Clear Cell Renal Cell Carcinoma Treatment

Abstract

The present invention provides methods of treating patients with ccRCC and other VHL(-) cancers, comprising administering to the patient a therapeutically effective amount of a protein kinase inhibitor and a therapeutically effective amount of at least one second agent comprising bisantrene or a derivative thereof, or a pharma- ceutical acceptable salt of bisantrene or a derivative thereof. The present invention also provides pharmaceutical compositions and kits for treating ccRCC and other VHL(-) cancers, and the use of such compositions in the manufacture of medicaments for treating ccRCC and other VHL(-) cancers.

Description

[Technical field]

[0001] The present invention relates to the use of bisantrene in combination with a protein kinase inhibitor to treat clear cell renal cell carcinoma (ccRCC) and other cancers with an inactive von Hippel-Lindau (VHL) tumor suppressor gene. The present invention also discloses pharmaceutical compositions and kits for this treatment, and the use of such compositions in the manufacture of medicaments for such treatment. [Background technology]

[0002] Kidney cancer is one of the 10 most common types of cancer. Renal cell carcinoma (RCC) accounts for 3.8% of all new cancers. Approximately 80%-85% of RCCs are clear cell renal cell carcinoma (ccRCC). The remaining 15%-20% are non-clear cell RCCs with a diverse group of histological subtypes, each with a distinct molecular profile. According to the American Cancer Society, the lifetime risk of developing kidney cancer is 1 in 47 for men and 1 in 82 for women.

[0003] Smoking and obesity are well-established risk factors for the development of RCC. Several hereditary forms of RCC also exist, with von Hippel-Lindau (VHL) disease being the most common. VHL disease is caused by autosomal dominant mutations in the VHL gene that predispose individuals to ccRCC and various other cancers. VHL loss of function has also been shown to be important in sporadic ccRCC. Many studies have reported that the VHL loss-of-function alteration VHL(-) in more than 90% of sporadic ccRCC is caused by allelic deletion, mutation, and promoter methylation.

[0004] In the United States, there were approximately 65,000 new cases of kidney cancer and 15,000 deaths in 2019. Patients with advanced disease are often asymptomatic, and approximately 20% of patients have metastatic disease at the time of diagnosis. The most important prognostic determinants of 5-year survival are tumor stage, grade, local extent of tumor, presence of regional lymph node metastasis, and evidence of metastatic disease at the time of presentation. RCC primarily metastasizes to the lungs, lymph nodes, bone, liver, adrenal glands, and brain. The 5-year survival rate for metastatic ccRCC is low at 12%.

[0005] Surgery is the first choice of treatment for stage I-III ccRCC, first with cytoreductive nephrectomy, followed by systemic therapy commonly used to treat metastatic disease. RCC does not respond highly to cytotoxic chemotherapy or radiotherapy. Targeted therapy has benefited an increasing number of RCC patients. In recent years, targeted agents such as vascular endothelial growth factor (VEGF)-binding monoclonal antibodies (bevacizumab), oral tyrosine kinase inhibitors with strong activity against VEGF receptors (e.g., axitinib, cabozantinib, lenvatinib, pazopanib, sorafenib, sunitinib, and tivozanib), and mammalian target of rapamycin inhibitors (everolimus and temsirolimus) have provided new therapeutic alternatives in recent years.

[0006] Most ccRCC tumor cells express PD-L1 on the cell membrane, which helps ccRCC tumor cells escape immune attack. Immune checkpoint inhibitors, PD-1 blockade or PD-L1 blockade, block this pathway, releasing the immune system's "off switch" and enhancing the ability of cytotoxic T cells to kill tumor cells. CTLA-4 blockade supports effector T cell activation and proliferation by shutting down autoreactive T cells during the immune priming phase. The FDA has approved two PD-1 inhibitors (nivolumab and pembrolizumab), one PD-L1 inhibitor (avelumab), and one CTLA-4 inhibitor (ipilimumab) for use in ccRCC. Other PD-1 / PD-L1 immune checkpoint inhibitors of potential consideration include atezolizumab, bevacizumab, cemiplimab, dostarlimab, and durvalumab. Summary of the Invention [Problem to be solved by the invention]

[0007] Despite these new treatment options, many patients demonstrate disease progression due to drug resistance or the development of bypass pathways. Therefore, new drugs, new combinations of drugs, or new combination drug therapies are needed to treat ccRCC and other VHL(-) cancers. [Means for solving the problem]

[0008] The present invention provides a new paradigm for treating ccRCC or other VHL(-) cancers by administering a combination of bisantrene and a protein kinase inhibitor, which meets the need for new therapies that improve clinical outcomes for patients with ccRCC or other VHL(-) cancers, especially those with protein kinase inhibitor-resistant ccRCC.

[0009] Bisantren is an antitumor agent with multiple mechanisms of action, including DNA intercalation, inhibition of topoisomerase, fat mass and obesity-associated protein (FTO), and activation of the immune system.

[0010] Surprisingly, through the course of these studies, it was found that bisantrene and its pharma- ceutically acceptable salts act synergistically with protein kinase inhibitors against clear cell renal cell carcinoma cancer cells.

[0011] Thus, one aspect of the invention provides a method of treating a patient having ccRCC or other VHL(-) cancer, the method comprising administering to the patient a therapeutically effective amount of a protein kinase inhibitor, which may be a tyrosine kinase inhibitor, and a therapeutically effective amount of at least one second agent comprising bisantrene or a derivative thereof, or a pharma- ceutical acceptable salt of bisantrene or a derivative thereof.

[0012] According to certain embodiments, the protein kinase inhibitor inhibits one or more of VEGFR1, VEGFR2, VEGFR3, DDR1, DDR2, RET, and RIPK2. According to certain embodiments, the protein kinase inhibitor inhibits mTOR.

[0013] According to certain embodiments, the at least one protein kinase inhibitor is (a) pazopanib, (b) lenvatinib, (c) cabozantinib, (d) everolimus, (e) sorafenib, (f) sunitinib, (g) temsirolimus, (h) mitomycin C, (i) axitinib, (j) tivozanib, and (k) Belzchifan is selected from the group consisting of:

[0014] According to certain embodiments, the at least one protein kinase inhibitor is (a) pazopanib, (b) lenvatinib, and (c) Cabozantinib is selected from the group consisting of:

[0015] In certain embodiments, the second agent is bisantrene or a pharma- ceutically acceptable salt thereof.

[0016] According to certain embodiments, the method comprises administering to the patient a therapeutically effective amount of a protein kinase inhibitor and a therapeutically effective amount of bisantrene or a pharma- ceutically acceptable salt thereof.

[0017] According to certain embodiments, the method comprises treating protein kinase inhibitor-resistant ccRCC.

[0018] The method may further comprise determining the VHL mutation and epigenetic status of the patient's cancer. The VHL mutation and epigenetic status may be determined before the start of treatment or alternatively during treatment as a marker of progress of treatment. The VHL mutation and epigenetic status of the cancer may be determined by next generation sequencing, RT-PCR, ELISA assay using a suitable antibody against VHL, RNA hybridization assay, or any other method known in the art.

[0019] According to certain embodiments, the method may further comprise administering at least one additional therapeutic agent, optionally a checkpoint inhibitor or an immunomodulatory agent, for treating ccRCC or other VHL(-) cancer, wherein the at least one additional therapeutic agent is selected from the group consisting of atezolizumab, avelumab, bevacizumab, cemiplimab, dostallimab, durvalumab, interleukin-2, ipilimumab, nivolumab, pembrolizumab, and proleukin.

[0020] According to certain embodiments, the methods may comprise administering to the patient at least one said protein kinase inhibitor prior to, simultaneously with, or after administration of the second agent to the patient.

[0021] According to other embodiments, the methods may comprise administering to the patient at least one said protein kinase inhibitor and said second agent simultaneously, optionally in a single composition.

[0022] According to certain embodiments, the treatment has a synergistic effect against ccRCC and other VHL(-) cancers compared to methods in which the at least one protein kinase inhibitor or the second agent is administered alone, in certain embodiments, the dosage of the at least one protein kinase inhibitor is at least 20% lower than the dosage required to achieve the same target result without administration of the second agent.

[0023] Another aspect of the invention provides a pharmaceutical composition for treating ccRCC or other VHL(-) cancers according to the method of treatment embodiments as described above, said composition comprising at least one protein kinase inhibitor, which may be a tyrosine kinase inhibitor as described above, and a second agent comprising bisantrene or a derivative thereof or a pharma- ceutically acceptable salt of bisantrene or a derivative thereof as described above. The composition may further comprise an active agent as described above.

[0024] Another aspect of the present invention provides the use of a composition according to the invention as described above in the manufacture of a medicament for treating ccRCC and other VHL(-) cancers, optionally protein kinase inhibitor resistant ccRCC, in a patient.

[0025] Another aspect of the invention provides a kit for treating ccRCC or other VHL(-) cancer, optionally protein kinase inhibitor resistant ccRCC, according to an embodiment of the method of the invention as described above, said kit comprising at least one protein kinase inhibitor, optionally a tyrosine kinase inhibitor as described above, and at least one second agent comprising bisantrene or a derivative thereof or a pharma- ceutically acceptable salt of bisantrene or a derivative thereof as described above. The kit according to the invention comprises instructions for administering said at least one protein kinase inhibitor to said patient before, simultaneously with or after administration of said second agent to said patient. Alternatively, the kit according to the invention may comprise instructions for administering to said patient at least one protein kinase inhibitor and said second agent simultaneously. [Brief description of the drawings]

[0026] The following invention will become better understood with reference to the specification, the appended claims and the accompanying drawings, in which the terms "Zan" or "Zantrene" are alternative terms for Bisantrene.

[0027] [Figure 1]Webb analysis of bisantrene-everolimus or bisantrene-sunitinib drug combinations in 786-O cells. A) Cell viability for everolimus, sunitinib and Zantrene combinations at different dose ranges as indicated. Experimental data for each drug alone and in combination are shown. "Expected" is calculated using Webb's method and indicates the expected value if the drug combination is additive. Thus, any experimental values ​​below this line are considered synergistic. Those at or close to the line are additive and those above the line are counterproductive. B) Webb analysis of all drug combination doses measured, where a result of <-0.1 indicates synergy (*), results between -0.1 and 0.1 indicate additive (&) and results >0.1 indicate counterproductive (#). [Diagram 2] Webb analysis of drug combinations of bisantrene-sorafenib or bisantrene-pazopanib in 786-O cells. A) Cell viability for different dose ranges of sorafenib, pazopanib in combination with Zantrene as indicated. Experimental data for each drug alone and in combination are shown. "Expected" is calculated using Webb's method and indicates the expected value if the drug combination is additive. Thus, any experimental values ​​below this line are considered synergistic. Those at or close to the line are additive and those above the line are counterproductive. B) Webb analysis of all drug combination doses measured, where results <-0.1 indicate synergy (red), results between -0.1 and 0.1 indicate additive (green), and results >0.1 indicate counterproductive (yellow). [Diagram 3]Webb analysis of bisantrene-lenvatinib or bisantrene-cabozantinib drug combinations in 786-O cells. A) Cell viability for Lenvatinib, Cabozantinib and Zantrene combinations at different dose ranges as indicated. Experimental data for each drug alone and in combination are shown. "Expected" is calculated using Webb's method and indicates the expected value if the drug combination is additive. Thus, any experimental value below this line is considered synergistic. Those at or close to the line are additive and those above the line are counterproductive. B) Webb analysis of all drug combination doses measured, where a result of <-0.1 indicates synergy (*), results between -0.1 and 0.1 indicate additive (&), and results >0.1 indicate counterproductive (#). [Figure 4] Webb analysis of bisantrene-everolimus or bisantrene-sunitinib drug combinations in Caki-1 cells. Cell viability for different dose ranges of everolimus, sunitinib and Zantrene combinations as indicated. Experimental data for each drug alone and in combination are shown. "Expected" is calculated using Webb's method and indicates the expected value if the drug combination is additive. Thus, any experimental value below this line is considered synergistic. Those at or close to the line are additive and those above the line are countervailing. B) Webb analysis of all drug combination doses measured, where a result of <-0.1 indicates synergy (*), results between -0.1 and 0.1 indicate additive (&) and results >0.1 indicate countervailing (#). [Diagram 5]Webb analysis of drug combinations of bisantrene-sorafenib or bisantrene-pazopanib in Caki-1 cells. A) Cell viability for different dose ranges of sorafenib, pazopanib and Zantrene as indicated. Experimental data for each drug alone and in combination are shown. "Expected" is calculated using Webb's method and indicates the expected value if the drug combination is additive. Thus, any experimental values ​​below this line are considered synergistic. Those at or close to the line are additive and those above the line are counterproductive. B) Webb analysis of all drug combination doses measured, where a result of <-0.1 indicates synergy (*), results between -0.1 and 0.1 indicate additive (&), and results >0.1 indicate counterproductive (#). [Figure 6] Webb analysis of bisantrene-lenvatinib or bisantrene-cabozantinib drug combinations in Caki-1 cells. A) Cell viability for Lenvatinib, Cabozantinib and Zantrene combinations at different dose ranges as indicated. Experimental data for each drug alone and in combination are shown. "Expected" is calculated using Webb's method and indicates the expected value if the drug combination is additive. Thus, any experimental values ​​below this line are considered synergistic. Those at or close to the line are additive and those above the line are countervailing. B) Webb analysis of all drug combination doses measured, where results <-0.1 indicate synergy (*), results between -0.1 and 0.1 indicate additive (&), and results >0.1 indicate countervailing (#). [Figure 7]Figure 2 shows Webb analysis of bisantrene-everolimus or bisantrene-sunitinib drug combinations in Caki-2 cells. As shown, cell viability for different dose ranges of everolimus, sunitinib and Zantrene in combination. Experimental data for each drug alone and in combination are shown. "Expected" is calculated using Webb's method and indicates the expected value if the drug combination is additive. Thus, any experimental value below this line is considered synergistic. Those at or close to the line are additive and those above the line are countervailing. B) is Webb analysis of all drug combination doses measured, where a result of <-0.1 indicates synergy (*), results between -0.1 and 0.1 indicate additive (&) and results >0.1 indicate countervailing (#). [Figure 8] Webb analysis of drug combinations of bisantrene-sorafenib or bisantrene-pazopanib in Caki-2 cells. A) Cell viability for different dose ranges of sorafenib, pazopanib and Zantrene as indicated. Experimental data for each drug alone and in combination are shown. "Expected" is calculated using Webb's method and indicates the expected value if the drug combination is additive. Thus, any experimental values ​​below this line are considered synergistic. Those at or close to the line are additive and those above the line are countervailing. B) Webb analysis of all drug combination doses measured, where a result of <-0.1 indicates synergy (*), results between -0.1 and 0.1 indicate additive (&), and results >0.1 indicate countervailing (#). [Figure 9]Webb analysis of bisantrene-lenvatinib or bisantrene-cabozantinib drug combinations in Caki-2 cells. A) Cell viability for Lenvatinib, Cabozantinib and Zantrene combinations at different dose ranges as indicated. Experimental data for each drug alone and in combination are shown. "Expected" is calculated using Webb's method and indicates the expected value if the drug combination is additive. Thus, any experimental value below this line is considered synergistic. Those at or close to the line are additive and those above the line are countervailing. B) Webb analysis of all drug combination doses measured, where a result of <-0.1 indicates synergy (*), results between -0.1 and 0.1 indicate additive (&), and results >0.1 indicate countervailing (#). [Figure 10] Webb analysis of bisantrene-everolimus or bisantrene-sunitinib drug combinations in RCC4 EV cells. Cell viability for different dose ranges of everolimus, sunitinib and Zantrene combinations as indicated. Experimental data for each drug alone and in combination are shown. "Expected" is calculated using Webb's method and indicates the expected value if the drug combination is additive. Thus, any experimental value below this line is considered synergistic. Those at or close to the line are additive and those above the line are countervailing. B) Webb analysis of all drug combination doses measured, where a result of <-0.1 indicates synergy (*), results between -0.1 and 0.1 indicate additive (&) and results >0.1 indicate countervailing (#). [Figure 11]Webb analysis of bisantrene-sorafenib or bisantrene-pazopanib drug combinations in RCC4 EV cells. A) Cell viability for sorafenib, pazopanib and Zantrene combinations at different dose ranges as indicated. Experimental data for each drug alone and in combination are shown. "Expected" is calculated using Webb's method and indicates the expected value if the drug combination is additive. Thus, any experimental values ​​below this line are considered synergistic. Those at or close to the line are additive and those above the line are counterproductive. B) Webb analysis of all drug combination doses measured, where results <-0.1 indicate synergy (*), results between -0.1 and 0.1 indicate additive (&), and results >0.1 indicate counterproductive (#). [Figure 12] Webb analysis of bisantrene-lenvatinib or bisantrene-cabozantinib drug combinations in RCC4 EV cells. A) Cell viability for Lenvatinib, Cabozantinib and Zantrene combinations at different dose ranges as indicated. Experimental data for each drug alone and in combination are shown. "Expected" is calculated using Webb's method and indicates the expected value if the drug combination is additive. Thus, any experimental value below this line is considered synergistic. Those at or close to the line are additive and those above the line are countervailing. B) Webb analysis of all drug combination doses measured, where a result of <-0.1 indicates synergy (*), results between -0.1 and 0.1 indicate additive (&), and results >0.1 indicate countervailing (#). [Figure 13]Figure 2 shows Webb analysis of drug combinations of bisantrene-everolimus or bisantrene-sunitinib in RCC4 VHL cells. Cell viability for different dose ranges of everolimus, sunitinib and Zantrene as indicated. Experimental data for each drug alone and in combination are shown. "Expected" is calculated using Webb's method and indicates the expected value if the drug combination is additive. Thus, any experimental value below this line is considered synergistic. Those at or close to the line are additive and those above the line are countervailing. B) is Webb analysis of all drug combination doses measured, where a result of <-0.1 indicates synergy (*), results between -0.1 and 0.1 indicate additive (&) and results >0.1 indicate countervailing (#). [Figure 14] Webb analysis of drug combinations of bisantrene-sorafenib or bisantrene-pazopanib in RCC4 VHL cells. A) Cell viability for different dose ranges of sorafenib, pazopanib in combination with Zantrene as indicated. Experimental data for each drug alone and in combination are shown. "Expected" is calculated using Webb's method and indicates the expected value if the drug combination is additive. Thus, any experimental value below this line is considered synergistic. Those at or close to the line are additive and those above the line are counterproductive. B) Webb analysis of all drug combination doses measured, where a result of <-0.1 indicates synergy (*), results between -0.1 and 0.1 indicate additive (&), and results >0.1 indicate counterproductive (#). [Figure 15]Webb analysis of bisantrene-lenvatinib or bisantrene-cabozantinib drug combinations in RCC4 VHL cells. A) Cell viability for Lenvatinib, Cabozantinib and Zantrene combinations at different dose ranges as indicated. Experimental data for each drug alone and in combination are shown. "Expected" is calculated using Webb's method and indicates the expected value if the drug combination is additive. Thus, any experimental value below this line is considered synergistic. Those at or close to the line are additive and those above the line are countervailing. B) Webb analysis of all drug combination doses measured, where results <-0.1 indicate synergy (*), results between -0.1 and 0.1 indicate additive (&), and results >0.1 indicate countervailing (#). [Figure 16] Bisantrene and targeted or chemotherapeutic agents are presented to overcome the therapeutic resistance of ccRCC and other VHL(-) cancers. [Figure 17] We present a study design for combining bisantrene with checkpoint inhibitors to overcome immunotherapy resistance in ccRCC and other VHL(-) cancers. [Figure 18] Figure 1 shows the association of bisantrene sensitivity with VHL status of syngeneic kidney cancer cell lines RCC4 EV (VHL(-) mutant) and RCC4 VHL (VHL rescue line). (A) IC50 values ​​based on VHL status compared using t-test. (B) Direct comparison of Zantrene sensitivity of RCC4 syngeneic cell lines pair. Mean ± SEM, n=3. [Figure 19] Association of bisantrene sensitivity with VHL status assessed using colony formation assay. (A) IC50 values ​​compared between VHL mutant and wild type cell lines. NS, not significant, unpaired t-test. (B) Colonies observed for RCC4 EV and RCC4 VHL rescue cell lines. n=4, *p<0.05, paired t-test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] definition As used herein, "treating" means affecting a subject / patient, tissue or cell to achieve a desired pharmacological and / or physiological effect, including inhibiting a pathology, i.e., slowing or arresting its development, or alleviating or ameliorating the effects of a pathology, i.e., causing a reversal or reduction in the effects of a pathology.

[0029] As used herein, "preventing" means to bar a disease state from occurring in a cell, tissue or subject that may be at risk of having the disease state, but does not necessarily mean that the disease state will not eventually develop or that the subject will not eventually develop the disease state. Preventing includes delaying the onset of the disease state in a cell, tissue or subject.

[0030] As used herein, the term "subject" or "patient" refers to a mammal. A mammal may be human or non-human. Examples of non-humans include primates, livestock animals (e.g., sheep, cows, horses, donkeys, pigs), pets (e.g., dogs, cats), laboratory test animals (e.g., mice, rabbits, rats, guinea pigs, hamsters), and captive wild animals (e.g., foxes, deer). Typically, the mammal is a human or a non-human primate. More typically, the mammal is a human.

[0031] The term "composition" includes compositions and formulations that include an active pharmaceutical ingredient (e.g., a "protein kinase inhibitor" and a "second agent that includes bisantrene or a derivative of bisantrene or a pharma- ceutically acceptable salt of bisantrene or a cardioprotective derivative of bisantrene) with an excipient or carrier, and also includes compositions and formulations that have a carrier. In a pharmaceutical composition, the excipient or carrier is "pharmaceutical acceptable," meaning that it is not biologically or otherwise undesirable, i.e., the material may be incorporated into a pharmaceutical composition administered to a patient without causing undesirable biological effects or interacting adversely with any other components of the composition in which it is contained. Supplementary active ingredients may also be incorporated into the composition.

[0032] As used herein, "pharmaceutical acceptable," for example in the description of a "pharmaceutical acceptable salt" or a "pharmaceutical acceptable excipient or carrier," means a material that is not biologically or otherwise undesirable, i.e., the material may be incorporated into a pharmaceutical composition administered to a patient without causing undesirable biological effects or interacting adversely with any other components of the composition in which it is contained.

[0033] The term "effective amount" or "therapeutically effective amount" refers to an amount of an active pharmaceutical ingredient sufficient to produce a desired therapeutic response. The specific effective amount or therapeutically effective amount will vary depending on factors such as the particular condition being treated, the age, weight, general health, physical condition, sex and diet of the subject, the duration of treatment, the nature of concomitant therapy (if any), and the severity of the particular condition.

[0034] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, fillers, carrier solutions, suspensions, colloids, and molding and binding agents, which may include other pharmaceutical excipients known in the art, including lubricants, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, antioxidants, and other stabilizers, including physical stabilizers such as thickeners and viscosity enhancers, colorants, flavorings, and sweeteners. The use of such media and agents in pharmaceutical active ingredients is well known in the art. Unless any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated.

[0035] As used herein, "administration," "administering," or "administering" refers to the dosing, application, or provision of one or more agents to a subject. Administration can be accomplished using any of several methods known in the art. For example, as used herein, "administering" means via injection (intravenous (iv)), parenteral administration. "Parenteral" means intravenous, subcutaneous, or intramuscular administration.

[0036] Detailed Description of the Invention The present disclosure relates to methods of treating ccRCC or other VHL(-) cancers, particularly protein kinase inhibitor-resistant ccRCC, comprising administering a therapeutically effective amount of bisantrene in combination with a protein kinase inhibitor, particularly a tyrosine kinase inhibitor ("TKI").

[0037] Protein Kinase Inhibitors Protein kinases are enzyme proteins that phosphorylate the side chains of hydroxyl-bearing amino acids, i.e., serine, threonine, and tyrosine, on target proteins and typically on other targets in signal transduction pathways. Indeed, several signal transduction pathways include cascades of protein kinases. In many cases, this phosphorylation leads to a conformational change in the target protein, which in turn can lead to activation or inhibition of the target protein's activity, or to changes in its physical properties (thereby modulating interactions). Thus, protein phosphorylation and dephosphorylation events can activate or inhibit specific physiological pathways. Dysregulation of such signal transduction, such as by mutations that cause constitutive expression or unregulated activity of specific proteins or pathways, can contribute to and / or enable cancer survival. Dysregulation of expression and / or activity of various protein kinases, such as EGFR, VEGFR, HER, ALK, RAS, and RAF (to name a few), is associated with increased growth, proliferation, motility, and survival of various tumor cells, including ccRCC cells.

[0038] The range of protein kinases known in the art is very wide.In fact, human genome is known to contain 518 protein kinase genes.A large number of protein kinases are known to be related to cancer, including ccRCC, which is not surprising, since protein kinases are related to signal transduction pathways and the activation or inhibition of pathways related to growth, especially angiogenesis, and transmembrane signal transduction.

[0039] Protein kinases associated with cancer, particularly renal cancer such as ccRCC, include, for example, tyrosine kinases, serine / threonine kinases, cyclin-dependent kinases, Aurora kinases, mTOR, mitogen-activated protein kinases, among others, which may include kinases such as AATK, ABL1, AKT2, ALK, AURKA, AXL, CHK2, DDR1, DDR2, EML4-ALK, EPHA2, EPHA5, ERBB2, EGFR, FIP1L1-PDGFRA, FTL3, HER, HIF, KIT, MET, p21Cip1, p27Kip1, PDGFR, PKD1, RET, retinoblastoma protein (RB), RIPK2, SCH1, VEGFR1, VEGFR2 and VEGFR3.

[0040] It is important to note that kinases such as those mentioned above may have a primary target, but may phosphorylate other targets / sites. For example, a tyrosine kinase may phosphorylate serine and / or threonine hydroxyl sites. Similarly, a serine / threonine kinase may phosphorylate tyrosine hydroxyl sites. Similarly, a kinase may primarily target one protein, but may also phosphorylate other proteins. Thus, a tyrosine kinase may primarily target, or be known to target, tyrosine hydroxyl groups on a particular protein (usually a receptor), but may also be capable of phosphorylating serine and / or threonine and / or hydroxyl sites on other proteins. As known in the art, this variability in target specificity is incorporated herein in the context of protein kinases.

[0041] A variety of protein kinase inhibitors have been developed for the treatment of various cancers, including imatinib, the first rationally designed protein kinase inhibitor to receive FDA approval in 2001. Since then, protein kinase inhibitors such as gefitinib, erlotinib, dasatinib, nilotinib, lapatinib, osimertinib, olmutinib, lorlatinib, capmatinib, tepotinib, selpercatinib, pralsetinib, selumetinib, trametinib, afatinib, ibrutinib, dabrafenib, ponatinib, bosutinib, radotinib, idelalisib, crizotinib, vemurafenib, ruxolitinib, olmutinib, mitomycin C, temsirolimus, everolimus, axitinib, sorafenib, sunitinib, cabozantinib, lenvatinib, pazopanib, and tivozanib, to name a few.

[0042] Protein kinase inhibitors currently approved to treat ccRCC include, for example, temsirolimus, axitinib, velzutifan, cabozantinib, everolimus, lenvatinib, mitomycin C, pazopanib, sorafenib, sunitinib, and tivozanib.

[0043] Axitinib, cabozantinib, pazopanib, sorafenib, and sunitinib are small molecule tyrosine kinase inhibitors whose targets include the VEGFR family. Belzutifan is an inhibitor of hypoxia-inducible factor (HIF)-2 alpha (HIF-2a). Everolimus and temsirolimus are inhibitors of the mammalian target of rapamycin (mTOR) complex. Lenvatinib is a multikinase inhibitor that acts on VEGFR1, 2, and 3, as well as fibroblast growth factor receptors (FGFR) 1, 2, 3, and 4, platelet-derived growth factor receptor (PDGFR) alpha, c-Kit, and RET proto-oncogenes. Mitomycin C is a potent DNA cross-linking chemotherapeutic agent. Tivozanib is a multikinase inhibitor that acts on VEGFR1, 2, and 3.

[0044] In addition to the VEGFR family of protein kinases, common targets of lenvatinib, cabozantinib and pazopanib include DDR1 and DDR2 (discoidin domain receptor 1 and 2), RET ("REarranged during Transfection") and RIPK2 (receptor-interacting protein serine / threonine kinase 2). RET has also been identified as a target for sunitinib and axitinib, and AXL and MET have been identified as additional cabozantinib targets.

[0045] Although protein kinase inhibitors have been successful in treating a variety of cancers, including ccRCC and other VHL(-) cancers, many patients experience disease progression due to drug resistance or the development of bypass pathways.

[0046] This study demonstrated a surprising synergy between bisantrene and protein kinase inhibitors used to treat ccRCC and other VHL(-) cancers. Such synergy is expected to at least help reverse or reduce, if not eliminate, the development of resistance, and is also expected to provide the added benefit of allowing lower doses of protein kinase inhibitors and / or bisantrene, thereby reducing any side effects.

[0047] Bisantrene and its derivatives or analogues, and pharma- ceutically acceptable salts Bisantren has immunological properties that may be responsible for some of its activity, making it a useful tool in combinatorial supercoiling, causing strand breaks associated with DNA-associated proteins. This is due to inhibition of the enzyme topoisomerase II, which relaxes the coiling of DNA during replication. The drug is inactive orally, intravenously (iv), intraperitoneally (ip) or subcutaneously (sc), but was found to be effective in cancer models using colon 26, Lewis lung, Ridgway osteosarcoma, B16, Lieberman plasma cell, P388 or L1210 cancer cells. Colony formation assay activity from 684 patients was seen in breast, small cell lung, large cell lung, squamous cell lung, ovarian, pancreatic, renal, adrenal, head and neck, sarcoma, gastric, lymphoma and melanoma tumor cells, but not in colorectal cancer. Importantly, no cross-resistance with adriamycin and mitoxantrone was found. Macrophages could be isolated from peritoneal exudates with cytostatic antiproliferative activity in cultures of P815 (mastocytoma) tumor cells after treatment with bisantrene and for 4 weeks thereafter. Supernatants from macrophages activated with bisantrene also have protective cytostatic effects in tumor cell cultures. Further studies showed that macrophages activated with bisantrene and adoptively transferred into mice bearing EL-4 lymphoma increased median survival by more than two-fold, with 7 out of 10 mice in the group being cured. Repeated administration of activated macrophages was more effective than a single administration. Bisantrene is a potent inhibitor (IC) of RNA N6-methyladenosine (m6A) demethylase, a protein associated with fat mass and obesity (FTO). 50142 nM) (Su, R., Dong, L., Li, Y., Gao, M., Han, L., Wunderlich, M. et al. (2020), "Targeting FTO Suppresses Cancer Stem Cell Maintenance and Immune Evasion", Cancer Cell, 38(1): 79-96.e11).

[0048] The chemical name of bisantrene is 9,10-anthracenedicarboxaldehyde bis[(4,5-dihydro-1H-imidazol-2-yl)hydrazine] dihydrochloride, and it was initially classified as an anthracycline chemotherapy agent. As used herein, the term "bisantrene" refers to bisantrene, or bisantrene dihydrochloride, as well as any pharmacologically compatible salt form, unless the dihydrochloride or another particular pharmacologically compatible salt form is specifically indicated. Typically, the pharmacologically compatible salt of bisantrene is bisantrene dihydrochloride for most pharmacological applications. These are drugs with planar structures based on a resonant aromatic ring structure that intercalates into the helix of DNA, disrupting various functions, including replication, possibly due to their inhibitory effect on the enzyme topoisomerase II. As with other anthracyclines, they were found to be able to kill tumor cells in colony formation assays and inhibit DNA and RNA synthesis by intercalating into DNA. The main chemotherapeutic mechanism of bisantrene is to preferentially bind AT-rich regions, which affect the transformation.

[0049] Recent studies have confirmed that bisantrene suppresses immune checkpoint gene expression and immune evasion through the enzymatic inhibition of FTO RNA demethylase, as described in R. Su. et al. (2020) "Targeting FTO Suppresses Cancer Stem Cell Maintenance and Immune Evasion", Cancer Cell, 38(1):79-96.e11.

[0050] Bisanthren was also found to achieve a non-immunological telomere effect. Bisanthren binds to DNA at a site called the G-quadruplex, where four guanines are associated by folding. Stabilization of the G-quadruplex interferes with the telomere-telomerase interaction and inhibits the activity of telomerase in various ways, including the displacement of telomerase-binding proteins. Because the level of topoisomerase II inhibition does not necessarily correlate with cytotoxic efficacy, alternative mechanisms may play a role in the action of bisantren. To enhance anti-telomerase activity, analogs of bisantren have been produced, which are further described below. Human melanoma (SK-Mel5) and colon cancer (LoVo) tumor cells were observed to lose their ability to proliferate in the presence of these drugs. No apoptosis was observed. However, a loss of immortality was seen, and treated cells again acquired the ability to senesce, mature, and die.

[0051] As mentioned above, in addition to the direct antitumor effect related to bisantrene's activity as a DNA intercalator, bisantrene also has other mechanisms of action, including immune enhancement. These mechanisms are described in (i)-(v). (i) NR West et al. (2011), "Tumor-Infiltrating Lymphocytes Predict Response to Anthracycline-Based Chemotherapy in Estrogen-Resistant Breast Cancer", Breast Canc. Res.13: R126, concluded that the level of tumor-infiltrating lymphocytes correlates with the response to anthracycline drug administration, and markers associated with tumor-infiltrating lymphocytes (TIL) include CD19, CD3D, CD48, GZMB, LCK, MS4A1, PRF1 and SELL; (ii) L. Zitvogel et al. (2008), "Immunological Aspects of Cancer Chemotherapy", Nature Rev. Immunol. 8:59-73, it is described that DNA damage, for example damage caused by intercalating agents such as bisantrene, induces the expression of NKG2D ligands on tumor cells in an ATM-dependent and CHK1-dependent (but p53-independent) manner, and that NKG2D is an activating receptor involved in tumor immune surveillance of NK cells, NK T cells, γδ T cells and resting (in mice) and / or activated (in humans) CD8+ T cells, and that anthracycline drugs function as immunostimulants, particularly in combination with IL-12, and that these drugs also promote the release of HMGB1 and activate T cells; (iii) DVKrysko et al. (2011), "TLR2 and TLR9 Are Sensors of Apoptosis in a Mouse Model of Doxorubicin-Induced Acute Inflammation", Cell Death Different. 18: 1316-1325, it is described that anthracycline antibiotics induce an immunogenic form of apoptosis with immune stimulatory properties mediated by MyD88, TLR2 and TLR9, and (iv) C.Ferraro et al. (2000), "Anthracyclines Trigger Apoptosis of Both G0-G1 and Cycling Peripheral Blood Lymphocytes and Induce Massive Deletion of Mature T and B Cells", Cancer Res. 60: 1901-1907, describes that anthracyclines induce apoptosis and ceramide production, activate caspase-3 in quiescent and cycling cells, and that the induced apoptosis is independent of CD95-L / CD95 and TNF / TNF-R; (v) K. Lee et al. (2009), "Anthracycline Chemotherapy Inhibits HIF-1 Transcriptional Activity and Tumor-Induced Mobilization of Circulating Angiogenic Cells", Proc. Natl. Acad. Sci. USA 106:2353-2358, it is described that anthracycline antibiotics provide another antitumor mechanism, namely, inhibition of HIF-1-mediated gene transcription, which in turn inhibits the transcription of VEGF, which is necessary for angiogenesis, and that HIF-1 also activates the transcription of genes encoding the glucose transporter GLUT1 and hexokinases HK1 and HK2, which are necessary for the high levels of glucose uptake and phosphorylation observed in metastatic cancer cells, and pyruvate dehydrogenase kinase 1 (PDK1), which shunts pyruvate from mitochondria and increases lactate production, and that patients with HIF-1α overexpression based on immunohistochemistry results are suggested to be good candidates for treatment with anthracycline antibiotics.

[0052] Several clinical trials have investigated the pharmacokinetics of bisantren in humans. In a study conducted in patients receiving a 90-minute infusion of 260 mg / m2, biphasic elimination was observed with an early half-life of 65 ± 15 minutes, a terminal half-life of 1142 ± 226 minutes, and a steady-state volume of distribution (Vdss) of 1845 L / m2. Plasma clearance in this study was 735 mL / min / m2, and 11.3% of the administered dose was excreted unchanged in the urine over 24 hours. In another study evaluating doses of 80 to 250 mg / m2, the early and terminal half-lives were 0.6 and 24.7 hours, respectively, with clearance of 1045.5 ± 51.0 mL / kg / hour and a calculated volume of distribution of 42.1 ± 5.9 L / kg. In this study, only 3.4 ± 1.1% of the administered dose was found in the urine over 96 hours. Three other single-dose studies reported triphasic elimination, and one study showed t 1 / 2 In one study, the clearance times were 3.44 min, 1.33 h, and 26.13 h for α, β, and γ, respectively; in another study, they were 3 min, 1 h, and 8 h, respectively; and in the last study, the clearance times shown were 0.1 h, 1.9 h, and 43.9 h, respectively. In one report, the large volume of distribution (687 L / m2) was interpreted as tissue sequestration of the drug with a subsequent depot effect. In a 72-h infusion study, plasma concentrations of 12 ± 6 ng / mL were observed at a dose of 56 mg / m2, and plasma concentrations of 55 ± 8 ng / mL were obtained at a dose of 260 mg / m2. In this study, the plasma clearance was 1306 ± 179 mL / min / m2, and the 24-h urinary excretion was 4.6% of the dose. Finally, in another study, a 60-min infusion schedule for 5 days showed t 1 / 2 The α and β periods were 0.9 and 9.7 hours, respectively, and 7.1% of the administered dose was excreted in the urine.

[0053] The structure of bisantrene dihydrochloride is shown in formula (I). [ka]

[0054] Bisantrene dihydrochloride is a tricyclic aromatic compound with the chemical name 9,10-anthracenedicarboxaldehyde bis[(4,5-dihydro-1H-imidazol-2-yl)hydrazine] dihydrochloride. Bisantrene dihydrochloride has the molecular formula C 22 H 22 It is N8·2HCl and has a molecular weight of 471.4. The alkylimidazole side chain is highly basic and positively charged at physiological pH, which is thought to facilitate electrostatic attraction to the negatively charged ribose phosphate groups in DNA.

[0055] Bisantren is typically administered intravenously, either centrally or peripherally.

[0056] Various formulations suitable for administering bisantrene or its derivatives or analogues are known in the art. U.S. Patent No. 4,784,845 to Desai et al. discloses a composition for delivering a hydrophobic drug, comprising (i) a hydrophobic drug (i.e., bisantrene or its derivatives or analogues), (ii) an oily excipient or oil phase substantially free of butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT), (iii) a co-surfactant or emulsifier, (iv) a co-surfactant or co-emulsifier, and (v) benzyl alcohol as a co-solvent. U.S. Patent No. 4,816,247 to Desai et al. discloses a method for the preparation of a glycerol-based emulsion comprising: (i) a hydrophobic drug (e.g., bisantrene or a derivative or analog thereof); (ii) a pharma- ceutically acceptable oily vehicle or oil selected from the group consisting of (a) naturally occurring vegetable oils and (b) semi-synthetic mono-, di-, and triglycerides, which does not contain BHT or BHA; (iii) a surfactant or emulsifier; (iv) a co-surfactant or emulsifier; and (v) a C6-C7 surfactant or emulsifier if the hydrophobic drug is basic. 20Disclosed is a composition for intravenous, intramuscular or intraarticular delivery of hydrophobic drugs, comprising an ion pairing agent selected from saturated or unsaturated fatty acids, and, if the hydrophobic drug is acidic, a pharma- ceutically acceptable aromatic amine, and (vi) water.Lawter et al., U.S. Patent No. 5,000,886 and Lawter et al., U.S. Patent No. 5,143,661 disclose compositions for delivery of pharmaceuticals, such as bisantrene or its derivatives or analogs, comprising microcapsules, the microcapsules comprising a curing agent that is a volatile silicone fluid.Murdock et al., U.S. Patent No. 5,070,082, Murdock et al., U.S. Patent No. 5,077,282, and Murdock et al., U.S. Patent No. 5,077,283 disclose prodrug forms of poorly soluble hydrophobic drugs, including bisantrene and derivatives and analogs, that are salts of phosphoramidic acids. No. 5,116,827 to Murdock et al. and U.S. Patent No. 5,212,291 to Murdock et al. disclose prodrug forms of poorly soluble hydrophobic drugs including quinoline carboxylic acid derivatives, bisanthrene and derivatives and analogs. U.S. Patent No. 5,378,456 to Tsou includes compositions containing anthracene antitumor agents such as bisanthrene or its derivatives or analogs conjugated or mixed with divinyl ether maleic acid (MVE) copolymers. U.S. Patent No. 5,609,867 to Tsou discloses polymeric 1,4-bis derivatives of bisanthrene and copolymers of bisanthrene with another monomer such as a dianhydride.

[0057] The methods and compositions described herein may use derivatives or analogs of bisantrene instead of bisantrene itself, such as those described in U.S. Patent No. 10,500,19 and U.S. Patent No. 10,548,876 to Garner et al.

[0058] Methods of Treating ccRCC and Other VHL(-) Cancers Surprisingly, this study shows that protein kinase inhibitors and bisantrene (or a pharma- ceutically acceptable salt thereof) act synergistically in ccRCC cell lines. In the methods and compositions of the present invention, bisantrene derivatives having substantially the same activity are also expected to act synergistically with protein kinases.

[0059] Thus, one aspect of the invention provides a method of treating a patient with ccRCC or other VHL(-) cancer, particularly protein kinase inhibitor-resistant ccRCC, comprising administering to the patient a therapeutically effective amount of a protein kinase inhibitor, such as a tyrosine kinase inhibitor, and a therapeutically effective amount of at least one second agent comprising bisantrene or a derivative thereof, or a pharma- ceutical acceptable salt of bisantrene or a derivative thereof.

[0060] The protein kinase inhibitor can inhibit one or more potential protein kinase targets associated with ccRCC and other VHL(-) cancers. According to certain embodiments, the protein kinase inhibitor inhibits. According to certain embodiments, the protein kinase inhibitor inhibits at least one or more of VEGFR1, VEGFR2, VEGFR3, DDR1, DDR2, RET, and RIPK2. According to certain embodiments, the protein kinase inhibitor inhibits at least one or more of VEGFR1, VEGFR2, and VEGFR3.

[0061] According to certain embodiments, the at least one protein kinase inhibitor is selected from the group consisting of pazopanib, lenvatinib, cabozantinib, everolimus, sorafenib, sunitinib, temsirolimus, mitomycin C, axitinib, tivozanib, and velzutifan.

[0062] According to specific embodiments, the at least one protein kinase inhibitor is selected from the group consisting of pazopanib, lenvatinib, cabozantinib, sorafenib, sunitinib, mitomycin C, axitinib, tivozanib, and velzutifan.

[0063] According to more specific embodiments of the present invention, the at least one protein kinase inhibitor is selected from the group consisting of pazopanib, lenvatinib, cabozantinib, sorafenib, sunitinib, axitinib, and tivozanib.

[0064] According to even more specific embodiments of the present invention, the at least one protein kinase inhibitor is selected from the group consisting of pazopanib, lenvatinib, and cabozantinib.

[0065] According to an alternative embodiment, the at least one protein kinase inhibitor is an inhibitor of mTOR optionally selected from temsirolimus and everolimus.

[0066] According to a specific embodiment of the invention, the second agent is bisantrene or a pharma- ceutically acceptable salt thereof.

[0067] According to certain embodiments, bisantrene can be administered as a drug compound or as a component of a pharmaceutical composition, as further described below.

[0068] According to certain specific embodiments, the method comprises administering to the patient a therapeutically effective amount of a protein kinase inhibitor and a therapeutically effective amount of bisantrene or a pharma- ceutically acceptable salt thereof.

[0069] The pharmaceutical compositions and medicaments of the present invention may be administered to a subject by standard enteral or parenteral routes, including but not limited to injection (e.g., intravenous, subcutaneous, intramuscular, bolus administration, etc.), or by routes of administration, such as topical, oral, sublingual, nasal, pulmonary, otic, rectal, or vaginal. In some embodiments, the pharmaceutical compositions of the present invention may be administered to a subject alone or in combination with other pharmaceutical compositions. In the latter case, administration may be simultaneous or sequential, or administration of the pharmaceutical compositions may be independent of one another.

[0070] In certain embodiments, bisantrene (or a derivative, or a pharma- ceutically acceptable salt thereof) is administered intravenously, whether centrally or peripherally, by intramuscular, subcutaneous and / or intradermal injection.

[0071] In general, the pharmaceutical compositions and medicaments of the present invention can be administered in a manner that is compatible with the route of administration and the physical characteristics (including health status) of the subject, and that induces the desired effect (i.e., therapeutic efficacy and / or prophylactic effect). For example, the appropriate dosage may depend on a variety of factors, including, but not limited to, the physical characteristics of the subject (e.g., age, weight, sex), whether the composition or medicament is used as a single agent, the progression of the disease or disorder being treated (i.e., pathological state), and other factors that will be readily apparent to one of skill in the art.

[0072] The appropriate dosage, frequency, duration and route of administration of the chemotherapeutic agent are known in the art. As suggested by FIG. 16, bisantrene, derivatives of bisantrene or pharmaceutically acceptable salts thereof may be administered in the same pharmaceutical composition as the protein kinase inhibitor, or in a separate composition from the protein kinase inhibitor at the same time or at a different time. When bisantrene, derivatives of bisantrene or pharmaceutically acceptable salts of bisantrene or derivatives of bisantrene are administered at a different time from the protein kinase inhibitor, they may be administered before or after the protein kinase inhibitor, and / or at a different time, according to a different time and / or frequency scheme. Those skilled in the art can determine the appropriate administration schedule based on variables such as the subject's age, weight and sex, the subject's susceptibility to the active agent side effects, genetic markers, the dosage of the active agent, the subject's past history of using the active agent, and other pharmacokinetic parameters such as cardiac, hepatic or renal function.

[0073] The methods and compositions provided herein allow subjects to receive treatment more frequently without significantly changing the administration scheme due to the risk of side effects such as cardiac toxicity.The dosage of protein kinase inhibitor, bisantrene, derivatives of bisantrene, or pharmaceutically acceptable salts of bisantrene or derivatives of bisantrene may be administered to the subject once or multiple times per day.In some cases, the daily dosage of chemotherapy may be administered in a single dose with bisantrene, derivatives of bisantrene, or pharmaceutically acceptable salts thereof.

[0074] The pharmaceutical compositions described herein may be administered to a patient once or multiple times per day. In some cases, the pharmaceutical compositions may be administered to a patient once per day. In some cases, the pharmaceutical compositions may be administered to a patient at least two, three, four, five or six times per day. For example, the pharmaceutical compositions may be administered to a patient three times per day.

[0075] In the methods described herein, the appropriate dosage of bisantrene (or a derivative of bisantrene, or a pharma- ceutically acceptable salt thereof) can be determined by one skilled in the art. The selected dosage level depends on various pharmacokinetic factors, including the amount of active agent being administered, the route of administration, the time of administration, the excretion rate of the particular compound being used, the severity of the disease state, other health considerations affecting the subject, and the state of the liver and kidney function of the subject. The selected dosage level also depends on the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular therapeutic agent being used, as well as the age, weight, disease state, general health, medical history, and similar factors of the subject being treated. Methods for determining optimal dosages are described in the art, for example, in Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th Edition, 2000, and Gilman et al. (eds.), (1990), "Goodman And Gilman's: The Pharmacological Bases of Therapeutics", Pergamon Press. Optimal dosages for a given set of disease conditions can be determined by one of skill in the art using conventional dosage-ranging tests in view of the drug's experimental data.

[0076] According to certain embodiments, the administration of bisantrene is about 0.1 mg / m 2 / day ~ approx. 400mg / m 2 / day, e.g., about 0.2 mg / m 2 / day ~ approx. 300mg / m 2 / day, about 0.5mg / m 2 / day~about 200mg / m 2 / day, about 0.5mg / m 2 / day~about 100mg / m 2 / day, about 0.5mg / m 2 / day~about 50mg / m 2 / day, about 0.5mg / m 2 / day ~ approx. 30mg / m 2 / day, about 0.5mg / m 2 / day~about 20mg / m 2 / day, approximately 1.0 mg / m 2 / day~about 10mg / m 2 / day, approximately 1.0 mg / m 2 / day~about 8mg / m 2 / day, approximately 1mg / m 2 / day, about 2mg / m 2 / day, about 3mg / m 2 / day, approximately 4mg / m 2 / day, about 5mg / m 2 / day, approximately 6mg / m 2 / day, approximately 7mg / m 2 / day, or approximately 10 mg / m 2 In some embodiments, bisantren is administered daily or weekly, once every two weeks, once every three weeks, once every four weeks, for example, over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 35, 42, 49, 56, or 63 days. In certain embodiments, bisantren is administered at a dose of about 20-50 mg / m2 once or more times over a period of 28 days, optionally once or more times daily or weekly, once every two weeks, once every three weeks, once every four weeks. 2 / 28 day dosage. Administration of bisantren or a pharma- ceutically acceptable salt thereof or a pharma- ceutically acceptable salt thereof may be carried out at similar dosage rates and adjusted for molar equivalence.

[0077] Dosing schemes for protein kinase inhibitors will depend on the particular protein kinase inhibitor and have been published for known protein kinase inhibitors, including for treating ccRCC and other VHL(-) cancers, and appropriate dosages for a given protein kinase inhibitor can be readily determined by one of skill in the art using the known approaches and techniques described above for bisantrene.

[0078] According to certain embodiments, as a result of the synergistic biological activity observed when bisantrene (or a derivative of bisantrene, or a pharma- ceutically acceptable salt thereof) is administered with a protein kinase inhibitor, a therapeutic result can be achieved more effectively for a given dosage ratio of the protein kinase inhibitor. Such biological activity of bisantrene (or a derivative of bisantrene, or a pharma- ceutically acceptable salt thereof) may also enable the use or extended use of protein kinase inhibitors that may have adverse side effects, such as cardiotoxicity, at normal dosages.

[0079] Alternatively, when administered in combination with bisantrene (or a derivative of bisantrene, or a pharma- ceutically acceptable salt thereof), the protein kinase inhibitor can be administered to a patient for an extended period of time at a lower dose than would normally be administered, while still maintaining a relatively sustained therapeutic effect. This is particularly important for active agents that have adverse side effects, such as cardiotoxicity. Thus, in certain embodiments of the method of the present invention, when administered in combination with bisantrene (or a derivative of bisantrene, or a pharma-ceutically acceptable salt thereof), the protein kinase inhibitor can be administered at a rate that is significantly lower than the recommended dosage rate of the protein kinase inhibitor when administered alone, for example, at least 10%, 20%, 30%, 40%, 50%, 60% or 70% lower than the dosage rate of the protein kinase inhibitor when administered alone.

[0080] Additional activators Programmed cell death 1 ligand 1 (PDL1) and its receptor programmed cell death 1 (PD1) regulate the activation of immune cells. Checkpoint inhibitors can help suppress this regulation so that immune cells can destroy tumors. One emerging strategy is to combine checkpoint inhibitors with protein kinase inhibitors, particularly those that target VEGFR and other kinases involved in angiogenesis, the underlying principle being that angiogenesis is important in creating an immunosuppressive environment. Known checkpoint inhibitors include antibodies that target PD1 or PDL1.

[0081] The FDA has approved two PD-1 inhibitors (nivolumab and pembrolizumab), one PD-L1 inhibitor (avelumab), and one CTLA-4 inhibitor (ipilimumab) for use in ccRCC. Other PD-1 / PD-L1 immune checkpoint inhibitors of potential consideration include atezolizumab, bevacizumab, cemiplimab, dostallimab, and durvalumab.

[0082] Additionally, some studies have shown that protein kinase inhibitors themselves can stimulate immune responses against tumors, potentially resulting in synergistic responses when combined with checkpoint inhibitors.

[0083] Similarly, immunomodulatory agents such as cytokines that regulate the maturation, growth and activation of immune cells, including interleukin-2 (Aldesleukin / Proleukin®), interferon alpha-2a, interferon alpha-2b (and pegylated interferon alpha-2b), and granulocyte-macrophage colony-stimulating factor (GM-CSF), have been approved for the treatment of various cancers through immune stimulation. Interleukin-2 (or the recombinant human version, Aldesleukin / Proleukin®) has been approved for the treatment of RCC.

[0084] Thus, the methods of the present invention further comprise administering at least one additional therapeutic agent for treating ccRCC or other VHL(-) cancer.

[0085] According to certain embodiments, the additional therapeutic agent is a checkpoint inhibitor or an immunomodulatory agent, such as those described above. According to certain embodiments, the at least one additional therapeutic agent comprises a checkpoint inhibitor selected from the group including nivolumab, pembrolizumab, avelumab, ipilimumab, atezolizumab, bevacizumab, cemiplimab, dostallimab and durvalumab, or an immunomodulatory agent selected from interleukin-2 (Aldesleukin / Proleukin®), interferon alpha-2a, interferon alpha-2b (and pegylated interferon alpha-2b) and GM-CSF. According to certain embodiments, the at least one additional therapeutic agent comprises a checkpoint inhibitor selected from the group including nivolumab, pembrolizumab, avelumab and ipilimumab. According to certain embodiments, the at least one additional therapeutic agent comprises interleukin-2 (Aldesleukin / Proleukin®).

[0086] The appropriate dosages, frequency, duration and routes of administration of these additional therapeutic agents are known in the art. As suggested by FIG. 17, these additional therapeutic agents can be administered simultaneously with bisantrene or a derivative or analog of bisantrene, or at a different time than bisantrene, a derivative or analog of bisantrene. If the additional therapeutic agents are administered at a different time than bisantrene or a derivative or analog of bisantrene, they can be administered before or after, and / or at a different time, and can be administered according to a different time and / or frequency scheme. Similarly, these additional therapeutic agents may be administered simultaneously with the protein kinase inhibitor, or at a different time than the protein kinase inhibitor, independent of consideration of bisantrene. If the additional therapeutic agents are administered at a different time than the protein kinase inhibitor, they can be administered before or after, and / or at a different time, and can be administered according to a different time and / or frequency scheme. Those skilled in the art can determine the appropriate administration schedule based on variables such as the age, weight and sex of the patient, the severity of the cancer, genetic markers as further described below, and pharmacokinetic parameters of liver and kidney function.

[0087] FIG. 16 shows a rational example of a bisantrene and targeted drug combination study design to overcome treatment resistance in ccRCC and other VHL(−) cancers.

[0088] FIG. 17 shows a rational example of a bisantrene and checkpoint inhibitor combination trial design to overcome immunotherapy resistance in ccRCC and other VHL(−) cancers.

[0089] Pharmaceutical compositions for treating ccRCC and other VHL(-) cancers The present invention also provides pharmaceutical compositions for treating ccRCC or other VHL(-) cancers, comprising at least one protein kinase inhibitor as described above and a second agent comprising bisantrene or a derivative thereof, or a pharma- ceutical acceptable salt of bisantrene or a derivative thereof. As described above, the compositions of the present invention may comprise an additional therapeutic agent selected from a checkpoint inhibitor and an immunomodulatory agent.

[0090] According to certain embodiments, the protein kinase inhibitor is a tyrosine kinase inhibitor. According to certain embodiments, the protein kinase inhibitor inhibits at least one or more of VEGFR1, VEGFR2, VEGFR3, DDR1, DDR2, RET and RIPK2. According to certain embodiments, the protein kinase inhibitor inhibits at least one or more of VEGFR1, VEGFR2 and VEGFR3.

[0091] According to certain embodiments, the at least one protein kinase inhibitor is selected from the group consisting of pazopanib, lenvatinib, cabozantinib, everolimus, sorafenib, sunitinib, temsirolimus, mitomycin C, axitinib, tivozanib, and velzutifan.

[0092] According to a specific embodiment of the invention, the at least one protein kinase inhibitor is selected from the group consisting of pazopanib, lenvatinib, and cabozantinib.

[0093] According to a specific embodiment of the invention, the second agent is bisantrene or a pharma- ceutically acceptable salt thereof.

[0094] According to certain specific embodiments of the invention, the composition may comprise a therapeutically effective amount of one protein kinase inhibitor and a therapeutically effective amount of bisantrene or a pharma- ceutically acceptable salt thereof.

[0095] The compositions of the present invention may further comprise administration of at least one additional therapeutic agent for the treatment of ccRCC or other VHL(-) cancers.

[0096] According to certain embodiments, the additional therapeutic agent is a checkpoint inhibitor or an immunomodulatory agent, such as those described above. According to certain embodiments, the at least one additional therapeutic agent comprises a checkpoint inhibitor selected from the group including nivolumab, pembrolizumab, avelumab, ipilimumab, atezolizumab, bevacizumab, cemiplimab, dostallimab and durvalumab, or an immunomodulatory agent selected from interleukin-2 (Aldesleukin / Proleukin®), interferon alpha-2a, interferon alpha-2b (and pegylated interferon alpha-2b) and GM-CSF. According to certain embodiments, the at least one additional therapeutic agent comprises a checkpoint inhibitor selected from the group including nivolumab, pembrolizumab, avelumab and ipilimumab. According to certain embodiments, the at least one additional therapeutic agent comprises interleukin-2 (Aldesleukin / Proleukin®).

[0097] The compositions of the present invention may be administered by any route, in a form suitable for this route, as known in the art. Thus, the compositions of the present invention may be suitable for administration by enteral or parenteral routes, including injection (e.g., intravenous, subcutaneous, intramuscular, bolus administration, etc.), or by, for example, topical, oral, sublingual, nasal, pulmonary, otic, rectal or vaginal administration routes.

[0098] Typically, the pharmaceutical compositions described herein include at least one pharma- ceutically acceptable carrier or excipient and / or diluent. For preparing pharmaceutical compositions and medicaments, the inert, pharma- ceutically acceptable carrier may be either solid or liquid. Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water-propylene glycol solutions for parenteral injection. Also included are solid form preparations, such as tablets, or amorphous or crystalline powders, including lyophilized preparations, intended to be converted to liquid form preparations immediately prior to use for oral or injectable administration. Such liquid forms include solutions, suspensions, and emulsions. Examples of pharma- ceutical acceptable carriers and methods of preparation of various compositions may be found, for example, in "Remington: The Science and Practice of Pharmacy", Mack Publishing Co., 20th Edition, 2000, and Gilman et al. (eds.), (1990), "Goodman And Gilman's: The Pharmacological Bases of Therapeutics", Pergamon Press.

[0099] Pharmaceutically acceptable carriers and excipients include (i) a liquid carrier; (ii) an isotonic agent; (iii) a wetting or emulsifying agent, (iv) preservatives, (v) a buffering agent, (vi) acidifying agent; (vii) antioxidants, (viii) an alkalizing agent, (ix) a carrier agent, (x) a chelating agent, (xi) colorants, (xii) a complexing agent, (xiii) a solvent, (xiv) suspending and / or thickening agents, (xv) oil; (xvi) penetration enhancers, (xvii) a polymer, (xviii) a curing agent, (xix) a protein, (xx) carbohydrates, (xxi) fillers, and (xxii) Lubricants Includes.

[0100] Other pharma- ceutically acceptable carriers and excipients known in the art may also be used.

[0101] Carriers, diluents, excipients and adjuvants must be "acceptable" in terms of compatibility with other ingredients of the composition or pharmaceutical preparation and generally are not harmful to the subject. Non-limiting examples of pharma- ceutically acceptable carriers or diluents include demineralized or distilled water; saline; vegetable oils, such as peanut oil, safflower oil, olive oil, cottonseed oil, corn oil; sesame oil, such as peanut oil, safflower oil, olive oil, cottonseed oil, corn oil, sesame oil, peanut oil or coconut oil; silicone oils, including polysiloxanes, such as methylpolysiloxane, phenylpolysiloxane and methylphenylpolysorboxane; volatile silicones; mineral oils, such as liquid paraffin, soft paraffin or squalane; cellulose derivatives, such as methylcellulose, ethylcellulose, carboxymethyl ... Examples of suitable carriers include sodium cellulose or hydroxypropyl methylcellulose; lower alkanols such as ethanol or isopropanol; lower aralkanols such as ethanol or isopropanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols such as polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrrolidone; agar; gum tragacanth or gum acacia, and petrolatum. Typically, the carrier will constitute from about 10% to about 99.9% by weight of the composition, vaccine or medicament.

[0102] For administration as an injectable solution or suspension, non-toxic parenterally acceptable diluents or carriers can include Ringer's solution, isotonic saline, phosphate buffered saline, ethanol and 1,2 propylene glycol. Methods for preparing parenterally administrable pharmaceutical compositions and medicaments are clear to those skilled in the art and are described in detail, for example, in "Remington: The Science and Practice of Pharmacy", Mack Publishing Co., 20th Edition, 2000, Gilman et al. (eds.), (1990), "Goodman And Gilman's: The Pharmacological Bases of Therapeutics", Pergamon Press.

[0103] For oral administration, some examples of suitable carriers, diluents, excipients and adjuvants include peanut oil, liquid paraffin, sodium carboxymethylcellulose, methylcellulose, sodium alginate, gum acacia, gum tragacanth, dextrose, sucrose, sorbitol, mannitol, gelatin and lecithin.These oral preparations may also contain suitable flavorings and colorings.When used in capsule form, capsules can be coated with compounds such as glyceryl monostearate or glyceryl stearate that delay disintegration.

[0104] Solid forms for oral administration may contain binders, sweeteners, disintegrants, diluents, flavorings, coatings, preservatives, lubricants and / or time delay agents acceptable to human and veterinary pharmaceutical practice. Suitable binders include gum acacia, gelatin, corn starch, gum tragacanth, sodium alginate, carboxymethylcellulose or polyethylene glycol. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharin. Suitable disintegrants include corn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate. Suitable flavorings include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavorings. Suitable coating agents include polymers or copolymers of acrylic and / or methacrylic acid and / or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methylparaben, propylparaben or sodium bisulfite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.

[0105] Liquid forms for oral administration can contain, in addition to the above-mentioned agents, liquid carriers.Suitable liquid carriers include water, oils such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, peanut oil, coconut oil, liquid paraffin, ethylene glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol, glycerol, fatty alcohols, triglycerides or mixtures thereof.

[0106] Suspensions for oral administration may further include dispersing agents and / or suspending agents. Suitable suspending agents include sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, polyvinylpyrrolidone, sodium alginate or acetyl alcohol. Suitable dispersing agents include lecithin, polyoxyethylene esters of fatty acids such as stearic acid, polyoxyethylene sorbitol monooleate or dioleate, polyoxyethylene sorbitol monostearate or distearate or polyoxyethylene sorbitol monolaurate or dilaurate, polyoxyethylene sorbitan monooleate or dioleate, polyoxyethylene sorbitan monostearate or distearate or polyoxyethylene sorbitan monolaurate or dilaurate, and the like.

[0107] In certain embodiments, the pharmaceutical composition may include liposomes. A suitable liposome formulation for bisantrene or its cardioprotective derivatives comprises small unilamellar or multilamellar liposomes in the size range of 0.01-100 μM, and about 50-95% liposome-encapsulated bisantrene, and consists of hydrogenated soy phosphatidylcholine, distearoylphosphatidylglycerol, and cholesterol of natural or synthetic origin lipids, which in aqueous solution can be reconstituted from a lyophilized form into an injectable liposomal suspension. The composition is prepared by reconstituting the lyophilized bisantrene / liposome composition into a liposome concentrate, and then diluting the concentrate for parenteral administration for the treatment of melanoma.

[0108] In yet another embodiment, the pharmaceutical composition may include a complex with β-cyclodextrin. A suitable liposomal formulation for bisantrene, a therapeutically active derivative of bisantrene, or a pharma- ceutically acceptable salt of bisantrene or its derivatives includes a complex formed in an aqueous solution, which can be reconstituted from a lyophilized form into an injectable suspension. One such composition is prepared by reconstituting a lyophilized bisantrene / β-cyclodextrin composition into a concentrate, and then diluting the concentrate for parenteral administration. β-cyclodextrin complexes and methods for preparing the complexes are known in the art and are described, for example, in WO 2019 / 073296 to Rothman.

[0109] Various formulations suitable for administration of bisantrene, therapeutically active derivatives of bisantrene, or pharma- ceutically acceptable salts of bisantrene or its derivatives are known in the art. U.S. Patent No. 4,784,845 to Desai et al. discloses a composition for delivering a hydrophobic drug, comprising: (i) a hydrophobic drug (i.e., bisantrene or its cardioprotective derivatives), (ii) an oily excipient or oil phase substantially free of butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT), (iii) a cosurfactant or emulsifier, (iv) a cosurfactant or coemulsifier, and (v) benzyl alcohol as a cosolvent. U.S. Patent No. 4,816,247 to Desai et al. discloses a method for the preparation of a glycerol-based emulsion comprising: (i) a hydrophobic drug (e.g., bisantrene or its cardioprotective derivatives or analogs); (ii) a pharma- ceutically acceptable oily vehicle or oil selected from the group consisting of (a) naturally occurring vegetable oils and (b) semi-synthetic mono-, di-, and triglycerides, which does not contain BHT or BHA; (iii) a surfactant or emulsifier; (iv) a co-surfactant or emulsifier; and (v) a C6-C7 surfactant or emulsifier if the hydrophobic drug is basic. 20 Disclosed is a composition for delivering a hydrophobic drug via an intravenous, intramuscular or intra-articular route, comprising: (i) an ion pairing agent selected from a saturated or unsaturated fatty acid, and if the hydrophobic drug is acidic, a pharma- ceutically acceptable aromatic amine; and (ii) water.

[0110] According to a particular embodiment, the compositions of the present invention have a concentration of about 0.1 mg / m 2 / day~about 100mg / m 2 / day, e.g., about 0.2 mg / m 2 / day~about 50mg / m 2 / day, about 0.5mg / m 2 / day~about 20mg / m 2 / day, approximately 1.0 mg / m 2 / day~about 10mg / m 2 / day, approximately 1.0 mg / m 2 / day~about 8mg / m 2 / day, approximately 1mg / m 2 / day, about 2mg / m 2 / day, about 3mg / m 2 / day, approximately 4mg / m 2 / day, about 5mg / m 2 / day, approximately 6mg / m 2 / day, approximately 7mg / m 2 / day, or approximately 10 mg / m 2 In some embodiments, the compositions of the present invention may be suitable for administration of bisantrene at a dosage of about 20-50 mg / m2 / day, optionally once or more times per day or week, once every two weeks, once every three weeks, or once every four weeks, for a period of, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, 56 days, or 63 days. In certain embodiments, the compositions of the present invention may be suitable for administration of bisantrene at a dosage of about 20-50 mg / m2 / day, optionally once or more times per day or week, once every two weeks, once every three weeks, or once every four weeks, for a period of 28 days. 2 Compositions according to the invention comprising a pharma- ceutically acceptable salt of bisantrene or a therapeutically active derivative or pharma- ceutically acceptable salt thereof may be suitable for delivery at similar dosage rates and may be adjusted for molar equivalence.

[0111] The dosing scheme of protein kinase inhibitors depends on the specific protein kinase inhibitor and has been published for known protein kinase inhibitors, including for treating ccRCC. Also, the appropriate dose of a given protein kinase inhibitor can be easily determined by those skilled in the art using the known approaches and techniques described above for bisantrene. The compositions of the present invention can be prepared to contain protein kinases at their recommended dose ratios or different dose ratios as described below.

[0112] In certain embodiments, as a result of the synergistic biological activity observed when bisantrene (or a derivative of bisantrene, or a pharma- ceutically acceptable salt thereof) is administered with a protein kinase inhibitor, the dosage of the protein kinase in the compositions of the present invention may be lower than the dosage when administered alone to a patient. This is particularly important for active agents that have adverse side effects, such as cardiotoxicity. Thus, in certain embodiments of the compositions of the present invention, the protein kinase inhibitor may be included at a dosage significantly lower than the recommended dosage rate of the protein kinase inhibitor when administered alone, for example at least 10%, 20%, 30%, 40%, 50%, 60% or 70% lower than the dosage rate of the protein kinase inhibitor when administered alone.

[0113] Similarly, when a composition of the present invention includes an additional therapeutic agent that is a checkpoint inhibitor and / or an immunomodulatory agent, this additional therapeutic agent may be included in the composition at its known recommended ratio for treating ccRCC and other VHL(-) cancers, or even at a lower ratio if a synergistic effect between the components is determined.

[0114] Use of the composition for the manufacture of a medicament for treating ccRCC and other VHL(-) cancers The invention also provides the use of a composition according to the invention in the manufacture of a medicament for treating ccRCC or other VHL(-) cancer, particularly protein kinase inhibitor resistant ccRCC, in a patient.

[0115] The parameters of such manufacture are described above, including the protein kinase inhibitors, bisantrene, bisantrene derivatives and salts thereof, and checkpoint inhibitors and immunomodulators as optional additional therapeutic agents, as well as dosage / administration, dosage forms and other ingredients of all active agents.

[0116] Kits for treating ccRCC and other VHL(-) cancers The present invention also provides a kit for treating ccRCC or other VHL(-) cancers, particularly protein kinase-resistant ccRCC, comprising at least one protein kinase inhibitor and at least one second agent comprising bisantrene or a derivative thereof, or a pharma- ceutically acceptable salt of bisantrene or a derivative thereof.

[0117] The parameters of such kits, including protein kinase inhibitors, bisantrene, bisantrene derivatives and salts thereof, and checkpoint inhibitors and immunomodulators as optional additional therapeutic agents, as well as dosage / administration, dosage forms and other components of all active agents, are described above.

[0118] In some cases, the kit may also contain vials, tubes, needles, packaging or other materials.

[0119] Kits are provided that contain unit doses, usually injectable doses, of one or more of the active agents described herein.Such kits may include a container with the unit dose, an information insert that describes the use and associated benefits of the drug in treating disease, and optionally an instrument or device for delivering the composition.

[0120] The kit may further comprise any device suitable for administration of the composition. For example, the kit may comprise a needle suitable for intravenous administration.

[0121] In some cases, the kit may include instructions. The instructions may be provided on the kit or may be accessed electronically. The instructions may provide information on how to use the compositions of the present disclosure. The instructions may further provide information on how to use the devices of the present disclosure. The instructions may provide information on how to perform the methods of the present disclosure. In some cases, the instructions may provide administration information. The instructions may provide drug information such as mechanism of action, drug formulation, risk of complications, contraindications, etc. In some cases, the kit is purchased by a physician or medical professional and used for administration in a clinic or hospital. In some cases, the kit is purchased by a laboratory and used to screen candidate compounds.

[0122] Preferred forms of the invention will now be described, by way of example only, with reference to the following examples including comparative data which should not be construed as in any way limiting the scope or spirit of the invention. EXAMPLES

[0123] Example 1 Cell viability assays and analysis Cell viability was measured using a resazurin metabolic activity assay. Cells were plated at 1 × 10 in duplicate wells of a 96-well microtiter plate. 3 Cells / well (786-O, RCC4 EV, RCC4 VHL) or 3 × 10 3 Cells / well (Caki-1, Caki-2) were seeded and cultured for 24 hours.

[0124] Zantrene (bisantrene dihydrochloride) was reconstituted at 20 mM in dimethyl sulfoxide (DMSO) or 15 mM in 5% Captisol solution. Everolimus (RAD001), sunitinib, sorafenib, pazopanib, lenvatinib (mesylate), cabozantinib (BMS-907351) were reconstituted at 17.2 mM (lenvatinib) or 100 mM (all others) in DMSO.

[0125] Drugs were then diluted in medium, added to the wells and the cells cultured for an additional 72 hours.

[0126] Human kidney cell lines were cultured in the media shown in Table 1 below in a humidified chamber at 37° C. and 5% CO2.

[0127] [Table 1]

[0128] Viability was measured using the fluorescent viability dye resazurin (Ex 544 nm, Em 590 nm; 0.6 mM resazurin, 78 μM methylene blue, 1 mM potassium hexacyanoferrate(III), 1 mM potassium hexacyanoferrate(II) trihydrate (Sigma-Alldrich) dissolved in PBS [3]). Resazurin is metabolized to red fluorescent resorufin by metabolically active cells. Fluorescence was measured 5 h after addition of resazurin solution (1:10, v / v) under 544 nm excitation / 590 nm emission on a FLUOstar OPTIMA plate reader (BMG LabTechnologies). Graphs were generated using Graphpad Prism 9 software. Drug IC values ​​were calculated by cubic spline-lowess regression analysis using Prism 9. 50 Values ​​were measured. At least three independent replicates were performed for each cell line and each drug combination. Data are expressed as mean ± standard error (SEM). Culture conditions for each cell line were as follows:

[0129] Colony formation assay - A colony formation assay was used to measure the colony forming ability of cells treated with Zantrene. RCC4 EV and RCC4 VHL cells were seeded in 6-well plates at 1000 cells / well and allowed to attach for 24 hours. Zantrene was diluted in culture medium, added to the wells, and cells were cultured for 96 hours. Drug-containing medium was removed and replaced with fresh medium (without drug) and cultured for an additional 96 hours to allow cell colonies to form. At the end point, medium was removed from the wells and cells were washed twice with cold PBS. Cells were fixed with ice-cold methanol on ice for 10 minutes and then immediately stained with crystal violet solution (0.5% crystal violet, 25% methanol in PBS) at room temperature. Excess crystal violet solution was washed away and pictures of the plates were captured on a ChemiDoc MP imaging system (Bio-Rad). Images were analyzed using the ColonyArea plugin [Guzman C. et al. (2014), ColonyArea: an ImageJ plugin to automatically quantify colony formation in clonogenic assays, PLoS One, 9(3):e92444] for ImageJ [Schneider CA et al. (2012), NIH Image to ImageJ: 25 years of image analysis, Nat Methods 9(7):671-675], and the percentage of well area filled by colonies was measured using Prism 9 and expressed as % of untreated cells. Four independent replicates were performed for each cell line. Data are presented as mean ± SEM.

[0130] Synergy analysis - for drug combination therapy, Webb's fractional product method (Webb J(1963), "Effect of more than one inhibitor", Hochster ER, Quastel J.(eds), "Enzymes and metabolic inhibitors", Academic Press: New York), Chou-Talalay method (combination index, CI) (Chou TC._(2020), "Theoretical Basis, Experimental Design, and Computerized Simulation of Synergism and Antagonism in Drug Combination Studies", Pharmacological Reviews, 58(3):621-681), and SynergyFinder 2.0 software (Ianevski A, Giri AK, Aittokallio T(2020): "SynergyFinder 2.0: visual analytics of multi-drug combination synergies", Nucleic Acids Res., 48(W1):W488-W493) and the BLISS synergy method (Bliss (1939) "CI: The toxicity of poisons applied jointly", Ann App Biol, 26:585-615). Details of each method are provided below.

[0131] Webb's fractional product method: Webb introduced a method in 1963 (Web J(1963), "Effect of more than one inhibitor", in Hochster ER, Quastel J. (eds.), "Enzymes and metabolic inhibitors", Academic Press: New York), later called the "fractional product" method (Chou TC.(2020), "Theoretical Basis, Experimental Design, and Computerized Simulation of Synergism and Antagonism in Drug Combination Studies", Pharmacological Reviews, 58(3):621-681). It estimates the expected additive effect of two drugs using the fractional product of the effects of each drug alone, i.e., 1-Fa(drug 1 + drug 2) = (1-Fa drug 1) x (1-Fa drug 2) Fa = fraction of cells affected, expressed as a decimal

[0132] A result in which the observed Fa(drug 1 + drug 2) value is greater than the expected Fa(drug 1 + drug 2) value represents a synergistic effect, whereas a result in which the observed Fa(drug 1 + drug 2) value is less than the expected value represents an antagonistic effect. Thus, Webb's method takes into account the efficacy of each drug at a particular dose, but does not take into account the overall shape of the dose-response curve.

[10] The results are considered to be: A result value <-0.1 represents a synergistic effect. Results values ​​between −0.1 and 0.1 represent additive effects. A result value >0.1 indicates a countervailing effect.

[0133] Chou-Talalay method: In 1984, Chou and Talalay proposed the most widely used method of evaluating synergy to date (Chou TC and Talalay P (1984), "Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Advances in enzyme regulation", 1984, 22:27-55). Chou and Talalay derived the "median effective" equation using the Henderson-Hasselbalch, Michaelis-Menten, Scatchard, and Hill equations, and used the input dose and the corresponding dose effect to model the median effective dose and sigmoidality of the dose-response curve. Chou and Talalay also coined the term "combination index (CI)" to evaluate the degree of synergy between two compounds and defined it as follows:

number

[0134] The CI for any Fa can be estimated using the median effect equation.

[0135] Both the Chou Talalay and Webb methods are widely used and robust methods for measuring synergy. The Chou Talalay method is considered a more complex model, but it suggests using a fixed dose ratio for the combination of two drugs. Therefore, if this assumption is not met, we suggest using the Webb method.

[0136] Bliss method: SynergyFinder (version 2.0) is a standalone web application for interactively analyzing and visualizing multi-drug (two or more drugs) combination analysis data (Ianevski A, Giri AK, Aittokallio T (2020): "SynergyFinder 2.0: visual analytics of multi-drug combination synergies", Nucleic Acids Res., 48(W1):W488-W493). The degree of combination synergy or counteraction is quantified by comparing the observed drug combination response with the expected response, calculated using a reference model that assumes no interactions between drugs. In SynergyFinder, the Bliss model was used. This model quantifies the degree of synergy as a multiplicative effect of a single drug, as if the drugs acted independently. This independence model assumes a stochastic process in which the two drugs exert their effects independently and the expected combination effect can be calculated based on the probability of independent events. The synergy scores are as follows: Less than -10: The interaction between the two drugs is likely to be counteractive. -10 to 10: The interaction between the two drugs is likely to be additive. >10: The interaction between the 2 drugs is likely to be synergistic.

[0137] Example 2 Combination of Zantrene with currently used protein kinase inhibitors Three different methods were used to measure the effect of combination drug treatment. The Webb analysis method revealed the synergistic effect of multi-drug doses of Zantrene and protein kinase inhibitors in the 786-O cell line (Figures 1-3). The highest synergistic effect was observed for the combination of Zantrene with lenvatinib, cabozantinib, and pazopanib. Synergistic effects of doses of sunitinib, sorafenib, and everolimus were also observed, mostly at low doses. Similar results were observed in the other four cell lines, with Zantrene consistently showing the highest synergistic effect with lenvatinib and cabozantinib, followed by pazopanib (Figures 4-15).

[0138] Analysis using the Bliss method similarly revealed that Zantrene had an overall synergistic effect with Lenvatinib and Pazopanib in all five cell lines (Table 2). This method further revealed that Everolimus was synergistic in all five cell lines, and Cabozantinib was synergistic in all cell lines (but this was only marginal for Caki-2 cells). Sunitinib and Sorafenib were observed to have a marginal synergistic effect (Table 2). Bliss analysis can also provide a score of the most synergistic region among drug doses, which suggests the region of synergy of the tested drugs in all cell lines (Table 3). Individual Bliss scores for each dose combination further revealed multiple doses that were synergistic in the majority of cell lines.

[0139] [Table 2] Values ​​>10 are synergistic ( * ), and values ​​between -10 and 10 are considered additive ( & ), and values ​​less than -10 are considered to be offset ( # ) is considered to be

[0140] [Table 3] Values ​​>10 are synergistic ( *), and values ​​between -10 and 10 are considered additive ( & ), and values ​​less than -10 are considered to be offset ( # ) is considered to be

[0141] Finally, the Chou-Talalay analysis was used to quantify the combined effects of zantrene with each anti-RCC drug. This analysis was less rigorous than the Webb or Bliss analysis and confirmed the synergistic effects of almost all combinations at most doses. The IC 50 When considering the synergy scores in the study, synergy between Zantrene and sunitinib, pazopanib, lenvatinib, and cabozantinib was predicted (Table 4).

[0142] [Table 4] Combination index was calculated from ED50 using Chou-Talalay analysis. # ), additive effect ( & ), moderate synergy ( * ).

[0143] Zantrene showed the highest synergy with lenvatinib, cabozantinib and pazopanib in the ccRCC cells tested. This may indicate that these drugs with synergistic lethality with zantrene have a common cellular target. All of these drugs are VEGFR inhibitors that inhibit angiogenesis. In addition to inhibiting VEGFR on endothelial cells, these multikinase inhibitors also inhibit tumor growth due to the endogenous inhibition of other kinases by tumor cells. As our assays consisted of tumor cells only, the synergistic cytotoxicity observed with zantrene is undoubtedly due to kinase inhibition within tumor cells. Common targets of lenvatinib, cabozantinib and pazopanib include DDR2, DDR1, RET and RIPK2. RET was also identified as a target of sunitinib. The Caki-1 cell line was further added to the four cell line pool to analyze several drugs including cabozantinib, which further revealed that AXL and MET are targets of cabozantinib.

[0144] Example 3 Zantrene kills clear cell renal cell carcinoma and is more toxic to VHL(-) cells The results shown in Example 2 indicate that the combination results in greater synergy between bisantrene and protein kinase inhibitors when treating mutant VHL(-) cells (RCC4 EV cells) compared to the VHL rescue line (RCC4 VHL). As shown in Table 5 and Figure 18, these results are consistent with earlier observations that showed that bisantrene was not only toxic to all renal cancer cell lines (data not shown), but also surprisingly more toxic to VHL(-) mutant cells, RCC4 EV cells, compared to isogenic rescue (VHL(+)) RCC4 VHL cells.

[0145] [Table 5]

[0146] Using the long-term colony formation cell growth assay (cell colony formation) described in Example 1, which better measures the effect of a drug on cancer cell growth rather than cell killing, a higher lethality was also observed between VHL loss and bisantrene in RCC4 EV cells compared to RCC4 VHL cells.

[0147] The sensitivity of RCC-4 EV (VHL mutant) cells to bisantrene was significantly higher (2.9-fold) than wild-type VHL rescue cells (Table 6 and FIG. 19).

[0148] [Table 6]

[0149] Effect of the Invention The present invention provides a new paradigm for treating ccRCC and other VHL(-) cancers by administration of bisantrene, an antitumor agent with multiple mechanisms of action including DNA intercalation, inhibition of topoisomerase, inhibition of fat and obesity associated protein (FTO), and activation of the immune system. Bisanthrene is well tolerated and, in particular, has little cardiotoxicity, which is characteristic of some other anthracene derivatives. Bisanthrene can be used with other therapeutic agents used to treat ccRCC and other VHL(-) cancers.

[0150] The invention illustratively described herein can suitably be practiced without any elements and limitations not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," etc., should be read broadly and without limitation. As used herein, the use of the term "comprising" as a transitional phrase in the claims is intended to include the use of the narrower transitional phrases "consisting essentially of" or "consisting of," unless those transitional phrases are expressly excluded. Although the invention has been specifically disclosed by preferred embodiments and optional features, those skilled in the art may modify and vary the invention disclosed herein, and these modifications and variations are considered to be within the scope of the invention disclosed herein. The invention is described broadly and generically herein. Each narrower species and subgeneric grouping falling within the scope of the general disclosure also forms part of the invention. This includes the general description of the invention with a condition or negative limitation excluding any subject of that genus, regardless of whether the excluded material is specifically recited herein.

Claims

1. A composition for use in a method of treating a patient having ccRCC or other VHL(-) cancer, wherein the composition comprises bisanthren or a derivative thereof or a pharmaceutically acceptable salt of bisanthren or a derivative thereof, and the method comprises administering to the patient a therapeutically effective amount of the composition and a therapeutically effective amount of a protein kinase inhibitor.

2. The protein kinase inhibitor is a tyrosine kinase inhibitor, preferably inhibiting one or more of VEGFR1, VEGFR2, VEGFR3, DDR1, DDR2, RET, and RIPK2, preferably at least one of the protein kinase inhibitors. (a) Pazopanib, (b) Lenvatinib, (c) Cabozantinib, (d) Everolimus, (e) Sorafenib, (f) Sunitinib, (g) Temsirolimus, (h) Mitomycin C, (i) Axitinib, (j) tivozanib, and (k) Berzchifan Selected from the group consisting of, more preferably at least one of the protein kinase inhibitors, (a) Pazopanib, (b) Lenvatinib, and (c) Cabozantinib A composition for use according to claim 1, selected from the group consisting of the following.

3. The composition for use according to claim 1, comprising bisanthren or a pharmaceutically acceptable salt thereof.

4. The composition for use according to any one of claims 1 to 3, wherein the method comprises treating ccRCC and optionally treating protein kinase inhibitor-resistant ccRCC.

5. The method further comprises administering at least one additional therapeutic agent for treating ccRCC or other VHL(-) cancer, preferably the additional therapeutic agent being a checkpoint inhibitor or an immunomodulator, and more preferably at least one additional therapeutic agent is (a) Atezolizumab, (b) Avelumab, (c) Bevacizumab, (d) Semiprimab, (e) Dostallumab, (f) Durvalumab, (g) Interleukin-2, (h) Ipilimumab, (i) Nivolumab, (j) Pembrolizumab, and (k) Proleukin A composition for use according to any one of claims 1 to 3, selected from the group consisting of the following.

6. The composition for use according to any one of claims 1 to 3, wherein at least one of the protein kinase inhibitors is administered to the patient before, simultaneously with, or after the administration of the composition to the patient.

7. The composition for use according to claim 5, wherein at least one protein kinase inhibitor and the composition are administered to the patient simultaneously, and optionally, at least one protein kinase inhibitor is provided to the composition.

8. A pharmaceutical composition for treating ccRCC or other VHL(-) cancer, comprising at least one protein kinase inhibitor and a second agent comprising bisanthren or a derivative thereof, or a pharmaceutically acceptable salt of bisanthren or a derivative thereof.

9. The protein kinase inhibitor is a tyrosine kinase inhibitor, preferably inhibiting one or more of VEGFR1, VEGFR2, VEGFR3, DDR1, DDR2, RET, and RIPK2, and more preferably at least one of the protein kinase inhibitors is (a) Pazopanib, (b) Lenvatinib, (c) Cabozantinib, (d) Everolimus, (e) Sorafenib, (f) Sunitinib, (g) Temsirolimus, (h) Mitomycin C (i) Axitinib, (j) tivozanib, and (k) Berzchifan Selected from the group consisting of, more preferably at least one of the protein kinase inhibitors, (a) Pazopanib, (b) Lenvatinib, and (c) Cabozantinib A pharmaceutical composition according to claim 8, selected from the group consisting of the following.

10. The present invention further comprises at least one additional therapeutic agent for treating ccRCC or other VHL(-) cancer, preferably at least one of the additional therapeutic agents being a checkpoint inhibitor or an immunomodulator, and more preferably at least one of the additional therapeutic agents being (a) Atezolizumab, (b) Avelumab, (c) Bevacizumab, (d) Semiprimab, (e) Dostallumab, (f) Durvalumab, (g) Interleukin-2, (h) Ipilimumab, (i) Nivolumab, (j) Pembrolizumab, and (k) Proleukin A pharmaceutical composition according to claim 8, selected from the group consisting of the following.

11. A pharmaceutical composition according to any one of claims 8 to 10, comprising a therapeutically effective amount of one protein kinase inhibitor and a therapeutically effective amount of bisanthren or a pharmaceutically acceptable salt thereof.

12. A kit for treating ccRCC or other VHL(-) cancer, comprising at least one protein kinase inhibitor and at least one second agent comprising bisanthren or a derivative thereof, or a pharmaceutically acceptable salt of bisanthren or a derivative thereof.

13. The protein kinase inhibitor is a tyrosine kinase inhibitor, preferably inhibiting one or more of VEGFR1, VEGFR2, VEGFR3, DDR1, DDR2, RET, and RIPK2, and more preferably at least one of the protein kinase inhibitors is (a) Pazopanib, (b) Lenvatinib, (c) Cabozantinib, (d) Everolimus, (e) Sorafenib, (f) Sunitinib, (g) Temsirolimus, (h) Mitomycin C (i) Axitinib, (j) tivozanib, and (k) Berzchifan Selected from the group consisting of, more preferably at least one of the protein kinase inhibitors, (a) Pazopanib, (b) Lenvatinib, and (c) Cabozantinib A kit according to claim 12, selected from the group consisting of the following.

14. The kit according to claim 12, comprising one protein kinase inhibitor and bisantrene or a pharmaceutically acceptable salt thereof.

15. The present invention further comprises at least one additional therapeutic agent for treating ccRCC or other VHL(-) cancer, preferably the additional therapeutic agent being a checkpoint inhibitor or an immunomodulator, and more preferably at least one additional therapeutic agent is (a) Atezolizumab, (b) Avelumab, (c) Bevacizumab, (d) Semiprimab, (e) Dostallumab, (f) Durvalumab, (g) Interleukin-2, (h) Ipilimumab, (i) Nivolumab, (j) Pembrolizumab, and (k) Proleukin A kit according to any one of claims 12 to 14, selected from the group consisting of the following.

16. The kit according to any one of claims 12 to 14, comprising instructions for administering at least one of the protein kinase inhibitors to the patient before, simultaneously with, or after administering the second agent to the patient.

17. A kit according to any one of claims 12 to 14, comprising instructions for use for simultaneously administering at least one of the protein kinase inhibitors and the second agent to a patient.

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