Neuropilin antagonists in combination with p38 alpha-kinase inhibitors for the treatment of cancer

Abstract

Neuropilin-1 is an important target in cancer treatment in the future, but its mechanism of action remains elusive, and therefore the development of inhibitory small molecules is needed for its investigation. 165 We report that two small neuropilin antagonists (NRPa-47 and NRPa-48), which are NRP-1 / NRP-1 binding inhibitors, can reduce VEGF-R phosphorylation and modulate its downstream cascade in a triple-negative breast cancer cell line (MDA-MB-231). In particular, we demonstrated for the first time how NRPas can alter tumor cell signaling and contribute to down-modulation of p38α-kinase phosphorylation, a key factor in cancer therapy. More importantly, the combination of NRPas with p38α inhibitors results in additive and / or synergistic effects (depending on the dose used) of these drugs in significantly reducing breast cancer cell proliferation. Therefore, an efficient combination of NRPas with p38α-kinase inhibitors is effective for cancer treatment.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to the field of medicine, particularly oncology. [Background technology]

[0002] 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, which are involved in axon guidance and repulsion [2]. Neuropilins are multifunctional non-tyrosine kinase receptors for some members of the VEGF (vascular endothelial growth factor) family. VEGF-A, a splice form of VEGF-A, has been reported to be upregulated in some tumor tissues. 165 VEGF-A is considered one of the most efficient pro-angiogenic factors. 165 NRPs bind to structurally related tyrosine kinase receptors, such as VEGF-R1 (Flt-1) and VEGF-R2 (Flk-2), and to NRPs, co-receptors that 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 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 treatment resistance in patients with generally poor responses have led to the need to develop new antiangiogenic strategies.

[0004] Drug development against emerging NRP targets has led to new 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 in inhibitory small molecules that reduce VEGF binding to NRPs and tumor growth in vivo and in vitro [14, 15]. Our research team is the first in this field to develop completely nonpeptide inhibitory molecules, so-called neuropilin antagonists (NRPa)

[15] . However, the molecular mechanisms by which NRPs modulate cancer progression remain poorly understood. NRPa should provide additional data regarding theoretical understanding 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 cancer treatment in the future, but its mechanism of action remains elusive, and therefore the development of inhibitory small molecules is needed for its investigation. 165We report that two small neuropilin antagonists (NRPa-47 and NRPa-48), which are inhibitors of VEGF-R / NRP-1 binding, can reduce VEGF-R phosphorylation and modulate their downstream cascades in triple-negative breast cancer cell lines (MDA-MB-231). Nevertheless, NRPa induces divergent pathway regulation of MAPK phosphorylation, including JNK-1 / -2 / -3, ERK-1 / -2, and p38β / γ / δ-kinase, and their respective downstream targets. However, NRPa-47 and NRPa-48 exert a common downregulation of p38α-kinase phosphorylation and its downstream targets, reinforcing its central regulatory role. More importantly, none of the 40 selected kinases, including SAPK2a / p38α, was affected by NRPa in vitro, confirming their specificity. In summary, NRPa induced cell death by down-modulating pro- and anti-apoptotic proteins, 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 demonstrated for the first time how NRPa alters tumor cell signaling and can contribute to the down-modulation of p38α-kinase phosphorylation, an important factor in cancer therapy. Therefore, the efficient combination of NRPa with a p38α-kinase inhibitor could be effective in cancer treatment.

[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, and rats; primates, such as chimpanzees and monkeys; and economically important animals, such as cows, pigs, rabbits, horses, sheep, and goats.

[0009] As used herein, the term "cancer" has its common 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 originating from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, digestive tract, gums, head, kidney, liver, lung, nasopharynx, cervix, ovary, prostate, skin, stomach, testicles, tongue, or uterus. Cancer may also be of the following specific histological types, but is not limited to: 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; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenocortical 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 of the breast;Acinic cell carcinoma;Adenosquamous carcinoma;Adenocarcinoma with squamous metaplasia;Thymoma, malignant;Ovarian stromal tumor, malignant Gender;Theca cell tumor, malignant;Granulosa cell tumor, malignant;And neuroblastoma, malignant;Sertoli cell carcinoma;Leydig cell tumor, malignant;Lipocyte tumor, malignant;Paragaglioma, 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; stromal sarcoma; mixed tumor, malignant; mixed Müllerian 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;Ganglioneuroblastoma;Neuroblastoma;Retinoblastoma;Olfactory neuronal tumor;Meningioma, malignant;Neurofibrosarcoma;Neurilemoma, malignant;Granular cell tumor, malignant;Malignant lymphoma;Hodgkin's disease;Hodgkin's lymphoma;Side 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 methods of the present invention are particularly suitable for treating breast cancer, and in particular, triple-negative breast cancer. As used herein, the term "triple-negative breast cancer" has its general meaning in the art, meaning that the breast cancer lacks receptors for the hormones estrogen (ER-negative) and progesterone (PR-negative) and 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 treatment and curative or disease-modifying treatment, including treatment of unhealthy patients or patients diagnosed with a disease or condition, as well as treatment of patients at risk of or suspected of having a disease, including the suppression of clinical recurrence. Treatment may be administered to patients with a medical disorder or who may ultimately acquire a disorder to prevent, cure, or delay the onset of the disorder or recurrent disorder, reduce its severity, or ameliorate one or more symptoms thereof, or to extend the patient's survival beyond that expected in the absence of such treatment. "Therapeutic regimen" refers to a pattern of disease treatment, e.g., the pattern of medication used during treatment. The therapeutic regimen can include induction regimens and maintenance regimens. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or portion of a therapeutic regimen) used in 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 phase of the treatment regimen. The induction regimen may (partially or entirely) employ a "loading regimen," which may involve the physician administering a higher dose of drug than during the maintenance regimen, administering the drug more frequently than 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 a patient in remission for an extended period (months or years). A maintenance regimen may employ continuous treatment (e.g., administering a 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 specific 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: neuropilin-1 (NRP-1) and neuropilin-2 (NRP-2), which share 44% sequence homology. Neuropilins are involved in the regulation of VEGF-A. 165 It is a multifunctional non-tyrosine kinase receptor for some members of the VEGF (vascular endothelial growth factor) family, including VEGF-1, VEGF-2, and VEGF-3.

[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 antagonists 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 RNAs, catalytic RNAs, 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 are directed against neuropilin proteins (e.g., NRP-1) and their partners, particularly VEGF-A. 165inhibits the interaction between

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

[0016] [Table 1] TIFF0007718989000002.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 specifically describes 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 comprising 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 salts and esters thereof, 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, act to directly block the translation of NRP-1 mRNA by binding to it, thereby preventing protein translation or increasing mRNA degradation, thereby reducing the level and thus 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 encoding NRP-1 can be synthesized, for example, by conventional phosphodiester techniques. The method of using antisense technology to specifically inhibit the gene expression of known gene sequences is 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 expression inhibitor for use in the present invention.NRP-1 gene expression can be reduced by contacting patient or cell with small double-stranded RNA (dsRNA) or 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 oligonucleotide, siRNA, shRNA and ribozyme of the present invention can be delivered in vivo alone or in combination with vector. 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 that expresses NRP-1. Typically, the vector transports the nucleic acid into the 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 with the insertion or incorporation of antisense oligonucleotides, siRNAs, shRNAs, or ribozymes. 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 viruses; polyomaviruses; Epstein-Barr virus; papillomaviruses; herpesviruses; vaccinia viruses; polioviruses; and RNA viruses such as retroviruses. Other vectors not named but known in the art can readily be 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, etc. A p38α-kinase inhibitor can be any type of molecule that interferes with at least p38α-related signal transduction, for example, by reducing the transcription or translation of a nucleic acid encoding p38α, or by inhibiting or blocking p38α kinase activity, or both. In some examples, a p38α-kinase inhibitor is an agent that interferes with p38α-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 the interaction between the p38α-kinase inhibitor and p38α results in the reduction or cessation of p38α kinase activity or expression. For example, a desirable p38α-kinase inhibitor for use in certain methods herein is one that binds to p38α and blocks p38α signaling, and that has no or minimal effect on any other members of the p38 MAPK family, e.g., p38-β, p38-γ, and / or p38-δ. It will be recognized that the p38α-kinase inhibitors described herein can 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 100 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; rosmapimod; GlaxoSmithKline Plc or Selleckchem), SB-681323 (dilmapimod; GlaxoSmithKline Plc), and KC706 (Kemia). 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 (dramapimod; 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 any dosage form that provides a first drug together with an additional (second, third, etc.) drug. The drugs may be administered simultaneously, separately, or sequentially, and in any order. Drugs administered in combination have biological activity in the patient to whom they are delivered. Within the context of the present invention, a combination therefore includes 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 a dosage and for a period of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of a drug can vary according to factors such as the individual's disease state, age, sex, and weight, as well as the drug's ability 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 a drug depend on the disease or condition to be treated and can be determined by one of ordinary skill in the art. A physician with 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 dosage 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 subject's size, 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, e.g., about 0.1-50 mg / kg, e.g., about 0.1-20 mg / kg, e.g., about 0.1-10 mg / kg, e.g., about 0.5, e.g., 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 present invention is 0.02-100 mg / kg, e.g., about 0.02-30 mg / kg, e.g., about 0.05-10 mg / kg, or 0.1-3 mg / kg, e.g., about 0.5-2 mg / kg. Administration can be, for example, intravenous, intramuscular, intraperitoneal, or subcutaneous, but can be administered proximal to the target site. The dosage regimen in the above-described methods and uses of treatment is adjusted to produce the optimum desired response (e.g., a therapeutic response).For example, a single bolus may be administered, or 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 throughout treatment, e.g., at predetermined time points. As a non-limiting example, treatment according to the present invention may be administered at a daily dose of about 0.1 to 100 mg / kg per day, e.g., 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 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 of an agent of the present invention, 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, or 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, It may be provided 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 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 a subject in the form of a pharmaceutical composition containing a pharmaceutically acceptable carrier.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 a subject, the composition is formulated for administration to a 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 term "use" herein encompasses 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 in 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. Any sterile, fixed oil, including synthetic mono- or diglycerides, may be used for this purpose. 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 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 formulating pharmaceutically 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 pharmaceutically acceptable solid, liquid, or other dosage forms, may also be used for formulation purposes. The compositions of the present invention can be orally administered in any orally acceptable dosage form, including, but not limited to, capsules, tablets, aqueous suspensions, or solutions. For 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 an aqueous suspension is required for oral use, the active ingredient is combined with an emulsifier and suspending agent. If desired, certain sweeteners, flavorings, or coloring agents 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, thereby melting in the rectum and releasing the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol. The compositions of the present invention may also be administered topically, particularly 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 composition 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 composition may be formulated into a suitable lotion or cream containing the active ingredient suspended or dissolved in one or more pharmaceutically 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 achieved in a rectal suppository formulation (see above) or a suitable enema formulation. Patches may also be used. The compositions of the present 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 solutions in saline using 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 present 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 suitable dosage range for the antibody in the pharmaceutical composition of the invention is about 1 mg / mL. 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 antibody 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 explanation of the drawings]

[0032] [Figure 1]NRPa-47 induces VEGF downstream signaling modifications. Histograms show pixel intensities of p38α / p38β / p38γ / p38δ in untreated (control, open histogram) and NRPa-47-treated MDA-MB-231 cells at 10 minutes (hatched histogram) and 60 minutes (black histogram). Histograms represent the mean ± SD of a representative selection of previous experiments analyzed using Image-J software to quantify pixel intensities (□, P<0.05; **, P<0.01; ***, P<0.001; NS=not significant). [Figure 2] Mechanism of action of NRPa-48 and its derivative NRPa-47 on downstream VEGF signaling. Histograms show pixel intensities of p38α / p38β / p38γ / p38δ at 10 minutes (hatched histogram) and 60 minutes (black histogram) in untreated (control, open histogram) and NRPa-48-treated (IC50 = 0.4 μM) MDA-MB-231 cells. Histograms represent the mean ± SD of a representative selection of previous experiments analyzed using Image-J software to quantify pixel intensities (□, 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-receptors 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-receptors and related biochemical kinase signaling observed during this study. [Figure 4A]The NRPa / Ralimetinib® combination increased anti-breast cancer cell proliferation. A wide range of concentrations of Ralimetinib® was tested on the proliferation of MDA MB 231 cells, either alone or in combination with NRPa (IC50) (A) or a suboptimal NRPa IC50 (0.1 μM) (B). The NRPa IC50 / Ralimetinib® combination showed an additive effect (AE) at high Ralimetinib® concentrations and a synergistic effect (SE) at low Ralimetinib® concentrations (A). The suboptimal NRPa IC50 / Ralimetinib® combination showed an additive effect (AE) at high Ralimetinib® concentrations and a synergistic effect (SE) at low Ralimetinib® concentrations. Data represent the mean ± SD of three separate experiments (NS: not significant). [Figure 4B] The NRPa / Ralimetinib® combination increased anti-breast cancer cell proliferation. A wide range of concentrations of Ralimetinib® was tested on the proliferation of MDA MB 231 cells, either alone or in combination with NRPa (IC50) (A) or a suboptimal NRPa IC50 (0.1 μM) (B). The NRPa IC50 / Ralimetinib® combination showed an additive effect (AE) at high Ralimetinib® concentrations and a synergistic effect (SE) at low Ralimetinib® concentrations (A). The suboptimal NRPa IC50 / Ralimetinib® combination showed an additive effect (AE) at high Ralimetinib® concentrations and a synergistic effect (SE) at low Ralimetinib® concentrations. Data represent the mean ± SD of three separate experiments (NS: not significant). [Example]

[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: RNA from MDA-MB-231 was extracted using the NucleoSpinRNA II kit (Macherey-Nagel, France) and quantified using a Nanodrop (ND-1000 spectrophotometer). One microgram of each RNA sample was reverse transcribed into cDNA using the iScript cDNA Synthesis Kit (Bio-Rad, France) according to the manufacturer's instructions. PCR amplification was performed in a 25-μL reaction mixture containing 200 μM of each dNTP, 1 μg of cDNA, 1 μM primer, and 0.625 U of GoTaq DNA Polymerase (Promega, France), for 30 cycles of denaturation at 95°C for 45 seconds, annealing at 60°C for 45 seconds, and extension at 72°C for 1 minute. 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 Cells were incubated with or without ATP, and protein lysates were prepared and quantified as previously described [17, 18]. Biochemical signaling detection was assessed 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 onto nitrocellulose membranes. Cell extracts were incubated on the membranes overnight, washed to remove unbound proteins, and then incubated with a cocktail of biotinylated detection antibodies. Streptavidin-HRP and chemiluminescent detection reagents were applied, and the 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 cultured in a 96-well plate at 200 μL / well for 10 min. 3 Plated at 100 cells / well and incubated with NRPa-47 (IC 50 ), NRPa-48(IC 50 Cells were treated with or without 5-FU, oxaliplatin, and ralimetinib, either alone or in combination at different concentrations. 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, IC values were 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 transfected with IC 50 MDA-MB-231 cells were cultured in the presence of NRPa-47 or NRPa-48 for 5–60 min, and then total tyrosine phosphorylation of VEGF-R1 and VEGF-R2 was detected using MDA-MB-231 lysates using an ELISA assay (R&D system).

[0038] 2.6-Molecular Docking: The binding site was defined within 4 Å of 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 covering the entire binding site. Docking poses were evaluated using an empirical scoring function. ICM is based on Monte Carlo simulations of internal coordinates, which optimize the molecular position 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 scoring function.

[0039] 2.7-Protein kinase profiling: For protein kinase profiling against a selection of 40 protein kinases, NRPa-47 and NRPa-48 specificity profiling assays were performed at Eurofins Pharma Discovery Services (Dundee, UK). Results for protein kinases assayed at 1 μM of each NRPa are presented as percentages 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 accordance 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 days. 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 force-fed with NRPa-48 (50 mg / kg each) or vehicle every 3 days for 39 days. During treatment, tumor growth and body weight were measured every 3 days. Mice were weighed periodically to assess the toxicity of the 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. The statistical significance of the results was assessed by ANOVA, and probability values were * 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 demonstrated that VEGF-A 165We previously described a neuropilin antagonist (NRPa), so-called Compound-1, which inhibited VEGF / NRP-1 binding, tumor survival, and tumor growth in vivo, and this compound is hereby designated NRPa-47

[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 VEGF-A signaling pathways. 165 Because VEGF-R1 interacts with both VEGF-R2 and VEGF-R2 in the presence of VEGF-R1 to mediate intrinsic tyrosine kinase activity, we first investigated the half-maximal inhibitory antiproliferative concentration (IC) previously reported for MDA-MB-231. 50 We investigated the phosphorylation status of VEGF-R1 / VEGF-R2 in the presence of NRPa-47 (at concentrations of 0.6 ± 0.03 μM)

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

[15] . Supporting this result, we investigated the interaction of HIF-1α and VEGF-A as a negative feedback loop. 165 We verified the efficient abrogation of VEGF-R1 and VEGF-R2 phosphorylation mediated by NRPa-47 by tracking the expression of both HIF-1α and VEGF-A mRNA. 165 Both mRNAs were reduced in a time-dependent manner in the presence of their antagonists, with the effect being maximal 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 our study using a biochemical membrane platform targeting 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 hyperphosphorylation events were consistent with 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 conclusion, even though NRPa-47 induced dephosphorylation of VEGF-R1 / -R2, their downstream kinases became hyperphosphorylated. Prior to these intriguing results, we focused on a third MAPK in the p38 pathway (including p38α, p38β, p38γ, and p38δ). In this pathway, no significant changes in p38 phosphorylation were observed, except for p38α, which was significantly reduced 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 effects 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 novel structural docking analysis. To this end, 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 unexpectedly constrain the unconventional carboxythiourea linker geometrically. Therefore, we decided to remove this methyl group to study this new structurally related compound, referred to herein as NRPa-48 (data not shown). We also examined the ability of NRPa-48 to inhibit the 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 inhibitory activity against tyrosine VEGF-R1 and VEGF-R2 kinases (data not shown), but was less effective than NRPa-47 (data not shown). Despite this, NRPa-48 was also more effective 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 in VEGF-R downstream signaling.

[0045] In contrast to NRPa-47, NRPa-48 significantly inhibited all tested MAPKs (data not shown), such as ERK-1 / -2 (data not shown), JNK-1 / -2 / -3 / pan (data not shown), and p38α / p38β / p38γ / p38δ, in a time-dependent manner (Figure 2). Their 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 by 10 to 60 minutes (data not shown). In summary, removal of the methyl group from the chemical structure of NRPa-47 allows NRPa-48 to inhibit the VEGF-A pathway of all MAPKs and downstream kinases studied. 165 This conferred interesting efficiency in blocking the activity induced by β-glucan.

[0046] To confirm the specificity of our hits, we evaluated the activity of NRPa-47 and NRP-48 in vitro against 40 selected kinases, including growth factor receptors, cell cycle kinases, and insulin receptors. 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, we did not observe any reduction in kinase activity of NRP coreceptors, such as VEGF-R1 / -R2 / -R3, TGF-β1-R1, FGF-R1 / -R2 / -R3 / -R4, or EGF-R, or downstream VEGF-R signaling (Figures 3A-3B). More importantly, in contrast to the observed modulation of kinase phosphorylation induced by these hits in treated tumor cells, none of the kinase activities tested in vitro were blocked by these hits. Thus, this study supports the impact of these hits on the inhibition of p38α, a key factor in cancer therapy, which is a specific downstream consequence of this treatment.

[0048] Apoptotic pathway induced by 3.4-NRPa-47 Because both NRPa-47 and NRPa-48 differentially regulated MAPK phosphorylation, we investigated the impact of this differential effect on cell death. Therefore, we focused our research on the NRPa-induced apoptotic cascade. To elucidate this mechanism of action, we performed short-term (60 min) and long-term (48 h) apoptotic proteome array experiments (data not shown). We first analyzed death receptors, such as TRAIL-R1 / DR4, TRAIL-R2 / DR5, FAS / TNFSF6, and TNF-R1 / TNSFRSF1, and their adaptor protein FADD. All of these parameters were significantly down-modulated from 60 min onward, and to a lesser extent at 48 h (data not shown). This result suggested that NRPa-47-induced cell death 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, except for the induction of caspase-3 cleavage, a rapid decrease in these proteins was observed after short drug exposure and remained reduced even after prolonged exposure (data not shown). However, antiapoptotic proteins, such as Bcl-2, Bcl-x, cIAP-1, cIAP-2, XIAP, survivin, livin, and clusterin, as well as heat shock proteins (HSP-27, HSP-60, and HSP-70), were significantly reduced from 60 min onward and remained reduced at 48 h (data not shown).

[0049] Furthermore, NRPa-47 comprehensively induced the expression of cell cycle proteins such as p21 / CIP1 / CDNK1A and p27 / kip1, as well as a reduction in Rad-17 phosphorylation (data not shown). Furthermore, all phosphorylation sites of p53 were inhibited from 60 min onward (data not shown). Surprisingly, NRPa-47 induced rapid oxidative stress at 60 min, as evidenced 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 and death receptors. However, NRPa-47 rapidly caused oxidative stress, as 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 after 60 min (data not shown). Cell death may be due to a decrease in both HIF-1α and survivin (data not shown).

[0051] 3. Mechanism of 5-NRPa-48-induced cell death To clarify the opposing effects of NRPa-48 compared with NRPa-47 on MAPK regulation, we expanded our study to elucidate its mechanism of action on the apoptotic pathway (data not shown). In contrast to the early effects of NRPa-47, NRPa-48 had a delayed effect on the regulation of the apoptotic pathway, observed at 48 hours but not at 60 minutes (data not shown). Specifically, except for the induction of caspase-3 cleavage, proapoptotic proteins such as Bad, Bax, SMAC / Diablo, HTRA2 / Omi, and cytochrome c were significantly reduced (data not shown). Antiapoptotic proteins such as Bcl-2, Bcl-x, cIAP-1, cIAP-2, XIAP, survivin, livin, and clusterin, heat shock proteins (HSP-27, HSP-60, and HSP-70), and death receptors such as TRAIL-R1 / DR4, TRAIL-R2 / DR5, FAS / TNFSF6, and TNF-R1 / TNSFRSF1A and their adaptor protein FADD were also down-modulated (data not shown). NRPa-48 induced a reduction in the expression of cell cycle proteins such as p21 / CIP1 / CDNK1A and p27 / kip1, as well as the phosphorylation of Rad-17 and claspin (data not shown). In addition, all phosphorylation sites of p53 were inhibited (data not shown). In contrast to NRPa-47, NRPa-48 induced late oxidative stress, as detected by elevated catalase levels, while PON-2 levels remained unchanged (data not shown). Interestingly, NRPa-48 possesses 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. 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 do not have the same effect 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 in xenografted NOG mice NRPa-48 was used to carry out in vivo experiments on MDA-MB-231 xenografted NOG mice to compare its efficacy in tumor growth inhibition with that of NRPa-47, which our team has previously described (PMID 24752068). One group was treated with 50 mg / kg of NRPa-48 by gavage 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 NRPa-48 does not exhibit acute toxicity at this concentration (data not shown). On day 38 (21 days after treatment began), the tumor size was reduced by approximately 29% compared to the reference group ( *** p<0.001), NRPa-48 strongly reduced tumor growth (data not shown). The 50 mg / kg group remained largely effective on day 45, showing a 34% reduction in tumor size (data not shown). More interestingly, treatment of mice with 50 mg / kg NRPa-48 significantly increased survival time compared with the control group (data not shown). Thus, median survival time was 35 days for control animals and over 56 days for NRPa-48-treated animals (p=0.008) (data not shown). In summary, NRPa-48-mediated in vivo tumor growth inhibition was efficient, with 62% of treated mice still surviving at the end of treatment.

[0055] Consideration The development of NRPas has provided new tools for cancer treatment and knowledge of the biochemical pathways involved in this process. In this report, we observed that a very small structural change in the structure of two structurally related NRPas (NRPa-47 and NRPa-48), i.e., suppression of a methyl group, induced a significant change in the nature of the affected signaling pathway. NRP-1 inhibitors rapidly inhibited not only HIF-1α protein and mRNA expression but also VEGF mRNA, thus potentially contributing to the disruption of the autocrine HIF-1α / VEGF feedback loop. This result is particularly interesting, as HIF-1α is an important cancer drug target

[22] . In this context, NRPas may induce VEGF starvation in tumor cells, impairing their growth and survival. In addition, high oxidative stress, reflected by increased catalase expression, was induced in NRPa-47 at 60 minutes and in NRPa-48 at 48 hours. Although both NRPas can induce dephosphorylation of VEGF-R tyrosine kinase, their downstream targets are not affected to the same extent. The main difference observed between the two NRPas is the restriction to MAPK (ERK, JNK, and p38 excluding 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 investigation is needed to identify and / or clarify alternative downstream pathway-mediated MAPK phosphorylation in this setting.

[0056] Nevertheless, this opposing effect on MAPK regulation, inducing not only hyperphosphorylation but also dephosphorylation, can lead to tumor cell death. Although down-modulation of death receptors, pro-apoptotic, and anti-apoptotic proteins can result in an apoptosis / survival imbalance (which can also lead to cell death), no distinct apoptotic pathway emerged between the two. The most important events responsible for apoptosis induction are the reduction of survivin expression, the induction of oxidative stress (catalase), the down-regulation of heme oxygenase, and the down-modulation of HIF-1α. The expression of other HIF-1α-inducing factors, such as HO-1 / HMOX1 / HSP32, was inhibited, as was the phosphorylation of p38α, which is also an activator of HO-1. Inhibition of the p38α pathway resulted in p53 dephosphorylation, aberrant phosphorylation of HSP27, GSK-3β, and MSK2, and the down-modulation of survivin (data not shown). Disruption of survivin expression increased apoptosis and reduced tumor growth.

[0057] Overall, 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 whose progression depends on p38 MAPK activity. 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] . Furthermore, p38α inhibitors increase the sensitivity of tumor cells to chemotherapy drugs such as 5-fluorouracil (5-FU) and oxaliplatin

[24] . In this regard, the combination of both NRPas with 5-FU or oxaliplatin in breast cancer cells increased their sensitivity to these respective drugs, as expected (data not shown). More importantly, the combination of NRPa with Ralemitinib® (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 summary, NRPa could be used alone or in combination with other drugs to treat cancer. This observation has led to 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 which are incorporated by reference into this disclosure. [Table 2] TIFF0007718989000008.tif246165 TIFF0007718989000009.tif238165 TIFF0007718989000010.tif92165

Claims

1. 1. A pharmaceutical composition for treating cancer in a subject in need thereof, comprising at least one neuropilin-1 (NRP-1) antagonist that induces a decrease in NRP-1 activity or expression, in combination with at least one p38α-kinase inhibitor that partially or completely blocks, inhibits or neutralizes the biological activity or expression of p38α protein, A pharmaceutical composition, wherein the NRP-1 antagonist is N-[5-(1H-benzimidazol-2-yl)-2-methylphenyl]-N'-(2,3-dihydro-1,4-benzodioxin-6-ylcarbonyl)thiourea (NRPa-47) or N-[3-(1H-benzimidazol-2-yl)phenyl]-N'-(2,3-dihydro-1,4-benzodioxin-6-ylcarbonyl)thiourea (NRPa-48).

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

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

4. The pharmaceutical composition according to claim 1 , wherein the cancer is a hematopoietic cancer or a non-hematopoietic cancer.

5. The pharmaceutical composition according to claim 1, wherein the cancer is breast cancer.

6. The pharmaceutical composition of claim 1, wherein the cancer is triple-negative breast cancer.

7. The pharmaceutical composition according to claim 1, wherein the cancer is neuropilin-positive.

8. An NRP-1 antagonist inhibits the interaction between the NRP-1 protein and its partner. The pharmaceutical composition of claim 1, which inhibits the activity of 9. The pharmaceutical composition of claim 1, wherein the p38α-kinase inhibitor is selected from the group consisting of 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, and p38α-binding peptides, such that interaction between the p38α-kinase inhibitor and p38α results in reduction or cessation of p38α kinase activity or expression.

10. 2. The pharmaceutical composition 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, SCIO-469, PH-797804, VX-702, SB-202190, SB-203580, SB-239063, BIRB-796, BMS-582949, and pamapimod.

11. 2. The pharmaceutical composition of claim 1, wherein the NRP-1 antagonist is N-[5-(1H-benzimidazol-2-yl)-2-methylphenyl]-N'-(2,3-dihydro-1,4-benzodioxin-6-ylcarbonyl)thiourea (NRPa-47) and the p38α-kinase inhibitor is ralimetinib.

12. 2. The pharmaceutical composition of claim 1, wherein the NRP-1 antagonist is N-[3-(1H-benzimidazol-2-yl)phenyl]-N'-(2,3-dihydro-1,4-benzodioxin-6-ylcarbonyl)thiourea (NRPa-48) and the p38α-kinase inhibitor is ralimetinib.

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