WO2014087240A2 - Compositions, methods and kits for preventing, reducing, and eliminating cancer metastasis - Google Patents

Abstract

Compositions, kits and methods for preventing and treating cancer in a subject (e.g., human) by preventing, reducing or eliminating cancer (e.g., cancerous tumor) metastasis include administration of an anti-platelet agent that inhibits metastasis of cancer cells in a subject having cancer, and optionally, one or more additional anti-cancer agents. The compositions, methods and kits can be used for preventing and treating any cancer with metastatic potential such as, for example, metastatic melanoma and breast cancer. The compositions can be used as adjuvant therapy against cancer in combination with chemotherapy and/or radiation therapy (e.g., chronic or short term adjuvant therapy).

Description

COMPOSITIONS, METHODS AND KITS FOR PREVENTING, REDUCING, AND

ELIMINATING CANCER METASTASIS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of Provisional Application Serial No. 61/733,363 filed December 4, 2012, Provisional Application Serial No. 61/798,309 filed March 15, 2013, and Provisional Application Serial No. 61/888,579 filed October 9, 2013, which are herein incorporated by reference in their entireties.

FIELD OF THE INVENTION

[0002] The invention relates generally to the fields of pharmacology, molecular biology, and oncology. More particularly, the invention relates to preventing or reducing cancer metastasis by modulating platelet activity.

BACKGROUND

[0003] Cancer remains one of the world's deadliest diseases. Most cancer deaths are caused by the ability of cancer cells to spread, or metastasize, to different parts of the body. To spread, cancer cells must travel through the circulation. Research has shown that cancer cells can avoid being detected and killed by the body's defenses by covering themselves in a layer of platelets, the cells that normally help to form clots and stop bleeding. Cancer cells can also use platelets to stick to the inner walls of blood vessels in order to exit the bloodstream and enter different organs. Finally, cancer cells can take up molecules produced by platelets to help accelerate their own growth.

[0004] Although anti-platelet agents have been reported to inhibit cancer growth and metastasis, recent clinical trial results (The FDA Prasugrel Secondary Review - available for download from the FDA website) suggesting that the potent anti-platelet prasugrel (a thienopyridine in the same class as the anti-platelet clopidogrel) increased cancer risk or progression has led researchers to question the safety of platelet therapy, particularly for the similar anti-platelet clopidogrel. There is some data that suggests clopidogrel use accelerates cancer progression, and presently at least one clinical trial is underway with clopidogrel to examine the cancer risk ("Beta-blocker/clopidogrel usage and cancer progression in patients with colorectal cancer," Queen's University Belfast, Dr C Cardwell). In one particular clinical trial, cancer risks after prasugrel therapy are growing over time, especially in women, and results in 27% increase in colorectal, lung, and breast solid malignancies is discouraging (The FDA Prasugrel Secondary Review - available for download from the FDA website; the FDAA Prasugrel Action Package - available for download from the FDA website; and CAPRIE Steering Committee - A randomized, blinded, trial of clopidogrel versus aspirin in patients at risk of ischaemic events. Lancet 1996; 348: 1329-13). In other studies when testing the effectiveness and safety of prasugrel and ticagrelor for heart disease, it was found that both drugs caused an increase risk of cancer. (Victor L. Serebruany. "The TRrfON versus PLATO trials: Differences beyond platelet inhibition." Thrombosis and Haemostasis. Feb. 2010). Such findings discourage the consideration of such anti-platelet agents as potential cancer therapies. Therapies that prevent and eliminate metastasis in cancer patients remain elusive and are greatly needed.

SUMMARY

[0005] Described herein are compositions, methods and kits for preventing and treating cancers with metastatic potential such as all solid tumors including but not limited to lung, liver, prostate, breast, metastatic melanoma, pancreatic and glial-based tumors including glioblastoma multiforme. The compositions, methods and kits involve administration of an anti-platelet agent that inhibits metastasis of cancer cells in a subject having cancer or a subject predisposed to having cancer, and optionally, one or more additional anti-cancer agents. Currently, the drugs clopidogrel (Plavix^®) and ticagrelor (Brilinta™) are used to reduce the ability of platelets to stick to each other and form clots in patients who are at risk of heart attack and stroke caused by abnormal clot formation. As described herein, these drugs may be used to prevent platelets from sticking to cancer cells, thereby preventing or reducing metastases and improving patient survival. The compositions can be used as adjuvants against cancer (e.g., chronic or short term adjuvant therapy). For example, a composition as described herein can include an anti-platelet agent and an adjuvant. As another example, a composition including an anti-platelet agent is used as an adjuvant in adjuvant therapy.

[0006] Accordingly, described herein is a method of treating cancer in a subject (e.g., human) having cancer cells. The method includes administering to the subject a composition including a pharmaceutically acceptable carrier and at least one anti-platelet agent in a therapeutically effective amount for inhibiting cancer cell-platelet interactions and inhibiting metastasis of cancer cells in the subject, wherein the anti-platelet agent is an anti-cancer agent. The cancer cells can be, for example, lung cancer cells, liver cancer cells, prostate cancer cells, breast cancer cells, melanoma cells, pancreatic cells or glial-based tumor cells. In the method, the at least one anti-platelet agent can be, for example, one or more of: ticagrelor or a derivative thereof, clopidogrel or a derivative thereof, ticlopidine or a derivative thereof and prasugrel or a derivative thereof. The composition can further include an additional anti-cancer agent (e.g., chemotherapy drug). The composition can be administered in combination with at least one of: chemotherapy, radiation therapy and surgery. Typically, administration of the composition inhibits at least one of: platelet activation in the subject, platelet-induced cancer cell proliferation in the subject, activity of ADP receptor P2Y12 in the subject, and cancer cell adhesion to and transmigration across endothelial cells in the subject. Administration of the composition improves survival in the subject. The composition is administered to the subject by any suitable route, e.g., intratumoral, intravenously (i.v.), intraperitoneal (i.p.), or orally.

[0007] Also described herein is a kit for treating cancer in a subject. The kit includes: a composition including a pharmaceutically acceptable carrier and a therapeutically effective amount of an anti-platelet agent for inhibiting metastasis of cancer cells in a subject having cancer, wherein the anti-platelet agent is an anti-cancer agent; instructions for use; and packaging. In a kit, the anti-platelet agent can be, for example, one or more of: ticagrelor or a derivative thereof, clopidogrel or a derivative thereof, ticlopidine or a derivative thereof and prasugrel or a derivative thereof. The kit can further include an additional anti-cancer agent.

[0008] Further described herein is a composition including a pharmaceutically acceptable carrier and a therapeutically effective amount of at least one anti-platelet agent for inhibiting metastasis of cancer cells in a subject (e.g., human) having cancer, wherein the anti-platelet agent is an anti-cancer agent. The at least one anti-platelet agent can be, for example, one or more of: ticagrelor or a derivative thereof, clopidogrel or a derivative thereof, ticlopidine or a derivative thereof and prasugrel or a derivative thereof. The composition can further include an additional anti-cancer agent. The cancer cells can be, for example, lung cancer cells, liver cancer cells, prostate cancer cells, breast cancer cells, melanoma cells, pancreatic cells or glial-based tumor cells (e.g., glioblastoma multiforme tumor cells). In a typical embodiment, the additional anticancer agent is a chemotherapy drug. [0009] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0010] As used herein, "protein" and "polypeptide" are used synonymously to mean any peptide-linked chain of amino acids, regardless of length or post-translational modification, e.g., glycosylation or phosphorylation.

[0011] By the term "gene" is meant a nucleic acid molecule that codes for a particular protein, or in certain cases, a functional or structural RNA molecule.

[0012] As used herein, a "nucleic acid" or a "nucleic acid molecule" means a chain of two or more nucleotides such as RNA (ribonucleic acid) and DNA (deoxyribonucleic acid).

[0013] By the term "anti-platelet agent" is meant any molecule, chemical entity, composition, drug, or biological agent having the ability to down-regulate, decrease, reduce, suppress, or inactivate at least partially the activity and/or function of platelets, e.g., inhibit platelet activation and/or inhibit cancer cell-platelet interactions. In a preferred embodiment, an anti-platelet agent is capable of inhibiting metastasis of cancer cells, inhibiting platelet-induced cancer cell proliferation, and improving the ability of immune cells to kill cancer cells. Examples of anti-platelet agents include clopidogrel, ticagrelor, ticlopidine, prasugrel, etc.

[0014] As used herein, the term "anti-cancer agent" relates to any agent which is administered to a patient with cancer or at risk for having cancer for the purpose of preventing or treating the cancer. The anti-platelet agents described herein are examples of anti-cancer agents. Standard and experimental chemotherapy drugs are also considered anti-cancer agents.

[0015] By the term "tumor" is meant an abnormal benign or malignant growth of tissue that possesses no physiological function and arises from uncontrolled, usually rapid, cellular proliferation. The term "tumor" includes solid and non-solid tumors.

[0016] The terms "patient," "subject" and "individual" are used interchangeably herein, and mean a mammalian (e.g., human, rodent, non-human primates, canine, bovine, ovine, equine, feline, etc.) subject to be treated and/or to obtain a biological sample from.

[0017] As used herein, "bind," "binds," or "interacts with" means that one molecule recognizes and adheres to a particular second molecule in a sample or organism, but does not substantially recognize or adhere to other structurally unrelated molecules in the sample. [0018] The term "labeled," with regard to a probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody.

[0019] When referring to a nucleic acid molecule or polypeptide, the term "native" refers to a naturally-occurring (e.g., a wild type or WT) nucleic acid or polypeptide.

[0020] As used herein, the terms "regulating", "regulation", "modulating" or "modulation" refer to the ability of an agent to either inhibit or enhance or maintain activity and/or function of a molecule (e.g., a platelet, a receptor). For example, an inhibitor of the ADP receptor P2Y12 would down-regulate, decrease, reduce, suppress, or inactivate at least partially the activity and/or function of the receptor. Up-regulation refers to a relative increase in function and/or activity.

[0021] The phrases "isolated" and biologically pure" refer to material, which is substantially or essentially free from components which normally accompany it as found in its native state.

[0022] By the terms "analog" and "derivative" is meant any molecule modified, relative to a parent molecule, that retains at least some partial structure and biological function (or improved biological function) of the parent molecule. A biological function, for example, is the ability to inhibit tumor metastasis. Another biological function, for example, is the ability to inhibit cancer cell-platelet interactions. When referring to a derivative of ticagrelor, such a derivative may be derived from the following compound:

[0023] In the compound above: Ri is a Ci-6 alkyl, C₂-6 alkenyl, C₂_₆ alkynyl, C₃_8-cycloalkyl or a phenyl group, each group being optionally substituted by one or more substituents selected from halogen, OR⁸, NR⁹R¹⁰, SR¹¹ or Ci_6 alkyl (itself optionally substituted by one or more halogen atoms);

R₂ is Ci-8 alkyl optionally substituted by one or more substituents selected from halogen, OR , NR⁹R¹⁰, SR¹¹, C₃_8-cycloalkyl, aryl (optionally substituted by one or more alkyl groups and/or halogen atoms), or Ci-6-alkyl; or R₂ is a C₃_g-cycloalkyl group optionally substituted by one or more substituents selected from halogen, OR⁸, NR⁹R¹⁰, SR¹¹, Ci-6-alkyl or phenyl, the latter two groups being optionally substituted by one or more substituents selected from halogen, N0₂,

C(0)R 8 , OR 8 , SR 11 , NR 12 R 13 , a fused 5- or 6-membered saturated ring containing one or two oxygen atoms, phenyl or Ci-6-alkyl the latter two groups being optionally substituted by OR , NR⁹R¹⁰ or one or more halogen atoms; one of R³ and R⁴ is hydroxy and the other is hydrogen, hydroxy or NR⁹R¹⁰;

R is a group (CR⁵R⁶)_mOR⁷ where m is 0 or 1, R⁵ and R⁶ are independently hydrogen, Ci_6 alkyl or phenyl the latter two groups being optionally substituted by halogen, and R is hydrogen, C_1-6 alkyl or (CR⁵R⁶)_nR¹⁴ where R⁵ and R₆ are as defined above, n is 1 to 3 and R¹⁴ is COOH, OR¹⁵, NR¹⁶R¹⁷ or CONR¹⁶R¹⁷; or R is a C_1-4 alkyl or C₂_₄ alkenyl group, each of which is substituted by one or more groups selected from =S, =0, =NR 20 or OR 21 and optionally substituted by one or more groups selected from halogen, C_1-4 alkyl, phenyl, SR²¹, N0₂ or NR²²R²³ (where R²¹, R²² and R²³ are independently hydrogen, C_1-4 alkyl or phenyl; R 20 is OR 24 or NR 25 R 26 , where R 24 is hydrogen, Ci_ 4 alkyl or phenyl, and R²⁵ and R²⁶ are independently hydrogen, C_1-4 alkyl, aryl, C_1-6 acyl, arylsulphonyl or arylcarbonyl);

R 8 is hydrogen, C_1-6 alkyl optionally substituted by halogen or R 8 is phenyl optionally substituted by one or more substituents selected from halogen, N0₂, C(0)R⁶, OR⁶, SR⁹, NR¹⁰Rⁿ;

R⁹, R¹⁰ and R¹¹ are independently hydrogen or Ci_6 alkyl; R and R are independently hydrogen, C_1-6 alkyl, acyl, alkyl sulfonyl optionally substituted by halogen, or phenyl sulfonyl optionally substituted by C C₄ alkyl; and

R¹⁵, R¹⁶ and R¹⁷ are independently hydrogen or C_1-6 alkyl; or a pharmaceutically acceptable salt or solvate thereof. Methods of making such compounds and derivatives thereof are well known in the art and are described, for example, in U.S. Patent No. 6,251,910, the entirety of which is incorporated herein by reference.

[0024] As another example of a derivative of ticagrelor, such a derivative may be derived from the following compound:

[0025] In the compound above:

R¹ is C3..5 alkyl optionally substituted by one or more halogen atoms; R² is a phenyl group, optionally substituted by one or more fluorine atoms; R and R⁴ are both hydroxy; R is XOH, where X is CH₂, OCH₂CH₂ or a bond; or a pharmaceutically acceptable salt or solvate thereof, or a solvate of such provided that: when X is CH₂ or a bond, R^f is not propyl; when X is CH₂ and R^! is CH₂CH₂CF₃, butyl or pentyl, the phenyl group at R" must be substituted by fluorine; when X is OCH₂CH₂ and R¹ is propyl, the phenyl group at I must be substituted by fluorine;

[0026] Methods of making such compounds and derivatives thereof are well known in the art and are described, for example, in U.S. Patent No. 6,525,060, the entirety of which is incorporated herein by reference.

[0027] The term "antibody" is meant to include polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, humanized antibodies, anti-idiotypic (anti-Id) antibodies to antibodies that can be labeled in soluble or bound form, as well as fragments, regions or derivatives thereof, provided by any known technique, such as, but not limited to, enzymatic cleavage, peptide synthesis or recombinant techniques.

[0028] As used herein, the terms "diagnostic," "diagnose" and "diagnosed" mean identifying the presence or nature of a pathologic condition (e.g., cancer, tumor metastasis).

[0029] The term "sample" is used herein in its broadest sense. A sample including polynucleotides, polypeptides, peptides, antibodies and the like may include a bodily fluid, a soluble fraction of a cell preparation or media in which cells were grown, genomic DNA, RNA or cDNA, a cell, a tissue, skin, hair and the like. Examples of samples include saliva, serum, blood, urine and plasma.

[0030] As used herein, the term "treatment" is defined as the application or administration of a therapeutic agent to a patient, or application or administration of the therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease, or the predisposition toward disease. Treatment can include, for example, preventing, reducing or eliminating cancer metastasis in a subject, decreasing platelet activation in a subject, improving patient (subject) survival, etc.

[0031] As used herein, the term "safe and effective amount" refers to the quantity of a component, which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention. By "therapeutically effective amount" is meant an amount of a composition of the present invention effective to yield the desired therapeutic response, for example, an amount effective to delay the growth of or to cause a cancer (e.g., lung, liver, prostate, breast, melanoma, pancreatic and glial-based tumors including glioblastoma multiforme) to shrink or prevent metastasis. The specific safe and effective amount or therapeutically effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal or animal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives.

[0032] As used herein, the term "therapeutic agent" is meant to encompass any molecule, chemical entity, composition, drug, or biological agent capable of preventing or treating cancer. An example of a therapeutic agent is a drug (e.g., clopidogrel, ticagrelor, ticlopidine, prasugrel, a derivative thereof, etc). The term includes small molecule compounds, antisense reagents, nucleic acids, siRNA reagents, antibodies, enzymes, polypeptides, peptides, organic or inorganic molecules, natural or synthetic compounds and the like.

[0033] Although compositions, kits, and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable compositions, kits, and methods are described below. All publications, patent applications, and patents mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. The particular embodiments discussed below are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE FIGURES

[0034] Figure 1 is a graph and a pair of photographs showing that oral ticagrelor administration (10 mg/kg/d) results in decreased B16 melanoma lung metastasis. Depicted are representative lung surfaces and percentage of total lung surface covered in tumour (n=5; **P<0.01; PBS, phosphate-buffered saline).

[0035] Figure 2 is a photograph of an electrophoretic gel showing results from a reverse transcription polymerase chain reaction performed on brain (positive control), platelets (Pit), B16 melanoma, and 4T1 breast cancer cells demonstrating no discernible mRNA expression of the P2Y12 receptor (500 base pair band) on tumour cells. Hypoxanthine-guanine polyribosyltransferase (HPRT) served as internal calibrating control (249 Bp). [0036] Figure 3 is a graph showing results from an experiment demonstrating that ticagrelor is associated with decreased lung metastasis in a 4T1 breast cancer model.

[0037] Figure 4: Ticagrelor administration protects mice from B16-F10 melanoma metastases. (A) Calculated percentage of total lung surface area involved with tumor nodules, and corresponding photographs of representative lung surfaces, for PBS-, clopidogrel- and ticagrelor-treated mice, ^***P<.001, n=9 per group. Images were visualized using a Leica S6D dissecting microscope equipped with a lOx/0.32 Plan Apo lens (Leica, Richmond Hill, ON) and a Qimaging Micropublisher 3.3 RTV digital camera (Qlmaging, Surrey, BC). Images were captured with QCapture Pro 5.0 software (Qlmaging) and lung surface area covered by tumor nodules calculated using Simple PCI digital image analysis (Compix Inc., Sewickley, PA). (B) Survival curves comparing PBS-treated (solid line) and ticagrelor-treated (dashed line) mice over 30 days following intravenous injection of B16-F10 melanoma cells, P = .02, n=10 per group. (C) Calculated percentage of total liver surface area involved with tumor nodules, and corresponding photographs of representative liver surfaces, for PBS- and ticagrelor-treated mice, ^***P < .001 compared to PBS, n=10 per group. (D) Weights of livers isolated from uninoculated mice compared to tumor-inoculated PBS- and ticagrelor-treated mice, P < .01 compared to uninoculated control; ^#P < .01 compared to tumor- inoculated PBS-treated. A similar difference was observed if liver metastasis was assessed by measuring total nodule number (data not shown). For each group, n=12. (E) Survival curves comparing PBS-treated (solid line) and ticagrelor-treated (dashed line) mice over the month following intrasplenic injection of B16-F10 melanoma cells, P <.001, n=10 per group.

[0038] Figure 5: Ticagrelor prevents platelet binding to B16-F10 tumour cells and reduces tumor cell-platelet adhesion to endothelial monolayers. (A) Expression of P2Y12 transcripts by RT-PCR in brain tissue (positive control), platelets, B16-F10 melanoma, and bEnd.3 cells. HPRT was used as internal standard. (B) Viability of B16-F10 melanoma cells treated with ticagrelor (0-100 μg/mL; black bars, normalized to untreated group) or serum derived from PBS- or ticagrelor-treated mice (white bars, normalized to PBS-treated serum group) was assessed by MTT assay. ^***P <.001 compared with untreated group. Each dose was tested 16 times per experiment. Data representative of 3 independent experiments. (C) CFDA-SE-labeled platelets from PBS- or ticagrelor-treated mice were preincubated in the presence or absence of a blocking monoclonal antibody against GPIIbllla and applied to confluent B16-F10 monolayers. After 1 hour, wells were washed and adherent fluorescent foci counted. P < .001 compared to all other groups. No significant difference was noted between groups treated with ticagrelor and/or monoclonal antibody against GPIIbllla. Data is representative of 3 independent experiments. (D) CFDA-SE-labeled B16-F10 melanoma cells were co-incubated with or without platelet-rich plasma (PRP, 5x10 platelets) isolated from PBS- or ticagrelor-treated mice (10 mg/kg daily for three days). The cell mixture was then added to a confluent bEnd.3 monolayer. One hour later, wells were washed and adherent fluorescent cells counted. P < .01 compared to B16-F10 group; ^# P<.01 compared to B16-F10 and PBS-treated PRP. Data representative of 3 independent experiments. Pretreatment of B16-F10 melanoma cells or bEnd.3 with ticagrelor or serum from ticagrelor-treated mice resulted in no statistically significant difference in adhesion to that observed between untreated B16-F10 and bEnd.3 cells (data not shown).

[0039] Figure 6: a series of graphs showing MTT data.

DETAILED DESCRIPTION

[0040] Compositions, kits and methods for inhibiting metastasis of cancer cells in a subject having cancer or in a subject predisposed to having cancer include an anti-platelet agent in a therapeutically effective amount for inhibiting metastasis of cancer cells (e.g., cancerous tumor) in the subject (e.g., a human subject). The ability of anti-platelet agents such as clopidogrel, ticagrelor, ticlopidine, prasugrel, etc., as well as derivatives thereof, for example, to prevent cancer metastasis in animal models (e.g., well-established mouse models), and by what mechanisms this beneficial effect is mediated, are examined by investigating the following: the role of anti-platelet agents (e.g., ticagrelor) in inhibiting platelet-induced cancer cell proliferation and expression of genes implicated in cancer spread; the effect of anti-platelet agents (e.g., ticagrelor) on cancer cell adhesion to and transmigration across endothelial cells (the cells that line the inner wall of blood vessels); the role of anti-platelet agents (e.g., clopidogrel and ticagrelor) in disrupting cancer cell-platelet interactions and improving the ability of immune cells called natural killer cells to kill cancer cells; the effectiveness of anti-platelet agents (e.g., clopidogrel and ticagrelor) in reducing metastases and improving survival in mammalian experimental metastasis models (e.g., established mouse experimental metastasis models); and the role of anti-platelet agents (e.g., clopidogrel and ticagrelor) in inhibition of new blood vessel growth (known as angiogenesis) in metastases. [0041] The below described preferred embodiments illustrate adaptations of these compositions, kits, and methods. Nonetheless, from the description of these embodiments, other aspects of the invention can be made and/or practiced based on the description provided below.

Biological Methods

[0042] Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises such as Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; and Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Biochemistry techniques are generally known in the art and are described in detail in methodology treatises such as A Manual for Biochemistry Protocols (Manuals in Biomedical Research) M. R. Wenk, A. Z. Fernandis, World Scientific Publishing Company; 1st edition (March 30, 2007). Immunology techniques are generally known in the art and are described in detail in methodology treatises such as Advances in Immunology, volume 93, ed. Frederick W. Alt, Academic Press, Burlington, MA, 2007; Making and Using Antibodies: A Practical Handbook, eds. Gary C. Howard and Matthew R. Kaser, CRC Press, Boca Raton, Fl, 2006; Medical Immunology, 6^th ed., edited by Gabriel Virella, Informa Healthcare Press, London, England, 2007; and Harlow and Lane ANTIBODIES: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988.

The Role of Platelets In Cancer Metastasis

[0043] A link between cancer metastasis and the hemostatic and coagulation systems (Varki. Blood. 2007;110: 1723-1729.) is known. Previous studies examined the interplay between cancer and thrombosis by examining the role of the anticoagulant activated protein C in inhibiting metastasis (Bezuhly et al. Blood. 2009;113:3371-3374.). Three key mechanisms have been identified whereby the hemostatic and coagulation systems cooperate with cancer cells: (i) the interaction of platelets and coagulation factors with tumor cells to make platelet-tumor cell aggregates which aid entry of cancer cells into distant body sites, (ii) the formation of a platelet cloak around tumor cells that protects them from being killed by immune cells such as natural killer cells, and (iii) the release of various growth factors and small molecules by platelets which promote cancer growth and invasion (Gay and Felding-Habermann. Nat Rev Cancer. 2011 ; 11 : 123- 134, Timar et al. Oncology 2005 ;69 : 185-201.).

[0044] When cancer cells enter the circulation from a primary tumor they come into contact with platelets and immune cells such as natural killer (NK) cells. Platelets are the main cells responsible for forming clots. In cancer, normal platelet function is necessary for disease progression. This has been illustrated by the observation that mice depleted of platelets have markedly fewer metastases (Gasic et al. Proc Natl Acad Sci U S A. 1968;61:46-52., Camerer et al. Blood. 2004;104:397-401.). After activation, platelets release molecules involved in hemostasis and inflammation. Chief among these is adenosine diphosphate (ADP) (Gay and Felding-Habermann. Nat Rev Cancer. 2011 ;11: 123-134., Labelle et al. Cancer Cell. 2011;20:576-590.). Small amounts of ADP bind to the ADP receptor P2Y12 on the platelet' s surface, leading to further platelet activation and ADP release. Platelet activation augments expression of other surface receptors (eg. P-selectin) on both platelets and cancer cells that augment their ability to bind to each other and to endothelial cells that line the inner surface of blood vessels (Bezuhly et al. Blood. 2009;113:3371-3374.). The formation of cancer cell-platelet aggregates thereby allows cancer cells to enter distant sites (Palumbo et al. Blood. 2005;105: 178-185.). Natural killer cells are largely responsible for eliminating cancer cells from the circulation before they enter distant sites (Hanna. Biochim Biophys Acta. 1985;780:213- 226.). Depletion of NK cells in mice makes them more susceptible to metastases (Nieswandt et al. Cancer Res. 1999;59: 1295-1300.). It has been suggested that platelets make a cloak around tumor cells, shielding them from NK cells (Palumbo et al. Blood. 2005;105: 178-185., Nieswandt et al. Cancer Res. 1999;59: 1295-1300.). Cancer cells also use the factors released by activated platelets to stimulate their own and new blood vessel growth (Pinedo et al. Lancet. 1998;352: 1775-1777.). One of the most powerful triggers for platelet release of these molecules is ADP (Battinelli et al. Blood. 2011;118: 1359-1369.).

Preventing, Reducing and Eliminating Cancer Metastasis Using Anti-Platelet Agents

[0045] Given the ability of cancer cells to exploit activated platelets to their own ends, antiplatelet agents currently in clinical use are investigated for their ability to reduce cancer cell metastasis. Given the pivotal role of ADP in platelet activation and tumor cell-platelet interactions, agents in clinical use that specifically target the ADP pathway, namely clopidogrel (Plavix ) and ticagrelor (Brilinta™; AstraZeneca Pharmaceuticals LP; Wilmington, DE), are first investigated. Clopidogrel is currently the most widely used inhibitor of the ADP receptor P2Y12. It is recommended for prevention of heart attack and stroke. Clopidogrel is an oral prodrug that exerts its antiplatelet effects after metabolism by the liver (Savi et al. Thromb Haemost. 2000; 84:891-6.). The active metabolite binds irreversibly to and inhibits P2Y12. Ticagrelor is a novel P2Y12 inhibitor (Health Canada. Summary Basis of Decision (SBD) PrBrilinta™, Ticagrelor, 90 mg tablet, AstraZeneca Canada Inc. Submission Control Number: 132218.). In comparison to clopidogrel, ticagrelor binds P2Y12 reversibly and does not require metabolic conversion for its activity (Husted S and van Giezen JJJ. Cardiovasc Ther. 2009; 27:259-274.). It has been shown to have a more rapid onset of activity and to be a more effective anti-thrombotic than clopidogrel with equivalent bleeding risk (Wallentin et al. N Engl J Med. 2009 Sep 10; 361(11): 1045-57.). As described below, however, any suitable anti-platelet agent(s) can be used in the compositions, kits and methods described herein.

Compositions for Preventing, Reducing or Eliminating Tumor Metastasis In A Subject

[0046] Described herein are compositions for preventing and treating cancer in a subject (e.g., a human subject). The compositions described herein reduce the ability of platelets to stick to each other and are theorized to prevent platelets and endothelial cells from adhering to cancer cells, and thus may be used to prevent, reduce or eliminate tumor metastasis and improve patient survival. A non-limiting list of examples of cancers with metastatic potential that can be treated using the compositions include those of the breast, the squamous epithelium, the bladder, the stomach, the kidneys, of head and neck, the oesophagus, the cervix, the thyroid, the intestine, the liver, the brain, the prostate, the urogenital tract, the lymphatic system, the stomach, the larynx, the lung, the skin, as well as monocytic leukaemia, lung adenocarcinoma, small-cell lung carcinoma, pancreatic cancer, glioblastoma, acute myeloid leukaemia, chronic myeloid leukaemia, acute lymphatic leukaemia, chronic lymphatic leukaemia, Hodgkin's lymphoma, and non-Hodgkin's lymphoma. In one embodiment, a composition includes a therapeutically effective amount of an anti-platelet agent for inhibiting metastasis of cancer cells in a subject having cancer, and a pharmaceutically acceptable carrier. In the compositions, the anti-platelet agent is an anti-cancer agent. Any suitable anti-platelet agent can be used. Examples of antiplatelet agents include ticagrelor, clopidogrel, ticlopidine, prasugrel, etc., and derivatives thereof. Any suitable form of an anti-platelet agent or prodrug, precursor, or derivative thereof, can be used. In general, a derivative of one of ticagrelor, clopidogrel, ticlopidine, or prasugrel is a compound that reduces the ability of platelets to stick to each other and that prevents platelets and endothelial cells from adhering to cancer cells. In a preferred embodiment, a derivative as described herein makes cancer cells more susceptible to chemotherapy, and inhibits tumor metastasis.

[0047] In addition to an anti-platelet agent, a composition can include an additional anticancer agent. Examples of additional anti-cancer drugs include standard chemotherapy drugs such as paclitaxel, cytarabine or doxorubicin or similar classes of drugs. Anti-platelet agents and additional anti-cancer agents that may find particular use in the compositions and methods described herein are those that inhibit upregulation of mediators of tumor cell adhesion and angiogenesis induced by PRP and ADP, and those that inhibit cancer cell proliferation. Examples of mediators of tumor cell adhesion and angiogenesis induced by PRP and ADP include P- selectin, ICAM-1, PECAM-1, and others. Inhibiting cancer cell proliferation includes inducing death (killing of) of the cancer cells, and/or modulation expression of genes implicated in cancer cell proliferation. In such embodiments, when administered to a subject, the composition inhibits platelet-cancer cell interactions. Typically, when combining an anti-platelet agent with an additional anti-cancer agent (e.g., chemotherapeutic agent), by adding the antiplatelet agent one can reduce the dose and hence toxicity of the chemotherapeutic agent with the same clinical efficacy. For a discussion of an P2Y12 inhibitor enhancing the cytotoxic effects of cisplatin, see Sarangi et al., Med Oncol, vol. 30:567, 2013.

[0048] In the compositions described herein, an anti-platelet agent or other anti-cancer agent can be obtained commercially or synthesized according to known methods. Compositions may include pharmaceutically usable precursors, prodrugs, derivatives, solvates, tautomers and stereoisomers of known anti-platelet agents and additional anti-cancer agents, including mixtures thereof in all ratios, for the preparation of a medicament for the prevention or treatment of cancer in which platelet activation and metastasis play a role.

[0049] Two or more therapeutic agents (e.g., one anti-platelet agent and one additional anticancer agent) may be mixed together in a composition. For example, two or more therapeutic agents may be mixed together in a tablet, or they may be partitioned in a tablet. In one example of a partitioned tablet, the first therapeutic agent is contained on the inside of the tablet, and the second therapeutic agent is on the outside, such that a substantial portion of the second therapeutic agent is released prior to the release of the first therapeutic agent. In another embodiment, rather than being mixed together in a single composition, two different therapeutic agents may be formulated in two different compositions.

[0050] Optionally, a composition as described herein may be administered in combination with any other standard or experimental anti-cancer therapy; such methods are known to the skilled artisan. In one example, an effective amount of a composition including an anti-platelet agent (e.g., clopidogrel, ticagrelor, ticlopidine, prasugrel, etc.) is administered in combination with radiation therapy and/or surgery. In another embodiment, an effective amount of a composition including an anti-platelet agent (e.g., clopidogrel, ticagrelor, ticlopidine, prasugrel, etc.) is administered in combination with chemotherapy. For example, a single composition can include both an anti-platelet agent and a chemotherapy drug. In another example, a first composition including an anti-platelet agent is administered and a second composition including a chemotherapy is administered (in such embodiments, the first composition can be administered prior to administration of the second composition, or the first composition can be administered subsequent to administration of the second composition). The anti-platelet agent can be administered prior to the radiation therapy and/or surgery or chemotherapy, concomitant to the radiation therapy and/or surgery or chemotherapy, or subsequent to the radiation therapy and/or surgery or chemotherapy. Combinations are expected to be advantageously synergistic. Typically, combining an anti-platelet agent with standard chemotherapy or radiation therapy renders the cancer cells more susceptible to the chemotherapy or radiation therapy. In such embodiments, the anti-platelet agent acts as an adjuvant to the chemotherapy or radiation therapy (e.g., the anti-platelet agent enhances the effects of the chemotherapy or radiation therapy). Thus, the compositions described herein can be used as adjuvants against cancer. In addition to standard chemotherapy and radiation therapies, an anti-platelet agent as described herein can be used as an adjuvant to any experimental anti-cancer therapies. The compositions can be used in short term or chronic adjuvant therapy for preventing and treating cancer. Therapeutic combinations that inhibit cancer (e.g., lung, liver, prostate, breast, melanoma, and glial-based tumors including glioblastoma multiforme) metastasis, cell growth and/or induce apoptosis of cancer cells are identified as useful in the methods described herein. [0051] The compositions, methods, and kits described herein have both prophylactic and treatment applications, i.e., can be used as a prophylactic to prevent onset of a disease or condition in a subject, as well as to treat a subject having a disease or condition. In prophylactic applications, the compositions described herein are administered to an individual at risk of developing (e.g., genetically predisposed to, and/or environmentally exposed to) cancer. In some embodiments, when risk of metastasis is high, the compositions described herein can be used in peri- operative treatment.

Effective Doses

[0052] The compositions (e.g., pharmaceutical formulations) described above are preferably administered to a mammal (e.g., rodent, human, non-human primates, canine, bovine, ovine, equine, feline, etc.) in an effective amount, that is, an amount capable of producing a desirable result in a treated subject (e.g., preventing, reducing or eliminating metastasis of a cancer in the subject, rendering cancer cells more susceptible to chemotherapy and/or radiation therapy). Toxicity and therapeutic efficacy of the compositions utilized in methods of the invention can be determined by standard pharmaceutical procedures. As is well known in the medical and veterinary arts, dosage for any one animal depends on many factors, including the subject's size, body surface area, body weight, age, the particular composition to be administered, time and route of administration, general health, the clinical symptoms of the cancer and other drugs being administered concurrently. A composition as described herein is typically administered at a dosage that inhibits cancer cell-platelet interactions and prevents or reduces cancer cell (e.g., tumor) metastasis, as assayed by, for example, identifying a reduction in tumor metastases or tumor burden based on imaging modalities. Pharmaceutical formulations can be administered in the form of dosage units which include a predetermined amount of active ingredient per dosage unit. Such a unit can include, for example, 1 mg/kg to 100 mg/kg daily of a compound (antiplatelet agent, additional anti-cancer agent such as a chemotherapy drug) according to the invention, depending on the condition treated, the method of administration and the age, weight and condition of the patient. The formulation can be prepared in such a way that the release is extended or retarded, such as, for example, by coating or embedding of particulate material in polymers, wax and the like. Methods of Treating Cancer

[0053] Described herein are methods of preventing and treating cancer (e.g., metastatic cancers such as lung, liver, prostate, breast, melanoma, pancreatic and glial-based tumors including glioblastoma multiforme) and/or disorders or symptoms thereof. The methods include administering to the subject a composition as described herein, e.g., a pharmaceutically acceptable carrier and an anti-platelet agent in a therapeutically effective amount for inhibiting cancer cell-platelet interactions and inhibiting metastasis of cancer cells in the subject (e.g., a mammal such as a human). In the methods, the composition can further include a therapeutically effective amount of one or more additional anti-cancer drugs or standard or experimental chemotherapy. The therapeutic methods of the invention (which include prophylactic treatment) in general include administration of a therapeutically effective amount of the compositions described herein to a subject in need thereof, including a mammal, particularly a human, or to a subject predisposed to having cancer. The subject or patient can belong to any mammalian species, for example a primate species, particularly humans, rodents, including mice, rats and hamsters, rabbits, horses, cows, dogs, cats, etc. Animal models are of interest for experimental investigations, where they represent a model for the treatment of human disease.

[0054] The administration of a composition including an anti-platelet agent and/or other anticancer agent or a precursor, prodrug, or derivative thereof, for the prevention or treatment of cancer (e.g., lung, liver, prostate, breast, melanoma, pancreatic, and glial-based tumors including glioblastoma multiforme, etc.) may be by any suitable means that results in a concentration of the therapeutic that, (e.g., in some embodiments, when combined with other components), is effective in reducing, preventing or eliminating metastasis of the cancer, or otherwise ameliorating, reducing, or stabilizing the cancer. The anti-platelet agent, the optional additional anti-cancer agent, or prodrug, precursor, or derivative(s) thereof, may be contained in any appropriate amount in any suitable carrier substance, and are generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for local or systemic administration (e.g., oral, parenteral, subcutaneous, intravenous, intramuscular, intratumoral, or intraperitoneal). The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

[0055] Compositions as described herein may be administered parenterally by injection, infusion or implantation (subcutaneous, intratumoral, intravenous, intramuscular, intraperitoneal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, supra. Compositions as described herein may be delivered directly to a target site, e.g., directly to a tumor or neoplasm or to a site near a tumor or neoplasm.

[0056] Compositions for parenteral use may be provided in unit dosage forms (e.g., in single- dose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below). The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the active agent(s) that reduces or ameliorates a cancer (e.g., cancer metastasis), the composition may include suitable parenterally acceptable carriers and/or excipients. The active therapeutic agent(s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing agents.

[0057] As indicated above, the pharmaceutical compositions described herein may be in a form suitable for sterile injection. To prepare such a composition, the suitable active therapeutic(s) is dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the compounds is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like. [0058] The dosage unit formulations for oral administration can, if desired, be encapsulated in microcapsules. Materials for use in the preparation of microspheres and/or microcapsules are, e.g., biodegradable/bioerodible polymers such as polygalactin, poly-(isobutyl cyanoacrylate), poly(2-hydroxyethyl-L-glutam- nine) and, poly(lactic acid). Biocompatible carriers that may be used when formulating a controlled release parenteral formulation are carbohydrates (e.g., dextrans), proteins (e.g., albumin), lipoproteins, or antibodies. Materials for use in implants can be non-biodegradable (e.g., polydimethyl siloxane) or biodegradable (e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid) or poly(ortho esters) or combinations thereof).

[0059] Formulations for oral use include tablets containing the active ingredient(s) (e.g., ticagrelor, ticlopidine, clopidogrel) in a mixture with non-toxic pharmaceutically acceptable excipients. Such formulations are known to the skilled artisan. Excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like.

[0060] The tablets may be uncoated or they may be coated by known techniques, optionally to delay disintegration and absorption in the gastrointestinal tract and thereby providing a sustained action over a longer period. The coating may be adapted to release the active drug(s) in a predetermined pattern (e.g., in order to achieve a controlled release formulation) or it may be adapted not to release the active drug until after passage of the stomach (enteric coating). The coating may be a sugar coating, a film coating (e.g., based on hydroxypropyl methylcellulose, methylcellulose, methyl hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers, polyethylene glycols and/or polyvinylpyrrolidone), or an enteric coating (e.g., based on methacrylic acid copolymer, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, shellac, and/or ethylcellulose). Furthermore, a time delay material, such as, e.g., glyceryl monostearate or glyceryl distearate may be employed.

[0061] As described above, a composition as described herein may be administered in combination with any other anti-cancer agent or therapy; such methods are known to the skilled artisan and described in Remington: The Science and Practice of Pharmacy, supra. In one example, an effective amount of a composition including an anti-platelet agent (e.g., clopidogrel, ticagrelor, ticlopidine, prasugrel, etc.) is administered in combination with radiation therapy and/or surgery. In another embodiment, an effective amount of a composition including an antiplatelet agent (e.g., clopidogrel, ticagrelor, ticlopidine, prasugrel, etc.) is administered in combination with chemotherapy. For example, a single composition can include both an antiplatelet agent and a chemotherapy drug. In another example, a first composition including an anti-platelet agent is administered and a second composition including a chemotherapy is administered (in such embodiments, the first composition can be administered prior to administration of the second composition, or the first composition can be administered subsequent to administration of the second composition). The anti-platelet agent can be administered prior to the radiation therapy and/or surgery or chemotherapy, concomitant to the radiation therapy and/or surgery or chemotherapy, or subsequent to the radiation therapy and/or surgery or chemotherapy.

[0062] Prior to or after administration of a composition as described herein (e.g., a composition including an anti-platelet agent and a chemotherapy drug) to a subject, the subject's response to the therapy can be analyzed or measured. In one embodiment, the invention provides a method of monitoring treatment progress. In one example of a method of monitoring treatment progress, the method includes the step of determining a level of changes in a marker(s) such as circulating tumor cells, and/or the number and size of tumor metastases on imaging parameters or diagnostic measurement (e.g., screen, assay) in a subject suffering from cancer (e.g., lung, liver, prostate, breast, melanoma, and glial-based tumors including glioblastoma multiforme) in which the subject has been administered a therapeutic amount of a composition as described herein. The level of marker(s) determined in the method can be compared to known levels of marker(s) in either healthy normal controls or in other afflicted patients to establish the subject's disease status. In preferred embodiments, a second level of marker(s) in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain preferred embodiments, a pre-treatment level of marker(s) in the subject is determined prior to beginning treatment according to the methods described herein; this pre-treatment level of marker(s) can then be compared to the level of marker(s) in the subject after the treatment commences, to determine the efficacy of the treatment. Any suitable biological sample can be tested for analyzing or measuring a subject's response to one of: an anti-platelet agent, an anti-platelet agent and a chemotherapy drug, and an anti-platelet agent and radiation therapy. Examples of biological samples include blood, serum, plasma, urine, saliva and tissue. The sample may be tested using any suitable protocol or assay. Examples of suitable assays include enzyme-linked immunosorbent assays (ELISAs), Western blots, flow cytometry assays, immunofluorescence assays, qPCR, microarray analysis, etc.

Kits for Preventing and Treating Cancer In a Subject

[0063] Described herein are kits for preventing and treating cancer in a subject by preventing, reducing or eliminating cancer metastasis. A typical kit includes a composition including a therapeutically effective amount of an anti-platelet agent for inhibiting metastasis of cancer cells in a subject having cancer or a subject predisposed to cancer. Such a kit can further include packaging and instructions for use. In one embodiment of a kit, the composition may further include a pharmaceutically acceptable carrier in unit dosage form. If desired, the kit also contains an effective amount of an additional anti-cancer agent (e.g., experimental or standard chemotherapy drugs such as cytarabine or doxorubicin or similar classes of drugs). In such a kit, the additional anti-cancer agent may be formulated in the same composition as the antiplatelet agent, or may be formulated as a separate composition. Typically, the kit includes a sterile container which contains a therapeutic or prophylactic composition; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

EXAMPLES [0064] The present invention is further illustrated by the following specific examples. The examples are provided for illustration only and should not be construed as limiting the scope of the invention in any way.

Example 1 - Determine if Clopidogrel and Ticagrelor Can Prevent Tumor Metastasis In Mouse Models and By What Mechanism This Effect Is Mediated

[0065] Whether clopidogrel and ticagrelor can prevent tumor metastasis in well-established mouse models, and by what mechanisms this beneficial effect is mediated, was determined. To this end, both in vitro and in vivo experiments were conducted. The effect of clopidogrel was not assessed in vitro as it must be metabolized by the liver into its active metabolite (Savi et al. Thromb Haemost. 2000; 84:891-6.).

[0066] To examine the role of ticagrelor in inhibition of platelet-mediated tumour cell proliferation, MTT cell viability assays were performed as previously described (Mosmann. J. Immunol Methods. 1983;65:55-63.). Melanoma (SK-MEL-28 human and B16-F10 mouse) and breast cancer (MDA-231 human and E0771 mouse) cell lines were cultured in the absence or presence of species-specific platelet-rich plasma (PRP) generated as previously described (Langer et al. Arterioscler. Thromb. Vase. Biol. 2007;27: 1463-1470, Massberg et al. J. Exp. Med. 2006;203: 1221-1233.). Along with PRP, cancer lines were also cultured in the absence or presence of varying concentrations of ticagrelor with or without ADP (Husted S and van Giezen JJJ. Cardiovasc Ther. 2009;27:259-274.). Protein levels of P2Y12 in each cancer and endothelial cell line were assessed by Western blot analysis following treatment with PRP, ticagrelor and/or ADP (Czajkowski et al. J. Pharmacol. 2004; 141: 497-507.). Expression levels of mRNA for mediators of thrombosis and metastatic potential were assessed by quantitative real-time polymerase chain reaction (RT-PCR).

[0067] To assess the ability of ticagrelor to inhibit cancer cell adhesion to an endothelial cell layer, human umbilical vein endothelial cells (HUVEC) or bEnd.3 mouse endothelial monolayers were left untreated or pretreated with or without species-specific PRP in the presence or absence of ticagrelor and/or ADP. Fluorescence-labeled cancer cells were also pretreated with combinations of PRP, ticagrelor and ADP and added to the endothelial monolayers (Chambers et al. J Natl Cancer Inst. 1992;84:797-803.). One hour later, wells were washed and adherent fluorescent cells counted. For transmigration assays, endothelial monolayers grown on Transwell inserts (Corning-Costar, Corning, NY) were treated as above. To induce chemo taxis, fetal calf serum or the chemokine CXCL12 were added to lower chambers. Fluorescence-labeled cancer cells pretreated as above were added to upper chambers. After 16 hours, fluorescent cells in lower chambers were counted.

[0068] To study the effect of antiplatelet drug treatment on in vivo tumor cell-platelet interactions in real-time, intravital microscopy was performed (Bonder et al. J Immunol. 2004; 172:45-53.). C57BL/6 mice were treated with intraperitoneal clopidogrel (Apotex, Toronto, ON), ticagrelor or PBS for one week. Rhodamine-6G was administered to allow for platelet visualization. To induce a dilutional thrombocytopenia, mice received a PBS injection prior to intrasplenic injection of fluorescence-labeled tumor cells. One hour following tumor cell injection, the right liver lobe was exteriorized and positioned onto a Plexiglas microscope stage for image capture of cell transit.

[0069] To assess whether platelets can impede NK cell killing of tumor cells, cytotoxic activity was tested in a standard 4-h 51Cr-release assay (Nieswandt et al. Cancer Res. 1999; 59: 1295-1300.). Briefly, B16-F10 or E0771 target cells labeled with 51Cr were cocultured in the presence of combinations of PRP, ticagrelor and/or ADP prior to being plated with splenic effector cells from C57BL/6 mice at various effectontarget ratios. Label release into the supernatant was used to calculate cytotoxicity.

[0070] To evaluate the effectiveness of clopidogrel and ticagrelor in reducing metastases and improving survival, three well-established mouse experimental metastasis models were used. The first two models involve the injection of tumor cells directly into the portal or systemic circulation without a primary site. Mice received intrasplenic or tail vein injections of B16-F10 cells as previously described (Ludwig et al. Cancer Res. 2004;64:2743-2750., Stevenson et al. Clin Cancer Res. 2005;11:7003-7011., Hiraoka et al.. Clin Cancer Res. 2006;12:7108-16.). Intraperitoneal injections of clopidogrel, ticagrelor, or saline were initiated two days prior, one day after or at the time of cancer cell inoculation and repeated daily for 2 weeks. Clopidogrel and ticagrelor dosing regimens may be altered during the study, as current dosing is based on the cardiovascular literature and no oncologic studies have been previously performed (Schulz et al. Thromb Haemost 2008;99: 190-5., Hoving et al.. Radiother Oncol. 2011; 101: 100-108.). After two weeks, or sooner if moribund, mice were euthanized. Groups were examined for differences in survival time using surrogate endpoints. Livers, spleens, and lungs were weighed, photographed, and biopsied for histology and RT-PCR. Images of liver or lung surface area covered by tumor nodules were visualized and digitally quantified. The third model involved the development of a primary tumor and more closely approximated clinical cancer management. The transplantable 4T1 tumor model mimics metastatic breast cancer in that tumor cells spontaneously metastasize to other tissues (Pulaski and Ostrand-Rosenberg. Cancer Res. 1998;58: 1486-1493., Lelekakis et al. Metastasis 1999;17: 163-170.). BALB/c mice received a subcutaneous inoculation of 4T1 mammary carcinoma cells into a mammary fatpad. When tumors reached 3-4 mm diameter they were excised, analogous to surgical resection in human breast cancer. As 4T1 cells were resistant to 6-thioguanine, tumor burden was examineded using a clonogenic colony plating assay (Aslakson and Miller. Cancer Res. 1992;52: 1399-1405.).

[0071] To assess the ability of clopidogrel and ticagrelor to inhibit angiogenesis, a well- established model of endothelial tubule formation was used. Briefly, HUVEC or bEnd.3 cells were plated in 96-well plates coated with growth factor-reduced Matrigel (BD Biosciences, Mississauga, ON) and were incubated in the presence or absence of ticagrelor, PRP and/or ADP (Khoo et al. Tissue Eng Part C Methods. 2011;17:895-906.). After incubation, media was removed and cells fixed for photomicroscopy. To assess the effect of soluble mediators produced by tumor cells on tubule formation in this model, experiments were repeated with Transwell inserts containing human and mouse breast cancer and melanoma cells placed into each Matrigel well to serve as an upper chamber.

[0072] Statistical methods: Survival data was analyzed by log-rank (Mantel-Cox) analysis. All other data was analyzed by two-tailed ANOVA with the Fisher posthoc test. All data is reported as mean with standard error. Statistical significance is set at P values < .05.

Example 2 - Evaluation of Results

[0073] These in vitro studies shed light on the potential mechanisms by which ticagrelor may inhibit tumor metastasis. Firstly, it was not anticipated that P2Y12 would be expressed on cancer cells, and to this end it was unlikely that ticagrelor alone would directly inhibit cancer cell proliferation. Instead, it was hypothesized that ticagrelor will inhibit PRP- and ADP-induced cancer cell proliferation. Similarly, it was not anticipated that P2Y12 would be expressed on endothelial cells and to this end ticagrelor may only be seen to reduce cancer cell adhesion to and transmigration across an endothelial monolayer that is otherwise increased in the presence of PRP or ADP. It was hypothesized that ticagrelor treatment would similarly inhibit the upregulation of mediators implicated in tumor cell adhesion and angiogenesis induced by PRP and ADP to untreated levels. Prior to conducting any of these experiments, adequate PRP was obtained. Human PRP was isolated from volunteers.

[0074] These in vivo studies were the first to examine the use of antiplatelet agents in reducing tumor metastasis. It is anticipated that the use of both clopidogrel and ticagrelor will significantly reduce tumor burden and improve animal survival. In addition, this improvement in survival and reduced metastases was also characterized by decreased cancer cell adhesion and transmigration observed by intravital microscopy.

Example 3 - Effect of Ticagrelor Administration Initiated Prior to Surgical Excision of Invasive Mammary Tumours in Mice on Metastasis and Survival

[0075] Researchers have long recognized a link between increased proliferation and metastasis of malignancies, including breast cancer, and the hemostatic system. Three key mechanisms have been identified whereby the hemostatic system cooperates with cancer cells: interaction of activated platelets with tumour cells to make platelet-tumour cell aggregates which aid cancer cell extravasation into metastatic sites; formation of platelet cloaks around tumour cells that protect against natural killer (NK) cell tumouricidal activity; and release of growth factors and molecules by activated platelets which promote tumour growth and invasion. It is hypothesized that an agent that blocks platelet activation may reduce cancer spread by inhibiting tumour cell-platelet interactions. Ticagrelor, a novel antithrombotic that prevents platelet activation by reversibly binding platelet adenosine diphosphate (ADP) P2Y12 receptors may be just such an agent. It is speculated that ticagrelor administration initiated prior to surgical excision of invasive mammary tumours in mice will lead to decreased metastasis and enhanced survival.

[0076] To examine the role of ticagrelor in reducing metastases in 4T1 and E0771 tumour models, the anti-metastatic potential of ticagrelor in mice bearing transplantable 4T1 and E0771 mammary carcinomas was examined. These tumours mimic advanced human breast cancer as they do not elicit a robust immune response, and undergo spontaneous metastasis from the primary tumour. Once mice inoculated with tumour cells developed a palpable primary mammary tumour, ticagrelor therapy was initiated and held immediately prior to primary tumour resection. Following tumour resection, ticagrelor was restarted. Tumour nodules on liver and lungs were assessed at 28 days. Additional survival studies were performed using surrogate endpoints.

[0077] The role of ticagrelor in inhibition of NK cell-mediated tumour killing was examined. To determine whether platelets can impede NK cell killing of tumour cells, standard cytotoxicity assays were performed in the presence or absence of platelets isolated from ticagrelor-treated and untreated animals. As it has been shown that NK cell inhibition is mediated by the interaction of glucocorticoid-induced TNF-related ligand (GITRL) on platelets adherent to tumour cells with GITR on NK cells, flow cytometry was used to assess whether treatment with ticagrelor changes surface expression of these proteins on platelets and NK cells.

[0078] The effect of ticagrelor on platelet-cancer cell aggregation and cancer cell invasion was examined. The ability of ticagrelor to inhibit formation of platelet-tumour cell aggregates was assessed by flow cytometry. To examine ticagrelor' s ability to inhibit breast cancer cell adhesion to and transmigration across an endothelial monolayer, well-described adhesion and Transwell transmigration experiments were performed. Additionally, intravital microscopy was used to directly assess interactions between fluorescence-labeled platelets and tumour cells with endothelial cells in the hepatic vasculature in vivo. Given its critical role in metastasis, P-selectin expression levels on platelets isolated from ticagrelor-treated and untreated mice, as well as bEnd.3 endothelial cells treated with ticagrelor in vitro was assessed by flow cytometry.

[0079] The role of ticagrelor in prevention of tumour proliferation and angiogenesis was examined. To examine the effect of ticagrelor on platelet-mediate tumour cell proliferation, CFSE proliferation assays were performed after coculturing tumour cells with platelets from ticagrelor-treated and untreated mice. Levels of angiogenic mediators from ticagrelor-treated and untreated mouse sera were assessed by proteomic antibody array. Finally, the ability of ticagrelor to inhibit angiogenesis was assessed using an established Matrigel endothelial tubule formation model.

[0080] These studies provide insights into the anti-metastatic effects of ticagrelor. Understanding by what mechanisms these beneficial effects are mediated allows for better design of clinical trials and may provide new therapeutic approaches to prevent cancer metastasis in breast cancer patients. Example 4 - Ticagrelor for Prevention of Cancer Metastasis

[0081] In vitro experiments and in vivo experiments in rodents were performed to evaluate the ability of ticagrelor to prevent cancer metastasis. Referring to Figure 1, oral ticagrelor administration (10 mg/kg/d) resulted in decreased B 16 melanoma lung metastasis. Depicted are representative lung surfaces and percentage of total lung surface covered in tumour (n=5; **P<0.01; PBS, phosphate-buffered saline).

[0082] Referring to Figure 2, reverse transcription polymerase chain reaction performed on brain (positive control), platelets (Pit), B16 melanoma, and 4T1 breast cancer cells demonstrated no discernible mRNA expression of the P2Y12 receptor (500 base pair band) on tumour cells. Hypoxanthine-guanine polyribosyltransferase (HPRT) served as an internal calibrating control (249 Bp).

[0083] Referring to Figure 3, ticagrelor is associated with decreased lung metastasis in a 4T1 breast cancer model. BALB/c mice were inoculated in the mammary fatpad with 4T1 breast cancer cells. Once a tumour was palpable, mice received daily oral ticagrelor (10 mg/kg/d) or PBS alone. Ticagrelor was held 2 days prior to and resumed 2 days after tumour excision to avoid bleeding risk, mirroring clinical practice. When tumours reached 3-4 mm (2-3 weeks) they were excised, analogous to tumour resection in humans. Mice were sacrificed at 28 days. As 4T1 cells are resistant to 6-thioguanine, tumour burden was assessed by a clonogenic colony plating assay (n=4; P=0.057; CFU, colony-forming unit).

Example 5 - Role of the reversible P2Y12 inhibitor ticagrelor in inhibition of tumor metastasis

[0084] Tumor cells use activated platelets to promote their proliferation and metastatic potential. Platelet activation is largely mediated through ADP engagement of purinergic P2Y12 receptors on platelets. We investigated the potential of the reversible P2Y12 inhibitor ticagrelor to inhibit tumor adhesion and metastasis. In B16-F10 metastasis models, mice treated with a clinical dose of ticagrelor (10 mg/kg) exhibited marked reductions in lung (84%) and liver (86%) metastases and improved survival compared to saline-treated animals. In vitro, B16-F10 cells exhibited decreased aggregation with platelets from ticagrelor-treated as compared to saline- treated mice, an effect similar to that seen with blockade of glycoprotein lib Ilia. Similarly, B16- F10 cells co-incubated with platelets from ticagrelor-treated mice exhibited reduced adhesion to endothelial monolayers compared to those co-incubated with platelets from saline-treated animals. These findings support a role for P2Y12-mediated platelet activation in promoting tumor cell invasiveness, and a potential benefit of ticagrelor in inhibiting metastasis.

[0085] Study design

[0086] Animals and cell lines: Wildtype C57BL/6 mice were purchased from Charles River Laboratories (St-Constant, QC). Experiments were performed in accordance with Canadian Council on Animal Care guidelines. B16-F10 mouse melanoma and bEnd.3 mouse brain microvascular endothelial lines were purchased from ATCC (Manassas, VA).

[0087] Metastasis models: Mice received intrasplenic or tail vein injections of B16-F10 cells (2.5 x 10⁵ cells) in 50 μΐ PBS as previously described (Cullen et al., J Immunol. 2009;183:5807- 15; Stevenson et al., Clin Cancer Res. 2005;11:7003-7011; Bezuhly et al. Blood 2009;113:3371- 3374). Three days prior to tumor cell inoculation and thereafter, mice received daily intraperitoneal injections of phosphate buffered saline (PBS), ticagrelor (10 mg/kg; Astra-Zeneca Inc., Mississauga,ON) or clopidogrel (10 mg/kg; Bristol-Myers Squibb Inc., Montreal, QC). Dosing (10 mg/kg) was determined based on a doubling of bleeding time following a standard tail cut (Podrez et al., Nat.Med. 2007;13: 1086-1095). Two weeks later, or sooner if moribund, mice were euthanized. Livers, spleens, and lungs were weighed and photographed. Liver or lung surface area covered by tumor nodules was calculated using Simple PCI digital image analysis (Compix Inc., Sewickley, PA). For survival experiments, animals were maintained for up to thirty days.

[0088] Tumor-platelet and tumor-endothelial adhesion assays: For tumor-platelet adhesion assays, confluent B16-F10 monolayers were grown on 24-well polystyrene plates. Platelets isolated from PBS- or ticagrelor-treated mice and labeled with CFDA-SE (Invitrogen, Burlington, ON) were added to each well and incubated for 30 minutes. Wells were washed three times and adherent fluorescent cells counted. To examine the role of glycoprotein (GP) lib Ilia in platelet- tumor cell aggregation, isolated platelets were pretreated with 10 g/ml anti-GPIIbllla blocking antibody (Biolegend, San Diego, CA) as previously described (Ashkar et al., Science 2000; 287: 860-864). For tumor-endothelial adhesion assays, CFDA-SE-labeled B16-F10 cells

(2 x 10 5^J) were co-incubated with platelet-rich plasma (PRP, 5x107 platelets) isolated from PBS- or ticagrelor-treated mice (10 mg/kg daily for three days) as previously described (Aslam et al., Blood 2012; 120: 2127-2132). The cell mixture was then added to a confluent bEnd.3 monolayer grown on 24-well polystyrene plates (BD Biosciences, Mississauga, ON). One hour later, wells were washed and adherent fluorescent cells counted.

[0089] RT-PCR: Total RNA was isolated from B16-F10 and bEnd.3 cells, platelets, and mouse brain by TRIzol reagent (Invitrogen, Burlington, ON) and chloroform extraction, and cDNA was prepared using Superscript III Reverse Transcriptase (Invitrogen). Polymerase chain reaction (PCR) was performed in duplicate with an Eppendorf Mastercycler EP thermocycler (Eppendorf, Mississauga, ON) using 1 μL· of cDNA. Primer pairs were designed for mouse P2Y12, with hypoxanthine-guanine phosphoribosyltransferase (HPRT) used as an internal normalizing standard.

[0090] Statistical analysis: Survival data were analyzed by log-rank (Mantel-Cox) analysis. All other data were analyzed by ANOVA with the Fisher posthoc test. All data are reported as mean with standard error. Statistical significance was set at P values < .05.

[0091] Results and Discussion

[0092] To assess whether systemic P2Y12 inhibition can decrease metastasis, well- established mouse models were used (Cullen et al., J Immunol. 2009;183:5807-15; Stevenson et al., Clin Cancer Res. 2005;11:7003-7011; Bezuhly et al. Blood 2009;113:3371-3374). Treatment with clinically relevant doses of ticagrelor, a reversible cyclo-pentyl-triazolo-pyrimidine P2Y12 inhibitor, or clopidogrel, an irreversible thienopyridine P2Y12 inhibitor, led to significant reductions in lung metastasis (Figure 4A). Compared to clopidogrel, ticagrelor does not require in vivo metabolism for its antiplatelet activity (Floyd et al., Clin Pharmacokinet. 2012; 51:429- 442) and has a greater perisurgical safety profile (Wallentin et al., N Engl J Med. 2009;361: 1045-57). Furthermore, there are concerns regarding potential toxicity of clopidogrel' s metabolites (Masaneni et al., Toxicology. 2012 Sep 28;299: 139-45). To this end, subsequent experiments were undertaken with ticagrelor alone. Ticagrelor led to improved survival following intravenous tumor inoculation (Figure 4B). Thirty percent of ticagrelor- treated mice were euthanized due to dyspnea, a documented side effect of ticagrelor in humans (Wallentin et al., N Engl J Med. 2009;361: 1045-57); however, on post-mortem assessment, ticagrelor animals demonstrated reduced metastasis (75.4 +2.5 in PBS vs. 33.3 +4.4 in ticagrelor; P<.05). Ticagrelor treatment following intrasplenic tumor cell injection led to reduced liver metastasis as assessed by both surface tumor coverage (Figure 4C), and liver weight (Figure 4D). Improved survival (Figure 4E) was observed in ticagrelor-treated animals compared to PBS-treated animals. Dyspnea was not observed in liver metastasis experiments, suggesting that lung metastases exacerbate this effect. Mean splenic tumor weights in PBS- and ticagrelor-treated mice did not differ significantly, indicating the difference in metastasis was not due to inhibition of primary tumor growth. No bleeding complications were noted.

[0093] As P2Y12 is predominantly expressed on platelets, we hypothesized that the observed protection from B16-F10 melanoma metastasis was due to ticagrelor' s action on platelets. In support of this, P2Y12 was undetectable in tumor or endothelial cell lines (Figure 5A). Furthermore, pretreatment of B16-F10 melanoma cells with serum from ticagrelor-treated mice or with peak drug concentrations observed in humans (<1.3 μg/mL) (Floyd et al., Clin Pharmacokinet. 2012; 51:429-442) did not decrease cell viability (Figure 5B) (Mosmann T., J. Immunol Methods. 1983;65:55-63). To assess the role of ticagrelor on platelet-tumor cell interactions, we incubated platelets from PBS- and ticagrelor-treated mice with B16-F10 cells. Ticagrelor treatment resulted in a 75% reduction in platelet-tumor cell binding (Figure 5C). As platelet aggregation is promoted by a conformational change in glycoprotein (GP) Ilbllla following ADP engagement of P2Y12, we next examined whether blockade of GPIIbllla could similarly inhibit platelet-tumor interaction. A monoclonal antibody against GPIIbllla reduced platelet-tumor cell binding to levels observed with ticagrelor-treated platelets. No further reduction was noted when the antibody was combined with platelets from ticagrelor-treated animals, suggesting that ticagrelor prevents platelet-tumor cell interactions by inhibiting GPIIbllla activity. This supports the earlier observation that tumor cell-platelet interactions are mediated through engagement of (Χνβ₃ integrin on B16-F10 cells with GPIIbllla on platelets (Lonsdorf et al., J Biol Chem. 2012;287:2168-78). We next examined the effect of ticagrelor on tumor cell-endothelial cell interactions. Compared to B16-F10 cells alone, tumor cells preincubated with PBS-treated mouse PRP had increased binding to endothelial monolayers. In contrast, tumor cells preincubated with PRP from ticagrelor-treated mice exhibited adhesion similar to that between untreated B16-F10 and bEnd.3 cells (Figure 5D). Pretreatment of either tumor cells or endothelial monolayers with ticagrelor (1 μg/mL) or serum from PBS- or ticagrelor-treated mice did not affect adhesion, confirming a platelet-mediated effect.

[0094] In summary, we provide direct evidence that the P2Y12 inhibitor ticagrelor protects against tumor metastasis. Our data supports the notion that this anti-metastatic effect is due to inhibition of P2Y12 activation on platelets. Blockade of platelet P2Y12 leads to decreased GPIIbllla activity critical to platelet interactions both with other platelets, and with B16-F10 cells, likely through interactions with (¾νβ₃ integrin (Lonsdorf et al., J Biol Chem. 2012;287:2168-78). This study provides proof-of-concept for the strategy of using ticagrelor in the prevention of tumor metastasis, and underscores the complex interplay between thrombosis and cancer progression.

Example 6 - Direct cytotoxicity of the drug in cancer cells that express P2Y12

[0095] In Figure 6, MTT data is shown. Of the four cell lines tested, the glioblastoma has the highest P2Y12 expression and hence appears to be most sensitive to direct cytotoxicity. P2Y12 inhibition makes the cancer cells more susceptible to chemotherapy.

Other Embodiments

[0096] Any improvement may be made in part or all of the compositions, kits, and method steps. All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended to illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. Any statement herein as to the nature or benefits of the invention or of the preferred embodiments is not intended to be limiting, and the appended claims should not be deemed to be limited by such statements. More generally, no language in the specification should be construed as indicating any non-claimed element as being essential to the practice of the invention. This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contraindicated by context.

Claims

What is claimed is:

1. A method of treating cancer in a subject having cancer cells comprising administering to the subject a composition comprising a pharmaceutically acceptable carrier and an anti-platelet agent in a therapeutically effective amount for inhibiting cancer cell-platelet interactions and inhibiting metastasis of cancer cells in the subject, wherein the anti-platelet agent is an anticancer agent.

2. The method of claim 1, wherein the cancer cells are lung cancer cells, liver cancer cells, prostate cancer cells, breast cancer cells, melanoma cells, pancreatic cells or glial-based tumor cells.

3. The method of claim 1, wherein the subject is a human.

4. The method of claim 1, wherein the anti-platelet agent is ticagrelor or a derivative thereof.

5. The method of claim 1, wherein the anti-platelet agent is clopidogrel or a derivative thereof.

6. The method of claim 1, wherein the anti-platelet agent is ticlopidine or a derivative thereof.

7. The method of claim 1, wherein the anti-platelet agent is prasugrel or a derivative thereof.

8. The method of claim 1, wherein the composition further comprises an additional anticancer agent.

9. The method of claim 1, wherein the composition is administered in combination with at least one of: chemotherapy, radiation therapy and surgery.

10. The method of claim 1, wherein administration of the composition inhibits at least one of: platelet activation in the subject, platelet-induced cancer cell proliferation in the subject, activity of ADP receptor P2Y12 in the subject, and cancer cell adhesion to and transmigration across endothelial cells in the subject.

11. The method of claim 1, wherein administration of the composition improves survival in the subject.

12. The method of claim 1, wherein the composition is administered to the subject by a route selected from the group consisting of: intratumoral, i.v., i.p., and oral.

13. A kit for treating cancer in a subject, the kit comprising:

i) a composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of an anti-platelet agent for inhibiting metastasis of cancer cells in a subject having cancer, wherein the anti-platelet agent is an anti-cancer agent;

ii) instructions for use; and

iii) packaging.

14. The kit of claim 13, wherein the anti-platelet agent is selected from the group consisting of: ticagrelor or a derivative thereof, clopidogrel or a derivative thereof, ticlopidine or a derivative thereof, and prasugrel or a derivative thereof.

15. The kit of claim 13, further comprising an additional anti-cancer agent.

16. A composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of an anti-platelet agent for inhibiting metastasis of cancer cells in a subject having cancer, wherein the anti-platelet agent is an anti-cancer agent.

17. The composition of claim 16, wherein the anti-platelet agent is ticagrelor or a derivative thereof.

18. The composition of claim 16, wherein the anti-platelet agent is clopidogrel or a derivative thereof.

19. The composition of claim 16, wherein the anti-platelet agent is ticlopidine or a derivative thereof.

20. The composition of claim 16, wherein the anti-platelet agent is prasugrel or a derivative thereof.

21. The composition of claim 16, further comprising an additional anti-cancer agent.

22. The composition of claim 16, wherein the cancer cells are selected from the group consisting of: lung cancer cells, liver cancer cells, prostate cancer cells, breast cancer cells, melanoma cells, pancreatic cells and glial-based tumor cells.

The composition of claim 22, wherein the cancer cells are glioblastoma multiforme

24. The composition of claim 21, wherein the additional anti-cancer agent is a chemotherapy drug.

25. The composition of claim 16, wherein the subject is a human.