Bcl2 inhibitor and peptide combination therapy for treatment of proliferative diseases

Abstract

The present disclosure relates to combination therapies comprising a Bcl2 inhibitor and a peptide. The disclosure also relates to compositions, methods and kits comprising a Bcl2 inhibitor and a peptide for treating proliferative diseases.

Description

Technical Field

[0001] The present disclosure relates to combination therapies of B-cell lymphoma 2 (Bcl2) inhibitors and peptides for the treatment of proliferative diseases. Background Art

[0002] The following is a list of references that are believed to be relevant to the background art of the presently disclosed subject matter:

[0003] -[1]Lessene G, Czabotar PE, Colman PM. BCL-2family antagonists for cancer therapy. Nat Rev Drug Discov. 2008;7(12):989-1000.

[0004] -[2] WO 2006 / 046239

[0005] -[3] WO 2007 / 122622

[0006] -[4] WO 2007 / 091240

[0007] -[5] WO 2008 / 075349

[0008] -[6]Sandler,U.et al.,2010,Recent advances in clinical medicine,ISSN:1790-5125.

[0009] -[7]Sandler,U.et al.,2010,J Experimental Therapeutics and Oncology8:327-339.

[0010] -[8] WO2015 / 083167

[0011] -[9] WO2017 / 134668

[0012] Acknowledgment herein of the above references should not be inferred as an intention that these references are in any way relevant to the patentability of the presently disclosed subject matter. Background of the Invention

[0014] BCL2-related proteins are a large family of proteins that regulate the intrinsic or mitochondrial pathway of apoptosis through non-enzymatic protein:protein interactions, thereby preventing or promoting apoptosis.

[0015] Pro-survival proteins include, for example, BCL-2 itself, B-cell lymphoma-extra large (BCL-xL), myeloid cell leukemia-1 (MCL-1), B-cell lymphoma-w (BCL-w), and BCL-2-related gene 1 expressed in fetal liver.

[0016] On the other hand, proapoptotic proteins are further divided into (a) proapoptotic BH3-only proteins, including BCL-2-interacting mediator of cell death (BIM), p53-upregulated regulator of apoptosis, and BCL-2-associated death promoter, and (b) proapoptotic effector proteins BCL-2-associated X protein (BAX) and BCL-2 homologous antagonist killer protein (BAK).

[0017] The balance of activity between pro-survival and BH3-only pro-apoptotic proteins determines whether a cell survives or undergoes apoptosis, and they work together to integrate multiple extracellular and intracellular signals that promote survival (e.g., growth factors, nutrients) or death induced by stress (e.g., oncogenesis / proliferation, DNA damage, etc.).

[0018] BH3 mimetics are a new class of anticancer drugs that mimic the effects of BH3-only proteins because they bind to pro-survival proteins such as BCL2 in the same manner and inhibit the ability of BCL2 to bind to BAX or BAK [1]. Since BCL2 also binds to native BH3-only proteins, BH3 mimetics can also replace these endogenous activators of apoptosis. For example, venetoclax is a BCL2-selective BH3 mimetic and can effectively trigger apoptosis when added to cancer cells that overexpress BCL2 in vitro.

[0019] A peptide called "T101" encoded by a cDNA unique to the human thymus has been previously identified. This peptide and its derivatives are used to treat cancer, particularly through the use of T101 as an immune system stimulator (WO 2006 / 046239, [2]). WO 2006 / 046239 demonstrated that T101 was able to stimulate the immune system and reduce tumor size, indicating that the peptide affects the proliferation of cancer cells. WO 2006 / 046239 also proposed immune-based effects of T101, such as protecting patients during standard chemotherapy.

[0020] The treatment of cancer by using T101 is also proposed in WO 2007 / 122622 [3], which in particular demonstrates the effect of T101 on the development of various types of tumors. The peptide T101 is also described in WO 2007 / 091240 [4] (related to the treatment of immune diseases) and WO 2008 / 075349 [5] and in publications by Sandler et al. [6-7], which are related to the treatment or prevention of diseases involving cells with T1 / ST2 receptors.

[0021] In addition, a peptide derivative of T101 called "Nerofe" has been reported to reduce the secretion of proteins known to be associated with cancer metastasis by cancer cells and directly inhibit the migration of cancer cells in vitro. In addition, the peptide has been shown to affect serum levels of vascular endothelial growth factor (VEGF) in cancer patients (WO2015 / 083167, [8]) and has been suggested for use in methods for preventing or treating cancer metastasis.

[0022] In addition, the above-mentioned peptide called "Nerofe" was also shown in WO2017 / 134668 [9] to be involved in the induction of ER stress to promote cell death, and it was suggested to reduce the standard of care dose of anti-cancer agents administered to cancer patients undergoing treatment. Summary of the invention

[0023] In a first aspect, the present disclosure provides a combined composition comprising at least one inhibitor of B-cell lymphoma 2 (Bcl2) pro-survival protein and an isolated peptide comprising the amino acid sequence shown in SEQ ID NO.1 or any functional derivative thereof, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent, excipient and / or additive.

[0024] In a second aspect, the present disclosure provides a method for treating, preventing, ameliorating, reducing or delaying the onset of at least one proliferative disorder in a subject in need thereof, the method comprising the step of administering a therapeutically effective amount of at least one inhibitor of a Bcl2 pro-survival protein and an isolated peptide comprising the amino acid sequence shown in SEQ ID NO.1 or any functional derivative thereof, or a pharmaceutically acceptable salt thereof, or any composition, kit or combination thereof.

[0025] In a third aspect, the present disclosure provides at least one inhibitor of Bcl2 pro-survival protein and at least one isolated peptide comprising the amino acid sequence shown in SEQ ID NO.1 or any functional derivative thereof, or a pharmaceutically acceptable salt thereof, for use in a method of treating, preventing, ameliorating, reducing or delaying the onset of at least one proliferative disorder.

[0026] In a fourth aspect, the present disclosure provides a kit comprising:

[0027] (a) at least one inhibitor of a Bcl2 pro-survival protein, or any pharmaceutically acceptable salt thereof, optionally in a first dosage form;

[0028] (b) optionally in a second dosage form an isolated peptide comprising the amino acid sequence shown in SEQ ID NO. 1 or any functional derivative thereof, or a pharmaceutically acceptable salt thereof.

[0029] In a fifth aspect, the present disclosure provides a method for treating, preventing, ameliorating, reducing or delaying the onset of at least one proliferative disorder in a subject treated with at least one inhibitor of a Bcl2 pro-survival protein, the method comprising the step of administering to the subject a therapeutically effective amount of an isolated peptide comprising the amino acid sequence shown in SEQ ID NO.1 or any functional derivative thereof, or a pharmaceutically acceptable salt thereof, or any composition, kit or combination thereof.

[0030] In a sixth aspect, the present disclosure provides a method for treating, preventing, ameliorating, reducing or delaying the onset of at least one proliferative disorder in a subject treated with at least one isolated peptide comprising the amino acid sequence shown in SEQ ID NO.1, the method comprising the step of administering to the subject a therapeutically effective amount of at least one inhibitor of Bcl2 pro-survival protein or any functional derivative thereof, or a pharmaceutically acceptable salt thereof, or any composition, kit or combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to better understand the subject matter disclosed herein and to illustrate how it may be implemented in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0032] Figure 1 is a graph showing that a drug called Nerofe and ABT-737 act synergistically to induce apoptosis in U937 cells as measured by activation of caspase 3.

[0033] Figure 2 is a graph showing that Nerofe synergistically induces death of U937 cells with ABT-737, as measured by the percentage of cell viability using Resazurin.

[0034] Figure 3 is a graph showing that Nerofe does not synergize with Azacitidine to induce death of U937 cells as measured by the percentage of cell viability using Resazurin.

[0035] Figure 4 is a graph showing that Nerofe synergizes with ABT-737 to reduce CT26 cell viability as measured by the percentage of cell viability using resazurin.

[0036] Figure 5 are pictures of Western blot staining of cleaved caspase 3 in CT26 cells treated with Nerofe in the presence / absence of ABT-737. DETAILED DESCRIPTION

[0037] Bcl2 pro-survival protein inhibitors, a new class of anti-cancer drugs, are shown herein to act synergistically with a peptide called "Nerofe" to induce apoptosis and cell death in malignant cells.

[0038] Therefore, in a first aspect, the present disclosure provides a combined composition comprising at least one inhibitor of B-cell lymphoma 2 (Bcl2) pro-survival protein and an isolated peptide comprising the amino acid sequence shown in SEQ ID NO.1 or any functional derivative thereof, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent, excipient and / or additive.

[0039] The term "isolated peptide" as defined herein encompasses an isolated peptide comprising the following items: the amino acid sequence shown in SEQ ID NO.1 (the amino acid sequence of the full D configuration Trp Trp Thr Phe Phe Leu Pro Ser Thr Leu Trp GluArg Lys), referred to herein as "dTCApFs" or "Nerofe", and a functional derivative of the amino acid sequence shown in SEQ ID NO.1, or a pharmaceutically acceptable salt of the isolated peptide. The present disclosure encompasses any pharmaceutically acceptable salt of the isolated peptide as defined herein, particularly the acetate of the peptide.

[0040] The term "peptide" as defined herein refers to a molecular chain of amino acid residues which may be modified, if desired, at each of its amino acid residues, for example by mannosylation, glycosylation, amidation (e.g., C-terminal amide), carboxylation or phosphorylation. Peptides may be obtained synthetically by genetic engineering methods, expression in a host cell or by any other suitable method. Methods for producing peptides are well known in the art.

[0041] More specifically, "amino acid molecule", "amino acid sequence" or "peptide sequence" refers to the chain order of amino acid residues connected by peptide bonds in peptides and proteins. The sequence is usually reported from the N-terminus containing the free amino group to the C-terminus containing the amide. If the amino acid sequence represents the primary structure of the protein, the amino acid sequence is usually called a peptide, protein sequence, however, one must distinguish the terms "amino acid sequence" or "peptide sequence" and "protein" because proteins are defined as amino acid sequences folded into a specific three-dimensional configuration and usually undergo post-translational modifications such as phosphorylation, acetylation, glycosylation, mannosylation, amidation, carboxylation, sulfhydryl bond formation, cleavage, etc.

[0042] As used herein, amino acids refer to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those amino acids encoded by the genetic code, as well as those amino acids that are subsequently modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. "Amino acid analogs" refer to compounds having the same basic chemical structure as naturally occurring amino acids, i.e., α carbon bound to hydrogen, carboxyl groups, amino groups, and R groups, such as homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. Such analogs have modified R groups or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. "Amino acid mimetics" refer to compounds having a structure different from the general chemical structure of amino acids but functioning in a manner similar to naturally occurring amino acids. Amino acids may be referred to herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0043] The term "isolated" refers to molecules, such as amino acid sequences or peptides, that are removed, separated or isolated from their natural environment.

[0044] As used herein, the term "amino acid" refers to naturally occurring amino acid residues and synthetic amino acid residues, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are subsequently modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine.

[0045] The term amino acid encompasses L-amino acids and D-amino acids, the latter being the mirror image of the L-amino acids in which the chirality at the alpha carbon has been inverted. D-amino acids are highly resistant to protease-mediated degradation and have a low immunogenic response.

[0046] The term "amino acid sequence" or "peptide sequence" also refers to the chain order of amino acid residues connected by peptide bonds in peptides and proteins. The sequence is usually reported from the N-terminus containing the free amino group to the C-terminus containing the free carboxyl group.

[0047] The term "comprising" means that the isolated peptide according to the present disclosure includes the peptide shown in SEQ ID NO: 1, but may further include additional amino acid residues at the N-terminus or C-terminus of the peptide, or at both termini.

[0048] As described above, the present disclosure also encompasses isolated peptides, including derivatives of peptides having the amino acid sequence shown in SEQ ID NO. 1. The term "derivative" is intended to include peptides that contain the amino acid sequence shown in SEQ ID NO: 1, but have one or more amino acids that differ in their overall sequence, i.e., have a deletion, substitution (e.g., at least one amino acid is replaced by another amino acid), inversion, or addition within the overall sequence of SEQ ID NO: 1. The term also encompasses at least one amino acid residue in the overall sequence being replaced by its corresponding L amino acid residue.

[0049] In particular embodiments, the modified isolated peptide of the present disclosure or composition of the invention has at least 70%, or 80%, or 90%, or 95%, or in particular 99% identity to the corresponding sequence of SEQ ID NO:1.

[0050] In some embodiments, compared to an unmodified peptide having the amino acid sequence of SEQ ID NO.1, a peptide comprising the amino acid sequence shown in SEQ ID NO.1 may include one or more amino acid residues replaced by conservative substitutions without significantly affecting the biological characteristics of the modified peptide.

[0051] An amino acid "substitution" is the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties, i.e., a conservative amino acid substitution. Amino acid substitutions can be made based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues involved. For example, each of the following eight groups contains amino acids, and the amino acids in each group are conservative substitutions for each other:

[0052] 1) Alanine (A), Glycine (G);

[0053] 2) Aspartic acid (D), Glutamic acid (E);

[0054] 3) Asparagine (N), glutamine (Q);

[0055] 4) Arginine (R), Lysine (K);

[0056] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0057] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W);

[0058] 7) serine (S), threonine (T); and

[0059] 8) Cysteine ​​(C), methionine (M).

[0060] It should be understood that these peptide derivatives must not alter the biological activity of the original peptide. The term "functional" means that the modified (i.e., derivative) retains a qualitatively similar biological activity to that of the unmodified peptide. The biological activity of the derivative can be determined as described herein, i.e., by monitoring the effects of the derivative after administration to an animal model, as known in the art.

[0061] In a specific embodiment, the present disclosure relates to a functional derivative or functional fragment of an isolated polypeptide comprising the amino acid sequence shown in SEQ ID NO.1, wherein the functional derivative or functional fragment has an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90% or 95%, in particular 99% identical to the amino acid sequence of the unmodified isolated polypeptide of the present invention (i.e., the amino acid sequence shown in SEQ ID NO:1), and retains a biological activity similar in quality to the unmodified peptide.

[0062] In some specific embodiments, the isolated peptide consists of the amino acid sequence of SEQ ID NO:1.

[0063] In addition, as used herein, the term Bcl-2 pro-survival protein refers to a proto-oncogene protein known to act as an inhibitor of apoptosis. Bcl-2 proteins form the basis of a growing family of related proteins, which are collectively referred to herein as Bcl-2 family proteins. These proteins are known to control apoptotic cell death through the mitochondrial pathway.

[0064] As understood in the art, members of the Bcl-2 family are either pro-survival or pro-apoptotic, but regardless of their activity, they all share significant sequence and structural homology. Specifically, the Bcl-2 family of proteins is characterized by up to four regions of sequence homology, termed Bcl-2 homology (BH) domains.

[0065] The Bcl-2 protein family includes three different groups of proteins: the first group is the pro-survival or anti-apoptotic group referred to herein as "Bcl-2 pro-survival proteins", the second group is the pro-apoptotic group including BAX and BAK; and the third group is referred to herein as BH3-only proteins, which exhibit pro-apoptotic activity.

[0066] "Bcl-2 pro-survival proteins" or "anti-apoptotic" or "Bcl-2-like" as used herein refers to a group of proteins responsible for protecting cells from apoptotic stimuli and characterized by containing all four BH domains in sequence.

[0067] It has been previously shown that the balance between pro-survival Bcl-2 proteins and pro-apoptotic Bcl-2 proteins and the specific interactions between them determine the activity of the protein family in response to apoptotic stimuli. For example, in contrast to Bcl-2 pro-survival proteins, BH3-only proteins initiate apoptosis in response to a variety of cellular stresses including DNA damage, growth factor deprivation, and intracellular stress. Activation of BH3-only proteins can involve transcriptional upregulation or modification.

[0068] Several mechanisms have been proposed in the art to explain the multiple activities and tight control of apoptosis in the Bcl-2 family, some of which involve Bax and Bak. Regardless of the exact mechanism, it is clear that the balance of pro-survival proteins and pro-apoptotic (BH3 only) proteins is key to this activation.

[0069] According to some embodiments, the Bcl2 inhibitor may antagonize at least one of the following: any Bcl-2 pro-survival protein, such as Bcl-2, Bcl-xL, Mcl-1, Bcl-w, Bcl2A1, and Bcl-B / Bcl2L10.

[0070] In some additional embodiments, the Bcl-2 pro-survival protein of the composition of the present invention is at least one of the following: Bcl-2, B-cell lymphoma-extra large (Bcl-xL), myeloid cell leukemia-1 (Mcl-1), B-cell lymphoma-w (Bcl-w), B-cell lymphoma 2-associated protein A1 (Bcl2A1), and B-cell lymphoma 2-like 10 (Bcl-B / Bcl2L10).

[0071] More specifically, in some embodiments, Bcl2 inhibitors can bind to and antagonize Bcl-2 proteins. As used herein, Bcl-2 (B cell CLL / lymphoma 2) is an integral outer mitochondrial membrane protein that blocks apoptotic death of some cells such as lymphocytes. Bcl-2 inhibits apoptosis in a variety of cell systems, including factor-dependent lympho-hematopoietic cells and neural cells. It regulates cell death by controlling mitochondrial membrane permeability. Bcl-2 appears to work in a feedback loop system with caspases, inhibiting caspase activity by preventing cytochrome c from being released from mitochondria and / or by binding to apoptosis activating factor (APAF-1). It should be noted that in certain embodiments, the present invention relates to the human Bcl-2 protein represented by GenBank Accession No. NP 000624 and SEQ ID NO:2 and NP 000648 of SEQ ID NO:3, which is encoded by the Bcl-2 gene of GenBank Accession No. NM_000633 of SEQ ID NO:4 and NM_000657 of SEQ ID NO:5.

[0072] In another embodiment, a Bcl2 inhibitor can bind to and antagonize Bcl-xL. As used herein, B-cell lymphoma-extra large (Bcl-xL) is a transmembrane molecule in mitochondria. It is a member of the Bcl-2 protein family and acts as a pro-survival protein by preventing the release of mitochondrial contents such as cytochrome c, which will lead to caspase activation. In certain embodiments, the present invention relates to human Bcl-xL protein (GenBank Accession No. CAA80661 SEQ ID NO: 6), which is encoded by the Bcl-xL gene shown in GenBank Accession No. Z23115 and SEQ ID NO: 7.

[0073] In yet another embodiment, the Bcl2 inhibitor may bind to and antagonize any of the human Bcl-2 pro-survival proteins Mcl-1, Bcl-w, Bcl2A1, and Bcl-B / Bcl2L10 represented by the following accession numbers, respectively: NP_068779.1, AAB09055, NP_004040.1, and NP_001293097.1.

[0074] In some other embodiments, the at least one inhibitor of a Bcl2 pro-survival protein is at least one Bcl-2 homology 3 (BH3) mimetic compound.

[0075] As mentioned above, BH3 mimetics are a class of anticancer drugs that mimic the effects of BH3-only proteins in that they bind pro-survival proteins such as BCL2 in the same manner and inhibit the ability of BCL2 to bind BAX or BAK.

[0076] In some embodiments, the BH3 mimetic compound is at least one of the following or any combination thereof: 4-{4-[(4'-chloro[1,1'-biphenyl]-2-yl)methyl]piperazin-1-yl}-N-(4-{[(2R)-4-(dimethylamino)-1-(phenylthio)butan-2-yl]amino}-3-nitrobenzene-1-sulfonyl)benzamide (ABT-737), 4-(4-{[2-(4-chlorophenyl)-4,4-dimethyl-1-cyclohexen-1-yl]methyl}-1-piperazinyl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)-amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]-pyridin-5-yloxy)benzamide (ABT-199), 4-(4-{[2-(4-chlorophenyl)-4,4-dimethyl-1-cyclohexen-1-yl]methyl}-1-piperazinyl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)-amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]-pyridin-5-yloxy)benzamide (ABT-199), {[2-(4-chlorophenyl)-5,5-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-(4-{[(2R)-4-(morpholin-4-yl)-1-(phe-nylthio)butan-2-yl]amino}-3-(trifluoromethanesulfonyl)benzene-1-sulfonyl)benzamide (ABT-263), 2-(2-((3,5-dimethyl-1H -pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-yl)-1H-indole (GX15-070), or 3-[1-(1-adamantylmethyl)-5-methylpyrazol-4-yl]-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydro-1H-isoquinolin-2-yl]pyridine-2-carboxylic acid (A-1331852).

[0077] In some additional embodiments, the at least one inhibitor of a Bcl2 pro-survival protein or a BH3 mimetic compound is 4-{4-[(4'-chloro[1,1'-biphenyl]-2-yl)methyl]piperazin-1-yl}-N-(4-{[(2R)-4-(dimethylamino)-1-(phenylthio)butan-2-yl]amino}-3-nitrobenzene-1-sulfonyl)-benzamide (ABT-737), or any pharmaceutically acceptable salt or hydrate thereof, or any stereoisomer or salt thereof.

[0078] ABT-737 is a small molecule drug that inhibits Bcl-2 and Bcl-xL, two members of the evolutionarily conserved Bcl-2 protein family that share a Bcl-2 homology (BH) domain. ABT-737 is not bioavailable after oral administration, so navitoclax (ABT-263) was developed as an orally available derivative with similar activity against small cell lung cancer (SCLC) cell lines.

[0079] ABT-737 has the following chemical structure, as shown in Formula I:

[0080]

[0081] The systematic (TUPAC) name of ABT-737 is 4-{4-[(4'-chloro[1,1'-biphenyl]-2-yl)methyl]piperazin-1-yl}-N-(4-{[(2R)-4-(dimethylamino)-1-(phenylthio)butan-2-yl]amino}-3-nitrobenzene-1-sulfonyl)benzamide (C 42 H 45 CIN6O5S2; CAS number: 852808-04-9). The molecular weight of ABT-737 is 813.43 g / mol.

[0082] In some additional embodiments, the at least one inhibitor of Bcl2 pro-survival protein or BH3 mimetic compound is 4-(4-{[2-(4-chlorophenyl)-4,4-dimethyl-1-cyclohexen-1-yl]methyl}-1-piperazinyl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)-amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]-pyridin-5-yloxy)benzamide (ABT-199), or any pharmaceutically acceptable salt or hydrate thereof, or any stereoisomer or salt thereof.

[0083] ABT-199 or venetoclax, sold under the brand names Venclexta and Venclyxto, is a drug used to treat adults with chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), or acute myeloid leukemia (AML). Venetoclax binds to the Bcl-2 protein, which is present in large quantities in CLL cancer cells, where it helps the cancer cells survive longer in the body and makes them resistant to anticancer drugs. By binding to Bcl-2 and blocking its action, venetoclax causes cancer cells to die, thereby slowing disease progression. Venetoclax is the first selective BCL2 inhibitor approved for routine clinical practice, specifically in chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML).

[0084] ABT-199 has the following chemical structure, as shown in Formula II:

[0085]

[0086] The systematic (IUPAC) name of ABT-199 is 4-(4-{[2-(4-chlorophenyl)-4,4-dimethyl-1-cyclohexen-1-yl]methyl}-1-piperazinyl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)-amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide (C45H50C1N7O7S; CAS No.: 1257044-40-8). The molecular weight of ABT-199 is 868.45 g / mol.

[0087] In some additional embodiments, the at least one inhibitor of a Bcl2 pro-survival protein or a BH3 mimetic compound is 4-(4-{[2-(4-chlorophenyl)-5,5-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-(4-{[(2R)-4-(morpholin-4-yl)-1-(phe-nylthio)butan-2-yl]amino}-3-(trifluoromethanesulfonyl)benzene-1-sulfonyl)benzamide (ABT-263), or any pharmaceutically acceptable salt or hydrate thereof, or any stereoisomer or salt thereof.

[0088] ABT-263 or Navitoc is an orally active anticancer drug, i.e., a Bcl-2 inhibitor. ABT-263 has the following chemical structure, as shown in Formula III:

[0089]

[0090] The systematic (IUPAC) name of ABT-263 is 4-(4-{[2-(4-chlorophenyl)-5,5-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-(4-{[(2R)-4-(morpholin-4-yl)-1-(phe-nylthio)butan-2-yl]amino}-3-(trifluoromethanesulfonyl)benzene-1-sulfonyl)benzamide (C 47 H 55 CIF3N5O6S3; CAS number: 923564-51-6). The molecular weight of ABT-263 is 974.61 g / mol.

[0091] In some additional embodiments, the at least one inhibitor of Bcl2 pro-survival protein or BH3 mimetic compound is 2-(2-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-yl)-1H-indole (GX15-070), or any pharmaceutically acceptable salt or hydrate thereof, or any stereoisomer or salt thereof.

[0092] GX15-070 or obatoclax is a drug used to treat various types of cancer. Obatoclax is an inhibitor of the Bcl-2 family of proteins. This inhibition can induce apoptosis in cancer cells, thereby preventing tumor growth. GX15-070 has the following chemical structure, as shown in Formula IV:

[0093]

[0094] The systematic (IUPAC) name of GX15-070 is 2-(2-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-yl)-1H-indole (C 20 H 19 N3O; CAS No.: 803712-7-6). The molecular weight of GX15-070 is 317.392 g / mol.

[0095] In some additional embodiments, the at least one inhibitor of Bcl2 pro-survival protein or BH3 mimetic compound is 3-[1-(1-adamantylmethyl)-5-methylpyrazol-4-yl]-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydro-1H-isoquinolin-2-yl]pyridine-2-carboxylic acid (A-1331852), or any pharmaceutically acceptable salt or hydrate thereof, or any stereoisomer or salt thereof.

[0096] A-1331852 is the first class of orally active BCL-XL inhibitors that selectively and effectively induce apoptosis in BCL-XL-dependent tumor cells. A-1331852 has the following chemical structure, as shown in Formula V:

[0097]

[0098] The systematic (IUPAC) name of A-1331852 is 3-[1-(1-adamantylmethyl)-5-methylpyrazol-4-yl]-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydro-1H-isoquinolin-2-yl]pyridine-2-carboxylic acid (C 38 H 38 N6O3S; CAS number: 1430844-80-6). The molecular weight of A-1331852 is 658.27 g / mol.

[0099] In some embodiments, the composition may comprise at least one additional therapeutic agent, such as an anti-cancer agent. In some additional embodiments, the composition does not comprise taxol (also known as paclitaxel) or doxorubicin or an anti-PDL1 antibody.

[0100] In yet other embodiments, the compositions of the present disclosure are used in a method of treating, preventing, ameliorating, reducing, or delaying the onset of at least one proliferative disorder.

[0101] In some embodiments, the proliferative disorder associated with the compositions of the invention is associated with overexpression of Bcl2.

[0102] In some additional embodiments, the proliferative disorder is at least one hematological cancer or neoplasm.

[0103] In some embodiments, the hematological cancer or neoplasm is at least one of chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), a mutated KRAS tumor, a lymphoma, multiple myeloma, a blastic plasmacytoid dendritic cell neoplasm, T-cell prolymphocytic leukemia, or a myelodysplastic disorder.

[0104] As shown in the examples below, the combined composition exhibits a synergistic effect to induce apoptosis and cell death in U937 cells and CT26 cells, which are cell lines derived from histiocytic lymphoma and colorectal cancer, respectively.

[0105] In some embodiments, the proliferative disorder is at least one of histiocytic lymphoma or colorectal cancer.

[0106] It should be noted that the combined compositions of the present invention can induce or enhance apoptosis of malignant cells / cancer cells. More specifically, the combined compositions of the present invention can lead to an increase, enhancement, induction or elevation of apoptosis of treated cells, and the increase, induction or elevation of apoptosis can be about 1% to 99.9%, specifically about 1% to about 95%, about 5% to 90%, about 10% to 85%, about 15% to 80%, about 20% to 75%, about 25% to 70%, about 30% to 65%, about 35% to 60%, about 40% to 55%, about 45% to 50% increase. More specifically, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99.9%. More specifically, an increase of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% compared to an untreated control.

[0107] With respect to the above, it should be understood that, where provided, percentage values, such as, for example, 10%, 50%, 120%, 500%, etc., are interchangeable with "fold change" values, i.e., 0.1, 0.5, 1.2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc., respectively.

[0108] The term "apoptosis" refers to a regulatory network of biochemical events that results in a selective form of cellular suicide and is characterized by readily observable morphological and biochemical phenomena. Cells undergoing apoptosis display characteristic morphological features and

[0109] Biochemical features. These features include chromatin aggregation or condensation, DNA fragmentation, nuclear and cytoplasmic condensation, and segmentation of the cytoplasm and nucleus into membrane-bound vesicles (apoptotic bodies) containing ribosomes, morphologically intact mitochondria, and nuclear material. Release of cytochrome C from mitochondria is considered an indicator of mitochondrial dysfunction accompanying apoptosis.

[0110] As mentioned above, apoptosis is a tightly controlled form of active cell death that is essential for development and organismal homeostasis. Death via the apoptotic pathway is achieved, inter alia, by activation of a family of highly potent and specific proteases called caspases (cysteine-aspartic acid proteases).

[0111] The activity of caspases is tightly regulated, and cells maintain several "checkpoints" to control the activity of these enzymes. The first level of regulation is intrinsic to the caspases themselves. Caspases are initially transcribed as weakly active zymogens, which are cleaved to form active enzymes only under appropriate stimulation. The second level of caspase regulation is achieved by inhibitors, namely a family of proteins known as IAPs (Inhibitors of Apoptosis Proteins).

[0112] Therefore, in a second aspect, the present disclosure provides a method for treating, preventing, ameliorating, reducing or delaying the onset of at least one proliferative disorder in a subject in need thereof, the method comprising the step of administering a therapeutically effective amount of at least one inhibitor of a Bcl2 pro-survival protein and an isolated peptide comprising the amino acid sequence shown in SEQ ID NO.1 or any functional derivative thereof, or a pharmaceutically acceptable salt thereof, or any composition, kit or combination thereof.

[0113] In some embodiments, a modified isolated peptide suitable for the present disclosure or methods of the present invention has at least 70%, or 80%, or 90%, or 95% identity to the corresponding sequence of SEQ ID NO:1.

[0114] In some specific embodiments, the isolated peptide consists of the amino acid sequence of SEQ ID NO:1.

[0115] In other embodiments, the Bcl-2 pro-survival protein relevant to the methods of the present invention is at least one of the following: Bcl-2, Bcl-xL, Mcl-1, Bcl-w, Bcl2A1, and Bcl-B / Bcl2L10.

[0116] In some additional embodiments, at least one inhibitor of a Bcl2 pro-survival protein suitable for the methods of the present invention is at least one BH3 mimetic compound.

[0117] In some additional specific embodiments, the BH3 mimetic compound is at least one of the following: 4-{4-[(4'-chloro[1,1'-biphenyl]-2-yl)methyl]piperazin-1-yl}-N-(4-{[(2R)-4-(dimethylamino)-1-(phenylthio)butan-2-yl]amino}-3-nitrobenzene-1-sulfonyl)benzamide (ABT-737), 4-(4-{[2-(4-chlorophenyl)-4,4-dimethyl-1-cyclohexen-1-yl]methyl}-1-piperazinyl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)-amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]-pyridin-5-yloxy)benzamide (ABT-199), 4-(4-{[2 -(4-chlorophenyl)-5,5-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-(4-{[(2R)-4-(morpholin-4-yl)-1-(phe-nylthio)butan-2-yl]amino}-3-(trifluoromethanesulfonyl)benzene-1-sulfonyl)benzamide (ABT-263), 2-(2-((3,5-dimethyl-1H-pyrrolidone)-1H-pyrrolidone)-2-(4-{[(2R)-4-(morpholin-4-yl)-1-(phe-nylthio)butan-2-yl]amino}-3-(trifluoromethanesulfonyl)benzene-1-sulfonyl)benzamide (ABT-263), 4-dihydro-1H-isoquinolin-2-yl]pyridine-2-carboxylic acid (A-1331852).

[0118] In yet another specific embodiment, the at least one inhibitor of the Bcl2 pro-survival protein is 4-{4-[(4'-chloro[1,1'-biphenyl]-2-yl)methyl]piperazin-1-yl}-N-(4-{[(2R)-4-(dimethylamino)-1-(phenylthio)butan-2-yl]amino}-3-nitrobenzene-1-sulfonyl)-benzamide (ABT-737), or any pharmaceutically acceptable salt or hydrate thereof, or any stereoisomer or salt thereof.

[0119] As used herein, "proliferative disorders" are disorders that exhibit excessive proliferation. The term means cell division and growth that is not part of normal cell renewal, metabolism, growth or reproduction of the entire organism. Unwanted cell proliferation is seen in tumors and other pathological proliferations of cells, which do not provide normal functions, and in the absence of external intervention, most cells will continue to grow unconstrained at a growth rate that exceeds that of cells of normal tissues. Pathological states that occur due to unwanted cell proliferation are referred to herein as "hyperproliferative diseases" or "hyperproliferative disorders". It should be noted that the terms "proliferative disorders", "cancer", "tumor" and "malignant tumor" all equally relate to excessive proliferation of tissues or organs. In general, the compositions and methods of the present invention can be used to treat non-solid tumors and solid tumors.

[0120] The malignant tumor contemplated in the present invention may be any of lymphoma, leukemia, carcinoma, melanoma, myeloma and sarcoma.

[0121] Lymphoma is a cancer in the lymphocytes of the immune system. Typically, lymphomas occur as solid tumors of lymphoid cells. These malignant cells often originate in the lymph nodes, where they appear as enlargements (tumors) of the nodes. It can also affect other organs, in which case it is called extranodal lymphoma. Non-limiting examples of lymphomas include Hodgkin's disease, non-Hodgkin's lymphoma, and Burkitt's lymphoma.

[0122] Leukemia refers to a progressive malignant disease of the blood-forming organs and is generally characterized by abnormal proliferation and development of white blood cells and their precursors in the blood and bone marrow. Leukemias are usually clinically classified based on: (1) the duration and characteristics of the disease, whether acute or chronic; (2) the cell type involved; myeloid (myelocytic), lymphoid (lymphoblastic), or monocytic; and (3) the presence or absence of an increase in the number of abnormal cells in the blood - leukemia or leukopenia (subleukemia).

[0123] As used herein, carcinoma refers to an invasive malignancy composed of transformed epithelial cells. Alternatively, it refers to a malignancy composed of transformed cells of unknown tissue origin, but which has specific molecular or histological features associated with epithelial cells, such as the production of cytokeratins or intercellular bridges.

[0124] Melanoma, as used herein, is a malignant tumor of melanocytes. Melanocytes are cells that produce the dark pigment melanin, which is responsible for the skin color. They are found primarily in the skin, but also in other parts of the body, including the intestines and eyes. Melanoma can occur in any part of the body that contains melanocytes.

[0125] Sarcomas are cancers that arise from transformed connective tissue cells. These cells originate from the mesoderm, or middle layer, of the embryo that forms bone, cartilage, and fat tissue. This is in contrast to cancers that originate from the epithelium. The epithelium lines the surfaces of structures throughout the body and is the origin of breast, colon, and pancreatic cancers.

[0126] Myeloma as mentioned herein is a cancer of plasma cells, which are a class of white blood cells that are usually responsible for producing antibodies. The collection of abnormal cells accumulates in the bones, where they cause bone damage, and accumulates in the bone marrow, where they interfere with the production of normal blood cells. Most myeloma cases are also characterized by the production of paraproteins, a kind of abnormal antibodies that can cause kidney problems and interfere with the production of normal antibodies that cause immunodeficiency. Hypercalcemia (high calcium levels) is often encountered.

[0127] Other malignancies that can be used in the present invention may include, but are not limited to, hematological malignancies (including lymphomas, leukemias, and myeloproliferative disorders), aplastic and aplastic anemias (viral-induced and spontaneous), myelodysplastic syndromes, all types of tumor-like syndromes (immune-mediated and spontaneous), and solid tumors (including gastrointestinal tract, colon, lung, liver, breast, prostate, pancreas, and Kaposi's sarcoma). More specifically, the malignant disorder may be a lymphoma. Non-limiting examples of cancers treatable according to the present invention include hematopoietic malignancies, such as all types of lymphomas, leukemias, e.g., acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), mutated KRAS tumors, chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), mast cell leukemia, hairy cell leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, Burkitt's lymphoma, and multiple myeloma, as well as for the treatment or inhibition of solid tumors, such as tumors of the lip and oral cavity, pharynx, , larynx, paranasal sinuses, major salivary glands, thyroid, esophagus, stomach, small intestine, colon, colorectum, anal canal, liver, gallbladder, extrahepatic bile duct, ampulla of Vater, exocrine pancreas, lung, pleural mesothelioma, bone, soft tissue sarcoma, skin cancer and malignant melanoma, breast, vulva, vagina, cervix, uterine corpus, ovary, fallopian tube, gestational trophoblastic tumor, penis, prostate, testis, kidney, renal pelvis, ureter, bladder, urethra, eyelid cancer, conjunctival cancer, conjunctival malignant melanoma, uveal malignant melanoma, retinoblastoma, lacrimal gland cancer, orbital sarcoma, brain, spinal cord, vascular system, angiosarcoma and Kaposi's sarcoma.

[0128] In particular, studies have shown that the Bcl-2 protein, an anti-apoptotic protein, controls pro-survival pathways and is overexpressed in a variety of tumor types such as small cell lung cancer, melanoma, prostate cancer, and breast cancer.

[0129] In some embodiments, the proliferative disorder associated with the methods of the invention is associated with Bcl2 overexpression or a Bcl-2 overexpression disorder or a Bcl-2 mediated disorder.

[0130] The terms "disorders associated with Bcl2 overexpression" or "Bcl-2 overexpression disorders" and "Bcl-2 mediated disorders" refer to pathological conditions and disease conditions in which Bcl-2 proteins are overexpressed as described above. In addition, the term also encompasses conditions in which Bcl-2 plays a role. Such a role may be directly related to the pathological condition or may be indirectly related to the condition. A common feature of such conditions is that they can be improved by inhibiting the expression, activity, function or association with the Bcl-2 protein.

[0131] The term "overexpression" refers to an increase in the measurable expression level of a Bcl-2 gene in a sample (measured by the amount of RNA and / or the amount of protein) compared to the measurable expression level of the Bcl-2 gene in a second sample (particularly a control sample). "Overexpression of Bcl-2" can be measured and assessed using a ratio of the Bcl-2 expression level in a sample to the average expression level of Bcl-2 in a control sample, wherein the ratio is not equal and is specifically greater than 1.0. When overexpression is determined based on the ratio, if the ratio of the expression level in the first sample to the expression level in the second sample is greater than 1.0, the RNA or protein is overexpressed. For example, a ratio greater than 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more.

[0132] Thus, a Bcl-2 overexpression pathological disorder refers to a disorder characterized by overexpression of Bcl-2 in a subject or a diseased tissue of the subject compared to a healthy subject or a healthy tissue of the same subject.

[0133] It should be noted that Bcl-2 overexpression disorders may be caused by chromosomal translocation, hypomethylation, and downregulation of microRNAs targeting Bcl-2.

[0134] The B-cell lymphoma 2 (BCL2) protein is known to be highly expressed in many hematological malignancies to varying degrees, providing cells with protection from oncogenic stress and external stress-induced cell death. For example, in chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML), venetoclax is the first selective BCL2 inhibitor and the first anticancer drug of its class (BH3 mimetic) approved for routine clinical practice.

[0135] In some specific embodiments, the compositions or methods of the invention are used to treat, prevent, ameliorate, reduce or delay the onset of chronic lymphocytic leukemia (CLL).Chronic lymphocytic leukemia (CLL) is a type of cancer in which the bone marrow produces excess lymphocytes.

[0136] In some embodiments, the compositions or methods of the invention are used to treat, prevent, ameliorate, reduce or delay the onset of acute myeloid leukemia (AML). Acute myeloid leukemia (AML) is a cancer of myeloid blood cells characterized by the rapid growth of abnormal cells that accumulate in the bone marrow and blood and interfere with the production of normal blood cells.

[0137] In some embodiments, the compositions or methods of the invention are used to treat, prevent, ameliorate, reduce or delay the onset of mutant KRAS tumors. Mutated KRAS tumors or KRAS-driven tumors refer to a class of cancer tumors in which the Kirsten rat sarcoma viral oncogene (KRAS) is mutated. The KRAS oncogene has the highest mutation rate among all cancers and is associated with a range of highly lethal cancers, such as pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC) and colorectal cancer (CRC).

[0138] Thus, in some embodiments, the proliferative disorder is at least one hematological cancer or neoplasm.

[0139] In yet other specific embodiments, the hematological cancer or neoplasm is at least one of chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), a mutated KRAS tumor, a lymphoma, multiple myeloma, a blastic plasmacytoid dendritic cell neoplasm, T-cell prolymphocytic leukemia, or a myelodysplastic disorder.

[0140] In some specific embodiments, the proliferative disorder associated with the methods of the invention is histiocytic lymphoma or colorectal cancer.

[0141] In some additional embodiments, the methods of the present invention enhance or induce apoptosis in malignant / cancer cells of the subject.

[0142] In yet another embodiment, the methods of the invention further comprise administering at least one therapeutic agent, such as at least one additional anti-cancer agent.

[0143] In some specific embodiments, the additional anti-cancer agent is not taxol or doxorubicin or an anti-PDL1 antibody.

[0144] In some embodiments of the methods of the present invention, at least one inhibitor of Bcl2 pro-survival protein and the isolated peptide comprising the amino acid sequence shown in SEQ ID NO. 1 are administered simultaneously or sequentially.

[0145] In a third aspect, the present disclosure provides at least one inhibitor of Bcl2 pro-survival protein and at least one isolated peptide comprising the amino acid sequence shown in SEQ ID NO.1 or any functional derivative thereof, or a pharmaceutically acceptable salt thereof, for use in a method of treating, preventing, ameliorating, reducing or delaying the onset of at least one proliferative disorder.

[0146] In some embodiments, a modified isolated peptide suitable for use according to the present disclosure or invention has at least 70%, or 80%, or 90%, or 95% identity to the corresponding sequence of SEQ ID NO:1.

[0147] In some specific embodiments, the isolated peptide consists of the amino acid sequence of SEQ ID NO:1.

[0148] In other embodiments, the Bcl-2 pro-survival protein relevant for use according to the invention is at least one of the following: Bcl-2, Bcl-xL, Mcl-1, Bcl-w, Bcl2A1 and Bcl-B / Bcl2L10.

[0149] In some further embodiments, the at least one inhibitor of a Bcl2 pro-survival protein suitable for use according to the present invention is at least one BH3 mimetic compound.

[0150] In some additional specific embodiments, the BH3 mimetic compound is at least one of the following: 4-{4-[(4'-chloro[1,1'-biphenyl]-2-yl)methyl]piperazin-1-yl}-N-(4-{[(2R)-4-(dimethylamino)-1-(phenylthio)butan-2-yl]amino}-3-nitrobenzene-1-sulfonyl)benzamide (ABT-737), 4-(4-{[2-(4-chlorophenyl)-4,4-dimethyl-1-cyclohexen-1-yl]methyl}-1-piperazinyl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)-amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]-pyridin-5-yloxy)benzamide (ABT-199), 4-(4-{[2 -(4-chlorophenyl)-5,5-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-(4-{[(2R)-4-(morpholin-4-yl)-1-(phe-nylthio)butan-2-yl]amino}-3-(trifluoromethanesulfonyl)benzene-1-sulfonyl)benzamide (ABT-263), 2-(2-((3,5-dimethyl-1H-pyrrolidone)-1H-pyrrolidone)-2-(4-{[(2R)-4-(morpholin-4-yl)-1-(phe-nylthio)butan-2-yl]amino}-3-(trifluoromethanesulfonyl)benzene-1-sulfonyl)benzamide (ABT-263), 4-dihydro-1H-isoquinolin-2-yl]pyridine-2-carboxylic acid (A-1331852).

[0151] In yet another specific embodiment, the at least one inhibitor of the Bcl2 pro-survival protein is 4-{4-[(4'-chloro[1,1'-biphenyl]-2-yl)methyl]piperazin-1-yl}-N-(4-{[(2R)-4-(dimethylamino)-1-(phenylthio)butan-2-yl]amino}-3-nitrobenzene-1-sulfonyl)-benzamide (ABT-737), or any pharmaceutically acceptable salt or hydrate thereof, or any stereoisomer or salt thereof.

[0152] In some embodiments, at least one inhibitor of the Bcl2 pro-survival protein for use according to the present invention and the isolated peptide comprising the amino acid sequence shown in SEQ ID NO. 1 are administered simultaneously or consecutively.

[0153] In yet another embodiment, the use of at least one inhibitor of Bcl2 pro-survival protein and an isolated peptide comprising the amino acid sequence shown in SEQ ID NO. 1 according to the present invention further comprises administering at least one additional anti-cancer agent.

[0154] In some specific embodiments, the additional anti-cancer agent is not taxol or doxorubicin or an anti-PDL1 antibody.

[0155] In some embodiments, the proliferative disorder associated with the use according to the invention is associated with Bcl2 overexpression.

[0156] In some embodiments, the proliferative disorder is at least one hematological cancer or neoplasm.

[0157] In yet other specific embodiments, the hematological cancer or neoplasm is at least one of chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), a mutated KRAS tumor, a lymphoma, multiple myeloma, a blastic plasmacytoid dendritic cell neoplasm, T-cell prolymphocytic leukemia, or a myelodysplastic disorder.

[0158] In some specific embodiments, proliferative disorders suitable for use in the present invention are histiocytic lymphoma and colorectal cancer, respectively.

[0159] In some embodiments, the isolated peptide or Bcl2 inhibitor or their pharmaceutically acceptable salts as defined herein and / or suitable for the methods and uses disclosed herein may be included in a pharmaceutical composition. The term "pharmaceutical composition" as defined herein refers to an isolated peptide and / or a Bcl2 inhibitor and optionally at least one pharmaceutically acceptable excipient or carrier known in the art. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial agents and antifungal agents, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. In addition to any conventional media or agents that are incompatible with the active ingredient, the use of media and agents in therapeutic compositions is considered. The pharmaceutical composition for treating a subject in need according to the present disclosure may optionally further include a buffer, an agent for adjusting its molar osmotic pressure concentration, and optionally one or more pharmaceutically acceptable additives known in the art. The pharmaceutical composition for treating a subject in need according to the present invention may be conveniently present in a unit dosage form and may be prepared according to conventional techniques well known in the pharmaceutical industry, such as those described in detail in the examples below. It should be understood that in addition to the ingredients particularly mentioned herein, compositions according to the present disclosure may include other agents conventional in the art having regard to the type of formulation in question.

[0160] The isolated peptide and / or Bcl2 inhibitor comprising the amino acid sequence shown in SEQ ID NO.1 as defined herein can be administered by any route of administration known to those skilled in the art, such as intravenous (iv) or any suitable additional route, including intraperitoneal, subcutaneous, transdermal, topical, intramuscular, intraarticular, subconjunctival or mucosal, such as oral, intranasal or intraocular administration.

[0161] The isolated peptide or Bcl2 inhibitor as defined herein can be administered in an "effective amount" so as to achieve the desired therapeutic result. The "effective amount" is determined by the severity of the disease in combination with the therapeutic purpose, the route of administration and the general condition of the patient (age, sex, weight and other considerations known to the attending physician). The effective amount for this purpose is generally in the range of 0.001 mg / Kg to 1000 mg / Kg, in particular 0.001 mg / Kg to 0.1 mg / Kg, 0.02 mg / Kg to 0.1 mg / Kg, 0.03 mg / Kg to 0.1 mg / Kg, 0.04 mg / Kg to 0.1 mg / Kg, 0.05 mg / Kg to 0.1 mg / Kg, 0.06 mg / Kg to 0.1 mg / Kg, 0.07 mg / Kg to 0.1 mg / Kg, 0.08 mg / Kg, 0.09 mg / Kg to 0.1 mg / Kg, 0.10 mg / Kg to 0.10 mg / Kg, 0.11 mg / Kg to 0.11 mg / Kg, 0.12 mg / Kg to 0.13 mg / Kg, 0.14 mg / Kg to 0.15 mg / Kg, 0.16 mg / Kg to 0.17 mg / Kg, 0.18 mg / Kg to 0.19 mg / Kg, 0.20 mg / Kg to 0.21 mg / Kg, 0.22 mg / Kg to 0.23 mg / Kg, 0.24 mg / Kg to 0.25 mg / Kg, 0.26 mg / Kg to 0.27 mg / Kg, 0.28 mg / Kg to 0.29 mg / Kg, 0.30 mg / Kg to 0.31 mg / 0.1mg / Kg to 1mg / Kg, 0.2mg / Kg to 1mg / Kg, 0.3mg / Kg to 1mg / Kg, 0.4mg / Kg to 1mg / Kg, 0.5mg / Kg to 1mg / Kg, 0.6mg / Kg to 1mg / Kg, 0.7mg / Kg to 1mg / Kg, 0.8mg / Kg to 1mg / Kg, 0.9mg / Kg to 1mg / Kg, 1mg / Kg to 10mg / Kg, 2mg / Kg to 10mg / Kg, 3mg / Kg to 10mg / Kg, 4mg / Kg to 10mg / Kg g, 5mg / Kg to 10mg / Kg, 6mg / Kg to 10mg / Kg, 7mg / Kg to 10mg / Kg, 8mg / Kg to 10mg / Kg, 9mg / Kg to 10mg / Kg, 10mg / Kg to 100mg / Kg, 20mg / Kg to 100mg / Kg, 30mg / Kg to 100mg / Kg, 40mg / Kg to 100mg / Kg, 50mg / Kg to 100mg / Kg, 60mg / Kg to 100mg / Kg, 70mg / Kg to 100mg / Kg, 80mg / Kg to 100mg / Kg, 90mg / Kg to 100mg / Kg, or 100mg / Kg to 1000mg / Kg, 200mg / Kg to 1000mg / Kg, 300mg / Kg to 1000mg / Kg, 400mg / Kg to 1000mg / Kg, 500mg / Kg to 1000mg / Kg, 600mg / Kg to 1000mg / Kg, 700mg / Kg to 1000mg / Kg, 800mg / Kg to 1000mg / Kg, 900mg / Kg to 1000mg / Kg.In some specific embodiments, the amount or dosage is 1mg / Kg, 2mg / Kg, 3mg / Kg, 4mg / Kg, 5mg / Kg, 6mg / Kg, 7mg / Kg, 8mg / Kg, 9mg / Kg, 10mg / Kg, 15mg / Kg, 20mg / Kg, 30mg / Kg, 40mg / Kg, 50mg / Kg, 60mg / Kg, 70mg / Kg, 80mg / Kg, 90mg / Kg or 100mg / Kg. In some specific embodiments, the amount or dosage of Nerofe is 15mg / Kg. In some other embodiments, the amount of Bcl2 inhibitor is 50mg / Kg.

[0162] In some additional embodiments, the effective amount refers to the amount of Nerofe and the Bcl2 inhibitor used to achieve a synergistic effect.

[0163] Single or multiple daily, weekly or monthly administration regimens can be performed according to the dosage level and mode selected by the treating physician. In addition, the administration of the composition of the present invention can be periodic, for example, the periodic administration can be twice a day, three times a day or at least once a day, for at least about three days to three months. The advantages of lower dosages are obvious to those skilled in the art. These factors include, in particular, a lower risk of side effects, particularly in long-term use, and a lower risk of desensitization of the patient to treatment. In another embodiment, the treatment performed using the composition of the present invention can be performed after at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 14 days, 30 days, 60 days, 90 days of treatment, and lifelong treatment is performed. It should be noted that treatment may indicate the use of different dosages or different time periods, which are obvious to skilled practitioners.

[0164] For preventive applications, the composition of the present invention may include a preventive effective amount of active ingredients. The term "preventive effective amount" is intended to mean that the pharmaceutical composition will prevent or reduce the amount of risk that researchers, veterinarians, doctors or other clinicians attempt to prevent the occurrence or recurrence of biological or medical events in tissues, systems, animals or humans. In preventive applications, the composition of the present invention is applied to patients at risk of developing disease states to enhance the resistance of patients. Such amounts are defined as "preventive effective doses". In this use, the exact amount depends again on the patient's health and overall immune level, but is typically within the range of 0.001 mg / dose to 1000 mg / dose.

[0165] The phrases "combination therapy" or "adjuvant therapy" or when defining the uses of the peptides described herein, specifically, an isolated peptide comprising the amino acid sequence as shown in SEQ ID NO: 1 (also referred to as "Nerofe") and at least one Bcl2 inhibitor, particularly an inhibitor of Bcl-2 pro-apoptotic protein, are intended to include the administration of each agent in a sequential manner according to a regimen that will provide the beneficial effects of the drug combination, and are also intended to include the co-administration of these agents in a substantially simultaneous manner, such as in a single formulation having a fixed ratio of these active agents, or in multiple separate formulations of each agent.

[0166] Therefore, in another aspect, in a fifth aspect, the present disclosure provides a method for treating, preventing, ameliorating, reducing or delaying the onset of at least one proliferative disorder in a subject treated with at least one inhibitor of a Bcl2 pro-survival protein, the method comprising the step of administering to the subject a therapeutically effective amount of an isolated peptide comprising the amino acid sequence shown in SEQ ID NO.1 or any functional derivative thereof, or a pharmaceutically acceptable salt thereof, or any composition, kit or combination thereof.

[0167] In yet another aspect, the present disclosure provides a method for treating, preventing, ameliorating, reducing or delaying the onset of at least one proliferative disorder in a subject treated with at least one isolated peptide comprising the amino acid sequence shown in SEQ ID NO. 1, the method comprising the step of administering to the subject a therapeutically effective amount of at least one inhibitor of Bcl2 pro-survival protein or any functional derivative thereof, or a pharmaceutically acceptable salt thereof, or any composition, kit or combination thereof.

[0168] With respect to the above aspects of the present disclosure, it is directed to a method of treating, preventing, ameliorating, reducing or delaying the onset of at least one proliferative disorder in a subject treated with at least one inhibitor of a Bcl2 prosurvival protein, or to a method of treating, preventing, ameliorating, reducing or delaying the onset of at least one proliferative disorder in a subject treated with at least one isolated peptide comprising the amino acid sequence shown in SEQ ID NO.1, in some embodiments, the modified isolated peptide has at least 70%, or 80%, or 90%, or 95% identity with the corresponding sequence of SEQ ID NO:1. In some embodiments, the isolated peptide consists of the amino acid sequence of SEQ ID NO:1. In some embodiments, the Bcl-2 prosurvival protein is at least one of the following: Bcl-2, Bcl-xL, Mcl-1, Bcl-w, Bcl2A1 and Bcl-B / Bcl2L10. In some embodiments, the at least one inhibitor of a Bcl2 prosurvival protein is at least one BH3 mimetic compound. In some embodiments, the BH3 mimetic compound is at least one of the following: 4-{4-[(4'-chloro[1,1'-biphenyl]-2-yl)methyl]piperazin-1-yl}-N-(4-{[(2R)-4-(dimethylamino)-(phenylthio)butan-2-yl]amino}-3-nitrobenzene-1-sulfonyl)benzamide (ABT-737), 4-(4-{[2-(4-chlorophenyl)-4,4-dimethyl-1-cyclohexen-1-yl]methyl}-1-piperazinyl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)-amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]-pyridin-5-yloxy)benzamide (ABT-199), 4-(4-{[2-( 4-( ... -2-yl)methylene)-3-methoxy-2H-pyrrol-5-yl)-1H-indole (GX15-070), or 3-[1-(1-adamantylmethyl)-5-methylpyrazol-4-yl]-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydro-1H-isoquinolin-2-yl]pyridine-2-carboxylic acid (A-1331852).In some embodiments, the at least one inhibitor of Bcl2 pro-survival protein is 4-{4-[(4'-chloro[1,1'-biphenyl]-2-yl)methyl]piperazin-1-yl}-N-(4-{[(2R)-4-(dimethylamino)-1-(phenylthio)butan-2-yl]amino}-3-nitrobenzene-1-sulfonyl)-benzamide (ABT-737), or any pharmaceutically acceptable salt or hydrate thereof, or any stereoisomer or salt thereof. In some embodiments, the at least one inhibitor of Bcl2 pro-survival protein and the isolated peptide comprising the amino acid sequence shown in SEQ ID NO.1 are administered simultaneously or sequentially. In some embodiments, the method further comprises administering at least one additional anti-cancer agent. In some embodiments, the proliferative disorder is associated with Bcl2 overexpression. In some embodiments, the proliferative disorder is at least one hematological cancer or neoplasm. In some embodiments, the hematological cancer or neoplasm is at least one of chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), a mutated KRAS tumor, a lymphoma, multiple myeloma, a blastic plasmacytoid dendritic cell neoplasm, T-cell prolymphocytic leukemia, or a myelodysplastic disorder.

[0169] Therefore, in another aspect, the present disclosure provides an isolated peptide comprising the amino acid sequence shown in SEQ ID NO.1, which is used in a method for treating, preventing, ameliorating, reducing or delaying the onset of at least one proliferative disorder in a subject treated with at least one inhibitor of Bcl2 pro-survival protein, the method comprising the step of administering to the subject a therapeutically effective amount of the following items: an isolated peptide comprising the amino acid sequence shown in SEQ ID NO.1 or any functional derivative thereof, or a pharmaceutically acceptable salt thereof, or any composition, kit or combination thereof.

[0170] In yet another aspect, the present disclosure provides a Bcl2 pro-survival protein for use in a method for treating, preventing, ameliorating, reducing or delaying the onset of at least one proliferative disorder in a subject treated with at least one isolated peptide comprising the amino acid sequence shown in SEQ ID NO.1, the method comprising the step of administering to the subject a therapeutically effective amount of: the at least one inhibitor of the Bcl2 pro-survival protein or any functional derivative thereof, or a pharmaceutically acceptable salt thereof, or any composition, kit or combination thereof.

[0171] In some embodiments, the modified isolated peptide suitable for the use of the present invention has at least 70%, or 80%, or 90%, or 95% identity to the corresponding sequence of SEQ ID NO: 1. In some embodiments, the Bcl-2 pro-survival protein suitable for the use of the present invention is at least one of the following: Bcl-2, Bcl-xL, Mcl-1, Bcl-w, Bcl2A1, and Bcl-B / Bcl2L10. In some embodiments, the at least one inhibitor of a Bcl2 pro-survival protein suitable for the use of the present invention is at least one BH3 mimetic compound. In some embodiments, the BH3 mimetic compound suitable for use in the present invention is at least one of the following: 4-{4-[(4'-chloro[1,1'-biphenyl]-2-yl)methyl]piperazin-1-yl}-N-(4-{[(2R)-4-(dimethylamino)-1-(phenylthio)butan-2-yl]amino}-3-nitrobenzene-1-sulfonyl)benzamide (ABT-737), 4-(4-{[2-(4-chlorophenyl)-4,4-dimethyl--cyclohexen-1-yl]methyl}-1-piperazinyl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)-amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]-pyridin-5-yloxy)benzamide (ABT-199), 4-(4-{[2-(4-chlorophenyl)-4,4-dimethyl--cyclohexen-1-yl]methyl}-1-piperazinyl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)-amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]-pyridin-5-yloxy)benzamide (ABT-199), {[2-(4-chlorophenyl)-5,5-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-(4-{[(2R)-4-(morpholin-4-yl)-1-(phe-nylthio)butan-2-yl]amino}-3-(trifluoromethanesulfonyl)benzene-1-sulfonyl)benzamide (ABT-263), 2-(2-((3,5-dimethyl-1H -pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-yl)-1H-indole (GX15-070), or 3-[1-(1-adamantylmethyl)-5-methylpyrazol-4-yl]-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydro-1H-isoquinolin-2-yl]pyridine-2-carboxylic acid (A-1331852). In some embodiments, the at least one inhibitor of Bcl2 pro-survival protein suitable for use of the present invention is 4-{4-[(4'-chloro[1,1'-biphenyl]-2-yl)methyl]piperazin-1-yl}-N-(4-{[(2R)-4-(dimethylamino)-1-(phenylthio)butan-2-yl]amino}-3-nitrobenzene-1-sulfonyl)-benzamide (ABT-737), or any pharmaceutically acceptable salt or hydrate thereof, or any stereoisomer or salt thereof. In some embodiments, the at least one inhibitor of Bcl2 pro-survival protein suitable for use of the present invention and the isolated peptide comprising the amino acid sequence shown in SEQ ID NO.1 are administered simultaneously or sequentially.In some embodiments, the use according to the present invention further comprises administering at least one additional anticancer agent. In some embodiments, the proliferative disorder associated with the use of the present invention is associated with Bcl2 overexpression. In some embodiments, the proliferative disorder associated with the use of the present invention is at least one hematological cancer or neoplasm. In some embodiments, the hematological cancer or neoplasm associated with the use of the present invention is at least one of the following: chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), mutated KRAS tumors, lymphomas, multiple myeloma, blastic plasmacytoid dendritic cell neoplasms, T-cell prolymphocytic leukemia, or myelodysplasia.

[0172] As described above, the present invention involves the use of different active ingredients, such as a peptide called "Nerofe" and at least one Bcl2 inhibitor, which can be administered by different routes, dosages and combinations. More specifically, the treatment of Bcl-2 overexpression diseases and conditions with a combination of active ingredients may involve the separate administration of each active ingredient. Therefore, providing a kit in a convenient modular format will allow the desired flexibility in the above parameters.

[0173] More specifically, the kit described further below herein may include the composition directly applied as a prepared liquid topical, nasal or oral dosage form or in a separate multiple dose unit form, or alternatively, may include the composition as a solid pharmaceutical composition that can be reconstructed with a solvent to provide a liquid dosage form. When the kit includes a solid pharmaceutical composition that can be reconstructed with a solvent to provide a liquid dosage form (e.g., for oral administration), the kit may optionally include a reconstructed solvent. In this case, the composition solvent or reconstructed solvent is merged with the active ingredient to provide a liquid dosage form of each active ingredient or a combination thereof. Typically, the active ingredient is soluble in a solvent and forms a solution. The solvent can be, for example, water, a non-aqueous liquid, or a combination of a non-aqueous component and an aqueous component. Suitable non-aqueous components include, but are not limited to, oils, alcohols (such as ethanol, glycerol) and glycols (such as polyethylene glycol and propylene glycol). In some embodiments, the solvent is phosphate buffered saline (PBS).

[0174] In another aspect, the present disclosure provides a kit comprising:

[0175] (a) at least one inhibitor of a Bcl2 pro-survival protein, or any pharmaceutically acceptable salt thereof, optionally in a first dosage form;

[0176] (b) optionally in a second dosage form an isolated peptide comprising the amino acid sequence shown in SEQ ID NO. 1 or any functional derivative thereof, or a pharmaceutically acceptable salt thereof.

[0177] In some embodiments, a modified isolated peptide suitable for the disclosure or kits of the invention has at least 70%, or 80%, or 90%, or 95% identity to the corresponding sequence of SEQ ID NO:1.

[0178] In some specific embodiments, the isolated peptide consists of the amino acid sequence of SEQ ID NO:1.

[0179] In some additional embodiments, the Bcl-2 pro-survival protein associated with the kit of the present invention is at least one of the following: Bcl-2, Bcl-xL, Mcl-1, Bcl-w, Bcl2A1, and Bcl-B / Bcl2L10.

[0180] In yet other specific embodiments, the at least one inhibitor of a Bcl2 pro-survival protein is at least one BH3 mimetic compound.

[0181] In some more specific embodiments, the BH3 mimetic compound suitable for the kit of the present invention is at least one of the following: 4-{4-[(4'-chloro[1,1'-biphenyl]-2-yl)methyl]piperazin-1-yl}-N-(4-{[(2R)-4-(dimethylamino)-1-(phenylthio)butan-2-yl]amino}-3-nitrobenzene-1-sulfonyl)benzamide (ABT-737), 4-(4-{[2-(4-chlorophenyl)-4,4-dimethyl--cyclohexen-1-yl]methyl}-1-piperazinyl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)-amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]-pyridin-5-yloxy)benzamide (ABT-199), 4-(4 -{[2-(4-chlorophenyl)-5,5-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-(4-{[(2R)-4-(morpholin-4-yl)-1-(phe-nylthio)butan-2-yl]amino}-3-(trifluoromethanesulfonyl)benzene-1-sulfonyl)benzamide (ABT-263), 2-(2-((3,5-dimethyl-1H -pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-yl)-1H-indole (GX15-070), or 3-[1-(1-adamantylmethyl)-5-methylpyrazol-4-yl]-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydro-1H-isoquinolin-2-yl]pyridine-2-carboxylic acid (A-1331852).

[0182] In some specific embodiments, the at least one inhibitor of the Bcl2 pro-survival protein is 4-{4-[(4'-chloro[1,1'-biphenyl]-2-yl)methyl]piperazin-1-yl}-N-(4-{[(2R)-4-(dimethylamino)-1-(phenylthio)butan-2-yl]amino}-3-nitrobenzene-1-sulfonyl)-benzamide (ABT-737), or any pharmaceutically acceptable salt or hydrate thereof, or any stereoisomer or salt thereof.

[0183] In some additional embodiments, kits according to the present disclosure are used in methods of treating, preventing, ameliorating, reducing or delaying the onset of at least one proliferative disorder.

[0184] In some embodiments, at least one inhibitor of Bcl2 pro-survival protein and said isolated peptide comprising the amino acid sequence shown in SEQ ID NO. 1 are in separate unit dosage forms in a kit for use according to the present invention.

[0185] In some additional embodiments, separate unit dosage forms of at least one inhibitor of Bcl2 pro-survival protein and an isolated peptide comprising the amino acid sequence shown in SEQ ID NO. 1 are administered simultaneously or sequentially.

[0186] In some other embodiments, the proliferative disorder associated with the kits for use in the present invention is associated with overexpression of Bcl2.

[0187] In some additional embodiments, the proliferative disorder is at least one hematological cancer or neoplasm.

[0188] In some embodiments, the hematological cancer or neoplasm is at least one of chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), a mutated KRAS tumor, a lymphoma, multiple myeloma, a blastic plasmacytoid dendritic cell neoplasm, T-cell prolymphocytic leukemia, or a myelodysplastic disorder.

[0189] The term "treat or prevent" refers to the full range of therapeutic benefits administered to a subject, including inhibition, reduction, alleviation, and relief of undesirable side effects of proliferative disorder symptoms or disorders associated with such proliferative disorders. More specifically, treatment or prevention includes preventing or delaying the development of the disease, preventing or delaying the development of symptoms, and / or reducing the severity of such symptoms that will develop or are expected to develop. These also include amelioration of existing symptoms, prevention of additional symptoms, and amelioration or prevention of underlying metabolic causes of symptoms.

[0190] As used herein, "disease," "disorder," "condition," and the like are used interchangeably in reference to a subject's health condition and have the meaning ascribed to each and all such terms.

[0191] The present invention relates to treating subjects or patients in need. "Patient" or "subject in need" means any organism that may be affected by the above-mentioned morbidity and is desired to be treated as described herein, including humans, domestic mammals and non-domestic mammals (such as canine and feline subjects, cattle, apes, horses and rat subjects), rodents, poultry, aquaculture, fish and exotic ornamental fish. It should be understood that the subject receiving treatment can also be any reptile or zoo animal. More specifically, the methods and compositions of the present invention are intended for mammals. "Mammalian subject" means any mammal that is desired to be proposed for therapy, including humans, equines, canines and feline subjects, most particularly humans. It should be noted that, particularly in the case of non-human subjects, the methods of the present invention can be administered by injection, drinking water, feeding, spraying, tube feeding, and directly administered to the digestive tract of subjects in need. It should be further noted that, particularly in the case of human subjects, administering the compositions of the present invention to the patient includes both self-administration and administration to the patient by another person.

[0192] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0193] As used herein, the term "about" means that the value may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% above or below the referenced value, the deviation range including the integer values ​​constituting the continuous range, and, if applicable, non-integer values. In some embodiments, the term "about" refers to ± 10%.

[0194] Unless expressly indicated to the contrary, the indefinite articles "a" and "an" as used herein in the specification and claims should be understood to mean "at least one". It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0195] The phrase "and / or" as used herein in the specification and claims should be understood to mean "either or both" of the elements so combined (i.e., elements that exist in combination in some cases and separately in other cases). Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so combined. Other elements other than the elements specifically designated by the "and / or" clause may optionally be present, whether related or unrelated to those elements specifically designated. Thus, as a non-limiting example, in one embodiment, when used in conjunction with open language such as "including", a reference to "A and / or B" may refer to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); and so on.

[0196] As used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, i.e., including multiple elements or at least one element in a list of elements, but also including more than one element therein, and optionally including additional unlisted items. Only when the term clearly indicates the opposite meaning, such as "only one of..." or "only one of...", or when used in the claims, "consisting of..." will refer to including multiple elements or only one element in a list of elements. Generally speaking, the term "or" as used herein, when used in the claims, when followed by exclusive terms such as "any of...", "one of...", "only one of..." or "only one of...", should only be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both"). "Substantially consisting of...", when used in the claims, should have its ordinary meaning used in the field of patent law.

[0197] As used herein in the specification and claims, the phrase "at least one" with respect to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of each element specifically listed in the list of elements, nor does it exclude any combination of elements in the list of elements. This definition also allows that some elements other than the elements specifically specified in the list of elements referred to by the phrase "at least one" may optionally be present, whether related or unrelated to those elements specifically specified. Thus, as a non-limiting example, in one embodiment, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently, "at least one of A and / or B") may refer to at least one, optionally including more than one A, without the presence of B (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one B, without the presence of A (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements); and so on.

[0198] It should also be understood that in any method claimed herein that includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are recorded unless clearly indicated to the contrary.

[0199] Throughout this specification and the subsequent examples and claims, all transitional phrases such as "comprising", "including", "carrying", "having", "containing", "involving", "holding", "consisting of", etc. should be understood to be open-ended, that is, meaning including but not limited to. Specifically, it should be understood that the inclusion of the stated integer or step or group of integers or steps is implied, but any other integer or step or group of integers or steps is not excluded. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases as set forth in the Manual of Patent Examining Procedure of the United States Patent Office, respectively. More specifically, the terms "comprising", "containing", "including", "having" and their variations all mean "including but not limited to". The term "consisting of" means "including and limited to". The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only when these additional ingredients, steps and / or parts do not substantially change the basic characteristics and novel features of the claimed composition, method or structure.

[0200] It should be noted that various embodiments of the present invention can be presented in a range format. It should be understood that the description of the range format is only for convenience and simplicity, and should not be interpreted as a non-flexible limitation on the scope of this aspect. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges, and each numerical value in the range. For example, the description of the range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, and each number in the range, such as 1, 2, 3, 4, 5 and 6. Regardless of the width of the range, this applies. Whenever a numerical range is indicated herein, any quoted number (fractional number or integer number) within the indicated range is intended to be included. The two quoted phrases "range between" the first indicating number and the second indicating number and "range from" the first indicating number "to" the second indicating number are interchangeably used herein, and are intended to include the first indicating number and the second indicating number, and all fractional numbers and integer numbers between the two.

[0201] As used herein, the term "method" refers to ways, means, techniques and procedures for accomplishing a given task, including but not limited to those ways, means, techniques and procedures known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine or readily developable from ways, means, techniques and procedures known to practitioners in these fields.

[0202] It should be understood that certain features of the invention described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for simplicity may also be provided individually or in any suitable subcombination, or, where appropriate, in any other described embodiment of the invention. Certain features described in the context of various embodiments are not considered essential features of those embodiments, unless the embodiment cannot be implemented without those elements.

[0203]

[00136] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0204] Although disclosed and described, it should be understood that the present invention is not limited to the specific embodiments, method steps and compositions disclosed herein, because such method steps and compositions can be slightly changed. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to be limiting, because the scope of the present invention will be limited only by the appended claims and their equivalents.

[0205] The following examples represent techniques employed by the inventors in carrying out various aspects of the present invention. It should be understood that although these techniques are examples of preferred embodiments for practicing the present invention, those skilled in the art will recognize, based on this disclosure, that many modifications may be made without departing from the spirit and intended scope of the present invention.

[0206] Example

[0207] Experimental Procedure

[0208] Preparation of dTCApFs peptide and composition containing the same

[0209] Peptide dTCApFs or NEROFE TM (these two terms are used interchangeably herein and refer to the same peptide as described above) is a 14 amino acid residue long peptide in which all of the amino acid residues are in their D configuration, having an amino acid sequence of Trp TrpThr Phe Phe Leu Pro Ser Thr Leu Trp Glu Arg Lys (or WWTFFLPSTLWERK in single letter code, as shown in SEQ ID NO: 1).

[0210] The peptide was synthesized as follows. The final drug product, dTCApFs acetate, for clinical studies was manufactured, packaged, tested, labeled and distributed by Nextar Ltd., Israel, in accordance with Good Manufacturing Practice (GMP).

[0211] Western blot of cleaved caspase 3 in U937 cells treated with Nerofe and ABT-737

[0212] Material:

[0213] For cell growth:

[0214] -Growth Medium-

[0215] oRPMI medium 1640 (Gibco 21875-034)

[0216] o 50 ml heat-inactivated FBS (Biological industries cat. no. 04-121-1A).

[0217] o 0.5 ml Amphotericin B 2500 pg / ml (Biological industries cat. no. 03-029-1).

[0218] o Penicillin-Streptomycin Solution (Biological Industries Cat. No. 03-031)

[0219] - Treatment medium -

[0220] oGrowth medium

[0221] ο 10% mannitol (Sigma catalog number M4125-500G)

[0222] After adding mannitol, the medium was filtered using a 0.2 pm filter.

[0223] -175cm 2 Culture flask

[0224] -20mg / ml Nerofe in mannitol

[0225] -ABT-737 (Cayman 11501)

[0226] -U937 cell line

[0227] For Western Blot

[0228] - a cleavage buffer comprising:

[0229] οRIPA buffer (Sigma R0278)

[0230] o Protease inhibitor cocktail (Sigma P2714).

[0231] O-phosphatase inhibitor (Sigma P5726)

[0232] ο Nuclease, purity>90% (70746 Millipore) 25U / ml

[0233] -X4 LDS sample buffer (Invitrogen catalog number NP0007)

[0234] -Sample reducing agent (Invitrogen catalog number NP0009, 10x concentrated)

[0235] -4%-12% Bis-Tris gel (Millipore catalog number MMMP41G10)

[0236] -10x MES transfer buffer (Millipore MMMPTRB)

[0237] -PVDF membrane (immobilon-P sq 0.2μm)

[0238] -TBST buffer: TBS (J640 Amersco) + 0.05% Tween-20

[0239] -5% skim milk in TBST

[0240] - Dilute the primary antibody 1:500 in 5% skim milk in TBST:

[0241] ο Anti-cleaved caspase 3 antibody (ab32042 abeam)

[0242] - Dilute the secondary antibody 1:10,000 in 5% skim milk in TBST:

[0243] ο Anti-rabbit IgG, HRP-linked antibody (Cells signaling 7074)

[0244] -SuperSignal TM West Femto Maximum Sensitivity Substrate(ThermoScientific 34095)

[0245] Cell Culture - Passaging:

[0246] The cells were grown at 37°C, 5% CO2, 100% humidity until they reached a density of 70% to 80%. The cells were resuspended in fresh medium and then grown for 2 to 3 days until they reached a density of 70% to 80%. This passaging procedure was repeated.

[0247] Cell treatment:

[0248] Cells were seeded in 13 ml of growth medium in 4 different flasks at a density of 700K cells / 75CM2 flask and incubated overnight. Mannitol sp was added to a final mannitol concentration of 5% mannitol. Cells were then treated as follows:

[0249] 1-Control (untreated)

[0250] 2-50μg / ml Nerofe

[0251] 3-No treatment (for ABT-737 on the second day)

[0252] 4-50μg / ml Nerofe (for ABT-737 on the second day)

[0253] The cells were then incubated for 24 hours and treated as follows:

[0254] 1-Control (untreated)

[0255] 2- No treatment (treated with Nerofe the day before)

[0256] 3-1μM ABT-737

[0257] 4-1μM ABT-737 (treated with Nerofe the day before)

[0258] The cells were then incubated for 24 hours and collected for Western blotting as previously described (Mahmood, T., & Yang, PC (2012). Western blot: technique, theory, and trouble shooting. North American journal of medical sciences, 4(9), 429-434).

[0259] Cell viability assay of U937 cells treated with Nerofe and Azacitidine (Resazurin)

[0260] Material:

[0261] - Cell culture medium:

[0262] ο10% FBS (Biological industries, catalog number 04-121-1A)

[0263] o DMEM (Gibco Cat. No. 41965)

[0264] ο1 mM sodium pyruvate (Biological Industries)

[0265] ο100 μg / ml penicillin and 100 μg / ml streptomycin (Biological Industries)

[0266] ο250 ng / ml amphotericin B (Biological Industries)

[0267] - Treatment medium -

[0268] o Culture medium

[0269] ο 10% mannitol (Sigma catalog number M4125-500G)

[0270] After adding mannitol, the medium was filtered using a 0.2 μm filter.

[0271] -Cytotoxicity assay kit (colorimetric analysis): abl 12118abeam

[0272] o Cell line: U937

[0273] ο2 mg / ml Nerofe stock solution: dilute 100 μl of 20 mg / ml Nerofe in 900 μl cell treatment medium

[0274] o5-Azacitidine (Sigma A2385, freshly diluted to 50 mg / mlm in treatment medium)

[0275] Solution:

[0276]

[0277]

[0278] Cells were seeded in a black 96-well plate with a transparent bottom at a density of 20,000 cells / well, 100 μl of growth medium was added, and incubated for 24 hours. Cells were treated with Nerofe and azacitidine in 100 μl cell treatment medium containing 10% mannitol, and the final volume was 200 μl and the final mannitol concentration was 5%. Cells were incubated for 48 hours. A certain amount of 20 μl cytotoxicity assay kit (colorimetric analysis) abl 12118 was added to each well, and cells were incubated for 4 hours. The Biotek Synergy-HT plate reader was used to read the plate at 530 excitation, 590 emission, and 40 sensitivity. Compared with the control, the cell viability percentage was calculated.

[0279] Cell viability assay of U937 cells treated with Nerofe and ABT-737 (Resazurin)

[0280] Material

[0281] The materials were the same as above except that ABT-737 (Cayman 11501) was used instead of 5-azacitidine.

[0282] Solution:

[0283]

[0284] Cells are seeded in black 96-well plates with a transparent bottom at a density of 20,000 cells / well, 100 μl of growth medium is added, and incubated for 24 hours. In 100 μl of cell treatment medium containing 10% mannitol, cells are treated with Nerofe, and the final volume is 200 μl and the final mannitol concentration is 5%. Cells are incubated for 24 hours. The ABT-737 treatment cells that are 225 pl in a final volume are treated in the following manner: 25 μl of treatment medium containing 5% mannitol and not containing FBS are added to each well accordingly. Cells are incubated for 24 hours. A certain amount of 25 μl of cytotoxicity assay kit (colorimetric analysis) abl 12118 is added to each well, and cells are incubated for 4 hours. Use Biotek Synergy-HT plate reader to read plates under excitation 530, emission 590, sensitivity 40. Compared with the control, calculate the cell viability percentage.

[0285] · Western blot of cleaved caspase 3 in CT26 cells treated with Nerofe and ABT-737

[0286] Material:

[0287] For cell growth:

[0288] -Growth Medium-

[0289] oRPMI medium 1640 (Gibco 21875-034)

[0290] o 50 ml heat-inactivated FBS (Biological industries cat. no. 04-121-1A).

[0291] o 0.5 ml Amphotericin B 2500 pg / ml (Biological industries cat. no. 03-029-1).

[0292] o Penicillin-Streptomycin Solution (Biological Industries Cat. No. 03-031)

[0293] - Treatment medium -

[0294] oGrowth medium

[0295] ο 5% mannitol (Sigma catalog number M4125-500G)

[0296] After adding mannitol, the medium was filtered using a 0.2 pm filter.

[0297] -Trypsin EDTA (Biological industries cat. no. 03-052-1B).

[0298] -175cm 2 Culture flask

[0299] - 20 mg / ml Nerofe in mannitol (use aliquots to avoid repeated freeze-thaw cycles).

[0300] -ABT-737 (Cayman 11501)

[0301] -CT26 cell line

[0302] For Western Blot

[0303] - a cleavage buffer comprising:

[0304] οRIPA buffer (Sigma R0278)

[0305] o Protease inhibitor cocktail (Sigma P2714).

[0306] O-phosphatase inhibitor (Sigma P5726)

[0307] ο Nuclease, purity>90% (70746 Millipore) 25U / ml

[0308] -X4 LDS sample buffer (Invitrogen catalog number NP0007)

[0309] -Sample reducing agent (Invitrogen catalog number NP0009, 10x concentrated)

[0310] -4%-12% Bis-Tris gel (Millipore catalog number MMMP41G10)

[0311] -10x MES transfer buffer (Millipore MMMPTRB)

[0312] -PVDF membrane (immobilon-P sq 0.2μm)

[0313] -TBST buffer: TBS (J640 Amersco) + 0.05% Tween-20

[0314] -5% skim milk in TBST

[0315] - Dilute the primary antibody 1:1000 in 5% skim milk in TBST:

[0316] o Anti-cleaved caspase 3 antibody (asp 175) Cell Signaling Cat. No. 9661

[0317] - Dilute the secondary antibody 1:10,000 in 5% skim milk in TBST:

[0318] ο Anti-rabbit IgG, HRP-linked antibody (Cells signaling 7074)

[0319] -SuperSignal TM West Femto Maximum Sensitivity Substrate(ThermoScientific 34095)

[0320] Cell Culture - Passaging:

[0321] The cells were grown at 37°C, 5% CO2, 100% humidity until they reached a density of 70% to 80%. The cells were trypsinized. The cells were resuspended in fresh medium and then grown for 2 to 3 days until they reached a density of 70% to 80%. The passaging procedure was repeated.

[0322] Cell treatment:

[0323] The cells were seeded in 25 ml of growth medium in 4 different flasks at a density of 1 M cells / 75CM2 flask and incubated overnight. The medium was changed. The cells were then treated as follows:

[0324] 1-Control (untreated)

[0325] 2-50μg / ml Nerofe

[0326] 3-No treatment (for ABT)

[0327] 4-50μg / ml Nerofe (for ABT)

[0328] 5-No treatment (for ABT)

[0329] 6-50μg / ml Nerofe (for ABT)

[0330] The cells were then incubated for 24 hours and treated as follows:

[0331] 1-Control (untreated)

[0332] 2-No treatment

[0333] 3-No treatment

[0334] 4-No treatment

[0335] 5-1μM ABT

[0336] 6-1μM ABT

[0337] The cells were then incubated for 24 hours and treated as follows:

[0338] 1-Control (untreated)

[0339] 2-50μg / ml Nerofe

[0340] 3-1μM ABT

[0341] 4-1μM ABT + 50μg / ml Nerofe

[0342] 5-No treatment

[0343] 6-50μg / ml Nerofe

[0344] The cells were then collected for Western blotting as previously described (Mahmood, T., & Yang, PC (2012). Western blot: technique, theory, and trouble shooting. North American journal of medical sciences, 4(9), 429-434).

[0345] Cell viability assay of CT26 cells treated with Nerofe and ABT-737 (Resazurin)

[0346] Material

[0347] - Cell culture medium:

[0348] ο10% FBS (Biological industries, catalog number 04-121-1A)

[0349] o DMEM (Gibco Cat. No. 41965)

[0350] ο1 mM sodium pyruvate (Biological Industries)

[0351] ο100 μg / ml penicillin and 100 μg / ml streptomycin (Biological Industries)

[0352] ο250 ng / ml amphotericin B (Biological Industries)

[0353] - Treatment medium -

[0354] o 5% mannitol in FBS-containing medium (Sigma catalog number M4125-500G) or

[0355] o 5% mannitol in medium without FBS (Sigma catalog number M4125-500G)

[0356] After adding mannitol, the medium was filtered using a 0.2 μm filter.

[0357] -Cytotoxicity assay kit (colorimetric analysis): abl 12118abeam

[0358] o Cell line: CT26

[0359] ο2 mg / ml Nerofe stock solution: dilute 100 μl of 20 mg / ml Nerofe in 900 μl cell treatment medium

[0360] οABT-737 (Cayman 11501)

[0361] Solution:

[0362]

[0363] Cells were seeded at a density of 5000 cells / well in a black 96-well plate with a clear bottom and 100 μl of growth medium was added. The cells were incubated for 24 hours. The medium was removed and the cells were treated with Nerofe in 100 μl of cell treatment medium containing FBS. The cells were incubated for 24 hours. The cells were then treated with ABT-737 in a final volume of 125 μl in the 48-hour treatment wells by adding 25 μl of treatment medium containing 5% mannitol without FBS to each well, respectively. The cells were incubated for 24 hours. The cells were treated with ABT-737 in the 24-hour treatment wells and treated with Nerofe in a final volume of 150 μl by adding 25 μl of treatment medium containing 5% mannitol without FBS to each well, respectively. The cells were incubated for 24 hours.

[0364] A certain amount of 25 μl of Cytotoxicity Assay Kit (Colorimetric Assay) abl 12118 was added to each well and the cells were incubated for 4 hours. The plate was read using a Biotek Synergy-HT plate reader at excitation 530, emission 590, and sensitivity 40. The percentage of cell viability was calculated compared to the control.

[0365] Example 1

[0366] Synergistic effect of Nerofe and BCL2 inhibitor in inducing apoptosis in U937 cells

[0367] As mentioned above, a peptide called "T101" encoded by a cDNA unique to the human thymus has been specifically identified and reported to reduce cancer tumor size. In addition, peptide derivatives of T101, referred to herein as dTCApFs (or "Nerofe"), have been reported to induce cancer cell migration [8] and participate in the induction of ER stress, which helps promote cell death [9].

[0368] Specifically, Nerofe is a peptide having an all-D amino acid sequence of Trp Trp Thr Phe Phe Leu Pro SerThr Leu Trp Glu Arg Lys as shown in SEQ ID NO: 1, and is prepared as described above.

[0369] The effects of Nerofe and ABT-737 alone or in combination on the activation of caspase 3 in U937 cells were examined. Figure 1 As shown, when Nerofe is combined with the BCL2 inhibitor, ABT-737, a strong synergistic effect on the activation of caspases, a marker of apoptosis, is observed. Without wishing to be bound by theory, it is believed that the BCL2 protein is the main inhibitor of the apoptotic effect of Nerofe.

[0370] Example 2

[0371] Synergistic effect of Nerofe and BCL2 inhibitor in inducing cell death in U937 cells

[0372] The effects of Nerofe and the BCL2 inhibitor, ABT-737, alone or in combination, on the percentage of cell viability of U937 cells were examined at different concentrations of ABT-737, i.e., 0.5 μM, 1 μM, or 2 μM. Figure 2 As shown, when Nerofe was combined with ABT-737, a strong synergistic effect on the induction of cell death was observed.

[0373] Example 3

[0374] No synergistic effect of Nerofe and azacitidine in inducing cell death was observed

[0375] The effects of Nerofe and azacitidine alone or in combination on the percentage of cell viability of U937 cells were examined at different concentrations of azacitidine (i.e., 50 nM or 100 nM). Figure 3As shown, when Nerofe is combined with azacitidine, no synergistic effect on inducing cell death is observed. Without wishing to be bound by theory, it seems that the anti-tumor activity of Nerofe is not through the same mechanism of action as azacitidine. Azacitidine is a chemical analog of the nucleoside cytidine, which is present in DNA and RNA and has anti-tumor activity through two mechanisms: at low doses, by inhibiting DNA methyltransferases, causing hypomethylation of DNA; and at high doses, by producing direct cytotoxicity by incorporation into DNA and RNA, leading to cell death. On the other hand, it is believed that Nerofe works by generating free oxygen radicals that damage the Golgi apparatus and induces unrepaired ER stress in cancer cells that overexpress T1 / ST2.

[0376] Example 4

[0377] Synergistic effect of Nerofe and BCL2 inhibitor in inducing cell death in CT26

[0378] The effects of Nerofe and the BCL2 inhibitor, ABT-737, alone or in combination, on the percentage of cell viability of CT26 cells were examined at a concentration of 1 μM ABT-737. Figure 4 As shown, when Nerofe was combined with ABT-737, a strong synergistic effect on the induction of cell death was observed.

[0379] Example 5

[0380] Synergistic effect of Nerofe and BCL2 inhibitor in inducing apoptosis in CT26 cells

[0381] The effects of Nerofe and ABT-737 alone or in combination on the activation of cleaved caspase 3 in CT26 cells were examined by Western blot analysis. Figure 5 As shown, when Nerofe was combined with the BCL2 inhibitor, ABT-737, a strong synergistic effect on the activation of caspases was observed.

[0382] Example 6

[0383] Effects of Nerofe and additional Bcl2 inhibitors

[0384] The effects of Nerofe and additional Bcl2 inhibitors such as ABT-199 (venetoclax), ABT-263 (nervitoclax), GX15-070 (obatoclax) or A-1331852 on the induction of apoptosis and / or cell death, for example, as described above, were further examined.

[0385] Example 7

[0386] Effects of the combination of Nerofe and BCL2 inhibitors on mouse models

[0387] 100,000 CT26 cells were inoculated subcutaneously (SC) into 32 Blab / C female mice. Once the tumor volume reached 50 mm 3 , the mice were divided into the following groups:

[0388] -8 mice - control

[0389] -8 mice - Nerofe (15 mg / Kg) intraperitoneally administered (IP) 3 times a week

[0390] -8 mice - ABT-737 (50 mg / Kg) IP 3 times a week

[0391] - 8 mice - Nerofe (15 mg / Kg) 3 times per week IP + ABT-737 (50 mg / Kg) 3 times per week IP.

[0392] The weight of mice was measured twice a week, and the tumor volume was also measured twice a week.

[0393] Example 8

[0394] Effects of the combination of Nerofe and additional BCL2 inhibitors in mouse models

[0395] 100,000 CT26 cells were inoculated subcutaneously (SC) into 32 Blab / C female mice. Once the tumor volume reached 50 mm 3 , the mice were divided into the following groups:

[0396] -8 mice - control

[0397] -8 mice - Nerofe (15 mg / Kg) intraperitoneally administered (IP) 3 times a week

[0398] -8 mice – ABT-199 (Venetoclax) 3 times per week IP

[0399] -8 mice - ABT-263 (Navitoclax) 3 times per week IP 3 times per week IP

[0400] -8 mice - GX15-070 (Obatok) 3 times per week IP 3 times per week IP

[0401] -8 mice - A-1331852 3 times per week IP 3 times per week IP

[0402] -8 mice - Nerofe 3 times per week IP + ABT-199 (Venetoclax) 3 times per week IP

[0403] -8 mice - Nerofe 3 times per week IP + ABT-263 (Navitoclax) 3 times per week IP

[0404] -8 mice – Nerofe 3 times per week IP + GX15-070 (Obatok) 3 times per week IP

[0405] -8 mice - Nerofe 3 times per week IP + A-1331852 (Obatok) 3 times per week IP

[0406] The weight of mice was measured twice a week, and the tumor volume was also measured twice a week.

Claims

1. A combined composition comprising at least one inhibitor of B-cell lymphoma 2 (Bcl2) pro-survival protein and an isolated peptide comprising the amino acid sequence shown in SEQ ID NO.1 or any functional derivative thereof, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent, excipient and / or additive.

2. The composition of claim 1, wherein the modified isolated peptide has at least 70%, or 80%, or 90%, or 95% identity to the corresponding sequence of SEQ ID NO:

1.

3. The composition according to any of the preceding claims, wherein the Bcl-2 pro-survival protein is at least one of the following: Bcl-2, B-cell lymphoma-extra large (Bcl-xL), myeloid cell leukemia-1 (Mcl-1), B-cell lymphoma-w (Bcl-w), B-cell lymphoma 2-associated protein A1 (Bcl2A1), and B-cell lymphoma 2-like 10 (Bcl-B / Bcl2L10).

4. The composition of any one of the preceding claims, wherein the at least one inhibitor of a Bcl2 pro-survival protein is at least one Bcl-2 homology 3 (BH3) mimetic compound.

5. The composition of claim 4, wherein the BH3 mimetic compound is at least one of the following or any combination thereof: 4-{4-[(4'-chloro[1,1'-biphenyl]-2-yl)methyl]piperazin-1-yl}-N-(4-{[(2R)-4-(dimethylamino)-1-(phenylthio)butan-2-yl]amino}-3-nitrobenzene-1-sulfonyl)benzamide (ABT-737), 4-(4-{[2-(4-chlorophenyl)-4,4-dimethyl-1-cyclohexen-1-yl]methyl}-1-piperazinyl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)-amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]-pyridin-5-yloxy)benzamide (ABT-199), -(4-{[2-(4-chlorophenyl)-5,5-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-(4-{[(2R)-4-(morpholin-4-yl)-1-(phe-nylthio)butan-2-yl]amino}-3-(trifluoromethanesulfonyl)benzene-1-sulfonyl)benzamide (ABT-263), 2-(2-((3,5-dimethyl- 1H-pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-yl)-1H-indole (GX15-070), or 3-[1-(1-adamantylmethyl)-5-methylpyrazol-4-yl]-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydro-1H-isoquinolin-2-yl]pyridine-2-carboxylic acid (A-1331852).

6. The composition of claim 5, wherein the at least one inhibitor of the Bcl2 pro-survival protein is 4-{4-[(4'-chloro[1,1'-biphenyl]-2-yl)methyl]piperazin-1-yl}-N-(4-{[(2R)- 4-(Dimethylamino)-1-(phenylthio)butan-2-yl]amino}-3-nitrobenzene-1-sulfonyl)-benzamide (ABT-737), or any pharmaceutically acceptable salt or hydrate thereof, or any stereoisomer or salt thereof.

7. A composition according to any one of the preceding claims for use in a method of treating, preventing, ameliorating, reducing or delaying the onset of at least one proliferative disorder.

8. The composition for use according to claim 7, wherein the proliferative disorder is associated with Bcl2 overexpression.

9. The composition for use according to claim 7 or 8, wherein the proliferative disorder is at least one hematological cancer or neoplasm.

10. The composition of claim 9, wherein the hematological cancer or neoplasm is at least one of chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), a mutated KRAS tumor, a lymphoma, multiple myeloma, a blastic plasmacytoid dendritic cell neoplasm, T-cell prolymphocytic leukemia, or a myelodysplastic disorder.

11. A method for treating, preventing, ameliorating, reducing or delaying the onset of at least one proliferative disorder in a subject in need thereof, the method comprising the step of administering a therapeutically effective amount of at least one inhibitor of a Bcl2 pro-survival protein and an isolated peptide comprising the amino acid sequence shown in SEQ ID NO.1 or any functional derivative thereof, or a pharmaceutically acceptable salt thereof, or any composition, kit or combination thereof.

12. The method of claim 11, wherein the modified isolated peptide has at least 70%, or 80%, or 90%, or 95% identity to the corresponding sequence of SEQ ID NO:

1.

13. The method of claim 11 or 12, wherein the Bcl-2 pro-survival protein is at least one of the following: Bcl-2, Bcl-xL, Mcl-1, Bcl-w, Bcl2A1, and Bcl-B / Bcl2L10.

14. The method according to any one of claims 11 to 13, wherein the at least one inhibitor of a Bcl2 pro-survival protein is at least one BH3 mimetic compound.

15. The method of claim 14, wherein the BH3 mimetic compound is at least one of: 4-{4-[(4'-chloro[1,1'-biphenyl]-2-yl)methyl]piperazin-1-yl}-N-(4-{[(2R)-4-(dimethylamino)-1-(phenylthio)butan-2-yl]amino}-3-nitrobenzene-1-sulfonyl)benzamide (ABT-737), 4-(4-{[2-(4-chlorophenyl)-4,4-dimethyl-1-cyclohexen-1-yl]methyl}-1-piperazinyl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)-amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]-pyridin-5-yloxy)benzamide (ABT-199), -{[2-(4-chlorophenyl)-5,5-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-(4-{[(2R)-4-(morpholin-4-yl)-1-(phe-nylthio)butan-2-yl]amino}-3-(trifluoromethanesulfonyl)benzene-1-sulfonyl)benzamide (ABT-263), 2-(2-((3,5-dimethyl-1H -pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-yl)-1H-indole (GX15-070), or 3-[1-(1-adamantylmethyl)-5-methylpyrazol-4-yl]-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydro-1H-isoquinolin-2-yl]pyridine-2-carboxylic acid (A-1331852).

16. The method of claim 15, wherein the at least one inhibitor of the Bcl2 pro-survival protein is 4-{4-[(4'-chloro[1,1'-biphenyl]-2-yl)methyl]piperazin-1-yl}-N-(4-{[(2R)-4-(dimethylamino)-1-(phenylthio)butan-2-yl]amino}-3-nitrobenzene-1-sulfonyl)-benzamide (ABT-737), or any pharmaceutically acceptable salt or hydrate thereof, or any stereoisomer or salt thereof.

17. The method according to any one of claims 11 to 16, wherein the at least one inhibitor of Bcl2 pro-survival protein and the isolated peptide comprising the amino acid sequence shown in SEQ ID NO. 1 are administered simultaneously or sequentially.

18. The method of any one of claims 11 to 17, further comprising administering at least one additional anticancer agent.

19. The method of any one of claims 11 to 18, wherein the proliferative disorder is associated with Bcl2 overexpression.

20. The method of any one of claims 11 to 19, wherein the proliferative disorder is at least one hematological cancer or neoplasm.

21. The method of claim 20, wherein the hematological cancer or neoplasm is at least one of chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), a mutated KRAS tumor, a lymphoma, multiple myeloma, a blastic plasmacytoid dendritic cell neoplasm, T-cell prolymphocytic leukemia, or a myelodysplastic disorder.

22. At least one inhibitor of a Bcl2 pro-survival protein and at least one isolated peptide comprising the amino acid sequence shown in SEQ ID NO. 1 or any functional derivative thereof, or a pharmaceutically acceptable salt thereof, for use in a method of treating, preventing, ameliorating, reducing or delaying the onset of at least one proliferative disorder.

23. A kit, comprising: (a) at least one inhibitor of a Bcl2 pro-survival protein, or any pharmaceutically acceptable salt thereof, optionally in a first dosage form; (b) optionally in a second dosage form an isolated peptide comprising the amino acid sequence shown in SEQ ID NO. 1 or any functional derivative thereof, or a pharmaceutically acceptable salt thereof.

24. The kit of claim 23, wherein the modified isolated peptide has at least 70%, or 80%, or 90%, or 95% identity to the corresponding sequence of SEQ ID NO:

1.

25. The kit of claim 23 or 24, wherein the Bcl-2 pro-survival protein is at least one of the following: Bcl-2, Bcl-xL, Mcl-1, Bcl-w, Bcl2A1, and Bcl-B / Bcl2L10.

26. The kit according to any one of claims 23 to 25, wherein the at least one inhibitor of a Bcl2 pro-survival protein is at least one BH3 mimetic compound.

27. The kit of claim 26, wherein the BH3 mimetic compound is at least one of: 4-{4-[(4'-chloro[1,1'-biphenyl]-2-yl)methyl]piperazin-1-yl}-N-(4-{[(2R)-4-(dimethylamino)-1-(phenylthio)butan-2-yl]amino}-3-nitrobenzene-1-sulfonyl)benzamide (ABT-737), 4-(4-{[2-(4-chlorophenyl)-4,4-dimethyl-1-cyclohexen-1-yl]methyl}-1-piperazinyl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)-amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]-pyridin-5-yloxy)benzamide (ABT-199), -{[2-(4-chlorophenyl)-5,5-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-(4-{[(2R)-4-(morpholin-4-yl)-1-(phe-nylthio)butan-2-yl]amino}-3-(trifluoromethanesulfonyl)benzene-1-sulfonyl)benzamide (ABT-263), 2-(2-((3,5-dimethyl-1H -pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-yl)-1H-indole (GX15-070), or 3-[1-(1-adamantylmethyl)-5-methylpyrazol-4-yl]-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydro-1H-isoquinolin-2-yl]pyridine-2-carboxylic acid (A-1331852).

28. The kit of claim 27, wherein the at least one inhibitor of Bcl2 pro-survival protein is 4-{4-[(4'-chloro[1,1'-biphenyl]-2-yl)methyl]piperazin-1-yl}-N-(4-{[(2R)-4-(dimethylamino)-1-(phenylthio)butan-2-yl]amino}-3-nitrobenzene-1-sulfonyl)-benzamide (ABT-737), or any pharmaceutically acceptable salt or hydrate thereof, or any stereoisomer or salt thereof.

29. The kit of any one of claims 23 to 28, for use in a method of treating, preventing, ameliorating, reducing or delaying the onset of at least one proliferative disorder.

30. The kit for use according to claim 29, wherein said at least one inhibitor of Bcl2 pro-survival protein and said isolated peptide comprising the amino acid sequence shown in SEQ ID NO. 1 are in separate unit dosage forms.

31. The kit for use according to claim 30, wherein said separate unit dosage forms of at least one inhibitor of Bcl2 pro-survival protein and the isolated peptide comprising the amino acid sequence shown in SEQ ID NO. 1 are administered simultaneously or sequentially.

32. A kit for use according to any one of claims 29 to 31 , wherein the proliferative disorder is associated with overexpression of Bcl2.

33. A kit for use according to any one of claims 29 to 32, wherein the proliferative disorder is at least one hematological cancer or neoplasm.

34. The kit for use according to claim 33, wherein the hematological cancer or neoplasm is at least one of chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), a mutated KRAS tumor, a lymphoma, multiple myeloma, a blastic plasmacytoid dendritic cell neoplasm, T-cell prolymphocytic leukemia, or a myelodysplastic disorder.

35. A method for treating, preventing, ameliorating, reducing or delaying the onset of at least one proliferative disorder in a subject treated with at least one inhibitor of a Bcl2 pro-survival protein, the method comprising the step of administering to the subject a therapeutically effective amount of an isolated peptide comprising the amino acid sequence shown in SEQ ID NO.1 or any functional derivative thereof, or a pharmaceutically acceptable salt thereof, or any composition, kit or combination thereof.

36. A method for treating, preventing, ameliorating, reducing or delaying the onset of at least one proliferative disorder in a subject treated with at least one isolated peptide comprising the amino acid sequence shown in SEQ ID NO.1, the method comprising the step of administering to the subject a therapeutically effective amount of at least one inhibitor of a Bcl2 prosurvival protein or any functional derivative thereof, or a pharmaceutically acceptable salt thereof, or any composition, kit or combination thereof.

Images