Compositions of cannabinoids and kinase inhibitors for treatment of cancer

Abstract

Compositions of cannabinoid and kinase inhibitors are useful for the treatment of hepatocellular carcinoma (HCC). Comprising cannabinolic acid (CBNA) and sorafenib synergistically acts on HepG2 cells, an in vitro liver cancer model and an in vitro human xenograft (PDX) HCC model. All of the disclosed compositions have superior effects than sorafenib used alone, while sorafenib is a first and second line chemotherapeutic agent selected for the treatment of HCC.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to novel compositions comprising a cannabinoid and a kinase inhibitor, and methods of making and using the same to treat cancer, in particular, hepatocellular carcinoma. BACKGROUND

[0002] Various cancers are treated with kinase inhibitors that target vascular endothelial growth factor (VEGF) or vascular endothelial growth factor receptor (VEGFR). Hepatocellular carcinoma (HCC) is a particularly malignant disease with poor patient prognosis. The need for effective treatments for cancers such as HCC has long been unmet in the art. Of those diagnosed with this disease, less than 35% survive 5 years. This number drops to less than 12% if the cancer has spread to nearby tissues, and to less than 2% if the cancer has metastasized to other organs (Kitisin, Packiam et al. 2011). To date, no approved phytocannabinoid-derived HCC chemotherapy regimen exists. HepG2 cells, which were originally isolated from a 15-year-old boy in 1975, have been well differentiated and characterized and have long served as a model for hepatocellular carcinoma. PDX models 575, 658, and LI-011 are patient-derived, human xenograft (PDX) models established in nude mice and are available for research through contract research organization (CRO) Charles River Laboratories.

[0003] The medicinal use of cannabis dates back to antiquity, as evidenced by gold vessels unearthed in Scythian tombs that contained cannabis residues. Today, cannabis has been 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 anticancer properties of cannabinoids and other components in cannabis, such as terpenes, flavonoids, etc., have been investigated (Blasco-Benito, Seijo-Vila et al. 2018). In the cannabis field, tetrahydrocannabinol (THC) and cannabidiol (CBD) have been the focus of cancer research. CBD has garnered more attention for its lack of central nervous system (CNS) “side effects.”

[0005] Decarboxylated cannabinoids such as THC and CBD are known to have poor oral bioavailability and drug-like properties (Meyer, Langos et al. 2018). Additionally, their low solubility and high first-pass metabolism make them unsuitable for administration by oral means. For example, the oral bioavailability of CBD ranges from 9% to 30% with large inter-individual differences in plasma profiles. In contrast, carboxylated forms of cannabinoids have significantly increased solubility and bioavailability compared to their decarboxylated counterparts, making them better candidates for oral administration, provided they are biologically active. For example, cannabidiolic acid (CBDA) is more soluble in water than its decarboxylated counterpart CBD, approximately 25,000 times more soluble (17.5 mg / ml vs. 0.7 pg / ml).

[0006] One emerging therapeutic target is agonistic or antagonistic compounds that alter the heterodimer formation within the many C-coupled protein receptors to which cannabinoid receptors belong. As this receptor superfamily accounts for approximately 4% of the protein-coding genome (Moreno, Cavic et al. 2019), this opens up a large number of new disease pathways. The new targets can provide mechanistic descriptions for the interaction of cannabinoids and / or other compounds.

[0007] The effects of cannabinoids can be explained by their variable binding affinities to a variety of G-coupled protein heterodimer receptors such as CB1, CB2, GPR55, and TPRV1, which transmit multiple downstream pathways, resulting in variable drug responsiveness (Moreno, Cavic et al. 2019). In fact, Zhong (2020) reported that cannabinol (CBN) mediated apoptosis through the MAPK / ERK and PI3K-ATK pathways and cell cycle arrest through downregulation of P21. Interestingly, Zhong’s work also showed that CBN treatment resulted in downregulation of CB2 and GPR55 receptors.

[0008] It is not known whether these effects are retained with their more soluble acid precursor forms, cannabinoid acids (CBNAs). Furthermore, Torres, Lorente et al. (2011) demonstrated that THC, when administered with the selective ALK inhibitor TAE-684, produced a synergistic response, suggesting that additive or synergistic effects can be enhanced by targeting convergent pathways. On the other hand, prior to this finding, it was not known whether these effects could be replicated with cannabinoids such as CBN / CBNA and compounds with a mechanism of convergence. SUMMARY

[0009] Aspects of the present disclosure generally relate to synergistic anticancer activity of cannabinoids and kinase inhibitors. The cannabinoids can include carboxylated or decarboxylated forms. The kinase inhibitors can include sorafenib or regorafenib. In one embodiment, the synergistic anticancer activity of CBN or CBNA with sorafenib or regorafenib in treating cancer is described in the present disclosure.

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

[0011] In another aspect, an oral dosage form comprising a synergistic composition comprising CBNA and a kinase inhibitor for treating cancer is disclosed. The oral dosage form can include tablets, solutions, suspensions, powders, and nano-encapsulated dosage forms. In such oral dosage forms, the molar ratio of CBNA to kinase inhibitor in the composition is between about 1 : 1 and about 6: 1. Preferably between about 2: 1 and about 5: 1, most preferably about 3: 1 or 4: 1.

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

[0013] In some embodiments, the cancer that can be treated with the synergistic combination disclosed herein is a cancer that is effectively treated with sorafenib or regorafenib, such as hepatocellular carcinoma, renal cell carcinoma, thyroid cancer, metastatic colorectal cancer, or gastrointestinal stromal tumor. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 . Chemical structures of carboxylated form of cannabigerol (cannabinolic acid, CBNA), sorafenib, and regorafenib.

[0015] Figure 2A . Comparison of observed versus predicted additive IC values at different CBNA and sorafenib ratios determined by three independent experiments (n=3) in DMEM medium containing 1% FBS. 50 Figure 2B . Observed (n=1) and predicted / calculated additive IC values at different CBNA and sorafenib ratios determined in DMEM medium containing 20% FBS.​50 Comparison of values.

[0016] Figure 3 CBNA: Sorafenib vs. Sorafenib IC50 in different culture media (DMEM with 1% FBS vs. IMDM with 20% FBS) 50 Histogram of values ​​(μM).

[0017] Figure 4 HepG2 spheres were formed in 96-well ultra-low adhesion round-bottom plates after culturing in William's E medium with 10% FBS at 37°C and 5% CO2 for 2.5 days.

[0018] Figure 5 In DMEM medium with 1% FBS, sorafenib and CBNA:sorafenib at a 3:1 ratio showed an IC50 effect on HepG2 spheroids. 50 Value curve graph.

[0019] Figure 6A SCI-0502 IC for 3 PDX models and HepG2 50 Value graph. Figure 6B Sorafenib's IC for 3 PDX models and HepG2 50 Value graph. Figure 6C : Figure 6A and 6B IC calculated in 50 Comparison of values. Figure 6D IC50 of PDX models treated with SCI-0502 and sorafenib 50 Comparison between values.

[0020] Figure 7 IC50 of SCI-0502 and sorafenib on normal hepatocytes (HC3_23) from 20 donor pools 50 Value graph.

[0021] Figure 8A HepG2 cells were administered sorafenib, 8B:CBNA, and 8C:SCI-0502 at their respective IC50 values. 50 Changes in protein expression in cells after treatment with a certain concentration for 6 hours using Western blot.

[0022] Figure 9 A schematic diagram illustrating the defined mechanisms of action of sorafenib (C1) and CBNA (C2).

[0023] Figure 10A and 10BPDX tumor model (575) compared to in vivo immunoprecipitation results with nano-spikes (Kibur Medical Inc.) implanted, a tumor device loaded with sorafenib, CBNA and CBNA:sorafenib (4:1).

[0024] Figure 11 DAB staining of Ki67 and cell proliferation prediction plot of 575 PDX tumor tissue treated with nano-spikes loaded with sorafenib, CBNA and CBNA:sorafenib (4:1).

[0025] Figure 12 Apoptosis index (%) of SCI-0502 in PDX (575) model loaded with nano-spikes device in NCG mice, compared to (vs.) its positive controls sorafenib and doxorubicin.

[0026] Figure 13 Plasma and liver concentration profiles of sorafenib in subjects orally administered 400 mg sorafenib (Jain, Woo et al. 2011). Solid and dashed lines are obtained from simulations, and open symbols are from 4 patients. DETAILED DESCRIPTION

[0027] It will be appreciated that, for clarity, certain features of the application that are described herein in the context of separate embodiments can also be provided in combination in a single embodiment. That is, unless explicitly stated otherwise, each individual embodiment is considered to be combinable with any other embodiment, and such combinations are considered to be another embodiment. Conversely, various features of the application described in the context of a single embodiment can also be provided independently of that single embodiment or in any sub-combination. Finally, although the embodiments can be described as a series of steps or as a series of structural elements, each of the steps themselves can also be considered an independent embodiment, combinable with other steps.

[0028] Some terms

[0029] The transitional terms "comprise," "comprising," "consist essentially of," and "consisting of" are intended to use their plain, ordinary meaning; that is, (i) "comprising" is to be interpreted inclusively or open-ended, and does not exclude additional, unrecited elements or method steps; (ii) "consisting of" is a closed or restrictive transition term and excludes any element, step, or ingredient not specified in the claim; and (iii) "consisting essentially of" limits the scope of a claim or the embodiment to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed application or embodiment. More specifically, the basic and novel characteristic(s) relate to the ability of the method or use to provide at least one benefit described herein, including but not limited to the ability to improve the survivability of a population relative to a comparative population described elsewhere herein. Embodiments described in a phrase "comprising" (or its counterpart) also provide those embodiments described in "consisting of" and "consisting essentially of."

[0030] When a value is expressed as an approximation by use of the term "about," it is understood that the particular value forms another embodiment. The term "about" means ±10% variance of the associated value, unless otherwise stated, but other embodiments include those in which the variance can be ±5%, ±15%, ±20%, ±25%, or ±50%. In particular, the term "about" means ±5% or ±10% variance of the associated value, more particularly ±5% variance.

[0031] When presenting a list, unless otherwise specified, it is understood that each individual element of the list, as well as each combination of the list, is an independent embodiment. For example, a list of embodiments expressed as "A, B, or C" should be interpreted to include the embodiments "A," "B," "C," "A or B," "A or C," "B or C," or "A, B, or C."

[0032] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0033] As used herein, "patient" means any animal, particularly a mammal. Thus, the methods or uses are applicable to both human and non-human animals, although most preferably to humans. The terms "patient" and "subject" and "human" are used interchangeably.

[0034] The terms "treat" and "treatment" refer to the treatment of a patient afflicted with a pathological condition, and refer to an effect that alleviates symptoms, combats the effects of the pathological condition, and / or combats the underlying causes of the pathological condition, whether the pathology is primary or secondary. Also included are treatments that have prophylactic or preventative effects, i.e., preventing the pathology.

[0035] “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 can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic or combination of therapeutics to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic or combination of therapeutics include, for example, improvement in the patient’s health status.

[0036] The term “dosage” refers to information about the amount of therapeutic administered to a subject and the frequency of the number of times the therapeutic is administered to the subject. The term “dose” refers to the amount or quantity of therapeutic administered at each time.

[0037] The term “cancer” as used herein refers to an abnormal growth of cells that tend to proliferate in an uncontrolled way and, in some cases, to metastasize (spread).

[0038] The term “combination therapy” and the like as used herein includes administration of selected therapeutic agents to a single patient and is intended to embrace the administration of these agents by the same or different dosing routes or at the same or different times.

[0039] The term “pharmaceutical combination” as used herein refers to a product that comprises two or more active ingredients in admixture or association. It includes both fixed and non-fixed combinations of the active ingredients. The term “fixed combination” refers to active ingredients, such as a kinase inhibitor and a cannabinoid, that are both administered to a patient simultaneously in the form of a single unit or single dosage form. The term “non-fixed combination” means that the active ingredients, such as a kinase inhibitor and a cannabinoid, are administered to a patient as separate units or separate dosage forms that are intended to be administered simultaneously, concurrently or sequentially, with no specific intervening time limits, wherein such administration provides therapeutically effective levels of the two active ingredients in the body of the patient. The above principles apply equally to cocktail therapy, for example, the administration of three or more active ingredients.

[0040] Compositions comprising a cannabinoid and a kinase inhibitor, exemplified by CBN or CBNA and sorafenib or regorafenib, are described herein. The inventors have determined that the compositions are more effective against a number of hepatocellular carcinoma models with a greater safety margin compared to the kinase inhibitor alone (e.g., sorafenib) when compared to a normal pool of hepatocytes. In vitro cell hepatocellular carcinoma models (HepG2), 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 intracellular Western blot expression studies were used to assess the relative potency, mechanism of action, and interaction of the test compounds and combinations thereof.

[0041] In some embodiments, the cannabinoid can include a decarboxylated form of a cannabinoid, such as cannabigerol (CBG).

[0042] Sorafenib or regorafenib is used to treat various cancers, including hepatocellular carcinoma, renal cell carcinoma, thyroid cancer, metastatic colorectal cancer, gastrointestinal stromal tumors. Accordingly, the synergistic combinations disclosed herein can be used to treat any of the cancers for which sorafenib or regorafenib is known to be an effective treatment.

[0043] In some embodiments, sorafenib or regorafenib can be administered as a pharmaceutically acceptable salt. In preferred embodiments, sorafenib is administered as the base form. One of skill in the art would be able to determine the effective amount of a pharmaceutically acceptable salt that corresponds to its base equivalent.

[0044] Salts can be prepared, for example, by reacting sorafenib or regorafenib with the appropriate acid in the appropriate solvent. Acid addition salts can be formed with both inorganic acids and organic acids. Examples of acid addition salts include salts formed with acids selected from the group consisting of acetic, hydrochloric, hydroiodic, phosphoric, nitric, sulfuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulfonic, toluenesulfonic, methanesulfonic (mesylate), ethanesulfonic, naphthalenesulfonic, valeric, acetic, propionic, butyric, malonic, glucuronic acid, and lactobionic acid. Another group of acid addition salts includes salts formed from acetic, adipic, ascorbic, aspartic, citric, DL-lactic, fumaric, gluconic, glucuronic, hippuric, hydrochloric, glutamic, DL-malic, methanesulfonic, sebacic, stearic, succinic, and tartaric acids.

[0045] The pharmaceutical compositions provided herein comprise an effective amount of a cannabinoid and a kinase inhibitor. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human as appropriate. A pharmaceutically acceptable composition can produce tolerable side effects. The preparation of a pharmaceutical composition comprising at least a kinase inhibitor and a cannabinoid will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Furthermore, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by regulatory offices, such as the U.S. Food and Drug Administration Office of Biological Standards.

[0046] In some embodiments, the synergistic combination of cannabinoids and kinase inhibitors can be effective for a broad population, i.e., not for personalized or precision medicine approaches. In other embodiments, the synergistic combination of cannabinoids and kinase inhibitors can be effective for an identifiable patient subpopulation that can be identified by appropriate testing.

[0047] As used herein, "pharmaceutically acceptable carrier" includes 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, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like which are physiologically compatible and which are known to one of ordinary skill in the art (e.g., see Remington's Pharmaceutical Sciences, 18th Ed. 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.

[0048] Almost all kinase inhibitors are effective orally. In certain preferred embodiments, the pharmaceutical composition is administered orally to treat cancer, such as HCC. In one embodiment, the effective oral dose of the CBNA / sorafenib combination is estimated for treating a patient with HCC.

[0049] In certain embodiments, the actual dose of the composition administered to a patient can be determined by physical and physiological factors such as body weight, severity of the disease, type of disease being treated, previous or concurrent therapeutic interventions, specific disease of the patient, and route of administration. In any event, the concentration of active ingredient in the composition and appropriate dose for an individual subject are determined by those of ordinary skill in the art.

[0050] In certain embodiments, the pharmaceutical composition can comprise, for example, at least about 0.1% of the active compound. In other embodiments, the active compound can comprise between about 2% and about 75%, or for example, between about 25% and about 60%, by weight of the unit, or any range derivable therein.

[0051] In other non-limiting examples, the active compound can also comprise a dose of about 1 milligram (mg) per kg of body weight, about 2.0 mg / kg of body weight, about 3.0 mg / kg of body weight, about 4.0 mg / kg of body weight to about 5.0 mg / kg of body weight or more per administration, and any range derivable therein. In non-limiting examples of ranges derivable from the numbers listed above, a range of about 2.5 mg / kg of body weight to about 5.5 mg / kg of body weight is derivable based on the numbers

[0052] In one embodiment, human pharmacokinetic data is used to assess the drug-like properties of sorafenib in humans.

[0053] In one embodiment, the drug-like properties of CBNA are assessed by in silico.

[0054] Embodiments

[0055] These examples are provided for illustrative purposes only and do not limit the scope of the claims provided herein.

[0056] Materials and methods

[0057] Pure cannabichromene acid was obtained from Cerilliant (CBNA, C-153-1ML, batch FE10222002; and Kinetochem, NGC1025, batch CBNA-RD-210826). Sorafenib was purchased from Selleckchem (S7397, batch S739707). Sorafenib was reconstituted to 10 mM in DMSO and subsequently diluted to working concentrations. CBNA was dissolved in 95% ethanol (10 mg / ml). Compounds were added directly to the culture medium at the highest concentration (100 μg / ml) and serially diluted to subsequent working concentrations.

[0058] The name SCI-0502 refers to the combination of CBNA and sorafenib.

[0059] Cellular assays

[0060] HepG2 cells were obtained from the American Type Culture Collection (ATCC). Normal hepatocyte pool cells were purchased from Xenotech. Cells cryopreserved in DMEM containing 5% DMSO were thawed and diluted 10-fold in phenol red-free DMEM medium containing 10% fetal bovine serum, 100 μg / ml penicillin / streptomycin, supplemented with Glutamax TM and sodium pyruvate. Subsequently, cells were centrifuged at 20 g for 10 minutes. Cells were resuspended in the same medium and added to a concentration of 2 x 10 5 cells / ml. 7.5 x 10 3One cell was seeded into a 96-well plate with cell culture treatment, 100 μl per well, and cultured at 37 °C, 7.5% CO2, and >95% relative humidity for 18 hours to allow cell adhesion. The original medium was then replaced with a similar medium (DMEM, IMDM, Williams E) containing 1% to 20% FBS and various test compounds (10 μg / ml to 100 ng / ml). Forty-eight hours later (cells doubled), sodium 2-(2-methoxy-4-nitro-5-sulfonatophenyl)-3-(2-methoxy-4-nitro-5-sulfophenyl)-N-phenyltetrazol-3-ium-5-carboximidate (XTT) (300 μg / ml) was added, and cells were incubated for 2 hours. Formazan yield was measured at 450 nm using a Varioskan Lux spectrophotometer. Nonspecific absorbance was measured at 660 nm and subtracted from the reference measurement at 450 nm. The experiment was performed three times (n=3). After blank subtraction, the data were averaged, and a four-point logistic regression curve was fitted to obtain the IC50. 50 Values. Blank samples included an equal volume of solvent concentration as a negative control. The ethanol or DMSO concentration of the blank standards reflected the ethanol or DMSO concentration used in the sequential cannabinoid dilutions. Preliminary studies were conducted to evaluate the activity of a single cannabinoid on HepG2 cell proliferation.

[0061] Spheroid (solid tumor) assays :

[0062] HepG2 cells were thawed and rehydrated in Williams E medium supplemented with Gibco hepatocyte maintenance supplement. Cells were seeded at a density of 1500 cells / well. Cells were cultured in 96-well microplates with black walls, transparent round bottoms, and ultra-low adsorption. The plates were rotated at 10xG for 20 minutes to allow cells to aggregate at the center of the wells. After 60 hours of incubation at 37°C and 5% CO2, the cells were treated with CBNA and sorafenib at a ratio of 3:1 for 48 hours before the addition of calcein-AM (1 μM). Cell viability was measured at excitation / emission at 508 nm / 527 nm after 60 minutes of incubation at 37°C. The experiment was performed three times (n=3). After blank subtraction, the data were averaged and fitted with a four-point logistic regression curve to obtain the IC50. 50 value.

[0063] Normal hepatocyte line studies

[0064] To assess the safety range of the compounds prior to clinical trials, the toxicity of the compounds on normal hepatocyte pools was investigated in-house using hepatocyte pools obtained from Xenotech (HC3_23). Cells were thawed and plated in 96-well, black-walled, gamma-irradiated plates (100 μl) after reconstitution in William's E medium with Gibco hepatocyte maintenance supplement. After an 18-hour recovery period, the medium was changed to the same medium containing 9 serial dilutions of the test compound mixture (8 μg / ml to 2000 μg / ml). The experiment was performed in triplicate. After 48 hours of incubation, calcein-AM was added to each well to provide a final concentration of 7 ng / ml, and readings were obtained after 60 minutes of reaction (excitation 494 nm, emission 517 nm). Data were fitted to sigmoidal curves using four-point logistic curve fitting software, and the 50% inflection point was used for IC 50 values.

[0065] Clonogenic assays

[0066] Tumor xenografts (patient-derived: 575, 685 and LI-011; and cell line-derived: HepG2) were passaged as subcutaneous xenografts in NMRi nu / nu mice. When the tumor volume reached 600 mm 3 to 1000 mm 3 , tumor harvesting was performed according to FELASA and GV-SOLAS guidelines for animal welfare. Tumors were mechanically disaggregated and subsequently 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 for 60 to 120 minutes at 37°C. Cells were filtered through 100 μm and 40 μm pore size screens, washed with the same medium without enzymes, and stored in the liquid nitrogen vapor phase.

[0067] Cloning 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. 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 before use. 100 μl of the same soft agar medium was layered into the test wells followed by 50 μl of medium containing tumor cells. After solidification, the soft agar layer was overlaid with 90 μl of the same medium without agar. After 24 hours, 10 μl of test compound or control medium was added and left for 14 days (continuous exposure, 100 μl of drug overlay). Incubation parameters were 37°C, 7.5% CO2 and >95% humidity. Study compounds, including sorafenib, were serially diluted in medium with 1% ethanol. Sunitinib (positive control) was serially diluted in DMSO and transferred into cell culture medium before being added to the assay plates. To determine the effect, each 96-well plate included 6 vehicle-treated control wells and drug-treated wells in triplicate (sunitinib in duplicate), as well as 9 concentrations of test compound combinations. At maximum colony formation, important colonies were stained with INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride, 1 mg / mL, 25 μl / well) in sterile water for 48 hours and colony counts were performed with a Biosys 5000Vα Bioreader set to colony diameter > 50 μm (area > 2000 μm 2 ) using the default settings. The Sigmoidal concentration-response curve was fitted to the data points (test versus control, T / C values) obtained for each tumor model using a four-parameter non-linear curve fit. IC 50 values are reported as absolute total IC 50 values (μM) for individual and combination therapy, i.e. the concentration of test compound at which the concentration-response curve intersects with T / C = 50%. For calculation of mean IC 50 values, the geometric mean was used.

[0068] Mass spectrometry

[0069] HPLC-DAD / MS technique was used to confirm the concentration and ratio of individual cannabinoids. After 30 minutes of incubation, samples were taken randomly (50 μΐ) from the test groups and disrupted with 150 μΐ of methanol to precipitate the proteins in the culture medium. The samples were centrifuged at 21910 RCF for 5 minutes and the supernatant was collected in a 2 mL glass vial with insert. Quantitative data were acquired using an Agilent 6410 triple quadrupole mass spectrometer coupled with a 1260 series HPLC and UV detector. Five μΐ of sample was injected into a Phenomenex Kinetex 5 μιη XB-C18 250 x 4.6 mm column equipped with a C18 Security Guard ULTRA Cartridge. Mobile phase A contained 0.05% ammonium acetate in water and mobile phase B consisted of methanol and 0.05% ammonium acetate. UV detection was performed at 215 nm using a 4 nm band width. Selected ion monitoring (SIM) LC / MS analysis was performed in negative ion mode using the following parameters: source gas temperature set to 320 °C, flow rate of 12 L / min, capillary voltage held at -3.8 kV, dwell time set to 200 ms, fragmentation voltage set to 135 V, and gas nebulizer pressure set to 35.0 psi. Concentrations were determined by quantifying the area response of the samples against a standard calibration curve, with a range of 0.025 μg / ml to 10 μg / ml for the MS detector and 0.5 μg / ml to 15 μg / ml for the UV detector.

[0070] Gene expression analysis

[0071] Custom designed Array cards that interrogate 90 unique genes associated with cancer-related pathways (Kanehisa, Goto et al. 2010) were analyzed for target cancer gene pathways.

[0072] Two million HepG2 cells in IMDM medium containing 20% FBS and 1% ethanol were seeded in T75 flasks and incubated at 37 °C, 7.5% C02until 70-80% confluency (approximately 7.5 x 10 5 Cells were supplemented with culture medium containing their predetermined IC 50 concentrations of test compounds (sorafenib 12 μΜ, SCI-0502 12 μΜ and CBNA 20 μΜ). Untreated cells were included as a negative control. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was included as an endogenous control and for normalization of data.

[0073] After 6 hours of incubation with the test compound, RNA was extracted using Invitrogen’s Dynabead mRNA DIRECT kit following the recommended protocol to provide 50 pL of purified mRNA solution. 50 pL of RT master mix was mixed to the mRNA solution and the mixture was allowed to react at 37°C for 60 minutes. Reverse transcriptase inactivation was performed at 95°C for 5 minutes and the resulting cDNA sample was stored at 4°C until use. 55 microliters of TaqMan Fast Advance master mix was added to an equal amount of cDNA sample containing 1 pg / 100 mΐ of cDNA, which was loaded into each cartridge. The cartridge was centrifuged at 1200 RPM for 3 minutes in a Legend XFR centrifuge and then an rtPCR reaction of 40 amplification cycles was performed in a QuantStudio 7 PCR instrument using the recommended settings. Data processing was performed using ThermoFisher’s Connect TM The application is complete.

[0074] Intracellular western blot analysis

[0075] Protein expression was evaluated in 1000 HepG2 cells / well seeded from cryogenic frozen stocks into Thermo-Scientific (model 142761) 384 well optical bottom black plates in IMDM media containing 20% FBS and 1% ethanol. After 48 hours of recovery, the original media was replaced with the same media containing Sorafenib 12 mM, CBNA 20 mM or SCI-0502 12 mM. After 6 hours of treatment, cells were fixed with 4% formaldehyde for 15 minutes, permeabilized with 0.1% Triton-x100 for 10 minutes and placed in blocking buffer (phosphate buffered saline (PBS) containing 3% bovine serum albumin (BSA)) overnight at 4 degrees Celsius. The next day, cells were rinsed with PBS and incubated with 25 mΐ of PBS containing primary antibodies (Table 1) for 3 hours at 25°C. Subsequently, primary antibodies were removed, cells were rinsed twice with PBS and incubated with 25 mΐ of PBS containing secondary antibodies (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 normalization of data. After one hour of incubation with secondary antibodies, cells were rinsed with PBS and readouts at 700 nm and 800 nm were acquired on a Licor Odyssey fluorescent imager with a resolution of 21 pm. Subsequent analysis was performed with Empiria Studio 2.1. A total of 5 samples were collected for each antibody.

[0076] Table 1. List of primary antibodies selected for western blot analysis.

[0077]

[0078] In vivo PDX model evaluation

[0079] The candidate drug was mixed with FITC-labeled PEG-5000 in a 2:8 milligram ratio in a suitable solvent and lyophilized to dryness. A sample (10 mg) of the resulting mixture was loaded into a nanospike (Kibur Medical Ltd.), a device for testing multiple samples in a single tumor. The cylindrical device, which measures 4 mm x 820 pm, contains 18 reservoirs that are 200 pm wide and 250 pm deep. The loaded device was kept at -80°C until tumor implantation. NCG mice (female, 8-12 weeks old) from Charles River were injected subcutaneously with 100 pL of LIXF-575 cells / mouse. Body weight measurements and caliper measurements of the tumor were performed every 3 days until the tumor reached 300 mm 3 to 400 mm3 (about 12 weeks). Nine mice were used for intratumoral implantation. The nanospike units were implanted directly into the tumor tissue using a 23 gauge bevelled metal needle (Electron Microscopy Sciences) and left for 72 hours, after which the mice were euthanized and the nanospike units and tissue material were recovered for immunofluorescence studies. The tumors containing the nanospikes were excised and stored in 10% formalin for 24 hours at room temperature. The tissues were embedded in paraffin, flash frozen, and 8 pm orthogonal sections were cut onto slides for staining. In addition to the 9 immunological targets, as well as Ki67 and Caspase 3, 15 primary antibodies were chosen (Table 2) to validate the previous western blot data for the calculation of the apoptosis index. After permeabilization with 1% sodium dodecyl sulfate for 15 minutes, the sections were stained with primary antibodies at a 1 :500 to 1 :2000 ratio in PBS for 24 hours at 4 degrees Celsius, followed by washing and incubation with fluorescently labeled secondary antibodies (1 : 1000) overnight at 4 degrees Celsius, followed by imaging and statistical analysis (Ahn, Ferland et al. 2021).

[0080] Table 2: In addition to the 9 immunological targets, 15 in vivo targets were chosen from PDX 575 to validate the in vitro obtained pathway responses.

[0081] Target Method / pathway HER2 Receptor MET Receptor / Hepatic growth factor MAP2K Ras / RAF / MEK PTEN ATK pathway PI3K ATK pathway AKT ATK pathway SMAD2 / 3 TGF signaling CC3 Apoptosis Ki67 Cell proliferation DVL2 Wnt / cell adhesion / metastasis Frizzled Receptor / Wnt / cell adhesion / metastasis WWTR1 HIPPO MYC Cell cycle / TGFb Cyclin E Cell cycle / TGFb SMO Hedge Hog FoxP3 Immune system CD8 Immune system CD3 Immune system CSF1R Immune system MHC-II Immune system CD11b Immune system Arginase-1 Immune system F4 80 Immune system CD45 Immune system

[0082] Example 1

[0083] The purpose of this example was, first, to establish the procedure for two-component interaction studies; second, to establish the analytical method to determine the potential interaction between the two components / compounds. For these interaction assays, experiments were performed in 96-well plates. Serial dilutions were made to obtain total concentrations of various CBNA:sorafenib ratios, ranging from 50 mM to 25 nM. After 48 hours of incubation, the IC 50 values of the various mixtures were obtained and compared to the IC 50 values of the pure compounds and the predicted / calculated IC 50 values of the various mixtures. A comparison plot of these data is shown in Figure 2A and 2B . Figure 2A The overall results of three iterations of the experiment are shown, where the predicted data shows the expected window of values based on the individual IC 50 values obtained from the pure compounds. In each experiment, assuming an additive response of the two compounds, the observed / experimental IC 50Values fell below the expected range of results. These data suggest that CBNA and sorafenib have a synergistic effect. In addition, a potential synergistic effect was observed for all of the different ratios explored. The data analysis section of this example outlines a more detailed explanation of the data processing.

[0084] Data analysis:

[0085] Serially diluted samples were analyzed by LC-MS / MS to orthogonally confirm concentrations. The ratios of the two components were organized to predict the IC 50 values for each individual concentration ratio within the data array.

[0086]

[0087] where y 最小 and y 最大 are the minimum and maximum responses, respectively, d is the dose tested, d m is the dose that produces 50% of the response (IC 50 ), and m is the Hill coefficient, which indicates the degree of interaction between the target and the chemical, and can be any value. In addition, according to the unified theory of Chou’s analysis of multiple compounds’ interactions, the IC 50 value of a mixture of compounds that do not interact with each other can be written as a function of the IC 50 and concentration ratios of each compound, as:

[0088]

[0089] where r 1i = d i / d1 is the concentration ratio of compound i to compound 1. From this value and the IC 50 50 预期 of the mixture, three possible types of interactions are obtained:

[0090]

[0091] To assess potential interactions, the IC 50 values of the cannabinoids alone in the control lanes were calculated and used to predict the IC 50 values of the sum of their individual components.

[0092] These experiments were performed in media containing only 1% FBS, and a matrix effect was observed between the cannabinoids (including CBNA) and the FBS component. Figure 3). Therefore, in media containing 20% FBS, a search for the reproduction of these results was sought. In fact, for sorafenib and CBNA, an increase in IC 50 values was observed Figure 2B , while at higher CBNA ratios the synergistic activity was also lost. 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 media used in vitro.

[0093] Example 2

[0094] The results of paired tests of 1% FBS and 20% FBS ( Figure 2A and 2B ) indicate that the CBNA:sorafenib ratio has synergistic activity between 1:1 and 4:1. To further validate these findings, a HepG2 solid three-dimensional spheroid / tumoroid model with a CBNA:sorafenib molar ratio of 3:1 was chosen for study. Since these models introduce diffusion limitations, a better understanding of how the therapy works in a more relevant biological context can be obtained. An example of the spheroids used in the test 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 2B , it is noteworthy that the similar response produced by the CBNA:sorafenib mixture resulted in only one quarter of the sorafenib dose. These data suggest that there can be overlapping and complementary mechanisms of action between the two components and that these dominate the observed dose response data.

[0095] Example 3

[0096] The candidate combination drugs described in the present invention are designed to disrupt multiple oncogenic pathways, and these drugs are more effective in patients with heterogeneous oncogenic genetic features. The hypothesis is that therapies against key pathways are more effective than single target therapies. This hypothesis was tested in the combination therapies described in the present invention. To test this hypothesis, the efficacy of SCI-0502 was compared to sorafenib in 575, 658, and LI-011 HCC PDX models with diverse genetic composition and in the HepG2 HCC cell line ( Figure 6A to 6C ). In agreement with the hypothesis, SCI-0502 demonstrated better response than sorafenib for all the PDX models explored ( Figure 6C ). In addition, despite the genetic heterogeneity of the selected models, the efficacy of SCI-0502 (IC 50 values) was very consistent across the three HCC PDX models. The comparison of SCI-0502 and sorafenib in all the PDX models examined showed that the IC50 The value increased by almost 20% ( Figure 6D (11.66 μM vs 14.34 μM, P = 0.023). SCI-0502 has been shown to be effective against a variety of hepatocellular carcinoma cell lines and has a better response than sorafenib, thus making it a viable candidate for further development in the treatment of hepatocellular carcinoma.

[0097] Example 4

[0098] The purpose of this embodiment is to demonstrate the IC50 observed in HepG2 hepatocellular carcinoma cells and normal hepatocytes. 50 Qualitative evaluation was conducted to explore the potential selectivity of the compound for cancer cells. This study is qualitative in nature because the two cell lines have different recommended culture media types, different amounts of FBS, and the FBS concentration directly affects the obtained IC50. 50 value( Figure 3 ).

[0099] Despite these limitations, preliminary data suggesting that combination therapy may be better tolerable than sorafenib caught the authors' attention. Sorafenib is a generic polyserine / threonine kinase inhibitor with a number of side effects, including bleeding, dyspnea, and skin blistering. CBNA, on the other hand, is a cannabinoid with a putative low toxicity. A dose-response study comparing CBNA:sorafenib in a 4:1 ratio with sorafenib in HepG2 hepatocellular carcinoma and normal hepatocyte pools (HC3_23) was conducted to better understand whether combination therapy was better tolerable than sorafenib monotherapy. It was presumed that lower doses of sorafenib would produce a lower response and potentially better tolerability. However, this remains unclear given the additive response observed in paired matrix studies. The results of this study were compared with IC50 obtained from previous XTT experiments on HepG2 cells (DMEM medium with 20% FBS, compound treatment at 37°C and 5% CO2 for 48 hours). 50 The values ​​were compared. These data indicate that, compared with the observed IC50 values ​​of HepG2 cancer cells... 50 Compared to the value, sorafenib's IC 50 The value is 7 times higher, while the IC of SCI-0502 is... 50 The value is 11 times higher ( Figure 7 (Table 2). In normal cells, the IC50 of SCI-0502 is... 50 The value is approximately higher than that of sorafenib's IC 50 The value is 1.6 times higher, which indicates that its selectivity is superior to sorafenib.

[0100] Table 3. IC50 values of sorafenib and SCI-0502 on both HepG2 HCC cells and normal hepatocyte pool (HC3_23) 50 Comparison of values.

[0101]

[0102] Example 5

[0103] The purpose of this study was to understand the mechanism of action of CBNA and sorafenib explored in this disclosure, and the most effective ratio of these compounds to inhibit HepG2 cell proliferation in vivo.

[0104] An evaluation of cancer gene pathways was performed using a custom designed TaqMan array card to explore 90 genes associated with many cancer related pathways. These pathways include top G-coupled protein receptors and downstream proteins and enzymes associated with apoptosis, autophagy, cell cycle arrest, and pathways interfered with by sorafenib and CBNA discussed in this disclosure. These AACt data are shown in Table 3. Over 1 / 3 (37 genes) of the 90 genes explored showed significant changes in their Ct values. While sorafenib presented the largest number of affected genes, the data for CBNA showed that many of the same genes involved in overlapping pathways were affected in both monotherapies.

[0105] In addition to new findings, CBNA, sorafenib, and SCI-0502 were compared to the negative control, and results were consistent with previous reports in the literature. In fact, this disclosure demonstrates that sorafenib alters several MAP kinase and PI3K pathway genes, but also alters many genes involved in cellular processes such as angiogenesis, cell adhesion, and cell cycle arrest.

[0106] We observed that CBNA-like CBNs act through the MAPK / PI3K pathway (Zhong 2020). However, our data further suggest that this mechanism is mediated through PTEN in addition to MAPK / Ras control alone. These data suggest that both compounds alter AKT via PI3K by affecting the Ras / Raf / MEK pathway; in addition, CBNA also affects AKT via PI3K through PTEN and directly through CRK. These converging mechanisms can lead to the observation of antagonistic anticancer responses. In addition to this mechanism, converging pathways affecting apoptosis were also observed. This can be through the MAP kinase pathway directly affecting cell cycle processes (through BCL2 / BCL-xl) where both compounds interact. These effects are shown in Figure 2. Figure 9Many of the pathways shown in this figure were maintained under treatment with SCI-0502 at the 6 hour time point examined (Table 3). In addition, we also observed (not reported) activation of TGF signaling and SMAD transcription factors, which are involved in metastatic pathways and expression of the catenin gene (CTNNB1). No effect on CB1 or CB2 receptors was observed in the 6 hour time frame of this experiment. However, a 8-fold downregulation of the ERBB2 / HER2 receptor was observed with sorafenib and CBNA, but the ERBB2 / HER2 receptor was reduced by more than 16-fold with the combination therapy SCI-0502. It is not clear from these data whether CBNA is directly binding to these receptors or they are affecting their expression through a feedback mechanism. Regardless, ERBB2 is an important target in its own right, as its overexpression is observed in many other cancer types including breast, ovarian, gastric and lung cancer, and is also targeted by other currently FDA-approved therapies such as trastuzumab (sold by Genentech) or lapatinib (sold by GlaxoSmithKline) for breast cancer.

[0107] Table 4. rtPCR of genes with changes in Ct values compared to untreated cells.

[0108] Change in AACt relative to negative control. Sorafenib CBNA SCI-0502

[0109]

[0110] Example 6

[0111] The disclosed PCR changes were orthogonally validated by in-cell Western blot analysis to examine whether the expected changes in protein expression correlated with the gene expression differences observed in the same time frame (6 hours). These data are shown in Figure 6. Figure 8A to 8C Fourteen targets were selected, which represent 11 proteins involved in the pathways identified from the PCR data. Three of these proteins were evaluated in their phosphorylated (active) and non-phosphorylated forms. These data confirm the changes in MAPK / PI3K pathway gene expression observed in the PCR study. Specifically, SCI-0502 exposure to HepG2 cells affected the AKT pathway through the PIK3CA and PTEN regulatory proteins. In addition, the confirmation of HER2 / ERBB2 gene expression was maintained. Activation of the MAP kinase protein (MEK2) has been shown to occur through ERBB2 transduction in breast cancer. This association was clearly maintained and perturbed in the hepatocellular carcinoma cell line HepG2.

[0112] Overlapping metabolic pathways were observed in both rtPCR and western blot studies of HepG2 cells treated with sorafenib and CBNA. The synergistic effects observed in in vitro and ex vivo assays can be due to pathway potentiation caused by convergence of pathways. Sorafenib directly acts on many of the kinase proteins involved in these pathways, while CBNA is known to act through cell surface receptors. Intrinsic and extrinsic perturbation of common pathways appears to result in synergistic effects.

[0113] Example 7

[0114] The objective of this example was to evaluate the predictability of the mechanism of action in vivo from patient-derived xenografts (PDX).

[0115] In vivo validation experiments were performed in NCG mice engrafted with hepatocellular carcinoma cells (575) to generate tumor xenografts and treated with drug-loaded nanospikes as described in paragraph

[65] . These data validated previous in vitro results and confirmed efficacy and mechanism of action in addition to determining preliminary immune system activation. Figure 10A and 10B The data shown in Figures 6 and 7 confirm the involvement of the PI3K / AKT pathway as a mechanism of action of CBNA. In addition to this, we further validated our finding that the Wnt and HIPPO pathways are affected by CBNA treatment. Activation of these pathways is associated with metastasis. Furthermore, we confirmed the role of macrophage activity in the clearance of apoptotic cell debris by positive identification of the F4 / 80 cell marker in tumor tissue in the vicinity of the nanospikes device. Figure 10B These data not only validate previously determined mechanisms, but also confirm superior activity of the combination candidate drugs in animal studies. Figure 11 The safety of CBNA was further demonstrated where treatment with CBNA alone did not significantly affect cell proliferation, however, when combined with sorafenib, using only 1 / 5thof the total drug amount, still resulted in increased tumor death compared to sorafenib alone.

[0116] The disclosure described in this invention illustrates the synergistic effect with CBNA and sorafenib. The mechanism of action derived from HepG2 in vitro can be extrapolated to an in vivo scalable human PDX model, which is of great significance for the development of combination cancer candidate drugs.

[0117] Example 8

[0118] The objective of this example was to estimate the appropriate clinical dose, route of administration, and dosage of SCI-0502.

[0119] To better estimate the human first-in-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® from the open-source systems pharmacology toolbox was used for this purpose. Version 11.2 was used for the simulations (Lippert, Burghaus et al. 2019). Computer simulation estimates of the pharmacokinetic parameters for CBNA and sorafenib are shown in Table 5. This set of parameters was used as the initial setup for further simulations.

[0120] Table 5: Computer simulation estimates of the pharmacokinetic parameters for CBNA and sorafenib

[0121]

[0122]

[0123] F: oral bioavailability; F g : fraction of dose absorbed from the gut; F l : fraction of absorbed portion escaping the liver; FaSSIF: fasted state simulated intestinal fluid; Cl TB : systemic clearance; Cl H : hepatic clearance; Cl r : renal clearance; V d,ss : volume of distribution at steady state; t 1 / 2 : half-life;

[0124] V d : organ volume distribution; P tp : tissue / plasma partition coefficient.

[0125] Sorafenib clinical data from Jain, Woo et al. (2011) was used for pharmacokinetic modeling of sorafenib. Sorafenib dose 400 mg, twice daily, was used for the simulations. The pharmacokinetic characteristics of sorafenib in humans are enterohepatic circulation (EHC), and the drug is a substrate for the canalicular efflux transporter multidrug resistance protein 2 (MRP2). These parameters were included in the simulations. In addition, parameters including passive permeability including intracellular space of organs, intestinal permeability, hepatic clearance, and dissolution rate were optimized to reproduce the clinical observations (Table 6 and Figure 13 ). This optimization established a PBPK model for sorafenib that appropriately described the plasma profiles of patients. Since there were no available clinical data for CBNA, the PBPK model was optimized to produce results using ADME Tox predictors.

[0126] Table 6 Pharmacokinetic parameters for sorafenib

[0127] Mock Clinical studies Total body clearance (l / h / kg) 0.11 0.11 Volume of distribution (l / kg) 1.26 2.66 Half-life (h) 8.63 NA T 最大 (h)]]> 5.5 2-9.5 AUC 0-24 (mg*h / l) 29.44 NA AUC 0-24 @liver (mg*h / l) 21.0 NA

[0128] Tmax: time to reach maximum concentration

[0129] AUC 0-24 : area under the plasma concentration curve from 0 to 24 hours

[0130] AUC 0-24@肝 : area under the liver tissue concentration curve from 0 to 24 hours.

[0131] Sorafenib is marketed as an oral drug, CBNA has a higher oral bioavailability than sorafenib, in addition, the liver clearance values between CBNA and sorafenib are comparable (Table 5), therefore it can be concluded that CBNA can also be administered orally.

[0132] The final objective of this example is to estimate the dose of SCI-0502 that can have a clinical effect in humans. Therefore, our goal is to propose an effective dose of SCI-0502 that reaches a synergistic ratio in the site of action (liver). As a target, the AUC ratio of CBNA to sorafenib in the liver is in the range of 1 : 1 to 6: 1. The dose of SCI-0502 is calculated using both PBPK models of CBNA and sorafenib, assuming a patient is a Caucasian male, weighing 73 kg. The dose is administered with food. The results of the simulation are shown in Table 7.

[0133] Table 7: Combination doses of sorafenib and CBNA provide equivalent or better efficacy than 200 mg or 400 mg oral doses of sorafenib.

[0134]

[0135] Since the safety of CBNA has been proven to be superior to the safety of sorafenib Figure 7 and Figure 11 , it can be concluded that the mixture of sorafenib and CBNA is a better candidate drug to treat HCC, since the dose of sorafenib can be as low as one sixth of the clinical dose of sorafenib.

[0136] REFERENCES

[0137] The following references are indicative of the level of skill in the art and are hereby incorporated by reference in their entirety, to the extent permitted. Ahn, S. W., B. Ferland, and O. H. Jonas (2021). "An Interactive Pipeline for Quantitative Histopathological Analysis of Spatially Defined Drug Effects in Tumors." Cancers 13(19). J Pathol Inform 12:34.

[0138] Blasco-Benito, S., M. Seijo-Vila, M. Caro-Villalobos, I. Tundidor, C. Andradas, E. Garcia-Taboada, J. Wade, S. Smith, M. Guzman, E. Perez-Gomez, M. Gordon, and C. Sanchez (2018). "Appraising the "entourage effect": Antitumor action of a pure cannabinoid versus a botanical drug preparation in preclinical models of breast cancer." Oncotarget 9(48): 28764-28779. Biochem Pharmacol 157:285-293.

[0139] Jain, L., S. Woo, E. R. Gardner, W. L. Dahut, E. C. Kohn, S. Kummar, D. R. Mould, G. Giaccone, R. Yarchoan, J. Venitz, and W. D. Figg (2011). "Population pharmacokinetic analysis of sorafenib in patients with solid tumours." Br J Cancer 105(8): 992-997. Br J Clin Pharmacol 72(2):294-305.

[0140] Kanehisa, M., S. Goto, M. Furumichi, M. Tanabe, and M. Hirakawa (2010) "KEGG for representation and analysis of molecular networks involving diseases and drugs." Nucleic Acids Res. Nucleic Acids Res 38 (Data Pool Problem): D355-360.

[0141] Kitisin, K., V. Packiam, J. Steel, A. Humar, T. C. Gamblin, D. A. Geller, J. W. Marsh, and A. Tsung (2011) "Presentation and outcomes of hepatocellular carcinoma patients at a western centre." HPB (Oxford) 13(10): 712-722.

[0142] Kleckner, A. S., I. R. Kleckner, C. S. Kamen, M. A. Tejani, M. C. Janelsins, G. R. Morrow, and L. J. Peppone (2019) "Opportunities for cannabis in supportive care in cancer." Ther Adv Med Oncol 11:17588359 19866362.

[0143] Meyer, P., M. Langos, and R. Brenneisen (2018) "Human Pharmacokinetics and Adverse Effects of Pulmonary and Intravenous THC-CBD Formulations." Medical Cannabis and Cannabinoids 1(1): 36-43.

[0144] Lippert, J., R. Burghaus, A. Edginton, S. Frechen, M. Karlsson, A. Kovar, T. Lehr, P. Milligan, V. Nock, S. Ramusovic, M. Riggs, S. Schaller, J. Schlender, S. Schmidt, M. Sevestre, E. Sjogren, J. Solodenko, A. Staab, and D. Teutonico (2019) "Open Systems Pharmacology Community - An Open Access, Open Source, Open Science Approach to Modeling and Simulation in Pharmaceutical Sciences." CPT Pharmacometrics Syst Pharmacol 8(12): 878-882.

[0145] Moreno, E., M. Cavic, A. Krivokuca, V. Casado, and E. Canela (2019) "The Endocannabinoid System as a Target in Cancer Diseases: Are We There Yet?" Front Pharmacol 10: 339.

[0146] Torres, S., M. Lorente, F. Rodriguez-Fornes, S. Hernandez-Tiedra, M. Salazar, E. Garcia-Taboada, J. Barcia, M. Guzman, and G. Velasco (2011) "A combined preclinical therapy of cannabinoids and temozolomide against glioma." Mol Cancer Ther 10(1): 90-103.

[0147] Zhong, N. (2020) Cannabinol inhibits proliferation and induces cell cycle arrest and apotosis in glioblastoma, hepatocellular carcinoma and breast cancer cells ​ ​ ​ . Master of Science, Lesley University.

Claims

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

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

1.

3. The composition according to claim 2, wherein, The molar ratio is between about 2:1 and about 5:

1.

4. The composition according to claim 3, wherein, The molar ratio is approximately 3:1 or approximately 4:

1.

5. The composition according to any one of claims 1 to 4, wherein, The combination includes sorafenib.

6. The composition according to any one of claims 1 to 4, wherein, The combination includes regorafenib.

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

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

9. The method according to claim 8, wherein, The cancers mentioned are hepatocellular carcinoma, renal cell carcinoma, and thyroid cancer.

10. The use of CBN or CBNA in synergistic combination with sorafenib or regorafenib for the treatment of cancer, wherein... The cancers mentioned are hepatocellular carcinoma, renal cell carcinoma, thyroid cancer, metastatic colorectal cancer, or gastrointestinal stromal tumor.

11. The use according to claim 10, wherein, CBNA: The molar ratio of sorafenib or regorafenib is between approximately 1:1 and approximately 6:

1.

12. The method according to claim 11, wherein, The molar ratio is between about 2:1 and about 5:

1.

13. The method according to claim 12, wherein, The molar ratio is approximately 3:1 or approximately 4:

1.

14. The use according to any one of claims 10 to 13, wherein, The cancer in question is hepatocellular carcinoma, renal cell carcinoma, thyroid cancer, metastatic colorectal cancer, or gastrointestinal stromal tumor.

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

16. The use according to claim 15, wherein, The cancer in question is hepatocellular carcinoma, renal cell carcinoma, or thyroid cancer.

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

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