Combination therapy of crocin and sorafenib for liver cancer

Abstract

A pharmaceutical combination for the treatment of liver cancer, comprising crocin or a pharmaceutically acceptable prodrug and sorafenib.

Description

[Technical Field]

[0001] The present invention relates to therapeutic formulations and methods for treating cancer using crocin alone or in combination with sorafenib. [Background technology]

[0002] Liver cancer varies depending on the cell type involved. Liver cancers include hepatocellular carcinoma (HCC), cholangiocarcinoma, and angiosarcoma. Liver cancer progression begins with frequent liver injury caused by several factors. Repeated liver injury leads to acute inflammation, which, if sustained, can lead to fibrosis, cirrhosis, and ultimately the formation of liver neoplasia. HCC is the most common type of primary liver cancer, with 782,000 cases diagnosed and 746,000 deaths in the United States alone in 2021. HCC is the fifth most common cancer worldwide and the third leading cause of cancer-related deaths. The incidence of liver cancer in the UAE is rising due to the increasing prevalence of liver disease, which, if left untreated, can progress to liver cancer. Liver cancer is the fourth leading cause of cancer-related deaths in both men and women in the UAE (Figure 2; Department of Health - Abu-Dhabi, 2015). The rising incidence of liver cancer is attributed to multiple risk factors, including alcohol consumption, fatty liver disease, and viral hepatitis C and B. As with most cancers, chemotherapy is the first line of defense. Chemotherapy drugs used to treat HCC include cisplatin, doxorubicin, and mitomycin. All of these drugs aim to improve survival rates and rarely reverse the cancer process. One major challenge with chemotherapy is its nonselective cytotoxicity. Patients with the best prognosis are those who detect the cancer early and choose to undergo surgical tumor resection and liver transplantation. However, in most cases, patients are diagnosed with liver cancer at an advanced stage, when opportunities for intervention are limited and often ineffective. Summary of the Invention [Problem to be solved by the invention]

[0003] Sorafenib is the only targeted therapy for HCC approved by the U.S. Food and Drug Administration (FDA). Sorafenib is an oral mircrokinase inhibitor that inhibits tumor cell proliferation by targeting Raf / MEK / ERK signaling and angiogenesis by targeting vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptors. Despite the efficacy of sorafenib, further advanced HCC treatments are needed. Compared with monotherapy, combination therapy targeting multiple signaling pathways may offer a better treatment option by potentially avoiding resistance, feedback activation, and compensatory activation of pro-survival pathways.

[0004] Crocetin digentiobiose ester, known as crocin, is known to be a major compound in saffron. Crocin is one of the few water-soluble carotenoids. Crocin possesses numerous pharmacological properties, including potent antioxidant and anti-inflammatory properties. Crocin and cisplatin, administered alone or in combination, to osteosarcoma MG63 and OS732 cells demonstrated potent cell killing, inhibited MG63 cell invasion, and increased the expression of caspase-3 and caspase-8, markers of apoptosis activation. Furthermore, administration of crocin-coated nanoparticles to precancerous livers in mice resulted in significant regression of precancerous lesions and increased apoptosis. Another study demonstrated that crocin prevented early liver cancer in HepG2 cells and Wistar rats. Crocin has been shown to inhibit the growth of colorectal cancer (HCT116 wild-type and HCT116 p53- / - cell lines), and crocin treatment induces classical programmed cell death that is independent of autophagy. [Means for solving the problem]

[0005] Original Clearly According to the study, the method treats, inhibits, or reduces the severity of liver cancer in a subject. Pharmaceutical composition for And, No. 1 dose of sorafeni Bu and , No. 2 amount of crocin or a pharmaceutically acceptable prodrug thereof and a pharmaceutical composition comprising crocin or a pharmaceutically acceptable prodrug thereof and sorafenib in a mass ratio of 50:1 to 5:1, wherein the prodrug is selected from the group consisting of crocin salts, hydrates, hemiacetals, acetals, thioacetals, silyl ethers, tautomers, and combinations thereof. In one embodiment, the crocin is α-crocin. Crocin or a prodrug thereof and sorafenib are combined together in a composition containing both compounds. It has been Alternatively, crocin or a prodrug thereof and sorafenib may be administered in separate pharmaceutical compositions. provided Crocin or a prodrug thereof and sorafenib may be administered together or sequentially. For example, crocin or a prodrug thereof may be administered first to sensitize cancer cells before exposure to sorafenib, and sorafenib may be administered later. . liver The cancer may be hepatocellular carcinoma (HCC), fibrolamellar HCC, cholangiocarcinoma, angiosarcoma, and / or metastatic liver cancer.

[0006] Non In limited embodiments, the crocin is selected from the group consisting of monoglycosyl polyene esters of crocetin, diglycosyl polyene esters of crocetin, and combinations thereof. .Ma Ta, As a reference embodiment of the present invention, Also provided is a method for treating, inhibiting, or reducing the severity of liver cancer in a subject. Pharmaceutical Composition to the subject in a therapeutically effective amount of

[0007] nine The rosin and sorafenib may be formulated together in the same single pharmaceutical composition containing both compounds, or may be formulated separately, for example in separate containers included in a kit.

[0008] The present invention may be better understood with reference to the following figures and description, in which the components are not necessarily to scale and are not intended to accurately represent molecules, cells, organelles, tissues, or their interactions, emphasis instead being placed upon illustrating the principles of the invention. [Brief explanation of the drawings]

[0009] [Figure 1]1 shows the experimental design carried out in the study reported in this application. [Figure 2] FIG. 1 shows a schematic diagram of the blood and liver samples used to assess some parameters. [Figure 3] Representative images of the livers of animals used to demonstrate the therapeutic antitumor effects of drugs are shown: PBS, DEN-induced HCC rats (HCC), or rats treated with crocin (HCC CR), sorafenib (HCC SB), separately or in combination (HCC CR+SB). [Figure 4] Quantitative analysis of the number of liver nodules from DEN-induced HCC in rats untreated (HCC alone group) or treated with sorafenib (HCC SB), crocin (HCC CR), separately, or in combination (HCC CR+SB). Statistical significance was determined using the Microsoft Excel Data Analysis ToolPak t-test, assuming equal variance between the two groups. (a: vs. PBS, b: vs. HCC; *P<0.05, **<0.01). [Figure 5] Images of reticulin-stained liver sections are shown. Sections were taken from control (PBS), untreated rats (HCC), or DEN-induced HCC treated with crocin (HCC CR), sorafenib (HCC SB), or their combination (HCC CR SB). High magnification: 40x; low magnification: 10x. Arrows indicate reticular fibers. [Figure 6]This shows that crocin inhibits the growth of induced HCC. (a) Western blot images of PCNA were obtained in control (PBS) and DEN-induced HCC (HCC), or in experiments treated with crocin (HCC CR), sorafenib (HCC SB), separately, or in combination (HCC CR SB). (b) Quantification plots were performed using ImageJ to quantify the signal and normalize it according to total liver protein. Results are expressed as the mean ± SD for n = 4 animals per group. Statistical significance was determined using a t-test using the Microsoft Excel Data Analysis ToolPak, assuming equal variance between the two groups. a: vs. PBS; b: vs. HCC; *P < 0.05, **P < 0.01, ***P < 0.001. [Figure 7] Western blot analysis of key proteins involved in the intrinsic apoptosis pathway in the liver (PARP, procaspase-9, procaspase-3, Bax, Bcl-2) in the control and experimental groups is shown. The results indicate that crocin activates the intrinsic apoptosis pathway in DEN-induced HCC. Control (PBS), untreated rats (HCC), or DEN-induced HCC treated with crocin (HCC CR) and sorafenib (HCC SB), either separately or in combination (HCC CR SB), were analyzed. [Figure 8] Quantitative analysis of procaspase-3, procaspase-3, and PARP in the control and experimental groups was performed. Control (PBS), untreated rats (HCC), or DEN-induced HCC treated with crocin (HCC CR) and sorafenib (HCC SB), separately or in combination (HCC CR SB), were analyzed. Band intensity was quantified using ImageJ and normalized according to total liver protein. Results are presented as the mean ± SD for n = 4 animals in each group. Statistical significance was determined using a t-test in Microsoft Excel Data Analysis ToolPak, assuming equal variance between the two groups. a: vs. PBS; b: HCC; *P < 0.05, **P < 0.01, ***P < 0.001. [Figure 9]Quantification of Bax, Bcl-2, and the Bax / Bcl ratio from control and experimental groups is shown. Sections were analyzed from control (PBS), untreated (HCC), or DEN-induced HCC treated with crocin (HCC CR) and sorafenib (HCC SB), either separately or in combination (HCC CR SB). Band intensity was quantified using ImageJ and normalized according to total liver protein. Results are presented as the mean ± SD for n = 4 animals in each group. Statistical significance was determined using a t-test using the Microsoft Excel Data Analysis Tool Pack, assuming equal variance between the two groups. a: vs. PBS; b: vs. HCC; *P < 0.05, **P < 0.01, ***P < 0.001. [Figure 10] Representative images of histopathological evaluation of the livers of the control and experimental groups are shown. Samples were taken from the control group (PBS), untreated rats (HCC), or DEN-induced HCC treated with crocin (HCC CR), sorafenib (HCC SB), or in combination (HCC CR SB). High magnification is 40x, and low magnification is 10x. Arrows indicate representative areas of AHF. [Figure 11] The activity of liver enzymes (AST, ALT) measured in serum is shown (n=8). Statistical significance was determined using the Microsoft Excel Data Analysis Tool Pack and t-test, assuming equal variance between the two groups. (a: vs. PBS, b: vs. HCC, c: vs. HCC+SB; *P<0.05, **P<0.01, P<0.001) [Figure 12] A table reporting human equivalent dose (HED) dosing factors based on body surface area for other species is presented, based on data from the U.S. Food and Drug Administration draft guidelines. DETAILED DESCRIPTION OF THE INVENTION

[0010] A study was conducted to investigate the therapeutic effect of crocin on chemically induced hepatocellular carcinoma (HCC). Male Wistar rats were first chemically induced with HCC. Crocin was then administered, and biomarkers of liver function were assessed. Liver tissue was then evaluated histopathologically, and key tumor markers were examined by Western blotting. Based on the biochemical, histological, and molecular markers evaluated in this study, saffron-based crocin proved to be a potent novel therapeutic candidate for DEN-induced HCC.

[0011] This study also revealed that the combination of crocin and sorafenib can achieve surprisingly good therapeutic effects. Therefore, the present invention is based, at least in part, on the discovery that crocin and sorafenib exert synergistic anticancer effects against HCC in experimental rats. This therapeutic effect can be utilized in the treatment of liver cancer. Compared with sorafenib monotherapy, the combination of sorafenib and crocin, which targets multiple signaling pathways, offers a better treatment option. Without being bound by theory, it is believed that administration of crocin activates the intrinsic apoptotic pathway in liver cancer cells. Interestingly, the combination of crocin and sorafenib appears to be more effective at activating the intrinsic apoptotic pathway than either crocin or sorafenib monotherapy. These findings indicate that the specific combination of sorafenib and crocin has a synergistic, or greater than additive, effect, resulting in greater improvement in liver cancer than monotherapy with either crocin or sorafenib.

[0012] In exemplary embodiments, the present application provides a therapeutic combination of drugs comprising a first amount of crocin (or a pharmaceutically acceptable prodrug thereof) and a second amount of sorafenib. Essentially, the combination of crocin and sorafenib can be a more effective therapeutic combination than either drug alone or the simple sum of the two drugs. Furthermore, different doses of the combination may provide additional benefits in treating liver cancer compared to either crocin or sorafenib alone. Thus, in some embodiments, the present application provides unexpectedly advantageous methods and compositions for treating liver cancer, whereby sorafenib and crocin are administered in a particularly effective ratio (e.g., synergistic or greater than additive).

[0013] [Crocin] Chemically, crocin is a diester formed from the disaccharide gentiobiose and the dicarboxylic acid crocetin. [ka]

[0014] When isolated as a pure compound, crocin is deep red in color and forms crystals with a melting point of 186 °C. When dissolved in water, it forms an orange solution. The broad term crocin can also refer to any member of a series of related hydrophilic carotenoids, which are monoglycosyl or diglycosyl polyene esters of crocetin. The crocin responsible for saffron's aroma is α-crocin (trans-crocetin di(β-D-gentiobiosyl) ester), with the systematic name (IUPAC) 8,8-diapo-8,8-carotenoic acid. The primary active component of saffron is the yellow pigment crocin 2, which contains gentiobiose (disaccharide) groups at both ends of the molecule; three other derivatives with different glycosylation levels are also known.

[0015] Compositions containing crocin can be formulated in a specific form for administration, such as a solid or liquid form, including those adapted for: (1) oral administration, e.g., buccal, sublingual, or systemic absorption, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, or pastes for application to the tongue; (2) parenteral administration, e.g., sterile solutions, suspensions, or sustained-release formulations for subcutaneous, intramuscular, intravenous, or epidural injection; (3) topical administration, e.g., creams, ointments, controlled-release patches, or sprays applied to the skin; (4) vaginal or rectal administration, e.g., pessaries, creams, foams, or formulations adapted for (5) sublingual, (6) ocular, (7) transdermal, or (8) nasal administration.

[0016] The compositions may also include wetting agents such as sodium lauryl sulfate and magnesium stearate, emulsifiers, lubricants, coloring agents, release agents, coating agents, sweeteners, flavorings and perfuming agents, preservatives and antioxidants.

[0017] Formulations of crocin include those suitable for parenteral (including subcutaneous, intravenous, intramedullary, intraarticular, intramuscular, or intraperitoneal injection), rectal, topical, transdermal, or oral (e.g., capsules, suspensions, or tablets) administration. The formulations are conveniently presented in unit dosage amounts and can be prepared by any method well known in the art of pharmacy. The amount of crocin or a pharmaceutically acceptable prodrug thereof that can be combined with a carrier material to produce a single dosage form varies depending on the therapeutic target and mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will typically be that amount of compound having a therapeutic effect. Typically, this amount will range from about 1% to about 99% by weight of the active ingredient, preferably from about 5% to about 70% by weight, and most preferably from about 10% to about 30% by weight, of one hundred percent.

[0018] Regardless of the route of administration selected, crocin or its prodrugs can be formulated into a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art. Crocin, like other pharmaceuticals, may be formulated for administration in any convenient way for use in human or veterinary medicine.

[0019] [Sorafenib] In various embodiments, the present invention encompasses the use of sorafenib, such as sorafenib tosylate and other pharmaceutically acceptable forms, salts, and esters of sorafenib. Sorafenib is commercially available as NEXAVAR®, the tosylate salt of sorafenib. The IUPAC chemical name for sorafenib tosylate is 4-(4-{3-[4-chloro-3(trifluoromethyl)phenyl]ureido}phenoxy)N-methylpyridine-2-carboxamide 4-methylbenzenesulfonate, and the structural formula is: [ka]

[0020] The recommended daily dose of sorafenib tosylate is 800 mg, administered orally at 400 mg (two tablets) twice daily. However, treatment interruption and / or dose reduction may be necessary to manage suspected side effects. In such cases, the dose can be reduced to 400 mg once daily or 400 mg every other day. Those skilled in the art will understand that the dosage and administration method of sorafenib not only follow medically accepted guidelines, but can also deviate from or be modified from medically accepted guidelines.

[0021] [Liver cancer] In one aspect, the present invention provides a method for treating liver cancer cells, including in vitro treatment of cancer cells in a subject or isolated cancer cells. When the cancer cells are in the subject's body, the subject may be a primate, such as a human, with liver cancer. The subject may be a mammal. The subject may be an adult (i.e., 18 years of age or older) or a juvenile (under 18 years of age). In various embodiments, the liver cancer may be hepatocellular carcinoma (HCC), fibrolamellar HCC, cholangiocarcinoma, angiosarcoma, or metastatic liver cancer.

[0022] In some embodiments, the liver cancer is not resistant to sorafenib. Alternatively, the liver cancer may exhibit primary or secondary resistance to sorafenib. The subject may respond to sorafenib in the absence of crocin. The subject may not respond to sorafenib in the absence of crocin. In some embodiments, the subject has been previously treated with sorafenib for at least 1, 2, 4, 6, 8, 10 months or more. In other embodiments, the subject is a patient who experiences one or more serious adverse side effects to sorafenib and therefore requires a dose reduction.

[0023] In some embodiments, the liver cancer is intermediate, advanced, or end-stage. It may be metastatic or non-metastatic, resectable or unresectable. It may include a single tumor, multiple tumors, or poorly defined tumors with an infiltrative growth pattern (into the portal or hepatic vein). It may include fibrolamellar, pseudoglandular (adenoid), pleomorphic (giant cell), or clear cell tumors. It may include well-differentiated tumors, where tumor cells resemble hepatocytes, form trabeculae, cords, and nests, and / or contain bile pigments in the cytoplasm. It may include poorly differentiated tumors, where malignant epithelial cells are non-cohesive, pleomorphic, undifferentiated, and / or giant. In some embodiments, the liver cancer may be associated with hepatitis B, hepatitis C, cirrhosis, or type 2 diabetes.

[0024] In some embodiments, the subject is a human with an Eastern Cooperative Oncology Group (ECOG) performance status of 2 or less. In some embodiments, the subject is a human with acceptable liver function, as defined as: (i) total bilirubin 1.5 times the upper limit of normal (ULN) or less, or for patients with hepatocellular carcinoma only, total bilirubin 3 mg / dL (i.e., Child-Pugh for bilirubin 2 or less), (ii) aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) 5 times the ULN or less, or (iii) acceptable renal function: serum creatinine 1.5 times the ULN or less, or calculated creatinine clearance 60 mL / min / 1.73 m for patients with creatinine levels greater than 1.5 times normal. 2 That's all.

[0025] In some embodiments, the subject is a human with an acceptable hematological status as defined below: (i) absolute neutrophil count (ANC) > 1500 cells / mm 3 (ii) Platelet count ≥100,000plts / mm 3 (without transfusion), and for patients with hepatocellular carcinoma only, platelet count ≥ 75,000 plts / mm 3 , or (iii) hemoglobin ≥ 9 g / dL.

[0026] In some embodiments, the subject is a human with a prothrombin time (PT) or international normalized ratio (INR) ≦1.25×ULN, an INR<1.7, or a prothrombin time (PT) less than 4 seconds above the ULN (Child-Pugh score of ≦1 for coagulation parameters), or a serum albumin >2.8 g / dL (Child-Pugh score for albumin ≦2).

[0027] [Combination therapy] Combination therapy or polytherapy is the use of multiple drugs or other therapies, as opposed to monotherapy, which is a therapy that uses a single drug.In one aspect of the present invention, provided herein is a therapeutic combination of drugs for the treatment of liver cancer, which combination comprises crocin and sorafenib.In an exemplary embodiment of the combination, crocin and sorafenib are formulated together in the same single pharmaceutical composition that contains both compounds.In another exemplary embodiment, crocin and sorafenib are in separate pharmaceutical compositions.Also provided is a method for treating, suppressing, or reducing the severity of liver cancer in a subject by administering a therapeutic amount of the combination to the subject.

[0028] In some embodiments, a therapeutic combination refers to the use of a specific combination (e.g., ratio and / or dosing schedule) of crocin and sorafenib. More specifically, the present invention provides therapeutic combinations and methods for treating liver cancer in which crocin and sorafenib are administered in a particularly effective ratio (e.g., synergistic or greater than additive). In exemplary embodiments, the mass ratio of crocin to sorafenib is about 50:1, 40:1, 30:1, 25:1, 20:1, 10:1, 5:1, 2:1, 1:2, 1:5, 1:10, 1:20, 1:30, 1:40, or 1:50. In some embodiments, the ratio is at least about 1, 2, 5, 10, 12, 15, 20, or 50. In some embodiments, the ratio is less than about 5, 10, 15, 20, 30, 40, 50, 60, or 70. Exemplary weight pairs are about 1, 2, 5, 8, 10, 15, 20, 25, 30, 40, and 50.

[0029] The mass ratio of crocin:sorafenib can be measured over various time periods. For example, the mass ratio can be based on the amount of crocin and sorafenib administered to a subject over a period of 1 day, 1 week, 14 days, 21 days, or 28 days.

[0030] The dosage and / or administration schedule of sorafenib can be in accordance with clinically approved or experimental guidelines. In various embodiments, the dose of sorafenib is about 800, 600, 400, or 200 mg / day. The 200 mg / day dose can be administered as a 400 mg dose every other day.

[0031] Similarly, the dosage and / or administration schedule of crocin can follow clinically approved or experimental guidelines. Additionally, data obtained from animal studies can be used to formulate a dosage range of crocin for use in humans. For example, an effective dosage achieved in one animal species can be extrapolated for use in other animals, including humans, as shown in the conversion table in Figure 12, which reports human equivalent dose (HED) dosing coefficients based on body surface area for other species. In exemplary embodiments, the dose of crocin ranges from about 0.001, 0.01, 0.1, 0.5, 1, 10, 15, 20, 25, 50, 100, 200, 300, 400, 500, 600, or 750 to 1000 mg / kg of subject body weight per day. In certain embodiments, the dose of crocin is typically in the range of about 100 mg / kg to about 1000 mg / kg of subject body weight per day, specifically about 200 mg / kg to about 750 mg / kg per day. More specifically, it is in the range of about 250 mg / kg to about 500 mg / kg per day. In one embodiment, the dose is in the range of about 50 mg / kg to about 250 mg / kg per day. In a further embodiment, the dose is in the range of about 100 mg / kg to about 200 mg / kg per day. In one embodiment, the dose is in the range of about 15 mg / kg to about 60 mg / kg per day. In a further embodiment, the dose is in the range of about 20 mg / kg to about 50 mg / kg per day. In an additional embodiment, the dose is in the range of about 25 mg / kg to about 45 mg / kg per day.

[0032] The dosage of crocin can be set within a therapeutically effective range based on the selected ratio and the dosage of sorafenib. As described above, the ratio can be determined using the amount of sorafenib administered to the subject for 1 day, 1 week, 14 days, 21 days, or 28 days.

[0033] In some embodiments, crocin is administered to a subject in 1, 2, 3, 4, 5, 6, or 7 daily doses over a 1-week (7-day) period. Crocin may also be administered to a subject in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 daily doses over a 14-day period. Crocin may also be administered to a subject in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 daily doses over a 21-day period. Crocin may be administered to a subject in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 daily doses over a 28 day period.

[0034] In various embodiments, crocin is administered for the following periods: 2 weeks (14 days total), 1 week on with 1 week off (14 days total), 3 consecutive weeks on (21 days total), 2 weeks on with 1 week off (21 days total), 1 week on with 2 weeks off (21 days total), 4 consecutive weeks on (28 days total), 3 weeks on with 1 week off (28 days total), 2 weeks on with 2 weeks off (28 days total), 1 week on with 3 weeks off (28 days total).

[0035] In further embodiments, crocin is administered on day 1 of a 7, 14, 21, or 28 day dosing cycle, or on days 1 and 15 of a 21 or 28 day dosing cycle, or on days 1, 8, and 15 of a 21 or 28 day dosing cycle, or on days 1, 2, 8, and 15 of a 21 or 28 day cycle. Crocin can be administered once every 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

[0036] The course of crocin-sorafenib therapy can be continued until clinical endpoints are met. In some embodiments, treatment is continued until disease progression or unacceptable toxicity occurs. In some embodiments, treatment is continued until a pathological complete response rate is achieved, defined as the absence of liver cancer (e.g., HCC). In some embodiments, treatment is continued until partial or complete remission of liver cancer. Administration of crocin and sorafenib to multiple subjects with liver cancer may improve overall survival (OS), progression-free survival (PFS), disease-free survival (DFS), response rate (RR), quality of life (QoL), or a combination thereof.

[0037] In various embodiments, treatment reduces the size and / or number of liver cancer tumors, treatment can prevent liver cancer tumors from increasing in size and / or number, or treatment can prevent liver cancer tumor metastasis.

[0038] In the methods of the present invention, administration of crocin and sorafenib is not limited to a particular delivery system and may include, but is not limited to, parenteral (including subcutaneous, intravenous, intramedullary, intraarticular, intramuscular, or intraperitoneal injection), rectal, topical, transdermal, or oral (such as capsules, suspensions, or tablets). Administration to an individual can be a single dose or multiple doses, and may be administered in any of a variety of physiologically acceptable prodrug or salt forms, and / or as part of a pharmaceutical composition, with acceptable pharmaceutical carriers and / or additives. Physiologically acceptable salt forms and standard pharmaceutical formulation techniques, dosages, and excipients are well known to those skilled in the art. Furthermore, effective dosages achieved in one animal can be extrapolated for use in other animals, including humans, using conversion factors known in the art.

[0039] The combination therapy of the present invention is not particularly limited to a particular course or regimen and can be used separately or in combination with other therapies (eg, chemotherapy or radiation therapy).

[0040] In some embodiments, crocin is administered before, concurrently with, or after sorafenib, or a combination thereof. Crocin is administered systemically or locally.

[0041] Combination therapies according to the present invention can include additional therapies other than crocin and sorafenib (e.g., pharmaceuticals, radiation, etc.). Similarly, the present invention can be used as adjuvant therapy (e.g., when combined with surgery). In various embodiments, the subject is also treated with surgical resection, percutaneous ethanol or acetic acid injection, transcatheter arterial chemoembolization, radiofrequency ablation, laser ablation, cryoablation, focused external beam stereotactic radiotherapy, selective internal radiotherapy, intra-arterial iodine-131 lipiodol administration, and / or high-intensity focused ultrasound.

[0042] The combination of crocin and sorafenib can be used as adjuvant, neoadjuvant, combination, concurrent, or palliative therapy.The combination of crocin and sorafenib can be used as first-line, second-line, or crossover therapy.

[0043] In some embodiments, the therapeutically effective dose of sorafenib is reduced by combining with crocin. For example, the daily, weekly, or monthly dose of sorafenib can be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the maximum recommended dose or maximum tolerated dose. In other embodiments, sorafenib is administered at an effective dose that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or more lower than the dose required for efficacy in the absence of crocin. In some embodiments, the IC50 of sorafenib is reduced by at least 2, 4, 5, 10, 20, 30, 40, 50, or 100 times compared to the IC50 in the absence of crocin.

[0044] [kit] The present invention also provides a kit for treating liver cancer. For example, the kit may include one or more pharmaceutical compositions of crocin and sorafenib as described above. The composition may be a pharmaceutical composition containing a pharmaceutically acceptable excipient. In another embodiment, the kit includes a first pharmaceutically acceptable composition containing crocin, a second pharmaceutically acceptable composition containing sorafenib, and, optionally, instructions for their use in treating liver cancer. In yet another embodiment, the kit includes one or more pharmaceutical compositions and one or more devices for administering such compositions. For example, the subject kit may include a pharmaceutical composition and a catheter for direct intra-arterial injection of the composition into the cancer. In one embodiment, the device is an intra-arterial catheter.

[0045] [Experimental Results] (In vivo model: Animal) Male Wistar rats were obtained from the Animal Research Facility at the UAE University Faculty of Medicine and Health Sciences. Initially, animals weighed approximately 110 g and were housed at 24–26°C under a 12-h light / dark cycle. They were maintained on a standard laboratory diet with free access to food and water. All animal experiments were approved by the UAE University Faculty of Medicine and Health Sciences Animal Research Ethics Committee (approval number A8-15).

[0046] (Experimental Plan) Carcinogenesis This study used 4-week-old male Wistar rats. The rats were randomly divided into two groups: a control group (n = 8) that received 1x PBS via intraperitoneal (ip) injection, and an experimental group that received 50 mg / kg body weight of DEN (Sigma-Aldrich, USA) in PBS via ip injection once a week for 16 weeks. This HCC induction protocol was modified from protocols described by (DePeralta et al., 2016; Schiffer et al., 2005).

[0047] Treatment Protocol At week 16, HCC was established in the experimental group, and the animals were divided into four groups (n = 8): HCC alone, HCC + crocin, HCC + sorafenib, and HCC + crocin + sorafenib (see Figure 1). From week 17 to week 19, crocin and / or sorafenib were administered once daily, 5 days a week. All medications were administered orally via intragastric tube (oral gavage). The dosage and route of administration were in accordance with those previously reported in the literature.

[0048] During the first week of the experiment, the PBS group received an i.p. injection of 1x PBS for the entire duration of the experiment. Meanwhile, the HCC experimental group received 50 mg / kg body weight of DEN (Sigma-Aldrich, USA) dissolved in PBS once a week for 16 weeks. From the 17th week, crocin and sorafenib were administered orally by gavage five times a week until the 19th week.

[0049] In the HCC + crocin group, crocin (Sigma Aldrich, USA) was administered at a dose of 200 mg / kg body weight. In the HCC + sorafenib group, sorafenib (Carbosynth Limited) was administered at a dose of 10 mg / kg body weight. Animals in the HCC + crocin + sorafenib group received 200 mg / kg body weight of crocin followed immediately by 10 mg / kg body weight of sorafenib. Crocin was diluted in 1x PBS. Sorafenib was dissolved in 0.3% DMSO. The oral LD50 of crocin when administered intraperitoneally in male Wistar rats is 1-5 g / kg body weight. At the end of the experimental period, 24 hours after the last drug administration, the animals were euthanized using diethyl ether and dissected under controlled conditions. Blood and liver tissue samples were collected and stored in buffered formalin at room temperature for histological analysis or in PBS at −80°C for biochemical and immunoblotting analysis.

[0050] (Sample preparation) blood sample To collect blood samples, rats were euthanized and decapitated, and blood was collected into collection tubes (BD Vacutainer). To separate serum from whole blood, samples were centrifuged at 1200 rpm for 10 minutes. Serum was collected, flash-frozen in liquid nitrogen, and stored at -80°C for further investigation.

[0051] biochemical analysis Levels of the liver enzymes alanine transaminase (ALT) and aspartate aminotransferase (AST) were assessed using commercially available colorimetric assays. Kits were purchased from Abeam (Cambridge, United Kingdom), and ALT and AST concentrations were measured using a Promega GloMax Discover microplate reader according to the provided protocol (see Figure 2).

[0052] Liver samples The liver tissue was washed using PBS, and photographs of the entire liver were taken. Next, each harvested liver was divided into two parts. One part was stored in an empty Eppendorf tube, immediately flash-frozen in liquid nitrogen, and stored at -80°C for further investigation. The other liver part was stored at room temperature in 10% neutral buffered formalin for histopathological examination (see Figure 2).

[0053] Histopathological preparation To maintain tissue integrity, liver tissue samples were fixed in 10% neutral buffered formalin solution. Subsequently, the tissues were cut into 3-μm-thick sections and dehydrated to remove moisture. The dehydration process was achieved using a series of increasing concentrations of ethanol solutions. Xylene was then used to remove any remaining ethanol from the tissue. Next, the tissue sections were embedded in paraffin wax to prepare paraffin blocks, allowing the wax to penetrate the tissue. The resulting blocks were then cut into 3-μm-thick sections. Two stains, H&E and reticulin, were used to observe morphological changes at the cellular level. Tissue slides were observed and examined under a light microscope according to the manufacturer's instructions (Abeam). To assess the validity of the carcinogenesis model and the effects of the treatments used in this study, all liver tissue samples were examined blindly by a pathologist at Tawam Hospital, UAE.

[0054] Western blot analysis Liver tissue samples (10 mg) were homogenized in chilled RIPA buffer (Sigma-Aldrich, USA) with 2 mL of protease and phosphatase inhibitors. The protein concentration of each liver sample was measured using the Bradford method with a Promega GloMax Discover (Bio-Rad, Hercules, CA). To blot proteins onto the gel, the cell lysate obtained by homogenizing the liver tissue was supplemented with the loading dye 2-mercaptoethanol. The mixture of cell lysate and loading dye was then loaded onto an SDS-PAGE gel. The gel percentage was varied depending on the protein size to be determined. After separating the protein content of each sample on the SDS-PAGE gel, the proteins were transferred to a PVDF membrane and blocked with 5% BSA in TBST or 5% (w / v) non-fat milk for 1 hour at room temperature. After the blocking step, the membrane was incubated with primary antibodies overnight at 4°C. In this study, the primary antibodies selected here were anti-proliferating cell nuclear antigen (PCNA), anti-caspase-3 (Cell Signaling Technology Inc.), anti-caspase-9 (Nous Biologicals), anti-poly (ADP-ribose) polymerase (PARP), anti-Bcl-2, and anti-Bax (Santa Cruz). After overnight incubation with the primary antibodies, the membranes were washed extensively with TBST and reprobed with secondary antibodies, such as anti-rabbit IgG (Cell Signaling Technology, Inc., MA, USA) or anti-mouse IgG, which were incubated at room temperature for 1 hour to conjugate horseradish peroxidase. Protein bands were then detected using a chemiluminescent solution known as WesternSure PREMIUM, and the signal was detected and visualized using a Bio-Rad ChemiDoc XRS+ system. Band intensity was measured using ImageJ software. Total protein was stained using SYPRO Ruby protein gel stain according to the manufacturer's protocol (Thermo Fisher Scientific) and used as an internal control.

[0055] Total protein Housekeeping proteins are used as controls in all Western blot analyses. Because housekeeping proteins are stably expressed in tissues, they are an excellent indicator for verifying the integrity of protein samples loaded onto SDS-PAGE gels and accurately reflect the amount of protein in the sample. Current studies are investigating whether these housekeeping proteins can maintain stability under various experimental conditions. In this study, various housekeeping proteins, such as GAPDH, b-actin, and b-tubulin, were used as loading controls and found to be inconsistent. A research paper published in 2003 showed that gene expression of commonly used loading controls, such as GAPDH and b-actin, was increased, varying between 7-fold and 23-fold. Interestingly, this was demonstrated in cancer tissues (Kim & Kim, 2003). Therefore, a different approach was used to solve this problem: total protein was used as a loading control. This protocol measures the protein content of the entire sample rather than relying on a single protein, and has proven to be a reliable loading control in colorectal cancer and HCC.

[0056] [result] (liver enzymes in serum) Liver function analyses were performed to assess the levels of enzymes released from hepatocytes into the bloodstream. These levels reflect the integrity and functionality of the liver. Upon liver injury, these enzymes are widely released from hepatocytes. ALT and AST are the enzymes most commonly checked to assess liver efficiency.

[0057] Effect of treatment on serum enzymes ALT (P<0.01) and AST levels were significantly increased in the HCC group compared with the control group, indicating the severity of liver damage due to tumor formation. Crocin alone and in combination with sorafenib significantly reduced ALT levels compared with the HCC group (P<0.01). Crocin alone and in combination with sorafenib significantly reduced ALT levels compared with the sorafenib alone group (HCC + sorafenib) (P<0.05).

[0058] (Anti-cancer effect of crocin on DEN-induced HCC in rats) Gross appearance of the liver The livers of animals in the PBS group exhibited normal structure, size, texture, and a shiny dark brown color, with no visible lesions. The livers of HCC animals exhibited a pronounced pale color and appearance with multiple lesions evident to the naked eye (see Figure 3). The treated livers appeared less stressed compared to the HCC group. Therapeutic administration of drugs to DEN-induced HCC rats restored normal liver morphology compared to the HCC-only group. Crocin alone (HCC+CR) and combined crocin and sorafenib (HCC+CR+SB) animals showed a reduced number of lesions compared to HCC animals. Crocin-treated animals showed significantly fewer lesions compared to sorafenib-treated (HCC+SB) livers (see Figure 4).

[0059] Histopathological evaluation of the liver To further investigate the therapeutic effect of crocin on DEN-induced HCC rats, histopathological examination of liver tissue was performed (see Figure 10). Liver sections were observed under a light microscope, and representative images were obtained. To visualize cellular components and tissues under the microscope, sections were stained with hematoxylin (H), which stains nuclei blue, and eosin (E), which stains cytoplasm pink. H&E is a standard stain used in histology. The control PBS group showed normal liver structure and organization, normal hepatic lobules with a central vein, and hepatocytes arranged in normal radial spans from the central vein. Histological examination also revealed an intact core and normal vascular relationships between the portal tract and hepatic venules. Histological evaluation of the HCC group revealed abnormal cellular morphology consistent with HCC progression. Hepatic cords appeared wider than normal hepatic plates, and the normal structure of the lobules was lost. Administration of crocin alone or in combination with sorafenib significantly restored normal liver structure in the HCC group.

[0060] Reticulin staining Reticulin is an immunostaining method primarily used to investigate liver histopathology. It aids in the visualization of reticular fibers and is useful for diagnosing well-differentiated HCC. Liver sections from all groups were processed, stained with reticulin, and observed under a light microscope (see Figure 5). The control PBS group showed a normal reticular network. The experimental HCC group showed obvious disruption of the reticular network. Administration of crocin alone (HCC + crocin) and in combination with sorafenib (HCC + crocin + sorafenib) reversed the DEN-induced cancer damage, with fiber disruption less frequent than in the HCC group.

[0061] Western blot analysis and PCNA quantification showed that the expression level of PCNA in the liver of HCC animals was significantly (P<0.01) increased compared with the control group, and that administration of crocin alone or in combination with sorafenib significantly (P<0.01) decreased the expression level of PCNA. Furthermore, the combination of crocin and sorafenib significantly (P<0.001) decreased the PCNA level compared with administration of crocin or sorafenib alone (see Figure 6).

[0062] (The role of crocin in apoptosis) The role of crocin in the apoptotic pathway of DEN-induced HCC was investigated by assessing the expression of key markers, particularly those involved in mitochondrial (intrinsic) apoptosis. Crocin treatment significantly (P<0.05) reduced the expression of the anti-apoptotic protein Bcl-2 compared to the HCC group, while significantly (P<0.05) increased the expression of the pro-apoptotic protein Bax, which plays a key role in activating the apoptotic pathway. Furthermore, the combination of sorafenib and crocin had a stronger (P<0.05) effect on DEN-induced HCC compared with either crocin or sorafenib alone. To thoroughly investigate the role of crocin in apoptosis, multiple Western blot experiments were performed, confirming that procaspase-3, procaspase-9, and PARP activate the intrinsic apoptotic pathway. The expression of these proteins was significantly (P<0.01, P<0.01, P<0.001, respectively) decreased in the crocin-treated group and in the crocin-sorafenib combination group compared with the HCC group alone (see Figures 8 and 9).

[0063] [Consideration] The liver is a complex organ that performs essential functions to maintain homeostasis. Major biological processes occurring in the liver include the uptake, metabolism, and excretion of various xenobiotics. The liver is also involved in immune response, phagocytosis, and elimination of microorganisms. It is also the site of protein, carbohydrate, and fat metabolism. Routine liver function analysis checks the levels of biomarkers such as ALT and AST. These levels largely reflect the level of liver damage and dysfunction. ALT is present in high concentrations in the liver but is also present in other sites, such as the kidney and heart. In the cytoplasm of hepatocytes, ALT plays an important role as a catalyst for transamination reactions. ALT is also involved in transamination reactions and is present in the mitochondria and cytoplasm of hepatocytes, but is primarily concentrated in cardiac tissue. Serum ALT and AST levels are significantly elevated with slight liver damage.

[0064] We investigated the chemopreventive effect of saffron on DEN-induced HCC and found that it significantly restored ALT and AST levels in rats. Coadministration with sorafenib had a similar effect on these liver enzyme levels. ALT and AST levels were significantly lower in the treatment group, indicating improved liver function. This study also found that the hepatic serum enzyme levels of both ALT and AST were higher in the DEN-induced HCC group compared with the PBS control group, directly linked to severe liver damage. The data obtained from this study show that administration of crocin alone had a significant effect on reducing both serum ALT and AST levels compared with DEN-induced HCC rats (see Figure 11). Combination therapy with crocin and sorafenib had a significantly stronger effect on reducing these serum enzyme levels compared with sorafenib alone (P<0.05). Without being bound by any particular theory, this evidence suggests that crocin and sorafenib act synergistically, resulting in a better effect on ameliorating DEN-induced liver damage (see Figure 11).

[0065] (Effect of crocin on improving histological changes) The liver is divided into lobules, each with a hexagonal structure, containing a central vein, a branching hepatic artery, and bile ducts. Hepatocytes are typically arranged in single-cell plates radiating from the central vein. To evaluate the efficacy of crocin, liver biopsies were analyzed. Liver biopsies are traditionally performed as a diagnostic tool to identify the cause and severity of liver disease. In this study, macroscopic nodules were observed in livers collected from DEN-treated animals, as shown in Figure 3. Histopathological examination of the livers of DEN-induced animals revealed clear characteristics of HCC lesions, characterized by a fascicular morphology and clear cytoplasm. The normal hexagonal shape of hepatocytes was lost. Plates were no longer arranged in single-cell thicknesses. Lymphocyte infiltration was observed, all of which are indicative of HCC development. Similar results have been observed in other studies. Administration of crocin alone and as an adjunct to sorafenib significantly reduced the number of observed hepatic nodules (see Figure 4). A study using crocin-coated D-MNPs nanoparticles to treat DEN-induced precancerous mouse liver in mice revealed that the livers of animals administered crocin showed obvious liver histology recovery.

[0066] (Effect of crocin on reticulated networks) Connective tissue is one of the four major types of tissue in the body. It plays an important role in immune defense, growth, repair, and providing mechanical support. It serves as the backbone of epithelial tissue. Connective tissue is primarily composed of extracellular matrix and various cell types. There are three types of extracellular fibers, including collagen, elastin, and reticular fibers. Reticulum fibers are primarily composed of type III collagen and form a reticular network found primarily in liver and muscle fibers. Reticulin staining has been shown to be useful in the differential diagnosis of well-differentiated hepatocellular carcinoma (HCC) and benign liver lesions. In HCC, the reticular network is either completely lost or exhibits an abnormal pattern, including cord-like expansion associated with increased cell layer thickness.

[0067] A rat study was conducted to clarify changes in reticular fiber patterns during liver fibrosis induced by thioacetamide and bile duct ligation. One week after fibrosis induction, a significant decrease in reticular fibers was observed, suggesting disruption of the hepatic reticular network in response to injury. A review of immunohistochemical stains currently used in the differential diagnosis of HCC found that reticulin staining of biopsies from HCC patients showed a loss of reticulin, confirming that this stain can be reliably used to diagnose HCC and differentiate between different phenotypes of liver cancer.

[0068] In this study, liver sections were processed, stained with reticulin, and observed under an optical microscope (see Figure 5). The PBS group showed an intact reticulin network with thin plates. The experimental HCC group showed an abnormal pattern in the HCC nodule area, with the reticulin network clearly disrupted and thicker plates observed. Crocin administered alone (HCC + crocin) and as an adjuvant to sorafenib (HCC + crocin + sorafenib) significantly reversed the damage caused by cancer induction with DEN. Fiber breakage was less frequent compared to the HCC group. The results of this study are consistent with the aforementioned studies.

[0069] Anticancer and antiproliferative effects of crocin in DEN-induced HCC rats. Effect of crocin on growth PCNA has been shown to play important roles in many cellular processes, including DNA replication, DNA repair, and cell cycle progression. Mutations during DNA replication are reflected in post-translational modifications of PCNA, thereby altering its function. Because of its role during replication, PCNA has been considered a marker of proliferation. During replication, PCNA molecules form a sliding clamp around the DNA helix, creating a platform for controlling the replication process. PCNA expression increases in breast and liver cancer metastases. PCNA peaks during S phase, making it an indicator of cell proliferation. Evaluating PCNA expression levels is used as a diagnostic and prognostic tool for cancer assessment. A study conducted on breast cancer patients found that PCNA was highly expressed in cancer tissues compared with adjacent normal tissues. Another study using Sprague-Dawley rats to evaluate the antitumor effects of honeybees found higher levels of PCNA in DEN-induced animals compared with controls. In this study, Western blot analysis showed that PCNA levels were significantly higher in DEN-induced HCC animals compared with controls. Administration of crocin and the combination of crocin and sorafenib reduced PCNA expression (see Figure 6). Crocin has been shown to inhibit cell proliferation and induce apoptosis in A549 and SPC-A1 lung cancer cells. In another study of breast cancer cells, crocin inhibited proliferation by disrupting the microtubule network. A study investigating the antitumor effect of crocin on melanoma treatment in C57BL / 6 mice implanted with B16G10 cancer cells showed that 21-day administration of crocin resulted in tumor shrinkage and significantly prolonged survival.

[0070] Western blot analysis The role of crocin in the induction of apoptosis Apoptosis, or programmed cell death, is one of the most studied topics in cell biology. Apoptosis is a complex process that occurs in both physiological and pathological states. Apoptosis is essential for maintaining homeostasis and regulating cellular development. An imbalance in apoptosis leads to the development of many diseases, including autoimmune diseases and cancer. In cancer, the balance between cell death and cell division is lost. This problem occurs because cancer cells are highly intelligent and adaptive, possessing the ability to skip death signals and continue to proliferate indefinitely. This results in the formation of tumors, which are masses of mutated cells.

[0071] Two pathways are involved in the activation of apoptosis: the extrinsic pathway, which is switched on by different death receptors, and the intrinsic pathway, which is controlled by mitochondria and leads to the release of apoptotic factors. Enzymes involved in both pathways play important roles in the activation and deactivation of apoptosis. The most important family of these enzymes is called "caspases." Caspases primarily function to cleave and activate specific substrates. In this study, we focused on investigating the intrinsic apoptotic pathway.

[0072] The intrinsic pathway is activated by extracellular or intracellular stresses, including hypoxia, DNA damage, and oxidative stress. These stimuli induce changes in the permeability of the outer mitochondrial membrane, leading to the release of cytochrome c. The permeability of the outer mitochondrial membrane is regulated by the BCL-2 protein family, particularly Bcl-2 and Bax. Upon release, cytochrome c activates Apaf-1 and procaspase-9, forming the apoptosome. Accumulation of procaspase-9 activates inducible caspase-9. Under normal circumstances, the proapoptotic protein Bax binds to the antiapoptotic protein Bcl-2 to regulate mitochondrial membrane permeability. Upon release of cytochrome c, executioner caspase-3 and inducible caspase-9 are activated.

[0073] Under normal physiological conditions, PARP functions to detect and repair DNA damage and plays an essential role in regulating apoptosis. When activated, PARP transfers NAD+, allowing PAR to bind to DNA polymerase and repair problems that occur during DNA replication. When PARP is overactivated, the intrinsic apoptosis pathway is switched on. PARP is a pro-apoptotic signal, and after activation, PARP translocates from the nucleus to the cytoplasm and interacts with the outer membrane of mitochondria. During this process, apoptosis-inducing factors are released, causing cells to undergo apoptosis.

[0074] Apoptosis plays a key role in promoting carcinogenesis and rendering cancer cells chemoresistant to conventional treatments. The ability of cancer to skip apoptosis is one of its hallmarks. A study of lung cancer cells examined the effect of saffron on inducing apoptosis in A549 cells and found increased caspase levels in saffron-treated cells. This suggests that saffron treatment activates apoptosis in cancer cells. Another study demonstrated activation of apoptosis in DEN-induced HCC in Wistar rats treated with melatonin. This was indicated by increased levels of caspases and cleaved PARP, and melatonin led to an increase in the Bax / Bcl-2 ratio. A study investigating the effects of glycerol treatment in Wistar rats demonstrated increased apoptosis, which was also indicated by increased caspase-3 activation and an increased Bax / Bcl-2 ratio. A study investigating the effects of crocin, another major bioactive molecule found in saffron, showed that crocin induced apoptosis in HepG2 cells, increasing the ratio of Bax to Bcl-2 and upregulating caspase-3 and caspase-9. Another study, using crocin as a preventative measure against liver cancer lesions in Wistar rats, demonstrated a significant increase in apoptosis in the livers of crocin-treated animals using histological examination with the M30 CytoDeath antibody, confirming crocin's apoptosis-activating effect. A study using crocin-coated D-MNPs showed a significantly greater increase in cell death compared to DNE-injected mice. Crocin has also been shown to induce classical programmed cell death, independent of autophagy, in colorectal cancer cells, including HCT116 wild-type and HCT116 p53- / - cell lines.

[0075] In this study, Western blot analysis (see Figures 7, 8, and 9) demonstrated that treatment with crocin resulted in activation of the intrinsic apoptotic pathway. This was concluded by measuring the expression of key players in the intrinsic pathway, including Bax, Bcl-2, PARP, caspase-3, and caspase-9. Our results also showed that crocin treatment resulted in increased Bax expression and decreased Bcl-2 expression compared with HCC rats. Therefore, the increased Bax / Bcl-2 ratio appears to support this conclusion. Interestingly, adjunctive treatment with crocin and sorafenib appeared to have a superior effect on intrinsic pathway activation compared with treatment with crocin or sorafenib alone. Further investigation of the intrinsic pathway revealed significantly decreased expression of total PARP, procaspase-3, and procaspase-9 in crocin-treated rats compared with HCC animals. A study conducted to evaluate the anti-apoptotic effects of saffron on colorectal cancer cell lines showed that saffron induced p53-dependent caspase-3 activation in HCT116 cells.

[0076] [Definition] As used in this specification and the appended claims, the following terms shall have the following meanings, unless the context otherwise requires.

[0077] The term "treatment" as used herein is understood to refer to the administration of one or more drugs to a patient suffering from cancer.

[0078] As used herein, the term "therapeutically effective amount" refers to an amount of a drug or agent that elicits the biological or medical response of a tissue, system, animal, or human that is being sought, for example, by a researcher or clinician. Furthermore, the term "therapeutically effective amount" refers to any amount that results in improved treatment, cure, prevention, or amelioration of a disease, disorder, or side effects, or a reduction in the rate of progression of a disease or disorder, compared to a corresponding subject not receiving such amount. The term also includes within its scope an amount effective to enhance normal physiological function.

Claims

1. 1. A pharmaceutical composition for treating, inhibiting, or reducing the severity of liver cancer in a subject, comprising: a first amount of sorafenib; and a second amount of crocin or a pharmaceutically acceptable prodrug thereof; the crocin or a pharmaceutically acceptable prodrug thereof and the sorafenib are contained in a mass ratio of between 50:1 and 5:1; The prodrug is selected from the group consisting of crocin salts, hydrates, hemiacetals, acetals, thioacetals, silyl ethers, tautomers, and combinations thereof. A pharmaceutical composition comprising:

2. 2. The pharmaceutical composition of claim 1, wherein the crocin is α-crocin.

3. 10. The pharmaceutical composition of claim 1, wherein the crocin or a prodrug thereof and the sorafenib are formulated together in one composition containing both compounds.

4. 2. The pharmaceutical composition of claim 1, wherein the crocin or a prodrug thereof and the sorafenib are formulated in separate pharmaceutical compositions.

5. The pharmaceutical composition of claim 1, wherein the crocin or a prodrug thereof and the sorafenib are administered sequentially.

6. 2. The pharmaceutical composition of claim 1, wherein the crocin or a prodrug thereof is first administered to sensitize cancer cells prior to exposure to the sorafenib, and then the sorafenib is administered.

7. 2. The pharmaceutical composition of claim 1, wherein the liver cancer is selected from the group consisting of hepatocellular carcinoma (HCC), fibrolamellar HCC, cholangiocarcinoma, angiosarcoma, metastatic liver cancer, and combinations thereof.

8. The pharmaceutical composition according to claim 1, wherein the liver cancer is hepatocellular carcinoma.

9. 2. The pharmaceutical composition of claim 1, wherein the crocin is selected from the group consisting of monoglycosylpolyene esters of crocetin, diglycosylpolyene esters of crocetin, and combinations thereof.

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