CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/659,218, filed April 18, 2018, the content of which is hereby incorporated by reference in its entirety.

BACKGROUND

[0002] In 2012, there were 14 million cancer patients in the world, and the patient number is predicted to be 22 million over the next two decades. Liver cancers are among the most common types of cancers, which cause about estimated 695,900 deaths each year. Jemal et al, CA Cancer J Clin., 61:69-90 (2011). Liver cancers have the seventh highest age- adjusted incidence rate in the world, with about 1 million new patients diagnosed with liver cancers in 2012. Each year, about 695,900 patients die from liver cancer. The common risk factors for liver cancers include alcohol, viral hepatitis (e.g., hepatitis B virus (HBV) and hepatitis C virus (HCV)), and nonalcoholic fatty liver diseases.

[0003] Hepatocellular carcinoma (HCC) is a primary malignancy of liver cancer.

Additional risk factors for developing HCC include aflatoxin-contaminated food, diabetes, obesity, hereditary conditions (e.g., hemochromatosis), and metabolic disorders. HCC accounts for 70%-85% of primary liver cancer cases in some countries. El-Serag HB , N Engl JMed., 365(12): 1118-27 (2011). HCC is often asymptomatic in its early stages; thus, almost 85% of patients diagnosed with HCC are in intermediate or advanced stages, for which only limited treatment options are available. Villanueva A. et al, Gastroenterology, 140(5): 1410— 26 (2011). Poor prognosis of HCC poses further challenges for early detection of this malignancy.

[0004] Currently, Sorafenib remains the only systemic agent approved by FDA to treat hepatocellular carcinoma patients. Multiple trials to identify new treatments for patients with advanced unresectable hepatocellular tumors have failed to bring new therapeutic regimes against HCC to the market. Sun et al., J Gastrointest. Cancer, doi: 10.1007/S12029-018- 0065-8 (2018). For HCC, despite extensive application of targeted therapy, current therapies for advanced HCC fail to provide satisfactory results. In the meantime, HCC carries a significant economic burden on society at large, especially in the East Asian countries where HBV infection is endemic. Therefore, there remains an urgent medical need for novel methods and compositions for treating liver cancers or HCC.

SUMMARY OF THE INVENTION

[0005] The disclosure of the present invention meets this medical need by providing a pharmaceutical composition comprising a tyrosine kinase inhibitor, an antihistamine, and a pharmaceutically acceptable carrier. In one embodiment, the tyrosine kinase inhibitor comprises an epidermal growth factor receptor (EGFR) inhibitor, a vascular endothelial cell growth factor receptor (VEGFR) inhibitor, a platelet-derived growth factor receptor

(PDGFR) inhibitor, a c-KIT inhibitor, an FMS-like tyrosine kinase 3 (FLT-3) inhibitor, and a Breakpoint cluster region Abelson (BCR-ABL) inhibitor. In one embodiment, the tyrosine kinase inhibitor comprises one or more of imatinib, bafetinib, dasatinib, nilotinib, bosutinib, ponatinib, thiazol, thiadiazole, and their salts, derivatives, or analogs. In one embodiment the tyrosine kinase inhibitor comprises imatinib. In one embodiment, the antihistamine comprises one or more of perphenazine, sertralin, thioridazine, chlorpromazine, paroxetine, flupentixol, fluphenazine, hydroxyzine, promethazine, loratadine, cyproheptadine, and their salts, derivatives, or analogs.

[0006] In another aspect, the disclosure relates to methods of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a tyrosine kinase inhibitor and an antihistamine. In another embodiment, the tyrosine kinase inhibitor comprises an epidermal growth factor receptor (EGFR) inhibitor, a vascular endothelial cell growth factor receptor (VEGFR) inhibitor, a platelet-derived growth factor receptor

(PDGFR) inhibitor, a c-KIT inhibitor, an FMS-like tyrosine kinase 3 (FLT-3) inhibitor, and a Breakpoint cluster region Abelson (BCR-ABL) inhibitor. In another embodiment, the tyrosine kinase inhibitor comprises one or more of imatinib, bafetinib, dasatinib, nilotinib, bosutinib, ponatinib, thiazol, thiadiazole, and their salts, derivatives, or analogs. In another embodiment, the tyrosine kinase inhibitor comprises imatinib. In another embodiment, the antihistamine comprises one or more of perphenazine, sertralin, thioridazine, chlorpromazine, paroxetine, flupentixol, fluphenazine, hydroxyzine, promethazine, loratadine,

cyproheptadine, and their salts, derivatives, or analogs.

[0007] In another aspect, the disclosure relates to methods of reducing proliferation of a cancer cell comprising contacting the cancer cell with a therapeutically effective amount of a tyrosine kinase inhibitor and an antihistamine. In another embodiment, the tyrosine kinase inhibitor comprises an epidermal growth factor receptor (EGFR) inhibitor, a vascular endothelial cell growth factor receptor (VEGFR) inhibitor, a platelet-derived growth factor receptor (PDGFR) inhibitor, a c-KIT inhibitor, an FMS-like tyrosine kinase 3 (FLT-3) inhibitor, and a Breakpoint cluster region Abelson (BCR-ABL) inhibitor. In another embodiment, the tyrosine kinase inhibitor comprises one or more of imatinib, bafetinib, dasatinib, nilotinib, bosutinib, ponatinib, thiazol, gefitinib, afatinib, thiadiazole, and their salts, derivatives, or analogs. In another embodiment, the tyrosine kinase inhibitor comprises imatinib. In another embodiment, the antihistamine comprises one or more of perphenazine, sertralin, thioridazine, chlorpromazine, paroxetine, flupentixol, fluphenazine, hydroxyzine, promethazine, loratadine, cyproheptadine, and their salts, derivatives, or analogs.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1A shows the inhibition rates of HepG2 cell growth in response to treatment of imatinib in absence or presence of cyproheptadine. Figure 1B shows the inhibition rates of HepG2 cell growth in response to treatment of cyproheptadine in absence or presence of imatinib.

[0009] Figure 2 shows inhibition rates of Huh- 7 cell growth in response to treatment of imatinib in absence or presence of cyproheptadine.

[0010] Figure 3 shows inhibition rates of normal human L-02 cells in response to treatment of imatinib in absence or presence of cyproheptadine.

[0011] Figure 4 shows the tumor volumes in mice implanted with HepG2 cells.

[0012] Figure 5 shows the body weights of mice.

[0013] Figure 6 shows the survival rates mice. [0014] Figure 7 shows the survival rates of mice after treatments with CMC-Na, combination of cyproheptadine (6 mg/kg/day) and imatinib, and sorafenib.

[0015] Figure 8 shows the body weights of mice after treatments with CMC-Na, combination of cyproheptadine (6 mg/kg/day) and imatinib, and sorafenib.

[0016] Figure 9A shows the surviving rates of A549 cells after treatment of gefitinib alone, or in combination of cyproheptadine or loratadine. Figure 9B shows the surviving rates of A549 cells after treatment of afatinib alone, or in combination of cyproheptadine or loratadine.

DETAILED DESCRIPTION

[0017] After reading this description, it will become apparent to one skilled in the art how to implement the invention in various alternative embodiments and alternative applications. However, not all embodiments of the present invention are described herein. It will be understood that the embodiments presented here are presented by way of an example only, and not limitation. As such, this detailed description of various alternative embodiments should not be construed to limit the scope or breadth of the present invention as set forth below.

[0018] Before the present invention is disclosed and described, it is to be understood that the aspects described below are not limited to specific compositions, methods of preparing such compositions, or uses thereof as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

Definitions

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0020] In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings: [0021] The terminology used herein is for the purpose of describing particular

embodiments only and is not intended to be limiting the invention. As used herein, the singular forms“a,”“an,” and“the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0022] All numerical designations, e.g., pH, temperature, time, concentration, amounts, and molecular weight, including ranges, are approximations which are varied (+) or (-) by 10%, 1%, or 0.1%, as appropriate. It is to be understood, although not always explicitly stated, that all numerical designations may be preceded by the term“about.” It is also to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0023] The term“comprising” or“comprises” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of,” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. For example, a composition consisting essentially of the elements as defined herein would not exclude other elements that do not materially affect the basic and novel characteristic(s) of the claimed invention. “Consisting of’ shall mean excluding more than trace amount of other ingredients and substantial method steps recited. Embodiments defined by each of these transition terms are within the scope of this invention.

[0024] As used here, the term“inhibitor” refers to a substrate that blocks or suppresses the activity, function, or effect of a target. In some embodiments, the target is a compound, a protein, a gene, a cell, or an agent. In some embodiments, the target is a tyrosine kinase. In some embodiments, the inhibitor includes a compound that prevents binding of another molecule to an enzyme or molecular pump. In some embodiments, the inhibitor is a compound that causes downregulation of the enzyme. An inhibitor can be a competing or non-competing inhibitor. As used here, the term“non-competing inhibitor” refers to a type of inhibitor that binds to an enzyme or a target so that the enzyme or the target cannot bind to or act on another substrate. [0025] As used herein, the term“tyrosine kinase inhibitor” is a compound that can compete with ATP for the ATP binding site of a phospho tyrosine kinase and reduce tyrosine kinase phosphorylation. In one embodiment, the tyrosine kinase inhibitor comprises one or more of an epidermal growth factor receptor (EGFR) inhibitor, a vascular endothelial cell growth factor receptor (VEGFR) inhibitor, a platelet-derived growth factor receptor (PDGFR) inhibitor, a c-KIT inhibitor, an FMS-like tyrosine kinase 3 (FLT-3) inhibitor, and a BCR- ABL inhibitor. In one embodiment, the EGFR inhibitor includes one or more of gefitinib, erlotinib, canertinib, and their salts, derivatives, or analogs. In one embodiment, the VEGFR inhibitor includes one or more of vatalanib, semaxanib, sunitinib, and their salts, derivatives, or analogs. In one embodiment, the PDGFR inhibitor includes one or more of pazopanib, nilotinib, cediranib, and their salts, derivatives, or analogs. In one embodiment, the c-KIT inhibitor includes one or more of dasatinib, pazopanib, quzartinib, and their salts, derivatives, or analogs. In one embodiment, the FLT-3 inhibitor includes one or more of CEP701, sunitinib, PKC412, and their salts, derivatives, or analogs. In one embodiment, the BCR- ABL inhibitor includes one or more of imatinib, bafetinib, dasatinib, nilotinib, bosutinib, ponatinib, thiazol, thiadiazole, and their salts, derivatives, or analogs. In some embodiments, the BCR-ABL inhibitor is imatinib. The EGFR inhibitor, in one embodiment, comprises gefitinib and/or afatinib.

[0026] As used herein, the term“antihistamine” is a compound that antagonizes histamine induced responses. Histamine is a biogenic amine that exerts its physiologic action by binding to receptors belonging to the superfamily of seven transmembrane G protein-coupled receptors (GPCRs). Monczor et al., Mol Pharmacol. 90:640-648 (2016). Histamine exerts its multiple biological actions via one of several receptors, including the Hl receptor (H1R), H2 receptor (H2R), H3 receptor (H3R), and more recently, H4 receptors (H4R). The H1R signals through one or more of increased calcium signaling, cyclic guanosine monophosphate (cGMP) mediated signaling, nuclear factor kappaB (NFkappaB), increased phospholipase C (PLC) activity, increased phospholipase A2 and/or D activity, cyclic adenosine

monophosphate activity (cAMP), and/or nitric oxide synthase activity, and thus can activate a variety of cellular responses associated with these pathways, such as cell proliferation, cell differentiation, apoptosis, cytoskeleton remodeling, vesicular trafficking, ion channel conductance, endocrine function, and neurotransmission. See U.S. Patent 9,808,444. In some embodiments, the antihistamine comprises one or more of perphenazine, sertralin, thioridazine, chlorpromazine, paroxetine, flupentixol, fluphenazine, hydroxyzine, promethazine, loratadine, cyproheptadine, and their salts, derivatives, or analogs. In some embodiments, the antihistamine is cyproheptadine.

[0027] The terms“dosage” or“dosage regiment” is defined herein as the amount needed for effectiveness of each of the various disease states. Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single dosage may be administered or several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals. In some embodiments, dosage of a particular compound is provided as absolute weight. In some embodiments, the dosage of a particular compound is provided as mass ratio wherein the mass ratio is the fraction of a particular compound out of the total composition. In some embodiments, the dosage is provided as mg compound per kg total body weight of the subject to whom the composition is provided, and this dosage format is hereinafter designated mg/kg. In some embodiments, the dosage is provided in hourly, daily, weekly, or monthly dosage regimens.

[0028] The terms“patient,”“subject,”“individual,” and the like are used interchangeably herein and refer to any animal, or cells thereof, whether in vitro or in situ, amenable to the methods described herein. In a preferred embodiment, the patient, subject, or individual is a mammal. In some embodiments, the mammal is a mouse, a rat, a guinea pig, a non-human primate, a dog, a cat, or a domesticated animal (e.g. , horse, cow, pig, goat, sheep). In especially preferred embodiments, the patient, subject, or individual is a human. [0029] The term“cancer” is used herein to refer to conditions in which abnormal cells divide without control and can invade nearby tissues. There are several main types of cancer. Carcinoma is a cancer that begins in the skin or in tissues that line or cover internal organs. Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is a cancer that starts in blood-forming tissue, such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the blood. Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system. Central nervous system cancers are cancers that begin in the tissues of the brain and spinal cord. In some embodiments, the cancer is one or more of pancreatic cancer, renal cancer, small cell lung cancer, brain cancer, neural cancer, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer, and melanoma. In some embodiments, the cancer is liver cancer. In some embodiments, the liver cancer is one or more of hepatocellular carcinoma, bile duct cancer, angiosarcoma, hemangiosarcoma, hepatoblastoma, hemangioma, hepatic adenoma, and focal nodular hyperplasia.

[0030] The term“treating” or“treatment” covers the treatment of a cancer described herein in a subject, such as a human, and includes (i) inhibiting a cancer, i.e., arresting its development; (ii) relieving a cancer or disorder, i.e., causing regression of the cancer; (iii) slowing progression of the cancer; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the cancer. For example, treatment of a cancer includes, but is not limited to, elimination of the cancer or the condition caused by the cancer, remission of the tumor, inhibition of the cancer, reduction, or elimination of at least one symptom of the tumor.

[0031] The term“administering” or“administration” of an agent to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function.

A route of administration is the path by which a drug, fluid, poison, or other substance is taken into the body. Routes of administration are generally classified by the location at which the substance is applied. Administration can be carried out by any suitable route, including parenterally, orally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via minicells, via antibody conjugation, via cell targeting peptides, via inhalation, vaginally, intraoccularly, via local delivery by catheter or stent, subcutaneously, intraadiposally, intraarticularly, or intrathecally.

[0032] The phrase“concurrently administering” refers to administration of at least two agents to a patient over a period of time. Concurrent administration includes, without limitation, separate, sequential, and simultaneous administration.

[0033] The term“separate” administration refers to an administration of at least two active ingredients at the same time or substantially the same time by different routes.

[0034] The term“sequential” administration refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients.

[0035] The term“simultaneous” administration refers to the administration of at least two ingredients by the same route and at the same time or at substantially the same time.

[0036] The term“therapeutic” as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

[0037] The term“therapeutically effective amount” or“effective amount” refers to an amount of the agent that, when administered, is sufficient to cause the desired effect. For example, an effective amount of a composition may be an amount sufficient to treat, control, alleviate, or improve the conditions related to parasitic diseases. The therapeutically effective amount of the agent may vary depending on the pathogen being treated and its severity as well as the age, weight, etc., of the patient to be treated. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compounds may be administered to a subject having one or more signs or symptoms of a disease or disorder. [0038] The term“analog” refers to a compound in which one or more individual atoms or functional groups have been replaced, either with a different atom or a different functional, generally giving rise to a compound with similar properties. In some aspect, the analog refers to a structure that is similar to another but differs in one or two components.

[0039] The term“derivative” refers to a compound that is formed from a similar, beginning compound by attaching another molecule or atom to the beginning compound. Further, derivatives, according to the invention, encompass one or more compounds formed from a precursor compound through addition of one or more atoms or molecules or through combining two or more precursor compounds.

[0040] The term“pharmaceutically acceptable carrier” refers to a carrier that is

conventionally used in the art to facilitate the storage, administration, and/or the healing effect of a biologically active agent. Pharmaceutical carriers can also provide timed delayed release of the drug and targeted release of the drug to specific tissues. Many types of delivery systems for targeted release of drugs are available and known to those of ordinary skill in the art, including controlled-release biodegradable polymers, polymeric microsphere carriers and liposomes, as well as the co-administration of cytoprotective agents with antineoplastics as described in Chonn and Cullis, Curr Opinion in Biotechnology, 6:698-708 (1995); Kemp et al., J. Clin. Oncol, 14:2101-2112 (1996); Kumanohoso et al., Cancer Chemother.

Pharmacol, 40: 112-116 (1997); Schiller et al., J. Clin. Oncol, 14: 1913-1921 (1996); and Sipos et al., Cancer Chemother. Pharmacol, 39:383-389 (1997). The liposomes can be further coated with polyethene glycol (PEG) to prolong their circulation time. Furthermore, targeted delivery of the drugs can be achieved by using minicells as described in WO 2005/079854. The minicells comprise a first arm that carries specificity for a bacterially derived minicell surface structure and a second arm that carries specificity for a mammalian cell surface receptor, to target drug-loaded minicells to specific mammalian cells and to cause endocytosis of the minicells by the mammalian cells. Accordingly, in some embodiments, the pharmaceutical composition is administered in a targeted release system.

[0041] The term“cell targeting peptide” refers to a peptide that is conventionally used in the art to recognize and bind specific cells and tissues as described in Mousavizadeh et al, Colloids and Surfaces B: Biointerfaces 158:507-517 (2017). A person having ordinary skill in the art will know how to conjugate a small drug to a cell targeting peptide to achieve targeted delivery of the drug to specific cells and tissues.

[0042] The term“antibody” is used herein to refer to immunoglobulins conventionally used in the art to recognize and bind specific antigens and can be conjugated with small molecules for targeted delivery to specific cells and tissues as described in Tsuchikama et al, Protein Cell 9:33-46 (2018). A person having ordinary skill in the art will know how to conjugate small molecule drugs to antibodies.

Tyrosine kinase inhibitors

[0043] Protein tyrosine kinases (PTKs) are major regulators of cellular signal transduction. Jiao et aI., Moί Cancer, 17:36 (2018). PTKs catalyze transfer of ATR-g-phosphate to the tyrosine residues of the substrate protein, the phosphorylation of which initiates signaling pathways to regulate cell growth, differentiation, and cell death. PTKs are divided into receptor PTKs (RTKs) and non-receptor PTKs (NRTKs). The human genome includes at least 58 genes expressing RTKs and 32 genes expressing NRTKs.

[0044] Abnormal PTK activity usually leads to cell proliferation disorders, and is closely related to tumor invasion, metastasis and tumor angiogenesis. Overexpression of PTK genes cause abnormal PTK activity and activation of the signaling pathways that lead to cell proliferation and even tumor formation. Drake et al., Mol Cell Biol., 34: 1722-1732 (2014). Furthermore, genetic mutations in PTK genes often lead to constitutively activation of PTKs and unregulated cell growth. Prickett et al. , Nat Genet. , 41: 1127-1132 (2009).

[0045] Because PTKs-regulated signaling pathways are involved in cellular proliferation, differentiation, and cell death, the tyrosine kinase inhibitors (“TKIs”) can be used as anti tumor agents. In some embodiments, the TKIs inhibit the PTK-induced proliferation, differentiation, angiogenesis, metastasis, and/or cell death. TKIs can be used to inhibit abnormal proliferation of cancer cells, reverse the cancer cell’s abnormal differentiation, and/or induce apoptosis in the cancer cell. In some embodiments, tyrosine kinase inhibitors (TKIs) compete with ATP for the ATP binding site of PTK and reduce tyrosine kinase phosphorylation. Exemplary TKIs include but are not limited to EGFR inhibitors, VEGFR inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, and Breakpoint cluster region Abelson (BCR-ABL) inhibitors. More than 20 TKIs have been approved by FDA for treatment of pancreatic cancer, renal cancer, small cell lung cancer, brain cancer, neural cancer, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer, and melanoma. Arora et al., J

Pharmacol Exp Ther, 315:971-9 (2005). Initially approved by FDA in 2001, Imatinib is primarily used for treating chronic myeloid leukemia (CML) caused by constitutively active BCR-ABL tyrosine kinase due to chromosomal translocation.

[0046] Some features of TKIs, including high selectivity, high efficacy, low side effects, and ease of preparation, render them appealing as anti-cancer drugs. However, TKIs have not tested effectively against diseases other than CML. For example, TKI fails to effectively treat solid tumors. The incomplete elimination of tumor cell targets results in the development of drug-resistant cancer cells. Klecsko et al, Mol. Cancer, 17:60 (2018). Indeed, the median effective time for TKI therapy has been shown to be only 5 to 9 months. Furthermore, imatinib was less effective in treating unresectable human hepatocellular carcinoma. Lin et al., Am J Clin Oncol., 3l(l):84— 8 (2008).

[0047] Applicant discovered that the anti-cancer (e.g., anti-HCC) effects of TKIs are significantly augmented by administration of antihistamines. Accordingly, the present disclosure provides pharmaceutical compositions comprising a tyrosine kinase inhibitor, an antihistamine, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions comprise imatinib, cyproheptadine, and a pharmaceutically acceptable carrier.

[0048] In one embodiment, the TKI comprises one or more of an epidermal growth factor receptor (EGFR) inhibitor, a vascular endothelial cell growth factor receptor (VEGFR) inhibitor, a platelet-derived growth factor receptor (PDGFR) inhibitor, a c-KIT inhibitor, an FMS-like tyrosine kinase 3 (FLT-3) inhibitor, and a BCR-ABL inhibitor. In one embodiment, the EGFR inhibitor includes one or more of afatinib, gefitinib, erlotinib, canertinib, and their salts, derivatives, or analogs. In one embodiment, the VEGFR inhibitor includes one or more of vatalanib, semaxanib, sunitinib, and their salts, derivatives, or analogs. In one embodiment, the PDGFR inhibitor includes one or more of pazopanib, nilotinib, cediranib, and their salts, derivatives, or analogs. In one embodiment, the c-KIT inhibitor includes one or more of dasatinib, pazopanib, quzartinib, and their salts, derivatives, or analogs. In one embodiment, the FLT-3 inhibitor includes one or more of CEP701, sunitinib, PKC412, and their salts, derivatives, or analogs. In one embodiment, the BCR- ABL inhibitor comprises one or more of imatinib, bafetinib, dasatinib, nilotinib, bosutinib, ponatinib, thiazol, thiadiazole, and their salts, derivatives, or analogs. In some embodiments, the BCR-ABL inhibitor is imatinib.

[0049] All forms of the TKIs are contemplated herein, which includes their hydrates and co-crystals of such compounds and such salts. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid addition salts of basic residues such as amines; alkali or organic addition salts of acidic residues; and the like, and combinations comprising one or more of the foregoing salts. The pharmaceutically acceptable salts include non-toxic salts and the quaternary ammonium salts of the TKIs. For example, non-toxic acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; other acceptable inorganic salts include metal salts such as sodium salt, potassium salt, cesium salt, and the like; and alkaline earth metal salts, such as calcium salt, magnesium salt, and the like, and combinations comprising one or more of the foregoing salts. Pharmaceutically acceptable organic salts includes salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, HOOC-(CH2)n-COOH where n is 0-4, and the like; organic amine salts such as triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N,N’-dibenzylethylenediamine salt, and the like; and amino acid salts such as arginate, asparaginate, glutamate, and the like; and combinations comprising one or more of the foregoing salts; organic amine salts such as triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N,N’ dibenzylethylenediamine salt, and the like; and amino acid salts such as arginate, asparaginate, glutamate, and the like; and combinations comprising one or more of the foregoing salts.

[0050] The tyrosine kinase inhibitors can also be conjugated to peptides and/or antibodies that recognize specific cell markers for targeted delivery to specific cells and tissue.

Mousavizadeh et al, Biointerfaces, 158:507-517 (2017); Tsuchikama el al, Protein Cell, 9:33-46 (2018). A person having ordinary skill in the art will know how to conjugate small molecule drugs to cell targeting peptides and antibodies. Hence, in some embodiments, the tyrosine kinase inhibitor is conjugated to a cell targeting peptide or an antibody.

Antihistamines

[0051] Histamine is a biogenic amine that exerts its physiologic action by binding to receptors belonging to the superfamily of seven transmembrane G protein-coupled receptors (GPCRs). Monczor et al., Mol Pharmacol. 90:640-648 (2016). Histamine exerts its multiple biological actions via one of several receptors including the Hi receptor (HiR), ¾ receptor (H2R), H3 receptor (H3R), and ¾ receptor (H4R). The HiR signaling pathways are involved in calcium signaling, cyclic guanosine monophosphate (cGMP) mediated signaling, nuclear factor kappaB (NFkappaB) signaling, phospholipase C (PLC) activity, phospholipase A2 and/or D activities, cyclic adenosine monophosphate activity (cAMP) and/or nitric oxide synthase activities as described in U.S. Patent 9,808,444. Thus, histamines can regulate a variety of cellular responses associated with these pathways or activities, e.g., cell proliferation, cell differentiation, apoptosis, cytoskeleton remodeling, vesicular trafficking, ion channel conductance, endocrine function, and neurotransmission.

[0052] An antihistamine is used to antagonize one or more of these histamine-associated pathways. In some embodiments, the antihistamine comprises one or more of perphenazine, sertralin, thioridazine, chlorpromazine, paroxetine, flupentixol, fluphenazine, hydroxyzine, promethazine, loratadine, cyproheptadine, and their salts, derivatives, or analogs. In some embodiments, the antihistamine is cyproheptadine. In some embodiments, the antihistamine is loratadine. [0053] Cyproheptadine was tested for its potential anti-cancer functions. Cyproheptadine can inhibit expression of D cyclins and induce apoptosis. Mao et al, Blood, 112:760-769 (2008). Furthermore, cyproheptadine can function as a histone deacetylase inhibitor to exert antineoplastic activity. Paoluzzi et al., Br J Haematol., 146:656-659 (2009). Furthermore, cyproheptadine has been reported to activate p38 mitogen activated protein kinase (MAPK) and checkpoint kinase 2 (CHK2), resulting in cell cycle arrest. Feng et al., BMC Cancer, 15: 134 (2015).

[0054] Cyproheptadine was found to inhibit breast cancer cell growth. Takemoto et al. , J. Med. Chem., 59:3650-60 (2016). In addition, cyproheptadine may improve survival outcomes of sorafenib-treated advanced HCC patients. Feng et al, Jpn JClin Oncol., 45:336-342 (2015). Although these studies are promising, sorafenib remains currently the only systemic agent approved by FDA to treat HCC patients.

[0055] Inventors of the present disclosure surprisingly found that cyproheptadine can synergistically improve the anti-cancer properties of tyrosine kinase inhibitors. Thus, the present disclosure provides novel pharmaceutical compositions or methods for treatment of liver cancers ( e.g . , HCC). The present disclosure provides a pharmaceutical composition which comprises a tyrosine kinase inhibitor, an antihistamine, and a pharmaceutically acceptable carrier. In some embodiments, the antihistamine comprises one or more of perphenazine, sertralin, thioridazine, chlorpromazine, paroxetine, flupentixol, fluphenazine, hydroxyzine, promethazine, cyproheptadine, and their salts, derivatives, or analogs. In some embodiments, the antihistamine is cyproheptadine, and its salts, derivatives, or analogs.

[0056] All forms of the antihistamines are contemplated herein, which include their hydrates, and co-crystals of such compounds and such salts. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid addition salts of basic residues such as amines; alkali or organic addition salts of acidic residues; and the like, and combinations comprising one or more of the foregoing salts. The pharmaceutically acceptable salts include non-toxic salts and the quaternary ammonium salts of the antihistamines. For example, non-toxic acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; other acceptable inorganic salts include metal salts such as sodium salt, potassium salt, cesium salt, and the like; and alkaline earth metal salts, such as calcium salt, magnesium salt, and the like, and combinations comprising one or more of the foregoing salts. Pharmaceutically acceptable organic salts includes salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic,

hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, HOOC-(CH2)n-COOH where n is 0-4, and the like; organic amine salts such as triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N,N’-dibenzylethylenediamine salt, and the like; and amino acid salts such as arginate, asparaginate, glutamate, and the like; and combinations comprising one or more of the foregoing salts; organic amine salts such as triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N,N’ dibenzylethylenediamine salt, and the like; and amino acid salts such as arginate, asparaginate, glutamate, and the like; and combinations comprising one or more of the foregoing salts.

[0057] The antihistamines can be conjugated to peptides and/or antibodies that recognize specific cell markers for targeted delivery to cells and tissues. A person having ordinary skill in the art will know how to conjugate small molecule drugs to cell targeting peptides and antibodies. Hence, in some embodiments, the antihistamine is conjugated to a cell targeting peptide, or an antibody.

Pharmaceutical compositions

[0058] The disclosure provides that a combination of tyrosine kinase inhibitors and antihistamines can act synergistically to inhibit cancer cells. Applicant discovered that administration of a combination of the anti -histamine ( e.g ., cyproheptadine and loratadine) and tyrosine kinase inhibitor (e.g., imatinib) inhibited the cancer cells (e.g., HepG2 and Huh- 7) synergistically compared to treating the cells with either compound alone. This synergistic effect was specifically targeting cancer cells as the combination did not inhibit normal human cells more effectively than imatinib. Consistent with this observation, the combination treatment can also increase the survival rate of the mice suffering from liver cancer as compared to a single-agent treatment. Thus, this disclosure provides compositions that enhance the clinical utility of tyrosine kinase inhibitors, including imatinib, and

antihistamines such as cyproheptadine or loratadine.

[0059] In one aspect, the disclosure provides a pharmaceutical composition, wherein the pharmaceutical composition comprises, a tyrosine kinase inhibitor, an antihistamine, and a pharmaceutically acceptable carrier.

[0060] The dosages of the tyrosine kinase inhibitors can vary among patient. In some embodiments, the dosage to achieve the therapeutic effects of tyrosine kinase inhibitor in the pharmaceutical composition is from about 0.1 mg/kg/day to about 100 mg/kg/day, from about 0.5 mg/kg/day to about 75 mg/kg/day, from about 50 mg/kg/day to about 90 mg/kg/day, from about 1 mg/kg/day to about 50 mg/kg/day, from about 2 mg/kg/day to about 20 mg/kg/day, from about 2 mg/kg/day to about 15 mg/kg/day, or from about 4 mg/kg/day to about 10 mg/kg/day. In some embodiments, the dosage of imatinib is from about 0.1 mg/kg/day to about 100 mg/kg/day, from about 0.5 mg/kg/day to about 75 mg/kg/day, from about 1 mg/kg/day to about 50 mg/kg/day, from about 2 mg/kg/day to about 20 mg/kg/day, from about 2 mg/kg/day to about 15 mg/kg/day, or from about 4 mg/kg/day to about 10 mg/kg/day. In some embodiments, the dosage of imatinib is at least 0.1 mg/kg/day, at least 0.2 mg/kg/day, at least 0.3 mg/kg/day, at least 0.4 mg/kg/day, at least 0.5 mg/kg/day, at least, at least 1 mg/kg/day, at least 2 mg/kg/day, at least 3 mg/kg/day, at least 4 mg/kg/day, at least 5 mg/kg/day, at least 6 mg/kg/day, at least 7 mg/kg/day, at least 8 mg/kg/day, at least 9 mg/kg/day, or at least 10 mg/kg/day.

[0061] The dosages of the antihistamines can vary among patients. In some embodiments, the dosage to achieve the therapeutic effects of antihistamines in the pharmaceutical composition is from about from about 0.01 mg/kg/day to about 10 mg/kg/day, from about 0.05 mg/kg/day to about 7.5 mg/kg/day, from about 1 mg/kg/day to about 7 mg/kg/day, from about 0.1 mg/kg/day to about 5.0 mg/kg/day, from about 0.1 mg/kg/day to about 2 mg/kg/day, from about 0.02 mg/kg/day to about 1.5 mg/kg/day, or from about 0.1 mg/kg/day to about 1.0 mg/kg/day. In some embodiments, the dosage of cyproheptadine is from about 0.01 mg/kg/day to about 10 mg/kg/day, from about 0.05 mg/kg/day to about 7.5 mg/kg/day, from about 1 mg/kg/day to about 7 mg/kg/day, from about 0.1 mg/kg/day to about 5.0 mg/kg/day, from about 0.1 mg/kg/day to about 2 mg/kg/day, from about 0.02 mg/kg/day to about 1.5 mg/kg/day, or from about 0.1 mg/kg/day to about 1.0 mg/kg/day. In some embodiments, the dosage of cyproheptadine is at least 0.1 mg/kg/day, at least 0.2 mg/kg/day, at least 0.3 mg/kg/day, at least 0.4 mg/kg/day, at least 0.5 mg/kg/day, at least 0.6 mg/kg/day, at least 0.7 mg/kg/day, at least 0.8 mg/kg/day, at least 0.9 mg/kg/day, or at least 1 mg/kg/day.

[0062] In some embodiments, the dosage ratio of the tyrosine kinase inhibitor to the antihistamine is at least 2: 1, 5: 1, 10: 1, 20: 1, or 30: 1. In one embodiment, the dosage ratio of imatinib to cyproheptadine is at least 2: 1, 5: 1, 10: 1, 20: 1, or 30: 1.

[0063] In some embodiments, the pharmaceutical composition further comprises one or more of sorafenib, sunitinib, brivanib, bevacizumab, ramucirumab, vatalanib, linifanib, TSU- 68, cediranib, erlotinib, nintedanib, regorafenib, cetuximab, lapatinib, cixutumumab, everolimus, sirolimus, and tivantinib.

[0064] A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, parenterally, orally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via minicells, via antibody conjugation, via cell targeting peptides, via inhalation, vaginally, intraoccularly, via local delivery by catheter or stent, subcutaneously, intraadiposally, intraarticularly, or intrathecally. In a specific embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for parenterally, orally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via minicells, via antibody conjugation, via cell targeting peptides, via inhalation, vaginally, intraoccularly, via local delivery by catheter or stent, subcutaneously, intraadiposally, intraarticularly, or intrathecally administration to human beings. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocamne to ease pain at the site of the injection. In some embodiments, the composition may be formulated as a sterile aqueous solution suitable for injection intravenously, subcutaneously, intraperitoneally, or intramuscularly.

[0065] In one embodiment, compositions can be formulated orally in the form of tablets, capsules, cachets, gelcaps, solutions, or suspensions. The tablets may be coated by methods well-known in the art. Liquid preparations for oral administration may take the form of, but not limited to, solutions, syrups, or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.

[0066] In some embodiments, the composition further comprises one or more of binding agents, flavor agents, lubricating agents, flow agents, disintegration agents, delay agents, and organic solvents. In some embodiments, the binding agents comprise starch, modified starch, cellulose, modified cellulose, brewer’s yeast, sucrose, dextrose, whey, and dicalcium phosphate. In some embodiments, the lubricating agents comprise magnesium stearate, stearic acid, starch, modified starch, and modified cellulose. In some embodiments, the flow agents comprise silica dioxide, modified silica, fumed silica, and talc. In some embodiments, the disintegration agents comprise croscarmellose sodium, sodium starch glycolate, starch, and modified starch. In some embodiments, the delay agents comprise one or more of stearic acid, stearic acid salts, magnesium stearate, polyethylene glycols, starch, modified starch, and methacrylate polymers. In some embodiments, the organic solvents comprise propylene glycol, polyethylene glycols, ethanol, dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone, glycofurol, Solketal, glycerol formal, acetone, tetrahydrofurfuryl alcohol, diglyme, dimethyl isosorbide, and ethyl lactate. In some embodiments, the concentration of the organic solvent is 0.1% to about 35% of the total volume of the composition. In some embodiments, the concentration of the organic solvent is 2% of the total volume of the composition.

[0067] In some embodiments, the tyrosine kinase inhibitor and the antihistamine of the pharmaceutical composition are conjugated to synthetic peptides and/or antibodies that recognize specific cell markers. See, e.g.. Mousavizadeh el al, Colloids and Surfaces B: Biointerfaces 158:507-517 (2017), and Tsuchikama et al., Protein Cell 9:33-46 (2018). A person having ordinary skill in the art will know how to conjugate small molecule drugs to cell targeting peptides and antibodies. Hence, in some embodiments, the tyrosine kinase inhibitor is conjugated to a cell targeting peptide, or an antibody. In some embodiments, the antihistamine is conjugated to a cell targeting peptide, or an antibody.

Methods of treatment

[0068] In one aspect, the disclosure provides methods of treating a cancer in a subject, comprising, alternatively consisting essentially of, or yet consisting of administering to the subject an effective amount of a tyrosine kinase inhibitor and an effective amount of an antihistamine. In some embodiments, the subject is a human. In some embodiments, the subject is a mammal. In some embodiments, the subject is a non-mammal. In some embodiments, the mammal comprises a mouse, a rat, a guinea pig, a non-human primate, a dog, a cat, a horse, a cow, a pig, a goat, and/or a sheep. In some embodiments, the cancer is one or more of pancreatic cancer, renal cancer, small cell lung cancer, brain cancer, neural cancer, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer, and melanoma. In some embodiments, the cancer is liver cancer. In some embodiments, the liver cancer is one or more of hepatocellular carcinoma, bile duct cancer, angiosarcoma, hemangiosarcoma,

hepatoblastoma, hemangioma, hepatic adenoma, and focal nodular hyperplasia.

[0069] The described TKI (including its derivatives and analogs) can be formulated as pharmaceutical compositions for any cancer described herein. In one embodiment, the TKI comprises one or more of an epidermal growth factor receptor (EGFR) inhibitor, a vascular endothelial cell growth factor receptor (VEGFR) inhibitor, a platelet-derived growth factor receptor (PDGFR) inhibitor, a c-KIT inhibitor, an FMS-like tyrosine kinase 3 (FLT-3) inhibitor, and a BCR-ABL inhibitor. In one embodiment, the BCR-ABL inhibitor comprises one or more of imatinib, bafetinib, dasatinib, nilotinib, bosutinib, ponatinib, thiazol, thiadiazole, and their salts, derivatives, or analogs.

[0070] In some embodiments, the effective amount of TKI is from about 0.1 mg/kg/day to about 100 mg/kg/day, from about 0.5 mg/kg/day to about 75 mg/kg/day, from about 1 mg/kg/day to about 50 mg/kg/day, from about 2 mg/kg/day to about 20 mg/kg/day, from about 2 mg/kg/day to about 15 mg/kg/day, from about 3 mg/kg/day to about 12 mg/kg/day, or from about 4 mg/kg/day to about 10 mg/kg/day. In some embodiments, the dosage of imatinib is at least 0.1 mg/kg/day, at least 0.2 mg/kg/day, at least 0.3 mg/kg/day, at least 0.4 mg/kg/day, at least 0.5 mg/kg/day, at least 1 mg/kg/day, at least 2 mg/kg/day, at least 3 mg/kg/day, at least 4 mg/kg/day, at least 5 mg/kg/day, at least 6 mg/kg/day, at least 7 mg/kg/day, at least 8 mg/kg/day, at least 9 mg/kg/day, or at least 10 mg/kg/day.

[0071] The described antihistamine (including its derivatives and analogs) can be formulated as pharmaceutical compositions for any cancer described herein. In some embodiments, the antihistamine comprises one or more of perphenazine, sertralin, thioridazine, chlorpromazine, paroxetine, flupentixol, fluphenazine, hydroxyzine, promethazine, cyproheptadine, and their salts, derivatives, or analogs. In some embodiments, the antihistamine is cyproheptadine. In some embodiments, the effective amount of antihistamine or cyproheptadine is from about 0.01 mg/kg/day to about 10 mg/kg/day, from about 0.05 mg/kg/day to about 7.5 mg/kg/day, from about 0.1 mg/kg/day to about 5.0 mg/kg/day, from about 0.1 mg/kg/day to about 2 mg/kg/day, from about 0.02 mg/kg/day to about 1.5 mg/kg/day, or from about 0.1 mg/kg/day to about 1.0 mg/kg/day. In some embodiments, the dosage of cyproheptadine is at least 0.1 mg/kg/day, at least 0.2 mg/kg/day, at least 0.3 mg/kg/day, at least 0.4 mg/kg/day, at least 0.5 mg/kg/day, at least 0.6 mg/kg/day, at least 0.7 mg/kg/day, at least 0.8 mg/kg/day, at least 0.9 mg/kg/day, or at least 1 mg/kg/day.

[0072] In one embodiment, the tyrosine kinase inhibitors and/or antihistamines are administered parenterally, orally, intraperitoneally, intravenously, intraarterially,

transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via minicells, via antibody conjugation, via cell targeting peptides, via inhalation, vaginally, intraoccularly, via local delivery by catheter or stent, subcutaneously, intraadiposally, intraarticularly, or intrathecally.

[0073] In some embodiments, the method further comprises administering to the subject one or more of sorafenib, sunitinib, brivanib, bevacizumab, ramucirumab, vatalanib, linifanib, TSU-68, cediranib, erlotinib, nintedanib, regorafenib, cetuximab, lapatinib, cixutumumab, everolimus, sirolimus, and tivantinib.

[0074] In one embodiment, the tyrosine kinase inhibitor and the antihistamine are administered separately, simultaneously, or sequentially. Methods of reducing proliferation of a cancer cell

[0075] Also provided in the disclosure is a method of inhibiting cancer cell growth by contacting the cancer cell with a TKI and an antihistamine. Cancer cells often become resistant to the anti-proliferative effect of TKIs because the cancer cells may harbor mutations that allow them to escape the killing function of the TKI. Applicant found that cyproheptadine can sensitize heptacellular carcinoma cells to the antiproliferative effect of imatinib on the heptacellular carcinoma cells HepG2 and Huh-7. Thus, the combination treatment can be used to inhibit proliferation of cancer cells, especially those types that are resistant to conventional treatments.

[0076] The cancer cell targeted by the claimed method, in some embodiments, derives from liver cancer. The liver cancer comprises one or more of hepatocellular carcinoma, bile duct cancer, angiosarcoma, hemangiosarcoma, hepatoblastoma, hemangioma, hepatic adenoma, and focal nodular hyperplasia. In some embodiments, the liver cancer is hepatocellular carcinoma. In some embodiments, the tyrosine kinase inhibitor is a BCR-ABL inhibitor. In some embodiments, the BCR-ABL inhibitor is imatinib. In some embodiments, the antihistamine is cyproheptadine. In some embodiments, the cancer cell is a mammal cancer cell. In some embodiments, the mammal is human. In some embodiments, the mammal comprises a mouse, a rat, a guinea pig, a non-human primate, a dog, a cat, a horse, a cow, a pig, a goat, and/or a sheep.

[0077] In another aspect, the method of reducing proliferation of a cancer cell further comprises contacting the cancer cell with an effective amount of sorafenib, sunitinib, brivanib, bevacizumab, ramucirumab, vatalanib, linifanib, TSU-68, cediranib, erlotinib, nintedanib, regorafenib, cetuximab, lapatinib, cixutumumab, everolimus, sirolimus, tivantinib, or a combination thereof.

[0078] In some embodiments, the method of reducing proliferation of a cancer cell comprises contacting the cancer cell with the TKI and the antihistamine separately, simultaneously, or sequentially. In one embodiment, the cancer cell is contacted with imatinib and cyproheptadine separately, simultaneously, or sequentially. Dose and Administration

[0079] The compounds disclosed herein can be administered in combination or alternation with a second biologically active agent to increase its effectiveness against the target disorder. In combination therapy, effective dosages of two or more agents are administered together, whereas during alternation therapy an effective dosage of each agent is administered serially. The dosages will depend on absorption, inactivation, and elimination rates of the drug as well as other factors known to those with ordinary skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens and schedules should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. A person with ordinary skill in the art would understand that a proper dosage for administration and know how to convert a proper dosage from one specie to another. For example, in view of a dosage for a mouse, the person with ordinary skill in the art would understand how to convert the dosage to an equivalent dosage for human.

[0080] The pharmaceutical compositions, as described herein, are administered in effective amounts for treating the herein disclosed diseases. The effective amount will depend upon the mode of administration, the particular condition being treated, and the desired outcome.

It will also depend upon, as discussed above, the stage of the condition, the age and physical condition of the subject, the nature of concurrent therapy, if any, and like factors well known to the medical practitioner. For therapeutic applications, it is that amount sufficient to achieve a medically desirable result. A person of ordinary skill in the art will appreciate that dosages determined by animal experiments can be converted an equivalent dosage for a different animal species or human. See, e.g., Nair et al., J. Basic Clin. Pharm. 7:27-31 (2016). For example, a dosage for an animal species can be converted to an equivalent dosage for human based on the conversion table in Nair et al., J. Basic Clin. Pharm. 7:27-31 (2016).

[0081] Generally, the dose of the TKI is from about 0.1 mg/kg/day to about 100 mg/kg/day, from about 0.5 mg/kg/day to about 75 mg/kg/day, from about 1 mg/kg/day to about 50 mg/kg/day, from about 2 mg/kg/day to about 20 mg/kg/day, from about 2 mg/kg/day to about 15 mg/kg/day, from about 3 mg/kg/day to about 12 mg/kg/day, or from about 4 mg/kg/day to about 10 mg/kg/day. In some embodiments, the dosage of the tyrosine kinase is at least 0.1 mg/kg/day, at least 0.2 mg/kg/day, at least 0.3 mg/kg/day, at least 0.4 mg/kg/day, at least 0.5 mg/kg/day, at least, at least 1 mg/kg/day, at least 2 mg/kg/day, at least 3 mg/kg/day, at least 4 mg/kg/day, at least 5 mg/kg/day, at least 6 mg/kg/day, at least 7 mg/kg/day, at least 8 mg/kg/day, at least 9 mg/kg/day, or at least 10 mg/kg/day.

[0082] Generally, the dose of the antihistamine is from about 0.01 mg/kg/day to about 10 mg/kg/day, from about 0.05 mg/kg/day to about 7.5 mg/kg/day, from about 0.1 mg/kg/day to about 5.0 mg/kg/day, from about 0.1 mg/kg/day to about 2 mg/kg/day, from about 0.02 mg/kg/day to about 1.5 mg/kg/day, or from about 0.1 mg/kg/day to about 1.0 mg/kg/day. In some embodiments, the dosage of the antihistamine is at least 0.1 mg/kg/day, at least 0.2 mg/kg/day, at least 0.3 mg/kg/day, at least 0.4 mg/kg/day, at least 0.5 mg/kg/day, at least 0.6 mg/kg/day, at least 0.7 mg/kg/day, at least 0.8 mg/kg/day, at least 0.9 mg/kg/day, or at least 1 mg/kg/day.

[0083] In one embodiment, an effective amount of a pharmaceutical composition, TKI, and/or antihistamine is administered every 1 hour to every 24 hours, for example, every 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours. In some embodiments, the pharmaceutical composition, TKI, and/or antihistamine is administered every 1 day to every 21 days, for example, every 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days. In one embodiment, an amount of the pharmaceutical composition is administered every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks.

[0084] A variety of administration routes are available. The pharmaceutical composition of the invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active ingredients without causing clinically unacceptable adverse effects. Accordingly, the pharmaceutical compositions can be administered to a subject parenterally, orally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via minicells, via antibody conjugation, via cell targeting peptides, via inhalation, vaginally, intraoccularly, via local delivery by catheter or stent, subcutaneously, intraadiposally, intraarticularly, or intrathecally.

[0085] Modes of administration include oral, rectal, topical, nasal, intradermal, or parenteral routes. The term“parenteral” includes subcutaneous, intravenous, intramuscular, or infusion. Intravenous or intramuscular routes are not particularly suitable for long-term therapy and prophylaxis. Oral administration is used in prophylactic treatment because of the convenience to the patient as well as the dosing schedule.

[0086] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed 25 oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, inert gases, and the like. Lower doses will result from other forms of administration, such as intravenous administration. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.

[0087] Delivery systems that targets specific tissues is used for effective treatment of solid cancers such as liver cancer to reduce toxic side effects on unintended tissues. Many types of delivery systems for targeted release of drugs are available and known to those of ordinary skill in the art, including controlled-release biodegradable polymers, polymeric microsphere carriers and liposomes, as well as the co-administration of cytoprotective agents with antineoplastics as described in Chonn and Cullis, Curr. Opinion in Biotechnology, 6:698-708 (1995); Kemp et al., J. Clin. Oncol., 14:2101-2112 (1996); Kumanohoso et al., Cancer Chemother. Pharmacol, 40: 112-116 (1997); Schiller et al., J. Clin. Oncol, 14: 1913-1921 (1996); and Sipos et al., Cancer Chemother. Pharmacol, 39:383-389 (1997). The liposomes can be further coated with polyethene glycol (PEG) to prolong their circulation time.

Furthermore, targeted delivery of the drugs can be achieved by using minicells as described in WO 2005/079854. The minicells comprises a first arm that carries specificity for a bacterially derived minicell surface structure and a second arm that carries specificity for a mammalian cell surface receptor, to target drug-loaded minicells to specific mammalian cells and to cause endocytosis of the minicells by the mammalian cells. Accordingly, in some embodiments, the pharmaceutical composition is administered in a targeted release system.

[0088] Other systems for targeted delivery of drugs to specific cells or tissue involve conjugating the drugs to synthetic peptides and/or antibodies that recognize specific cell markers. A person having ordinary skill in the art will know how to conjugate small molecule drugs to cell targeting peptides and antibodies. Hence, in some embodiments, the TKI is conjugated to a cell targeting peptide or an antibody. In some embodiments, the antihistamine is conjugated to a cell targeting peptide or an antibody.

[0089] Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, or lozenges, each containing a predetermined amount of the active agent(s). Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir, or emulsion.

[0090] Other delivery systems can include time-release, delayed-release, or sustained- release delivery systems. Such systems can avoid repeated administrations of the pharmaceutical composition of this invention, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer-based systems such as poly (lactide- glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109. Delivery systems also include non-polymer systems that are lipids, including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-, di-, and tri-glycerides;

hydrogel release systems; sylastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.

[0091] In one embodiment, the pharmaceutical composition is administered in a time- release, delayed-release, or sustained-release delivery system. In one embodiment, the time- release, delayed-release, or sustained-release delivery system comprising the pharmaceutical composition of the invention is inserted directly into the tumor.

[0092] When administered, the pharmaceutical preparations of the invention are applied in pharmaceutically acceptable amounts and in pharmaceutically acceptable compositions.

Such preparations may routinely contain salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium, or calcium salts.

Kit of Parts

[0093] In one aspect, this invention relates to a kit of parts for treatment of a cancer in a subject, the kit comprising a TKI and an antihistamine. In one embodiment, the cancer is one or more of pancreatic cancer, renal cancer, small cell lung cancer, brain cancer, neural cancer, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer, and melanoma. In some embodiments, the cancer is liver cancer. In some embodiments, the liver cancer is one or more of hepatocellular carcinoma, bile duct cancer, angiosarcoma, hemangiosarcoma, hepatoblastoma, hemangioma, hepatic adenoma, and focal nodular hyperplasia. [0094] In another embodiment, the TKI comprises, alternatively consists essentially of, or yet consists of one or more of one or more of an epidermal growth factor receptor (EGFR) inhibitor, a vascular endothelial cell growth factor receptor (VEGFR) inhibitor, a platelet- derived growth factor receptor (PDGFR) inhibitor, a c-KIT inhibitor, an FMS-like tyrosine kinase 3 (FLT-3) inhibitor, and a BCR-ABL inhibitor. In one embodiment, the EGFR inhibitor includes one or more of gefitinib, erlotinib, canertinib, and their salts, derivatives, or analogs. In one embodiment, the VEGFR inhibitor includes one or more of vatalanib, semaxanib, sunitinib, and their salts, derivatives, or analogs. In one embodiment, the PDGFR inhibitor includes one or more of pazopanib, nilotinib, cediranib, and their salts, derivatives, or analogs. In one embodiment, the c-KIT inhibitor includes one or more of dasatinib, pazopanib, quzartinib, and their salts, derivatives, or analogs. In one embodiment, the FLT-3 inhibitor includes one or more of CEP701, sunitinib, PKC412, and their salts, derivatives, or analogs. In one embodiment, the BCR-ABL inhibitor includes one or more of imatinib, bafetinib, dasatinib, nilotinib, bosutinib, ponatinib, thiazol, thiadiazole, and their salts, derivatives, or analogs. In some embodiments, the BCR-ABL inhibitor is imatinib.

[0095] In another embodiment, the antihistamine comprises, alternatively consists essentially of, or yet consists of one or more of perphenazine, sertralin, thioridazine, chlorpromazine, paroxetine, loratadine, flupentixol, fluphenazine, hydroxyzine,

promethazine, cyproheptadine, and their salts, derivatives, or analogs. In some embodiments, the antihistamine is cyproheptadine.

[0096] In another embodiment, the kit further comprises sorafenib, sunitinib, brivanib, bevacizumab, ramucirumab, vatalanib, bnifanib, TSU-68, cediranib, erlotinib, nintedanib, regorafenib, cetuximab, lapatinib, cixutumumab, everolimus, sirolimus, and tivantinib.

[0097] In one embodiment, the kit further comprises instructions for treating the cancer. In one embodiment, the kit of parts comprises instructions for dosing and/or administration of the pharmaceutic composition of this invention. WORKING EXAMPLES

[0098] The following examples are for illustrative purposes only and should not be interpreted as limitations of the claimed invention. There are a variety of alternative techniques and procedures available to those of skill in the art which would similarly permit one to successfully perform the intended invention.

Example 1: In vitro treatment of hepatocellular carcinoma cells.

[0099] HCC cell lines Hep G2 and Huh-7 were maintained in DMEM containing 10% (v/v) FBS. Human normal liver cell line L-02 was maintained in RPMI1640 containing 10% (v/v) FBS. 48 hours after the treatment, MTT assay was performed and IC50 of the drugs was calculated by the Graphpad Prism 5 software. Cyproheptadine lowered the IC50 of imatinib from 22.2 mM to 0.3728 pM, thereby synergistically increasing the inhibitory effect of imatinib on HepG2 cells (Figure 1 A). Imatinib lowered the IC50 of cyproheptadine from 36.50 pM to 9.55 pM, thereby synergistically increasing the inhibitory effect of

cyproheptadine on HepG2 cells (Figure 1B). Moreover, cyproheptadine synergistically lowered the IC50 of imatinib on Huh-7 cells from 15.04 pM to 0.01299 pM (Figure 2). This synergistic effect was specifically targeting cancer cells because the combination of imatinib and cyproheptadine did not inhibit normal human cells (Figure 3).

Example 2: Treatment of mice implanted with hepatocellular carcinoma cells.

[0100] HpeG2 cells (IO ^c IO ⁶ ) were injected subcutaneously into the flank of BALB/c nude mice. When tumors reached a volume of ~ 1 OOmnT. mice were randomly divided into 4 groups with 10 mice per group:

1. Group 1 is the control in which the mice were administered 0.5% CMC-Na;

2. in Group 2, mice were administered imatinib at a dose of 80 mg/kg/day;

3. in Group 3, mice were administered cyproheptadine at a dose of 6 mg/kg/day; and

4. the mice in Group 4 were administered the combination of cyproheptadine (6

mg/kg/day) and imatinib (80 mg/kg/day). [0101] The control solution, imatinib, cyproheptadine, or combination of imatinib and cyproheptadine were administered orally. Tumor volume of different groups were measured and calculated with the formula as tumor volume (mm ³ ) = (W ² x L)/2. The body weights of mice in each group were measured weekly.

[0102] This in vivo study showed that after 19 days of treatment, the average tumor volumes of mice in Group 2-4 were significantly smaller than those of Group 1 with an inhibition rates as 29%, 33%, and 37% respectively as shown in Figure 4. After 65 days of treatment, the tumor volumes of the mice in Group 2-4 were still smaller than that of Group 1 with an inhibition rates as 12%, 22%, and 21% respectively. Thus, imatinib and

cyproheptadine can both inhibit the growth of hepatocellular carcinoma cells in vivo.

[0103] The mice in Group 4 that received the combination of imatinib and cyproheptadine exhibited the least reduction in body weights caused by tumor and/or drug toxicity (Figure 5). The mice in this group also exhibited the best daily activities among the groups (Data not shown). By the end of study, the survival rate for Group 4 was 40%; whereas the survival rates of Group 2 and Group 3 mice were 10% (Figure 6). The average survival period for 50% of the mice in Group 4 is 186 days which is significantly longer than those of Group 2 (120 days) and Group 3 (164 days). Thus, the combination of imatinib and cyproheptadine increased the survival rate of mice with implanted hepatocellular carcinoma cells.

Example 3: Treatment of hepatocellular carcinoma in orthotopic mouse model.

[0104] The tumors from mice with subcutaneous transplant of HepG2 cells (10* 10 ⁶ ) was removed and cut into about 2 mm ³ slices, which were implanted into the left lobe of the livers of mice. 18 days after the orthotopic implantation, the mice were randomly divided into three groups based on different treatments:

1. Group 1 : mice were administered with CMC-Na as negative control;

2. Group 2: mice were administered with combination of cyproheptadine (6 mg/kg/day) and imatinib (80 mg/kg/day); and

3. Group 3: mice were administered with sorafenib at a dose of 30 mg/kg/day as

positive control. [0105] The control solution, sorafenib, or the combination of imatinib and cyproheptadine were administered orally. The survival rate and body weights of mice in each group were measured at 169 days after the orthotopic implantation. The results showed that the average survival period for 50% of the mice in Group 2 was 141 days, which is significantly longer than Group 1 (71 days) and also longer than Group 3 (135 days).

[0106] By the end of the experiment, the survival rate for the mice in Group 1 is 10% and the survival rate for the mice both in Group 2 and Group 3 is 40% (Figure 7). The mice in Group 2 that received the combination of imatinib and cyproheptadine exhibited the best body weights (Figure 8) as well as daily activities.

Example 4: In vitro treatment of non-small-cell lung carcinoma.

[0107] A549 cells, a type of human carcinoma cells, were maintained in DMEM containing

10% (v/v) FBS. After the 48-hour treatment with different concentrations of EGFR inhibitor (gefitinib or afatinib), antihistamine (cyproheptadine or loratadine) or their combinations, MTT assays were performed to measure inhibitions of c A549 ell growth. After 48-hour treatment with loratadine (15 mM), A549 cell’s survival rate was 97.24%, which shows that loratadine by itself had little effect on inhibiting cell growth. However, when the same concentration of loratadine was used along with gefitinib or afatinib, the combination led to a synergy in inhibiting proliferation of A549 tumor cells (Figure 9).

Equivalents

[0108] It is to be understood that while the disclosure has been described in conjunction with the above embodiments, the foregoing description and examples are intended to illustrate and not limit the scope of the disclosure. Other aspects, advantages, and modifications within the scope of the disclosure will be apparent to those skilled in the art to which the disclosure pertains.

[0109] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. [0110] The embodiments illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms“comprising,”“including,” containing,” etc., shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure.

[0111] Thus, it should be understood that, although the present disclosure has been specifically disclosed by specific embodiments and optional features, modification, improvement, and variation of the embodiments therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements, and variations are considered to be within the scope of this disclosure. The materials, methods, and examples provided here are representative of particular embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure.

[0112] The scope of the disclosure has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0113] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that embodiments of the disclosure may also thereby be described in terms of any individual member or subgroup of members of the Markush group.

[0114] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control