirred until LC-MS analysis indicated the completion of the reaction (between 2 and 20 h). The solvent was removed under reduced pressure. In order to remove excess of coupling agents the crude peptide was redissolved in CH.sub.2Cl.sub.2 or Me-THF (50 mL) and organic layer was extracted with saturated NaHCO.sub.3 (2×20 mL) and brine (2×20 mL), dried with Na.sub.2SO.sub.4 and then evaporated under vacuum. After evaporation, TFA/H.sub.2O/TIS final deprotection cocktail (95/2.5/2.5, 20 mL) was smoothly added. The resulting mixture was stirred for 3 h and then precipitated 3 times using dry-ice cold EtO.sub.2 (3×30 mL) and recovered after centrifugations (3×5 min, 7800 rpm) and drying (under nitrogen flow).

(99) The resulting crude cyclic peptide was dissolved in aqueous (11% (v/v) TFA. Purification was conducted on reversed-phase HPLC Prep C18 column, eluting with 0.1% TFA in water (solvent A) and 0.1% TFA in acetonitrile (solvent B), using the following elution gradient of 15% to 35% B in 30 min, at a flow rate of 14 ml/min at 20° C. Peptide fractions from purification were analysed by analytical LC-MS (method C or D), pooled according to their purity and lyophilized on an Alpha 2/4 freeze dryer from Bioblock Scientific to get the expected macrocyclic peptide (mg, μmol) as a white powder with an overall yield of 7%.

(100) TABLE-US-00002 TABLE II (Peptides Molecular Weights given as free ammonium) Peptide Mw (g .Math. mol.sup.−1) m/z (ESI) t.sub.R (method) PKTD1 2289.8 764.2 [M + 3H].sup.3+ 2.33 (C) 573.5 [M + 4H].sup.4+ PKTD3 2261.7 1131.8 [M + 2H].sup.2+  2.25 (C) 754.9 [M + 3H].sup.3+ 566.4 [M + 4H].sup.4+ PKTD6 2275.7 1138.7 [M + 2H].sup.2+  2.50 (C) 749.4 [M + 3H].sup.3+ PKTD7 2101.6 701.5 [M + 3H].sup.3+ 2.94 (D) PKTD9 2424.8 809.3 [M + 3H].sup.3+ 4.69 (D) PKTD10 2275.8 1138.7 [M + 2H].sup.2+  2.35 759.4 [M + 3H].sup.3+ PKTD10-NMe 2289.8 1145.6 [M + 2H].sup.2+  2.52 764.0 [M + 3H].sup.3+ PKTD10-X-NMe 2271.7 1136.6 [M + 2H].sup.2+  2.62 758.0 [M + 3H].sup.3+ PKTD11 2491.0 1246.3 [M + 2H].sup.2+  2.45 831.1 [M + 3H].sup.3+ PKTD11-NMe 2505.0 1253.1 [M + 2H].sup.2+  2.40 838.5 [M + 3H].sup.3+ PKTD11-Q 2433.9 1217.7 [M + 2H].sup.2+  2.59 812.1 [M + 3H].sup.3+ PKTD11-S 2475.0 1238.4 [M + 2H].sup.2+  2.46 825.9 [M + 3H].sup.3+ PKTD12 2316.8 1159.3 [M + 2H].sup.2+  2.40 773.1 [M + 3H].sup.3+ PKTD16 2589.2 1295.3 [M + 2H].sup.2+  2.38 863.9 [M + 3H].sup.3+ 648.1 [M + 4H].sup.4+ PKTD18 2245.7 1123.6 [M + 2H].sup.2+  2.51 749.3 [M + 3H].sup.3+ PKTDi1 2275.8 1138.7 [M + 2H].sup.2+  2.37 759.4 [M + 3H].sup.3+ PKTDi2 2101.6 1151.6 [M + 2H].sup.2+  2.28 701.3 [M + 3H].sup.3+ PKTDi3 1831.3 1831.9 [M + 2H].sup.2+  2.85 916.4 [M + 3H].sup.3+ PKTDi4 1646.1 1646.7 [M + 2H].sup.2+  3.26 823.8 [M + 3H].sup.3+ PKD8 1646.1 1646.7 [M + 2H].sup.2+  3.57 823.8 [M + 3H].sup.3+ PKD9 1628.0 1628.7 [M + 2H].sup.2+  3.64 814.7 [M + 3H].sup.3+ PKD10 1643.1 1643.8 [M + 2H].sup.2+  3.12 822.3 [M + 3H].sup.3+ L-PKD10 1661.1 1661.7 [M + 2H].sup.2+  2.65 831.1 [M + 3H].sup.3+ PKD10-FF 1627.1 1627.8 [M + 2H].sup.2+  3.33 814.3 [M + 3H].sup.3+
2. In Vitro Activity of Cyclic Peptides According to the Invention

(101) The effects of several cyclic peptides of the invention on the proliferation of tumor cells were evaluated on 5 cell lines (MCF-7, human breast cancer cells; HCT-116, human colon cancer cells; BxPC3, human pancreas cancer cells and A549, human lung cancer cells) by cytotoxic assay and by counting directly the number of cells.

(102) 2.1. Materials

(103) The cyclic peptides of general formula (I), PKTD1, PKTD7, PKTD9, PKTD10, PKTD10-3, PKTD10-RNMe, PKTD10-X-RNMe, PKTD10-4, PKTD11, PKTD11RNMe, PKTD12, PKTD16, PKTD18, PKD8, PKD10 and PKD10-FF were synthesized as described in the experimental part.

(104) Those cyclic peptides were dissolved in DMSO at the following concentrations: 0, 5, 10, 25, 50 and 100 μM.

(105) PKC1 is a cyclic peptide that only comprise fragment of beta-strand No 7 of TSP-1 (SEQ ID NO:8); its structure is as follows:

(106) ##STR00048##

(107) Linear PKT16 peptide [(D)Lys-(N-Me)Arg-Phe-Tyr-Val-Val-Nle-Trp-Lys-(D)Lys] (SEQ ID NO:66) was used as positive control.

(108) Compound CTGG (also called 4NGG, a linear peptide of sequence KRFYGGMWKK) was used as negative control.

(109) Culture media used for these assays are: EMEM=10% SVF for MCF-7 and Wi38; RPMI 1640=10% SVF for HCT-116 and BxPC3 and F-12K=10% SVF for A549.

(110) 2.2. Methods

(111) 2.2.1. Analysis of Cell Viability and Proliferation

(112) 500 cells were seeded in 96-well plates, incubated at 37° C. for 24 hours, and treated by the cyclic peptides at the different concentrations and controls for 2 h.

(113) 2.2.2. Cell Death Analysis by Flow Cytometry

(114) To detect possible apoptotic processes, cells were seeded in 35 mm dishes and cultured in media containing each tested cyclic peptide for 2 hours following procedure A or B.

(115) Procedure A: Etopiside (40 nM) was used as a positive control to induce apoptosis. Cells were then trypsinized, washed in cold PBS, and stained with Annexin V-FrrC (BD Pharmingen) in Annexin buffer for 15 min at room temperature. Finally, they were counterstained with 50 μg/mL propidium iodide (Sigma) and analyzed with a FACSCalibur flow cytometer. Experiment on each cell type was repeated three times. 20,000 events per sample were analyzed in each experiment.

(116) Procedure B: Peptides were incubated for 2 hours on HCT-116 cells. Superkiller Trail (ALX-201-115C-010) was used as a positive pro-apoptotic control. Cells were analyzed by cytofluorometry using FITC Annexin V apoptosis detection kit with 7-AAD from BIOLEGEND (BLE640922).

(117) 2.3. Results

(118) Results of these assays are presented in FIGS. 7A to 7F and in the tables III to VI below:

(119) TABLE-US-00003 TABLE III Mean (%) Late Early apoptosis Apoptosis Viable Superkiller 100 ng/mL 1.79 52.55 45.59 PKTD10-3 0 2.04 2.83 94.96 12.5 4.35 8.17 86.83 25 7.06 13.16 79.30 50 6.85 13.13 79.67 PKTD10-4 0 2.04 2.83 94.96 12.5 4.39 9.27 85.90 25 5.54 11.91 82.41 50 7.53 15.68 76.52 PKTD10- 0 2.04 2.83 94.96 RNMe 12.5 2.17 5.97 91.72 25 2.77 6.04 91.10 50 8.10 14.95 76.73 PKTD12 0 2.04 2.83 94.96 12.5 3.11 5.78 90.87 25 3.26 7.15 89.42 50 6.80 19.16 73.78

(120) TABLE-US-00004 TABLE IV Mean (%) Late Early apoptosis Apoptosis Viable Superkiller 100 ng/mL 2.06 41.42 56.39 DMSO 0 7.41 5.72 86.35 PKD10 12.5 15.76 18.30 65.66 25 24.58 43.67 31.50 50 39.02 32.70 27.55 PKTD1 0 7.41 5.72 86.35 12.5 11.60 11.42 76.44 25 18.77 17.24 63.51 50 36.99 24.68 37.56 PKTD9 0 7.41 5.72 86.35 12.5 7.83 9.78 82.19 25 8.58 11.40 79.38 50 14.94 13.32 70.52

(121) TABLE-US-00005 TABLE V Mean (%) Late Early apoptosis Apoptosis Viable Superkiller 100 ng/mL 1.79 52.55 45.59 PKD10 0 2.04 2.83 94.96 12.5 10.07 40.48 49.30 25 16.97 59.50 23.28 50 56.90 28.42 12.74 PKTD10-X- 0 2.04 2.83 94.96 NMe 12.5 5.15 11.92 82.76 25 6.96 17.66 75.17 50 20.43 30.86 48.31 PKTD11RNMe 0 2.04 2.83 94.96 12.5 3.17 7.80 88.71 25 3.99 9.39 86.38 50 9.58 23.06 67.03 PKTD18 0 2.04 2.83 94.96 12.5 5.21 12.14 82.45 25 8.59 15.95 75.12 50 9.60 22.48 67.48

(122) TABLE-US-00006 TABLE VI Mean (%) Late Early apoptosis Apoptosis Viable Superkiller 100 ng/mL 1.54 48.43 49.92 DMSO 0 1.60 2.94 95.35 PKD10 12.5 5.14 11.41 83.31 25 7.36 45.90 46.71 50 24.87 50.77 23.93 PKD10-FF 0 6.79 93.2 12.5 74.66 25.225 25 69.29 30.445 50 67.35 31.97 PKC1 12.5 2.25 4.54 92.95 25 2.56 4.55 92.72 50 2.75 4.98 92.15

(123) All tested cyclic peptides PKTD1, PKTD7, PKTD9, PKTD10, PKTD10-3, PKTD10-RNMe, PKTD10-X-RNMe, PKTD10-4, PKTD11, PKTD11RNMe, PKTD12, PKTD16, PKTD18, PKD10 and PKD10-FF show a dose-dependent viability decrease in all cell strains (from 20 to 80% of PCD induction in 2 hours from 10 to 50 μM peptide concentration). This activity is significantly higher than positive control (PKT16) which is not efficient at the concentrations tested here (efficacy to induce PCD not observed at 100 μM).

(124) Cyclic peptide PKC1 shows no efficacy in triggering PCD whatever the concentration used (from 12.5 to 50 μM, same results are observed); this result demonstrates the importance of the cyclic hairpin structure involving the beta strands 6 and 7 or 7 and 8 of TSP-1, highlighting the fact that the simple cyclisation of the 4N1 CD47-binding epitope of TSP-1 is not sufficient to improve its potency.

(125) Cyclic peptide PKD10 induces a very significant decrease of cell viability at a low dose (25 μM) after 2 hours of incubation (FIG. 7F).

(126) These results thus allow considering cyclic peptide of the invention as very promising tools for treating diseases associated with defects in PCD such as tumors and immunological diseases (including diseases associated with chronic inflammation), either in animals or in human beings. Besides its therapeutic efficiency in reducing tumor size, those cyclic peptides do not show strong toxicity usually associated with cytotoxic drugs. It is therefore embodied as being the future therapeutic agent for treating patients suffering from tumors.

(127) 3. Binding Experiments

(128) Binding affinity measurements. The binding affinities of peptides, here PKT16, PKTD1, PKTD10, PKTD10-1, PKTD10-3, PKTD10-5, PKDT10-7 and PKTD10-8 for a membrane preparation from Jurkat and/or MEC-1 cells were measured by Microscale Thermophoresis.sup.xlv on a Monolith NT115-pico system (Nanotemper Technologies, Munich, Germany).

(129) Binding curve measured by MST. The measurement method is based on the directed movement of molecules along a temperature gradient, an effect termed “thermophoresis”. A local temperature difference ΔT leads to a local change in molecule concentration (depletion or enrichment), quantified by the Soret coefficient S.sub.T: c.sub.hot/c.sub.cold=exp(−S.sub.TΔT). MEC-1 or Jurkat membrane preparation is labeled using the Nanotemper NT-647 labeling kit as described elsewhere..sup.xlvi The labeled preparation is eluted with PBS and stored at 4° C. A stock solution of each peptide is prepared in DMSO (5 mM) and then diluted with PBS. For the peptides evaluated by MST, we have kept the concentration of the NT.115-labeled membrane constant, while the concentration of the ligand (peptide) was varied. After a short incubation the samples were loaded into MST premium glass capillaries and the MST analysis was performed using the Monolith NT.115-pico. FIG. 5: MST curve observed for PKT16 [(D)Lys-(N-Me)Arg-PheTyr-Val-Val-Nle-Trp-Lys-(D)Lys] (SEQ ID NO:66) a linear peptide derived from the C-terminal binding domain of TSP-1. The Kd=1600 nM. FIG. 6A to 6H: MST curve observed for PKTD1, PKTD10, PKTD10-1, PKTD10-3, PKTD10-4, PKTD10-5, PKTD10-7, PKTD10-8 [see table II] all cyclic peptide analogues of the C-terminal binding domain of TSP-1. The Kd (from 0.1 to 505 nM). The Kd ratio highlights the fact that these cyclic analogues are all much more efficient in CD47 ligation than the linear analogues designed from the beta-strand 7. Kd of these cyclic peptides is comprised between 2.8 and 50 nM; this value is highly variable depending the tumor cells and their preparation.

(130) 4. Stability Assays

(131) Proteolytic stability studies were performed by peptides incubation with proteases (such as Trypsin, Chymotrypsin and Proteinase K) or in Human Serum as described earlier (See Karoyan et al. J. Med. Chem. 2016). In these conditions, peptides such as PKD8, PKD9 or PKD10 appeared to be fully stable toward proteases whereas PKC1 is degraded by Trypsin in less than 2 hours like the linear analogue of PKD10, i.e L-PKD10: cyclisation is not sufficient to improve the metabolic stability (PKC1) neither to improve the pharmacological profil (PKC1) but the development of a stable harpin mimetic of the C-terminal binding domain of TSP-1 led to stable analogues (at least PKD8, PKD9 and PKD10) with improved pharmacological properties and ability to induce programmed Cell death (PKD8, PKD10, PKD10-FF at least).

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