Treatment of clear cell renal cell carcinoma

Abstract

The present invention provides a method of treating a patient with ccRCC and other VHL (-) cancer, the method comprising administering to the patient a therapeutically effective amount of a protein kinase inhibitor and a therapeutically effective amount of at least a second agent comprising a Bispheng or a derivative thereof, or a pharmaceutically acceptable salt of a Bispheng or a derivative thereof. The invention also provides pharmaceutical compositions and kits for the treatment of ccRCC and other VHL (-) cancers, as well as the use of such compositions for the preparation of medicaments for the treatment of 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 for the treatment of clear cell renal cell carcinoma (ccRCC) and other cancers with inactive von Hippel-Lindau (VHL) tumor suppressor genes. The present invention also contemplates pharmaceutical compositions and kits for such treatments, and the use of such compositions for the manufacture of medicaments for such treatments. Background Art

[0002] Kidney cancer is one of the top ten most common cancers. Renal cell carcinoma (RCC) accounts for 3.8% of all new cancers. Approximately 80-85% of renal cell carcinomas (RCC) are clear cell renal cell carcinomas (ccRCC). Another 15% to 20% are non-clear cell RCC, which includes a diverse group of histological subtypes, each with different molecular characteristics. 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 established risk factors for the development of RCC. There are also several inherited types of RCC, of ​​which VHL disease is the most common. VHL disease is caused by autosomal dominant mutations in the VHL gene that predispose individuals to ccRCC and a range of other cancers. VHL loss of function has also been shown to be important in sporadic ccRCC. A large number of studies have reported VHL loss of function changes, VHL(-), caused by allelic loss, mutations, and promoter methylation in more than 90% of sporadic ccRCC.

[0004] In the United States, there will be 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 the tumor, presence of regional lymph node metastases, and evidence of metastatic disease at presentation. RCC metastasizes primarily to the lungs, lymph nodes, bones, liver, adrenal glands, and brain. The 5-year survival rate for metastatic ccRCC is as low as 12%.

[0005] Surgery is the first line of treatment for stage I to III ccRCC, with cytoreductive nephrectomy performed first, followed by systemic therapy usually used to treat metastatic disease. RCC is not highly responsive 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 potent activity against VEGF receptors (e.g., axitinib, cabozantinib, lenvatinib, pazopanib, sorafenib, sunitinib, and tivozanib), and oral tyrosine kinase inhibitors with potent activity against mammalian targets of rapamycin inhibitors (everolimus and temsirolimus) have provided new treatment options.

[0006] Most ccRCC tumor cells express PD-L1 on their cell membranes, helping them evade immune attack. Immune checkpoint inhibitors PD-1 inhibition or PD-L1 inhibition blocks this pathway, releasing the immune system’s “off switch” and enhancing the ability of cytotoxic T cells to kill tumor cells. CTLA-4 inhibition blocks autoreactive T cells during the immune sensitization phase, thereby supporting the activation and proliferation of effector T cells. The FDA has approved two PD-1 inhibitors (nivolumab and pembrolizumab), one PD-L1 inhibitor (avelumab), and one CTLA-4 inhibitor (ipilimumab) for ccRCC. Other PD-1 / PD-L1 immune checkpoint inhibitors that may be considered include atezolizumab, bevacizumab, cemiplimab, dostarlimab, and durvalumab.

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

[0008] The present invention provides a new way to treat ccRCC and other VHL(-) cancers by co-administering bisantrene with a protein kinase inhibitor. This meets the need for new therapies that improve clinical outcomes for patients with ccRCC and other VHL(-) cancers, particularly those with protein kinase inhibitor-resistant ccRCC.

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

[0010] Surprisingly, during the course of these studies it was discovered that bisantrene and pharmaceutically acceptable salts thereof act synergistically with protein kinase inhibitors against clear cell renal carcinoma cells.

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

[0012] According to certain embodiments, the protein kinase inhibitor inhibits one or more than one 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 selected from:

[0014] (a) Pazopanib;

[0015] (b) lenvatinib;

[0016] (c) cabozantinib;

[0017] (d) everolimus;

[0018] (e) sorafenib;

[0019] (f) sunitinib;

[0020] (g) temsirolimus;

[0021] (h) Mitomycin C;

[0022] (i) axitinib;

[0023] (j) tivozanib; and

[0024] (k) Belzutifan.

[0025] According to certain embodiments, the at least one protein kinase inhibitor is selected from:

[0026] (a) Pazopanib;

[0027] (b) lenvatinib; and

[0028] (c) Cabozantinib.

[0029] According to certain embodiments, the second agent is bisantrene or a pharmaceutically acceptable salt thereof.

[0030] 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 pharmaceutically acceptable salt thereof.

[0031] According to certain embodiments, the method comprises treating protein kinase inhibitor-resistant ccRCC.

[0032] The method may further include a step of determining the VHL mutation and epigenetic state of the patient's cancer. The VHL mutation or epigenetic state can be determined before treatment begins or during treatment as a marker of treatment progress. The VHL mutation or epigenetic state of the cancer can be determined by next generation sequencing, by RT-PCR, by ELISA assay using a suitable antibody for VHL, by RNA hybridization assay or any other method known in the art.

[0033] According to certain embodiments, the method may further include administering at least one additional therapeutic agent for treating ccRCC or other VHL(-) cancers, optionally a checkpoint inhibitor drug or an immunomodulator. The at least one additional therapeutic agent is selected from atezolizumab, avelumab, bevacizumab, cemiplimab, dostarlimab, durvaluma, interleukin-2, ipilimumab, nivolumab, pembrolizumab, and proleukin.

[0034] According to certain embodiments, the method may include administering to the patient the at least one protein kinase inhibitor prior to, simultaneously with, or after administering to the patient the second agent.

[0035] According to other embodiments, the method may comprise administering to the patient the at least one protein kinase inhibitor and the second agent simultaneously, optionally in a single composition.

[0036] According to certain embodiments, the treatment has a synergistic outcome for ccRCC and other VHL(-) cancers compared to a method of administering the at least one protein kinase or the second agent alone. In certain embodiments, the dose of the at least one protein kinase inhibitor is at least 20% lower than the dose of the at least one protein kinase inhibitor agent required to achieve the same target outcome when not administered with the second agent.

[0037] Another aspect of the present invention provides a pharmaceutical composition for treating ccRCC or other VHL(-) cancers by the embodiments of the treatment methods described above, the composition comprising at least one protein kinase inhibitor as described above and a second agent as described above, the protein kinase inhibitor may be a tyrosine kinase inhibitor, the second agent comprising bisantrene or a derivative thereof, or a pharmaceutically acceptable salt of bisantrene or a derivative thereof. The composition may comprise other active agents as described above.

[0038] Another aspect of the present invention provides the use of a composition according to the present invention as described above for the preparation of a medicament for treating ccRCC or other VHL(-) cancer, optionally protein kinase inhibitor-resistant ccRCC, in a patient.

[0039] Another aspect of the present invention provides a kit for treating ccRCC or other VHL(-) cancers, optionally protein kinase inhibitor-resistant ccRCC, by an embodiment of the method of the present invention as described above, the kit comprising at least one protein kinase inhibitor as described above (optionally a tyrosine kinase inhibitor) and at least one second agent as described above, the second agent comprising bisantrene or a derivative thereof, or a pharmaceutically acceptable salt of bisantrene or a derivative thereof. The kit according to the present invention may include instructions for administering the at least one protein kinase inhibitor to the patient before, simultaneously with, or after the administration of the second agent to the patient. Alternatively, the kit according to the present invention may include instructions for administering the at least one protein kinase inhibitor and the second agent to the patient simultaneously. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The following invention, in which the term "Zan" or "Zantrene" is an alternative term for bisantrene, will be better understood with reference to the specification, appended claims and drawings.

[0041] Figure 1 Weber analysis of bisantrene-everolimus or -sunitinib drug combinations in 786-O cells is shown. A) Cell viability in response to the indicated different dose ranges of everolimus and sunitinib in combination with bisantrene. Experimental data for each drug alone and the combination are shown. The "expected value" is calculated using the Weber method and shows the expected value if the drug combination is additive. Therefore, any experimental value below this line is considered synergistic. Equal to or close to the line is additive; above the line is antagonistic. B) Weber analysis of all drug combination doses tested, where results <-0.1 indicate synergy (*), between -0.1 and 0.1 are additive (&), and >0.1 are antagonistic (#).

[0042] Figure 2 Weber analysis of bisantrene-sorafenib or -pazopanib drug combinations in 786-O cells is shown. A) Cell viability in response to the indicated different dose ranges of sorafenib and pazopanib in combination with bisantrene. Experimental data for each drug alone and the combination are shown. The "expected value" is calculated using the Weber method and shows the expected value if the drug combination is additive. Therefore, any experimental value below this line is considered synergistic. Equal to or close to the line is additive; above the line is antagonistic. B) Weber analysis of all drug combination doses tested, where results <-0.1 indicate synergy (red), between -0.1 and 0.1 are additive (green), and >0.1 are antagonistic (yellow).

[0043] Figure 3 Weber analysis of bisantrene-lenvatinib or -cabozantinib drug combinations in 786-O cells is shown. A) Cell viability in response to the indicated different dose ranges of lenvatinib and cabozantinib combined with zancren. Experimental data for each drug alone and the combination are shown. The "expected value" is calculated using the Weber method and shows the expected value when the drug combination is additive. Therefore, any experimental value below this line is considered synergistic. Equal to or close to the line is additive; above the line is antagonistic. B) Weber analysis of all tested drug combination doses, where results <-0.1 represent synergy (*), between -0.1 and 0.1 are additive (&), and >0.1 are antagonistic (#).

[0044] Figure 4Weber analysis of bisantrene-everolimus or -sunitinib drug combinations in Caki-1 cells is shown. A) Cell viability in response to the indicated different dose ranges of everolimus and sunitinib in combination with bisantrene. Experimental data for each drug alone and the combination are shown. The "expected value" is calculated using the Weber method and shows the expected value if the drug combination is additive. Therefore, any experimental value below this line is considered synergistic. Equal to or close to the line is additive; above the line is antagonistic. B) Weber analysis of all drug combination doses tested, where results <-0.1 indicate synergy (*), between -0.1 and 0.1 are additive (&), and >0.1 are antagonistic (#).

[0045] Figure 5 Weber analysis of bisantrene-sorafenib or -pazopanib drug combinations in Caki-1 cells is shown. A) Cell viability in response to the indicated different dose ranges of sorafenib and pazopanib in combination with bisantrene. Experimental data for each drug alone and the combination are shown. The "Expected Value" is calculated using the Weber method and shows the expected value if the drug combination is additive. Therefore, any experimental value below this line is considered synergistic. Equal to or close to the line is additive; above the line is antagonistic. B) Weber analysis of all drug combination doses tested, where results <-0.1 indicate synergy (*), between -0.1 and 0.1 are additive (&), and >0.1 are antagonistic (#).

[0046] Figure 6 Weber analysis of bisantrene-lenvatinib or -cabozantinib drug combinations in Caki-1 cells is shown. A) Cell viability in response to the indicated different dose ranges of lenvatinib and cabozantinib in combination with bisantrene. Experimental data for each drug alone and the combination are shown. The "expected value" is calculated using the Weber method and shows the expected value if the drug combination is additive. Therefore, any experimental value below this line is considered synergistic. Equal to or close to the line is additive; above the line is antagonistic. B) Weber analysis of all tested drug combination doses, where results <-0.1 indicate synergy (*), between -0.1 and 0.1 are additive (&), and >0.1 are antagonistic (#).

[0047] Figure 7Weber analysis of bisantrene-everolimus or -sunitinib drug combinations in Caki-2 cells is shown. A) Cell viability in response to the indicated different dose ranges of everolimus and sunitinib in combination with bisantrene. Experimental data for each drug alone and the combination are shown. The "expected value" is calculated using the Weber method and shows the expected value if the drug combination is additive. Therefore, any experimental value below this line is considered synergistic. Equal to or close to the line is additive; above the line is antagonistic. B) Weber analysis of all drug combination doses tested, where results <-0.1 indicate synergy (*), between -0.1 and 0.1 are additive (&), and >0.1 are antagonistic (#).

[0048] Figure 8 Weber analysis of bisantrene-sorafenib or -pazopanib drug combinations in Caki-2 cells is shown. A) Cell viability in response to the indicated different dose ranges of sorafenib and pazopanib in combination with bisantrene. Experimental data for each drug alone and the combination are shown. The "Expected Value" is calculated using the Weber method and shows the expected value if the drug combination is additive. Therefore, any experimental value below this line is considered synergistic. Equal to or close to the line is additive; above the line is antagonistic. B) Weber analysis of all drug combination doses tested, where results <-0.1 indicate synergy (*), between -0.1 and 0.1 are additive (&), and >0.1 are antagonistic (#).

[0049] Fig. 9 Weber analysis of bisantrene-lenvatinib or -cabozantinib drug combinations in Caki-2 cells is shown. A) Cell viability in response to the indicated different dose ranges of lenvatinib and cabozantinib in combination with zanetrin. Experimental data for each drug alone and the combination are shown. The "expected value" is calculated using the Weber method and shows the expected value if the drug combination is additive. Therefore, any experimental value below this line is considered synergistic. Equal to or close to the line is additive; above the line is antagonistic. B) Weber analysis of all tested drug combination doses, where results <-0.1 indicate synergy (*), between -0.1 and 0.1 are additive (&), and >0.1 are antagonistic (#).

[0050] Fig.10Weber analysis of bisantrene-everolimus or -sunitinib drug combinations in RCC4 EV cells is shown. A) Cell viability in response to the indicated different dose ranges of everolimus and sunitinib in combination with bisantrene. Experimental data for each drug alone and the combination are shown. The "expected value" is calculated using the Weber method and shows the expected value if the drug combination is additive. Therefore, any experimental value below this line is considered synergistic. Equal to or close to the line is additive; above the line is antagonistic. B) Weber analysis of all drug combination doses tested, where results <-0.1 indicate synergy (*), between -0.1 and 0.1 are additive (&), and >0.1 are antagonistic (#).

[0051] Fig.11 Weber analysis of bisantrene-sorafenib or -pazopanib drug combinations in RCC4 EV cells is shown. A) Cell viability in response to the indicated different dose ranges of sorafenib and pazopanib in combination with bisantrene. Experimental data for each drug alone and the combination are shown. The "expected value" is calculated using the Weber method and shows the expected value if the drug combination is additive. Therefore, any experimental value below this line is considered synergistic. Equal to or close to the line is additive; above the line is antagonistic. B) Weber analysis of all drug combination doses tested, where results <-0.1 indicate synergy (*), between -0.1 and 0.1 are additive (&), and >0.1 are antagonistic (#).

[0052] Fig.12 Weber analysis of bisantrene-lenvatinib or -cabozantinib drug combinations in RCC4 EV cells is shown. A) Cell viability in response to the indicated different dose ranges of lenvatinib and cabozantinib in combination with bisantrene. Experimental data for each drug alone and the combination are shown. The "expected value" is calculated using the Weber method and shows the expected value if the drug combination is additive. Therefore, any experimental value below this line is considered synergistic. Equal to or close to the line is additive; above the line is antagonistic. B) Weber analysis of all tested drug combination doses, where results <-0.1 indicate synergy (*), between -0.1 and 0.1 are additive (&), and >0.1 are antagonistic (#).

[0053] Fig.13Weber analysis of bisantrene-everolimus or -sunitinib drug combinations in RCC4 VHL cells is shown. A) Cell viability in response to the indicated different dose ranges of everolimus and sunitinib in combination with bisantrene. Experimental data for each drug alone and the combination are shown. The "expected value" is calculated using the Weber method and shows the expected value if the drug combination is additive. Therefore, any experimental value below this line is considered synergistic. Equal to or close to the line is additive; above the line is antagonistic. B) Weber analysis of all tested drug combination doses, where results <-0.1 represent synergy (*), between -0.1 and 0.1 are additive (&), and >0.1 are antagonistic (#).

[0054] Fig.14 Weber analysis of bisantrene-sorafenib or -pazopanib drug combinations in RCC4 VHL cells is shown. A) Cell viability in response to the indicated different dose ranges of sorafenib and pazopanib in combination with bisantrene. Experimental data for each drug alone and the combination are shown. The "expected value" is calculated using the Weber method and shows the expected value if the drug combination is additive. Therefore, any experimental value below this line is considered synergistic. Equal to or close to the line is additive; above the line is antagonistic. B) Weber analysis of all tested drug combination doses, where results <-0.1 indicate synergy (*), between -0.1 and 0.1 are additive (&), and >0.1 are antagonistic (#).

[0055] Fig.15 Weber analysis of bisantrene-lenvatinib or -cabozantinib drug combinations in RCC4 VHL cells is shown. A) Cell viability in response to the indicated different dose ranges of lenvatinib and cabozantinib combined with zanzun. Experimental data for each drug alone and the combination are shown. "Expected value" is calculated using the Weber method and shows the expected value when the drug combination is additive. Therefore, any experimental value below this line is considered synergistic. Equal to or close to the line is additive; above the line is antagonistic. B) Weber analysis of all tested drug combination doses, where results <-0.1 represent synergy (*), between -0.1 and 0.1 are additive (&), and >0.1 are antagonistic (#).

[0056] Fig.16 Bisantrene plus targeted or chemotherapeutic agents are shown to overcome treatment resistance in ccRCC and other VHL(-) cancers.

[0057] Fig.17 A bisantrene plus checkpoint inhibitor combination trial design is shown to overcome immunotherapy resistance in ccRCC and other VHL(-) cancers.

[0058] Fig.18 The association between bisantrene sensitivity and VHL status of the isogenic renal cancer cell lines RCC4 EV (VHL(-) mutant) and RCC4VHL (VHL rescue line) is shown. (A) IC based on VHL status 50 Values ​​were compared using t-test. (B) Direct comparison of sensitivity of RCC4 isogenic cell lines to centromeric groups. Mean + / - SEM, n=3.

[0059] Fig.19 The association between bisantrene sensitivity and VHL status assessed using the clonogenicity assay is shown. (A) IC 200 was compared between VHL mutant and wild-type cell lines. 50 Values. NS, not significant, unpaired t-test. (B) Colonies observed for RCC4EV and RCC4 VHL rescued cell lines. n=4, *p<0.05, paired t-test. DETAILED DESCRIPTION

[0060] definition

[0061] As used herein, "treat" means affecting a subject / patient, tissue or cell to obtain a desired pharmacological and / or physiological effect, and includes inhibiting a disorder, i.e., slowing or arresting its development; or alleviating or ameliorating the effects of the disorder, i.e., causing reversal or regression of the effects of the disorder.

[0062] As used herein, "prevention" means preventing the occurrence of a disorder in a cell, tissue or subject that may be at risk of the disorder, but does not necessarily mean that the disorder will not eventually occur or that the subject will not eventually develop the disorder. Prevention includes delaying the onset of a disorder in a cell, tissue or subject.

[0063] As used herein, the term "subject" or "patient" refers to a mammal. The mammal can be human or non-human. Examples of non-humans include primates, livestock (e.g., sheep, cows, horses, donkeys, pigs), companion animals (e.g., dogs, cats), laboratory test animals (e.g., mice, rabbits, rats, guinea pigs, hamsters), 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.

[0064] The term "composition" encompasses compositions and formulations comprising an active pharmaceutical ingredient (e.g., a "protein kinase inhibitor" and a "second agent comprising a pharmaceutically acceptable salt of bisantrene or a bisantrene derivative, or a cardioprotective derivative of bisantrene") and an excipient or carrier, as well as compositions or formulations containing a carrier. In pharmaceutical compositions, the excipient or carrier is "pharmaceutically acceptable," meaning that it is not biologically or otherwise undesirable, i.e., the material may be incorporated into a pharmaceutical composition for administration to a patient without causing any undesirable biological effect and without interacting in a deleterious manner with any other component of the composition in which it is contained. Supplementary active ingredients may also be incorporated into the composition.

[0065] "Pharmaceutically acceptable", for example, in the description of "pharmaceutically acceptable salts" or "pharmaceutically acceptable excipients or carriers", means herein a material that is not biologically or otherwise undesirable, i.e., the material can be incorporated into a pharmaceutical composition administered to a patient without causing any undesirable biological effect or interacting in a deleterious manner with any other component of the composition containing it.

[0066] The term "effective amount" or "therapeutically effective amount" refers to the amount of active pharmaceutical ingredient sufficient to produce the desired therapeutic response. The specific effective amount or therapeutically effective amount will vary with factors such as the specific condition being treated, the age, weight, general health, physical condition, sex and diet of the subject, the duration of treatment, the nature of concurrent therapy (if any), and the severity of the specific condition.

[0067] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, extenders, carrier solutions, suspensions, colloids, and molding agents and binders, any and all of which may include other pharmaceutical excipients known in the art, including lubricants, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, antioxidants, other stabilizers (including physical stabilizers such as thickeners and viscosity enhancers), colorants, flavorings and sweeteners, etc. Such media and agents are well known in the art for pharmaceutically active substances. Unless any conventional media or agents are incompatible with the active ingredient, it is contemplated that they will be used in therapeutic compositions.

[0068] As used herein, "administering" or "administration" refers to distributing, applying or providing one or more agents to a subject. Administration can be performed using any of a variety of methods known in the art. For example, as used herein, "administering" means administration by infusion (intravenous administration (iv)), parenteral administration. "Parenteral" means intravenous, subcutaneous and intramuscular administration. DETAILED DESCRIPTION OF THE INVENTION

[0070] The present disclosure relates to a method of treating ccRCC or other VHL(-) cancers, particularly protein kinase inhibitor-resistant ccRCC, comprising administering a therapeutically active amount of bisantrene in combination with a protein kinase inhibitor, particularly a tyrosine kinase inhibitor ("TKI").

[0071] Protein kinase inhibitors

[0072] Protein kinases are protein enzymes that phosphorylate amino acid side chains (i.e., serine, threonine, and tyrosine) with hydroxyl groups on target proteins, as well as other targets (usually in signal transduction pathways). In fact, many signal transduction pathways contain cascades of protein kinases. In many cases, this phosphorylation leads to conformational changes in the target protein, which in turn may lead to activation or inhibition of the target protein's activity, or change its physical properties (thereby regulating its interactions). Therefore, protein phosphorylation and dephosphorylation events may activate or inhibit specific physiological pathways. Disorders of this signal transduction, such as mutations that lead to constitutive expression or unregulated activity of specific proteins or pathways, may be associated with cancer and / or allow its survival. Disorders of expression and / or activity of various protein kinases such as EGFR, VEGFR, HER, ALK, RAS, and RAF (to name a few) have been associated with increased growth, proliferation, motility, and survival of various tumor cells, including ccRCC cells.

[0073] The range of protein kinases known in the art is very extensive. In fact, the human genome is known to include 518 protein kinase genes. A large number of protein kinases are known to be associated with cancers including ccRCC, which is not surprising because they are involved in activation or inhibition of signal transduction pathways and growth-related pathways (particularly angiogenesis) and transmembrane signaling.

[0074] 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 and mitogen-activated protein kinases, etc. These may include, for example, 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.

[0075] It is important to note that the kinases described above may have a primary target, but may also phosphorylate other targets / moieties. For example, tyrosine kinases may also phosphorylate serine and / or threonine hydroxyl moieties. Similarly, serine / threonine kinases may also phosphorylate tyrosine hydroxyl moieties. Similarly, although kinases may primarily target a protein, it may also phosphorylate other proteins. Therefore, tyrosine kinases may primarily target or be known to target tyrosine hydroxyl moieties on specific proteins (usually receptors), but may also be able to phosphorylate hydroxyl moieties on serine and / or threonine and / or other proteins. As known in the art, this variability of target specificity is incorporated herein in the context of protein kinases.

[0076] A variety of protein kinase inhibitors have been developed for the treatment of various cancers, including imatinib, which was the first rationally designed protein kinase inhibitor approved by the FDA in 2001. Since then, a large number of protein kinase inhibitors have been developed, such as gefitinib, erlotinib, dasatinib, nilotinib, lapatinib, osimertinib, olmutinib, lorlatinib, capmatinib, tepotinib, selpercatinib, pralsetinib, sirpatinib, and tadalafil. selumetinib, trametinib, afatinib, ibrutinib, dabrafenib, ponatinib, bosutinib, radotinib, idelalisib, crizotinib, vemurafenib, xolitinib, olmutinib, mitomycin C, temsirolimus, everolimus, axitinib, sorafinib, sunitinib, cabozantinib, lenvatinib, pazopanib, and tivozanib, to name a few.

[0077] Protein kinase inhibitors currently approved for the treatment of ccRCC include, for example, temsirolimus, axitinib, belzutifan, cabozantinib, everolimus, lenvatinib, mitomycin C, pazopanib, sorafenib, sunitinib, and tivozanib.

[0078] Axitinib, cabozantinib, pazopanib, sorafenib, and sunitinib are small molecule tyrosine kinase inhibitors, including targets such as the VEGFR family. Beztifan is an inhibitor of hypoxia-inducible factor (HIF)-2α (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 receptor (FGFR) 1, 2, 3, and 4, platelet-derived growth factor receptor (PDGFR) α, 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.

[0079] In addition to the VEGFR family of protein kinases, common targets of lenvatinib, cabozantinib, and pazopanib include DDR1 and DDR2 (discinodomain receptors 1 and 2), RET (“rearranged during transfection”), and RIPK2 (receptor-interacting serine / threonine kinase 2). RET has also been identified as a target of sunitinib and axitinib, and AXL and MET have been identified as additional targets of cabozantinib.

[0080] Despite the success in using protein kinase inhibitors to treat various cancers, including ccRCC and other VHL(-) cancers, many patients experience disease progression due to the development of drug resistance or bypass pathways.

[0081] The current study shows a surprising synergy between bisantrene and protein kinase inhibitors for the treatment of ccRCC and other VHL(-) cancers. This synergy is expected to at least help reverse or mitigate the development of resistance, if not eliminate it, and is expected to have the added benefit of allowing a reduction in the dose of the protein kinase inhibitor and / or bisantrene, thereby reducing any adverse side effects.

[0082] Bisantrene and its derivatives or analogs and pharmaceutically acceptable salts

[0083] Bisantrene has immunogenic properties that may account for some of its activity and may make this agent a useful tool in combinatorial supercoiling and initiating strand breaks associated with DNA binding proteins. This is due to inhibition of topoisomerase II, which relaxes the DNA coil during replication. The drug has been found to be effective in cancer models using colon 26, Lewis lung, Ridgway osteosarcoma, B16, Lieberman plasma cell, P388 or L1210 cancer cells, although inactive when administered orally, intravenously (iv), intraperitoneally (ip) or subcutaneously (sc). In a 684-patient clonogenic assay, activity was observed in breast, small cell lung cancer, large cell lung cancer, squamous cell lung cancer, ovarian cancer, pancreatic cancer, kidney cancer, adrenal cancer, head and neck cancer, sarcoma, gastric cancer, lymphoma and melanoma tumor cells, but not in colorectal cancer. Importantly, a lack of cross-resistance with adriamycin and mitoxantrone was found. After treatment with bisantrene and 4 weeks thereafter, macrophages could be isolated from peritoneal exudates that had cytostatic antiproliferative activity in cultures of P815 (mastocytoma) tumor cells. In addition, supernatants from bisantrene-activated macrophages also had a protective cytostatic effect in the tumor cell cultures. Further work revealed that macrophages activated with bisantrene and adoptively transferred into mice with EL-4 lymphoma more than doubled their median survival time, with 7 of 10 mice in the group being cured. Multiple dosing of activated macrophages was more effective than a single dose. Bisantrene is an unusual agent with direct cytotoxic effects as well as genomic and immunological methods of action, including being a highly potent inhibitor (ICP) of the RNA N6-methyladenosine (m6A) demethylase, the fat mass and obesity-associated protein (FTO). 50 142nM) (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).

[0084] Bisantrene, whose chemical name is 9,10-Anthracenedicarboxaldehyde bis[(4,5-dihydro-1H-imidazol-2-yl)hydrazine] dihydrochloride, was originally classified as an anthracycline chemotherapeutic agent. The term "bisantrene" as used herein refers to bisantrene or any pharmacologically compatible salt form, and not just bisantrene dihydrochloride, unless the dihydrochloride or another specific pharmacologically compatible salt form is specifically indicated. Generally, for most pharmacological applications, the pharmacologically compatible salt of bisantrene is bisantrene dihydrochloride. These drugs have a planar structure based around a resonant aromatic ring structure that intercalates within the helix of DNA and disrupts various functions, including replication, probably due to a strong inhibitory effect on topoisomerase II. It has been found, like other anthracyclines, to kill tumor cells in clonogenic assays and intercalate into DNA, where it inhibits DNA and RNA synthesis. The main chemotherapeutic mechanism of bisantrene is that it preferentially binds to AT-rich regions, where it affects alterations.

[0085] Recent studies have shown that bisantrene suppresses immune checkpoint gene expression and immune evasion through 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.

[0086] Bisantrene has also been found to have non-immune telomeric effects. Bisantrene binds to DNA at a site called a G-quadruplex, where four guanines are bound by a fold. Stabilization of the G-quadruplex can interfere with the telomere-telomerase interaction, thereby inhibiting the activity of telomerase in various ways, including displacement of telomerase binding proteins. Since the level of inhibition of topoisomerase II does not always correlate with cytotoxic efficacy, alternative mechanisms may play a role in the action of bisantrene. Analogs of bisantrene have been prepared in an attempt to enhance the anti-telomerase activity; these analogs are described further below. Human melanoma (SK-Mel5) and colon cancer (LoVo) tumor cells were observed to lose their ability to proliferate in the presence of these agents. No apoptosis was observed; however, a loss of immortality was observed, with the treated cells regaining the ability to become senescent, age, and die.

[0087] As detailed above, in addition to the direct anti-tumor effects associated with bisantrene's activity as a DNA intercalator, bisantrene has other mechanisms of action, including immune enhancement. These mechanisms are described in the following literature: (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, which concluded that the level of tumor-infiltrating lymphocytes is associated with the response to administration of anthracycline-based agents; markers associated with tumor-infiltrating lymphocytes (TILs) 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, which indicates that DNA damage, such as that produced by intercalating agents such as bisantrene, induces expression of NKG2D ligands on tumor cells in an ATM-dependent and CHK1-dependent (but p53-independent) manner; NKG2D is an activating receptor involved in tumor immunosurveillance by NK cells, NK T cells, γδ T cells, and resting (in mice) and / or activated (in humans) CD8+ T cells, and also indicates that anthracycline-based agents can act as immunostimulants, particularly when combined with IL-12; such agents also promote HMGB1 release and activate T cells; (iii) DV Krysko 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, which indicates that anthracycline-based antibiotics induce an immunogenic form of apoptosis with immunostimulatory properties mediated by MyD88, TLR2, and TLR9; (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, which indicates that anthracyclines induce apoptosis and ceramide production and activate caspase-3 in resting and cycling cells; 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". Cells), Proc. Natl. Acad. Sci. USA 106: 2353-2358, which provides an alternative antitumor mechanism for anthracyclines, namely, inhibition of HIF-1-mediated gene transcription, which in turn inhibits the transcription of VEGF required for angiogenesis; HIF-1 also activates the transcription of genes encoding the glucose transporter GLUT1 and hexokinase HK1 and HK2 (which are required for the high levels of glucose uptake and phosphorylation observed in metastatic cancer cells) and pyruvate dehydrogenase kinase 1 (PDK1) (which shunts pyruvate away from mitochondria, thereby increasing lactate production); based on immunohistochemical results, patients with HIF-1α overexpression are considered good candidates for treatment with anthracycline-based antibiotics. .

[0088] Several clinical trials have investigated the pharmacokinetics of bisantrene in humans. 2 Patients were infused for 90 minutes, and biphasic elimination occurred, with an initial 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 / m 2 In this study, the plasma clearance was 735 mL / min / m 2 , 11.3% of the administered dose was excreted unchanged in the urine within 24 hours. In another trial, 80-250 mg / m 2The initial and terminal half-lives were 0.6 h and 24.7 h, respectively, with a clearance of 1045.5 ± 51.0 mL / kg / h 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 within 96 h. Triphasic elimination was reported in three other single-dose studies, with t 1 / 2 The α, β, and γ were 3.44 min, 1.33 h, and 26.13 h, respectively, another was 3 min, 1 h, and 8 h, and the last revealed a clearance period of 0.1 h, 1.9 h, and 43.9 h, respectively. In one report, a large volume of distribution (687 L / m 2 ) was interpreted as tissue sequestration of the drug followed by a storage effect. In a 72-h infusion study, a 2 The plasma concentration was 12±6 ng / mL at a dose of 260 mg / mL. 2 The dose produced a plasma concentration of 55 ± 8 ng / mL. In this study, the plasma clearance was 1306 ± 179 mL / min / m 2 , with 4.6% of the dose excreted in urine within 24 hours. Finally, in another study, a 60-minute infusion schedule over 5 days showed t 1 / 2 The durations of α and β are 0.9 h and 9.7 h respectively, and 7.1% of the dose is excreted in the urine.

[0089] The structure of bisantrene dihydrochloride is shown in formula (I)

[0090]

[0091] Bisantrene dihydrochloride is a tricyclic aromatic compound with the chemical name 9,10-anthracene dicarboxaldehyde bis[(4,5-dihydro-1H-imidazol-2-yl)hydrazine] dihydrochloride. The molecular formula of Bisantrene dihydrochloride is C 22 H 22 N 8 2HCl, molecular weight 471.4. The alkylimidazole side chain is very basic and positively charged at physiological pH. This is thought to facilitate electrostatic attraction to the negatively charged ribose phosphate groups in DNA.

[0092] Bisantrene is usually administered intravenously, either centrally or peripherally.

[0093] Various formulations suitable for administration of bisantrene or its derivatives or analogs are known in the art. U.S. Patent No. 4,784,845 to Desai et al. discloses a composition of matter for delivery of a hydrophobic drug (i.e., bisantrene or its derivatives or analogs) comprising: (i) the hydrophobic drug; (ii) an oily carrier 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. Desai et al., U.S. Pat. No. 4,816,247, discloses a composition of matter for delivering a hydrophobic drug (e.g., bisantrene or a derivative or analog thereof) via intravenous, intramuscular, or intraarticular routes, comprising: (i) the hydrophobic drug; (ii) a pharmaceutically acceptable oily vehicle or oil selected from: (a) naturally occurring plant oils and (b) semisynthetic mono-, di-, and tri-glycerides, wherein the oily vehicle and oil do not contain BHT or BHA; (iii) a surfactant or emulsifier; (iv) a co-surfactant or emulsifier; (v) when the hydrophobic drug is basic, a pharmaceutically acceptable solvent or oil selected from C 6 -C 20 A saturated or unsaturated aliphatic acid, and an ion pair former selected from pharmaceutically acceptable aromatic amines when the hydrophobic drug is acidic; and (vi) water. U.S. Pat. No. 5,000,886 to Lawter et al. and U.S. Pat. No. 5,143,661 to Lawter et al. disclose compositions comprising microcapsules for delivering a pharmaceutical agent such as bisantrene or a derivative or analog thereof, wherein the microcapsules include a hardening agent which is a volatile silicone fluid. U.S. Pat. No. 5,070,082 to Murdock et al., U.S. Pat. No. 5,077,282 to Murdock et al., and U.S. Pat. No. 5,077,283 to Murdock et al. disclose prodrug forms of poorly soluble hydrophobic drugs including bisantrene and its derivatives and analogs, which are salts of phosphamide acid. U.S. Pat. Nos. 5,116,827 to Murdock et al. and 5,212,291 to Murdock et al. disclose prodrug forms of poorly soluble hydrophobic drugs including bisantrene and its derivatives and analogs, which are quinolinecarboxylic acid derivatives. U.S. Pat. No. 5,378,456 to Tsou includes compositions containing anthracene antineoplastic agents such as bisantrene or its derivatives or analogs, wherein the bisantrene or its derivatives or analogs are coupled or mixed with divinyl ether-maleic acid (MVE) copolymers. U.S. Pat. No. 5,609,867 to Tsou discloses polymeric 1,4-bis derivatives of bisantrene and copolymers of bisantrene and another monomer (e.g., dianhydride).

[0094] The methods and compositions described herein may use derivatives or analogs of bisantrene in place of bisantrene itself. Derivatives and analogs of bisantrene are described in U.S. Pat. Nos. 10,500,19 and 10,548,876 to Garner et al.

[0095] Methods for treating ccRCC and other VHL(-) cancers

[0096] Surprisingly, the present study shows that protein kinase inhibitors and bisantrene (or a pharmaceutically acceptable salt thereof) have a synergistic effect on ccRCC cell lines. It is envisaged that in the methods and compositions according to the present invention, bisantrene derivatives having substantially the same activity will also act synergistically with protein kinases.

[0097] Therefore, one aspect of the present invention provides a method for treating a patient suffering from ccRCC or other VHL(-) cancers, particularly protein kinase inhibitor-resistant ccRCC, the method comprising administering to the patient a therapeutically effective amount of a protein kinase inhibitor (e.g., a tyrosine kinase inhibitor) and a therapeutically effective amount of at least one second agent, the second agent comprising bisantrene or a derivative thereof, or a pharmaceutically acceptable salt of bisantrene or a derivative thereof.

[0098] 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, VEGFR3.

[0099] According to certain embodiments, the at least one protein kinase inhibitor is selected from pazopanib, lenvatinib, cabozantinib, everolimus, sorafenib, sunitinib, temsirolimus, mitomycin C, axitinib, tivozanib, and beztifan.

[0100] According to certain embodiments, the at least one protein kinase inhibitor is selected from pazopanib, lenvatinib, cabozantinib, sorafenib, sunitinib, mitomycin C, axitinib, tivozanib and beztifan.

[0101] According to a more specific embodiment of the present invention, said at least one protein kinase inhibitor is selected from the group consisting of pazopanib, lenvatinib, cabozantinib, sorafenib, sunitinib, axitinib and tivozanib.

[0102] According to an even more particular embodiment of the present invention, said at least one protein kinase inhibitor is chosen from pazopanib, lenvatinib and cabozantinib.

[0103] According to an alternative embodiment, the at least one protein kinase inhibitor is an inhibitor of mTOR, optionally selected from temsirolimus and everolimus.

[0104] According to certain embodiments of the invention, the second agent is bisantrene or a pharmaceutically acceptable salt thereof.

[0105] According to certain embodiments, the bisantrene may be administered as a pharmaceutical compound or as a component of a pharmaceutical composition, as discussed further below.

[0106] 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 pharmaceutically acceptable salt thereof.

[0107] The pharmaceutical composition and medicine of the present invention can be administered to the subject by standard enteral or parenteral routes, including but not limited to injection (e.g., intravenous, subcutaneous, intramuscular, push injection, etc.), or by, for example, local, oral, sublingual, nasal, pulmonary, ear, rectal or vaginal routes of administration. In some embodiments, the pharmaceutical composition of the present invention can be administered to the subject by itself or in combination with other pharmaceutical compositions. In the latter case, administration can be simultaneous or sequential, or the administration of the pharmaceutical composition can be independent of each other.

[0108] In certain embodiments, bisantrene (or a derivative, or a pharmaceutically acceptable salt of either) is administered centrally or peripherally intravenously, including by intramuscular, subcutaneous, and / or intradermal injection.

[0109] In general, the pharmaceutical compositions and drugs of the present invention can be administered in a manner compatible with the route of administration and the physical characteristics of the subject (including health conditions), and in a manner that induces the desired effect (i.e., therapeutically effective and / or preventive). For example, a suitable 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 drug is used as a single agent, the progression of the disease or condition being treated (i.e., pathological state), and other factors apparent to those of ordinary skill in the art.

[0110] Suitable dosages, dosage frequencies, dosage durations, and routes of administration of chemotherapeutic agents are known in the art. Fig.16As proposed, bisantrene, a bisantrene derivative, or a pharmaceutically acceptable salt of either may be administered in the same pharmaceutical composition as the protein kinase inhibitor, in a separate composition but simultaneously with the protease inhibitor, or at a different time. If the bisantrene, a bisantrene derivative, or a pharmaceutically acceptable salt of bisantrene or a bisantrene derivative is administered at a different time than the protein kinase inhibitor, it may be administered before or after the protein kinase inhibitor, and / or at different times according to different time and / or frequency schedules. One of ordinary skill in the art can determine the appropriate dosing schedule based on variables such as the age, weight, and sex of the subject, the subject's susceptibility to side effects of the active agent, genetic markers, the dose of the active agent, the subject's previous medical history with the active agent, and other pharmacokinetic parameters (e.g., cardiac, liver, or kidney function).

[0111] The methods and compositions provided herein enable subjects to receive therapy more frequently without significantly changing the dosage regimen due to the risk of side effects (e.g., cardiotoxicity). The dosage of the protein kinase inhibitor and bisantrene, a bisantrene derivative, or a pharmaceutically acceptable salt of bisantrene or a bisantrene derivative can be administered to the subject in one or more doses per day. In some cases, the daily dosage of the chemotherapeutic agent can be administered in a single dose together with bisantrene, a bisantrene derivative, or a pharmaceutically acceptable salt of either.

[0112] The pharmaceutical compositions described herein can be administered to a patient once or more per day. In some cases, the pharmaceutical composition can be administered to a patient once per day. In some cases, the pharmaceutical composition can be administered to a patient at least 2, 3, 4, 5, or 6 times per day. For example, the pharmaceutical composition can be administered to a patient 3 times per day.

[0113] In the methods described herein, one of ordinary skill in the art can determine the appropriate dosage of bisantrene (or a bisantrene derivative or a pharmaceutically acceptable salt of either). The selected dosage level depends on various pharmacokinetic factors, including the amount of active agent administered, the route of administration, the time of administration, the excretion rate of the specific compound used, the severity of the condition, other health factors affecting the subject, and the state of the subject's liver and kidney function. It also depends on factors such as the duration of treatment, other drugs, compounds and / or materials used in combination with the specific therapeutic agent used, and the age, weight, condition, general health and previous medical history of the subject being treated. Methods for determining optimal dosages are described in the art, for example, 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. Those skilled in the art can determine the optimal dosage for a given set of conditions using routine dosage determination tests based on experimental data for the agent.

[0114] According to certain embodiments, bisantrene is administered at about 0.1 mg / m 2 / day to about 400 mg / m 2 / day, for example, about 0.2 mg / m 2 / day to about 300 mg / m 2 / day, about 0.5mg / m 2 / day to about 200 mg / m 2 / day, about 0.5mg / m 2 / day to about 100 mg / m 2 / day, about 0.5mg / m 2 / day to about 50 mg / m 2 / day, about 0.5mg / m 2 / day to about 30 mg / m 2 / day, about 0.5mg / m 2 / day to about 20 mg / m 2 / day, about 1.0mg / m 2 / day to about 10 mg / m 2 / day, about 1.0mg / m 2 / day to about 8 mg / m 2 / day, about 1mg / m 2 / day, about 2mg / m2 / day, about 3mg / m 2 / day, about 4mg / m 2 / day, about 5mg / m 2 / day, about 6mg / m 2 / day, about 7mg / m 2 / day or about 10 mg / m 2 In some embodiments, bisantrene is administered once a day or once a week, once every two weeks, once every three weeks, once every four weeks, over 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, bisantrene is administered once or more over a period of 28 days, optionally daily or once or more weekly, once every two weeks, once every three weeks, once every four weeks, at a dose of about 20-50 mg / m 2 Administration of a pharmaceutically acceptable salt of bisantrene or a derivative thereof or a pharmaceutically acceptable salt thereof may be carried out at similar dosage rates and adjusted for molar equivalents.

[0115] The dosing regimen for the protein kinase inhibitor will depend on the specific protein kinase inhibitor and has been published for known protein kinase inhibitors, including for the treatment of ccRCC and other VHL(-) cancers. In addition, one of ordinary skill in the art can readily determine the appropriate dosage for a given protein kinase inhibitor using known methods and techniques discussed above with respect to bisantrene.

[0116] According to certain embodiments, and as a result of the synergistic biological activity observed when bisantrene (or a bisantrene derivative or a pharmaceutically acceptable salt of either) is administered with a protein kinase inhibitor, for a given dose rate of the protein kinase inhibitor, the therapeutic result can be more effectively achieved. This biological activity of bisantrene (or a bisantrene derivative or a pharmaceutically acceptable salt of either) can also allow for the use or expand the use of protein kinase inhibitors that may have deleterious side effects (e.g., cardiotoxicity) at normal doses.

[0117] Alternatively, when co-administered with bisantrene (or a bisantrene derivative or a pharmaceutically acceptable salt of either), the protein kinase inhibitor can be administered to a patient at a lower dose than is typically administered over a longer period of time while maintaining comparable sustained therapeutic results. This is particularly important for active agents that have deleterious side effects (e.g., cardiotoxicity). Thus, in certain embodiments of the methods according to the invention, when co-administered with bisantrene (or a bisantrene derivative or a pharmaceutically acceptable salt of either), the protein kinase inhibitor can be administered at a rate significantly lower than the recommended dose rate of the protein kinase inhibitor when administered alone, for example, at a rate that is at least 10%, 20%, 30%, 40%, 50%, 60%, or 70% lower than the dose rate of the protein kinase inhibitor when administered alone.

[0118] Additional active agents

[0119] 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 inhibit this regulation, allowing immune cells to destroy tumors. An emerging strategy is to combine checkpoint inhibitors with protein kinase inhibitors, particularly those targeting VEGFR and other kinases involved in angiogenesis, with the rationale that angiogenesis is important in establishing an immunosuppressive environment. Known checkpoint inhibitors include antibodies that target PD1 or PDL1.

[0120] The FDA has approved two PD-1 inhibitors (nivolumab and pembrolizumab), one PD-L1 inhibitor (avelumab), and one CTLA-4 inhibitor (ipilimumab) for ccRCC. Other PD-1 / PD-L1 immune checkpoint inhibitors that may be considered include atezolizumab, bevacizumab, cemiplizumab, dotalimumab, and durvalumab.

[0121] In addition, some studies have shown that protein kinase inhibitors themselves can stimulate immune responses against tumors, which in turn can produce a synergistic response when combined with checkpoint inhibitors.

[0122] Likewise, immunomodulators such as cytokines that regulate immune cell maturation, growth and activation, including interleukin-2 (Aldesleukin / ), interferon α-2a, interferon α-2b (and pegylated interferon α-2b), and granulocyte-macrophage colony-stimulating factor (GM-CSF), have also been approved for the treatment of various cancers through immune stimulation. Interleukin-2 (or the recombinant human version aldesleukin / ) has been approved for the treatment of RCC.

[0123] Therefore, the method according to the present invention may further comprise administering at least one additional therapeutic agent for the treatment of ccRCC or other VHL(-) cancers.

[0124] According to certain embodiments, the additional therapeutic agent is a checkpoint inhibitor or an immunomodulator, such as those discussed above. According to certain embodiments, the at least one additional therapeutic agent comprises a checkpoint inhibitor selected from nivolumab, pembrolizumab, avelumab, ipilimumab, atezolizumab, bevacizumab, cemiplizumab, dotalimumab, and durvalumab, or a checkpoint inhibitor selected from interleukin-2 (aldesleukin / ), 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 nivolumab, pembrolizumab, avelumab, and ipilimumab. According to certain embodiments, the at least one additional therapeutic agent comprises interleukin-2 (aldesleukin / ).

[0125] Suitable dosages, dosage frequencies, dosage durations, and routes of administration for these additional agents are known in the art. Fig.17 As suggested, these additional agents may be administered at the same time as bisantrene or a derivative or analog of bisantrene, or at a different time than bisantrene, a derivative or analog of bisantrene. If the additional agent is administered at a different time than bisantrene or a derivative or analog of bisantrene, it may be administered before or after bisantrene or a derivative or analog of bisantrene, and / or at different times according to different time and / or frequency schedules. Similarly, and independent of bisantrene considerations, these additional agents may be administered at the same time as the protein kinase inhibitor, or at a different time than the protein kinase inhibitor. If the additional agent is administered at a different time than the protein kinase inhibitor, it may be administered before or after the protein kinase inhibitor, and / or at different times according to different time and / or frequency schedules. One of ordinary skill in the art can determine the appropriate dosing regimen based on variables such as the patient's age, weight and sex, the severity of the cancer, genetic markers such as those described further below, and pharmacokinetic parameters such as liver and kidney function.

[0126] Fig.16 A prophetic example of a trial design for bisantrene and targeted agent combinations to overcome therapy resistance in ccRCC and other VHL(-) cancers is shown.

[0127] Fig.17 A prophetic example of bisantrene and checkpoint inhibitor combination trial design is shown to overcome immunotherapy resistance in ccRCC and other VHL(-) cancers.

[0128] Pharmaceutical compositions for treating ccRCC and other VHL(-) cancers

[0129] The present invention also provides a pharmaceutical composition for treating ccRCC or other VHL(-) cancers, comprising at least one protein kinase inhibitor as described above and a second agent, wherein the second agent comprises bisantrene or a derivative thereof, or a pharmaceutically acceptable salt of bisantrene or a derivative thereof. As described above, the composition of the present invention may comprise an additional agent selected from a checkpoint inhibitor and an immunomodulator.

[0130] 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 than one of VEGFR1, VEGFR2, VEGFR3, DDR1, DDR2, RET and RIPK2. According to certain embodiments, the protein kinase inhibitor inhibits at least one or more than one of VEGFR1, VEGFR2, VEGFR3.

[0131] According to certain embodiments, the at least one protein kinase inhibitor is selected from pazopanib, lenvatinib, cabozantinib, everolimus, sorafenib, sunitinib, temsirolimus, mitomycin C, axitinib, tivozanib, and beztifan.

[0132] According to a particular embodiment of the invention, the at least one protein kinase inhibitor is selected from pazopanib, lenvatinib and cabozantinib.

[0133] According to certain embodiments of the invention, the second agent is bisantrene or a pharmaceutically acceptable salt thereof.

[0134] According to certain embodiments of the present invention, the composition may comprise a therapeutically effective amount of a protein kinase inhibitor and a therapeutically effective amount of bisantrene or a pharmaceutically acceptable salt thereof.

[0135] The composition according to the present invention may further comprise the administration of at least one additional therapeutic agent for the treatment of ccRCC or other VHL(-) cancers.

[0136] According to certain embodiments, the additional therapeutic agent is a checkpoint inhibitor or an immunomodulator, such as those discussed above. According to certain embodiments, the at least one additional therapeutic agent comprises a checkpoint inhibitor selected from nivolumab, pembrolizumab, avelumab, ipilimumab, atezolizumab, bevacizumab, cemiplizumab, dotalimumab, and durvalumab, or a checkpoint inhibitor selected from interleukin-2 (aldesleukin / ), 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 nivolumab, pembrolizumab, avelumab, and ipilimumab. According to certain embodiments, the at least one additional therapeutic agent comprises interleukin-2 (aldesleukin / ).

[0137] The composition according to the present invention can be administered by any route known in the art and in a form suitable for the route. Therefore, the composition according to the present invention can be adapted to be administered by enteral or parenteral routes, including by injection (e.g., intravenous, subcutaneous, intramuscular, push injection, etc.) or by, for example, local, oral, sublingual, nasal, pulmonary, ear, rectal or vaginal routes of administration.

[0138] Typically, the pharmaceutical compositions described herein include at least one pharmaceutically acceptable carrier or excipient and / or diluent. In order to prepare pharmaceutical compositions and drugs, inert, pharmaceutically acceptable carriers can be solid or liquid. Liquid preparations include solutions, suspensions and emulsions, such as water or water-propylene glycol solutions for parenteral injection. Also included are solid preparations, such as tablets or amorphous or crystalline powders, including lyophilized preparations, the purpose of which is to be converted into liquid preparations just before use for oral or injection administration. Such liquid forms include solutions, suspensions and emulsions. Examples of pharmaceutically acceptable carriers and methods for preparing various compositions can be found in, for example, Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th edition, 2000, and Gilman et al., ed., (1990), Goodman and Gilman: The Pharmacological Basis of Therapeutics, Pergamon Press.

[0139] Pharmaceutically acceptable carriers and excipients include:

[0140] (i) a liquid carrier;

[0141] (ii) isotonic agents;

[0142] (iii) wetting agents or emulsifiers;

[0143] (iv) preservatives;

[0144] (v) buffers;

[0145] (vi) acidifier;

[0146] (vii) antioxidants;

[0147] (viii) alkalizing agents;

[0148] (ix) carrier;

[0149] (x) chelating agents;

[0150] (xi) colorants;

[0151] (xii) complexing agents;

[0152] (xiii) solvent;

[0153] (xiv) suspending agents and / or viscosity increasing agents;

[0154] (xv) oils;

[0155] (xvi) penetration enhancers;

[0156] (xvii) polymers;

[0157] (xviii) hardener;

[0158] (xix) proteins;

[0159] (xx) carbohydrates;

[0160] (xxi) bulking agents; and

[0161] (xxii) Lubricants.

[0162] Other pharmaceutically acceptable carriers and excipients known in the art may be used.

[0163] The carrier, diluent, excipient and adjuvant must be "acceptable" in terms of compatibility with the other ingredients of the composition or medicament and generally not harmful to the subject thereof. Non-limiting examples of pharmaceutically acceptable carriers or diluents are desalted or distilled water; saline solution; plant-based oils, such as peanut oil, safflower oil, olive oil, cottonseed oil, corn oil; sesame oils, 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 methylphenylpolysiloxane; volatile silicones; mineral oils, such as liquid paraffin, soft paraffin or squalane; cellulose; Cellulose derivatives, such as methylcellulose, ethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, such as ethanol or isopropanol; lower aryl alkanols; lower polyalkylene glycols or lower alkylene glycols, such as polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerol; fatty acid esters, such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinyl pyrrolidone; agar; tragacanth gum or gum arabic and vaseline. Typically, one or more carriers will form about 10% to about 99.9% by weight of the composition, vaccine or medicament.

[0164] For administration as an injectable solution or suspension, nontoxic parenterally acceptable diluents or carriers may 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 apparent to those of ordinary skill in the art and are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th edition, 2000, and Gilman et al., eds., (1990), Goodman and Gilman: The Pharmacological Bases of Therapeutics, Pergamon Press.

[0165] For oral administration, some examples of suitable carriers, diluents, excipients and adjuvants include peanut oil, liquid paraffin, sodium carboxymethylcellulose, methylcellulose, sodium alginate, gum arabic, tragacanth, glucose, sucrose, sorbitol, mannitol, gelatin and lecithin. In addition, these oral preparations can contain suitable flavorings and coloring agents. When used in capsule form, the capsule can be coated with a compound that delays disintegration, such as glyceryl monostearate or stearyl glyceride.

[0166] Solid forms for oral administration can contain acceptable binders, sweeteners, disintegrants, diluents, flavoring agents, coating agents, preservatives, lubricants and / or time delay agents in human and veterinary pharmaceutical practice. Suitable binders include gum arabic, gelatin, corn starch, tragacanth, sodium alginate, carboxymethyl cellulose or polyethylene glycol. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharin. Suitable disintegrants include corn starch, methylcellulose, polyvinyl pyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar. Suitable diluents include lactose, sorbitol, mannitol, glucose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate. Suitable flavoring agents include peppermint oil, wintergreen oil, cherry, orange or raspberry flavoring. Suitable coating agents include polymers or copolymers of acrylic acid and / or methacrylic acid and / or its esters, wax, fatty alcohol, 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.

[0167] Liquid forms for oral administration may also contain a liquid carrier in addition to the above-mentioned agents. 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 alcohol, triglyceride or a mixture thereof.

[0168] Suspensions for oral administration may further comprise dispersants and / or suspending agents.Suitable suspending agents include sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, sodium alginate or acetyl alcohol.Suitable dispersants include lecithin, fatty acids such as stearic polyoxyethylene esters, monooleate or dioleate, stearate or laurate of polyoxyethylene sorbitan, monooleate or dioleate, stearate or laurate of polyoxyethylene alcohol sorbitan etc.

[0169] In certain embodiments, the pharmaceutical composition may comprise liposomes. Liposomal formulations suitable for bisantrene or its cardioprotective derivatives comprise small unilamellar or multilamellar liposomes ranging in size from 0.01 to 100 μM in aqueous solution, with liposomes entrapped between about 50-95% bisantrene, the liposomes being composed of hydrogenated soy phosphatidylcholine, distearoylphosphatidylglycerol, and cholesterol of lipids of natural or synthetic origin, which 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, which is then diluted for parenteral administration to treat melanoma.

[0170] In yet another embodiment, the pharmaceutical composition may comprise a complex with β-cyclodextrin. Liposomal formulations suitable for bisantrene, therapeutically active derivatives of bisantrene, or pharmaceutically acceptable salts of bisantrene or its derivatives comprise a complex formed in an aqueous solution that 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 of preparing such complexes are known in the art and are described, for example, in WO 2019 / 073296 to Rothman.

[0171] Various formulations suitable for administration of bisantrene, therapeutically active derivatives of bisantrene, or pharmaceutically acceptable salts of bisantrene or derivatives thereof are known in the art. U.S. Patent No. 4,784,845 to Desai et al. discloses a composition for delivery of a hydrophobic drug (i.e., bisantrene or a cardioprotective derivative thereof) comprising: (i) the hydrophobic drug; (ii) an oily vehicle or 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. Desai et al., U.S. Pat. No. 4,816,247, discloses a composition for delivering a hydrophobic drug (e.g., bisantrene or a cardioprotective derivative or analog thereof) by intravenous, intramuscular or intraarticular routes, comprising: (i) the hydrophobic drug; (ii) a pharmaceutically acceptable oily vehicle or oil selected from: (a) naturally occurring plant oils and (b) semisynthetic mono-, di- and tri-glycerides, wherein the oily vehicle or oil does not contain BHT or BHA; (iii) a surfactant or emulsifier; (iv) a co-surfactant or emulsifier; (v) when the hydrophobic drug is basic, a pharmaceutically acceptable solvent or oil selected from C 6 -C 20 a saturated or unsaturated fatty acid and, when the hydrophobic drug is acidic, an ion pair forming agent selected from pharmaceutically acceptable aromatic amines; and (vi) water.

[0172] According to certain embodiments, the composition according to the present invention can be adapted to be administered at about 0.1 mg / m 2 / day to about 100 mg / m 2 / day, for example, about 0.2 mg / m 2 / day to about 50 mg / m 2 / day, about 0.5mg / m 2 / day to about 20 mg / m 2 / day, about 1.0mg / m 2 / day to about 10 mg / m2 / day, about 1.0mg / m 2 / day to about 8 mg / m 2 / day, about 1mg / m 2 / day, about 2mg / m 2 / day, about 3mg / m 2 / day, about 4mg / m 2 / day, about 5mg / m 2 / day, about 6mg / m 2 / day, about 7mg / m 2 / day or about 10 mg / m 2 In some embodiments, the compositions according to the invention may be adapted to administer bisantrene daily or once a week, once every two weeks, once every three weeks, once every four weeks, over 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 according to the invention may be adapted to administer bisantrene over a period of 28 days, optionally daily or once a week, once every two weeks, once every three weeks, once every four weeks, at a dose of about 20-50 mg / m 2 The composition comprising a pharmaceutically acceptable salt of bisantrene or a therapeutically active derivative thereof or a pharmaceutically acceptable salt thereof according to the present invention can be adapted to deliver similar dosage rates, adjusted for molar equivalence.

[0173] The dosing regimen for the protein kinase inhibitor will depend on the specific protein kinase inhibitor and has been published for known protein kinase inhibitors, including for the treatment of ccRCC. In addition, one of ordinary skill in the art can readily determine the appropriate dose for a given protein kinase inhibitor using known methods and techniques discussed above with respect to bisantrene. The compositions according to the invention can be formulated to contain the protein kinase at a recommended dose rate or at a different dose rate as discussed below.

[0174] According to certain embodiments, and as a result of the synergistic biological activity observed when bisantrene (or a bisantrene derivative or a pharmaceutically acceptable salt of either) is administered together with a protein kinase inhibitor, the dosage of the protein kinase in the composition according to the invention may be lower than when it is administered alone to a patient. This is particularly important for active agents that have deleterious side effects (e.g., cardiotoxicity). Thus, in certain embodiments of the composition according to the invention, the protein kinase inhibitor may be included in the composition at a dosage significantly lower than the recommended dosage rate of the protein kinase inhibitor when administered alone, for example at a dosage rate that is at least 10%, 20%, 30%, 40%, 50%, 60% or 70% lower than the dosage rate of the protein kinase inhibitor when administered alone.

[0175] Similarly, when the composition of the present invention comprises an additional therapeutic agent (which is a checkpoint inhibitor and / or an immunomodulator), the additional therapeutic agent may be included in the composition at its known recommended dosage rate for the treatment of ccRCC and other VHL(-) cancers, or if a synergistic effect between the components is determined, it may be included in the composition at an even lower dosage rate.

[0176] Use of the composition for preparing a drug for treating ccRCC and other VHL(-) cancers

[0177] The present invention also provides the use of the composition according to the present invention for the preparation of a medicament for treating ccRCC or other VHL(-) cancers, in particular protein kinase-resistant ccRCC, in a patient.

[0178] The parameters of such preparations are described above, including the protein kinase inhibitor, bisantrene, bisantrene derivatives and salts thereof, and checkpoint inhibitors and immunomodulators as optional additional therapeutic agents, as well as the dosage / administration of all active substances, dosage forms and other components.

[0179] Kit for treating ccRCC and other VHL(-) cancers

[0180] The present invention also provides a kit for treating ccRCC or other VHL(-) cancers, particularly protein kinase-resistant ccRCC, the kit comprising at least one protein kinase inhibitor and at least one second agent, the second agent comprising bisantrene or a derivative thereof, or a pharmaceutically acceptable salt of bisantrene or a derivative thereof.

[0181] The parameters of such a kit are described above, including the protein kinase inhibitor, bisantrene, bisantrene derivatives and salts thereof, and checkpoint inhibitors and immunomodulators as optional additional therapeutic agents, as well as the dosages / administration of all active substances, dosage forms and other components.

[0182] In some cases, the kit may also include vials, test tubes, needles, packaging, or other materials.

[0183] Kits containing unit doses of one or more active agents described herein, typically injectable doses, are provided. Such kits may include a container containing the unit doses, an informational package insert describing the use of the drug in treating the disease and the attendant benefits, and optionally an implement or device for delivering the composition.

[0184] The kit may further comprise any device suitable for administering the composition. For example, the kit may comprise a needle suitable for intravenous administration.

[0185] In some cases, the kit may be provided with instructions. The instructions may be provided in the kit or may be accessed electronically. The instructions may provide information on how to use the composition of the present disclosure. The instructions may also provide information on how to use the device of the present disclosure. The instructions may provide information on how to perform the method of the present disclosure. In some cases, the instructions may provide dosage information. The instructions may provide drug information, such as mechanism of action, drug formulation, adverse risks, contraindications, etc. In some cases, the kit is purchased by a doctor or healthcare provider for administration in a clinic or hospital. In some cases, the kit is purchased by a laboratory and used to screen candidate compounds.

[0186] Preferred forms of the invention will now be described with reference to the following examples including comparative data, by way of example only, and the examples should not be construed as limiting the scope or spirit of the invention in any way.

[0187] Example

[0188] Example 1: Cell viability determination and analysis

[0189] Cell viability was determined using the resazurin metabolic activity assay. Cells were plated at 1 x 10 3 cells / well (786-O, RCC4 EV, RCC4 VHL) or 3x 10 3 Cells / well (Caki-1, Caki-2) were seeded in duplicate in wells of a 96-well microtiter plate and cultured for 24 h.

[0190] Zantran (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 in DMSO at 17.2 mM (lenvatinib) or 100 mM (all others).

[0191] Drugs were then diluted in culture medium and added to the wells, and cells were cultured for an additional 72 h.

[0192] Human kidney cell lines were cultured in a humidified incubator at 37 °C and 5% CO 2 The cells were cultured in the culture medium listed in Table 1 below:

[0193] Table 1 - Cell culture media and supplements used for renal cell lines

[0194]

[0195]

[0196] Viability was determined using the fluorescent 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 Aldrich), dissolved in PBS [3]). Resazurin is metabolized by metabolically active cells to red fluorescent resorufin. Fluorescence was measured 5 h after addition of the resazurin solution (1:10, v / v) at 544 nm excitation / 590 nm emission on a FLUOstar OPTIMA plate reader (BMG LabTechnologies). Graphs were generated using Graphpad Prism 9 software. Drug IC was determined by cubic spline-lowess regression analysis using Prism 9. 50 At least three independent replicates were performed for each cell line and each drug combination, and the data are presented as mean ± standard error of the mean (SEM). The culture conditions for each cell line are described below.

[0197] Clonogenicity assay - The colony-forming ability of cells treated with bisantrine was determined using a clonogenicity assay. RCC4 EV and RCC4 VHL cells were seeded in 6-well plates at a density of 1000 cells / well and allowed to adhere for 24 hours. Bisantrine was diluted in culture medium and added to the wells, and the cells were cultured for 96 hours. The medium containing the drug was removed, replaced with fresh medium (drug-free), and cultured for another 96 hours to allow cell colonies to form. At the endpoint, the medium was removed from the wells and the cells were washed twice with cold PBS. The cells were fixed on ice with ice-cold methanol 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 off and photos 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 inclonogenic assays, PLoS One, 9(3): e92444] of 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 the area of ​​the well filled with colonies was determined using Prism 9 and expressed as % of untreated cells. Four independent parallel experiments were performed for each cell line and data are expressed as mean ± SEM.

[0198] Synergy analysis - For combination drug treatments, three different synergy analyses have been performed, including Weber's fractional product method (Weber J (1963), "Effect of more than one inhibitor", in Hochster ER, Quastel J., eds., Enzymes and metabolic inhibitors, Academic Press: New York), Chou-Talalay (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 using SynergyFinder 2.0 software (Ianevski A, Giri AK, Aittokallio T, (2020), "SynergyFender The BLISS synergy method (BLISS, (1939), "CI: The toxicity of poisons applied jointly", Ann App Biol, 26: 585-615) was used in conjunction with SynergyFinder 2.0: visual analytics of multi-drug combination synergies, Nucleic Acids Res., 48(W1): W488-W493). Details of each method are provided below.

[0199] Weber's fractional product method: Weber introduced a method later known as the "fractional product" method in 1963 (Weber J (1963), "Effect of more than one inhibitor", in Hochster ER, Quastel J., eds., Enzymes and metabolic inhibitors, Academic Press: New York) (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 uses the fractional product of the effect of each drug alone to estimate the expected additive effect of two drugs, that is:

[0200] 1–Fa(drug 1+drug 2)=(1-Fa drug 1)*(1–Fa drug 2)

[0201] Where Fa = fraction of cells affected, expressed as a decimal.

[0202] An observed Fa(drug 1 + drug 2) value greater than the expected Fa(drug 1 + drug 2) value indicates synergy, while an observed Fa(drug 1 + drug 2) value less than the expected value indicates antagonism. Thus, Weber's method takes into account the potency of each drug at a specific dose, but does not consider the shape of the overall dose-response curve

[10] . The results are considered to be:

[0203] Result value <-0.1 indicates synergy

[0204] Result values ​​between -0.1 and 0.1 indicate additive

[0205] Result value > 0.1 indicates antagonism

[0206] Chou-Talalay method: In 1984, Chou and Talalay proposed the most widely used synergy assessment method 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). Using the Henderson-Hasselbalch, Michaelis-Menten, Scatchard and Hill equations, Chou and Talaly derived the "median effect" equation, using the input dose and the corresponding dose effect to model the median effect dose and S-shapedness of the dose-response curve. Chou and Talalay also coined the term "combination index (CI)" to evaluate the degree of synergy between two compounds, which is defined as:

[0207]

[0208] in:

[0209] DRI = drug reduction index, or the number of times the dose of drug A is reduced due to the presence of drug B.

[0210] ·CI less than 0.9 indicates synergy;

[0211] A CI between 0.9 and 1.1 indicates an additive interaction; and

[0212] A CI greater than 1.1 indicates antagonism.

[0213] The CI for any Fa can be estimated using the median effect equation.

[0214] Both Chou Talalay and Weber methods are widely used robust synergy determination methods. Although the Chou Talalay method is considered a more complex model, it recommends the use of a constant dose ratio between the two drugs in the combination. Therefore, when this assumption is not met, the Weber method is recommended.

[0215] Bliss method: SynergyFinder (version 2.0) is a stand-alone web application for interactive analysis and visualization of multi-drug (2 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 antagonism is quantified by comparing the observed drug combination response with the expected response, which is calculated using a reference model that assumes no interaction between the drugs. The Bliss model is used in SynergyFinder. The model quantifies the degree of synergy as the multiplicative effect of the single drugs as if they acted independently. This independence model assumes a random process in which the two drugs act independently and the expected combination effect can be calculated based on the probability of independent events. When the synergy score:

[0216] Less than -10: The interaction between the two drugs may be antagonistic;

[0217] -10 to 10: The interaction between the two drugs is likely to be additive; and

[0218] When greater than 10: the interaction between the two drugs may be synergistic.

[0219] Example 2: Combination of Zanqun with currently used protein kinase inhibitors

[0220] To determine the effect of the combined drug treatment, three different methods were used. The Weibull analysis method revealed Synergistic effects of zanquinone and protein kinase inhibitors in cell lines at various drug doses ( Figure 1-3 The strongest synergy was observed for the combination of Zantril with lenvatinib, cabozantinib, and pazopanib. Synergy was also observed for sunitinib, sorafenib, and everolimus doses, most often at lower doses. Similar results were observed in the other 4 cell lines, with lenvatinib and cabozantinib, followed by pazopanib, consistently showing the strongest synergy with Zantril ( Figure 4-15 ).

[0221] Analysis using the Bliss method also revealed overall synergy of Zanqun with lenvatinib and pazopanib in all 5 cell lines (Table 2). This method further revealed that everolimus was synergistic in all 5 cell lines and cabozantinib was synergistic in all cell lines (although this was marginal for Caki-2 cells). Slight synergy was observed for sunitinib and sorafenib (Table 2). Bliss analysis can also provide scores for the strongest synergistic areas among drug doses, which suggests synergistic areas for drugs tested in all cell lines (Table 3). The individual Bliss scores for each dose combination further revealed multiple doses that were synergistic in most cell lines.

[0222] Table 2 - Overall synergy scores for bisantrene-drug combinations determined by Bliss synergy analysis

[0223]

[0224] Values ​​> 10 are considered synergistic (*); values ​​between -10 and 10 are additive ( & ); values ​​below -10 are considered antagonistic ( # ).

[0225] Table 3 - Maximum synergy region scores for bisantrene-drug combinations determined by Bliss synergy analysis

[0226]

[0227] Values ​​> 10 are considered synergistic (*); values ​​between -10 and 10 are additive ( & ); values ​​below -10 are considered antagonistic ( # ).

[0228] Finally, Chou-Talalay analysis was used to quantify the combined effects of Zanqun and each anti-RCC drug. This analysis was less stringent than the Weber or Bliss analysis and identified synergistic effects at most doses for almost all combinations. 50 When the synergy score was set, synergy between Zanqun and sunitinib, pazopanib, lenvatinib, and cabozantinib was predicted (Table 4).

[0229] Table 4 - Combination Index ED for Bisantrene-Drug Combinations Determined by Chou Talalay Analysis 50

[0230]

[0231] Combination indices were calculated from ED50 using Chou-Talalay analysis; antagonism (#); additive (&); moderate synergism (*).

[0232] Among the ccRCC cells tested, lenvatinib, cabozantinib, and pazopanib showed the strongest synergy with zantriol. This may indicate that these drugs have common cellular targets that are synergistically lethal with zantriol. These drugs are all VEGFR inhibitors that inhibit angiogenesis. In addition to inhibiting VEGFR on endothelial cells, these multi-kinase inhibitors also inhibit tumor growth due to intrinsic inhibition of other kinases by tumor cells. Given that our assay system consists of only tumor cells, the synergistic cytotoxicity observed with zantriol must be due to kinase inhibition within tumor cells. Common targets for lenvatinib, cabozantinib, and pazopanib include DDR2, DDR1, RET, and RIPK2. RET has also been identified as a target of sunitinib. The Caki-1 cell line was further added to the pool of 4 cell lines for analysis of some drugs, including cabozantinib, which further revealed AXL and MET as targets of cabozantinib.

[0233] Embodiment 3: Zanqun kills clear cell renal cell carcinoma cells and is more toxic to VHL(-) cells

[0234] The results presented in Example 2 indicate that there is greater synergy between bisantrene and a combination of protein kinase inhibitors when treating mutant VHL(-) cells (RCC4 EV cells) compared to VHL rescued lines (RCC4 VHL). These results are consistent with earlier observations in which bisantrene was shown to be not only toxic to all renal cancer cell lines (data not shown), but surprisingly was more toxic to RCC4 EV cells, which are VHL(-) mutant cells, compared to isogenic rescued (VHL(+)) RCC4 VHL cells, as shown in Tables 5 and Fig.18 as shown in .

[0235] Table 5 - Cytotoxicity IC of bisantrene in renal cancer RCC4 EV (VHL(-); mutant / Mut) and VHL rescued RCC4VHL (wild type / WT) cell lines 50 value

[0236]

[0237] Greater lethality between VHL loss and bisantrine was also observed for RCC4 EV cells compared to RCC4 VHL cells using a long-term clonogenic cell growth assay (colony formation) as described in Example 1, which better measures the effects of drugs on cancer cell growth rather than cell killing.

[0238] RCC-4EV (VHL mutant) cells were significantly more sensitive to bisoprolol (2.9x) compared to wild-type VHL rescued cells (Tables 6 and Fig.19 ).

[0239] Table 6 - Clonogenic IC of Zan Group in Renal RCC4 EV (VHL(-); Mutant / Mut) and VHL-rescued RCC4 VHL (Wild Type / WT) Cell Lines 50

[0240]

[0241] Advantages of the present invention

[0242] The present invention provides a new way to treat ccRCC and other VHL(-) cancers by administering bisantrene, an antineoplastic agent with multiple mechanisms of action, including DNA intercalation, topoisomerase inhibition, inhibition of fat and obesity-associated protein (FTO), and activation of the immune system. Bisantrene is well tolerated, particularly lacking the cardiotoxicity characteristic of some other anthracene derivatives. Bisantrene can be used in conjunction with other therapeutic agents for the treatment of ccRCC and other VHL(-) cancers.

[0243] The invention illustratively described herein can be properly practiced in the absence of any one or more elements, limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. should be understood broadly and without restriction. As used herein, the use of the term "comprising" as a transitional phrase in the claims is intended to include the use of the transitional phrases "essentially consisting of..." or "consisting of..." therein, if a narrower transitional phrase is not explicitly excluded. Although the present invention has been specifically disclosed through preferred embodiments and optional features, modifications and changes may be made to the invention disclosed herein by those skilled in the art, and these modifications and changes are considered to be within the scope of the invention disclosed herein. The present invention has been described broadly and generally herein. Each narrower species and subgeneric grouping falling within the scope of the generic disclosure also constitutes a part of these inventions. This includes the generic description of each invention, but with a proviso or negative limitation that any subject matter is deleted from the generic, regardless of whether the deleted material is specifically present therein.

Claims

1. A method of treating a patient with ccRCC or other VHL(-) cancer, the method comprising administering to the patient a therapeutically effective amount of a protein kinase inhibitor and a therapeutically effective amount of at least a second agent, the second agent comprising bisantrene or a derivative thereof, or a pharmaceutically acceptable salt of bisantrene or a derivative thereof.

2. The method of claim 1, wherein the protein kinase inhibitor is a tyrosine kinase inhibitor.

3. The method of claim 1 or claim 2, wherein the protein kinase inhibitor inhibits one or more than one of VEGFR1, VEGFR2, VEGFR3, DDR1, DDR2, RET and RIPK2.

4. The method of claim 1, wherein the at least one protein kinase inhibitor is selected from: (a) Pazopanib; (b) Lenvatinib; (c) Cabozantinib; (d) Everolimus; (e) Sorafenib; (f) Sunitinib; (g) temsirolimus; (h) Mitomycin C; (i) Axitinib; (j) Tivozanib; and (k) Beizu Tifan.

5. The method of claim 1, wherein the at least one protein kinase inhibitor is selected from: (a) Pazopanib; (b) lenvatinib; and (c) Cabozantinib.

6. The method of any one of claims 1 to 5, wherein the second agent is bisantrene or a pharmaceutically acceptable salt thereof.

7. The method of any one of claims 1 to 6, comprising administering to the patient a therapeutically effective amount of a protein kinase inhibitor and a therapeutically effective amount of bisantrene or a pharmaceutically acceptable salt thereof.

8. The method according to any one of claims 1 to 7, comprising treating ccRCC, optionally protein kinase inhibitor-resistant ccRCC.

9. The method of any one of claims 1 to 8, further comprising administering at least one additional therapeutic agent for the treatment of ccRCC or other VHL(-) cancers.

10. The method of claim 9, wherein the additional therapeutic agent is a checkpoint inhibitor drug or an immunomodulatory agent.

11. The method of claim 10, wherein the at least one additional therapeutic agent is selected from: (a) Atezolizumab; (b) Avelumab; (c) bevacizumab; (d) cemiplimab; (e) Dotalimumab; (f) durvalumab; (g) interleukin-2; (h) Ipilimumab; (i) Nivolumab; (j) pembrolizumab; and (k) Aldesleukin.

12. The method of claim 1, comprising administering to the patient the at least one protein kinase inhibitor prior to, simultaneously with, or after administering to the patient the second agent.

13. The method of any one of claims 1 to 10, wherein the at least one protein kinase inhibitor and the second agent are administered to the patient simultaneously, optionally in a single composition.

14. The method of any one of claims 1 to 13, wherein the treatment has a synergistic outcome on ccRCC and other VHL(-) cancers compared to a method of administering the at least one protein kinase or the second agent alone.

15. The method of any one of claims 1 to 14, wherein the dosage of the at least one protein kinase inhibitor is at least 25% less than the dosage of the at least one protein kinase inhibitor agent required to achieve the same target result when administered without the second agent.

16. A pharmaceutical composition for treating ccRCC or other VHL(-) cancers, comprising at least one protein kinase inhibitor and a second agent, wherein the second agent comprises bisantrene or a derivative thereof, or a pharmaceutically acceptable salt of bisantrene or a derivative thereof.

17. The composition of claim 16, wherein the protein kinase inhibitor is a tyrosine kinase inhibitor.

18. The composition of claim 17, wherein the protein kinase inhibitor inhibits one or more than one of VEGFR1, VEGFR2, VEGFR3, DDR1, DDR2, RET, and RIPK2.

19. The composition of claim 16, wherein the at least one protein kinase inhibitor is selected from: (a) Pazopanib; (b) Lenvatinib; (c) Cabozantinib; (d) Everolimus; (e) Sorafenib; (f) Sunitinib; (g) temsirolimus; (h) Mitomycin C; (i) Axitinib; (j) Tivozanib; and (k) Beizu Tifan.

20. The composition of claim 16, wherein the at least one protein kinase inhibitor is selected from: (a) Pazopanib; (b) lenvatinib; and (c) Cabozantinib.

21. The composition of any one of claims 16 to 20, further comprising at least one additional therapeutic agent for the treatment of ccRCC and other VHL(-) cancers.

22. The composition of claim 21, wherein the additional therapeutic agent is a checkpoint inhibitor drug or an immunomodulator.

23. The composition of claim 22, wherein the at least one additional therapeutic agent is selected from: (a) Atezolizumab; (b) Avelumab; (c) bevacizumab; (d) cemiplimab; (e) Dotalimumab; (f) durvalumab; (g) interleukin-2; (h) Ipilimumab; (i) Nivolumab; (j) pembrolizumab; and (k) Aldesleukin.

24. The composition of any one of claims 16 to 23, wherein the second agent is bisantrene or a pharmaceutically acceptable salt thereof.

25. The composition of any one of claims 16 to 24 comprising a therapeutically effective amount of a protein kinase inhibitor and a therapeutically effective amount of bisantrene or a pharmaceutically acceptable salt thereof.

26. The composition according to any one of claims 16 to 25, for use in treating ccRCC, optionally protein kinase inhibitor-resistant ccRCC.

27. The composition of any one of claims 16, which has synergistic activity against ccRCC or other VHL(-) cancers compared to the at least one protein kinase or the second agent administered alone.

28. Use of a composition according to any one of claims 16 to 26 for the preparation of a medicament for treating ccRCC or other VHL(-) cancers in a patient.

29. The use according to claim 28, wherein the medicament is for treating ccRCC, optionally protein kinase inhibitor-resistant ccRCC.

30. The use according to claim 28 or claim 29, wherein the medicament comprises a protein kinase inhibitor and bisantrene or a pharmaceutically acceptable salt thereof.

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

32. The kit of claim 31, wherein the protein kinase inhibitor is a tyrosine kinase inhibitor.

33. The kit of claim 31 or claim 32, wherein the protein kinase inhibitor inhibits one or more than one of VEGFR1, VEGFR2, VEGFR3, DDR1, DDR2, RET and RIPK2.

34. The kit of claim 31, wherein the at least one additional therapeutic agent is selected from: (a) Pazopanib; (b) Lenvatinib; (c) Cabozantinib; (d) Everolimus; (e) Sorafenib; (f) Sunitinib; (g) temsirolimus; (h) Mitomycin C; (i) Axitinib; (j) Tivozanib; and (k) Beizu Tifan.

35. The kit of claim 31, wherein the at least one protein kinase inhibitor is selected from: (a) Pazopanib; (b) lenvatinib; and (c) Cabozantinib.

36. The kit of any one of claims 31 to 35, wherein the second agent is bisantrene or a pharmaceutically acceptable salt thereof.

37. A kit according to any one of claims 31 to 36 comprising a protein kinase inhibitor and bisantrene or a pharmaceutically acceptable salt thereof.

38. The kit of any one of claims 31 to 37, further comprising at least one additional therapeutic agent for the treatment of ccRCC or other VHL(-) cancers.

39. The kit of claim 38, wherein the additional therapeutic agent is a checkpoint inhibitor drug or an immunomodulatory agent.

40. The kit of claim 39, wherein the at least one additional therapeutic agent is selected from: (a) Atezolizumab; (b) Avelumab; (c) bevacizumab; (d) cemiplimab; (e) Dotalimumab; (f) durvalumab; (g) interleukin-2; (h) Ipilimumab; (i) Nivolumab; (j) pembrolizumab; and (k) Aldesleukin.

41. A kit according to any one of claims 31 to 40 for use in treating ccRCC, optionally protein kinase inhibitor-resistant ccRCC.

42. The kit of any one of claims 31 to 41, comprising instructions for administering the at least one protein kinase inhibitor to the patient prior to, simultaneously with, or after administration of the second agent to the patient.

43. The kit of any one of claims 31 to 42, comprising instructions for administering the at least one protein kinase inhibitor and the second agent simultaneously to the patient.

Images