WO2016166761A1

WO2016166761A1 - Combination therapies and uses thereof in the treatment of cancer

Info

Publication number: WO2016166761A1
Application number: PCT/IL2016/050397
Authority: WO
Inventors: Shmuel Ben-Sasson; Osnat Ohne
Original assignee: Tiltan Pharma Ltd
Current assignee: Tiltan Pharma Ltd
Priority date: 2015-04-14
Filing date: 2016-04-14
Publication date: 2016-10-20
Anticipated expiration: 2017-10-14

Abstract

The invention relates to a combination therapy comprising at least one angiogenesis inhibitor, at least one cytotoxic agent, at least one non-steroidal anti-inflammatory agent and a pharmaceutically acceptable carrier; and uses thereof in the treatment of cancer.

Description

COMBINATION THERAPIES AND USES THEREOF IN THE TREATMENT

OF CANCER

TECHNOLOGICAL FIELD

The present invention relates to the field of treatment and/or prevention of cancer using a combination of agents.

BACKGROUND ART

References considered to be relevant as background to the presently disclosed subject matter are listed below:

- US 2005/148521

- WO 2006/056889

- Saito A. et al. Endocrinology 148:1412-23 (2007)

- Powis G., Free Radic. Biol. Med. 6:63-101 (1989)

- Tetef M. et al. /. Cancer Res. Clin. Oncol. 121 : 103-6 (1995)

DeVita et al. Cancer: Principles and Practice of Oncology, 5^th Edition, pp 1171-5

Natori at al. Biomedicine & Pharmacotherapy, 59: 56-60 (2005)

- US Patent Application 20030158118

- Saarloos MN et al. Clin. Exp. Metastasis 11 : 275-83 (1993)

- US Patent Application 20030158118

- WO 2003/061566

- WO 2008/004231

- Sobrero AF. Am. Soc. Clin. Oncol, pp. 1939 - 1940 (2011)

Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.

BACKGROUND

Cancer generally refers to one of a group of more than 100 diseases caused by the uncontrolled, abnormal growth of cells that can spread to adjoining tissues or other parts of the body. Cancer cells can form a solid tumor, in which the cancer cells are massed together, or exist, as dispersed cells, as in leukemia. Normal cells divide until maturation is attained and then only as necessary for replacement of damaged or dead cells. Cancer cells are often referred to as "malignant" , because they divide endlessly, eventually crowding out nearby tissues and spreading to other parts of the body. The tendency of cancer cells to invade and spread from one organ to another or from one part of the body to another distinguishes them from benign tumor cells, which overgrow but do not spread to other organs or parts of the body. Malignant cancer cells eventually metastasize and spread to other parts of the body via the bloodstream or lymphatic system, where they can multiply and form new tumors. This sort of tumor progression makes cancer a deadly disease.

Although there have been great improvements in the diagnosis and treatment of cancer, many people die from cancer each year, and their deaths are typically due to metastases and cancers that are resistant to conventional therapies.

Most drug-mediated cancer therapies rely on poisons, called cytotoxic agents, selective for dividing cells. These drugs are effective because cancer cells generally divide more frequently than normal cells. However, such drugs almost inevitably do not kill all of the cancer cells in the patient. One reason is that cancer cells can acquire mutations that confer drug resistance. Another is that not all cancer cells divide more frequently than normal cells, and slowly- dividing cancer cells can be as, or even more, insensitive to such cytotoxic agents as normal cells. Some cancer cells divide slowly, because they reside in a poorly vascularized, solid tumor and are unable to meet the needs required for cell division. For example, cytotoxic agents such as cyclophosphamide have been used to treat cancer.

Although cancer chemotherapy has advanced dramatically in recent years, treating cancers with a single agent has had limited success. Firstly, any single agent may only target a subset of the total population of malignant cells present, leaving a subpopulation of cancerous cells to continue growing. Secondly, cells develop resistance upon prolonged exposure to a drug. Combination therapies, which employ two or more agents with differing mechanisms of action and differing toxicities, have been useful for circumventing drug resistance and increasing the target cell population, but have not proven effective in the treatment of all cancers. In addition, certain combinations of agents may be synergistic: their combined effect is larger than that predicted based on their individual activities. Thus, combining different agents can be a powerful strategy for treating cancer.

The most striking difference between malignant and healthy cells is the capacity of cancer cells for unrestricted proliferation. This difference is exploited by many cytotoxic agents, which typically disrupt cell proliferation by interfering with the synthesis or integrity of DNA. Examples of classes of cytotoxic agents which function in this manner include alkylating agents, such as cyclophosphamide, antimetabolites (e.g. purine and pyrimidine analogues), and platinum coordination complexes.

One problem with cytotoxic agents which function by disrupting cell division is that they do not discriminate between normal and malignant cells: any dividing cell is a potential target for their action. Thus, cell populations which normally exhibit high levels of proliferation (such as bone marrow) are affected, leading to the toxic side effects commonly associated with cancer treatments.

As a tumor grows, it requires blood supply and, consequently, growth of new vasculature. Angiogenesis is a process of tissue vascularization that involves the growth of new developing blood vessels into a tissue, and is also referred to as neovascularization. Blood vessels are the means by which oxygen and nutrients are supplied to living cells.

Inhibitors of pro-angiogenic growth factors are agents used to inhibit the signaling of known pro-angiogenic factors like VEGF or FGF. Currently, these agents by themselves failed to demonstrate sufficient efficacy in the treatment of cancer.

With only a few exceptions, no single drug or drug combination is curative for most cancers. Thus, new drugs or combinations that can delay the growth of life- threatening tumors and/or improve quality of life by further reducing tumor load are needed. GENERAL DESCRIPTION

The present invention provides a combination therapy comprising at least one angiogenesis inhibitor, at least one cytotoxic agent, at least one non-steroidal antiinflammatory agent and a pharmaceutically acceptable carrier.

The term "combination therapy" as used herein refers to a combination of therapeutically active agents (at least one angiogenesis inhibitor, at least one cytotoxic agent, at least one non-steroidal anti-inflammatory agent) encompassed in a single or multiple compositions. Thus, in some embodiments said combination therapy of the invention comprises at least one angiogenesis inhibitor, at least one cytotoxic agent, at least one non-steroidal anti-inflammatory agent being administered together at the same time to a patient in need thereof or separately (each or in any combinations thereof) in any sequential manner prescribed by a medical care taker.

In some embodiments, said at least one angiogenesis inhibitor is a protein kinase inhibitor. In other embodiments, said at least one angiogenesis inhibitor is an inhibitor of angiopoietin-1/2 signaling through the Tie2 receptor.

In further embodiments, said at least one angiogenesis inhibitor is selected from vatalanib, bevacizumab, aflibercept (VEGF-Trap), vandetanib, cediranib, axitinib, sorafenib, regorafenib, brivanib, sunitinib, motesanib, pazopanib, Apatinib, BIBF and any combination thereof.

In another aspect the invention provides a combination therapy comprising vatalanib, sorafenib or regorafenib, at least one cytotoxic agent, at least one nonsteroidal anti-inflammatory agent and a pharmaceutically acceptable carrier.

The term "angiogenesis inhibitor" relates to agents which are used to inhibit the signaling of known pro-angiogenic factors such as VEGF, FGF or PDGF. Without wishing to be bound by theory, it was shown that these agents can act extracellularly, by the inhibition of the interaction of an angiogenic factor with its receptor or can act intracellularly via the inhibition of the protein-kinase activity of the corresponding receptors. Non limiting examples of these agents include anti-VEGF or anti-VEGF- Receptor antibodies or inhibitors of the protein-kinase domain of VEGF-R, FGF-R or PDGF-R.

The term "cytotoxic agent" as used herein relates to any agent used for the treatment of abnormal and uncontrolled progressive cellular growth. Non limiting examples of cytotoxic agents include the alkylating agents cyclophosphamide (CTX) (Bristol-Meyers Squibb), ifosfamide (Bristol-Meyers Squibb), chlorambucil (Glaxo Wellcome), and carmustine (Bristol-Meyers Squibb); the anti-metabolites cytarabine (Pharmacia & Upjohn), 6-mercaptopurine (Glaxo Wellcome), 6-thioguanine (Glaxo Wellcome), and methotrexate (Immunex); the antibiotics doxorubicin (Pharmacia & Upjohn), daunorubicin (NeXstar), and mitoxantrone (Immunex); and miscellaneous agents such as vincristine (Lilly), vinblastine (Lilly), and paclitaxel (Bristol-Meyers Squibb). In one embodiment of the present invention, the cytotoxic agent may be selected from the group consisting of: cyclophosphamide, ifosfamide, cytarabine, 6- mercaptopurine, 6-thioguanine, vincristine, doxorubicin, daunorubicin, chlorambucil, carmustine, vinblastine, methotrexate, mitoxantrone, and paclitaxel or their pharmaceutically acceptable salts. In a further embodiment of the present invention, the cytotoxic agent may be cyclophosphamide or ifosfamide.

In some embodiments, said at least one cytotoxic agent is selected from cyclophosphamide, ifosfamide and any combination thereof.

The term "anti-inflammatory agent (drug)" as used herein relates to any agents capable of reducing and/or inhibiting and/or preventing inflammation disease cased by either response to infection, injury, irritation, or surgery. In another embodiment the anti-inflammatory drug is a steroidal anti-inflammatory drug selected from a group consisting of dexamethasone and betamethasone. In a further embodiment the antiinflammatory agent is a non-steroidal anti inflammatory drug. In a specific embodiment the non-steroidal anti-inflammatory drug may be selected from a COX-1 inhibitor, a COX-2 inhibitor and a non-selective COX-1 and COX-2 inhibitor. In yet a further embodiment of the invention, the COX-1 and COX-2 inhibitors may be selected from the group consisting of diclofenac, piroxicam and indomethacin. In another embodiment, in addition to the non-steroidal anti-inflammatory drug, the composition further comprises a steroidal anti-inflammatory drug such as, but not limited to, dexamethasone and betamethasone.

In some embodiments, said at least one non-steroidal anti-inflammatory agent is selected from diclofenac, piroxicam, indomethacin and any combinations thereof.

In the context of the present invention the term "pharmaceutically acceptable carrier" relates to pharmaceutically-acceptable, nontoxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. Such carriers may include, however not limited to, buffering agents, solubilizing agents, stabilizing agents or taste additives.

In some embodiments a combination therapy of the invention further comprises at least one of an NFkB inhibitor, an H2-blocker, levamisol, at least one agent that enhances intracellular accumulation of NADH + H⁺, an inhibitor of a matrix metalloproteinase, a redox quinine or any combination thereof.

In some embodiments a combination therapy of the invention further comprises an NFkB inhibitor.

The term "NFkB inhibitor" as used herein relates to any agent used for the inhibition of the Nuclear Factor kappa B (NFkB) intracellular transcription factor. In a specific embodiment, the NFkB inhibitor is selected from sulfasalazine, rapamycin, caffeic acid phenethylester, SN50 (a cell-permeable inhibitory peptide), parthenolide, triptolide, wedelolactone, lactacystin and MG-132 [Z-Leu-Leu-Leu-H]. In yet a further embodiment, the NFkB inhibitor is selected from sulfasalazine and rapamycin or their derivatives. In another embodiment rapamycin derivatives are selected from temsirolimus and everolimus. In some embodiments, said NFkB inhibitor is sulfasalazine.

In some embodiments a combination therapy of the invention further comprises an H2-blocker.

The term " H2-blocker" as referred to in the present invention relates to an Histamine type 2-receptor antagonist, used to block the action of histamine on parietal cells in the stomach, decreasing acid production by these cells. The H2-blocker may be any H2-blocker known to those skilled in the art. For example, the H2-blocker may be selected from the group consisting of Cimetidine, Ranitidine, Famotidine and Nizatidine. In some embodiments, said H-2 blocker is cimetidine.

In some embodiments a combination therapy of the invention further comprises levamisol.

In some embodiments a combination therapy of the invention further comprises at least one agent that enhances intracellular accumulation of NADH + H⁺.

In some embodiments a combination therapy of the invention further comprises an inhibitor of a matrix metalloproteinase.

As used herein, the phrase "matrix metalloproteinase (MMP) inhibitor" relates to any chemical compound that inhibits by at least 5%, the hydrolytic activity of at least one matrix metalloproteinase enzyme that is naturally occurring in a mammal.

The MMP inhibitor may be any MMP inhibitor known in the art, such as AG- 3340, RO 32-3555, RS 13-0830, Tissue Inhibitors of metalloproteinases (TIMPs) (e.g. TIMP-1, TIMP-2, TIMP-3, or TIMP-4), alpha 2- macroglobulin, tetracyclines (e.g., tetracycline, minocycline, and doxycycline), hydroxamates (e.g. batimastat, marimistat and trocade), chelators (e.g., EDTA, cysteine, acetylcysteine, D-penicillamine, and gold salts), synthetic MMP fragments, succinyl mercaptopurines, phosphonamidates, and hydroxaminic acids. In a specific embodiment, the MMP inhibitor is an MMP2 or an MMP9 inhibitor.

In some embodiments a combination therapy of the invention further comprises a redox quinone. In some embodiments said redox quinone is Vitamin ¾. In some other embodiments, said Vitamin ¾ is selected from a group consisting of menadione and menadione sodium bisulfite.

Quinones are compounds having a fully conjugated cyclic dione structure, such as that of benzoquinones, derived from aromatic compounds by conversion of an even number of -CH= groups into -C(=0)- groups with any necessary rearrangement of double bonds (polycyclic and heterocyclic analogues are included). Quinones are known for their ability to induce oxidative stress through redox cycling, hereby referred to as "Redox quinones" (Powis G., Free Radic. Biol. Med. 6:63-101 (1989)). Pharmaceutically acceptable redox quinones such as Vitamin ¾ have special therapeutic value since they are required for the bioactivation of proteins involved in hemostasis. Vitamin ¾ is a redox quinone , known as a prothrombogenic agent, mainly in supplement of veterinary diet. Studies have shown that Vitamin ¾ has failed to demonstrate beneficial anti-cancer properties (Tetef M. et al. /. Cancer Res. Clin. Oncol. 121: 103-6 (1995)).

In some embodiments said combination therapy of the invention is being contained in at least one container. In other embodiments, said combination therapy of the invention is being contained in at least two containers.

The term "container" as used herein refers to any receptacle capable of holding at least one component of a composition of the combination therapy of the invention. Such a container may be any jar, vial or box known to a person skilled in the art and may be made of any material suitable for the components contained therein and additionally suitable for short or long term storage under any kind of temperature. The present invention further provides a formulation consisting of an aqueous or oily suspension or solution comprising compositions of the combination therapy of the invention.

In one embodiment, the formulation is formulated for oral administration. Such oral administration may allow for treatment to take place, for example, at the patient's home.

In a further embodiment of the present invention, the formulation further comprises a flavoring agent (e.g. menthol, anethol and/or salt). In another embodiment of the present invention, part of the constituents of the aqueous or oily suspension or solution of the formulation may be supplied in a dry form and reconstituted (e.g. solubilized) prior to oral administration.

In one embodiment of the present invention compositions may be provided as sustained release or timed release formulations. The carrier or diluent may include any sustained release material known in the art, such as glyceryl monostrearate or glyceryl distearate, alone or mixed with a wax. Micro-encapsulation may also be used. The timed release formulation can provide a pharmaceutical composition of immediate and pulsed release throughout the day. The diluent is selected so as not to affect the biological activity of a composition of the combination therapy of the invention. Examples of such diluents are distilled water, physiological saline, Ringer's solution, dextrose solution, and Hank' s solution.

A composition or formulation of the combination therapy of the invention may include carriers, adjuvants and emulsifiers such as poloxamers, or nontoxic, non- therapeutic, non-immunogenic stabilizers and the like. Effective amounts of such diluent or carrier will be those amounts which are effective to obtain a pharmaceutically acceptable formulation in terms of solubility of components, biological activity, and the like. In one embodiment, the formulations include a controlled- release device or composition where one or several of the components comprised in a composition of a combination therapy of the invention are being released in a delayed fashion. Such formulation may be in the form of a tablet (or a pill) which releases different doses of components comprised in a composition of a combination therapy of the invention, in different time intervals after being administered orally.

A composition of a combination therapy of the invention may be formulated in a solid, semi-solid, or liquid form such as, e.g. suspensions, aerosols, or the like or any other formulation known to a person skilled in the art. In one embodiment, the compositions are administered in unit dosage forms suitable for single administration of precise dosage amounts. The compositions may also include, depending on the formulation desired, pharmaceutically-acceptable carriers as defined above.

In one embodiment of the present invention, a composition of a combination therapy of the invention may be administered in a single dosage form comprising all the therapeutically active agents together.

In another embodiment, when said combination therapy of the present invention comprises more than two compositions contained in separate containers, said at least two compositions/containers may be separately administered, simultaneously or sequentially.

The term "administered sequentially" refers to ordered and successive administration of said compositions of a combination therapy of the invention. Said sequential administration may be 1, 2 or 3 days apart.

In some embodiments, a combination therapy of the invention consists of: a first container comprising Vatalanib, sorafenib or regorafenib, cyclophosphamide, diclofenac, cimetidine and sulfasalazine; and a second container comprising Vatalanib, cimetidine and sulfasalazine. In some embodiments, said first container is administered twice a week and said second container is administered five times a week, on non- overlapping days. In other embodiments, said first and said second containers are formulated in a liquid form suitable for oral administration.

In some embodiments said first container comprises Vatalanib, Sorafenib or Regorafenib, in the range of 200-1, 500mg; cyclophosphamide in the range of 100-500 mg; diclofenac in the range of 100-300 mg; cimetidine in the range of 200-1,000 mg; sulfasalazine in the range of 200-1,000 mg. In a further embodiment said second container comprises Vatalanib in the range of 300-l,500mg; cimetidine in the range of 200-1,000 mg; sulfasalazine in the range of 400-2,000 mg.

In some embodiments the dose-range for Vatalanib, Sorafenib or Regorafenib, in a combination therapy of the invention is 100mg-2000mg administered once or twice a day. In some embodiments the total daily dose of Vatalanib, Sorafenib or Regorafenib, in a combination therapy of the invention is in the range of 500mg- 1250mg administered once or twice a day.

In another aspect the invention provides a combination therapy as disclosed herein above and below for use in the treatment of at least one type of cancer disease or any condition or symptom associated therewith.

In some embodiments, said at least one type of cancer disease is selected from lung cancer (e.g. adenocarcinoma and including non-small cell lung cancer), pancreatic cancers (e.g. pancreatic carcinoma such as, for example exocrine pancreatic carcinoma), colon cancers (e.g. colorectal carcinomas, such as, for example, colon adenocarcinoma and colon adenoma), nasopharyngeal cancer, gastric cancer, liver cancer (e.g. hepatocellular carcinoma), biliary-tract cancer, prostate cancer including the advanced disease, hematopoietic tumors of lymphoid lineage (e.g. acute lymphocytic leukemia, B-cell lymphoma, Burkitt's lymphoma), myeloid leukemias (for example, acute myelogenous leukemia (AML)), thyroid follicular cancer, myelodysplastic syndrome (MDS), tumors of mesenchymal origin (e.g. fibrosarcomas and rhabdomyosarcomas), melanomas, teratocarcinomas, neuroblastomas, gliomas, glioblastoma, benign tumor of the skin (e.g. keratoacanthomas), breast carcinoma (e.g. advanced breast cancer), kidney carcinoma, ovary carcinoma, bladder carcinoma, epidermal carcinoma and any combinations thereof.

Thus, the present invention provides a method of inhibiting and/or treating at least one type of cancer disease or any condition or symptom associated therewith in a mammal comprising administering to the mammal a combination therapy of the invention.

The term "cancer" as referred to in the present invention relates to any type of neoplastic disease which is characterized by abnormal and uncontrolled cell division causing malignant growth or tumor. Cancer cells, unlike benign tumor cells, exhibit the properties of invasion and metastasis and are highly anaplastic. Cancer includes the two broad categories of carcinoma and sarcoma. In one embodiment of the present invention the cancer is a solid tumor or tumor metastasis. In a further embodiment of the present invention, said cancer may be selected from, however not limited to, the group consisting of lung cancer (e.g. adenocarcinoma and including non-small cell lung cancer), pancreatic cancers (e.g. pancreatic carcinoma such as, for example exocrine pancreatic carcinoma), colon cancers (e.g. colorectal carcinomas, such as, for example, colon adenocarcinoma and colon adenoma), prostate cancer including the advanced disease, hematopoietic tumors of lymphoid lineage (e.g. acute lymphocytic leukemia, B-cell lymphoma, Burkitt's lymphoma), myeloid leukemias (for example, acute myelogenous leukemia (AML)), thyroid follicular cancer, myelodysplastic syndrome (MDS), tumors of mesenchymal origin (e.g. fibrosarcomas and rhabdomyosarcomas), melanomas, teratocarcinomas, neuroblastomas, gliomas, glioblastoma, benign tumor of the skin (e.g. keratoacanthomas), breast carcinoma (e.g. advanced breast cancer), kidney carcinoma, ovary carcinoma, bladder carcinoma, epidermal carcinoma and any combinations thereof.

The term "inhibiting any type of cancer disease or any condition or symptom associated therewith" and/or "treating any type of cancer disease or any condition or symptom associated therewith" as used in the context of the present invention relates to a decrease in tumor size; decrease in rate of tumor growth; stasis of tumor size; decrease in the number of metastasis; decrease in the number of additional metastasis; decrease in invasiveness of the cancer; decrease in the rate of progression of the tumor from one stage to the next, inhibition of tumor growth in a tissue of a mammal having a malignant cancer, control of establishment of metastases, inhibition of tumor metastases formation, regression of established tumors as well as decrease in the angiogenesis induced by the cancer. The term "inhibiting cancer" can also refer to prophylaxis such as prevention as cancer reoccurs after previous treatment (including surgical removal) and prevention of cancer in an individual prone (genetically, due to life style, chronic inflammation and so forth) to develop cancer.

The term "administering" or its other lingual forms as used in the context of the present invention relates to the path by which a pharmaceutically active component, a drug, fluid or other substance is brought into contact with the body. The pharmaceutical composition is transported from the site of entry to the part of the body where its action is desired to take place. According to one embodiment of the present invention, said administering may be achieved via any medically acceptable means suitable for a composition of a combination therapy of the invention or any component thereof, including oral, rectal, vaginal, nasal, topical, transdermal, or parenteral (including subcutaneous, intramuscular, intrasynovial, intraperitoneal, intradermal and intravenous) administration.

A composition of a combination therapy of the present invention can thus be administered by any means known in the art, such as oral (including buccal and sublingual), rectal, vaginal, nasal, topical, transdermal, or parenteral (including subcutaneous, intramuscular, intravenous, intrasynovial, intraperitoneal and intradermal) administration.

Compositions, methods and systems of the present invention may be used either alone, or in conjunction with other cancer treatment methods known to those of skill in the art. Such methods may include, but are not limited to chemotherapy, radiation therapy or surgery. The administration of a composition of a combination therapy of the present invention may be conducted before, during or after other cancer therapies. In addition, a composition of a combination therapy of the present invention may be administered concurrently with other cancer treatments known to those of skill in the art.

Typically, oral administration requires a higher dose than intravenous administration. Thus, the administration route will depend upon the situation: the skilled artisan must determine which form of administration is best in a particular case, balancing dose needed versus the number of times per month administration is necessary.

In one embodiment the components of a composition of a combination therapy of the invention are administered using the normal dose of each component as known to a person skilled in the art.

In another embodiment the components of compositions of a combination therapy of the invention are administered using a lower dose than the dose known in the art, of one or more component. For example, when administering a cytotoxic agent, in order to reduce side effects, it is possible to use a lower dose than used when administered as a single cytotoxic agent -typically 75% or less of the individual amount, more specifically 50% or less, still more specifically 40% or less.

In therapeutic applications, the dosages and administration schedule of components of a composition of a combination therapy of the invention may vary depending on the component, the age, weight, and clinical condition of the recipient patient, and the experience and judgment of the clinician or practitioner administering the therapy, among other factors affecting the selected dosage. Generally, the dose and administration scheduled should be sufficient to result in slowing and/or regressing, the growth of the tumor(s) and may also cause complete regression of the cancer. In some cases, regression may be monitored via direct imaging (e.g. MRI) or by a decrease in blood levels of tumor specific markers. An effective amount of the pharmaceutical composition is that which provides a medical benefit as noted by the clinician or other qualified observer. Regression of a tumor in a patient is typically measured with reference to the diameter of a tumor. Decrease in the diameter of a tumor indicates regression. Complete regression is also indicated by failure of tumors to reoccur after treatment has stopped. The present invention allows for the administration of a composition of a combination therapy of the present invention, either prophylactically or therapeutically or in the context of adjuvant or neo-adjuvant treatment.

When provided prophylactically, a composition of a combination therapy of the invention may be administered in advance of any symptom. Prophylactic administration of compositions may serve to prevent or inhibit cancer. A composition of a combination therapy of the invention may prophylactically be administered to a patient with, for example, a family history of cancer. The risk for developing cancer may be determined by measuring levels of cancer marker proteins in the biological fluids (i.e. blood, urine) of a patient or by genetic markers. Alternatively, administration of a composition of a combination therapy of the invention may be administered to a patient with rising cancer marker protein levels. Such markers include, for example, rising PSA, CEA, thymosin β-15, thymosin β-16, calcitonin, and matrix metalloproteinase (MMP). When provided prophylactically, the dose of a composition of a combination therapy of the invention may be reduced to the appropriate prophylactic dosage.

When provided therapeutically, a composition or compositions of a combination therapy of the invention may be administered at (or after) the onset of a symptom or indication of a cancer. Thus, a composition of a combination therapy of the present invention may be provided either prior to the anticipated tumor growth at a site or after the malignant growth has begun at a site.

The term "mammal" as used in the context of the present invention relates to warm blooded vertebrate animals characterized by the presence of mammary glands, which produce milk in females for the nourishment of young, and in addition are covered with hair or fur. In one embodiment, said mammal may be selected from the group consisting of a human, a cat, a dog and a horse.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

Fig. 1 shows the synergistic effect of combined TL-118 and Vatalanib treatment in comparison with individual TL-118 and Vatalanib treatments on the tumor volume over the course of 28 days of e preclinical experiment (Example 1).

Fig. 2 shows the effect of single SRF treatment in comparison to combination therapy of the invention with SRF+TL-118 on the tumor volume over the course of 35 days of preclinical experiment (Example 2).

Fig. 3 shows the effect of TL-118 treatment in comparison to combination therapy of the invention with SRF+TL-118 on the tumor volume over the course of 35 days of preclinical experiment (Example 2).

DETAILED DESCRIPTION OF EMBODIMENTS

It is understood that the foregoing detailed description and the following examples are illustrative only and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments, which will be apparent to a person versed in the art, may be made without departing from the spirit and scope of the present invention. Further, all patents, patent applications, and publications cited herein are incorporated herein by reference.

In the following exemplary embodiments of the present invention, different compositions of a combination therapy of the invention were tested in vivo for the purpose of suppression of tumor growth in mice. The control group received a vehicle containing only non-active ingredients which was compared to groups which received pharmaceutical compositions comprising different active ingredients as described in detail below.

Example 1: TL-118 synergy with Vatalanib for anti-cancer activity

Disease Model: EMT 6 /CTX, a breast carcinoma cells derived from Balb/c mice, known for Cyclophosphamide resistance (Dr. Beverly Teicher, Dena Farber Cancer Institute).

Animals-Species-Strain: Mouse-CB6 Fl. Gender /Number /Age: Female 1901 5-7 weeks. Source: Harlan Laboratories, Israel.

Materials

Name: EMT 6 /CTX

Name/Formulation : Mammary Carcinoma

Name: Dulbecco's Modified Eagle Medium (DMEM)

Manufacturer: Biological Industries

Storage conditions: 2-8^uC

Name: Penicillin-Streptomycin Solution

Physical State: Liquid

Supplied by: Biological Industries

Storage Conditions: (-25) - (-15)^UC

Name: Fetal Bovine serum

Physical State: Liquid

Supplied by: Biological Industries

Storage Conditions: (-25) - (-15)°C

Name: Trypsin

Physical State: Liquid

Supplied by: Biological Industries Storage Conditions: (-25) - (-15)^UC

Name: Vatalanib

Storage Conditions: (-25) - (-15)^UC

Name: TL-118

Physical State: Clear solution

Storage Conditions: Frozen at -20 C

Formulations:

Medium for EMi CTX: Ten % FBS, 1% Pen/Strep, in DMEM.

Tumor Cells Preparation: EMT6/CTX were maintained following Pharmaseed's SOPs 400 "Contamination prevention in Tissue Culture", SOP 402 "Freezing and Thawing Cells" and SOP 403 "Counting Cells with Hemocytometer" Cells were cultured in growth medium and diluted 1 :2 or 1 :4 every 2-3 days by harvesting in Trypsin. Mice were injected with 3.5 x 10⁵ EMT6/CTX mammary carcinoma cells in a volume of 0.1 ml per mouse at a concentration of 3.5 x 10⁶ cells/ml.

General dosing formulations: In general, the 100% dosing solutions of all test articles are prepared at a final concentration suitable for 0.2 ml per-os administration for 25g mouse (Groups 2, 4, 7).

For 50% dosing, the test article were diluted 1:2 with Vehicle or WFI, as indicated below, prior to administration (Groups 3, 5, 8). i.e. again, 0.2 ml per-os administration for 25g mouse. For 50% + 50% injection (Groups 9, 6), 0.1ml of each of the corresponding 100% dosing solution, were administered.

Preparation of 100% dosing solutions: For each 100% group a net of 2.0 ml is needed per day and for 50% group, a net of 1.0 ml. 25-50% extra volume were added for expected losses (void volume etc.).

Composition of TL-118 clinical batch: TL-118 comprised two sets of drug combinations, each formulated as a liquid dosage form. The one containing the cytotoxic agent is Green-labeled while the non-cytotoxic formulation is White-labeled. The Green-labeled drug-combination is administered twice a week while the White one is administered during the rest of the week, according to the schedule specified below.

TL-118 Green-labeled Vial content:

Drug Dose per ml

Cimetidine 20mg/ml

Cyclophosphamide 20mg/ml

Sulfasalazine 15mg/ml

Diclofenac Sodium lOmg/ml

TL-118 White-labeled Vial content:

Drug Dose per ml

Cimetidine 20mg/ml

Sulfasalazine 45mg/ml

For TL-118 test article, a net volume of 5.0ml plus 50% spare = 7.5ml, is needed each day. The 100% preparation for the mice is 1:5 dilution of the clinical batch of TL-118. Such 1 :5 dilutions were prepared from Green-labeled vials and from White- labeled vials and were administered according to the schedule specified below.

Vatalanib: Test article was obtained as Vatalanib, Dihydrochloride Salt (powder), supplied by LC Laboratories (Woburn, MA 01801 USA). Daily 100% dosing solution were prepared beforehand according to the following and stored frozen: Vatalanib: 5ml of lOmg/ml each day X 6 (per week) X 4 wks = 120ml of lOmg/ml. 50mg Vatalanib were dissolved in 5ml WFI to ensure solubility. In 200ml beaker, l,300mg Vatalanib dissolved, in 130ml WFI, mixed and aliquot to 25 test-tubes, 5ml in each and stored at -20C.

Prior to administration, the dosing solutions were brought to room temperature and mixed. The solutions were diluted with WFI as needed.

Group # Mice # Treatment

IF 10 Control (Vehicle) 2F 10 TL-118 100% dose

3F 10 TL-118 50% dose

7F 10 Vatalanib 100% dose 80mg/kg

8F 10 Vatalanib 50% dose 40mg/kg

9F 10 Vatalanib 50% (40mg/kg) + TL-118 50%

Subcutaneous Tumour: Tumor implantation was done on Wednesday to enable starting of treatment on Sunday. In this model, CB6F1 mice were injected SC into the right flank with EMT6/CTX cells in a volume of 100 μL·. Subcutaneous tumors were measured in two perpendicular diameters starting on Day 4 (1^st day of treatment), twice a week with at least two days apart, until study termination.

Vehicle (Group 1) - WFI, 0.2ml PO.

TL-118 - mice were treated by PO administration, Daily (six times a week) for 28 days, starting Day 4 after tumor inoculation (=Sunday). Weekly sets of 6 test-tubes each, On days 1 and 4 of each treatment week (i.e. Sundays and Wednesdays when treatment began on a Sunday), formulation from Green-labeled vials were used, on days 2,3,5,6 of each treatment week (e.g. Mon, Tue, Thurs and Fri when treatment began on a Sunday) formulation from White-labeledvials were used.

100% dosing solution was prepared by 1:5 dilution in WFI of the original Green or White clinical batch vials of TL-118, from which 0.2ml is administered PO. to 25g mouse. 50% dose will be diluted by WFI. The corresponding 100% dosing solutions are divided into daily test tubes that are stored frozen and brought to room temperature prior to administration.

When treated with Green labeled test-tubes, treating technicians followed Pharmaseed's SOP I.D. no. 220. Hazard class was assessed according to Pharmaseed SOP for Study Hazard Class Classification (SOP I.D. no. 220).

Vatalanib - mice were treated by PO administration, Daily (six times a week) for 28 days, starting Day 4 after tumor inoculation. 100% dose were 80mg/kg, i.e. dosing solution of lOmg/ml from which 0.2ml was administered PO to 25g mouse. 50% dose were 40mg/kg, to be diluted by WFI.

Duration of the experimental period: The day of tumour cells injection was defined as Day=l, treatment initiation as Day =5 and study termination day was planned as Day=28.

Body Weight: Body weight was monitored once during acclimation and then twice a week until termination.

Termination: At study termination, on Day 28, the tumor were harvested, weighed and measured. If a 20 mm length diameter was measured before, it was euthanized and termination was performed to that specific mouse.

Preclinical results: Using a murine tumor xenograft EMTe/CTX model, TL-118 and Vatalanib were evaluated for their anti-tumor activity as a stand-alone therapy (100 and 50% dose) and in combination of both drugs (50% dose each) to evaluate potential synergy following a daily oral dosing (PO). Results demonstrated a clear synergy between TL-118 and Vatalanib. The anti-tumor activity of the combination group was more efficacious compared to 50% dose of each drug stand-alone. Moreover, the combination group was more efficacious compared to 100% dose of each drug standalone. The results are depicted in Figure 1.

In the past, Vatalanib has failed to prove efficacy, in metastatic colorectal patients, in two large phase 3 clinical trials when combined with FOLFOX. Failure was related mainly to incompatible dosing (once vs. twice daily) in a matrix with incompatible companion chemotherapy; FOLFOX (Fluorouracil, Leucovorin, Oxaliplatin). FOLFOX regimen is known as a high toxic chemotherapy which is not well tolerated by advanced cancer patients. Most of the patients cannot tolerate the maximal tolerated dose of this regimen. In the CONFIRM trials, patients received an unacceptably low chemotherapy dose in combination with Vatalanib either once or twice daily dose. This compromised combination failed to show efficacy but exhibited notable toxicity. It is therefore surprising that although TL-118 contains a cytotoxic agent, the unique TL-118 drug combination and schedule of administration was able to complemented Vatalanib in its anti-cancer activity. In addition, no excessive toxicity was observed when TL-118 and Vatalanib were administered together.

Example 2: TL-118 synergy with Sorafenib for anti-cancer activity

In essence, the design of the following experiments is identical to that described in Example 1 above, with the following differences: (1) TL-118 and Sorafenib were injected i.p. (instead of p.o.). (2) Sorafenib at a dose of 20mg/kg was used instead of Vatalanib. Sorafenib was dissolved in 50% Solutol HS-15 50% absolute Ethanol solution and diluted in DDW prior to injection. (3) 8 mice per group. (4) The rate of tumor-growth was calculated by nonlinear regression, as y=ce^bx where b is the slope (derived by Matlab). The difference between slopes in each experiment was assessed for significance (p<0.05) by Mann- Whitney U-test. (5) Treatment started 5 days after tumor inoculation.

TL-118 and Sorafenib, each, were administered at a sub-optimal dose to enable clear demonstration of synergy, the way it was done with Sorafenib.

As demonstrated in Fig. 2, Sorafenib by itself has negligible effect, at the indicated dose. On the other hand, in combination with TL-118, a powerful synergy was achieved.

Fig. 3 is complementary to Fig. 2: TL-118 has only a mild effect, at the indicated dose. However, in combination with Sorafenib, a powerful anti-tumor activity is obtained.

Claims

CLAIMS:

1. A combination therapy comprising at least one angiogenesis inhibitor, at least one cytotoxic agent, at least one non-steroidal anti-inflammatory agent and a pharmaceutically acceptable carrier.

2. The combination therapy according to claim 1, wherein said at least one angiogenesis inhibitor is a protein kinase inhibitor.

3. The combination therapy according to claim 1, wherein said at least one angiogenesis inhibitor is an inhibitor of angiopoietin-1/2 signaling through the Tie2 receptor.

4. A combination therapy according to claim 1, wherein said at least one angiogenesis inhibitor is selected from vatalanib, bevacizumab, aflibercept (VEGF- Trap), vandetanib, cediranib, axitinib, sorafenib, regorafenib, brivanib, sunitinib, motesanib, pazopanib, Apatinib, BIBF and any combinations thereof.

5. A combination therapy comprising vatalanib, sorafenib or regorafenib, at least one cytotoxic agent, at least one non-steroidal anti-inflammatory agent and a pharmaceutically acceptable carrier.

6. The combination therapy of any one of the preceding claims, wherein said at least one cytotoxic agent is selected from cyclophosphamide, ifosfamide and any combinations thereof.

7. The combination therapy of any one of the preceding claims, wherein said at least one non-steroidal anti-inflammatory agent is selected from diclofenac, piroxicam, indomethacin and any combinations thereof.

8. The combination therapy of any one of the preceding claims, further comprising an NFkB inhibitor.

9. The combination therapy of claim 8, wherein said NFkB inhibitor is sulfasalazine.

10. The combination therapy of any one of the preceding claims, further comprising an H2-blocker.

11. The combination therapy of claim 10, wherein said H-2 blocker is cimetidine.

12. The combination therapy of any one of the preceding claims, further comprising levamisol.

13. The combination therapy of any one of the preceding claims, further comprising at least one agent that enhances intracellular accumulation of NADH + H⁺.

14. The combination therapy of any one of the preceding claims, further comprising an inhibitor of a matrix metalloproteinase.

15. The combination therapy of any one of the preceding claims, further comprising a redox quinone.

16. The combination therapy of claim 11, wherein the redox quinone is Vitamin ¾.

17. The combination therapy of claim 12, wherein Vitamin ¾ is selected from a group consisting of menadione and menadione sodium bisulfite.

18. The combination therapy according to any one of the preceding claims, being contained in at least one container.

19. The combination therapy according to any one of the preceding claims, being contained in at least two containers.

20. The combination therapy according to any one of the preceding claims consisting of: a first container comprising vatalanib, sorafenib or regorafenib, cyclophosphamide, diclofenac, cimetidine and sulfasalazine; and a second container comprising Vatalanib, Sorafenib or Regorafenib, cimetidine and sulfasalazine.

21. The composition according to claim 16, wherein said first container is administered twice a week and said second container is administered five times a week, on non-overlapping days.

22. The composition of according to claim 16, wherein said first and said second container are formulated in a liquid form suitable for oral administration.

23. The combination therapy according to any one of the preceding claims for use in the treatment of at least one type of cancer disease or any condition or symptom associated therewith.

24. The combination therapy of claim 19, wherein said at least one type of cancer disease is selected from lung cancer, pancreatic cancers, colon cancers, nasopharyngeal cancer, gastric cancer, liver cancer, biliary-tract cancer, prostate cancer, myeloid leukemias, thyroid follicular cancer, myelodysplastic syndrome, tumors of mesenchymal origin, melanomas, teratocarcinomas, neuroblastomas, gliomas, glioblastoma, benign tumor of the skin, breast carcinoma, kidney carcinoma, ovary carcinoma, bladder carcinoma, epidermal carcinoma or any combinations thereof.