Compositions of cannabinoids and kinase inhibitors for the treatment of cancer - Patent Application 20070122999

Abstract

Compositions containing cannabinoids and kinase inhibitors are useful for the treatment of hepatocellular carcinoma (HCC). Compositions containing cannabinolic acid (CBNA) and sorafenib act synergistically on HepG2 cells, an in vitro liver cancer model, and on ex vivo patient-derived xenograft (PDX) HCC models. The effects of all of the disclosed compositions are superior to sorafenib alone, which is the first- and second-line chemotherapeutic agent for the treatment of HCC.

Description

[Technical Field]

[0001] The present disclosure relates to novel compositions comprising cannabinoids and kinase inhibitors, and methods of making and using such compositions to treat cancer, particularly hepatocellular carcinoma. [Background technology]

[0002] Various cancers are treated with kinase inhibitors targeting vascular endothelial growth factor (VEGF) or vascular endothelial growth factor receptor (VEGFR). Hepatocellular carcinoma (HCC) is a particularly aggressive disease with unfavorable patient outcomes. There is a long-standing unmet need in the art for effective treatments for cancers such as HCC. Fewer than 35% of people diagnosed with this disease survive for five years. This number decreases to less than 12% if the cancer spreads to nearby tissues and to less than 2% if the cancer metastasizes to other organs (Kitisin, Packiam et al., 2011). To date, there are no approved phytocannabinoid-derived chemotherapy treatment options for HCC. HepG2 cells, first isolated from a 15-year-old boy in 1975, are well differentiated and characterized and have served for many years as a model for hepatocellular carcinoma. PDX models 575, 658, and LI-011 are patient-derived human xenograft (PDX) models in nude mice available for research by the contract research organization (CRO) Charles River.

[0003] The medicinal use of cannabis dates back to ancient times, as evidenced by gold vessels containing cannabis residues excavated in Scythian tombs. Today, cannabis is approved in the United States and Canada as an adjunct to chemotherapy to relieve nausea and vomiting, loss of appetite, and pain (Kleckner, Kleckner et al., 2019).

[0004] The anti-cancer properties of cannabinoids and other components in cannabis, such as terpenes and flavonoids, are being investigated (Blasco-Benito, Seijo-Vila et al., 2018). Within the cannabis field, tetrahydrocannabinol (THC) and cannabidiol (CBD) have been the focus of cancer research. CBD has attracted more attention due to its lack of "side effects" on the central nervous system (CNS).

[0005] Decarboxylated cannabinoids, such as THC and CBD, are known to have low oral bioavailability and drug-like properties (Meyer, Langos, et al., 2018). Furthermore, their low solubility and high first-pass metabolism make them poor candidates for oral administration. For example, the oral bioavailability of CBD ranges from 9–30%, which is accompanied by significant interindividual variability in plasma profiles. In contrast, carboxylated forms of cannabinoids have significantly increased solubility and bioavailability compared to their decarboxylated counterparts, making them better drug candidates for oral administration if bioactive. For example, cannabidiolic acid (CBDA) is approximately 25,000 times more water soluble than its decarboxylated counterpart, CBD (17.5 mg / ml vs. 0.7 μg / ml).

[0006] New therapeutic targets are agonist or antagonist compounds that modify heterodimer formation within the numerous C-protein coupled receptors, of which the cannabinoid receptors are a part. Because this superfamily of receptors accounts for approximately 4% of the protein-coding genome (Moreno, Cavic et al., 2019), this opens up numerous new disease pathways. New targets may provide mechanistic explanations for interactions with cannabinoids and / or other compounds.

[0007] The effects of cannabinoids can be explained by their variable binding affinity to multiple G protein-coupled heterodimeric receptors, such as CB1, CB2, GPR55, and TPRV1, which signal multiple downstream pathways resulting in variable drug responsiveness (Moreno, Cavic, et al., 2019). Indeed, Zhong (2020) reported that cannabinol (CBN) mediates apoptosis via the MAPK / ERK and PI3K-ATK pathways and cell cycle arrest by downregulating P21. Interestingly, Zhong's study also demonstrated downregulation of CB2 and GPR55 receptors by CBN treatment.

[0008] Whether these effects are maintained for the more water-soluble acid precursor form, cannabinolic acid (CBNA), remains to be evaluated. Furthermore, Torres, Lorente, et al. (2011) demonstrated a synergistic response when THC was administered with the selective ALK inhibitor TAE-684, indicating that additive or synergistic effects can be enhanced by targeting convergent pathways. Again, prior to this discovery, it was unclear whether these effects could be replicated with cannabinoids such as CBN / CBNA and compounds with convergent mechanisms of action. Summary of the Invention

[0009] Aspects of the present disclosure generally relate to the synergistic anti-cancer activity of cannabinoids and kinase inhibitors. The cannabinoids may include either carboxylated or decarboxylated forms. The kinase inhibitors may include sorafenib or regorafenib. In one embodiment, the present disclosure describes the synergistic anti-cancer activity of CBN or CBNA with sorafenib or regorafenib in the treatment of cancer.

[0010] In one aspect, a synergistic composition comprising CBNA and a kinase inhibitor for the treatment of cancer is disclosed. In some embodiments, the molar ratio of CBNA to kinase inhibitor is about 1:1 to about 6:1, preferably about 2:1 to about 5:1, and most preferably about 3:1 or 4:1.

[0011] In another embodiment, an oral dosage form is disclosed that includes a synergistic composition comprising CBNA and a kinase inhibitor. The oral dosage form may include tablets, solutions, suspensions, powders, and nanoencapsulated dosage forms. In such oral dosage forms, the molar ratio of CBNA to kinase inhibitor in the composition is about 1:1 to about 6:1, preferably about 2:1 to about 5:1, and most preferably about 3:1 or 4:1.

[0012] In another embodiment, a method of treating cancer is disclosed, comprising administering to a patient a therapeutically effective amount of a synergistic combination of CBNA and a kinase inhibitor. The molar ratio of CBNA to kinase inhibitor in the combination can be about 1:1 to about 6:1, preferably about 2:1 to about 5:1, and most preferably about 3:1 or 4:1. The method can include administering the combination as a fixed or non-fixed combination.

[0013] In some embodiments, cancers that can be treated with the synergistic combinations disclosed herein are cancers for which sorafenib or regorafenib is effective in treating, such as hepatocellular carcinoma, renal cell carcinoma, thyroid cancer, metastatic colorectal cancer, or gastrointestinal stromal tumors. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 shows the chemical structures of the carboxylated forms of cannabinol (cannabinolic acid (CBNA)), sorafenib, and regorafenib.

[0015] [Figure 2A] FIG. 1 shows a comparison of observed and estimated additive IC50 values ​​for different CBNA and sorafenib ratios measured in DMEM medium containing 1% FBS from three separate experiments (n=3). [Figure 2B] FIG. 1 shows a comparison of observed (n=1) and predicted / calculated additive IC50 values ​​for different CBNA and sorafenib ratios measured in DMEM medium containing 20% ​​FBS.

[0016] [Figure 3] 1 is a histogram plot of IC50 values ​​(μM) of CBNA:sorafenib versus sorafenib in different media (DMEM with 1% FBS versus IMDM media containing 20% ​​FBS).

[0017] [Figure 4] FIG. 1 shows HepG2 spheroids formed in 96-well ultra-low attachment round-bottom plates after incubation in William's E medium containing 10% FBS at 37° C. and 5% CO for 2.5 days.

[0018] [Figure 5] FIG. 1 shows IC50 value plots of sorafenib and a 3:1 ratio of CBNA:sorafenib against HepG2 spheroids in DMEM with 1% FBS.

[0019] [Figure 6A] FIG. 1 shows IC50 value plots of SCI-0502 against three PDX models and HepG2. [Figure 6B] FIG. 1 shows IC50 plots of sorafenib against three PDX models and HepG2. [Figure 6C] FIG. 6B shows a comparison of IC50 values ​​calculated in FIG. 6A and FIG. 6B. [Figure 6D] FIG. 1 shows a comparison between IC50 values ​​for PDX models treated with SCI-0502 and sorafenib.

[0020] [Figure 7] FIG. 1 shows a plot of IC50 values ​​for SCI-0502 and sorafenib against a 20 donor pool of normal hepatocytes (HC3_23).

[0021] [Figure 8A]For HepG2 cells, FIG. 8A shows changes in protein expression by in-cell Western blot after treatment with sorafenib at respective IC50 concentrations for 6 hours. [Figure 8B] For HepG2 cells, FIG. 8B shows the changes in protein expression by in-cell Western blot after treatment with CBNA at respective IC50 concentrations for 6 hours. [Figure 8C] For HepG2 cells, Figure 8C shows the changes in protein expression by in-cell Western blot after treatment with SCI-0502 at respective IC50 concentrations for 6 hours.

[0022] [Figure 9] FIG. 1 is a diagram of the mechanisms of action determined for sorafenib (C1) and CBNA (C2).

[0023] [Figure 10A] Comparison of in vivo immunoprecipitation results obtained from a PDX tumor model ( 575 ) implanted with sorafenib, CBNA, and the intratumoral device NanoNail (Kibur Medical Inc.), which is loaded with CBNA:sorafenib (4:1). [Figure 10B] Comparison of in vivo immunoprecipitation results obtained from a PDX tumor model ( 575 ) implanted with sorafenib, CBNA, and the intratumoral device NanoNail (Kibur Medical Inc.), which is loaded with CBNA:sorafenib (4:1).

[0024] [Figure 11] FIG. 1 shows DAB staining of Ki67 and projected cell proliferation plots in nanonail-implanted 575 PDX tumors treated with sorafenib, CBNA, and CBNA:sorafenib (4:1).

[0025] [Figure 12]FIG. 1 shows the apoptotic index (%) of SCI-0502 versus its positive controls, sorafenib and doxorubicin, in a PDX(575) model in NCG mice equipped with a nanonail device.

[0026] [Figure 13] Figure 1 shows the plasma and liver concentration profiles of sorafenib in subjects receiving 400 mg of sorafenib orally (Jain, Woo et al., 2011). Solid and dashed lines are derived from simulations, and open symbols are derived from four patients. DETAILED DESCRIPTION OF THE INVENTION

[0027] It should be understood that certain features of the invention that are described herein for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. That is, unless clearly incompatible or specifically excluded, each individual embodiment is considered to be combinable with any other embodiment, and such combinations are considered to be separate embodiments. Conversely, various features of the invention that are described for brevity in the context of a single embodiment may also be provided separately or in any subcombination. Finally, while embodiments may be described as part of a series of steps or as part of a more general structure, each such step may itself be considered an independent embodiment that may be combined with others.

[0028] Specific Terms The transitional phrases "comprising," "consisting essentially of," and "consisting" are intended to connote their generally accepted meanings in patent terminology: (i) "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps, (ii) "consisting of" excludes any element, step, or ingredient not recited in the claim, and (iii) "consisting essentially of" limits a claim or embodiment to particular materials or steps, and those that do not "materially affect the basic and novel characteristics" of the invention recited in the claim or embodiment. More specifically, the basic and novel features relate to the ability of the methods or uses to provide at least one of the benefits described herein, including, but not limited to, the ability to improve the survival of a human population relative to the survival of a comparative human population described elsewhere herein. Embodiments described with the phrase "comprising" (or equivalents) also provide, as embodiments, those described independently with the phrases "consisting of" and "consisting essentially of."

[0029] When values ​​are expressed as approximations, using the statement "about," it will be understood that the particular value forms another embodiment. Unless otherwise specified, the term "about" means a variance of ±10% of the associated value, although additional embodiments include those where the variance can be ±5%, ±15%, ±20%, ±25%, or ±50%, and particularly where the term "about" means a variance of ±5% or ±10%, more particularly ±5%, of the associated value.

[0030] When lists are presented, it is to be understood that each individual element of that list, and every combination of that list, is a separate embodiment unless otherwise stated. For example, a list of embodiments presented as "A, B, or C" should be interpreted as including the embodiments "A," "B," "C," "A or B," "A or C," "B or C," or "A, B, or C."

[0031] As used herein, the singular forms "a," "an," and "the" include plural forms.

[0032] As used herein, "patient" is intended to mean any animal, particularly a mammal. Thus, the method or use is applicable to humans and non-human animals, but most preferably to humans. The terms "patient" and "subject" and "human" can be used interchangeably.

[0033] The terms "treat" and "treatment" refer to the treatment of a patient suffering from a pathological condition, and refer not only to the effects of alleviating the condition by killing cancerous cells, but also to the effects of inhibiting the progression of the condition, including slowing the rate of progression, stopping the rate of progression, improving the condition, and curing the condition. Treatment as a preventative measure (i.e., prophylaxis) is also included.

[0034] A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount may vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, an improvement in the patient's health status.

[0035] The term "dosage" refers to information about the amount of a therapeutic agent a subject should take and how often a subject should take the therapeutic agent. The term "dose" refers to the amount or quantity of a therapeutic agent taken at each time.

[0036] As used herein, the term "cancer" refers to an abnormal growth of cells that tends to grow in an uncontrolled manner and in some cases metastasize (spread).

[0037] As used herein, terms such as "co-administration" encompass the administration of selected therapeutic agents to a single patient and are intended to include therapeutic regimens in which the agents are administered by the same or different routes of administration or at the same or different times.

[0038] The term "pharmaceutical combination" as used herein refers to the product resulting from the mixing or combination of more than one active ingredient, and includes both fixed and non-fixed combinations of active ingredients.The term "fixed combination" means that both active ingredients, such as kinase inhibitors and cannabinoids, are administered to patients simultaneously in the form of a single unit or a single dosage form.The term "non-fixed combination" means that active ingredients, such as kinase inhibitors and cannabinoids, are administered to patients simultaneously, in parallel, or sequentially as separate units or separate dosage forms, without any specific intervening time limit, and such administration provides safe and effective levels of the two active ingredients in the human body.The latter also applies to cocktail therapy, for example, the administration of three or more active ingredients.

[0039] Described herein are compositions containing cannabinoids and kinase inhibitors, exemplified by CBN or CBNA and sorafenib or regorafenib. The inventors have demonstrated that these compositions are more effective than kinase inhibitors (e.g., sorafenib) alone in multiple hepatocellular carcinoma models, and have a greater margin of safety when compared to pools of normal hepatocytes. Using an in vitro cellular hepatocellular carcinoma model (HepG2), a normal pool of human hepatocytes, ex vivo patient-derived xenograft hepatocellular carcinoma models (575, 658, and LI-011), real-time polymerase chain reaction (rtPCR) gene expression studies, and in-cell Western blot protein expression studies, the relative efficacy of test compounds and their combinations, as well as the mechanisms of action and interaction, were evaluated.

[0040] In some embodiments, the cannabinoid may include a decarboxylated form of a cannabinoid, such as cannabinol (CBN).

[0041] Sorafenib or regorafenib is used to treat various cancers, including hepatocellular carcinoma, renal cell carcinoma, thyroid cancer, metastatic colorectal cancer, gastrointestinal stromal tumor.Therefore, the synergistic combination disclosed herein can be used to treat any of these cancers for which sorafenib or regorafenib is known to be an effective treatment.

[0042] In some embodiments, sorafenib or regorafenib can be administered as pharmaceutically acceptable salt.In preferred embodiments, sorafenib is administered in base form.Those skilled in the art can estimate the effective amount of the pharmaceutically acceptable salt that corresponds to its base equivalent.

[0043] Salts can be prepared, for example, by reacting sorafenib or regorafenib with a suitable acid in a suitable solvent. Acid addition salts can be formed using both inorganic and organic acids. Examples of acid addition salts include salts formed with an acid selected from the group consisting of acetic acid, hydrochloric acid, hydroiodic acid, phosphoric acid, nitric acid, sulfuric acid, citric acid, lactic acid, succinic acid, maleic acid, malic acid, isethionic acid, fumaric acid, benzenesulfonic acid, toluenesulfonic acid, methanesulfonic acid (mesylate), ethanesulfonic acid, naphthalenesulfonic acid, valeric acid, acetic acid, propanoic acid, butanoic acid, malonic acid, glucuronic acid and lactobionic acid. Another group of acid addition salts includes salts formed from acetic acid, adipic acid, ascorbic acid, aspartic acid, citric acid, DL-lactic acid, fumaric acid, gluconic acid, glucuronic acid, hippuric acid, hydrochloric acid, glutamic acid, DL-malic acid, methanesulfonic acid, sebacic acid, stearic acid, succinic acid and tartaric acid.

[0044] The pharmaceutical compositions provided herein contain effective amounts of both a cannabinoid and a kinase inhibitor. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse allergic or other undesirable reactions when administered to an animal, e.g., a human, as appropriate. A pharmaceutically acceptable composition may produce acceptable side effects. The preparation of pharmaceutical compositions containing at least a kinase inhibitor and a cannabinoid is known to those skilled in the art in light of the present disclosure. For example, examples are provided in Remington's Pharmaceutical Sciences, 18th Edition, Mack Printing Company, 1990, incorporated herein by reference. Furthermore, for animal (e.g., human) administration, it will be understood that formulations should meet sterility, pyrogenicity, general safety, and purity standards required by regulatory agencies, such as the FDA Office of Biological Standards.

[0045] In some embodiments, the synergistic combination of cannabinoids and kinase inhibitors may be effective in large populations, i.e., not used in personalized or precision medicine approaches, hi other embodiments, the synergistic combination of cannabinoids and kinase inhibitors may be effective in identifiable subsets of patients, which can be identified by appropriate testing.

[0046] As used herein, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and similar materials, and combinations thereof, as known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Edition, Mack Printing Company, 1990, pp. 1289-1329). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0047] Almost all kinase inhibitors are effective when taken orally.In certain preferred embodiments, pharmaceutical compositions are orally administered to treat cancer such as HCC.In one embodiment, the effective oral dose of CBNA / sorafenib combination is estimated for the treatment of patients with HCC.

[0048] In certain embodiments, the actual dosage of the composition administered to a patient can be assessed according to physical and physiological factors, such as body weight, severity of the condition, type of disease being treated, previous or concurrent therapeutic interventions, idiopathic disease of the patient, and route of administration. The physician responsible for administration will, in any event, assess the concentration of active ingredient in the composition and the appropriate dose for the individual subject.

[0049] In certain embodiments, pharmaceutical compositions may contain, for example, at least about 0.1% of the active compound, hi other embodiments, the active compound may comprise from about 2% to about 75%, or from about 25% to about 60%, for example, of the weight of the unit, or any range derivable therein.

[0050] In other non-limiting examples, the dosage of the active compound can also include about 1 milligram / kg / body weight, about 2.0 milligrams / kg / body weight, about 3.0 milligrams / kg / body weight, about 4.0 milligrams / kg / body weight, up to about 5.0 mg / kg / body weight or more per administration, and any range derivable therein. In non-limiting examples of ranges derivable from the numbers recited herein, based on the above numbers, the dosage can be in the range of about 2.5 mg / kg / body weight to about 5.5 mg / kg / body weight.

[0051] In one embodiment, the drug-like properties of sorafenib in humans are predicted using human pharmacokinetic data.

[0052] In one embodiment, the drug-like properties of CBNA are predicted in silico.

[0053] example These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein.

[0054] material and method Pure cannabinolic acid was obtained from Cerilliant (CBNA, C-153-1ML, lot FE10222002, and Kinetochem, NGC1025, lot CBNA-RD-210826). Sorafenib was obtained from Selleckchem (S7397, lot S739707). Sorafenib was reconstituted in DMSO to 10 mM and then diluted to the working concentration. CBNA was dissolved in 95% ethanol (10 mg / ml). Compounds were added directly to the medium at the highest concentration (100 μg / ml) and serially diluted to the working concentration.

[0055] The name SCI-0502 refers to the combination of CBNA and sorafenib.

[0056] Cell assay HepG2 cells were obtained from the American Type Culture Collection (ATCC). A normal hepatocyte pool of cells was obtained from Xenotech. Cells cryopreserved in DMEM containing 5% DMSO were thawed and diluted 10-fold with phenol red-free DMEM medium containing 10% fetal bovine serum, 100 μg / ml penicillin / streptomycin, supplemented with Glutamax™ and sodium pyruvate. The cells were then centrifuged at 20 g for 10 minutes. Cells were then diluted to 2×10 in the same medium supplemented with Revitacell®. 5 The cells were resuspended to a concentration of 7.5 x 10 cells / ml in 100 μl in a 96-well cell culture-treated flat-bottom plate. 3 Cells were seeded and incubated at 37°C, 7.5% CO2, and >95% relative humidity for 18 hours to allow cell attachment. After that, the medium was replaced with similar medium (DMEM, IMDM, Williams E) containing 1–20% FBS and various test compounds (10 μg / ml–100 ng / ml). After 48 hours (two cell doublings), sodium 2-(2-methoxy-4-nitro-5-sulfonatophenyl)-3-(2-methoxy-4-nitro-5-sulfophenyl)-N-phenyltetrazol-3-ium-5-carboxyimidate (XTT) (300 μg / ml) was added and the cells were incubated for 2 hours. Formazan production was measured at 450 nm using a Varioskan lux spectrophotometer. Nonspecific absorbance was measured at 660 nm and subtracted from the baseline measurement at 450 nm. Experiments were performed in triplicate (n=3). After blank subtraction, data were averaged and fitted to a four-point logistic regression curve to determine IC 50 Values ​​were obtained. Blank samples contained equivalent solvent concentrations as negative controls. The concentrations of ethanol or DMSO in the blank standards reflected the concentrations used in the serial dilutions of the consecutive cannabinoids. Initial studies were performed to evaluate the activity of single cannabinoids on HepG2 cell proliferation.

[0057] Spheroid (solid tumor) assay HepG2 cells were thawed and reconstituted in Williams E medium supplemented with Gibco Hepatocyte Maintenance Supplement. Cells were seeded at a density of 1500 cells / well into Corning® Spheroid black-walled, clear round-bottom, ultra-low attachment, 96-well microplates. The plates were spun at 10×G for 20 minutes to concentrate the cells in the center of the well. After 60 hours of incubation at 37°C and 5% CO2, the spheroids were treated with sorafenib to CBNA at a 3:1 ratio for 48 hours, followed by the addition of Calcein-AM (1 μM). After 60 minutes of incubation at 37°C, spheroid viability was measured (excitation / emission: 508 / 527 nm). Experiments were performed in triplicate (n=3). After blank subtraction, data were averaged and fitted to a four-point logistic regression curve to determine IC. 50 got the value.

[0058] Normal liver cell line test To assess the safety margin of compounds prior to clinical trials, we investigated the toxicity of compounds in-house against a pool of normal hepatocytes using a hepatocyte pool obtained from Xenotech (HC3_23). Cells were thawed, reconstituted in William's E medium containing Gibco hepatocyte maintenance supplement, and then seeded into 96-well, black-walled, gamma-irradiated plates (100 μl). After an 18-hour recovery period, the medium was replaced with the same medium containing a 9-fold dilution of the test compound mixture (8–2000 μg / ml). Experiments were performed in triplicate. After 48 hours of incubation, Calcein-AM was added to each well to a final concentration of 7 ng / ml. After a 60-minute reaction, readings were taken (excitation 494 nm, emission 517 nm). Data were fitted to a sigmoidal curve using four-point logistic curve fitting software to determine the IC. 50 The 50% inflection point was used as the value.

[0059] Colony formation assay Tumor xenografts (patient-derived 575, 685, and LI-011, and cell line-derived HepG2) were passaged as subcutaneous xenografts in NMRI nu / nu mice. Tumor volumes ranged from 600 to 1000 mm. 3 Once the tumors reached maturity, they were harvested in accordance with FELASA and GV-SOLAS animal welfare guidelines. The tumors were mechanically disaggregated and then incubated with collagenase type IV (41 U / mL), DNase I (125 U / mL), hyaluronidase type III (100 U / mL), and dispase II (1 U / mL) in RPMI 1640 medium at 37°C for 60–120 min. The cells were filtered through 100 μm and 40 μm mesh sieves, washed with the same medium without the enzymes, and stored in the vapor phase of liquid nitrogen.

[0060] Colony formation assays were performed in 96-well ultra-low attachment plates. Frozen aliquots of tumor cells were prepared from HepG2 HCC cells and the three tumor xenografts described above. Before use, cells were thawed in Iscove's modified Dulbecco's medium (IMDM) supplemented with 20% (v / v) fetal bovine serum, 50 μg / ml gentamicin, 1% ethanol, and 0.4% (w / v) agar. Test wells were first overlaid with 100 μl of the same soft agar medium, followed by 50 μl of medium containing tumor cells. After solidification, the soft agar layer was covered with 90 μl of the same culture medium without agar. After 24 h, 10 μl of test compound or control medium was added and left for 14 days (continuous exposure, 100 μl drug overlay). Incubation parameters were 37°C, 7.5% CO2, and >95% humidity. Study compounds, including sorafenib, were serially diluted in medium containing 1% ethanol. Sunitinib (positive control) was serially diluted in DMSO, transferred to cell culture medium, and then added to the assay plate. For efficacy evaluation, each 96-well plate included six vehicle-treated control wells, triplicate drug-treated wells (duplicate sunitinib), and nine concentrations of test compound combinations. At maximum colony formation, vital colonies were stained with a sterile aqueous solution of INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride, 1 mg / ml, 25 μl / well) for 48 hours, and colonies with a diameter setting of >50 μm (area >2000 μm) were collected. 2 Colony numbers were measured using a Biosys 5000 Va Bioreader (Biosys). Data points (test vs. control, T / C values) obtained for each tumor model were fitted with sigmoidal concentration-response curves using a four-parameter nonlinear curve fit. IC 50 Values ​​are absolute total IC for monotherapy and combination therapy 50 The values ​​are reported as IC values ​​(μM). 50 The value is the concentration of test compound at the intersection of the concentration-response curve and T / C=50%. Mean IC 50 The geometric mean was used to calculate the values.

[0061] mass spectrometry HPLC-DAD / MS technology was used to confirm the concentrations and ratios of individual cannabinoids. After 30 min of incubation, random samples (50 μl) were taken from the test set and precipitated with 150 μl of methanol to precipitate media proteins. Samples were centrifuged at 21910 RCF for 5 min, and the supernatant was collected in a 2 mL glass vial with an insert. Quantitative data were acquired using an Agilent 6410 triple quadrupole mass spectrometer coupled to a 1260 series HPLC and UV detector. Using a thermostated autosampler at 4 °C, 5 μL of sample was injected onto a Phenomenex Kinetex 5 μm XB-C18 100 Å, 250 x 4.6 mm column equipped with a C18 Security Guard ULTRA Cartridge at 40 °C, with a flow rate of 1.00 mL / min, using a gradient of 72 to 100% methanol over 45 min. Mobile phase A consisted of water containing 0.05% ammonium acetate, and mobile phase B consisted of methanol containing 0.05% ammonium acetate. UV detection was performed at 215 nm with a signal bandwidth of 4 nm. Selected ion monitoring (SIM) LC / MS analysis was performed in negative ion mode with the following parameters: source gas temperature was set at 320 °C, flow rate was 12 L / min, capillary voltage was maintained at -3.8 kV, dwell time was set to 200 ms, fragment voltage was set to 135 V, and gas nebulizer pressure was set to 35.0 psi. Concentrations were measured by quantifying the area response of the sample against a standard calibration curve in the range of 0.025–10 μg / mL for the MS detector and 0.5–15 μg / mL for the UV detector.

[0062] Gene expression analysis Targeted cancer gene pathways were analyzed using custom Taqman® array cards that interrogate 90 unique genes associated with cancer-related pathways (Kanehisa, Goto et al. 2010).

[0063] Seed 2 million HepG2 cells in IMDM medium containing 20% ​​FBS and 1% ethanol into a T75 flask and incubate at 37 °C, 7.5% CO until they are 70–80% confluent (approximately 7.5 × 105 The test compound was added to the medium at a predetermined IC 50 The cells were supplemented with medium containing sorafenib at the following concentrations: 12 μM sorafenib, 12 μM SCI-0502, and 20 μM CBNA. Untreated cells were included as a negative control. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was included as an endogenous control and used for data normalization.

[0064] After 6 hours of incubation with the test compounds, RNA was extracted using Invitrogen's Dynabead mRNA DIRECT kit according to the recommended protocol, yielding 50 μL of purified mRNA solution. 50 μL of RT master mix was added to the mRNA solution, and the mixture was incubated at 37°C for 60 minutes. Reverse transcriptase was inactivated at 95°C for 5 minutes, and the resulting cDNA samples were kept at 4°C until use. 55 microliters of TaqMan Fast Advance master mix was added to an equal volume of cDNA sample containing 1 μg / 100 μl of cDNA, which was then loaded onto each card reservoir. The cards were centrifuged twice at 1200 RPM for 3 minutes in a Legend XFR centrifuge, and rtPCR reactions were performed on a QuantStudio7 PCR instrument using the recommended settings of 40 amplification cycles. Data processing was performed using ThermoFisher's Connect™ application.

[0065] In-cell Western blot analysis Protein expression was assessed by seeding 1,000 HepG2 cells / well from cryogenically frozen stocks into Thermo-Scientific 384-well optical-bottom black culture plates (Part Number 142761) in IMDM medium containing 20% ​​FBS and 1% ethanol. After 48 hours of recovery, the medium was replaced with the same medium containing either sorafenib 12 μM, CBNA 20 μM, or SCI-0502 12 μM. After 6 hours of treatment, cells were fixed with 4% formaldehyde for 15 minutes, permeabilized with 0.1% Triton-X100 for 10 minutes, and left overnight at 4°C in blocking buffer (phosphate-buffered saline (PBS) containing 3% bovine serum albumin (BSA)). The next day, cells were rinsed with PBS and incubated with 25 μl of PBS containing primary antibodies (Table 1) for 3 hours at 25°C. The primary antibody was then removed, and the cells were rinsed twice with PBS and incubated with 25 μl of PBS containing the secondary antibody (rabbit: Alexa Fluor 790 donkey anti-rabbit IgG (H+L), lot 2409042; mouse: Alexa Fluor 790 donkey anti-mouse IgG (H+L), lot 2300923) at a concentration of 1:2000. Draq5 (1:2000) was added for data normalization. After 1 hour of incubation with the secondary antibody, the cells were rinsed with PBS, and readings were taken at 700 and 800 nm using a Licor Odyssey fluorescent imager with a 21 μm resolution. Analysis was then performed using Empiria Studio 2.1. A total of five samples per antibody were collected. [Table 1]

[0066] In vivo PDX model evaluation Drug candidates were mixed with FITC-labeled PEG-5000 in an appropriate solvent at a 2:8 milligram ratio and lyophilized to dryness. A sample (10 mg) of the resulting mixture was loaded onto a NanoNail (Kibur Medical Inc.), a device invented for testing multiple samples in a single tumor. The cylindrical device measures 4 mm x 820 μm and contains 18 reservoirs, each 200 μm wide and 250 μm deep. The loaded device was kept at -80°C until tumor implantation. Charles River NCG mice (female, 8-12 weeks old) were subcutaneously injected with 100 μL of LIXF-575 cells per mouse. Mouse weights and tumor caliper measurements were recorded when tumors reached 300-400 mm. 3 The implantation was performed every three days until the tumor reached a maturity of approximately 12 weeks. Nine mice were used for intratumoral implantation. Nanonail units were directly implanted into tumor tissue using a 23-gauge tapered metal needle (Electron Microscopy Science). After 72 hours, the mice were euthanized, and the nanonail units and tissue material were collected for immunofluorescence analysis. The nanonail-containing tumors were excised and stored in 10% formalin at room temperature for 24 hours. The tissue was embedded in paraffin, snap-frozen, and 8 μm orthogonal microtome sections were mounted on slides for staining. To validate previous Western blot data, 15 primary antibodies (Table 2) were selected. In addition, Ki67 and caspase 3 were selected for calculation of the apoptotic index, along with nine other immunological targets. After permeabilization with 1% sodium dodecyl sulfate for 15 minutes, staining was performed for 24 hours at 4°C using primary antibodies at a ratio of 1:500 to 1:2000 in PBS. After rinsing, the sections were incubated overnight at 4°C with fluorescently labeled secondary antibodies (1:1000) before imaging and statistical analysis (Ahn, Ferland et al., 2021). [Table 2]

[0067] Example 1 The objectives of this example are, first, to establish a procedure for binary interaction studies and, second, to establish analytical methods to identify potential interactions between two components / compounds. For these interaction assays, experiments were set up in 96-well plates. Serial dilutions were performed to obtain total concentrations ranging from 50 μM to 25 nM for various CBNA:sorafenib ratios. After 48 h of incubation, the IC values ​​of the various mixtures were calculated. 50 values ​​and IC values ​​for the pure compounds 50 values ​​and predicted / calculated IC values ​​for various mixtures 50 Comparative plots of these data are shown in Figures 2A and 2B. Figure 2A shows aggregate results from three experimental replicates, and predicted data are compared to the individual IC values ​​obtained from the pure compounds. 50 Displays a window of expected values ​​based on the variation of the observed / experimented IC 50 The values ​​were below the window of expected results assuming an additive response from the two compounds. These data suggest that CBNA and sorafenib act synergistically. Furthermore, potential synergism was observed for all the various ratios investigated. A more detailed description of the data processing is outlined in the data analysis section of this example.

[0068] Data analysis Samples from the serial dilutions were analyzed by LC-MS / MS to orthogonally confirm concentrations. For each unique concentration ratio in the data array, the IC 50 The ratio of the two components was adjusted to predict values. The Hill equation was used to model the dose-response relationship.

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[0069] IC of individual cannabinoids in control lanes to assess potential interactions 50 Calculate the IC value relative to the sum of their individual components 50 was used to predict the values.

[0070] These experiments were performed in a medium containing only 1% FBS, and a matrix effect was observed between cannabinoids, including CBNA, and FBS components (Figure 3). Therefore, the results were replicated in a medium containing 20% ​​FBS. Indeed, the IC for both sorafenib and CBNA was significantly higher than that for the control group. 50 An increase in CBNA values ​​was observed (Figure 2B), accompanied by a loss of synergistic activity at higher CBNA ratios. However, when the ratio was kept below 4:1, an additive / synergistic effect was still observed. These results are reflected by the direct interaction of the compounds and components of the medium used in vitro.

[0071] Example 2 The results of the pairwise assays in both 1% and 20% FBS (Figure 2A and Figure 2B) indicate that CBNA:sorafenib ratios between 1:1 and 4:1 exhibit synergistic activity. To further validate these findings, a solid three-dimensional spheroid / tumoroid model of HepG2 with a CBNA:sorafenib molar ratio of 3:1 was selected for investigation. The diffusion restriction introduced in these models allows for a better understanding of how the treatment functions within a more relevant biological context. An example of a spheroid used in the assay is shown in Figure 4. Dose-response data were obtained using Calcein-AM fluorescence (Figure 5). While the data reflect an additive response compared to the synergistic effect observed in Figure 2A and Figure 2B, it is noteworthy that a similar response with the CBNA:sorafenib mixture was obtained with only 1 / 4 the dose of sorafenib. These data suggest that overlapping and complementary mechanisms of action exist between the two components, which may govern the observed dose-response data.

[0072] Example 3 The combination candidates described in this invention are designed to disrupt multiple oncogenic pathways, which is more effective in patients with heterogeneous tumor genetic landscapes. The hypothesis is that therapies targeting critical pathways are more effective than single-targeted therapies. This hypothesis is tested in the combination therapy described in this invention. To investigate this hypothesis, we compared the efficacy of 575, 658, and LI-011 HCC PDX models with diverse genetic makeups as well as the HepG2 HCC cell line (Figures 6A-6C). Consistent with this hypothesis, a better response was demonstrated for SCI-0502 compared to sorafenib (Figure 6C) for all PDX models investigated. Furthermore, despite the genetic heterogeneity of the selected models, the efficacy (IC) of SCI-0502 in the three HCC PDX models was significantly higher than that of sorafenib (Figure 6C). 50 Comparison of SCI-0502 with sorafenib for all PDX models investigated showed that IC 50A nearly 20% improvement in the IL-10 value was evident (Figure 6D; 11.66 μM vs. 14.34 μM, P=0.023). SCI-0502 was shown to be effective against various hepatocellular carcinoma cell lines and to have a better response than sorafenib, making it a viable candidate for further development in the treatment of hepatocellular carcinoma.

[0073] Example 4 The purpose of this example is to compare the IC observed for HepG2 hepatocellular carcinoma cells and normal liver cells. 50 The objective of this study was to investigate the selectivity that compounds may have against cancer cells by performing a qualitative evaluation between IC values. This study was performed on two cell lines with different recommended media types, various amounts of FBS, and FBS concentrations. 50 It is qualitative in nature, considering that it directly affects the values ​​(Figure 3).

[0074] Despite these limitations, the authors were intrigued by preliminary data that might indicate better tolerance of the combination therapy than sorafenib. Sorafenib is a common multiserine / threonine kinase inhibitor with numerous side effects, including bleeding, respiratory distress, and skin blistering. CBNA, on the other hand, is a putatively low-toxicity cannabinoid. A dose-response study comparing a 4:1 ratio of CBNA:sorafenib with sorafenib was performed on a pool of HepG2 hepatocellular carcinoma and normal hepatocytes (HC3_23) to better understand whether the combination therapy was better tolerated than sorafenib monotherapy. Presumably, lower sorafenib doses would result in a reduced response and perhaps better tolerance. However, this was unclear given the additive response observed in the pairwise matrix study. The results of this study were compared with the IC values ​​obtained from a previous XTT experiment performed on HepG2 cells (48 hours of compound treatment in DMEM medium containing 20% ​​FBS at 37°C and 5% CO2). 50 These data were compared with IC 50The IC values ​​for SCI-0502 were 7-fold higher for sorafenib and 11-fold higher for SCI-0502 than those observed for HepG2 cancer cells (Figure 7, Table 2). 50 The value was approximately 1.6-fold higher than that of sorafenib in normal cells, suggesting improved selectivity over sorafenib.

[0075] [Table 3]

[0076] Example 5 The purpose of this study is to understand the mechanism of action of CBNA and sorafenib investigated in this disclosure, as well as the optimal effective ratio of these compounds on the proliferation of HepG2 cells in vivo.

[0077] Cancer gene pathways were assessed using a custom-designed TaqMan array card that surveyed 90 genes associated with multiple cancer-related pathways. These pathways included the highest levels of G protein-coupled receptors and downstream proteins and enzymes associated with apoptosis, autophagy, cell cycle arrest, and pathways disrupted by sorafenib and CBNA discussed in this disclosure. These ΔΔCt data are shown in Table 3. Of the 90 genes surveyed, more than one-third (37 genes) showed significant changes in their Ct values. While sorafenib showed the highest number of affected genes, data for CBNA indicated many of the same genes involved in overlapping pathways affected by both monotherapies.

[0078] In addition to new findings, the comparison of CBNA, sorafenib, and SCI-0502 with the negative control yielded results consistent with previous reports in the literature. Indeed, the present disclosure confirmed that sorafenib alters not only several MAP kinase and PI3K pathway genes, but also numerous genes involved in cellular processes such as angiogenesis, cell adhesion, and cell cycle arrest.

[0079] We observed that CBNA, like CBN, acts through the MAPK / PI3K pathway (Zhong 2020). However, our data further demonstrate that this mechanism of action is mediated through PTEN in addition to MAPK / Ras regulation alone. These data indicate that in addition to the two compounds affecting the Ras / Raf / MEK pathway and altering AKT via PI3K, CBNA also directly affects AKT through CRK, PTEN, and PI3K. These convergent mechanisms of action likely result in the additive anti-oncogenic responses observed. In addition to this mechanism of action, we also observed convergent pathways affecting apoptosis. This is likely through a direct effect on cell cycle processes (through BCL2 / BCL-xl) via the MAP kinase pathway, with both compounds interacting. These effects are outlined in Figure 9. Many of the pathways outlined in this figure are maintained by SCI-0502 treatment at the 6-hour time point examined (Table 3). Furthermore, we observed activation of TGF signaling and SMAD gene transcription factors involved in metastatic pathways and catenin gene expression (CTNNB1) (not reported). No effects on either CB1 or CB2 receptors were observed within the 6-hour time frame of this experiment. However, an 8-fold downregulation of ERBB2 / HER2 receptors was observed with sorafenib and CBNA, whereas a greater than 16-fold reduction was observed with the combination therapy SCI-0502. It remains unclear from these data whether CBNA directly binds to these receptors or affects their expression via a feedback mechanism. Nevertheless, ERBB2 is an important target in its own right, as its overexpression has not only been observed in many other cancer types, including breast, ovarian, gastric, and lung, but is also targeted by other currently FDA-approved therapies, such as trastuzumab or Herceptin®, marketed by Genentech. [Table 4-1] [Table 4-2]

[0080] Example 6 Orthogonal validation of the disclosed PCR changes was performed by in-cell Western blot analysis to determine whether the predicted changes in protein expression correlated with the gene expression differences observed within the same time frame (6 hours). These data are shown in Figures 8A-8C. Fourteen targets representing 11 proteins involved in the pathways identified from the PCR data were selected. Three of these proteins were evaluated in both their phosphorylated (active) and unphosphorylated forms. These data confirm the changes in gene expression of the MAPK / PI3K pathway observed in the PCR studies. Specifically, SCI-0502 exposure to HepG2 cells affects the AKT pathway via the PIK3CA and PTEN regulatory proteins. Furthermore, confirmation of HER2 / ERBB2 gene expression is maintained. Activation of the MAP kinase protein (MEK2) has been shown to occur via ERBB2 transduction in breast cancer. This association is clearly maintained and disrupted in the hepatocellular carcinoma cell line, HepG2.

[0081] Both rtPCR and Western blot studies using sorafenib and CBNA treatment in HepG2 cells demonstrated overlapping metabolic pathways. The synergistic effects observed in the in vitro and ex vivo assays are likely the result of pathway enhancement resulting from their convergence. Sorafenib acts directly on many of the kinase proteins involved in these pathways, whereas CBNA is known to act through cell surface receptors. Intrinsic and extrinsic perturbations of common pathways appear to result in synergistic effects.

[0082] Example 7 The purpose of this example is to evaluate the predictability of in vivo mechanisms of action derived from patient-derived xenografts (PDX).

[0083] In vivo validation experiments were performed in NCG mice implanted with hepatocellular carcinoma cells (575) to generate tumor xenografts and treated with drug-loaded nanonails as described in

[65] . These data confirmed previous results confirming efficacy and mechanism of action in vitro, in addition to identifying preliminary immune system activation. Data shown in Figures 10A and 10B confirmed the involvement of the PI3K / AKT pathway as the mechanism of action of CBNA. In addition, we further validated our findings that the Wnt and HIPPO pathways are affected by CBNA treatment. Activation of these pathways is associated with metastasis. Furthermore, we confirmed the involvement of macrophage activity in the clearance of apoptotic cell debris by positive identification of the F4 / 80 cell marker in tumor tissue proximal to the nanonail device (Figure 10B). These data not only validate the previously identified mechanism of action, but also confirmed the superior activity of the combination candidate in animal studies. Figure 11 further demonstrates the safety of CBNA, in that cell proliferation was not significantly affected by treatment alone, but inclusion with sorafenib still resulted in increased tumor death at only 1 / 5 the total dose compared to sorafenib alone.

[0084] The present disclosure demonstrates the synergistic interaction of CBNA with sorafenib. The mechanism of action derived from HepG2 in vitro can be extrapolated to scalable human PDX models in vivo, which is important for the development of combination cancer candidates.

[0085] Example 8 The purpose of this example is to estimate the appropriate clinical dose and route of administration and dosage of SCI-0502.

[0086] To better estimate the initial human (FIH) dose of SCI-0502, a whole-body physiologically based pharmacokinetic (PBPK) model was used to simulate the time course of sorafenib and CBNA concentrations in humans. PK-Sim® version 11.2 from the Open Systems Pharmacology Suite was used for the simulations (Lippert, Burghaus et al., 2019). In silico estimates of the CBNA and sorafenib pharmacokinetic parameters are shown in Table 5. This set of parameters was used as the initial setting for further simulations. [Table 5]

[0087] To model the pharmacokinetics of sorafenib, we utilized the clinical data of sorafenib from Jain, Woo, et al. (2011). A dose of 400 mg sorafenib twice daily was used for the simulation. The pharmacokinetic characteristics of sorafenib in humans are enterohepatic circulation (EHC), and the drug is a substrate of the canalicular efflux transporter multidrug resistance protein 2 (MRP2). These parameters were incorporated into the simulation. Furthermore, parameters including passive permeability to the intracellular space of organs, intestinal permeability, hepatic clearance, and dissolution rate were optimized to reproduce clinical findings (Table 6 and Figure 13). Through this optimization, we established a PBPK model of sorafenib that adequately describes the patient's plasma profile. Because no clinical data was available for CBNA, the PBPK model was optimized and results were obtained using the ADMET predictor. [Table 6]

[0088] Sorafenib is commercially available as an oral medication, and CBNA has higher oral bioavailability than sorafenib. Furthermore, the hepatic clearance values ​​between CBNA and sorafenib are comparable (Table 5). Therefore, it can be concluded that CBNA can also be taken orally.

[0089] The ultimate goal of this example is to estimate the dose of SCI-0502 in humans that can be clinically effective. Therefore, the goal is to devise an effective dose of SCI-0502 that achieves a synergistic ratio in the liver, the site of action. A target AUC ratio of CBNA to sorafenib in the liver ranging from 1:1 to 6:1 was used. Using two PBPK models, CBNA and sorafenib, the dose of SCI-0502 was calculated assuming a Caucasian male patient weighing 73 kg. The dose was administered with a meal. The simulation results are shown in Table 7. [Table 7]

[0090] The safety of CBNA was shown to be better than that of sorafenib (Figures 7 and 11). Therefore, it can be concluded that the mixture of sorafenib and CBNA is a better candidate for the treatment of HCC, since the dose of sorafenib can be as low as 1 / 6 of the clinical dose of sorafenib.

[0091] References The following references are indicative of the level of skill of one of ordinary skill in the art and, where permitted, are incorporated herein by reference in their entirety.

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Claims

1. A pharmaceutical composition comprising a therapeutically effective amount of a synergistic combination of cannabinol (CBN) or cannabinolic acid (CBNA) with sorafenib or regorafenib for the treatment of cancer.

2. The composition of claim 1, wherein the combination comprises CBNA and the molar ratio of CBNA:sorafenib or regorafenib is from about 1:1 to about 6:

1.

3. The composition of claim 2, wherein the molar ratio is from about 2:1 to about 5:

1.

4. 4. The composition of claim 3, wherein the molar ratio is about 3:1 or about 4:

1.

5. The composition of any one of claims 1 to 4, wherein the combination comprises sorafenib.

6. The composition of any one of claims 1 to 4, wherein the combination comprises regorafenib.

7. An oral dosage form comprising the composition of any one of claims 1 to 6 and a pharmaceutically acceptable excipient.

8. A method for treating hepatocellular carcinoma, renal cell carcinoma, thyroid cancer, colorectal cancer with distant metastasis, or gastrointestinal stromal tumor, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of any one of claims 1 to 6 or the oral dosage form of claim 7, in a fixed or loose combination.

9. 9. The method of claim 8, wherein the cancer is hepatocellular carcinoma, renal cell carcinoma, and thyroid cancer.

10. Use of a synergistic combination of CBN or CBNA with sorafenib or regorafenib to treat cancer that is hepatocellular carcinoma, renal cell carcinoma, thyroid cancer, colon cancer with distant metastasis, or gastrointestinal stromal tumor.

11. The use of claim 10, wherein the molar ratio of CBNA to sorafenib or regorafenib is from about 1:1 to about 6:

1.

12. The method of claim 11, wherein the molar ratio is from about 2:1 to about 5:

1.

13. 13. The method of claim 12, wherein the molar ratio is about 3:1 or about 4:

1.

14. The use according to any one of claims 10 to 13, wherein the cancer is hepatocellular carcinoma, renal cell carcinoma, thyroid cancer, colorectal cancer with distant metastasis, or gastrointestinal stromal tumor.

15. The use according to any one of claims 10 to 13, wherein the combination is CBNA and sorafenib.

16. 16. The use of claim 15, wherein the cancer is hepatocellular carcinoma, renal cell carcinoma, or thyroid cancer.

17. 17. The use according to claim 16, wherein the cancer is hepatocellular carcinoma.

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