Patent Description:
Cancer cells face multiple cellular stresses such as hypoxia, increased metabolic demand, genomic instability, immune surveillance, lack of nutriments, changing environment after metastasis and stresses resulting to treatments such as radiotherapy, chemotherapies and targeted therapies.

NADPH oxidases (NOX) are a family of enzymes harbouring <NUM> trans-membrane domain and that transfer electrons across biological membranes. Those enzymes are dedicated reactive oxygen species-generating enzymes that broadly and specifically regulate redox-sensitive signaling pathways that are involved in cancer development and progression and act at specific cellular membranes and microdomains through the activation of oncogenes and the inactivation of tumour suppressor proteins. NOX enzymes are considered to be an essential part of adaptive stress response, in particular for cancer cells, thereby allowing those cells to adapt and survive (<NPL>).

Marked induction of NOX expression has been reported in cancer cells and in host cells within the tumor environment.

The interplay between tumor microenvironment and cancer cells is recognized to have a major role for tumor growth and metastasis. Cancer-associated-fibroblasts (CAFs) are the most abundant cells found in the tumour stroma. CAFs, and their fibroblast-to-myofibroblast transdifferentiation lead to tumor growth and generally correlate with poor prognosis in multiple cancer types. While CAF promote "many of the hallmarks of malignancy", recent studies have highlighted a role in promoting tumor immune evasion with CAF-rich cancers which are designated as being "immune cold" for their poor therapeutic response to cancer immunotherapies such as immune checkpoint inhibitors and cancer vaccines and their propensity to evolve to metastasis.

Furthermore, high CAF content induces a dense stroma and dense tumor microenvironment which increases interstitial fluid pressure and thereby acts as a barrier to drug delivery, leading to poor accumulation of chemotherapies in tumours.

In particular, melanoma is known as an exceptionally aggressive and treatment-resistant human cancer. Although progresses have been made in the past decade, including the development of immunotherapy using immune checkpoint inhibitors, treatment for unresectable stage III, stage IV, and recurrent melanoma is still challenging with limited response rate, severe side effects and poor prognosis. Melanoma is not only driven by malignant melanocytes, but also by the altered communication between neoplastic cells and non-malignant cell populations, including fibroblasts, endothelial and inflammatory cells, in the tumor stroma. CAFs remodel the extracellular matrix (ECM) and architecture of the diseased tissue and secrete chemical factors, which all together promote the transformation process by encouraging tumor growth, angiogenesis, inflammation and metastasis and contribute to drug resistance. If it has been recently shown that NOX4 regulates myofibroblastic CAF differentiation in multiple cancers (<NPL>), the origin of CAFs and precise mechanisms by which CAFs contribute to cancer progression and drug resistance still remain poorly understood. Further, Hanley et al. , <NUM> did not point towards any specific anti-cancer immunotherapeutic agent as adjunct treatment with NOX4 inhibition.

Immunotherapy continues to gain interest as an effective therapeutic strategy across several cancer types such as melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, renal cell cancer, bladder cancer, ovarian cancer, uterine endometrial cancer, uterine cervical cancer, uterine sarcoma, gastric cancer, esophageal cancer, colon cancer, hepatocellular carcinoma, breast cancer, Merkel cell carcinoma, thyroid cancer, Hodgkin lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, mycosisfungoides, peripheral T-cell lymphoma, and include various approaches, ranging from stimulating effector mechanisms to counteracting inhibitory and suppressive mechanisms. Strategies to activate effector immune cells include vaccination with tumor antigens or augmentation of antigen presentations to increase the ability of the patient's own immune system to increase the efficacy of the immune response against neoplastic cells (<NPL>). Additional stimulatory strategies encompass adoptive cellular therapy (ACT), the administration of oncolytic viruses (OVs) for the initiation of systemic antitumor immunity, and the use of antibodies targeting members of the tumor necrosis factor receptor superfamily to enhance T cell activity. Strategies to neutralize immunosuppressor mechanisms include chemotherapy (cyclophosphamide), antibodies to diminish regulatory T cells (CD25-targeted antibodies), and antibodies against immune-checkpoint molecules such as CTLA-<NUM>, PD1 and PD-L1.

The field of cancer immunotherapy has been recently encouraged primarily by the approval of the autologous cellular immunotherapy, sipuleucel-T for the treatment of prostate cancer in <NUM> (<NPL>) and the approval of the anti-cytotoxic T lymphocyte-associated protein <NUM> (CTLA-<NUM>) antibody, ipilimumab, and of anti-programmed cell death protein <NUM> (PD1) antibodies for the treatment of melanoma in <NUM> and <NUM> (<NPL>).

Successful anti-cancer effect has been demonstrated through the use of immune checkpoint blockade targeting cytotoxic T-lymphocyte associated protein <NUM> (CTLA-<NUM>) and programmed-death <NUM> (PD-<NUM>)/PD-<NUM> ligand (PD-L1), with the highest objective response rates observed in cancer types with a high mutational burden such as melanoma and non-small cell lung cancer (<NPL>). However significant limitations exist with these therapeutic agents with objective responses to PD-<NUM> blockade observed in only <NUM>-<NUM>% of patients and the majority of patients demonstrating innate resistance. Acquired resistance to anti-PD-<NUM> therapy is also a problem, with approximately one quarter of responders later demonstrating disease progression (<NPL>).

Further, resistance of solid tumors to anti-cancer treatment has also been observed to antiangiogenic therapies and has become a high concern for the use of anti-VEGF therapies (<NPL>, anti-PDGF agents) since despite their encouraging beneficial effects, patients inevitably develop resistance and frequently fail to demonstrate significantly better overall survival.

Therefore, in view of the recent developments of various strategies in cancer immunotherapy such as cancer vaccines, adoptive cellular immunotherapy, immune checkpoint blockade, and oncolytic viruses and antiangiogenic therapies but also the encountered limitations to their efficacy, there is a growing need of developing efficient anti-cancer therapies for solid tumor cancers, in particular for cancers prone for developing a resistance to immunotherapy or antiangiogenic therapies, which would allow restoring sensitivity to immunotherapy or antiangiogenic treatments or potentiate cancer vaccine treatments.

The present invention is directed towards <NUM>-(<NUM>-chlorophenyl)-<NUM>-[<NUM>-(dimethylamino)phenyl]-<NUM>-methyl-<NUM>-pyrazolo[<NUM>,<NUM>-c]pyridine-<NUM>,<NUM>(<NUM>,<NUM>)-dione, and the unexpected findings that the recently found ability of pharmacological inhibition of NOX4 to revert the myofibroblastic-CAF phenotype in different cancer cells lines and suppresses tumor growth in multiple CAF-rich tumor models (TC1+CAF [HNSCC model], 4T1+CAF [breast cancer], MMTV-PyVT (breast cancer), MMTV-Her2/neu (breast cancer) both in vitro and/or in vivo (<NPL>) is useful for synergistically potentiating cancer immunotherapy or reversing anti-VEGF treatment elicited resistance.

The present invention is directed towards the unexpected findings that <NUM>-(<NUM>-chlorophenyl)-<NUM>-[<NUM>-(dimethylamino)phenyl]-<NUM>-methyl-<NUM>-pyrazolo[<NUM>,<NUM>-c]pyridine-<NUM>,<NUM>(<NUM>,<NUM>)-dione is able to restore sensitivity to immunotherapy and/or improve response to immunotherapy.

The present invention is directed to compositions useful for the restoration of responsiveness to immunotherapy, in particular for the restoration of responsiveness to cancer vaccines such as HPV and immune checkpoint blockade such as with PD-<NUM> inhibitors, PD-L1 inhibitors, and CTLA-<NUM> inhibitors.

In particular, the present invention is directed towards the unexpected findings that <NUM>-(<NUM>-chlorophenyl)-<NUM>-[<NUM>-(dimethylamino)phenyl]-<NUM>-methyl-<NUM>-pyrazolo[<NUM>,<NUM>-c]pyridine-<NUM>,<NUM>(<NUM>,<NUM>)-dione is able to restore sensitivity to anti-tumour immunotherapy and/or improve response to immunotherapy.

A first aspect of the invention provides <NUM>-(<NUM>-chlorophenyl)-<NUM>-[<NUM>-(dimethylamino)phenyl]-<NUM>-methyl-<NUM>-pyrazolo[<NUM>,<NUM>-c]pyridine-<NUM>,<NUM>(<NUM>,<NUM>)-dione for use in the treatment of solid tumor cancers presenting or susceptible to present a resistance to immunotherapy, wherein <NUM>-(<NUM>-chlorophenyl)-<NUM>-[<NUM>-(dimethylamino)phenyl]-<NUM>-methyl-<NUM>-pyrazolo[<NUM>,<NUM>-c]pyridine-<NUM>,<NUM>(<NUM>,<NUM>)-dione is to be administered in combination with an anti-cancer immunotherapeutic agent selected from at least one cancer vaccine or at least one immune checkpoint inhibitor. Another aspect of the invention relates to a pharmaceutical composition containing <NUM>-(<NUM>-chlorophenyl)-<NUM>-[<NUM>-(dimethylamino)phenyl]-<NUM>-methyl-<NUM>-pyrazolo[<NUM>,<NUM>-c]pyridine-<NUM>,<NUM>(<NUM>,<NUM>)-dione, as well as tautomers, geometrical isomers, optically active forms and pharmaceutically acceptable salts thereof combined with at least one anti-cancer immunotherapeutic agent and at least one pharmaceutically acceptable carrier, wherein said at least one anti-cancer immunotherapeutic agent is selected from at least one cancer vaccine or at least one immune checkpoint inhibitor.

Other features and advantages of the invention will be apparent from the following detailed description.

The expression "NOX inhibitor" as used herein refers to any substances that are able to totally or partially inhibit, block, attenuate, or interfere with NOX4 and/or NOX1. The term directly is defined as that the compound affects the enzymatic activity of the enzyme, the cellular localization, the stability of the protein, the expression of the messenger RNA or the protein. Preferably, a NOX4/NOX1 inhibitor should be able to diminish enzyme activity and ROS production in a cell free assay using membrane expressing only the NOX isoform NOX4/<NUM> protein, such as recombinant protein NOX4/<NUM>. Thus, the term "inhibitors" is intended to include but is not limited to, molecules, which inhibit completely or partially the activity of NADPH oxidase <NUM> and/or NADPH oxidase <NUM>. According to a particular embodiment, NOX4/<NUM> inhibitors have a major NOX inhibitory activity component towards NOX4 and/or NOX1 compared to other NOX proteins, for example to NOX2 and/or NOX3/<NUM>. According to a particular embodiment, NOX4/<NUM> inhibitors have a major NOX inhibitory activity on NOX4/<NUM> about at least five times higher than on other NOX proteins.

For example, NOX4/<NUM> inhibitors include small molecules, peptides, peptidomimetics, chimeric proteins, natural or unnatural proteins, nucleic acid derived polymers (such as DNA and RNA aptamers, siRNAs, shRNAs, PNAs, or LNAs), fusion proteins with NOX4/<NUM> antagonizing activities, antibody antagonists such as neutralizing anti-NOX4/<NUM> antibodies, or gene therapy vectors driving the expression of such NOX4/<NUM> antagonists.

In particular, NOX4/<NUM> inhibitors are agents that present an inhibitory constant Ki of less than <NUM> micromolar in a functional ROS production assay such as those described in <NPL>. For example, NOX4/<NUM> inhibitors are agents that inhibit ROS production in a range of about less than <NUM> microM, such as between about <NUM> to <NUM> nanomolar in a cell free assay using membrane expressing only the NOX isoform NOX4 or NOX1 protein, such as recombinant protein NOX4 or NOX1.

The term "siRNA" refers to small interfering RNA, which are double stranded RNA (about <NUM>-<NUM> nucleotides) able to knock down or silence a targeted mRNA from a target gene. Artificial siRNAs can be either chemically synthesized as oligonucleotides or cloned into a plasmid or a virus vector (adenovirus, retrovirus or lentivirus) as short hairpin RNAs to generate a transient or stable transfection in any type of cells (<NPL>; <NPL>).

The expression "solid tumour cancer" includes, glioblastoma, lung cancer (small cell and non-small cell), breast cancer, ovarian cancer, cervical cancer, uterus cancer, head and neck cancer, melanoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal carcinoma, kidney cancer, prostate cancer, gastric cancer, bronchus cancer, pancreatic cancer, urinary bladder cancer, hepatic cancer and brain cancer, in particular glioblastoma.

As used herein, "treatment" and "treating" and the like generally mean obtaining a desired pharmacological and physiological effect. The term "treatment" as used herein covers any treatment of a disease in a mammal, particularly a human, and includes inhibiting the disease, i.e., arresting its development; or relieving the disease, i.e. causing regression of the disease and/or its symptoms or conditions such as tumor growth arrest or tumor regression.

The term "subject" as used herein refers to mammals. For examples, mammals contemplated by the present invention include human, primates, domesticated animals such as cattle, sheep, pigs, horses, laboratory rodents, dogs and the like.

The term "effective amount" as used herein refers to an amount of at least one particle or a pharmaceutical formulation thereof according to the invention that elicits the biological or medicinal response in a tissue, system, animal, or human that is being sought. In one embodiment, the effective amount is a "therapeutically effective amount" for the alleviation of the symptoms of the disease or condition being treated. Typically, an effective amount can be used to inhibit the growth of cancer cells, i.e. any slowing of the rate of cancer cell proliferation and/or migration, arrest of cancer cell proliferation and/or migration, or killing of cancer cells, such that the rate of cancer cell growth is reduced in comparison with the observed or predicted rate of growth of an untreated control cancer cell. The term "inhibits growth" can also refer to a reduction in size or disappearance of a cancer cell or tumor, as well as to a reduction in its metastatic potential. Preferably, such an inhibition at the cellular level may reduce the size, defer the growth, reduce the aggressiveness, or prevent or inhibit metastasis of a cancer in a patient. Those skilled in the art can readily determine, by any of a variety of suitable indicia, whether cancer cell growth is inhibited.

The term "efficacy" of a treatment according to the invention can be measured based on changes in the course of a disease in response to a use or a method according to the invention. The efficacy of a treatment of a cancer according to the invention can be measured by a reduction of tumour volume, and/or an increase of progression free survival time and/or increased health and well-being of the subject (e.g. repressing a cancer). Inhibition of cancer cell growth may be evidenced, for example, by arrest of cancer cells in a particular phase of the cell cycle, e.g., arrest at the G2/M phase of the cell cycle. Inhibition of cancer cell growth can also be evidenced using well known imaging methods such as magnetic resonance imaging, computerized axial tomography, PET, SPECT, photo-acoustic imaging, X-rays and fluorescence imaging/detection. Cancer cell growth can also be determined indirectly, for example by determining the levels of circulating carcino-embryonic antigen, prostate specific antigen or other cancer- specific antigens that are correlated with cancer cell growth.

In particular, efficacy of a combined treatment according to the invention can be assessed by reduction of tumour size, or disappearance of tumour or of any biomarker relevant for a cancer type.

Unless otherwise constrained by the definition of the individual substituent, the term "substituted" refers to groups substituted with from <NUM> to <NUM> substituents selected from the group consisting of "C<NUM>-C<NUM> alkyl," "C<NUM>-C<NUM> alkenyl," "C<NUM>-C<NUM> alkynyl," "C<NUM>-C<NUM>-cycloalkyl," "heterocycloalkyl," "C<NUM>-C<NUM> alkyl aryl," "C<NUM>-C<NUM> alkyl heteroaryl," "C<NUM>-C<NUM> alkyl cycloalkyl," "C<NUM>-C<NUM> alkyl heterocycloalkyl," "amino," "alkyl amino," "aminosulfonyl," "ammonium," "alkoxy," "acyl", "acyl amino," "amino carbonyl," "aryl," "heteroaryl," "sulfinyl," "sulfonyl," "sulphonamide", "alkoxy," "alkoxy carbonyl," "carbamate," "sulfanyl," "halogen," trihalomethyl, cyano, hydroxy, mercapto, nitro, and the like.

The term "pharmaceutically acceptable salts or complexes" refers to salts or complexes of the below-specified compounds of the invention. Examples of such salts include, but are not restricted, to base addition salts formed by reaction of compounds of the invention with organic or inorganic bases such as hydroxide, carbonate, bicarbonate or the like, of a metal cation such as those selected in the group consisting of alkali metals (sodium, potassium or lithium), alkaline earth metals (e.g. calcium or magnesium), or with an organic primary, secondary or tertiary alkyl amine. Other examples of such salts include, but are not restricted, to acid addition salts formed by reaction of compounds of the invention with organic or inorganic acids such as hydrochloric acid, hydrobromic acid, sulphuric acid, para-toluene sulfonic acid, <NUM>-naphtalene sulfonic acid, camphosulfonic acid, benzene sulfonic acid, oxalic acid or the like.

"Pharmaceutically active derivative" refers to any compound that upon administration to the recipient is capable of providing directly or indirectly, the activity disclosed herein.

The compound of Formula (I) for use according to the invention is
<CHM>
<NUM>-(<NUM>-chlorophenyl)-<NUM>-[<NUM>-(dimethylamino)phenyl]-<NUM>-methyl-<NUM>-pyrazolo[<NUM>,<NUM>-c]pyridine-<NUM>,<NUM>(<NUM>,<NUM>)-dione.

According to a particular aspect is provided <NUM>-(<NUM>-chlorophenyl)-<NUM>-[<NUM>-(dimethylamino)phenyl]-<NUM>-methyl-<NUM>-pyrazolo[<NUM>,<NUM>-c]pyridine-<NUM>,<NUM>(<NUM>,<NUM>)-dione for use in combination with a cancer vaccine or with at least one immune checkpoint inhibitor.

According to a further particular aspect is provided <NUM>-(<NUM>-chlorophenyl)-<NUM>-[<NUM>-(dimethylamino)phenyl]-<NUM>-methyl-<NUM>-pyrazolo[<NUM>,<NUM>-c]pyridine-<NUM>,<NUM>(<NUM>,<NUM>)-dione for use in combination with at least one immune checkpoint inhibitor.

According to another further particular aspect is provided <NUM>-(<NUM>-chlorophenyl)-<NUM>-[<NUM>-(dimethylamino)phenyl]-<NUM>-methyl-<NUM>-pyrazolo[<NUM>,<NUM>-c]pyridine-<NUM>,<NUM>(<NUM>,<NUM>)-dione for use in combination with a cancer vaccine.

An anti-cancer immunotherapeutic agent that can be used according to the invention encompass cancer vaccines such as oncolytic or anti-Herpes simplex virus vaccines such as described in <NPL> (e.g. talimogene laherparepvec (Imlygic)) or in <NPL>, adoptive cellular immunotherapy such as described in <NPL>, immune checkpoint inhibitors such as PD-<NUM> inhibitors like those described in <NPL> or <NPL> or <NPL> (e.g. such as Pembrolizumab (Keytruda), Nivolumab (Opdivo)), or PD-L1 inhibitors like Atezolizumab (Tecentriq), Avelumab (Bavencio), Durvalumab (Imfinzi) or CTLA-<NUM> inhibitors such as Ipilimumab (Yervoy).

According to another particular aspect, an immune checkpoint inhibitor according to the invention may be selected from T cell immunoglobulin and mucin domain <NUM> (TIM3), Lymphocyte activation gene-<NUM> (LAG3), T-cell immunoglobulin and ITIM domains (TIGIT) or B- and T-lymphocyte attenuator (BTLA) inhibitors.

According to a particular aspect, an immune checkpoint inhibitor according to the invention is a PD-<NUM> inhibitor.

According to a particular aspect, an anti-cancer vaccine according to the invention encompasses DNA, RNA, peptide and oncolytic virus vaccines.

Further, more generally, since infiltration of CD8+ T-cells into tumours is fundamental to most immunotherapies, combinations and combined uses according to the invention would also be useful in adoptive T-cell transfer therapies, including tumour infiltrating lymphocytes (TILs), T cell receptor (TCR) T-cells and chimeric antigen receptor (CAR)-T-cells such as described in <NPL>. TILs have been shown to induce durable, complete responses in patients with metastatic melanoma. CAR T-cells have produced significant benefit in the treatment of haematological malignancies (<NPL>; <NPL>; <NPL>; <NPL>), however, the tumour microenvironment remains a significant barrier to success in solid cancers.

Similarly, immunotherapeutic agent that can be used according to the invention encompass CD8+ T-cell agonists, such as α-CD40, α-CD27, α-41BB, α-OX40, GITR.

The invention provides pharmaceutical or therapeutic agents as compositions and uses in methods for treating a patient, preferably a mammalian patient, and most preferably a human patient who is suffering from a solid tumor cancer presenting or susceptible to present a resistance to immunotherapy.

Pharmaceutical compositions of the invention can contain one or more compound in any form described herein. Compositions of this invention may further comprise one or more pharmaceutically acceptable additional ingredient(s), such as alum, solubilizers, stabilizers, antimicrobial agents, buffers, coloring agents, flavoring agents, adjuvants, and the like.

The compounds of the invention, together with a conventionally employed adjuvant, carrier, diluent or excipient may be placed into the form of pharmaceutical compositions and unit dosages thereof, and in such form may be employed as solids, such as powder in sachets, tablets or filled capsules, or liquids such as solutions, suspensions, emulsions, elixirs, nasal spray, or capsules filled with the same, all for oral use, or in the form of sterile injectable solutions for parenteral (including subcutaneous) use. Such pharmaceutical compositions and unit dosage forms thereof may comprise ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed. Compositions according to the invention are preferably oral, sublingual, nasal and subcutaneous.

Compositions of this invention may also be liquid formulations, including, but not limited to, aqueous or oily suspensions, solutions, emulsions, syrups, spray and elixirs. Liquid forms suitable for oral administration may include a suitable aqueous or non-aqueous vehicle with buffers, suspending and dispensing agents, colorants, flavors and the like. The compositions may also be formulated as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain additives, including, but not limited to, suspending agents, emulsifying agents, non-aqueous vehicles and preservatives. Suspending agents include, but are not limited to, sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminum stearate gel, and hydrogenated edible fats. Emulsifying agents include, but are not limited to, lecithin, sorbitan monooleate, and acacia. Non aqueous vehicles include, but are not limited to, edible oils, almond oil, fractionated coconut oil, oily esters, propylene glycol, and ethyl alcohol. Preservatives include, but are not limited to, methyl or propyl p-hydroxybenzoate and sorbic acid. Further materials as well as processing techniques and the like are set out in<NPL>.

Solid compositions of this invention may be in the form of powder in sachets, tablets or lozenges formulated in a conventional manner. For example, sachets, tablets and capsules for oral or sublingual administration may contain conventional excipients including, but not limited to, binding agents, fillers, lubricants, disintegrants and wetting agents. Binding agents include, but are not limited to, syrup, accacia, gelatin, sorbitol, tragacanth, mucilage of starch and polyvinylpyrrolidone. Fillers include, but are not limited to, lactose, sugar, microcrystalline cellulose, maizestarch, calcium phosphate, and sorbitol. Lubricants include, but are not limited to, magnesium stearate, stearic acid, talc, polyethylene glycol, and silica. Disintegrants include, but are not limited to, potato starch and sodium starch glycollate. Wetting agents include, but are not limited to, sodium lauryl sulfate. Tablets may be coated according to methods well known in the art.

Injectable compositions are typically based upon injectable sterile saline or phosphate-buffered saline or other injectable carriers known in the art.

Compositions of this invention may also be formulated for parenteral administration, including, but not limited to, by injection or continuous infusion. Formulations for injection may be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents including, but not limited to, suspending, stabilizing, and dispersing agents. The composition may also be provided in a powder form for reconstitution with a suitable vehicle including, but not limited to, sterile, pyrogen-free water.

Compositions of this invention may also be formulated as a depot preparation, which may be administered by implantation or by intramuscular injection. The compositions may be formulated with suitable polymeric or hydrophobic materials (as an emulsion in an acceptable oil, for example), ion exchange resins, or as sparingly soluble derivatives (as a sparingly soluble salt, for example).

The compounds of this invention can also be administered in sustained release forms or from sustained release drug delivery systems. A description of representative sustained release materials can also be found in Remington's Pharmaceutical Sciences.

Compositions of this invention may be administered in any manner, including, but not limited to, orally, parenterally, sublingually, via buccal administration, nasally, intralesionally or combinations thereof. Parenteral administration includes, but is not limited to subcutaneous and intramuscular. The compositions of this invention may also be administered in the form of an implant, which allows slow release of the compositions as well as a slow controlled i. In a particular embodiment, one or more NOX4, NOX4/<NUM> or NOX1 inhibitor is administered orally.

The dosage administered, as single or multiple doses, to an individual will vary depending upon a variety of factors, including pharmacokinetic properties, patient conditions and characteristics (age, body weight, health, body size), extent of symptoms, frequency of treatment and the effect desired.

According to one embodiment of the invention, <NUM>-(<NUM>-chlorophenyl)-<NUM>-[<NUM>-(dimethylamino)phenyl]-<NUM>-methyl-<NUM>-pyrazolo[<NUM>,<NUM>-c]pyridine-<NUM>,<NUM>(<NUM>,<NUM>)-dione and pharmaceutical formulations thereof is to be administered in combination with an anti-cancer immunotherapeutic agent selected from an anti-cancer vaccine or at least one immune check point inhibitor such as at least one PD-<NUM>, PD-L1 or CTLA4 inhibitor.

The invention encompasses the administration of <NUM>-(<NUM>-chlorophenyl)-<NUM>-[<NUM>-(dimethylamino)phenyl]-<NUM>-methyl-<NUM>-pyrazolo[<NUM>,<NUM>-c]pyridine-<NUM>,<NUM>(<NUM>,<NUM>)-dione or a pharmaceutical formulation thereof, wherein the inhibitor or a pharmaceutical formulation thereof is administered to an individual prior to, or simultaneously with an anti-cancer immunotherapeutic agent, for example concomitantly through the same formulation or separately through different formulations, in particular through different formulation routes. According to a particular aspect of the invention, <NUM>-(<NUM>-chlorophenyl)-<NUM>-[<NUM>-(dimethylamino)phenyl]-<NUM>-methyl-<NUM>-pyrazolo[<NUM>,<NUM>-c]pyridine-<NUM>,<NUM>(<NUM>,<NUM>)-dione and pharmaceutical formulations thereof is to be administered chronically (e.g. daily or weekly) for the duration of treatment and prior to the administration of an anti-cancer immunotherapeutic agent.

According to another particular aspect of the invention, <NUM>-(<NUM>-chlorophenyl)-<NUM>-[<NUM>-(dimethylamino)phenyl]-<NUM>-methyl-<NUM>-pyrazolo[<NUM>,<NUM>-c]pyridine-<NUM>,<NUM>(<NUM>,<NUM>)-dione and pharmaceutical formulations thereof is to be administered concomitantly with an anti-cancer immunotherapeutic agent.

According to another particular aspect of the invention, the anti-cancer immunotherapeutic agent can be administered in combination with other therapeutic regimens or co-agents useful in the treatment of cancer (e.g. multiple drug regimens), in a therapeutically effective amount, such as in combination with substances useful for treating, stabilizing, preventing, and/or delaying cancer such as substances used in conventional chemotherapy directed against solid tumors and for control of establishment of metastases or any other molecule that act by triggering programmed cell death e.g. for example a co-agent selected from angiogenesis inhibitors (e.g. anti-VEGF agents such as described in Gardner et al. , <NUM>, supra), immunotherapy agents (e.g. recombinant cytokines, interferones, interleukin, recombinant antibodies such as herceptin®) and chemotherapeutic agents (e.g. cisplatin, paclitaxel, methotrexate, <NUM>-fluoruracil, Gemcitabin, Vincristin, Vinblastin, Doxorubicin, Temozolomide). In particular, According to another particular aspect of the invention, the anti-cancer immunotherapeutic agent can be administered in combination with other therapeutic regimens or co-agents useful in the treatment of cancer (e.g. multiple drug regimens), in a therapeutically effective amount, such as in combination with at least one inhibitor of vascular endothelial growth factor (VEGF) (e.g. bevacizumab, sunitinib inhibitors), at least one inhibitor of basic fibroblast growth factor (bFGF) or at least one inhibitor of hypoxia-inducible factor-<NUM> (HIF-<NUM>).

<NUM>-(<NUM>-chlorophenyl)-<NUM>-[<NUM>-(dimethylamino)phenyl]-<NUM>-methyl-<NUM>-pyrazolo[<NUM>,<NUM>-c]pyridine-<NUM>,<NUM>(<NUM>,<NUM>)-dione or the pharmaceutical formulations thereof that are administered simultaneously with said anti-cancer immunotherapeutic agent can be administered in or within the same or different composition(s) and by the same or different route(s) of administration.

In one embodiment, subjects according to the invention are subjects suffering from a solid tumor cancer, in particular a poorly responsive solid tumor cancer presenting or susceptible to present a resistance to immunotherapy.

In a particular embodiment, subjects according to the invention are subjects suffering from a solid tumor cancer selected from lung cancer (small cell and non-small cell), breast cancer, ovarian cancer, cervical cancer, uterus cancer, head and neck cancer, melanoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal carcinoma, kidney cancer, prostate cancer, gastric cancer, bronchus cancer, pancreatic cancer, urinary bladder cancer, hepatic cancer and brain cancer, in particular glioblastoma.

In a particular embodiment, subjects according to the invention are subjects suffering from a solid tumor cancer and have high α-smooth muscle actin (α-SMA) expression.

In another particular embodiment, subjects according to the invention are subjects suffering from hepatocellular carcinoma (HCC).

In another particular embodiment, subjects according to the invention are subjects suffering from head and neck tumors.

In another particular embodiment, subjects according to the invention are subjects suffering from melanoma.

In another particular embodiment, subjects according to the invention are subjects suffering from colon cancer.

In another particular embodiment, subjects according to the invention are subjects suffering from lung carcinoma.

In another particular embodiment, subjects according to the invention are subjects suffering from breast cancer.

In another particular embodiment, subjects according to the invention are subjects suffering from hepatocellular carcinoma or hepatic cancer.

In another particular embodiment, subjects according to the invention are subjects suffering from rectal cancer or colorectal carcinoma.

In another particular embodiment, subjects according to the invention are subjects suffering from kidney cancer.

In another particular embodiment, subjects according to the invention are subjects suffering from pancreatic cancer.

In another particular embodiment, subjects according to the invention are subjects suffering from brain cancer, in particular glioblastoma.

In another particular embodiment, subjects according to the invention are subjects with solid tumor cancer who are at risk of developing resistance or partial resistance to anti-cancer immunotherapy due to another concomitant treatment or a genetic pre-disposition.

The invention provides compounds, uses and compositions useful for the treatment of a solid tumor cancer in the form of a combination wherein <NUM>-(<NUM>-chlorophenyl)-<NUM>-[<NUM>-(dimethylamino)phenyl]-<NUM>-methyl-<NUM>-pyrazolo[<NUM>,<NUM>-c]pyridine-<NUM>,<NUM>(<NUM>,<NUM>)-dione is to be administered in combination with at least one anti-cancer immunotherapeutic agent.

The invention having been described, the following examples are presented by way of illustration, and not limitation.

The efficacy of NOX4/<NUM> inhibitors for restoring or increasing responsiveness to an anti-cancer immunotherapeutic agent can be tested as follows:.

In order to test the efficacy of a combination according to the invention, the following experiments are conducted in a mouse xenograft tumour models as described below.

Subcutaneous xenograft tumours composed of C38 cells (colon cancer), CT26 cells (colon cancer), LLC1 cells (lung carcinoma), B16F10 cells (melanoma), Hepa1-<NUM> cells (liver cancer) or Renca cells (renal cancer) are injected subcutaneously into the flank of C57Bl/<NUM> or Balb/c mice (<NUM>-<NUM> months old). Alternatively, MC-<NUM> Cell Line derived from C57BL6 murine colon adenocarcinoma cells or Mouse 4T1 breast tumor model are used.

The combined treatment starts when the tumours reach a mean volume of <NUM>-<NUM><NUM>. Mice are randomized according to their individual tumour volume into different groups of <NUM> to <NUM> mice. Each group receives either placebo, or a NOX4/<NUM> inhibitor alone, or a PD-<NUM> antibody alone or NOX4/<NUM> in combination with PD-<NUM> antibody.

The NOX4/<NUM> inhibitors <NUM>-(<NUM>-chlorophenyl)-<NUM>-[<NUM>-(dimethylamino)phenyl]-<NUM>-methyl-<NUM>-pyrazolo [<NUM>,<NUM>-c]pyridine-<NUM>,<NUM>(<NUM>,<NUM>)-dione or (R)-<NUM>-methoxy-<NUM>-(<NUM>-morpholino-<NUM>-phenylethoxy)-N-(<NUM>-(pyridin-<NUM>-yl)-<NUM>,<NUM>,<NUM>-thiadiazol-<NUM>-yl) benzamide are prepared daily (<NUM> days/week) in <NUM>% Methyl cellulose plus <NUM>% Polysorbate80 (Sigma) and are administered in the animals from the respective groups by oral gavage via gavage tube at a <NUM> and <NUM>/kg dose respectively.

As PD-<NUM> inhibitor, an anti-PD-<NUM> antibody (ref. : BE0146, BioXcell; clone: RMP1-<NUM>, reactivity: mouse; isotype: Rat IgG2a; storage conditions: +<NUM>) is injected into the peritoneal cavity of mice (Intraperitoneally, IP). The administration volume is <NUM>/Kg adjusted to the most recent individual body weight of mice.

Fourteen (<NUM>) days after randomization and if the antitumor activity of NOX4/<NUM> compounds alone or in combination is considered sufficient, tumors from <NUM> satellite mice per group are collected, weighed and the tumor is cut in <NUM> fragments. One fragment is cut into slices <NUM> thick and fixed in <NUM>% neutral buffered formalin for <NUM> to <NUM>, and then embedded in paraffin (Histosec®, Merck, Darmstadt, Germany). One fragment is embedded in tissue Freezing Medium (Microm Microtech, France), snap-frozen in isopentane cooled over liquid nitrogen and stored at <NUM> until processing. Immunohistochemical stains for CD3, CD4 and CD8 are performed on paraffin-embedded tissue sections using standard techniques (Biodoxis, France). The number of CD3, CD4 and CD8 immunopositive cells per field are counted.

Fourteen days after randomization, the tumour from <NUM> mice per group are collected.

All the tumours are collected in RPMI culture medium (ref: BE12-702F, Lonza, Verviers, Belgium). The tumour immune infiltrate cells are quantified by flow cytometry analysis from each collected sample. Then, the antibodies directed against the chosen markers are added, according to the procedure described by the supplier for each antibody. All the antibodies except FoxP3 will be for surface labeling and FoxP3 for intracellular labeling. The antibodies used for flow cytometry analysis for effector T-Cell lymphocytes (Teff: CD45, CD3, CD8) and regulatory T-Cell lymphocytes (Treg: CD45, CD3, CD4, FoxP3) on mouse samples are listed in the Table <NUM> below:.

The stained cells are analyzed with a BD™ LSR II flow cytometer (BD Biosciences) equipped with <NUM> excitation lasers at wavelengths <NUM>, <NUM> and <NUM>. Flow cytometry data is acquired until either <NUM>,<NUM> mCD45+ events are recorded for each sample, or for a maximum duration of <NUM> minutes.

All study data, including animal body weight measurements, tumor volume, clinical and mortality records, and treatment is scheduled and recorded. The viability and behavior is recorded every day. Body weights are measured twice a week. The length and width of the tumor is measured twice a week with calipers and the volume of the tumor is estimated by the formula: <MAT>.

Humane endpoints. Experiment is terminated after <NUM> weeks or if:.

The treatment efficacy is assessed in terms of the effects of the test substances on the tumor volumes of treated animals relative to control animals. The following evaluation criteria of antitumor efficacy are determined.

The optimal value is the minimal T/C% ratio reflecting the maximal tumor growth inhibition achieved. The effective criteria for the T/C% ratio according to NCI standards, is * <NUM>%. Volume V and time to reach V is calculated. Volume V is defined as a target volume deduced from experimental data and chosen in exponential phase of tumor growth. For each tumor, the closest tumor volume to the target volume V is selected in tumor volume measurements. The value of this volume V and the time for the tumor to reach this volume is recorded. For each group, the mean of the tumor volumes V and the mean of the times to reach this volume is calculated. Mice survival will also be monitored and used as an efficacy parameter. Survival curves are drawn.

When MC38 cancer cells (<NUM> x <NUM>) are used, those are injected in phosphate-buffered saline (PBS) subcutaneously (s. c) into the flank of C57BL/<NUM> female mice aged <NUM>-<NUM> weeks. MC38 cells are either injected on their own, or mixed with C57BL/<NUM> colon fibroblasts (<NUM> x <NUM>), pre-treated ex vivo prior to injection with <NUM> ng/ml of TGFβ1 for <NUM> days to induce a CAF phenotype.

When 4T1 cancer cells (<NUM> x <NUM>) are used, those are injected in PBS s. c into the upper mammary fat pad of female mice aged <NUM>-<NUM> weeks. Cells are either injected on their own, or mixed with <NUM>. 5x105 BALB/C breast CAFs isolated from transgenic BALBneuT spontaneous stromal-rich breast tumours.

The NOX4 inhibitor <NUM>-(<NUM>-chlorophenyl)-<NUM>-[<NUM>-(dimethylamino)phenyl]-<NUM>-methyl-<NUM>-pyrazolo [<NUM>,<NUM>-c]pyridine-<NUM>,<NUM>(<NUM>,<NUM>)-dione (GKT137831) was administered to mice when tumours were palpable. GKT137831 was reconstituted in <NUM>% Methyl Cellulose (Sigma) with <NUM>% Polysorbate (Sigma) and administered by oral gavage 5X/week at <NUM>/kg. Control mice received vehicle by oral gavage. For longer term dosing, <NUM> initial doses were given as stated, but reduced to 3X/week for <NUM> weeks at <NUM>/kg, then 2X/week for <NUM> weeks at <NUM>/kg. The anti PD-<NUM> antibody (Bioxcell; RMP1-<NUM>) was given via intraparietal (i. p) injection. <NUM>µg of the antibody or the IgG2a isotype control (Bioxcell) were given when tumours were palpable every other day, totalling <NUM> doses.

For the data presented under <FIG>, tumours were measured every <NUM>-<NUM> days by electronic skin calliper from longest width and length. Tumour volume was calculated using the formula <NUM>/3πXr3, where the radius (r) was calculated from tumour width and length measurement to provide an average diameter value. Mice were randomized into groups based on tumour volume so that no statistical difference occurred between mean tumour volumes between groups before treatments began. <FIG> shows that at day <NUM> i.e. after <NUM> days of treatment, tumours were significantly smaller when mice were treated with the NOX4 inhibitor than compared with vehicle alone. Further, since immunochemistry (carried out as described above) revealed, as represented on <FIG> and <FIG>, respectively, that the treatment with the NOX4 inhibitor significantly reduces SMA-positive CAF in tumours and results in relocation of CD8+ T-cells from the tumour edge into the centre of the tumour. Using the 4T1 breast cancer model, these results clearly show that treatment with GKT inhibits formation of CAFs as shown by the diminished myobibroblast (SMA-positive cells) population, allowing CD8+ T-cells access to the tumour and kill cancer cells, reducing the tumour size. It supports the beneficial effects of the combination of a NOX4 inhibitor and anti-cancer immunotherapeutic agent that would further activate the CD8+ T-cells.

The beneficial effects of such a combination is further supported by the results presented on <FIG> for the combination of a PD-<NUM> inhibitor (αPD1) with the NOX4 inhibitor GKT137831 which significantly improves therapeutic response in CAF-rich tumours: tumours were significantly smaller when mice were treated with αPD1/GKT831 combination compared with αPD1 alone (<FIG>) and following the administration of the αPD1/GKT831 combination, there is a significant relocation of CD8+ T-cells from the tumour edge into the centre of the tumour (<FIG>) and the survival outcome is also significantly increased (<FIG>), compared with αPD1 alone. Using the MC38 colon cancer model, the beneficial effect of GKT/αPD1 combination therapy was confirmed by showing a very significant decrease of tumour volume, which is accompanied by an increase in mouse survival. Moreover, it was shown that this effect results from an infiltration of CD8+ T-cells into the tumour of the NOX inhibitors. These results strongly suggest that the NOX4 inhibitors of the invention, in particular GKT137831, are strong candidates for PD1 co-therapy for all CAF-rich cancers.

In order to test the efficacy of a combination according to the invention, NOX4/<NUM> inhibitors are combined with the treatment with a vaccine such as an anti-HPV vaccine.

TC1 cancer cells (<NUM> x <NUM><NUM>) (prostate cancer) were injected in phosphate-buffered saline (PBS) subcutaneously (s. c) into the flank of C57BL/<NUM> female mice aged <NUM>-<NUM> weeks. TC1 cells were either injected on their own, or mixed with C57BL/<NUM> lung fibroblasts (<NUM> x <NUM><NUM>), pre-treated ex vivo prior to injection with <NUM> ng/ml of TGFβ1 for <NUM> days to induce a CAF phenotype.

Tumours were measured every <NUM>-<NUM> days by electronic skin calliper from longest width and length. Tumour volume measurements, mice randomized and oral gavage dosage were carried out as described above.

Vaccination with a DNA vaccine encoding tetanus Fragment C domain <NUM> (Dom) fused to the immunodominant CD8 epitope of E7 HPV RAHYNIVTF (RAH, E7<NUM>-<NUM>) (<NPL>; <NPL>) was administered via intramuscular injection (i. m) when tumours were palpable. One injection containing <NUM>µg of DNA in PBS was given and any repeat doses were given <NUM> weeks post initial immunisation. Treatment with a NOX4 inhibitor (GKT137831) reconstituted as described in Example <NUM>, was administered to mice when tumours were palpable.

<FIG> supports that the combination of an anti-tumour vaccination with a NOX4 inhibitor significantly improves therapeutic response in CAF-rich tumours since at day <NUM>, tumours were significantly smaller when mice were treated with the combination vaccine/ NOX4 inhibitor compared with the vaccine alone and following the administration of the combination vaccine/ NOX4 inhibitor, there is a significant relocation of CD8+ T-cells from the tumour edge into the centre of the tumour (<FIG>) and the survival outcome is also significantly increased (<FIG>), compared with vaccine alone. Effective immunotherapy, whether based on checkpoint inhibitors, T-cell agonists, vaccination or adoptive T-cell transfer, requires the presence of CD8+ effector T-cells in the tumour. Cancer-associated fibroblasts are found in most solid cancers, and play a major role in tumour immune evasion by excluding CD8+ T-cells from cancers, thereby rendering immunotherapies ineffective. Therefore, since NOX inhibitors of the invention, in particular GKT831, effectively target CAF as shown by the diminution of SMA-positive cells in the 4T1 model, it promotes CD8+ T-cell infiltration into tumours and restores response to vaccine-based and PD1-based immunotherapies. These data suggest that combination immunotherapy with NOX4 inhibitors of the invention, in particular GKT137831, may significantly improve response rates for this type of treatment.

In order to test the efficacy of a combination, NOX4/<NUM> inhibitors are combined with the treatment with an anti-VEGF agent.

MC38 xenograft mouse models of tumors were produced by injecting MC38 tumor cells diluted in PBS (<NUM><NUM> for MC38) subcutaneously either in Wild-Type C57/BL6 mice or NOX1 deficient (NOX1-KO) mice. When tumors reached <NUM><NUM>, intra-peritoneal administration of purified antibodies: either an anti-VEGF: DC101 or an irrelevant Rat IgG (as control) were performed twice a week. DC101 was given at a dose of <NUM>µg per injection per mouse. Vehicle (VL) (i.e. methylcellulose and Tween <NUM>) or a NOX1-selective inhibitor, (R) <NUM>-methoxy-<NUM>-(<NUM>-morpholino-<NUM>-phenylethoxy)-N-(<NUM>-(pyridin-<NUM>-yl)-<NUM>,<NUM>,<NUM>-thiadiazol-<NUM>-yl)benzamide (GKT2) (twice daily at <NUM>/kg) were given by oral gavage until the sacrifice of mice. Tumor size was measured with a caliper and tumor volume was determined according to the equation: (Length*width*thickness). Tumor size was measured in vivo by a caliper (D-<NUM> to D-<NUM>) every <NUM> days. After sacrifice, tumors were removed without fixation with PFA (paraformaldehyde), isolated and blood vascular endothelial cells (CD45-/CD31+/GP38-) were analyzed by flow cytometry.

<FIG> shows that the combination of a highly selective NOX1 inhibitor (GKT2) and an anti-VEGF-R2 blocking antibody (DC101) allows inhibiting angiogenesis. Moreover, GKT2 and DC101 act synergistically in enhancing inhibition of neo-vascularization.

<FIG> shows that tumors in NOX1-KO mice showed decreased growth kinetics as compared to tumors in WT mice indicating a clear involvement of NOX1. Further, treatment with the anti-VEGFR2 antibody (DC101) decreased tumor growth in NOX1 deficient mice and this effect was even more pronounced compared to WT mice. This clearly suggests different mechanisms of action between VEGFR2 and NOX1 signaling.

Claim 1:
<NUM>-(<NUM>-chlorophenyl)-<NUM>-[<NUM>-(dimethylamino)phenyl]-<NUM>-methyl-<NUM>-pyrazolo[<NUM>,<NUM>-c]pyridine-<NUM>,<NUM>(<NUM>,<NUM>)-dione for use in the treatment of solid tumor cancers presenting or susceptible to present a resistance to immunotherapy, wherein said <NUM>-(<NUM>-chlorophenyl)-<NUM>-[<NUM>-(dimethylamino)phenyl]-<NUM>-methyl-<NUM>-pyrazolo[<NUM>,<NUM>-c]pyridine-<NUM>,<NUM>(<NUM>,<NUM>)-dione is to be administered in combination with an anti-cancer immunotherapeutic agent selected from at least one cancer vaccine or at least one immune checkpoint inhibitor.