Combination of an inhibitor of intracellular nitrosative and / or oxidative stress and an activator of apoptosis for use in the treatment of invasiveness of a primary brain tumor

Abstract

The invention provides new pharmacological approaches to the treatment of benign and malignant primary brain tumors, and specifically glioblastomas (GBM) and astrocytomas in which treatment is complicated by resistance to conventional chemotherapies. To which end the invention provides compositions and methods using modulators of intracellular nitrosative / oxidative stress and modulators DNA replication and apoptosis, with examples of specific inhibitors of neuronal and inducible NO synthetases (nNOS and iNOS) that were found to be effective either when administered alone or more effective when administered in combination temozolomide (TMZ), even in tumors displaying TMZ resistance.

Description

[0001] COMBINATION OF AN INHIBITOR OF INTRACELLULAR NITROSATIVE AND / OR OXIDATIVE STRESS AND AN ACTIVATOR OF APOPTOSIS FOR USE IN THE TREATMENT OF INVASIVENESS OF A PRIMARY BRAIN TUMOR

[0002] TECHNOLOGICAL FIELD

[0003] The invention pertains to the field of pharmacological therapies for primary brain tumors, and especially tumors that are resistant to conventional chemotherapies. To which end the invention provides compositions and methods using modulators of intracellular nitrosative / oxidative stress and modulators DNA replication and apoptosis.

[0004] BACKGROUND

[0005] Primary tumors of the central nervous system (CNS) are a heterogeneous group of neoplasms that include benign and malignant tumors. Their etiology is explained by various contributing factors such as age, race, ethnicity, gender, environmental conditions, as well as genetics and hormones. They include more than 100 different tumors that are distinguished by their incidence and histopathology, among them gliomas, astrocytomas, embryonal tumors, meningiomas, and medulloblastomas, and pituitary and pineal gland tumors. Glioblastoma (GBM) is the most common malignant tumor of the CNS, with the highest mortality rate.

[0006] CNS tumors are not as common as gastrointestinal or breast cancers, but their incidence has been increasing over time. About half of them are benign, but they can still be equally fatal if not treated and are allowed to proliferate in the skull. CNS tumors, as a group, have been recognized as one of the leading causes of death in children and adults, the second leading cause in children and the third in adults, and as tumors related to the most harmful and negative primary and secondary complications.

[0007] Gliomas are a relatively common type of CNS tumors. They develop in glial cells that surround and support neurons, such as astrocytes, oligodendrocytes and ependymal cells. Glioblastoma (GBM, glioblastoma multiforme or grade IV astrocytoma) is the most aggressive type of gliomas, with a 5-year survival rate of about 5-10%. GBM is generally considered malignant, apart from low-grade astrocytomas, and not metastatic. GBM constitute about 14% of all brain tumors and about 48% of primary brain tumors, with annual incidence of about 2-5 cases / 100,000 persons and a median survival rate of 12-18 months, depending on age and gender. Their treatment typically involves combination of radiotherapy and chemotherapy with temozolomide (TMZ), and surgical procedures. Despite recent advances in therapeutic approaches for cancers, GBM remains a challenging cancer to deal with. Current pharmacological treatments for GBM suffer from significant drawbacks of drug efficacy either due to emerging drug resistance (e.g., TMZ resistance), or drug inability to cross blood-brain barrier (BBB), or due to the inherent molecular heterogeneity and involvement of multiple molecular pathways, which is characteristic of primary brain tumors. Therefore, finding safe and effective pharmacological treatment for GBM remains an urgent necessity, and especially agents with long-term therapeutic effects that can be used as first-line treatment and remain effective in drug-resistant tumors, which are common in GBM.

[0008] Previous studies, among them studies by the inventor, suggested the involvement of nitric oxide (NO) overproduction in gliomagenesis, showing that certain inhibitors of NO synthetases (NOS) and factors reducing intracellular NO production can have mitigating effects on proliferation of CNS tumors and GBM in particular [1-3],

[0009] REFERENCES

[0010] 1. Berges Resende FF et al 2018. Function of neuronal nitric oxide enzyme in temozolomode-induced damage of astrocytic tumor cells. Oncol Lett, 15: 4891-4899.

[0011] 2. Kruglyakov D et al 2023. Nitric Oxide synthase inhibition prevents cell proliferation in glioblastoma. J Mol Neurosci 73(11): 875-883.

[0012] 3. Ojha SK et al 2025. Blocking nitric oxide production for glioblastoma: A targeted therapeutic approach. Genomic Press DOI: 1(3):41-49. doi 10.61373 / bm025r.0050

[0013] GENERAL DESCRIPTION

[0014] Nitric oxide (NO) is a small gaseous molecule that influences many biological processes. It has a crucial role in regulation of vascular functions, neurotransmission, neural development, smooth muscle relaxation, and immune and cytotoxic responses. NO dysregulation was implicated in various disorders, including endothelial dysfunction, increased blood pressure, cardiovascular issues, neurodegenerative diseases, and cancer.

[0015] Intracellular NO can be synthesized by three different types of NO synthetases: inducible (iNOS), endothelial (eNOS) and neuronal (nNOS) isoforms. The notion that elevated intracellular NO production can lead to cancer has been long known but its mechanism of action remains elusive. It is generally recognized that elevated NO levels lead to increased nitrosative and oxidative stress and increased risk of DNA modifications by alkylation, deamination or methylation, and can further reduce or interfere with DNA repair enzymes activity. All these processes can prompt neoplastic transformation and dysregulation of apoptosis, which eventually leads to cancer development.

[0016] In line with this notion, several studies reported that NOS inhibitors can reverse neoplastic phenotypes of some cancers, with examples of metastatic melanoma, breast cancer, ovarian cancer, oral cell carcinoma, head and neck cancer, colon cancer, and others. In the context of brain tumor specifically, several studies reported on correlation between elevated NO production in tumor microenvironment and advanced staging, and reduced patient survival.

[0017] There is conflicting evidence regarding the role of NO in cancer, and specifically GBM, revealed in its pro- and anti -neoplastic effects in different physiological conditions. On one hand, elevated NO production by tumor cells was shown to support a pro-growth environment for tumor proliferation and neovascularization. It was also shown that iNOS and nNOS overactivity promotes growth of glioma stem cells (GSC) - a subset of cells that are key drivers of tumor growth, relapses and drug resistance (e.g., TMZ resistance). On the other hand, other studies showed that at high levels NO acts as a pro-apoptotic agent via S-nitrosylation or cyclic guanosine monophosphate (cGMP) dependent pathways and thus can also have anti -neoplastic activity. Therefore, due to this inherent complexity and the dual role of NO in tumorigenesis, there is a need for a more careful examination of NO specific effects in specific cancers, and in GBM in particular.

[0018] The incentive behind the invention has been to find a more effective and safe pharmacological solution to the treatment of GBM, and potentially other primary brain tumors, by targeting intracellular NO production and NO associated cellular pathways. The invention stems from previous findings by the inventor, showing that certain selective inhibitors of iNOS and nNOS (L-NIL and 7-NI, respectively) induced assessable anti-tumorigenic effects in a GBM cell model in vitro (U87-MG). In this application, the inventor re-applies and refines this approach, using an in vivo GBM xenograft mouse model and other quantifiable GBM cell models that were specifically designed for this purpose, where he explores the effects and applications of specific NOS inhibitors and their combinations with known antiproliferative agents such as TMZ. To that end the inventor presently shows that a specific nNOS inhibitor, 7-NI, was surprisingly effective in attenuating GBM growth and increasing animals’ survival in vivo and was even more effective when introduced in the form of combo therapy with TMZ - an inhibitor of DNA replication and an activator of apoptosis through DNA alkylation and methylation. Studying intracellular effects of 7-NI activity, the inventor showed that 7-NI acted on both reduction of intracellular nitrosative / oxidative stress and activation of apoptosis, which can explain its superiority over other NOS inhibitors. The inventor further showed that the effect of 7-NI / TMZ combo on GBM growth was even more potent and synergistic by quantifiable parameters of tumorigenic cell phenotypes such as cells invasiveness, colony formation and cell migration (wound healing). Most importantly, using TMZ resistant GBM cell model that was specifically designed and developed for his purpose, the inventor showed that 7-NI / TMZ combo was the most potent and effective suppressor of TMZ resistant GBM compared to 7-NI or TMZ alone.

[0019] More specifically, the invention stems from an initial notion that intracellular nitrosative / oxidative stress is involved in the development of primary brain tumors, specifically GBM, and that mitigation or reduction of this component can provide potential therapeutic approach for these types of cancer. This notion was presently corroborated by administration of two iNOS and nNOS inhibitors, N6-(l-iminoethyl)-L- lysine (L-NIL) and 7-Nitroindazole (7-NI) respectively, into a subcutaneous xenograft GBM model in SKID mice - a bona fide GBM model in vivo, where they exhibited strong anti -neoplastic effects by various metrics of tumor size and progression. While both agents proved to be sufficiently effective and safe in this model, 7-NI was more potent, which made it a candidate agent all subsequent studies. (EXAMPLE 1)

[0020] A further study showed that 7-NI was effective across relatively wide range of doses (20-80 mg / kg) in a consistent and dose-dependent manner, peaking at the highest tested dose (80mg / kg). Importantly, it was discovered that in high doses, 7-NI conferred a significant protective effect against GBM progression even after termination of treatment. (EXAMPLE 2)

[0021] Study of specific molecular pathways activated by 7-NI showed that it can act through a number of molecular mechanisms, most noticeably by down-regulating processes of cellular proliferation and nitrosative / oxidative stress, as revealed by elevated expression of Ki-67 - a widely used marker of cell proliferation, cancer severity and metastases, and 3 -Nitrotyrosine (3-Ntyr) - a direct marker of nitrosative / oxi dative stress; and further, by upregulating processes of controlled cell death and apoptosis, by elevated expression of cleaved caspase 3 - a proteolytically activated caspase partaking in apoptosis, and cleaved poly(ADP-ribose) polymerase-1 (PARP-1) - a pro-apoptotic protein and product of activated caspase 3. (EXAMPLE 3)

[0022] These studies provided initial proof of concept for the potential applicability of pharmacotherapy using selective inhibitors of intracellular nitrosative / oxidative stress in treating GBM, and potentially in a broader group of primary brain tumors.

[0023] In attempt to enhance the effect of 7-NI on GBM growth, it was tested in the same GBM model in vivo in the form of combo therapy with TMZ - a DNA alkylating / methylating agent and an activator of apoptosis, and until now the mainstream pharmacotherapy for GBM. This study showed that the effect of 7-NI / TMZ combo on GBM growth surpassed the effects of 7-NI and TMZ alone to the extent that it was beyond additive but synergistic. Importantly, the combo increased the overall survival rate long after termination of the treatment period (80 days), which yet again points to synergistic and prolonged mode of action. (EXAMPLE 4)

[0024] The effects of 7-NI / TMZ combo on GBM growth were further tested in two cell models LN-18 (considered TMZ resistant) and LN-229, and both well-established human glioma cell lines. To that end the inventor undertook two main initiatives: 1st, by developing specific assays for the assessment and quantification of GBM related cellular phenotypes; and 2nd, by developing a more robust TMZ resistant GBM cell model which was tested in the same phenotypic assays.

[0025] In LN-18 cell model, the effect of 7-NI / TMZ combo essentially reproduced previous findings in vivo, showing effective suppression of GBM compared to individual agents, now by specific quantifiable phenotypic presentations of GBM growth in vitro, i.e., cell invasion, colony formation and cell migration (wound healing). As in previous studies, the effect of the combo was associated with corresponding reduction in intracellular nitrosative / oxidative stress (NO assay, 3-Ntyr marker) and increase of apoptosis (cleaved caspase 3 marker), suggesting again a cross-functional versatile mode of action across multiple cellular pathways. (EXAMPLE 5)

[0026] The effect of 7-NI / TMZ combo in LN-229 cell model was even more pronounced by all phenotypic parameters, i.e., cell invasion, colony formation and cell migration (wound healing), compared to individual agents. Importantly, in subcutaneous xenograft GBM model inoculated with LN-229 cells, the combo showed more effective in suppression of tumor size and progression than individual agents, with no apparent effects on animals’ body weight. (EXAMPLE 6)

[0027] Ultimately, the effect of 7-NI / TMZ combo was tested in a robust TMZ resistant GBM cell model, specifically developed for this purpose by subjecting LN-229 cells to repeated rounds of TMZ selection pressure and enrichment of the TMZ-resistant subpopulation of cells. In this model, 7-NI / TMZ combo was even more effective in suppressing GBM growth by all quantifiable phenotypes of cell invasion, colony formation and cell migration compared to individual agents and previous findings in LN- 18 and non-selected LN-229 cells. (EXAMPLE 7)

[0028] Taken together, these studies of suppression effects of the combo corroborated the notion that combining modulation of intracellular nitrosative / oxidative stress and modulation of DNA replication / apoptosis can provide an effective first-line treatment for primary brain tumors, and specifically GBM, including drug resistant tumors (e.g., TMZ resistant GBM) that so far suffered from lack effective treatment.

[0029] One of the expected advantages of using 7-NI / TMZ combo, in addition to its apparent effectiveness and the ability to manage TMZ resistant GBM, is the prospect of reducing therapeutic TMZ dose relative to the conventional therapeutic regimens using TMZ alone. This is especially important in view of the known side effects associated with prolonged TMZ therapy, such as GI malfunctions, nausea, dizziness, headache, rashes and hair loss in some cases. Several projections of the expected 7-NI / TMZ dose and dose regimens are presently provided. (EXAMPLE 8)

[0030] In summary, the invention provides a new pharmacological approach for the treatment of primary brain tumors, which in many cases can be fatal, invasive and resistant to conventional therapies, by inducing a combined or joint action of inhibitors of intracellular nitrosative and / or oxidative stress and activators of apoptosis. A hallmark example of such agents is 7-NI which exhibits all these functionalities. Another example is 7-NI / TMZ combo in which the inherent effects of 7-NI are augmented and synergized by the inherent effects of TMZ on DNA replication and apoptosis.

[0031] Proof of concept for feasibility of the proposed approach was provided in the example of GBM, the most common and deadly of primary brain tumors in children and adults. Importantly, it was demonstrated that 7-NI / TMZ combo provides the most effective and persisting suppression GBM growth even after termination of treatment. Ultimately, the proposed compositions and methods can be further perfected and adapted for the treatment of specific brain tumors by incorporation of known therapeutic modalities, such as conventional chemotherapies and radiological or surgical procedures.

[0032] BRIEF DECSRIPTION OF THE DRAWINGS

[0033] To better understand the subject matter, certain embodiments of the invention are now described by way of examples with reference to the following figures.

[0034] Figs. 1A-1D illustrate the effect of nNOS and iNOS inhibitors on GBM growth in a GBM xenograft mouse model. Figures show analyses of animals treated with vehicle, L-NIL, 7-NI or L-NIL / 7-NI combo as tumor images (1A) and measurements of tumor volumes (IB), body weights (1C) and tumor growth curves (ID), presented as mean ± SEM in each group (statistical analysis by one-way ANOVA and Bonferroni correction, with **P < 0.001, *P < 0.05 as significant and ns not significant). Figures show that the inhibitory effect of 7-NI was superior to L-NIL, with no contribution of L-NIL to the effect of L-NIL / 7-NI combo.

[0035] Figs. 2A-2D illustrate the effect of 7-NI on GBM growth in the same model with elevated 7-NI doses. Figures show analyses of animals treated with vehicle or 20, 40 or 80 mg / kg 7-NI for 14 days as tumor images (2A) and measurements of tumor volumes (2B), tumor weights (2C) and tumor growth curves (2D), presented as mean ± SEM in each group (statistical methods as above). Figures show that the inhibitory effect of 7-NI was consistent and dose-dependent, peaking at 80 mg / kg.

[0036] Figs. 3A-3E illustrate the effect of 7-NI on specific molecular cues in this model, using molecular markers of cell proliferation (Ki-67), nitrosative stress (3 -Nitrotyrosine, 3-Ntyr) and apoptosis (cleaved PARP1 and cleaved caspase 3) by microscope immunofluorescence and western blots analyses. Figures show analyses of tumor sections stained with DAPI, Ki-67 (3 A) or 3-Ntyr (3B) in control (vehicle) and treatment groups, as confocal images (left, x40 magnification and 50 pm scale bar) and quantitative image analyses (right), and western blot images and quantitative analyses of 3-Ntyr (3C), cleaved PARP1 (3D) and cleaved caspase 3 (3E) relative to P actin, presented as mean ± SEM in each group (statistical methods as above). Figures show that the inhibitory effect of 7-NI in vivo is exerted via a complex set of molecular pathways, involving down-regulation of Ki-67 and 3-Ntyr - inhibition of proliferation and nitrosative / oxidative stress, and up-regulation of cleaved PARP1 and cleaved caspase 3 - increase of controlled cell death and apoptosis.

[0037] Figs. 4A-4D illustrate the effect of 7-NI / TMZ combo on GBM growth in the same model. Figures show analyses of animals treated with vehicle, 7-NI (80 mg / kg), TMZ (10 mg / kg) or 7-NI / TMZ combo as tumor images (4A) and measurements of tumor volume (4B), tumor growth curve (4C) and animals survival curves (Kaplan-Meier plot, end point tumor size >1.5cm) (4D), presented as mean ± SEM in each group (statistical methods as above). Figures show that the effect of 7-NI / TMZ combo was superior to the effects of individual agents, 7-NI or TMZ, with significant contribution to survival rate after treatment.

[0038] Figs. 5-7 illustrate the effects of 7-NI / TMZ combo in a GBM cell model in vitro, using quantifiable cellular phenotypes of cell invasion, colony formation and cell migration (wound healing or monolayer formation of 80% over a scratch) in LN- 18 cells (TMZ resistant) treated with 7-NI (200 pM), TMZ (200 pM) or 7-NI / TMZ combo (200 pM each) and control (untreated) compared by statistical methods as above.

[0039] Figs. 5A-5B show results of trans-well Matrigel cell invasion assays as light microscope images (6A) and quantitative image analyses (6B). Figures show that the suppression effect of 7-NI / TMZ combo on tumor cell invasiveness was superior to the effects of 7-NI or TMZ alone.

[0040] Figs. 6A-6B show results of colony formation assays as plate images (7 A) and quantitative image analyses (7B). Figures show significant suppression effect of 7-NI / TMZ combo on tumor colony formation.

[0041] Figs. 7A-7B show results of cell migration assays as microscope images (8A) and quantitative image analyses (8B). Figures show significant suppression effect of 7-NI / TMZ combo on tumor cell migration capability.

[0042] Figs. 8-9 illustrate the effect of 7-NI / TMZ combo on markers of nitrosative / oxidative stress (3-Ntyr and NO assay) and apoptosis (cleaved caspase 3) in LN-18 cell model, comparing the combo to individual treatments with 7-NI (BA-101) or TMZ 200pM each, DMSO 0.04% and non-treated cells (control). Figs. 8A-8C show results of western blot analyses of 3-Ntyr as images (8A) and quantitative analyses (8B) relative to P actin, and NO assays (8C). Figures show that the combo significantly reduced nitrosative / oxidative stress by both 3-Ntyr marker and intracellular NO production. See also Fig. 3C.

[0043] Figs. 9A-9B show western blot analyses of cleaved caspase 3 apoptotic marker as images (9A) and quantitative analyses (9B) relative to P actin. Figures show a significant up regulation of cleaved caspase 3 in cells treated with the combo compared untreated control, which is indicative of increased apoptosis. See also Fig. 3E.

[0044] Figs. 10-12 illustrate the effect of 7-NI / TMZ combo in another GBM cell model, using LN-229 cells and the same drug concentrations, test assays and statistical methods as in Figs. 5-9.

[0045] Figs. 10A-10B show significant suppression effect of 7-NI / TMZ combo on tumor cell invasiveness compared to individual drugs by light microscope images of cell invasion assays (10A) and quantitative image analyses (10B).

[0046] Figs. 11A-11B show significant suppression effect of 7-NI / TMZ combo on tumor colony formation by plate images of colony formation (11 A) and quantitative image analyses (11B).

[0047] Figs. 12A-12B show significant suppression effect of 7-NI / TMZ combo on tumor cell migration capability by microscope images of cell migration assays (12A) and quantitative analyses (12B).

[0048] Figs. 13A-13D illustrate the effect of 7-NI / TMZ combo in a subcutaneous xenograft GBM mouse model using LN-229 cells. Figures show analyses of animals treated with vehicle (control), TMZ (10 mg / kg), 7-NI (BA-101, 80 mg / kg) or 7-NI / TMZ combo as tumor images (13A) and measurements of tumor volumes (13B), animals body weight (13C) and tumor progression (13D), presented as mean ± SEM in each group (statistical methods as above). Figures provide additional support for suppression effect of 7-NI / TMZ combo on GBM growth in vivo.

[0049] Figs. 14-17 illustrate the effect of 7-NI / TMZ combo in a rigorous TMZ resistant LN-229 cell model, after selection and enrichment for TMZ-resistant phenotype, using the same drug concentrations, test assays and statistical methods as in Figs 5-9, 10-12. Fig. 14 shows the effect of rounds of selection and enrichment for TMZ-resistant phenotype in LN-229, evident by preservation of LN-cells viability with increasing concentrations of TMZ (100pM-1000 pM).

[0050] Figs. 15A-15B show that in this system, the suppression effect of 7-NI / TMZ combo on tumor cell invasiveness was even more pronounced compared to TMZ and 7-NI alone, by microscope images (15A) and quantitative image analyses (15B).

[0051] Figs. 16A-16B show that the same for the suppression effect of 7-NI / TMZ combo on colony formation by images (16A) and quantitative image analyses (16B).

[0052] Figs. 17A-17B show the same for the suppression effect of 7-NI / TMZ combo on cell migration, by images (17A) and quantitative image analyses (17B).

[0053] DETAILED DESCRIPTION OF EMBODIMENTS

[0054] In the broadest sense the invention can be articulated in terms of therapeutic compositions, regimens and methods for alleviating or treating primary neoplastic diseases in the CNS. The term “primary neoplastic disease" encompasses here all neoplastic tumors, malignant and benign, that arise in the CNS, the brain or the spinal cord, as opposed to secondary CNS tumors that metastasize in the CNS but originate outside the CNS (e.g., lung, breast, colon, melanoma cancers). Primary CNS tumors can be differentiated from secondary tumors by the origin of neoplastic cells, using various molecular, histopathological and imaging methods.

[0055] In some embodiments the invention is relevant for treating malignant CNS tumors. The term “malignant tumors" encompasses herein all CNS tumors the common characteristics of which is more rapid growth, rapid increase in tumor volume and invasiveness into the surrounding healthy brain tissues. Some examples are gliomas (brain), hemangioblastomas (spinal cord) and glioblastoma (GBM). The term “malignant” encompasses herein cancer grades I-IV by clinical and / or histopathological criteria. The term “benign” generally implies a slower growth which does not invade the surrounding brain tissues. Notwithstanding, benign CNS tumors could be equally harmful if allowed to proliferate in the skull and are not removed by surgical or radiotherapeutic methods. Benign and malignant CNS tumors can be differentiated by various molecular, histopathologic and imaging methods, and detailed clinical work-up. As CNS is a complex organ involving various types of tissues, the terms “primary CNS tumors’" or “primary brain tumors"" encompass herein various types of origin cells.

[0056] In some embodiments the invention is relevant for treating primary CNS tumors which are of glial origin, e.g., Glioblastoma (GBM), Astrocytoma, Oligodendroglioma, Ependymoma.

[0057] In some embodiments the invention is relevant for treating non-glial tumors or tumors developing in nerves, embryotic cells and glands, e.g., Meningioma, Medulloblastoma, Pituitary adenoma.

[0058] In some embodiments the invention is relevant for treating CNS tumors that originate from microglia, astrocytes, oligodendrocytes, ependymal or progenitor cells - all non-neuronal cells that support, protect, and nourish neurons.

[0059] In some embodiments the primary CNS tumor to the which this invention is applicable can be Glioblastoma (GBM), Anaplastic astrocytoma, Diffuse astrocytoma, Glioblastoma multiforme, Gliosarcoma, Diffuse intrinsic pontine glioma (DIPG), Pleomorphic xanthoastrocytoma (PXA), Medulloblastoma, Ependymoma, Anaplastic ependymoma, Mixed glioma, Oligodendroglioma, Anaplastic oligodendroglioma, Pilocytic astrocytoma, Subependymal giant cell astrocytoma (SEGA), or H3 K27M- mutant diffuse midline glioma, which represent various clinical presentations of glial tumors or neuroepithelial tumors that occur in children and adults.

[0060] In some embodiments the invention is relevant for treating GBM, IDH-wildtype, Astrocytoma, IDH-mutant Grade 4, or a molecular or histopathological subtype thereof, which together account for the majority of highly infiltrative, malignant diffuse gliomas in adult patients.

[0061] In some embodiments the invention is relevant for treating adult brain tumors.

[0062] In some embodiments the invention is relevant for treating pediatric brain tumors.

[0063] In some embodiments the invention is relevant for treating primary brain tumors which are diagnosed as GBM in children and adults.

[0064] It is presently understood that in all the above applications and clinical conditions, the invention can provide effective means of treating or alleviating (which herein are interchangeable) of one or more neurological symptoms associated with these conditions in terms of reduction of number of symptoms, symptoms severity, symptoms progression, tumor size and tumor invasiveness, and improvement of patients’ survival overall. Thus, one important advantage of the invention is revealed in its ability to provide demonstrable immediate and prolonged effects on the management and treatment of primary brain tumors. In other words, it provides new first-line therapy fro primary brain tumors that could potentially replace the conventional approaches, which in many cases proved to be ineffective with prolonged use.

[0065] The existing therapeutic approaches to the treatment of primary brain tumors predominantly involve chemotherapies, applied either alone or in combination with radiotherapies and surgical procedures. Some but not limiting examples are Temozolomide (TMZ), Carmustine (BCNU), Lomustine (CCNU), Irinotecan, Topotecan, Etoposide, Gemcitabine, Cisplatin, Carboplatin, 5 -Fluorouracil (5-FU), Hydroxyurea, Fludarabine, Venetoclax, Obatoclax, Navitoclax (ABT-263), ABT-737, Nutlin-3, SMAC mimetics (e.g., LCL161), and TRAIL agonists These agents generally interfere with DNA replication, transcription, cell division, or apoptotic signaling pathways, and include both FDA-approved drugs and investigational compounds under clinical or preclinical evaluation for glioma and other CNS malignancies.

[0066] The terms “DNA replication damage" and “apoptosis’" are used herein interchangeably, as they represent closely related intersecting processes in the regulation of cell cycle. Under normal controlled conditions, disruption in DNA replication or DNA damage often leads to activation of apoptosis to prevent propagation of damaged cells. Abnormal neoplastic process is often associated with disruption of balance between DNA integrity and apoptosis, revealed in increased DNA damage and / or reduced apoptosis. Therefore, the main rationale behind many anti -neoplastic drugs and chemotherapies is to induce DNA damage and to trigger apoptosis, which affect all cells but are most effective in rapidly replicating tumor cells.

[0067] One of the main problems with the use of chemotherapies in cancer is the emergence of chemoresistance due to accumulation of genetic and molecular changes and strong selection pressure toward rapidly proliferating cancer cells. In brain tumors, this situation is exacerbated by blood brain barrier that leads to subtherapeutic drug levels.

[0068] It has been presently demonstrated that one of the main advantages of the invention is that it overcomes the chemoresistance problem, thus providing a long-term solution for brain tumors at risk of developing and with already existing chemoresistance. Thus, another important advantage of the invention is revealed in its ability to provide first-line treatment for various clinical presentations of primary brain tumors, including those manifesting chemoresistance with prolonged chemotherapeutic use.

[0069] One of the more common manifestations of chemoresistance is resistance to TMZ, for being one of the common chemotherapies for treating primary brain tumors. It has been presently demonstrated that in some embodiments the invention can be further applicable and effective for treating TMZ resistant primary brain tumors. Some examples of TMZ resistant primary brain tumors are high-grade GBM, Anaplastic Astrocytoma, Anaplastic Oligodendroglioma and Relapsed Medulloblastoma.

[0070] In some embodiments the invention can be applicable to TMZ resistant GBM.

[0071] Essentially, the invention is based on a surprising finding that a combination of modulators acting as inhibitors of intracellular nitrosative and / or oxidative stress and as activators of apoptosis can provide effective means for treating primary brain tumors.

[0072] The terms “ nitrosative stress" and “oxidative stress" are used herein interchangeably, as they represent closely related intersecting processes that produce reactive nitrogen or oxygen species (RNS or ROS) and lead to DNA and cellular damage. These terms imply herein an imbalance between the formation of RNS and ROS, and their elimination by intracellular control systems that destroy reactive intermediates and prevent or repair the resulting damage, or in other words, overproduction of at least one of nitric oxide (NO), peroxynitrite (ONOO-), nitrogen dioxide (NO2) and / or at least one of superoxide anion (O?*-), hydrogen peroxide (H2O2), hydroxyl radicals (»OH). In many instances, these terms are used herein simultaneously to denote relationship therebetween - nitrosative stress can boost oxidative stress (e.g., via ONOO- formation and mitochondrial dysfunction) and vice versa, (e.g., activation of NO synthases, NOS). They are further used herein in connection with known markers of nitrosative / oxidative stress, e.g., by measuring levels of RNS and / or ROS or activities of producing RNS / ROS enzymes (NOS or NOX) or protein nitration / oxidation (e.g., 3 -Nitrotyrosine, 3-Ntyr).

[0073] In many embodiments the modulators of the invention act inhibit the intracellular nitrosative stress and / or oxidative stress by inhibiting the intracellular production of RNS and / or ROS.

[0074] The term "apoptosis" is broadly used herein to relate to molecular pathways underlying processes of programmed and controlled cell death. In many embodiments, apoptosis is closely linked to DNA damage (e.g., via DDR damage response mechanisms or p53 pathway) as protective mechanisms against transmission of mutations and tumorigenesis. In many embodiments, apoptosis is further linked to excessive DNA replication (e.g., ATM, ATR kinases and p53 and Rb tumor suppressor pathways acting in cell cycle progression checkpoints). Apoptosis is further referred to herein in connection with known markers of apoptosis (e.g., cleaved PARP1, cleaved caspase 3, DNA fragmentation markers or others).

[0075] In many embodiments the modulators of the invention activate apoptosis.

[0076] In many embodiments the invention implies a combination of activities across multiple molecular pathways, with at least one modulator acting as an inhibitor of intracellular nitrosative and / or oxidative stress and at least one is an activator of apoptosis.

[0077] In some embodiments certain modulators of the invention can exert both these activities - being an inhibitor of intracellular nitrosative and / or oxidative stress and an activator of apoptosis. One example of such modulators in the nNOS inhibitor 7-NI.

[0078] More specifically, according to the invention, in some embodiments the inhibitors of intracellular nitrosative and / or oxidative stress can be inhibitors of intracellular NO production, revealed by measuring levels of RNS and / or ROS or activities of producing RNS / ROS enzymes (NOS or NOX) or protein nitration / oxidation (e.g., 3 -Nitrotyrosine, 3-Ntyr).

[0079] In some embodiments effective inhibitors of intracellular nitrosative and / or oxidative stress can be inhibitors of one or more NO synthetases (NOS), which encompass herein the entire family of mammalian enzymes catalyzing NO production from L-arginine - inducible (iNOS), endothelial (eNOS) or neuronal (nNOS) isoforms.

[0080] To that end, the invention further provides specific inhibitors, i.e., N6-(l- iminoethyl)-L-lysine (L-NIL) - a lysine analog acting on NOS and specifically iNOS; and 7-Nitroindazole (7-NI) - an indazole derivative which is a selective inhibitor of nNOS. The invention further implies herein use of various derivatives of L-NIL and 7-NI, and combinations thereof.

[0081] In practical terms, the invention provides therapeutic compositions, regimens and methods that comprise, use or administer certain type of inhibitors, acting either on all or some NOS isoforms or selectively on specific NOS isoforms - iNOS, eNOS or nNOS. In some embodiments the therapeutic compositions, regimens and methods of the invention can comprise, use or administer L-NIL or 7-NI, or derivatives thereof.

[0082] In some embodiments the therapeutic compositions, regimens and methods of the invention can comprise, use or administer combination of NOS inhibitor, and specifically L-NIL or 7-NI or derivatives thereof.

[0083] In some embodiments the therapeutic compositions, regimens and methods of the invention can comprise, use or administer only 7-NI, possessing a dual activity as an inhibitor of nitrosative and / or oxidative stress and an activator of apoptosis.

[0084] In other words, the required combination of activities - the inhibition of nitrosative and / or oxidative stress and the activation of apoptosis can be provided by the same agent, e.g., 7-NI.

[0085] In many embodiments, however, the required combination of activities can be provided by distinct groups of agents - one responsible for inhibition of nitrosative and / or oxidative stress and the other for activation of apoptosis, wherein this latter can be provided by a large group of chemotherapeutic agents and / or radiosensitizers. Examples of applicable chemotherapeutic agents were provided above. Some of these therapeutic agents further act as radiosensitizers, i.e., enhancing the effectiveness of radiation therapy - a known example is TMZ.

[0086] Thus, in some embodiments the therapeutic compositions, regimens and methods of the invention can comprise, use or administer one or more chemotherapeutic agents and / or radiosensitizers, which herein encompass any agent capable of inducing DNA damage, DNA replication damage and apoptosis.

[0087] In many embodiments the applicable chemotherapeutic agents can be DNA alkylating and / or methylating agents, which herein encompass any agent capable of inducing DNA modifications via addition of alkyl or methyl groups to DNA bases, which affect DNA structure and function and ultimately lead to DNA replication and transcription damage and apoptosis.

[0088] In some embodiments the applicable chemotherapeutic agent and / or radiosensitizer can be TMZ.

[0089] In some embodiments the therapeutic compositions, regimens and methods of the invention can comprise, use or administer 7-NI or L-NIL, derivatives or combinations thereof, in conjunction with TMZ. According to the invention, in some embodiments the two types of modulators - the inhibitors of nitrosative and / or oxidative stress and the activators of apoptosis can be formulated or comprised in the same or different pharmaceutical compositions or administered via different administration routes simultaneously or in succession.

[0090] More specifically, in some embodiments the therapeutic compositions, regimens and methods of the invention can comprise, use or administer, for example, 7-NI and TMZ, which can be formulated or comprised in the same or different pharmaceutical compositions (e.g., oral compositions) administered simultaneously or in succession.

[0091] In many embodiments the pharmaceutical composition and methods of the invention can be formulated or adapted for oral, enteral, parenteral, intraperitoneal, subcutaneous or intramuscular administrations.

[0092] More specifically, in many embodiments the therapeutic compositions and methods of the invention can further involve use of pharmaceutically acceptable carriers, buffers and / or excipients, depending on choice of actives and modes of administration. For example, administrations by injection often use aqueous isotonic formulations, which can further include various antioxidants, buffers and bacteriostats. Oral formulations can include solubilizers, thickening agents, stabilizers, and preservatives. A composition can be administered in a physiologically acceptable sterile diluent in a pharmaceutical carrier, such water, saline, aqueous dextrose or other sugar solutions, an alcohol such as ethanol, isopropanol or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, glycerol ketals such as 2,2-dimethyl-l,3-dioxolane-4-methanol, ethers, such as poly(ethyleneglycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides with or without pharmaceutically acceptable surfactants such as soaps, detergents, suspending agents such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, emulsifying agents and other pharmaceutical adjuvants. Parenteral formulations can use specific components, with specific examples of petroleum, animal, vegetable, or synthetic oils as suitable oils; oleic acid, stearic acid, and isostearic acid as suitable fatty acids; ethyl oleate and isopropyl myristate as suitable fatty acid esters; and fatty alkali metal, ammonium, and triethanolamine salts as suitable soaps or detergents, including cationic (e.g., dimethyl dialkyl ammonium halides, and alkyl pyridinium halides), anionic (e.g., alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether), nonionic (e.g., fatty amine oxides, fatty acid alkanolamides, poly oxy- ethylenepolypropylene copolymers) and amphoteric (e.g., alkyl- aminopriopionates) detergents and mixtures thereof.

[0093] Yet another important advantage of invention is revealed in its ability to impact an effective drug dose, and especially the effective dose of the chemotherapeutic agent comprised, used or administered in the compositions and methods of the invention. Many chemotherapeutic agents can cause significant toxicity to normal tissues, revealed in one or more of the following manifestations: bone marrow suppression, alopecia and toxicities across gastrointestinal, renal, neuronal, hepatic, cardiovascular, pulmonary and reproductive systems. Therefore, the ability to reduce an effective dose of a chemotherapeutic agent without compromising overall drug effectiveness is important.

[0094] The terms “ effective dose" or ^therapeutically effective dose" are used herein the broadest sense to denote an amount of drug per kilo or an initial concentration of drug that are administered to a subject to achieve one or more of the following: reduction of severity of one or more symptoms, reduction in progression of one or more symptoms, reduction in number of symptoms, reduction of tumor size and tumor invasiveness, and improvement of patients’ survival overall.

[0095] In many embodiments, the chemotherapeutic agent comprised, used or administered in the therapeutic compositions, regimens and methods of the invention can be provided at an effective therapeutic dose which is lower than in a conventional dosage form of the same chemotherapeutic agent alone.

[0096] In the example of TMZ which is an oral drug, in some embodiments TMZ can be provided at an effective oral dose of less than 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 75, 50, 25 and 10 mg / m2

[0097] In some embodiments, TMZ can be provided in an effective oral dose in one or more of the above ranges which are administered daily or 1, 2, 3, 4, 5, 6, 7, times a week, or 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 or 30 times a month.

[0098] In some embodiments, TMZ can be provided in an effective oral dose in one or more of the above regimens for at least 1-5, 5-10, 10-20, 20-30 consecutive days or more, or for at least 1-5, 5-10, 10-20, 20-30, 30-40, 40-50 consecutive weeks or more, or for 1-5, 5-10, 10-20, 20-30 consecutive years, or through entire patient’s life. In some embodiments, TMZ effective doses can be provided in cycles, including for example interchanging daily, weekly or monthly regimens as above, with increasing or decreasing drug doses in the ranges of 1000-10, 900-10, 800-10, 700-10, 600-10, 500- 10, 400-10, 300-10, 200-10, or 100-10 mg / m2, or ranges of 100-25, 400-25, 300-25, 200- 25 and 500-25 mg / m2.

[0099] In some embodiments TMZ can be further provided by daily of weekly and monthly administrations in an effective dose in one or more of the above ranges.

[0100] In some embodiments, TMZ can be provided via oral, intravenous intraperitoneal, subcutaneous or intramuscular or other administration routes.

[0101] In the example of 7-NI, administered either alone or with a chemotherapeutic agent, in some embodiments 7-NI can be provided at an effective oral dose of less than 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 75, 50, 25 and 10 mg / m2

[0102] In some embodiments, 7-NI can be provided in an effective oral dose in one or more of the above ranges which are administered daily or 1, 2, 3, 4, 5, 6, 7, times a week, or 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 or 30 times a month.

[0103] In some embodiments, 7-NI can be provided in an effective oral dose in one or more of the above regimens for at least 1-5, 5-10, 10-20, 20-30 consecutive days or more, or for at least 1-5, 5-10, 10-20, 20-30, 30-40, 40-50 consecutive weeks or more, or for 1-5, 5-10, 10-20, 20-30 consecutive years, or through entire patient’s life.

[0104] In some embodiments, 7-NI effective doses can be provided in cycles, including for example interchanging daily, weekly or monthly regimens as above, with increasing or decreasing drug doses in the ranges of 1000-10, 900-10, 800-10, 700-10, 600-10, 500- 10, 400-10, 300-10, 200-10, or 100-10 mg / m2, or ranges of 100-25, 400-25, 300-25, 200- 25 and 500-25 mg / m2.

[0105] In some embodiments, 7-NI can be provided via oral, intravenous intraperitoneal, subcutaneous or intramuscular or other administration routes.

[0106] In some embodiments, the compositions and methods of the invention can use a therapeutically effective dose of 7-NI, or a derivative, in one or more of the above ranges and a therapeutically effective dose of TMZ in one or more of the above ranges, administered simultaneously or in succession in the same or different compositions. In some embodiments such compositions and methods can provide TMZ in a therapeutically effective dose which is lower than in the respective conventional dosage form of TMZ alone.

[0107] The invention can be further articulated in terms of use of a combination of modulators of which one or more are inhibitors of intracellular nitrosative and / or oxidative stress and one or more are activators of apoptosis, in the manufacture of a medicament for alleviating, treating a primary brain tumor, following the definitions, ranges and interpretations of specific terms as above.

[0108] In many embodiments, such medicament can be applied to a primary brain tumor which is resistant to chemotherapy.

[0109] To further enhance the efficacy of treatment, it is understood that the compositions, regimens and methods of the invention can be applied in conjunction with additional adjuvant procedures that can include radiotherapy and / or surgical procedures.

[0110] The terms “about” or “approximately” are meant to encompass herein a deviation of up to ±10% from the specified values or ranges of values, and more specifically up to ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9% or ± 10% deviation therefrom.

[0111] The invention further provides:

[0112] A pharmaceutical composition for alleviating, treating or reducing invasiveness of a primary brain tumor, comprising a combination of modulators of which (i) at least one is an inhibitor of intracellular nitrosative and / or oxidative stress and (ii) at least one is an activator of apoptosis.

[0113] In some embodiments of a pharmaceutical composition of the invention, said primary brain tumor being resistant to chemotherapy.

[0114] In some embodiments of a pharmaceutical composition of the invention, said primary brain tumor being resistant to the treatment with Temozolomide (TMZ).

[0115] In some embodiments of a pharmaceutical composition of the invention, the primary brain tumor is Glioblastoma (GBM), Anaplastic astrocytoma, Diffuse astrocytoma, Glioblastoma multiforme, Gliosarcoma, Diffuse intrinsic pontine glioma (DIPG), Pleomorphic xanthoastrocytoma (PXA), Medulloblastoma, Ependymoma, Anaplastic ependymoma, Mixed glioma, Oligodendroglioma, Anaplastic oligodendroglioma, Pilocytic astrocytoma, Subependymal giant cell astrocytoma (SEGA), or H3 K27M-mutant diffuse midline glioma. In some embodiments of a pharmaceutical composition of the invention, the primary brain tumor is Glioblastoma (GBM), IDH-wildtype, Astrocytoma, IDH-mutant Grade 4, or a molecular or histopathological subtype thereof.

[0116] In some embodiments of a pharmaceutical composition of the invention, said primary brain tumor is Glioblastoma (GBM) which is resistant to Temozolomide (TMZ).

[0117] In some embodiments of a pharmaceutical composition of the invention, said inhibitor of intracellular nitrosative and / or oxidative stress is an inhibitor of intracellular NO production.

[0118] In some embodiments of a pharmaceutical composition of the invention, the inhibitor of intracellular NO production is an inhibitor of at least one NO synthetase (NOS).

[0119] In some embodiments of a pharmaceutical composition of the invention, the at least one NOS is at least one of an inducible synthetase (iNOS), an endothelial synthetase (eNOS) or a neuronal synthetase (nNOS).

[0120] In some embodiments of a pharmaceutical composition of the invention, said inhibitor of at least one NOS is N6-(l-iminoethyl)-L-lysine (L-NIL), 7-Nitroindazole (7- NI), a derivative or a combination thereof.

[0121] In some embodiments of a pharmaceutical composition of the invention, said inhibitor of intracellular nitrosative and / or oxidative stress is the nNOS inhibitor 7-NI or a derivative thereof.

[0122] In some embodiments of a pharmaceutical composition of the invention, said activator of apoptosis is the nNOS inhibitor 7-NI or a derivative thereof.

[0123] In some embodiments of a pharmaceutical composition of the invention, said activator of apoptosis is a chemotherapeutic agent and / or a radiosensitizer.

[0124] In some embodiments of a pharmaceutical composition of the invention, said chemotherapeutic agent and / or radiosensitizer is a DNA alkylating and / or methylating agent.

[0125] In some embodiments of a pharmaceutical composition of the invention, said chemotherapeutic agent and / or radiosensitizer is TMZ.

[0126] In some embodiments of a pharmaceutical composition of the invention, the composition comprising 7-NI and TMZ, or a derivative of 7-NI and TMZ, or a combination thereof. In some embodiments of a pharmaceutical composition of the invention, said chemotherapeutic agent and / or radiosensitizer are provided at an effective therapeutic dose which is lower than in a conventional dosage form of the same chemotherapeutic agent and / or radiosensitizer alone.

[0127] In some embodiments of a pharmaceutical composition of the invention, the composition further comprising a pharmaceutically acceptable carrier, buffer or excipient.

[0128] In some embodiments of a pharmaceutical composition of the invention, the composition is for oral, enteral, parenteral, intraperitoneal, subcutaneous or intramuscular administrations.

[0129] Also provided is a method of alleviating, treating or reducing invasiveness of a primary brain tumor in a subject in need thereof, the method comprises administering to the subject a therapeutically effective dose of a combination of modulators of which at least one is an inhibitor of intracellular nitrosative and / or oxidative stress and at least one is an activator of apoptosis, which are formulated in the same or different pharmaceutical compositions administered simultaneously or in succession.

[0130] In some embodiments of a method of the invention, said primary brain tumor being resistant to chemotherapy.

[0131] In some embodiments of a method of the invention, said primary brain tumor being resistant to the treatment with Temozolomide (TMZ).

[0132] In some embodiments of a method of the invention, the primary brain tumor is Glioblastoma (GBM), Anaplastic astrocytoma, Diffuse astrocytoma, Glioblastoma multiforme, Gliosarcoma, Diffuse intrinsic pontine glioma (DIPG), Pleomorphic xanthoastrocytoma (PXA), Medulloblastoma, Ependymoma, Anaplastic ependymoma, Mixed glioma, Oligodendroglioma, Anaplastic oligodendroglioma, Pilocytic astrocytoma, Subependymal giant cell astrocytoma (SEGA), or H3 K27M-mutant diffuse midline glioma.

[0133] In some embodiments of a method of the invention, the primary brain tumor is Glioblastoma (GBM), IDH-wildtype, Astrocytoma, IDH-mutant Grade 4, or a molecular or histopathological subtype thereof.

[0134] In some embodiments of a method of the invention, said primary brain tumor is Glioblastoma (GBM) which is resistant to Temozolomide (TMZ). In some embodiments of a method of the invention, said inhibitor of intracellular nitrosative and / or oxidative stress is an inhibitor of intracellular NO production.

[0135] In some embodiments of a method of the invention, the inhibitor of intracellular NO production is an inhibitor of at least one NO synthetase (NOS).

[0136] In some embodiments of a method of the invention, the at least one NOS is at least one of an inducible synthetase (iNOS), an endothelial synthetase (eNOS) or a neuronal synthetase (nNOS).

[0137] In some embodiments of a method of the invention, said inhibitor of at least one NOS is N6-(l-iminoethyl)-L-lysine (L-NIL), 7-Nitroindazole (7-NI), a derivative or a combination thereof.

[0138] In some embodiments of a method of the invention, said inhibitor of intracellular nitrosative and / or oxidative stress is the nNOS inhibitor 7-NI, or a derivative thereof.

[0139] In some embodiments of a method of the invention, said activator of apoptosis is the nNOS inhibitor 7-NI, or a derivative thereof.

[0140] In some embodiments of a method of the invention, said activator of apoptosis is a chemotherapeutic agent and / or a radiosensitizer.

[0141] In some embodiments of a method of the invention, said chemotherapeutic agent and / or radiosensitizer is a DNA alkylating and / or methylating agent.

[0142] In some embodiments of a method of the invention, said chemotherapeutic agent and / or radiosensitizer is TMZ.

[0143] In some embodiments of a method of the invention, said inhibitor of intracellular nitrosative and / or oxidative stress and said activator of apoptosis are 7-NI and TMZ, or a derivative of 7-NI and TMZ, or a combination thereof.

[0144] In some embodiments of a method of the invention, the at least one chemotherapeutic agent and / or radiosensitizer is administered at a therapeutically effective dose which is lower than in a conventional dosage form of the same chemotherapeutic agent and / or radiosensitizer alone.

[0145] In some embodiments of a method of the invention, said inhibitor of intracellular nitrosative and / or oxidative stress and said activator of apoptosis are administered via at least one of oral, enteral, parenteral, intraperitoneal, subcutaneous or intramuscular administration routes. In some embodiments of a method of the invention, the method further comprising subjecting the subject to a radiotherapy and / or a surgical procedure before or after the administration of said combination of modulators.

[0146] A method is also provided for alleviating, treating or reducing invasiveness of TMZ resistant GBM in a subject in need thereof, comprising administering to the subject a therapeutically effective dose of 7-NI or a derivative thereof and a therapeutically effective dose of TMZ, which are formulated in the same or different pharmaceutical compositions administered simultaneously or in succession, and wherein the therapeutically effective dose of TMZ is lower than in the respective conventional dosage form of TMZ alone.

[0147] A pharmaceutical composition is provided for alleviating, treating or reducing invasiveness of TMZ resistant GBM, comprising an effective dose of 7-NI or a derivative thereof and an effective dose of TMZ, said effective dose of TMZ being lower than in the respective conventional dosage form of TMZ alone.

[0148] A use is provided of a combination of modulators of which at least one is an inhibitor of intracellular nitrosative and / or oxidative stress and at least one is an activator of apoptosis in the manufacture of a medicament for alleviating, treating or reducing invasiveness of a primary brain tumor, said primary brain tumor being resistant to chemotherapy.

[0149] In some cases, in the use of the invention, said inhibitor and activator are defined herein.

[0150] Also provided is a pharmaceutical composition for alleviating or treating a primary brain tumor, comprising a combination of at least one nitrative stress mitigating agent acting on at least one intracellular mechanism of NO production and at least one anti -neoplastic drug being a DNA damage inflicting agent.

[0151] In some embodiments of a pharmaceutical composition of the invention, the primary brain tumor is Glioblastoma (GBM) or Neuroblastoma.

[0152] In some embodiments of a pharmaceutical composition of the invention, said primary brain tumor is resistant to the conventional Temozolomide (TMZ) therapy.

[0153] In some embodiments of a pharmaceutical composition of the invention, the at least one nitrative stress mitigating agent is at least one inhibitor of at least one NO synthetase (NOS). In some embodiments of a pharmaceutical composition of the invention, the at least one NOS is an inducible (iNOS), an endothelial (eNOS) or a neuronal (nNOS) isoform.

[0154] In some embodiments of a pharmaceutical composition of the invention, the at least one NOS inhibitor is N6-(l-iminoethyl)-L-lysine (L-NIL), 7-Nitroindazole (7-NI) or a combination thereof.

[0155] In some embodiments of a pharmaceutical composition of the invention, the at least one NOS inhibitor is 7-Nitroindazole (7-NI).

[0156] In some embodiments of a pharmaceutical composition of the invention, the at least one anti -neoplastic drug is a DNA alkylating agent.

[0157] In some embodiments of a pharmaceutical composition of the invention, the DNA alkylating agent is Temozolomide (TMZ).

[0158] In some embodiments of a pharmaceutical composition of the invention, the at least one nitrative stress mitigating agent is 7-Nitroindazole (7-NI) and the at least one anti -neoplastic drug is TMZ.

[0159] In some embodiments of a pharmaceutical composition of the invention, the composition further comprising a pharmaceutically acceptable carrier, buffer or excipient.

[0160] In some embodiments of a pharmaceutical composition of the invention, the composition is formulated for oral, enteral, parenteral, intraperitoneal or subcutaneous administrations.

[0161] A method is provided for alleviating or treating a primary brain tumor in a subject in need thereof, the method comprises administering to said subject at least one nitrative stress mitigating agent acting on at least one intracellular mechanism of NO production and at least one anti -neoplastic drug which is a DNA damage inflicting agent.

[0162] In some embodiments of a method of the invention, the primary brain tumor is Glioblastoma (GBM) or Neuroblastoma.

[0163] In some embodiments of a method of the invention, the primary brain tumor is resistant to the conventional Temozolomide (TMZ) therapy.

[0164] In some embodiments of a method of the invention, the at least one nitrative stress mitigating agent and the at least one anti -neoplastic drug are formulated in the same pharmaceutical composition as defined herein. In some embodiments of a method of the invention, the at least one nitrative stress mitigating agent and the at least one anti -neoplastic drug are formulated in separate pharmaceutical compositions and are administrated to the subject simultaneously or in succession.

[0165] In some embodiments of a method of the invention, the at least one nitrative stress mitigating agent and / or the at least one anti -neoplastic drug are administrated to the subject via enteral, parenteral, intraperitoneal or subcutaneous routes.

[0166] In some embodiments of a method of the invention, the method further comprising administering to the subject, before or after the administration of the at least one nitrative stress mitigating agent and the at least one anti -neoplastic drug, a radiotherapy and / or a surgical procedure for alleviating or treating said primary brain tumor.

[0167] A treatment regimen is provided for alleviating or treating a primary brain tumor that comprises administering at least one nitrative stress mitigating agent acting on at least one intracellular mechanism of NO production in combination with at least one anti- neoplastic drug which is a DNA damage inflicting agent.

[0168] In some embodiments of a regimen of the invention, the primary brain tumor is Glioblastoma (GBM) or Neuroblastoma.

[0169] In some embodiments of a regimen of the invention, the primary brain tumor is resistant to the conventional Temozolomide (TMZ) therapy.

[0170] In some embodiments of a regimen of the invention, the at least one nitrative stress mitigating agent and the at least one anti -neoplastic drug are formulated in the same pharmaceutical composition as defined herein or in separate pharmaceutical compositions.

[0171] In some embodiments of a regimen of the invention, the at least one nitrative stress mitigating agent and the at least one anti -neoplastic drug are formulated in the separate pharmaceutical compositions and are used simultaneously or in succession.

[0172] In some embodiments of a regimen of the invention, the at least one nitrative stress mitigating agent and / or the at least one anti-neoplastic drug are formulated so as to be adapted for oral, enteral, parenteral, intraperitoneal or subcutaneous administrations.

[0173] In some embodiments of a regimen of the invention, the regimen further comprising, before or after the administration of the at least one nitrative stress mitigating agent and the at least one anti -neoplastic drug, use of a radiotherapy and / or a surgical procedure for alleviating or treating said primary brain tumor.

[0174] In some embodiments of a regimen of the invention, alleviating or treatment of the primary tumor continues after the administration regimen has ended.

[0175] A use is provided of the pharmaceutical composition of the invention in the manufacture of the medicament for alleviating or treating a primary brain tumor.

[0176] A further use is provided of the pharmaceutical composition comprising a combination of 7-NI and TMZ in the manufacture of a medicament for alleviating or treating a primary brain tumor.

[0177] A composition is provided which comprises 7-NI and TMZ for alleviating or treating a primary brain tumor.

[0178] A composition is provided which comprises 7-NI for treating a primary brain tumor resistant to conventional Temozolomide (TMZ) therapy.

[0179] A method is provided for treating or alleviating a brain tumor resistant to TMZ in a subject, the method comprising administrating to the subject an effective amount of 7- NI.

[0180] EXAMPLES

[0181] The figures and examples provided herein serve only illustrative purposes and are not intended to be limiting for the scope of the invention.

[0182] The following protocols pertain to the experiments in a subcutaneous xenograft GBM model in SCID mice described in EXAMPLES 1-4.

[0183] Materials and Methods

[0184] — Primary antibodies, Anti Ki -67, anti -cleaved caspase 3 and secondary antibodies, anti-rabbit Alexa fluor 594, anti-mouse Alexa Fluor 488, HRP-conjugated anti-rabbit, HRP-conjugated anti-mouse, ProLong Gold Antifade with DAP I, Protease phosphatase inhibitor cocktail were purchased from Cell Signaling Technology; Primary anti 3- Nitrotyrosine from Abeam, UK; and Primary, anti-PARP 1 from Biotechnology Inc; and other chemicals from Sigma Aldrich and Bio-Rad Labs.

[0185] — Animals were 6 weeks old NOD.CB17-Prkdc-scid / NCrHsd male mice.

[0186] — Uppsala 87 Malignant Glioma (U87-MG) cell line obtained from ATCC was grown in Dulbecco's modified eagle medium (DMEM, Gibco), 10% fetal bovine serum (FBS, Gibco), 1% penicillin-streptomycin, 10,000 U / ml (penstrep, Gibco) in the humidified atmosphere (37°C, 5% CO?.).

[0187] Generation of subcutaneous xenograft GBM model and drug treatments

[0188] Subcutaneous GBM-bearing mice were obtained by subcutaneous injection of lx 106U87MG cells in lOOpl PBS into the flanks of 6-week-old male NOD-SCID. In week

[0189] 2-3 after tumor cell implantation, with an average tumor size of about 30-50 mm3, mice were randomly divided into four groups (7-8 mice per group) and treated intraperitoneally with a vehicle or 7-NI, L-NIL (20-80 mg / kg) or TMZ (10 mg / kg) in 100 ul of PBS with 5% DMSO. Body weight and tumor size (tumor volume = (length x width2) / 2 by digital caliper) were measured individually. After tumor size reached 1.5 cm in one of the dimensions, mice were sacrificed, tumor tissues were surgically excised and stored in - 80°C or fixed with 4% paraformaldehyde solution and used for cryo-sectioning.

[0190] Western blots

[0191] Tissues were homogenized in RIPA buffer containing 30 mM HEPES (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 5 mM EDTA, 1 mM Na3VO4, 50 mM NaF, 1 mM PMSF, and 1% protease / phosphatase inhibitors cocktail (pH 7.7) on ice, using magnetic stirrer. Homogenates were subjected to centrifugation at 17,000G for 30 min at 4°C. Supernatant was collected, and protein concentration was determined by Bicinchoninic acid (BCA) protein assay (Sigma Aldrich). Samples were subjected to polyacrylamide gel electrophoresis and transferred onto PVDF membrane using wet transfer method (BioRad Labs), non-specific sites were blocked by 5% dried skimmed milk or 5% bovine serum albumin in Tris-buffered saline with Tween 20 (TBST solution: 135 mM NaCl, 50 mM Tris, and 0.1% Tween 20, with a pH of 7.4) for 2 h at RT. PVDF protein loaded membranes were incubated with primary antibodies on shaker overnight at 4°C, i.e., anti-

[0192] 3 -Ntyr (diluted 1 : 1000), anti-Ki-67 (1 :200), anti-cleaved caspase 3 (1 : 1000), anti-cleaved PARP1 (1 : 1000), and anti-P-Actin (1 : 1000). Membranes were washed with TBST and further incubated with anti-mouse / rabbit HRP -conjugated secondary antibody for 1 h at RT. Protein quantitation was performed by previously described methods using an ECL substrate and Bio-Rad Chemidoc imaging system (both by Bio-Rad Labs). Immunofluorescence and confocal microscopy

[0193] Tumors were preserved in 10% PF A solution for 2 days, after which they were gradually dehydrated with 10%, 20%, 30%, sucrose solution and dissected into 20 pM sections by cryostat. For dual immunofluorescence, sections were incubated in a blocking buffer and mouse anti-Ki-67 (1 :500), and anti-3-Ntyr (1 :200) primary antibodies. Sections were rinsed with PBS and incubated with anti-rabbit Alexa Fluor 594 (1 : 1000) and anti-mouse (Alexa Fluor 488 (1 : 1000) secondary antibodies for 2 hours in the dark. Sections were washed with PBS (3 times) and mounted on glass slides with DAPI. Images were captured 40X using a Nikon confocal microscope.

[0194] Statistical analysis

[0195] Statistical analysis was performed using Prism 9.3 (GraphPad Software). For groups comparisons, a one-way ANOVA test followed by Bonferroni’s post hoc multiple comparison test was used. Data are presented as means ± SEM.

[0196] EXAMPLE 1: NOS inhibition reduces tumor growth in GBM mouse model

[0197] GBM inoculated mice were treated with NOS inhibitors and vehicle (control). Animals were sacrificed at the end of study period, tumors were isolated and measured, and western blot and immunofluorescence analyses were performed. The results are shown in Figs 1A-1D.

[0198] Results and Conclusions

[0199] The results show that selective inhibition of iNOS or nNOS by L-NIL or 7-NI, respectively, was surprisingly effective in suppressing GBM. Animals treated with L-NIL or 7-NI exhibited significantly reduced tumor size and tumor progression (tumor growth) by visual and measurement analyses compared to animals treated with vehicle (control), in which tumors were gradually expanding. The inhibitory effect of 7-NI was more significant than the effect of L-NIL, with this latter adding no significant contribution to the effect of L-NIL / 7-NI combo (Figs 1A-1B). Moreover, the inhibitory effect persisted after treatment, more distinctively in 7-NI and combo treated groups (Fig. ID). None of these agents or the combo had significant effects on body weight (Fig. 1C).

[0200] Overall, the results suggested that the inhibition of intracellular nitrosative stress, and specifically selective inhibition of iNOS and / or nNOS, can be a promising pharmacological approach to GBM, and potentially other primary brain tumors. EXAMPLE 2: The inhibitory effect of 7-NI is specific and dose-dependent

[0201] GBM inoculated mice were treated with elevated doses of 7-NI (nNOS inhibitor) by peritoneal injections of 20, 40 and 80 mg / kg 7-NI for 14 days, using experimental setup as above. The results are shown in Figs 2A-2D.

[0202] Results and Conclusions

[0203] The results show that the inhibitory effect of 7-NI is manifested in a specific dose dependent manner, wherein increasing doses of 7-NI correspond to increasing suppression effect on tumor size and tumor progression, with maximum effect at the highest 7-NI dose (Figs 2A-2C). This maximal dose (80 mg / kg) was further effective in providing maximum inhibition of tumor progression even after treatment (Fig. 2D).

[0204] These results essentially supported the notion that a selective inhibition of nNOS by 7-NI can form the basis for effective pharmacological treatment for GBM.

[0205] EXAMPLE 3: 7-NI acts by reducing nitrosative stress and increasing apoptosis

[0206] GBM inoculated mice were treated with 80 mg / kg 7-NI, and after treatment tested for markers of cell proliferation (Ki-67), nitrosative stress (3 -Nitrotyrosine, 3-Ntyr) and apoptosis (cleaved PARP1 and cleaved caspase 3), using microscope images, western blots and quantitative image analyses. The results are shown in Figs 3A-3E.

[0207] Results and Conclusions

[0208] The results show that the inhibitory effects of 7-NI on GBM tumor size and progression were accompanied by significant reduction of Ki-67 (Fig. 3A) and 3-Ntyr (Figs 3B-3C) markers and significant increase of cleaved PARP1 and cleaved caspase 3 markers (Figs 3D-3E) compared to vehicle group, suggesting that 7-NI acts through multiple molecular pathways and complex modulatory effects.

[0209] Overall, the results suggest that on the molecular level, the inhibitory effect of 7-NI on GBM growth is governed by several intracellular mechanisms in which 7-NI exhibits dual activities, specifically pathways governing cell proliferation and intracellular nitrosative stress where it acts an inhibitor and the apoptotic pathway where it acts as an activator. EXAMPLE 4: 7-NI and TMZ inhibit tumor growth synergistically

[0210] GBM inoculated mice were treated with 7-NI (80 mg / kg), TMZ (10 mg / kg) or 7-NI / TMZ combo, using experimental setup as above. The results are shown in Figs 4A-4D.

[0211] Results and Conclusions

[0212] The results show that 7-NI / TMZ combo was more effective than the individual treatments by all parameters (Figs 4A-4C). Computation of survival curves at a predetermined endpoint (>1.5 cm tumor size in either dimension, length / width) showed that the combo had significant contribution to animals’ survival rate compared to individual agents or control (Fig. 4D).

[0213] The main conclusion from these results is that 7-NI and TMZ act synergistically in suppressing GBM growth and progression and increasing animals’ survival rate. Importantly, 7-NI / TMZ combo proved to be more efficient in suppressing GBM than TMZ alone (mainstream GBM treatment), which had led to the notion that it can provide a solution for the problem of TMZ resistance which is characteristic of many GBM.

[0214] The following EXAMPLES 5-7 pertain to the experiments in various GBM cell models in vitro, with the idea to better characterize the inhibitory effect of 7-NI / TMZ combo and individual agents by quantifiable phenotypic parameters of cell invasion, colony formation and cell migration (wound healing), using specifically designed protocols (see below). Studies further included testing of intracellular markers of nitrosative stress (NO production and 3-Ntyr) and apoptosis (cleaved caspase 3), using commercial NO assay and previously described methods and statistical analyses.

[0215] EXAMPLE 5: 7-NI / TMZ combo inhibits tumor behavior in LN-18 GBM model

[0216] Studies used LN- 18 cell line - an established GBM cell model typified as TMZ resistant. Cells were treated with individual agents, 7-NI (200 pM), TMZ (200 pM) or 7- NI / TMZ combo (200 pM each) vs. vehicle (0.04% DMSO) or untreated control, and tested by cell invasion, colony formation and cell migration (wound healing) assays.

[0217] Materials and Methods

[0218] Transwell Matrigel cell invasion assay

[0219] LN-18 cells (2xl04cells) were seeded with 500 pl serum-free DMEM on Matrigel-coated upper portion of transwell inserts in 24-well plates (n =15) and treated with 200pM 7-NI, 200pM TMZ, and the combo (200pM each), 700 pl complete DMEM (10% FBS, chemoattractant) was added to the lower chamber. After 24 h incubation, noninvading cells were removed, and invading cells were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet (1 h). Inserts were washed with PBS and dried overnight at RT. Cells were visualized by light microscope (Nikon Eclipse Ts2) and the number of invaded cells was counted by Image!

[0220] Colony formation (proliferation) assay

[0221] LN- 18 cells (1 x 104) were seeded in complete DMEM in 6-well plates (n=6). After 24 h, cells were treated with 7-NI, TMZ and the combo in the concentrations as above, media were changed every 3rdday. On day 12th, colonies were fixed with 4% paraformaldehyde for 30 min, stained with 0.5% crystal violet (1 h), rinsed with water and air-dried for 24 h. Images were captured, and colonies (> 50 cells) were counted by ImageJ software.

[0222] Cell migration (wound healing) assay

[0223] LN-18 cells (0.3xl06) were seeded in 6-well plates (n=6). The wound was created by scratching confluent cell monolayer (80%) with a pipette tip. Cells were washed with PBS and transferred to fresh serum-free DMEM. Images were taken at time-0 (before treatments) using light microscope (Nikon Eclipse Ts2, lOx magnification), and after treatment with 7-NI, TMZ and the combo at the concentrations as above in serum-free medium for 48 h. Cell migration was visualized by light microscope (Nikon Eclipse Ts2) and quantitated by measuring the wound healing area or the area gap (%) by ImageJ.

[0224] NO assay

[0225] For protein expression analysis, 7.9 x 105cells were seeded in 75 cm2flasks. After treatment, cells were lysed in RIPA buffer with protease and phosphatase inhibitors and sonicated. Lysates were clarified by centrifugation (10,000 g, 15 min, 4 °C), and protein concentrations were quantified using BCA assay. Proteins were resolved on SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against 3-Ntyr and P-actin. Detection was performed using HRP-conjugated secondary antibodies and ECL substrate, with visualization using Bio-Rad ChemiDoc system and quantification Image Lab v6.1.

[0226] Results and Conclusions

[0227] The results in LN- 18 cell model essentially reproduced previous findings of the superior inhibitory effect of 7-NETMZ combo, which was now corroborated by quantification of specific cellular phenotypes related to GBM growth. The results show that 7-NI / TMZ combo was more effective than the individual agents (7-NI=BA101, TMZ) or controls (DMSO or untreated cells) in inhibiting cell invasion (Figs 5A-5B), colony formation (Figs 6A-6B) and cell migration (7A-7B). The inhibitory effect of the combo was further associated with inhibition of intracellular nitrosative stress by NO assay and 3-Ntyr marker (Figs 8A-8C) and activation of apoptosis by cleaved caspase 3 marker (Figs 9A-9B).

[0228] Altogether, these findings, with specific examples of 7-NI / TMZ combo and its applicability in LN-18 cells which represent TMZ resistant GBM, provided proof of concept for the applicability of pharmacological strategy using a combination of modulators that act simultaneously on inhibition of intracellular nitrosative and / or oxidative stress and on activation of apoptosis to suppress GBM and TMZ resistant GBM.

[0229] EXAMPLE 6: Reproducing the effects of 7-NI / TMZ in LN-229 GBM model

[0230] The following protocols pertain to the experiments in LN-229 human glioma cell line, providing quantifiable assessment of treatments (7-NI, TMZ, 7-NI / TMZ combo or DMSO) by cell invasion, colony formation and cell migration assays.

[0231] Materials and Methods

[0232] Transwell Matrigel cell invasion assay

[0233] Transwell inserts were distributed in wells of 24-well plates and pre-wetted with serum-free DMEM. Matrigel coating was prepared by adding serum-free media to the inserts and incubating them for 2 h at 37°C. After incubation, the media was removed. LN-229 cells were trypsinized and neutralized by a complete medium (at least x3 trypsin vol). Cell suspension was centrifuged at 161 RCF for 5 min, pellet was resuspended in 1 mL serum-free DMEM, and cells were counted. 2 * 104cells were seeded onto the Matrigel-coated upper chamber of transwell inserts in 24-well plates. Cells were treated with 7-NI, TMZ, combo (n=3 per condition) and DMSO vehicle or untreated (control), total volume in the inserts was adjusted to 500 pL with serum-free DMEM. 700 pL complete medium (chemoattractant) was added to the lower chamber, ensuring no air bubbles are trapped underneath the insert. The plate was incubated at 37°C for 24 h to allow for cell migration and invasion. After incubation, transwell inserts were transferred to new well containing 600 pL 4% paraformaldehyde and incubating for 30 min at RT. The inserts were then transferred to another well containing 600 pL 0.1% crystal violet stain and incubated for 30 min at RT. After, the inserts were washed in PBS (x3), nonmigrated cells were removed from the insert inner chamber, and the invaded cells (located at the bottom of the membrane) were left to air-dry overnight. Stained cells from five random fields per insert were visualized and captured using a light microscope (Nikon Eclipse Ts2) and the number of invaded cells was counted using ImageJ software.

[0234] Colony formation (proliferation) assay

[0235] LN-229 cells (500 cells) were seeded in DMEM complete medium in 6-well plates (n=6), with even distribution across the well. Following incubation at 37° C for 48 h in CO2 incubator. Cells will be treated with 7-NI (200uM), TMZ (200uM), and the combo, while replenishing the complete media every third day and maintaining the media until day 12. The colonies were then fixed with 4% paraformaldehyde for 30 min and stained with 0.5% crystal violet for 1 h, washed with water and air-dried for 24 h. Images were captured and colonies (>50 cells) counted using ImageJ software.

[0236] Cell migration (wound healing) assay

[0237] LN-229 cells (0.3 x 106) were seeded in 6-well plates (n=6) and allowed to grow until an 80% confluent monolayer was formed. A wound was created by scratching a straight vertical line in each well using a pipette tip. Immediately after, cells were washed with PBS, and images were captured at time-0 using light microscope (Nikon Eclipse Ts2 at 10x magnification). Cells were treated with 7-NI (200uM), TMZ (200uM) or the combo for 24 and 48 h, after which wound healing images were captured at respective time points using light microscope. The area gap of wound healing was measured at time- 0 and 24 h and 48 h using ImageJ and the percentage of cell migration was calculated.

[0238] The results of these studies are shown in Figs 10-12. Additional studies tested the effect of the combo in subcutaneous xenograft GBM model inoculated with LN-229 cells, using 7-NI (80 mg / kg) and TMZ (10 mg / kg) doses and previously described methods and statistical analyses. The results are shown in Figs 13A-13D.

[0239] Results and Conclusions

[0240] The results in LN-229 cell model were more pronounced, showing consistent and significant inhibition of cell invasion (Figs 10A-10B), colony formation (Figs 11A-11B) and cell migration (12A-12B) by 7-NETMZ combo compared to individual agents (7-NI or TMZ) and controls (DMSO or untreated cells). The combo was also effective in the LN-229 xenograft GBM model, showing successful and more effective suppression of tumor size and tumor progression than to individual agents and control (Figs 13A-13B and 13D), with no apparent effects on animals’ body weight (Fig. 13C).

[0241] EXAMPLE 7: Effects of 7-NI / TMZ in TMZ resistant LN-229 GBM model

[0242] The following protocols pertain to the experiments in TMZ resistant LN-229 cell model, which was specifically designed and developed for this purpose by subjecting the cells to repeated rounds of selection and enrichment of TMZ resistant cells (see Fig. 14).

[0243] Materials and Methods

[0244] Generation of TMZ resistant LN-229 subline

[0245] In the first cycle, LN-229 cells were exposed to 200 pM TMZ for 72 h, after which damaged cells were removed by repeated washings. The remaining viable cells were divided - one part was re-cultured in a fresh medium (RG1 cells) and another part was subjected to a second selection cycle by exposure to 200 pM TMZ for 72 h, damaged cells were removed after treatment and surviving cells were re-cultured for additional 72 h (RG2 cells), thus forming TMZ-resistant LN229 subline which was used in vitro and in vivo studies up to passage 15.

[0246] Cells were then treated and tested by cell invasion, colony formation and cell migration assays as in EXAMPLE 6. The results are shown in Figs 15-17.

[0247] Results and Conclusions

[0248] The effect of 7-NI / TMZ combo in the TMZ resistant LN-229 GBM model was the most significant by all tested phenotypic parameters, showing significant suppression of cell invasion (Figs 15A-15B), colony formation (Figs 16A-16B) and cell migration (17A-17B) compared to treatments with individual agents (7-NI or TMZ) and controls (DMSO or untreated cells).

[0249] These last findings provide additional support for the presently proposed inventive concept of use of a combination of inhibitors of intracellular nitrosative and / or oxidative stress and activators of apoptosis for effective suppression TMZ resistant GBM, which so far was deprived from suitable and effective treatment.

[0250] EXAMPLE 8: Projected doses of 7-NI / TMZ combo human subjects

[0251] From the present experiments in mice and cell lines in vitro, it would be reasonable to expect 7-NI / TMZ combo to provide more effective treatment of GBM and TMZ resistant GBM by parameters of clinical severity, tumor invasiveness and time to remission, or measurable attenuation of progression. It would be also reasonable to expect that the use of 7-NI / TMZ combo can allow to reduce the therapeutic doses of TMZ relative to the conventional therapeutic regimens using TMZ alone. This is especially important in view of the known side effects associated with prolonged TMZ therapy, such as GI malfunctions, nausea, dizziness, headache, rashes and hair loss in some cases.

[0252] Bona fide regimens of therapeutic use of 7-NI / TMZ combo in human subjects should be evaluated in future clinical trials. The current projections are based on several known methods for translation of doses demonstrated in mice to humans, such as established allometric scaling methods using body surface area normalization (conversion factor 12.3 for mice) to establish Human Equivalent Dose (HED) (e.g., Reagan-Shaw S et al 2008. Dose translation from animal to human studies revisited. FASEB J 22(3):659- 661), and others (e.g., Anroop BN and Shery J 2016. A simple practice guide for dose conversion between animals and humans. J Basic Clin Pharm 7(2): 27-31).

[0253] Some examples are presently provided for calculation of the 7-NI projected dose and the TMZ projected dose in huma subjects. Regimen A-C relate to the projected 7-NI dose. Regimen D relates to the conventional TMZ for comparison:

[0254] Regimen A. Based on a mouse dosing regimen of 80 mg / kg administered intraperitoneally daily, the human equivalent dose is calculated as 80 mg / kg 12.3 = approximately 6.5 mg / kg / day. For a 70 kg human subject, this corresponds to approximately 455 mg / day, which may be administered orally, intravenously, or by other suitable routes of administration.

[0255] Regimen B. The human equivalent dose of 6.5 mg / kg / day may be administered as a single daily dose or divided into multiple doses throughout the day (e.g., 2.2 mg / kg three times daily, or 3.25 mg / kg twice daily).

[0256] Regimen C Alternative dosing schedules may include 45-50 mg / m2daily based on body surface area calculations, or intermittent dosing such as 13 mg / kg twice weekly or 20 mg / kg once weekly.

[0257] Regimen D The established standard-of-care TMZ regimens for GBM include: Concomitant Phase (Stupp Protocol) - 75 mg / m2orally daily for up to 6-7 weeks concurrent with radiation therapy (Stupp et al., 2005. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma N Engl J Med. 352(10):987-96). Adjuvant / Maintenance Phase - 150-200 mg / m2orally daily for 5 consecutive days every 28 days. The initial cycle typically uses 150 mg / m2(Cycle 1), which may be escalated to 200 mg / m2for subsequent cycles (Cycles 2-6 or 2-12) if well tolerated.

[0258] Alternative Dosing Schedules, such as:

[0259] — Dose-dense regimen - 75 mg / m2daily for 21 days every 28 days,

[0260] — Metronomic dosing - 50 mg / m2daily continuously,

[0261] — Extended adjuvant treatment - 6-12 cycles of maintenance dosing.

Claims

CLAIMS1. A pharmaceutical composition for alleviating, treating or reducing invasiveness of a primary brain tumor, comprising a combination of modulators of which (i) at least one is an inhibitor of intracellular nitrosative and / or oxidative stress and (ii) at least one is an activator of apoptosis.

2. The pharmaceutical composition of claim 1, said primary brain tumor being resistant to chemotherapy.

3. The pharmaceutical composition of claim 1 or 2, said primary brain tumor being resistant to the treatment with Temozolomide (TMZ).

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the primary brain tumor is Glioblastoma (GBM), Anaplastic astrocytoma, Diffuse astrocytoma, Glioblastoma multiforme, Gliosarcoma, Diffuse intrinsic pontine glioma (DIPG), Pleomorphic xanthoastrocytoma (PXA), Medulloblastoma, Ependymoma, Anaplastic ependymoma, Mixed glioma, Oligodendroglioma, Anaplastic oligodendroglioma, Pilocytic astrocytoma, Subependymal giant cell astrocytoma (SEGA), or H3 K27M- mutant diffuse midline glioma.

5. The pharmaceutical composition of any one of claims 1 to 4, wherein the primary brain tumor is Glioblastoma (GBM), IDH-wildtype, Astrocytoma, IDH-mutant Grade 4, or a molecular or histopathological subtype thereof.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein said primary brain tumor is Glioblastoma (GBM) which is resistant to Temozolomide (TMZ).

7. The pharmaceutical composition of any one of the preceding claims, wherein said inhibitor of intracellular nitrosative and / or oxidative stress is an inhibitor of intracellular NO production.

8. The pharmaceutical composition of claim 7, wherein the inhibitor of intracellular NO production is an inhibitor of at least one NO synthetase (NOS).

9. The pharmaceutical composition of claim 8, wherein the at least one NOS is at least one of an inducible synthetase (iNOS), an endothelial synthetase (eNOS) or a neuronal synthetase (nNOS).

10. The pharmaceutical composition of claim 8 or 9, wherein said inhibitor of at least one NOS is N6-(l-iminoethyl)-L-lysine (L-NIL), 7-Nitroindazole (7-NI), a derivative or a combination thereof.

11. The pharmaceutical composition of any one of the preceding claims, wherein said inhibitor of intracellular nitrosative and / or oxidative stress is the nNOS inhibitor 7-NI or a derivative thereof.

12. The pharmaceutical composition of any one of the preceding claims, wherein said activator of apoptosis is the nNOS inhibitor 7-NI or a derivative thereof.

13. The pharmaceutical composition of any one of the preceding claims, wherein said activator of apoptosis is a chemotherapeutic agent and / or a radiosensitizer.

14. The pharmaceutical composition of claim 13, wherein said chemotherapeutic agent and / or radiosensitizer is a DNA alkylating and / or methylating agent.

15. The pharmaceutical composition of claim 13 or 14, wherein said chemotherapeutic agent and / or radiosensitizer is TMZ.

16. The pharmaceutical composition of any one of the preceding claims, comprising 7-NI and TMZ, or a derivative of 7-NI and TMZ, or a combination thereof.

17. The pharmaceutical composition of any one of claims 13 to 16, wherein said chemotherapeutic agent and / or radiosensitizer are provided at an effective therapeutic dose which is lower than in a conventional dosage form of the same chemotherapeutic agent and / or radiosensitizer alone.

18. The pharmaceutical composition of any one of the preceding claims, further comprising a pharmaceutically acceptable carrier, buffer or excipient.

19. The pharmaceutical composition of any one of the preceding claims which is formulated for oral, enteral, parenteral, intraperitoneal, subcutaneous or intramuscular administrations.

20. A method of alleviating, treating or reducing invasiveness of a primary brain tumor in a subject in need thereof, the method comprises administering to the subject a therapeutically effective dose of a combination of modulators of which at least one is an inhibitor of intracellular nitrosative and / or oxidative stress and at least one is an activator of apoptosis, which are formulated in the same or different pharmaceutical compositions administered simultaneously or in succession.

21. The method of claim 20, said primary brain tumor being resistant to chemotherapy.

22. The method of claim 21, said primary brain tumor being resistant to the treatment with Temozolomide (TMZ).

23. The method of any one of claims 20 to 22, wherein the primary brain tumor is Glioblastoma (GBM), Anaplastic astrocytoma, Diffuse astrocytoma, Glioblastoma multiforme, Gliosarcoma, Diffuse intrinsic pontine glioma (DIPG), Pleomorphic xanthoastrocytoma (PXA), Medulloblastoma, Ependymoma, Anaplastic ependymoma, Mixed glioma, Oligodendroglioma, Anaplastic oligodendroglioma, Pilocytic astrocytoma, Subependymal giant cell astrocytoma (SEGA), or H3 K27M-mutant diffuse midline glioma.

24. The method of any one of claims 20 to 23, wherein the primary brain tumor is Glioblastoma (GBM), IDH-wildtype, Astrocytoma, IDH-mutant Grade 4, or a molecular or histopathological subtype thereof.

25. The method of any one of claims 20 to 24, wherein said primary brain tumor is Glioblastoma (GBM) which is resistant to Temozolomide (TMZ).

26. The method of any one of claims 20 to 25, wherein said inhibitor of intracellular nitrosative and / or oxidative stress is an inhibitor of intracellular NO production.

27. The method of claim 26, wherein the inhibitor of intracellular NO production is an inhibitor of at least one NO synthetase (NOS).

28. The method of claim 27, wherein the at least one NOS is at least one of an inducible synthetase (iNOS), an endothelial synthetase (eNOS) or a neuronal synthetase (nNOS).

29. The method of claim 27 or 28, wherein said inhibitor of at least one NOS is N6- (l-iminoethyl)-L-lysine (L-NIL), 7-Nitroindazole (7-NI), a derivative or a combination thereof.

30. The method of any one of claims 20 to 29, wherein said inhibitor of intracellular nitrosative and / or oxidative stress is the nNOS inhibitor 7-NI, or a derivative thereof.

31. The method of any one of claims 20 to 30, wherein said activator of apoptosis is the nNOS inhibitor 7-NI, or a derivative thereof.

32. The method of any one of claims 20 to 31, is wherein said activator of apoptosis is a chemotherapeutic agent and / or a radiosensitizer.

33. The method of claim 32, wherein said chemotherapeutic agent and / or radiosensitizer is a DNA alkylating and / or methylating agent.

34. The method of claim 32 or 33, wherein said chemotherapeutic agent and / or radiosensitizer is TMZ.

35. The method of any one of claims 20 to 34, wherein said inhibitor of intracellular nitrosative and / or oxidative stress and said activator of apoptosis are 7-NI and TMZ, or a derivative of 7-NI and TMZ, or a combination thereof.

36. The method of any one of claims 20 to 35, wherein the at least one chemotherapeutic agent and / or radiosensitizer is administered at a therapeutically effective dose which is lower than in a conventional dosage form of the same chemotherapeutic agent and / or radiosensitizer alone.

37. The method of any one of claims 20 to 36, wherein said inhibitor of intracellular nitrosative and / or oxidative stress and said activator of apoptosis are administered via at least one of oral, enteral, parenteral, intraperitoneal, subcutaneous or intramuscular administration routes.

38. The method of any one of claims 20 to 37, further comprising subjecting the subject to a radiotherapy and / or a surgical procedure before or after the administration of said combination of modulators.

39. A method of alleviating, treating or reducing invasiveness of TMZ resistant GBM in a subject in need thereof, comprising administering to the subject a therapeutically effective dose of 7-NI or a derivative thereof and a therapeutically effective dose of TMZ, which are formulated in the same or different pharmaceutical compositions administered simultaneously or in succession, and wherein the therapeutically effective dose of TMZ is lower than in the respective conventional dosage form of TMZ alone.

40. A pharmaceutical composition for alleviating, treating or reducing invasiveness of TMZ resistant GBM, comprising an effective dose of 7-NI or a derivative thereof and an effective dose of TMZ, said effective dose of TMZ being lower than in the respective conventional dosage form of TMZ alone.

41. Use of a combination of modulators of which at least one is an inhibitor of intracellular nitrosative and / or oxidative stress and at least one is an activator of apoptosis in the manufacture of a medicament for alleviating, treating or reducing invasiveness of a primary brain tumor, said primary brain tumor being resistant to chemotherapy.

42. The use according to claim 41, said inhibitor and activator are defined as in any one of claims 7 to 19.

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