Neuropilin antagonist in combination with p38alpha-kinase inhibitor for treatment of cancer

Abstract

SOLUTION: To treat cancer by administering to a patient a therapeutically effective combination comprising a neuropilin antagonist, e.g., NRPa-47, NRPa-48, and a p38α-kinase inhibitor, e.g., ralimetinib.EFFECT: Neuropilin antagonists can reduce VEGF-Rs phosphorylation and modulate their downstream cascades in triple-negative breast cancer cell lines (MDA-MB-231), and a combination of NRPa with p38α inhibitors results in additional and / or synergistic effects of these drugs (depending on doses used) for significantly reducing breast cancer cell proliferation. Therefore, the efficient combination of NRPa with p38α-kinase inhibitors is effective in the treatment of cancer, as a consequence.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to the field of medicine, in particular oncology. [Background technology]

[0002] 2. Background of the Invention Neuropilin-1 (NRP-1) and neuropilin-2 (NRP-2) are transmembrane type I glycoproteins that share 44% sequence homology [1]. Initially, neuropilins (NRPs) were identified as neuronal receptors for certain secreted members of the semaphorin III family involved in axon guidance and repulsion [2]. Neuropilins are multifunctional nontyrosine kinase receptors for some members of the VEGF (vascular endothelial growth factor) family of members. VEGF-A, a splice form of VEGF-A that has been reported to be upregulated in some tumor tissues, 165 VEGF-A is considered one of the most efficient angiogenic factors. 165 VEGF-R binds to structurally related tyrosine kinase receptors, such as VEGF-R1 (Flt-1), VEGF-R2 (Flk-2), and the co-receptors NRPs, which lack cytosolic catalytic activity [3, 1]. In many cancers, expression of one or both NRPs correlates with tumor progression and / or poor prognosis (see reviews) [4, 5]. Through their direct interaction with VEGF-R, NRPs have suddenly emerged as key regulators of angiogenesis and tumor progression.

[0003] These identified protein-protein interactions that trigger the angiogenic process have led to the development of extracellular VEGF traps such as monoclonal antibodies (e.g., Avastin®), aptamers (e.g., Macugen®) and small molecules that target the intracellular kinase activity of its tyrosine kinase receptor (e.g., Sutent®). Nevertheless, the existence of different pathways involved in angiogenesis and the emergence of therapeutic resistance in patients with generally poor responses has led to the need to develop new antiangiogenic strategies.

[0004] Drug development against the newly emerging NRP targets has led to modern tools such as antibodies [1, 6, 7], peptides (A7R, EG3287, NRP-1 transmembrane peptide) [8–12] and peptidomimetics (EG00229)

[13] . Recently, new approaches have highlighted the interest of inhibitory small molecules that reduce VEGF binding to NRPs and tumor growth in vivo and in vitro [14, 15]. In this field, we are the first research team to develop completely non-peptide inhibitory molecules, so-called neuropilin antagonists (NRPa)

[15] . However, the molecular mechanisms by which NRPs modulate cancer progression remain to be fully elucidated. NRPa should provide additional data regarding theoretical knowledge of cell signaling involved in tumor development and survival.

[0005] Summary of the Invention As defined by the claims, the present invention relates to a neuropilin antagonist in combination with a p38α-kinase inhibitor for the treatment of cancer.

[0006] Detailed Description of the Invention Neuropilin-1 is an important target in the future of cancer treatment, but its mechanism of action remains elusive and therefore requires the development of inhibitory small molecules for its investigation. 165We report that two neuropilin small antagonists (NRPa-47, NRPa-48), which are VEGF-R / NRP-1 binding inhibitors, can reduce VEGF-R phosphorylation and modulate their downstream cascades in triple-negative breast cancer cell line (MDA-MB-231). Nevertheless, NRPa leads to the phosphorylation of MAPKs such as JNK-1 / -2 / -3, ERK-1 / -2 and p38β / γ / δ-kinase and branched pathway regulation of their respective downstream targets. However, NRPa-47 and NRPa-48 exert a common downregulation of p38α-kinase phosphorylation and their downstream targets reinforcing its central regulatory role. More importantly, none of the 40 selected kinases, including SAPK2a / p38α, was affected by NRPa in vitro, supporting their specificity. In summary, NRPa induced cell death by down-modulating pro- and anti-apoptotic proteins, cell death receptors and adaptors, heat shock proteins (HSP-27 / -60 / -70), cell cycle proteins (p21, p27, phospho-RAD17) and transcription factors (p53, HIF-1α). In conclusion, we showed for the first time how NRPa can alter tumor cell signaling and contribute to the down-modulation of p38α-kinase phosphorylation, a key factor in cancer therapy. Therefore, an efficient combination of NRPa with p38α-kinase inhibitors would be effective in the treatment of cancer.

[0007] A further object of the present invention relates to a method for treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective combination comprising at least one neuropilin antagonist and at least one p38α-kinase inhibitor.

[0008] As used herein, the term "subject" or "patient" refers to a mammal, preferably a human. Examples of non-human mammals include pets such as dogs, cats, domestic pigs, rabbits, ferrets, hamsters, mice, rats, etc.; primates, such as chimpanzees, monkeys, etc.; and economically important animals, such as cows, pigs, rabbits, horses, sheep, goats, etc.

[0009] As used herein, the term "cancer" has its general meaning in the art and includes, but is not limited to, hematopoietic cancers (e.g., blood-borne tumors) and non-hematopoietic cancers (e.g., solid tumors). The term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood, and blood vessels. The term "cancer" further includes both primary and metastatic cancers. Examples of cancers that can be treated by the methods and compositions of the present invention include, but are not limited to, cancer cells derived from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, digestive tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testes, tongue, or uterus. The cancer may also be of the following specific histological types, but is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma of adenomatous polyps; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; bronchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophilic carcinoma; acidophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulated sclerosing carcinoma; adrenal cortical carcinoma; Endometrioid carcinoma;Cutaneous adnexal carcinoma;Apocrine gland carcinoma;Sebaceous gland carcinoma;Earwax gland carcinoma;Mucoepidermoid carcinoma;Cystadenocarcinoma;Papillary cystadenocarcinoma;Papillary serous cystadenocarcinoma;Colloid cystadenocarcinoma;Colloid adenocarcinoma;Signet ring cell carcinoma;Invasive ductal carcinoma;Medullary carcinoma;Lobular carcinoma;Inflammatory carcinoma;Paget's disease, breast;Acinic cell carcinoma;Adenosquamous carcinoma;Adenocarcinoma w / squamous metaplasia;Thymoma, malignant;Ovarian stromal tumor, malignant malignant;theca cell tumor, malignant;granulosa cell tumor, malignant;and neuroblastoma, malignant;Sertoli cell carcinoma;Leydig cell tumor, malignant;lipid cell tumor, malignant;paraganglioma, malignant;extramammary paraganglioma, malignant;pheochromocytoma;glomangiosarcoma;malignant melanoma;amelanotic melanoma;superficial spreading melanoma;malignant melanoma of giant pigmented nevus;epithelioid cell melanoma;blue nevus, malignant;sarcoma;Fibrosarcoma;Fibrous histiocytoma, malignant;Myxosarcoma;Liposarcoma;Leiomyosarcoma;Rhabdomyosarcoma;Embryonal rhabdomyosarcoma;Alveolar rhabdomyosarcoma;Stromatous sarcoma;Mixed tumor, malignant;Mullerian mixed tumor;Nephroblastoma;Hepatoblastoma;Carcinosarcoma;Mesenchymoma, malignant;Brenner tumor, malignant;Phyllodes tumor, malignant;Synovial sarcoma;Mesothelioma, malignant;Dysgerminoma;Embryonal carcinoma;Teratoma, malignant;Ovarian goiter, malignant;Choriocarcinoma; Mesonephroma, malignant;angiosarcoma;hemangioendothelioma, malignant;Kaposi's sarcoma;hemangiopericytoma, malignant;lymphangiosarcoma;osteosarcoma;parosteal osteosarcoma;chondrosarcoma;chondroblastoma, malignant;mesenchymal chondrosarcoma;giant cell tumor of bone;Ewing's sarcoma;odontogenic tumor, malignant;ameloblastic odontosarcoma;ameloblastoma, malignant;ameloblastic fibrosarcoma;pinealoma, malignant;chordoma;glioma, malignant;ependymoma;astrocytoma;protoplasmic astrocytoma astrocytoma;fibrous astrocytoma;astroblastoma;glioblastoma;oligodendroglioma;oligodendroglioma;primitive neuroectodermal tumor;cerebellar sarcoma;ganglioneoblastoma;neuroblastoma;retinoblastoma;olfactory neuronal tumor;meningioma, malignant;neurofibrosarcoma;schwannoma, malignant;granular cell tumor, malignant;malignant lymphoma;Hodgkin's disease;Hodgkin's lymphoma;lateral granuloma;malignant lymphoma, small lymphocytic;malignant lymphoma, large cell, diffuse; Malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. ;

[0010] In some embodiments, the method of the present invention is particularly suitable for treating breast cancer, in particular triple-negative breast cancer.As used herein, the term "triple-negative breast cancer" has its general meaning in the art, and means that the breast cancer lacks the receptors for the hormones estrogen (ER negative) and progesterone (PR negative) and the receptor for the protein HER2.

[0011] In some embodiments, the cancer has previously been screened as "neuropilin positive", i.e., the cancer cells express neuropilin protein. The expression can be assessed in the tumor by any routine method known in the art, such as immunohistochemistry (IHC), immunofluorescence, mass spectrometry, RT-PCR, fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH), silver in situ hybridization (SISH) or comparative genomic hybridization (CGH), RNAscope, etc.

[0012] As used herein, the term "treatment" or "treat" refers to both prophylactic or preventative and curative or disease-modifying treatments, including treatment of unhealthy patients or patients diagnosed with a disease or condition, as well as patients at risk of or suspected of having a disease, including suppression of clinical recurrence. Treatments may be administered to patients with a medical disorder or those who may eventually acquire a disorder, to prevent, cure, delay the onset of the disorder or recurrent disorder, to reduce its severity, or to ameliorate one or more symptoms thereof, or to extend the survival of the patient beyond that expected in the absence of such treatment. "Therapeutic regimen" refers to a pattern of treatment of a disease, e.g., a pattern of dosing used during treatment. The therapeutic regimen may include induction regimens and maintenance regimens. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or a portion of a therapeutic regimen) used for the initial treatment of a disease. The general goal of an induction regimen is to provide high levels of drug to the patient during the initial period of the treatment regimen. The induction regimen may use (partially or entirely) a "loading regimen", which may involve the physician administering a higher dose of the drug than he or she uses during the maintenance regimen, administering the drug more frequently than he or she administers during the maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or a portion of a treatment regimen) used to maintain a patient during disease treatment, for example, to keep the patient in remission for an extended period of time (months or years). A maintenance regimen may use continuous treatment (e.g., administering the drug at regular intervals, for example, once a week, once a month, once a year, etc.) or intermittent treatment (e.g., intermittent treatment, intermittent treatment, treatment upon relapse, or treatment upon reaching a certain predetermined criterion (e.g., pain, disease symptoms, etc.). In particular, in the case of AML, maintenance treatment may eradicate clinically invisible minimal residual disease.

[0013] As used herein, the term "neuropilin" or "NRP" has its general meaning in the art and typically refers to a transmembrane glycoprotein consisting of five domains: three extracellular domains (a1 a2, b1, b2 and c), a transmembrane domain and a cytoplasmic domain. There are two neuropilin members that share 44% sequence homology: neuropilin-1 (NRP-1) and neuropilin-2 (NRP-2). Neuropilins are a member of the VEGF-A family of VEGF-A receptors. 165 It is a multifunctional non-tyrosine kinase receptor for some members of the VEGF (vascular endothelial growth factor) family, including

[0014] As used herein, the term "neuropilin antagonist" refers to a molecule that partially or completely blocks, inhibits or neutralizes the biological activity or expression of neuropilin protein.Neuropilin antagonist can be any type of molecule that interferes with the signal transduction associated with at least one or more neuropilin family members (e.g., NRP-1 or NRP-2) in cells, for example, by reducing the transcription or translation of the nucleic acid encoding neuropilin, or by inhibiting or blocking neuropilin polypeptide activity, or both. Examples of neuropilin antagonists include, but are not limited to, antisense polynucleotides, interfering RNA, catalytic RNA, RNA-DNA chimeras, neuropilin-specific aptamers, anti-neuropilin antibodies, neuropilin-binding fragments of anti-neuropilin antibodies, neuropilin-binding small molecules, neuropilin-binding peptides, and other polypeptides that specifically bind to neuropilin (including, but not limited to, neuropilin-binding fragments of one or more neuropilin ligands, optionally fused to one or more additional domains), such that the interaction between the neuropilin antagonist and neuropilin results in the reduction or elimination of neuropilin activity or expression. In particular, neuropilin antagonists bind to neuropilin proteins (e.g., NRP-1) and their partners, particularly VEGF-A. 165It inhibits the interaction between

[0015] Neuropilin antagonists are well known in the art and typically include those described below:

[0016] [Table 1] TIFF2025060805000002.tif148165

[0017] In some embodiments, the neuropilin antagonist specifically binds to a neuropilin (e.g., NRP-1 or NRP-2) and neutralizes its activity in activating the neuropilin signaling pathway, particularly the interaction between neuropilin and VEGF-A. 165 In some embodiments, the antibody binds to the extracellular domain of a neuropilin. In some embodiments, the antibody binds to domain c of NRP-1. Examples of antibodies that are neuropilin antagonists include those described in WO2011 / 143408, which describes, inter alia, the anti-NRP-1 antibody MNRP1685A.

[0018] As used herein, the term "antibody" includes, but is not limited to, polyclonal, monoclonal, humanized, chimeric, Fab fragments, Fv fragments, F(ab') fragments and F(ab')2 fragments, as well as single chain antibodies (scFv), fusion proteins and other synthetic proteins that contain the antigen-binding site of an antibody. Antibodies can be synthesized by those skilled in the art using methods known in the art and commercially available services and kits. Methods for preparing monoclonal antibodies are well known in the art and include hybridoma and phage display technologies. Additional antibodies suitable for use in the present disclosure are described, for example, in the following publications: Antibodies A Laboratory Manual, Second edition. Edward A. Greenfield. Cold Spring Harbor Laboratory Press (Sep. 30, 2013); Making and Using Antibodies: A Practical Handbook, Second Edition. Eds. Gary C. Howard and Matthew R. Kaser. CRC Press (Jul. 29, 2013); Antibody Engineering: Methods and Protocols, Second Edition (Methods in Molecular Biology). Patrick Chames. Humana Press (Aug. 21, 2012); Monoclonal Antibodies: Methods and Protocols (Methods in Molecular Biology). Eds. Vincent Ossipow and Nicolas Fischer. Humana Press (Feb. 12, 2014); and Human Monoclonal Antibodies: Methods and Protocols (Methods in Molecular Biology). Michael Steinitz. Humana Press (Sep. 30, 2013)).

[0019] In some embodiments, the neuropilin antagonist is a small molecule, eg, a small organic molecule, typically having a molecular weight of less than 5,000 kDa.

[0020] Examples of small molecules that are neuropilin antagonists include those described in WO2012156289, which are: - N-[5-(1H-benzimidazol-2-yl)-2-methylphenyl]-N'-(2,3-dihydro-1,4-benzodioxin-6-ylcarbonyl)thiourea (also named NRPa-47): [ka] - N-[3-(1H-benzimidazol-2-yl)phenyl]-N'-(2,3-dihydro-1,4-benzodioxin-6-ylcarbonyl)thiourea (also named NRPa-48): [ka] and / or - N-[3-(1H-benzimidazol-2-yl)phenyl]-N'-(1,3-benzodioxol-5-ylcarbonyl)thiourea [ka] or their salts and esters, and mixtures thereof.

[0021] Another example includes N-(2-ethoxyphenyl)-4-methyl-3-(N-(p-tolyl)sulfamoyl)benzamide, which is described in WO2015004212 and has the following formula: [ka]

[0022] In some embodiments, the neuropilin antagonist is an inhibitor of neuropilin expression.

[0023] "Expression inhibitor" refers to a natural or synthetic compound that has the biological effect of inhibiting gene expression.In some embodiments, the gene expression inhibitor is an siRNA, an antisense oligonucleotide, or a ribozyme.For example, antisense oligonucleotides, including antisense RNA molecules and antisense DNA molecules, will act to directly block the translation of NRP-1 mRNA by binding to it, thereby preventing protein translation or increasing the degradation of mRNA, thus reducing the level and thus the activity of NRP-1 in cells.For example, antisense oligonucleotides of at least about 15 bases that are complementary to unique regions of the mRNA transcript sequence that codes for NRP-1 can be synthesized, for example, by conventional phosphodiester techniques. Methods for using antisense technology to specifically inhibit gene expression of genes whose sequences are known are well known in the art (see, for example, U.S. Patent Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Small interfering RNA (siRNA) can also function as an inhibitor of expression for use in the present invention. NRP-1 gene expression can be reduced by contacting a patient or cell with small double-stranded RNA (dsRNA) or a vector or construct that causes the production of small double-stranded RNA, so that NRP-1 gene expression is specifically inhibited (i.e., RNA interference or RNAi). The antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the present invention can be delivered in vivo alone or in combination with vectors. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of an antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid into a cell, typically a cell expressing NRP-1. Typically, a vector transports the nucleic acid into a cell with reduced degradation compared to the extent of degradation that occurs in the absence of the vector.In general, vectors useful in the present invention include, but are not limited to, plasmids, phagemids, viruses, and other vehicles derived from viral or bacterial origin that have been engineered by the insertion or incorporation of antisense oligonucleotides, siRNAs, shRNAs, or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to, nucleic acid sequences derived from the following viruses: retroviruses such as Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; adenoviruses, adeno-associated viruses; SV40 type viruses; polyoma viruses; Epstein-Barr virus; papilloma viruses; herpes viruses; vaccinia viruses; polio viruses; and RNA viruses such as retroviruses. Other vectors not named but known in the art can be readily used. In some embodiments, the inhibitor of expression is an endonuclease. In certain embodiments, the endonuclease is CRISPR-cas. In some embodiments, the endonuclease is CRISPR-cas9 from Streptococcus pyogenes. CRISPR / Cas9 systems are described in US 8697359 B1 and US 2014 / 0068797. In some embodiments, the endonuclease is CRISPR-Cpf1, which is the more recently characterized CRISPR (Cpf1) from Provotella and Francisella 1 by Zetsche et al. ("Cpf1 is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).

[0024] As used herein, the term "p38α-kinase" has its general meaning in the art and refers to a member of the p38 mitogen-activated protein kinase (MAPK). The p38 MAPK family includes four members, p38-α (MAPK14), p38-β (MAPK11), p38-γ (MAPK12 / ERK6) and p38-δ (MAPK13 / SAPK4), which are involved in signal transduction cascades that control cellular responses.

[0025] As used herein, the term "p38α-kinase inhibitor" refers to a molecule that partially or completely blocks, inhibits or neutralizes the biological activity or expression of p38α protein. Suitable inhibitor molecules specifically include antagonist antibodies or antibody fragments, natural polypeptides, peptide fragments or amino acid sequence variants, antisense oligonucleotides, small organic molecules, recombinant proteins or peptides, and the like. A p38α-kinase inhibitor can be any type of molecule that interferes with at least p38-α-related signal transduction, for example, by reducing the transcription or translation of nucleic acid encoding p38-α, or by inhibiting or blocking p38-α kinase activity, or both. In some examples, a p38α-kinase inhibitor is an agent that interferes with p38-α-related signal transduction. Examples of p38α-kinase inhibitors include, but are not limited to, antisense polynucleotides, interfering RNA, catalytic RNA, RNA-DNA chimeras, p38α-specific aptamers, anti-p38α antibodies, p38α-binding fragments of anti-p38α antibodies, p38α-binding small molecules, p38α-binding peptides, and other polypeptides that specifically bind to p38α (including, but not limited to, p38α-binding fragments of one or more p38α ligands, optionally fused to one or more additional domains), such that interaction between the p38α-kinase inhibitor and p38α results in reduction or elimination of p38α kinase activity or expression. For example, a desirable p38α-kinase inhibitor for use in certain methods herein is a p38α-kinase inhibitor that binds to p38-α and blocks p38α signaling, and that has no or minimal effect on any other member of the p38 MAPK family, e.g., does not bind to p38-β, p38-γ and / or p38-δ. It will be appreciated that the p38α-kinase inhibitors described herein may be potent inhibitors of p38α.For example, the p38α-kinase inhibitor has a binding inhibitory activity (IC50 value) against p38α of 1000 μM or less, 1000 nM or less, 100 nM or less, 10 nM or less, or especially 1 nM or less. In another example, the p38α-kinase inhibitor has a binding inhibitory activity (IC50 value) against p38α of between 1000 μM and 1 nM, between 1000 μM and 10 nM, between 1000 μM and 100 nM, between 1000 μM and 1000 nM, between 1000 nM and 1 nM, between 1000 nM and 10 nM, between 1000 nM and 100 nM, between 100 nM and 1 nM, or between 10 nM and 1 nM.

[0026] In particular, p38α-kinase inhibitors are small molecules, eg, small organic molecules, typically having a molecular weight of less than 5,000 kDa. Inhibitors of p38α include ARRY-371797 (ARRY-797; Array BioPharma Inc.), ARRY-614 (pexmetinib; Array BioPharma Inc. or Selleckchem), AZD-7624 (AstraZeneca Plc), LY-2228820 (ralimetinib dimesylate; Eli Lilly and Co. or Selleckchem), LY-3007113 (Eli Lilly and Co.), FX005 (Flexion Therapeutics Inc.), GSK610677 (GlaxoSmithKline Plc), GW856553 (GW856553X; losmapimod; GlaxoSmithKline Plc or Selleckchem), SB-681323 (dilmapimod; GlaxoSmithKline Plc), KC706 (Kemia Plc), and ALK10 (Altima Gel; GlaxoSmithKline Plc). Inc.), UR-13870 (Palau Pharma SA), PF-03715455 (PF-3715455; Pfizer Inc.), VX-745 (Vertex Pharmaceuticals Inc. or Selleckchem), SCID-469 (talmapimod; Scios Inc.), PH-797804 (Pfizer or Selleckchem), VX-702 (Selleckchem), SB-202190 (FHPI; Selleckchem), SB-203580 (Selleckchem), SB-239063, BIRB-796 (drampimod; Selleckchem), BMS-582949, and pamapimod.

[0027] In some embodiments, the p38α-kinase inhibitor is an inhibitor of p38α-kinase expression.

[0028] As used herein, the term "combination" is intended to refer to all dosage forms that provide a first drug together with an additional (second, third, ...) drug. The drugs may be administered simultaneously, separately or sequentially and in any order. The drugs administered in combination have biological activity in the patient to whom they are delivered. Within the context of the present invention, a combination thus comprises at least two different drugs, and one drug is at least one neuropilin antagonist and the other drug is at least one p38α-kinase inhibitor. In some cases, the combination of the present invention results in synthetic lethality of cancer cells.

[0029] A "therapeutically effective amount" refers to an amount effective at the dosage and duration required to achieve the desired therapeutic result. The therapeutically effective amount of a drug may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the drug to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. The effective dosage and dosing regimen of the drug depends on the disease or condition to be treated and can be determined by one of ordinary skill in the art. A physician of ordinary skill in the art can easily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician can start the dose of the drug used in the pharmaceutical composition at a level lower than required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. In general, a suitable dose of the composition of the present invention will be the amount of the compound that is the lowest dose effective to produce a therapeutic effect according to a particular dosing regimen. Such an effective dose will generally depend on the factors described above. For example, a therapeutically effective amount in therapeutic applications can be measured by its ability to stabilize the progression of a disease. A therapeutically effective amount of a therapeutic compound may reduce tumor size or otherwise ameliorate symptoms in a subject. One of skill in the art would be able to determine such amounts based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected. An exemplary, non-limiting range for a therapeutically effective amount of a drug is about 0.1-100 mg / kg, such as about 0.1-50 mg / kg, such as about 0.1-20 mg / kg, such as about 0.1-10 mg / kg, for example about 0.5, such as about 0.3, about 1, about 3 mg / kg, about 5 mg / kg or about 8 mg / kg. An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the invention is 0.02-100 mg / kg, such as about 0.02-30 mg / kg, such as about 0.05-10 mg / kg or 0.1-3 mg / kg, such as about 0.5-2 mg / kg. Administration may be, for example, intravenous, intramuscular, intraperitoneal or subcutaneous, but may be administered proximal to the target site. The dosage regimen in the above-mentioned methods and uses of treatment is adjusted to produce the optimum desired response (e.g., therapeutic response).For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the effectiveness of treatment is monitored during treatment, e.g., at predetermined time points. As a non-limiting example, treatment according to the invention may be administered at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg / kg per day, for example, at a daily dosage of the agent of the invention in an amount of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg / kg per day, or at a daily dosage ... at least once every 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days, or alternatively at least once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 weeks, or any combination thereof, using a single or divided doses every 24, 12, 8, 6, 4 or 2 hours, or using any combination thereof.

[0030] Typically, the drug of the present invention is administered to the subject in the form of a pharmaceutical composition containing a pharmaceutically acceptable carrier.The pharmaceutically acceptable carriers that can be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, phosphates, buffer substances such as glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.For use in administering to the subject, the composition is formulated for administration to the patient.The composition of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally or via an implanted reservoir. The use herein includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Sterile injectable forms of the compositions of the present invention may be aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, such as solutions of 1,3-butanediol. Among the acceptable solvents and solvents that may be used are water, Ringer's solution and isotonic sodium chloride solution. Sterile, fixed oils are also conveniently used as solvents or suspending media. For this purpose, any sterile, fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, particularly in the polyoxyethylated varieties.These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose, or similar dispersants commonly used in the formulation of pharma- ceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tween, Span, and other emulsifiers or bioavailability enhancers commonly used in the manufacture of pharma-ceutically acceptable solid, liquid, or other dosage forms, may also be used for formulation purposes. The compositions of the present invention may be orally administered in any orally acceptable dosage form, including, but not limited to, capsules, tablets, aqueous suspensions, or solutions. In the case of tablets for oral use, commonly used carriers include lactose and cornstarch. Lubricants such as magnesium stearate are also typically added. For oral administration in capsule form, useful diluents include, for example, lactose. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifiers and suspending agents. If desired, certain sweeteners, flavors, or colorants may be added. Alternatively, the compositions of the present invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, so that it melts in the rectum and releases the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol. The compositions of the present invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, skin, or lower intestinal tract. Suitable topical formulations are easily prepared for each of these areas or organs. For topical application, the compositions may be formulated into a suitable ointment containing the active ingredient suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of the present invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax, and water. Alternatively, the compositions can be formulated into a suitable lotion or cream containing the active ingredient suspended or dissolved in one or more pharma- ceutically acceptable carriers.Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Topical application for the lower intestinal tract can be accomplished in a rectal suppository formulation (see above) or a suitable enema formulation. A patch may also be used. The compositions of the invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as a solution in saline with benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other conventional solubilizers or dispersants. For example, the antibody present in the pharmaceutical composition of the invention can be supplied at a concentration of 10 mg / mL in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials. The product is formulated for IV administration in 9.0 mg / mL sodium chloride, 7.35 mg / mL sodium citrate dihydrate, 0.7 mg / mL polysorbate 80, and sterile water for injection. The pH is adjusted to 6.5. An exemplary preferred dosage range for the antibody in the pharmaceutical composition of the invention is about 1 mg / m. 2 ~500mg / m 2 However, it will be recognized that these schedules are exemplary and that optimal schedules and regimens can be adapted taking into account the affinity and tolerability of the particular antibodies in the pharmaceutical composition, which needs to be determined in clinical trials.

[0031] The present invention is further illustrated by the following figures and examples, which, however, should not be construed as limiting the scope of the present invention in any way. [Brief description of the drawings]

[0032] [Figure 1]NRPa-47 induces VEGF downstream signaling modifications. Histograms show pixel intensity of p38α / p38β / p38γ / p38δ at 10 min (hatched histogram) and 60 min (black histogram) in untreated (control, open histogram) and NRPa-47-treated MDA-MB-231. Histograms represent the mean ± SD of a representative selection of previous experiments analyzed using Image-J software to quantify pixel intensity (□, P<0.05; **, P<0.01; ***, P<0.001; NS=not significant). [Diagram 2] Mechanism of action of NRPa-48, derived NRPa-47, on downstream VEGF signaling. Histograms show pixel intensity of p38α / p38β / p38γ / p38δ at 10 min (hatched histogram) and 60 min (black histogram) in untreated (control, open histogram) and NRPa-48-treated (IC50=0.4 μM) MDA-MB-231. Histograms represent the mean ± SD of a representative selection of previous experiments analyzed using Image-J software to quantify pixel intensity (□, P<0.05; **, P<0.01; ***, P<0.001; NS=not significant). [Figure 3A] NRPa-47 and NRPa-48 Protein Kinase Profiling: Protein kinase profiling of NRPa-47 (A) and NRPa-48 (B) was performed at 1 μM against a rich panel of 40 kinases including neuropilin-1 co-receptor and related biochemical kinase signaling observed during this study. [Figure 3B] NRPa-47 and NRPa-48 Protein Kinase Profiling: Protein kinase profiling of NRPa-47 (A) and NRPa-48 (B) was performed at 1 μM against a rich panel of 40 kinases including neuropilin-1 co-receptor and related biochemical kinase signaling observed during this study. [Figure 4A]NRPa / Ralimetinib® combinations increased anti-breast cancer cell proliferation. A wide range of concentrations of Ralimetinib® are tested on the proliferation of MDA MB 231 cells, alone or in combination with NRPa (IC50) (A) or suboptimal NRPa IC50 (0.1 μM) (B). NRPa IC50 / Ralimetinib® combinations showed additive effects (AE) at high Ralimetinib® concentrations and synergistic effects (SE) at low Ralimetinib® concentrations (A). Suboptimal NRPa IC50 / Ralimetinib® combinations showed additive effects (AE) at high Ralimetinib® concentrations and synergistic effects (SE) at low Ralimetinib® concentrations. Data represent the mean ± SD of three separate experiments (NS: not significant). [Figure 4B] NRPa / Ralimetinib® combinations increased anti-breast cancer cell proliferation. A wide range of concentrations of Ralimetinib® are tested on the proliferation of MDA MB 231 cells, alone or in combination with NRPa (IC50) (A) or suboptimal NRPa IC50 (0.1 μM) (B). NRPa IC50 / Ralimetinib® combinations showed additive effects (AE) at high Ralimetinib® concentrations and synergistic effects (SE) at low Ralimetinib® concentrations (A). Suboptimal NRPa IC50 / Ralimetinib® combinations showed additive effects (AE) at high Ralimetinib® concentrations and synergistic effects (SE) at low Ralimetinib® concentrations. Data represent the mean ± SD of three separate experiments (NS: not significant). EXAMPLES

[0033] Materials & Methods 2.1- Chemical synthesis of compounds: Chemical reagents and solvents were purchased from Sigma Aldrich, Fluka and Carlo Erba. NRPa-47 and NRPa-48 were synthesized and characterized as previously reported by the inventors

[16] .

[0034] 2.2- Total RNA preparation and RT-PCR: MDA-MB-231 RNA was extracted with NucleoSpinRNA II kit (Macherey-Nagel, France) and quantified using Nanodrop (ND-1000 spectrophotometer). 1 μg of each RNA sample was reverse transcribed into cDNA using iScript cDNA Synthesis Kit (Bio-Rad, France) according to the manufacturer's instructions. PCR amplification was performed in a reaction mixture (25 μL) containing 200 μM of each dNTP, 1 μg of cDNA, 1 μM of primers and 0.625 U of GoTaq DNA Polymerase (Promega, France) for 30 cycles of denaturation at 95°C for 45 s, annealing at 60°C for 45 s and extension at 72°C for 1 min. PCR products were separated by 1% agarose gel electrophoresis, stained with ethidium bromide (Sigma, Germany) and analyzed using a Gel Doc 2000 System (Bio-Rad, France).

[0035] 2.3- Proteome Profiler Array: MDA-MB-231 cells were treated with NRPa-47 or NRPa-48 compounds (IC 50 ) and protein lysates were prepared and quantified as previously described [17, 18]. Biochemical signaling detection was assessed by using Human Proteome Profiler Arrays (Human Phosphokinase Array and Human Apoptosis Array) according to the manufacturer's instructions (R&Dsystems, France). Briefly, capture and control antibodies were spotted in duplicate on nitrocellulose membranes. Cell extracts were incubated on the membrane overnight, washed to remove unbound proteins, and subsequently incubated with a cocktail of biotinylated detection antibodies. Streptavidin-HRP and chemiluminescent detection reagents were applied, and signal intensity, corresponding to the amount of bound protein, was measured for each capture spot using ImageJ software.

[0036] 2.4-Cell culture conditions Human invasive and metastatic estrogen R- / progesterone R- / Her2- triple-negative breast cancer cell line (MDA-MB-231) purchased from ATCC (Molsheim France) was incubated at 200 μL / well in a 96-well plate for 10 min at 10 °C. 3 Plated at 10 cells / well and incubated with NRPa-47 (IC 50 ), NRPa-48(IC 50 ), 5-FU®, oxaliplatin® and ralimetinib® alone or in combination at different concentrations or not. WST-1 (Roche®, France) was added for 1-2 h, and then cell viability was determined by analyzing the optical density at 490 nm using a microplate reader (Microplate Manager 5.2, Bio-Rad). For each compound, the IC was calculated from sigmoidal dose-response curves using Graph-Pad Prism (GraphPad Software, San Diego, USA). 50 value was determined.

[0037] 2.5-VEGF-R Kinase Assay: Cells were then IC 50 MDA-MB-231 lysates were then used to detect total tyrosine phosphorylation of VEGF-R1 and VEGF-R2 using an ELISA assay (R&D system).

[0038] 2.6-Molecular Docking: The binding site is defined at 4 Å around co-crystallized tuftsin bound to NRP-1 (PDB code 2ORZ)

[19] . Consensus molecular docking was performed using Surflex dock v2.5

[20] and ICM-VLS-v3.4

[21] . Surflex dock is based on a modified hammerhead fragmentation / reconstruction algorithm that allows compounds to be flexibly docked into the binding site. The query molecule is decomposed into rigid fragments that are superimposed on the Surflex Protocol, i.e., molecular fragments that cover the entire binding site. Docking poses were evaluated by an empirical score function. ICM is based on Monte Carlo simulations of internal coordinates, where the position of the molecule is optimized using a stochastic global optimization procedure combined with a pseudo-Brownian position / torsion step and fast local gradient minimization. Docking poses were evaluated using the ICM VLS empirical score function.

[0039] 2.7-Protein kinase profiling: NRPa-47 and NRPa-48 specificity profiling assays were performed at Eurofins Pharma Discovery Services (Dundee, UK) for protein kinase profiling against a collection of 40 protein kinases. Results for protein kinases assayed at 1 μM of each NRPa are presented as percentage of kinase activity in DMSO control reactions.

[0040] 2.8- In vivo xenograft tumor mouse model: The protocol was approved by the INSERM Institutional Care and Use Committee in accordance with the European Communities Council Directive. NOD / scid / IL-2Rγ- / - (NOG) female mice were bred and housed in pathogen-free conditions in compliance with the Federation of European Animal Associations (FELSA) guidelines. MDA-MB-231 cells were washed twice in PBS and resuspended in DMEM. The cells were then transferred to NOG mice (6–7 weeks old) for 2.10 min. 6 The cells were injected subcutaneously at a concentration of 10 cells / 200 μL. Mice were randomly divided into different groups (10 mice / group). Mice were then fed NRPa-48 (50 mg / kg, respectively) or vehicle every 3 days for 39 days using force feeding. Tumor growth and body weight were measured every 3 days during treatment. Mice were weighed periodically to assess toxicity of treatment and tumors were measured with calipers (width x width x length x Pi / 6) to determine growth.

[0041] 2.9-Statistical analysis: Data are expressed as arithmetic mean values ​​+ / - SD of at least three different experiments. Statistical significance of the results was assessed by ANOVA, with probability values ​​indicated. * p<0.05, ** p<0.01, *** p<0.001 was considered significant.

[0042] result 3.1-Neuropilin antagonist 47 (NRPa-47) inhibits VEGF-R1 / -R2 phosphorylation The present inventors have 165We have previously described a neuropilin antagonist (NRPa), so-called compound-1, which we now designate as NRPa-47, that inhibited VEGF-A / NRP-1 binding, tumor survival and tumor growth in vivo

[15] . However, the mechanism of action of NRPa-47 remains elusive, and therefore we direct our report to explaining its function and regulation of cell signaling. NRP-1 mediates the upregulation of VEGF-A and VEGF-B signaling. 165 Since VEGF-R1 and VEGF-R2 interact with each other in the presence of VEGF to mediate intrinsic tyrosine kinase activity, we first investigated the half inhibitory antiproliferative concentration (IC) previously reported for MDA-MB-231. 50 We studied the phosphorylation state of VEGF-R1 / VEGF-R2 in the presence of NRPa-47 (T = 0.6 + / - 0.03 μM)

[15] . Here, we showed that NRPa-47 significantly reduced tyrosine phosphorylation of both VEGF-R1 (20-40%) and VEGF-R2 (40-45%) in MDA-MB-231 cells at 5-60 min (data not shown). Of note, this inhibition was not due to inhibition of intrinsic kinase activity as previously reported

[15] . To support this result, we proposed a mechanism for the inhibition of HIF-1α and VEGF-A as a negative feedback loop. 165 We verified the efficient abrogation of NRPa-47-mediated VEGF-R1 and VEGF-R2 phosphorylation by tracking the expression of both HIF-1α and VEGF-A. 165 Both mRNAs were reduced in a time-dependent manner in the presence of their antagonists, with maximal effects at 60 min (data not shown).

[0043] 3.2-NRPa-47-induced modulation of NRP-1 / VEGF-R downstream signaling To further understand the effects of NRPa-47 on MDA-MB-231, we expanded the study using a biochemical membrane platform that targets mitogen-activated protein kinases (MAPKs) and downstream kinases (data not shown). Surprisingly, NRPa-47 did not negatively modulate MAPKs such as the extracellular signal-regulated kinase (ERK-1 / -2) and c-Jun N-terminal kinase (JNK-1 / -2 / -3 / -pan) pathways, but contributed to their significant hyperphosphorylation at 10 and 60 minutes of drug exposure, respectively (data not shown). These MAPK hyperphosphorylations were consistent with the increased phosphorylation of their downstream substrates, such as p90 ribosomal S6 kinase (RSK-2), observed at 60 minutes (data not shown). However, RSK-1 remained unchanged (data not shown). Furthermore, phosphorylation of AKT-1 / -2 / -3 / -pan was upregulated, as was its downstream p70 ribosomal S6 kinase (p70S6K) (data not shown). In summary, even though dephosphorylation of VEGF-R1 / -R2 was induced by NRPa-47, their downstream kinases became hyperphosphorylated. Prior to these interesting results, we focused on a third MAPK present in the p38 pathway (including p38α, p38β, p38γ, and p38δ). In this pathway, no significant fluctuation in p38 phosphorylation was observed, except for p38α, which was significantly decreased at 60 min (Figure 1). p38α downstream substrates, including small heat shock protein 27 (HSP27) and mitogen- and stress-activated protein kinase 2 (MSK2), were consequently dephosphorylated (data not shown). In addition, only GSK-3β phosphorylation was affected due to impaired p38α phosphorylation but not the ERK / AKT pathway (data not shown). Taken together, NRPa-47 induces phosphorylation of the ERK, JNK and AKT pathways but inhibits p38α phosphorylation and its downstream kinases.

[0044] 3.3-Modified NRPa-47 (NRPa-48) and its effect on NRP-1 / VEGF-R downstream signaling Faced with these intriguing results between NRPa-47-mediated dephosphorylation of VEGF-R and hyperphosphorylation of downstream signaling, we performed a new structural docking analysis. For this purpose, we used the pocket defined in the NRP-1 b1 domain by Tuftsin docking (data not shown). As we note, the methyl group of the docked NRPa-47 is located outside the pocket (data not shown), which may geometrically constrain the unconventional carboxythiourea linker in an unexpected way. Therefore, we decided to remove this methyl group to study this new structurally related compound, now called NRPa-48 (data not shown). We also examined the ability of NRPa-48 to inhibit phosphorylation of both VEGF-R1 and VEGF-R2 using the half maximal inhibitory antiproliferative concentration (IC) previously reported for MDA-MB-231. 50 =0.4+ / -0.2 μM)

[16] . As expected, NRPa-48 exerted significant tyrosine VEGF-R1 and VEGF-R2 kinase inhibitory potential (data not shown), but less efficiently than NRPa-47 (data not shown). Despite this fact, NRPa-48 was also more efficient than NRPa-47 in blocking MDA-MB-231 proliferation (0.6 vs. 0.4 μM)

[16] . These results therefore prompted us to investigate its role on VEGF-R downstream signaling.

[0045] In contrast to NRPa-47, NRPa-48 significantly inhibited all tested MAPKs (data not shown), e.g., ERK-1 / -2 (data not shown), JNK-1 / -2 / -3 / pan (data not shown), p38α / p38β / p38γ / p38δ in a time-dependent manner (Figure 2). Their own respective downstream kinase substrates, such as RSK-1 / RSK-2, MSK-2, HSP-27, and GSK-3α / β, were also inhibited in a time-dependent manner (data not shown). In addition, the AKT pathway, including AKT-1 / -2 / -3 / -pan, was also inhibited from 10 to 60 min (data not shown). In summary, removal of the methyl group from the chemical structure of NRPa-47 allowed NRPa-48 to inhibit the VEGF-A pathway of all MAPKs and downstream kinases studied. 165 This conferred interesting efficiency in blocking induced activity.

[0046] To corroborate the specificity of our hits, we assessed in vitro the activity of NRPa-47 and NRP-48 against 40 selected kinases among growth factor receptors, cell cycle kinases, insulin receptor, etc. None of these were significantly affected by both hits, as the kinase activity results remained within the range of the inefficient hit analysis section (Figures 3A-3B).

[0047] Notably, no reduction in kinase activity of NRP co-receptors such as VEGF-R1 / -R2 / -R3, TGF-β1-R1, FGF-R1 / -R2 / -R3 / -R4, EGF-R, or VEGF-R downstream signaling was observed (Figures 3A-3B). More importantly, in contrast to the observation of kinase phosphorylation modulation induced by these hits in treated tumor cells, none of the kinase activities tested in vitro are blocked by these hits. Thus, this study supports the impact of these hits on the inhibition of p38α, a key cancer therapeutic factor, which is a specific downstream consequence of this treatment.

[0048] 3. Apoptotic pathways induced by 4-NRPa-47 Since both NRPa-47 and NRPa-48 resulted in differential regulation of MAPK phosphorylation, we investigated the impact of this differential effect on cell death. Therefore, we directed our research to the NRPa-induced apoptosis cascade. To elucidate this mechanism of action, we performed apoptosis proteome array experiments at short (60 min) and long (48 h) times (data not shown). We first analyzed cell death receptors such as TRAIL-R1 / DR4, TRAIL-R2 / DR5, FAS / TNFSF6, TNF-R1 / TNSFRSF1 and their adaptor protein FADD. All of these parameters were significantly down-modulated from 60 min onwards, and to a lesser extent at 48 h (data not shown). This result suggested that cell death induced by NRPa-47 does not appear to be death receptor-dependent. In addition, proapoptotic proteins including Bad, Bax, SMAC / Diablo, HTRA2 / Omi and cytochrome c were not induced by NRPa-47, in contrast to the rapid decrease in these proteins observed at short drug exposures and remaining decreased at longer times, except for the induction of caspase-3 cleavage (data not shown). However, antiapoptotic proteins such as Bcl-2, Bcl-x, cIAP-1, cIAP-2, XIAP, survivin, livin, clusterin, and heat shock proteins (HSP-27, HSP-60, HSP-70) were significantly reduced from 60 min onwards and remained decreased at 48 h (data not shown).

[0049] Furthermore, NRPa-47 comprehensively induced the expression of cell cycle proteins such as p21 / CIP1 / CDNK1A, p27 / kip1, and reduced phosphorylation of Rad-17 (data not shown). Moreover, all phosphorylation sites of p53 protein were inhibited from 60 min onwards (data not shown). Surprisingly, NRPa-47 induced rapid oxidative stress at 60 min as revealed by induction of serum catalase, but not paraoxonase / arylesterase 2 (PON-2) (data not shown).

[0050] In conclusion, NRPa-47 induced down-modulation of pro- and anti-apoptotic proteins as well as death receptors. However, NRPa-47 rapidly caused oxidative stress reflected by catalase induction. In addition, the expression of both inducible (HO-1 / HMOX1 / HSP32) and constitutive (HO-2 / HMOX2) heme oxygenase forms was reduced from 60 min onwards (data not shown). Cell death could be attributed to the reduction of both HIF-1α and survivin (data not shown).

[0051] 3. Mechanism of 5-NRPa-48-induced cell death To clarify the opposing effect of NRPa-48 compared with NRPa-47 on MAPK regulation, we extended the study to elucidate its mechanism of action on apoptotic pathways (data not shown). In contrast to the early NRPa-47 effect, NRPa-48 had a delayed effect on the regulation of apoptotic pathways, as observed at 48 hours but not at 60 minutes (data not shown). In detail, proapoptotic proteins such as Bad, Bax, SMAC / Diablo, HTRA2 / Omi and cytochrome c were significantly decreased, except for caspase-3 cleavage induction (data not shown). Antiapoptotic proteins such as Bcl-2, Bcl-x, cIAP-1, cIAP-2, XIAP, survivin, livin, clusterin, heat shock proteins (HSP-27, HSP-60, HSP-70), and cell death receptors such as TRAIL-R1 / DR4, TRAIL-R2 / DR5, FAS / TNFSF6, TNF-R1 / TNSFRSF1A and their adaptor protein FADD were also down-modulated (data not shown). NRPa-48 induced a reduction in cell cycle protein expression such as p21 / CIP1 / CDNK1A, p27 / kip1, and phosphorylation of Rad-17 and claspin (data not shown). In addition, all phosphorylation sites of p53 protein were inhibited (data not shown). In contrast to NRPa-47, NRPa-48 induced late oxidative stress detected by high catalase levels, whereas PON-2 levels remained unchanged (data not shown). Interestingly, NRPa-48 has the ability to inhibit HIF-1α expression as previously observed with NRPa-47 (data not shown).In addition, expression of both inducible (HO-1 / HMOX1 / HSP32) and constitutive (HO-2 / HMOX2) heme oxygenase forms was reduced at 48 h (data not shown).

[0052] Taken together, both NRPas led to similar down-modulation of proteins involved in apoptosis and induced oxidative stress, and interestingly, the most important proteins modulated in this pathway by NRPas were HO-1 / HMOX1 / HSP32, survivin and HIF-1α.

[0053] In conclusion, even though NRPa-47 and NRPa-48 did not have the same effects on MAPK signaling, both induce treated cells into the cell death program in a similar manner but with different timing.

[0054] 3-6 In vivo antitumor activity of NRPa-48 against xenografted NOG mice In vivo experiments were carried out on MDA-MB-231 xenografted NOG mice using NRPa-48 to compare its efficiency in tumor growth inhibition with NRPa-47, which our team has previously described (PMID 24752068). One group was treated with 50 mg / kg NRPa-48 by force feeding three times a week, and the remaining group served as a negative control. Interestingly, the treated animals did not show any weight loss, suggesting that at this concentration, NRPa-48 does not show acute toxicity (data not shown). At day 38 (21 days after treatment was started), the tumor size was reduced by approximately 29% compared to the reference group ( *** p<0.001), tumor growth was strongly reduced by NRPa-48 (data not shown). The 50 mg / kg group remained largely efficient on day 45, showing a 34% reduction in tumor size (data not shown). More interestingly, survival time was significantly increased when mice were treated with 50 mg / kg NRPa-48 compared to the control group (data not shown). Thus, median survival time was 35 days in control animals and more than 56 days in NRPa-48-treated animals (p=0.008) (data not shown). In summary, the in vivo tumor growth inhibition mediated by NRPa-48 was efficient, with 62% of treated mice still alive at the end of treatment.

[0055] Consideration The development of NRPa has brought new tools for cancer treatment and knowledge of the biochemical pathways involved in this process. In this report, we observed that very small structural changes in the structure of two structurally related NRPa (NRPa-47 and NRPa-48), i.e., suppression of methyl groups, induced large changes in the nature of the affected signaling pathways. NRP-1 inhibitors rapidly inhibit not only HIF-1α protein and mRNA expression but also VEGF mRNA, and thus may contribute to the occurrence of a disturbance in the autocrine HIF-1α / VEGF feedback loop. This result is extremely interesting, since HIF-1α is an important cancer drug target

[22] . In this context, NRPa can induce VEGF starvation of tumor cells, impairing their growth and survival. In addition, high oxidative stress, reflected by increased catalase expression, is induced at 60 min in NRPa-47 and at 48 h in NRPa-48. Even though both NRPas can induce dephosphorylation of VEGF-R tyrosine kinase, their downstream targets are not affected in the same way. The main difference observed for the two NRPas is the restriction to MAPK (ERK, JNK, p38 except p38α) regulation, whose phosphorylation is increased by NRPa-47 and decreased by NRPa-48 (data not shown). Similar differences are observed in the AKT pathway and its downstream target (p70S6 kinase). Further investigations are required to identify and / or clarify alternative downstream pathway-mediated MAPK phosphorylation in this context.

[0056] Nevertheless, this opposing effect on MAPK regulation, inducing not only hyperphosphorylation but also dephosphorylation, may lead to tumor cell death. Even though downmodulation of death receptors, proapoptotic and antiapoptotic proteins may result in an apoptosis / survival imbalance (which may also lead to cell death), no prominent apoptotic pathway emerged between the two. The most important events that are certain to induce apoptosis are the reduction of survivin expression, induction of oxidative stress (catalase), downregulation of heme oxygenase and downmodulation of HIF-1α. The expression of other HIF-1α inducers such as HO-1 / HMOX1 / HSP32 was inhibited as well as p38α phosphorylation, which is also an activator of HO-1. P38α pathway inhibition results in p53 dephosphorylation, aberrant phosphorylation of HSP27, GSK-3β and MSK2, and downmodulation of survivin (data not shown). Disruption of survivin expression increases apoptosis and reduces tumor growth.

[0057] Taken together, this report highlights the important role of p38α in the cell signaling cascade mediated by NRPa. Several studies have reported p38α as a drug target for developing specific inhibitors to treat cancers that depend on p38 MAPK activity for progression. The combination of p38α inhibitors with DNA-damaging chemotherapy can induce cancer cell death by p38α-mediated cell cycle arrest and impairment of DNA repair mechanisms

[23] . Moreover, p38α inhibitors increase the sensitivity of tumor cells to chemotherapy such as 5-fluorouracil (5-FU) and oxaliplatin®

[24] . In this regard, the combination of both NRPa with 5-FU® or oxaliplatin® for breast cancer cells increased their sensitivity to these respective drugs, as expected (data not shown). More importantly, the combination of NRPa with Ralemitininb® (a P-p38α inhibitor) supported this hypothesis, as the additive and / or synergistic effects of these drugs (depending on the dose used) significantly reduced breast cancer cell proliferation (Figures 4A-4B). In short, NRPa could be used alone or in combination with drugs to treat cancer. This observation has led to the current interest in the development of NRPa.

[0058] References Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are incorporated by reference into this disclosure. [Table 2] TIFF2025060805000008.tif246165 TIFF2025060805000009.tif238165 TIFF2025060805000010.tif92165

Claims

1. A method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective combination comprising at least one neuropilin antagonist and at least one p38α-kinase inhibitor.

2. The method of claim 1 , wherein the subject is a human.

3. The method of claim 1 , wherein the subject is a non-human mammal.

4. The method of claim 1 , wherein the cancer is a hematopoietic cancer or a non-hematopoietic cancer.

5. The method of claim 1 , wherein the cancer is breast cancer.

6. 2. The method of claim 1, wherein the cancer is triple-negative breast cancer.

7. The method of claim 1 , wherein the cancer is neuropilin positive.

8. The method of claim 1, wherein the neuropilin antagonist is selected from the group consisting of antisense polynucleotides, interfering RNA, catalytic RNA, RNA-DNA chimeras, neuropilin-specific aptamers, anti-neuropilin antibodies, neuropilin-binding fragments of anti-neuropilin antibodies, neuropilin-binding small molecules, neuropilin-binding peptides, and other polypeptides that specifically bind to neuropilin (including, but not limited to, neuropilin-binding fragments of one or more neuropilin ligands, optionally fused to one or more additional domains), such that interaction between the neuropilin antagonist and neuropilin results in reduced or abolished neuropilin activity or expression.

9. Neuropilin antagonists inhibit the activity of neuropilin proteins (e.g., NRP-1) and their partners, particularly VEGF-A. １６５ The method of claim 1 , wherein the interaction between

10. Neuropilin antagonists specifically bind to neuropilin (e.g., NRP-1 or NRP-2) and neutralize its activity in activating the neuropilin signaling pathway, particularly in the binding of neuropilin and VEGF-A. １６５ The method of claim 1, wherein the antibody is an antibody that inhibits binding to the

11. The method of claim 1, wherein the neuropilin antagonist is NRPa-47 or NRPa-48.

12. The method of claim 1, wherein the p38α-kinase inhibitor is selected from the group consisting of antisense polynucleotides, interfering RNA, catalytic RNA, RNA-DNA chimeras, p38-α-specific aptamers, anti-p38α antibodies, p38α-binding fragments of anti-p38α antibodies, p38α-binding small molecules, p38α-binding peptides, and other polypeptides that specifically bind to p38α (including, but not limited to, p38α-binding fragments of one or more p38α ligands, optionally fused to one or more additional domains), such that interaction between the p38α-kinase inhibitor and p38α results in reduced or abolished p38α kinase activity or expression.

13. 2. The method of claim 1, wherein the p38α-kinase inhibitor is selected from the group consisting of ARRY-371797, ARRY-614, AZD-7624, ralimetinib, LY-3007113, FX005, GSK610677, GW856553, SB-681323, KC706, UR-13870, PF-03715455, VX-745, SCID-469, PH-797804, VX-702, SB-202190, SB-203580, SB-239063, BIRB-796, BMS-582949, and pamapimod.

14. 2. The method of claim 1, wherein the neuropilin antagonist is NRPa-47 and the p38α-kinase inhibitor is ralimetinib.

15. 2. The method of claim 1, wherein the neuropilin antagonist is NRPa-48 and the p38α-kinase inhibitor is ralimetinib.

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