DESC:TECHNICAL FIELD
The present disclosure relates to the field of a combination therapy for treatment of cancer. In particular, the disclosure relates to a combination therapy comprising a therapeutic compound selected from calcimycin, romidepsin, panobinostat, or A-1331852 and one or more chemotherapeutic agents selected from a taxane and a platinum compound.

BACKGROUND OF THE DISCLOSURE
Epithelial ovarian carcinoma (EOC) spreads predominantly through the peritoneal cavity where ovarian cancer cells exist as suspended multicellular clusters known as spheroids. The spheroids are known to harbor chemo-resistant, dormant, and self-renewing cellular niches and are implicated in disease recurrence. These spheroids are carried passively within the peritoneum by the peritoneal fluid or ascites, where they seed multiple distal metastases by attaching to and clearing the mesothelial lining. Despite advances in the research on drugs used in the treatment of cancer, survival rates for ovarian cancer have changed only modestly for decades, with the diseases still recording the shortest interval between diagnosis and death among gynecological malignancies. Presently, treatment of ovarian cancer usually involves a combination of debulking surgery and chemotherapy consisting of platinum- and taxane-based drugs, discovered over 15 years ago. Despite improved clinical response to this combination of chemotherapy, numerous patients either undergo relapse or succumb to the disease as a result of chemotherapeutic resistance. Since EOC has no cancer-specific symptoms, there are no early screening and detection modalities; thus, about 75% of women are diagnosed directly at advanced stage disease (FIGO IIIc or IV). Therefore, there is an unmet need for new drugs that can efficiently eliminate ovarian cancer cells in vivo.

STATEMENT OF THE DISCLOSURE
The present disclosure relates to a combination comprising one or more therapeutic compounds selected from calcimycin, romidepsin, panobinostat, and A-1331852 and one or more chemotherapeutic agents selected from a platinum compound and a taxane. In some embodiments, the platinum compound is selected from carboplatin, cisplatin, and oxaliplatin. In some embodiments, the taxane is selected from paclitaxel, docetaxel, and cabazitaxel. In some embodiments, the combination is selected from the combinations disclosed in Table A. In some embodiments, the combination is a single formulation/composition comprising said therapeutic compound and said one or more chemotherapeutic agents. In some embodiments, the combination is a kit comprising said therapeutic compound and said one or more chemotherapeutic agents as separate formulations. The combinations of the present disclosure show a synergistic cytotoxic effect on cancer cells.

The present disclosure also provides a method treating cancer in a subject in need thereof, comprising administering to the subject one or more therapeutic compounds selected from calcimycin, romidepsin, panobinostat, and A-1331852 and one or more chemotherapeutic agents selected from a platinum compound and a taxane. In some embodiments, the cancer is an ovarian cancer, a uterine cancer, a cervical cancer, a colorectal cancer, or a gastric cancer. In some embodiments, the cancer comprises cells that form spheroids. In some embodiments, administration of the combinations of the present disclosure is useful for treating advanced stages of cancer.

The present disclosure provides use of the combinations of the present disclosure for treating cancer.

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
FIG. 1 shows a schematic for the3-dimensional spheroid assay used for screening of compounds. Panel “a” shows a schematic for generating three-dimensional (3D) cellular spheroids. Panel “b” shows 3D spheroids generated on Day 7 after seeding of cells. Panel “c” shows a schematic and timeline for the 3D-spheroid based assay employed for screening of a library of compounds. Panel “d” shows Z-scores calculated for the screening assays with different condition.

FIG. 2 shows the screening of compounds from the LOPAC library. Panel “a” shows cell viability of cells from the 3D-spheroids using resazurin (left graph) and APH assay (right graph) plotted for 1280 drugs. Panel “b” shows a correlation plot for resazurin and APH assay. Panel “c” shows compounds showing effects in resazurin and APH assay and also affecting the spheroid morphology which were selected for further studies. Panel “d” shows the inverted pyramid scheme showing the screening process to arrive at the lead compounds.

FIG. 3 shows the screening of compounds from the NIH library. Panel “a” shows cell viability of cells from the 3D-spheroids using resazurin (top graph) and APH assay (bottom graph) plotted for 147 drugs. Panel “b” shows compounds showing effects in resazurin and APH assay and also affecting the spheroid morphology which were selected for further studies. Panel “c” shows the inverted pyramid scheme showing the screening process to arrive at the lead compounds.

FIG.4 shows dose response curves for the selected compounds and standard chemotherapeutic agents. Panel “a” shows dose response curves for Calcimycin, Romidepsin, Panobinostat, and A-1331852 against 2D cultures and 3D spheroids of OVCAR3 and SKOV3. Panel “b” shows a morphology change in drug-treated spheroids and untreated control. Panel “c” shows dose response curves for the chemotherapeutic agents - Carboplatin, Paclitaxel, and Docetaxel - against 2D and 3D cultures of OVCAR3 and SKOV3.

FIGs. 5A-5D show results of the combination treatment studies in OVCAR3 and SKOV3. Combination index (CI) calculated is represented as heatmap. CI<1 is considered to be synergistic. FIG. 5A shows the heat map of combination index for a combination of calcimycin with carboplatin, paclitaxel, and docetaxel. FIG. 5B shows the heat map of combination index for a combination of romidepsin with carboplatin, paclitaxel, and docetaxel. FIG. 5C shows the heat map of combination index for a combination of Panobinostat with carboplatin. FIG. 5D shows the heat map of combination index for a combination of A-1331852 with carboplatin.

FIG. 6 shows the viability of OVCAR-3 and FT282 cells in response to the increasing concentration of calcimycin and carboplatin. Panel A shows the viability of OVCAR-3 cells in monolayers (2D) in response to the increasing concentration of calcimycin and carboplatin. Panel B shows the viability of FT282 cells in monolayers (2D) in response to the increasing concentration of calcimycin and carboplatin. Panel C shows the viability of OVCAR-3 cells in spheroids (3D) in response to the increasing concentration of calcimycin and carboplatin.

DETAILED DESCRIPTION OF THE DISCLOSURE
With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results. Throughout this specification, the word “comprise”, or variations such as “comprises” or “comprising” or “containing” or “has” or “having”, or “including but not limited to” wherever used, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Reference throughout this specification to “one embodiment”, “an embodiment”, “some embodiments” means that a particular feature, structure or characteristic described in connection with the embodiment may be included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases “in one embodiment” “in an embodiment”, on “in some embodimets” in various places throughout this specification may not necessarily all refer to the same embodiment. It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

Recently, repurposing of the established non-cancer/cancer drugs has become an important strategy in developing effective treatment due to relatively lower costs and faster drug development. Moreover, most of the current efforts at drug screening rely on cytotoxicity assays using established cancer cell lines grown as two-dimensional (2D) cultures that exhibited a rapid, uncontrolled growth phenotype. However, cytotoxicity assays involving 2D cultures have a major drawback -these assays do not always accurately reflect the phenotypic manifestations and morphologies of cancer epithelia in vivo. This may partially account for the high rate of clinical trial failures for new drug molecules. To overcome this drawback, the inventors developed a medium-throughput three-dimensional (3D) ovarian cancer multicellular tumor spheroid MCTS-based assay that simulates the tumor microenvironment of ovarian cancer to screen chemical libraries (FIG. 1). By employing these 3D assays, the inventors provide therapeutic combinations for targeted treatment of ovarian cancer as well as other cancers where cancer cells form spheroids.

As used herein, the term “therapeutic compound” refers to a compound identified from screening of a library of compounds for therapeutic effects (e.g., inducing death of cancer cells, inhibition of a chemical pathway in cancer cells) against cancer cells. In some embodiments, the therapeutic compound is selected from calcimycin, romidepsin, panobinostat, and A-1331852.

As used herein, the term “chemotherapeutic agent” refers to known chemotherapeutic drugs employed for treating a specific cancer. In some embodiments, a chemotherapeutic agent is a platinum compound including, but not limited to, carboplatin, cisplatin, and/or oxaliplatin. In some embodiments, a chemotherapeutic agent is a taxane compound including, but not limited to, paclitaxel, docetaxel, cabazitaxel, and/or derivatives thereof. In some embodiments, one or more chemotherapeutic agents are employed including, but not limited to, a platinum compound and a taxane compound. In some embodiments, one or more chemotherapeutic agents are selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, cabazitaxel, and derivatives thereof.

As used herein, the term “combination” refers to a single composition/formulation comprising the therapeutic compound and one or more chemotherapeutic agent; or a kit comprising the therapeutic compound and one or more chemotherapeutic agent as separate dosage forms; or the therapeutic compound and one or more chemotherapeutic agent provided as separate dosage forms (i.e., not in the form of a kit) as long as the effect achieved is commensurate with the intended purpose of the invention, i.e., to synergistically work for treatment of cancer.

The term “subject” or “patient” as used herein refers to any mammal including, without limitation, humans and other primates (e.g., chimpanzees and other apes and monkey species), farm animals (e.g., cattle, sheep, pigs, goats and horses), domestic mammals (e.g., dogs and cats), and laboratory animals (e.g., rodents such as mice, rats, and guinea pigs). In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

The term “about” as used herein encompasses variations of +/- 10% and more preferably +/- 5%, as such variations are appropriate for practicing the present invention.

The present disclosure provides a combination comprising a therapeutic compound and one or more chemotherapeutic agents. In some embodiments, the present disclosure provides a combination comprising a therapeutic compound selected from calcimycin, romidepsin, panobinostat, and A-1331852 and one or more chemotherapeutic agents selected from a platinum compound and a taxane compound. In some embodiments, the combination comprises a therapeutic compound selected from calcimycin, romidepsin, panobinostat, and A-1331852 and one or more platinum compounds (e.g., carboplatin, cisplatin, and oxaliplatin). In some embodiments, the combination comprises a therapeutic compound selected from calcimycin, romidepsin, panobinostat, and A-1331852 and one or more taxane compounds (e.g., paclitaxel, docetaxel, and cabazitaxel).

In some embodiments, provided herein is a combination comprising one or more therapeutic compounds selected from calcimycin, romidepsin, panobinostat, and A-1331852 and one or more chemotherapeutic agents selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel. In some embodiments, the combination comprises calcimycin and one or more chemotherapeutic agents selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel. In some embodiments, the combination comprises romidepsin and one or more chemotherapeutic agents selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel. In some embodiments, the combination comprises panobinostat and one or more chemotherapeutic agents selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel. In some embodiments, the combination comprises A-1331852 and one or more chemotherapeutic agents selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel.

In some embodiments, also contemplated herein is a combination comprising more than one therapeutic compound and one or more chemotherapeutic agents. That is, in some embodiments, the combination comprises two or more therapeutic compounds selected from calcimycin, romidepsin, panobinostat, and A-1331852 and one or more chemotherapeutic agents selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel.

In some embodiments, the present disclosure provides a combination selected from Table A. Accordingly, every single combination provided in Table A represents a separate embodiment of the present disclosure. However, the present disclosure also envisages a merger or mixture of these embodiments to provide for combinations, including compositions and kits of the present disclosure. Thus, for the purposes of the present disclosure, each of the combinations that are derivable from Table A below are envisaged to exist individually, all together or in different combinations within the ambit of the present disclosure.
Table A
Chemotherapeutic Agent Therapeutic compound
Calcimycin Romidepsin Panobinostat A-1331852
Carboplatin X X X X
Cisplatin X X X X
Oxaliplatin X X X X
Paclitaxel X X X X
Docetaxel X X X X
Cabazitaxel X X X X
Carboplatin + Paclitaxel X X X X
Carboplatin + Docetaxel X X X X
Carboplatin + Cabazitaxel X X X X
Cisplatin + Paclitaxel X X X X
Cisplatin + Docetaxel X X X X
Cisplatin + Cabazitaxel X X X X
Oxaliplatin + Paclitaxel X X X X
Oxaliplatin + Docetaxel X X X X
Oxaliplatin + Cabazitaxel X X X X
Carboplatin + Paclitaxel + Docetaxel X X X X
Carboplatin + Paclitaxel + Cabazitaxel X X X X
Carboplatin + Docetaxel + Cabazitaxel X X X X
Cisplatin + Paclitaxel + Docetaxel X X X X
Cisplatin + Paclitaxel + Cabazitaxel X X X X
Cisplatin + Docetaxel + Cabazitaxel X X X X
Oxaliplatin + Paclitaxel + Docetaxel X X X X
Oxaliplatin + Paclitaxel + Cabazitaxel X X X X
Oxaliplatin + Docetaxel + Cabazitaxel X X X X
Carboplatin + Paclitaxel + Docetaxel + Cabazitaxel X X X X
Cisplatin + Paclitaxel + Docetaxel + Cabazitaxel X X X X
Oxaliplatin + Paclitaxel + Docetaxel + Cabazitaxel X X X X
Paclitaxel + Carboplatin + Cisplatin X X X X
Docetaxel + Carboplatin + Cisplatin X X X X
Cabazitaxel + Carboplatin + Cisplatin X X X X
Paclitaxel + Carboplatin + Oxaliplatin X X X X
Docetaxel + Carboplatin + Oxaliplatin X X X X
Cabazitaxel + Carboplatin + Oxaliplatin X X X X
Paclitaxel + Cisplatin + Oxaliplatin X X X X
Docetaxel + Cisplatin + Oxaliplatin X X X X
Cabazitaxel + Cisplatin + Oxaliplatin X X X X
Paclitaxel + Carboplatin + Cisplatin + Oxaliplatin X X X X
Docetaxel + Carboplatin + Cisplatin + Oxaliplatin X X X X
Cabazitaxel + Carboplatin + Cisplatin + Oxaliplatin X X X X
Carboplatin + Cisplatin X X X X
Carboplatin + Oxaliplatin X X X X
Cisplatin + Oxaliplatin X X X X
Carboplatin + Cisplatin + Oxaliplatin X X X X
Paclitaxel + Docetaxel X X X X
Paclitaxel + Cabazitaxel X X X X
Docetaxel + Cabazitaxel X X X X
Paclitaxel + Docetaxel + Cabazitaxel X X X X
Paclitaxel + Docetaxel + Carboplatin + Cisplatin X X X X
Paclitaxel + Docetaxel + Carboplatin + Oxaliplatin X X X X
Paclitaxel + Docetaxel + Cisplatin + Oxaliplatin X X X X
Paclitaxel + Docetaxel + Carboplatin + Cisplatin + Oxaliplatin X X X X
Paclitaxel + Cabazitaxel + Carboplatin + Cisplatin X X X X
Paclitaxel + Cabazitaxel + Carboplatin + Oxaliplatin X X X X
Paclitaxel + Cabazitaxel + Cisplatin + Oxaliplatin X X X X
Paclitaxel + Cabazitaxel + Carboplatin + Cisplatin + Oxaliplatin X X X X
Docetaxel + Cabazitaxel + Carboplatin + Cisplatin X X X X
Docetaxel + Cabazitaxel + Carboplatin + Oxaliplatin X X X X
Docetaxel + Cabazitaxel + Cisplatin + Oxaliplatin X X X X
Docetaxel + Cabazitaxel + Carboplatin + Cisplatin + Oxaliplatin X X X X
Paclitaxel + Docetaxel + Cabazitaxel + Carboplatin + Cisplatin + Oxaliplatin X X X X

Embodiments covered by Table A are explained below with the example of the first row of the table. The first row of Table A shows carboplatin as the chemotherapeutic agent and calcimycin, romidepsin, panobinostat, and A-1331852 as therapeutic compounds. “X” mark is placed in each cell of the first row. This indicates the following combinations:

Carboplatin + calcimycin
Carboplatin + romidepsin
Carboplatin + panobinostat
Carboplatin + A-1331852
Carboplatin + calcimycin + romidepsin
Carboplatin + calcimycin + panobinostat
Carboplatin + calcimycin + A-1331852
Carboplatin + romidepsin + panobinostat
Carboplatin + romidepsin + A-1331852
Carboplatin + panobinostat + A-1331852
Carboplatin + calcimycin + romidepsin + panobinostat
Carboplatin + calcimycin + romidepsin + A-1331852
Carboplatin + calcimycin + panobinostat + A-1331852
Carboplatin + calcimycin + romidepsin + panobinostat + A-1331852

Thus, each row of Table A indicates all possible permutations and combinations of the chemotherapeutic agent(s) listed in that row with one or more therapeutic compounds listed in that row.

In some embodiments, the combination is a single formulation comprising a mixture of the therapeutic compound and the chemotherapeutic agent. In this embodiment, both the components - the therapeutic compound and the chemotherapeutic agent- are present in a single composition/formulation. That is, in this embodiment, the therapeutic compound and the chemotherapeutic agent are administered to the subject together as part of a single formulation. In the embodiments where more than one chemotherapeutic agent is present, the therapeutic compound and at least one chemotherapeutic agent are present in a single composition/formulation and additional chemotherapeutic agent(s) can be present in the same composition/formulation or can be administered as separate composition/formulation.

In some embodiments, the combination is a kit comprising the therapeutic compound and the one or more chemotherapeutic agents as separate formulations. In this embodiment, the therapeutic compound and the one or more chemotherapeutic agents are not mixed together to form a single formulation; rather, they are present as separate formulations or separate dosage forms in a kit. That is, in this embodiment, the therapeutic compound and the one or more chemotherapeutic agents are administered to the subject as separate formulations/dosage forms – either concurrently, or one after the other, or at different time intervals.

The combinations of the present disclosure comprising a therapeutic compound and one or more chemotherapeutic agents are effective in treating a cancer. Accordingly, provided herein are methods for treating a cancer comprising administering to a subject any combination disclosed herein comprising a therapeutic compound and one or more chemotherapeutic agents.
In some embodiments, a method for treating a cancer comprises administering to a subject a combination comprising a therapeutic compound selected from calcimycin, romidepsin, panobinostat, and A-1331852 and one or more chemotherapeutic agents selected from a platinum compound and a taxane compound. In some embodiments, a method for treating a cancer comprises administering to a subject a combination comprising a therapeutic compound selected from calcimycin, romidepsin, panobinostat, and A-1331852 and one or more chemotherapeutic agents selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel.
In some embodiments, a method for treating a cancer comprises administering to a subject one or more therapeutic compounds selected from calcimycin, romidepsin, panobinostat, and A-1331852 and one or more chemotherapeutic agents selected from a platinum compound and a taxane compound. In some embodiments, a method for treating a cancer comprises administering to a subject a therapeutic compound selected from calcimycin, romidepsin, panobinostat, and A-1331852 and one or more chemotherapeutic agents selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel.

In some embodiments, a method for treating a cancer comprises administering to a subject calcimycin and one or more chemotherapeutic agents selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel. In some embodiments, a method for treating a cancer comprises administering to a subject romidepsin and one or more chemotherapeutic agents selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel. In some embodiments, a method for treating a cancer comprises administering to a subject panobinostat and one or more chemotherapeutic agents selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel. In some embodiments, a method for treating a cancer comprises administering to a subject A-1331852 and one or more chemotherapeutic agents selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel.

In some embodiments, a cancer treated according to the methods of the present disclosure comprises cancer cells that form spheroids. In some embodiments, a cancer treated according to the methods of the present disclosure is selected from an ovarian cancer, a uterine cancer, a cervical cancer, a colorectal cancer, a gastric cancer, mesothelioma, and a peritoneal cancer. In some embodiments, the cancer treated according to the methods of the present disclosure is a primary tumor. In some embodiments, the cancer treated according to the methods of the present disclosure is a metastatic tumor. Accordingly, in some embodiments, the cancer treated according to the methods of the present disclosure is of any grade, status, or stage, as long as a person skilled in the art or a medical practitioner deems administration of the combinations of the present disclosure fit and/or necessary for treatment of such a cancer.

In some embodiments, the present disclosure provides a method for treating an aggressive metastatic ovarian cancer comprising administering to a subject one or more therapeutic compounds selected from calcimycin, romidepsin, panobinostat, and A-1331852 and one or more chemotherapeutic agents selected from a platinum compound (e.g., carboplatin, cisplatin, and oxaliplatin) and a taxane compound (e.g., paclitaxel, docetaxel, and cabazitaxel). The term “aggressive metastatic ovarian cancer” as used herein refers to stage 3/stage 4 ovarian cancer where the cancer has spread away from the ovary to other body organs in the pelvis and abdomen, such as the mesentery, liver or lungs.

In some embodiments, the present disclosure provides a method for treating a metastatic niche of ovarian carcinoma comprising administering to a subject one or more therapeutic compounds selected from calcimycin, romidepsin, panobinostat, and A-1331852 and one or more chemotherapeutic agents selected from a platinum compound (e.g., carboplatin, cisplatin, and oxaliplatin) and a taxane compound (e.g., paclitaxel, docetaxel, and cabazitaxel). The term “metastatic niche” as used herein refers to a tumor-supportive and immune suppressive environment which is molecularly and functionally different from the niche at the primary tumor site. It results from a co-evolution of cancer cells and the surrounding microenvironment.

In some embodiments, the methods of the present disclosure comprise administering romidepsin to a subject in an amount of about 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 mg/m2, including values and ranges therebetween.

In some embodiments, the methods of the present disclosure comprise administering panobinostat to a subject in an amount of about 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, or 25 mg per day, including values and ranges therebetween.

In some embodiments, the methods of the present disclosure comprise administering calcimycin to a subject in an amount of about 5 ng/ml to about 5 µg/ml, including values and ranges therebetween. For example, in some embodiments, calcimycin is administered to the subject in an amount of about 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, or 5000 ng/ml, including values and ranges therebetween.

In some embodiments, the methods of the present disclosure comprise administering A-1331852 to a subject in an amount of about 500 mg to about 1500 mg per day, including values and ranges therebetween. In some embodiments, A-1331852 is administered to a subject in an amount of about 500, 600, 700, 750, 800, 900, 1000, 1100, 1200, 1250, 1300, 1400, or 1500 mg per day, including values and ranges therebetween.

In some embodiments, the methods of the present disclosure comprise administering carboplatin to a subject in an amount of about 100, 110, 120, 125, 130, 140, 150, 160, 170, 175, 180, 190, 200, 210, 220, 225, 230, 240, 250, 260, 270, 275, 280, 290, 300, 310, 320, 325, 330, 340, 350, 360, 370, 375, 380, 390, 400, 410, 420, or 425 mg/m2, including values and ranges therebetween.

In some embodiments, the methods of the present disclosure comprise administering cisplatin to a subject in an amount of about 2.5, 5, 7.5, 10, 12.5, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 mg/m2, including values and ranges therebetween.

In some embodiments, the methods of the present disclosure comprise administering oxaliplatin to a subject in an amount of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg/m2, including values and ranges therebetween.

In some embodiments, the methods of the present disclosure comprise administering paclitaxel to a subject in an amount of about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 mg/m2, including values and ranges therebetween.

In some embodiments, the methods of the present disclosure comprise administering docetaxel to a subject in an amount of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg/m2, including values and ranges therebetween.

In some embodiments, the methods of the present disclosure comprise administering cabazitaxel to a subject in an amount of about 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, or 25 mg/m2, including values and ranges therebetween.

In some embodiments, calcimycin and the platinum compound are present in the combination or are administered to the patient in a ratio of about 1:50 to about 1:400, including values and ranges thereof, such as about 1:100, 1:150, 1:200, 1:250, 1:300, about 1:100 to about 1:300, and the like.

In some embodiments, calcimycin and the taxane compound are present in the combination or are administered to the patient in a ratio of about 1:1 to about 1:20, including values and ranges thereof, such as about 1:1, 1:2, 1:4, 1:5, 1:8, 1:10, 1:20, about 1:1 to about 1:10, about 1:1 to about 1:5, and the like. In some other embodiments, calcimycin and the taxane compound are present in the combination or are administered to the patient in a ratio of about 2:1 to about 10:1, including values and ranges thereof, such as about 5:1, 8:1, 10:1 and the like.

In some embodiments, romidepsin and the platinum compound are present in the combination or are administered to the patient in a ratio of about 1:50 to about 1:400, including values and ranges thereof, such as about 1:100, 1:150, 1:200, 1:250, 1:300, about 1:100 to about 1:300, and the like.

In some embodiments, romidepsin and the taxane compound are present in the combination or are administered to the patient in a ratio of about 2:1 to about 20:1, including values and ranges thereof, such as about 4:1, 5:1, 8:1, 10:1, 15:1, about 4:1 to about 10:1, and the like.

In some embodiments, panobinostat and the platinum compound are present in the combination or are administered to the patient in a ratio of about 1:50 to about 1:1500, including values and ranges thereof, such as about 1:50, 1:100, 1:200, 1:250, 1:300, 1:400, 1:500, 1:600, 1:700, 1:750, 1:800, 1:900, 1:1000, 1:1200, 1:400, about 1:100 to about 1:1000, about 1:100 to about 1:750, about 1:100 to about 1:500, and the like.

In some embodiments, panobinostat and the taxane compound are present in a combination or are administered to the patient in a ratio of about 1:1 to about 10:1, including values and ranges thereof, such as about 1:1, 2:1, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 7.5:1, 8:1, 10:1, about 1:1 to about 1:8, about 1:1 to about 1:5, about 1:1 to about 1:4, and the like.

In some embodiments, A-1331852 and the platinum compound are present in the combination or are administered to the patient in a ratio of about 1:400 to about 1:1500, including values and ranges thereof, such as about 1:500, 1:700, 1:750, 1:800, 1:1000, 1:1200, about 1:500 to about 1:1000, about 1:700 to about 1:1000, about 1:700 to about 1:1200 and the like.

In some embodiments, A-1331852 and the taxane compound are present in the combination or are administered to the patient in a ratio of about 1:1 to about 1:400, including values and ranges thereof, such as about 1:1, 1:5, 1:10, 1:20, 1:25, 1:50, 1:75, 1:100, 1:200, 1:250, 1:300, about 1:1 to about 1:10, about 1:1 to about 1:50, about 1:1 to about 1:100, about 1:1 to about 1:200, and the like.

As the present disclosure provides a combination therapy comprising administering one or more therapeutic compounds selected from calcimycin, romidepsin, panobinostat, and A-1331852 and one or more chemotherapeutic agents selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel; it would be understood that the dosing regimen disclosed herein for calcimycin, romidepsin, panobinostat, and A-1331852 is combined with the dosing regimen disclosed herein for one or more chemotherapeutic drugs selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel. For example, in one embodiment, the method for treating cancer comprises: (i) administering romidepsin to a subject in an amount of about 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 mg/m2; and (ii) administering carboplatin to the subject in an amount of about 100, 110, 120, 125, 130, 140, 150, 160, 170, 175, 180, 190, 200, 210, 220, 225, 230, 240, 250, 260, 270, 275, 280, 290, 300, 310, 320, 325, 330, 340, 350, 360, 370, 375, 380, 390, 400, 410, 420, or 425 mg/m2. Thus, any combination of the dosing regimen disclosed herein for the four therapeutic compounds (calcimycin, romidepsin, panobinostat, and A-1331852) with the dosing regimen disclosed herein for one or more chemotherapeutic drugs selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel is encompassed by the present disclosure.

In some embodiments, a method for treating cancer comprises administering to a subject any of the combinations disclosed herein as a single formulation comprising the therapeutic compound and the one or more chemotherapeutic agents. In some embodiments, a method for treating cancer comprises administering to a subject any of the combinations disclosed herein comprising the therapeutic compound and the one or more chemotherapeutic agents as separate formulations.
In some embodiments, the formulation(s)is/are administered to a subject orally, parenterally, or topically. In embodiments where the therapeutic compound and one or more chemotherapeutic agents are administered as separate formulations, all formulations can be administered using the same route of administration (e.g., orally, parenterally, or topically) or one formulation can be administered using one route of administration (e.g., orally, parenterally, or topically) and the other formulation(s) can be administered using the other routes of administration (e.g., if one is administered orally, the other is administered parenterally or topically or vice versa).

In some embodiments, the therapeutic compound (e.g., calcimycin, romidepsin, panobinostat, and A-1331852) is administered orally, parenterally, or topically. In some embodiments, one or more chemotherapeutic agents are administered orally, parenterally, or topically.

Parenteral administration comprises administration via injection or infusion. In some embodiments, parenteral administration is selected from intravenous, intramuscular, intradermal, subcutaneous, intratumoral, intralesional, intraperitoneal, and intrathecal administration. In some embodiments, parenteral administration is administration via intravenous infusion.

The combinations of the present disclosure comprising the therapeutic compound and one or more chemotherapeutic agents are cytotoxic to cancer cells. In some embodiments, the level of cytotoxicity exhibited by the combination is statistically significantly more than that exhibited by the therapeutic compound and one or more chemotherapeutic agents alone. In some embodiments, the combination comprising the therapeutic compound and one or more chemotherapeutic agents shows a synergistic effect against cancer cells compared to the sum of effects shown by the therapeutic compound and one or more chemotherapeutic agents alone.

In some embodiments, administration of the combinations disclosed herein comprising the therapeutic compound and one or more chemotherapeutic agents provides about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90% or 100%, including values and ranges therebetween, reduction in the number of cancer cells compared to the number of cancer cells prior to administration of the combination.

In some embodiments, reduction in the number of cancer cells provided by the combinations disclosed herein comprising the therapeutic compound and one or more chemotherapeutic agents is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90% or 100% higher than that provided by the therapeutic compound and the chemotherapeutic agents alone.

In some embodiments, administration of the combinations disclosed herein comprising the therapeutic compound and one or more chemotherapeutic agents provides about 2.5-fold, 3-fold, 4-fold, 5-fold, 7.5-fold, 8-fold, 10-fold, 12-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 80-fold, or 90-fold, including values and ranges therebetween, reduction in the number of cancer cells compared to the number of cancer cells prior to administration of the combination.

In some embodiments, reduction in the number of cancer cells provided by the combinations disclosed herein comprising the therapeutic compound and one or more chemotherapeutic agents is about 2.5-fold, 3-fold, 4-fold, 5-fold, 7.5-fold, 8-fold, 10-fold, 12-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 80-fold, or 90-fold higher than that provided by the therapeutic compound and the chemotherapeutic agents alone.

In some embodiments, the combinations of the present disclosure comprising the therapeutic compound and one or more chemotherapeutic agents exhibit the Combination Index (CI) values of less than 1 when measured by a method described by Chou and Talalay in an article entitled “Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors,” Adv Enzyme Regul. 1984;22:27–55. According to this method, the combination index equation for two drugs is: CI = (D)1/(Dx)1+(D)2/(Dx)2, where (Dx) 1, (Dx) 2 = the concentration of the tested Drug1 and Drug 2 used in the single treatment that was required to decrease the cell viability by x% and (D) 1, (D) 2 = the concentration of the Drug1 in combination with the concentration of the Drug2 that together decreased the cell viability by x%. The CI Value of less than 1 (CI<1) indicates synergism, the CI value equal to 1 (CI=1) indicates additive effect and the CI value of more than 1 (CI>1) indicates antagonism.

The single formulation or the separate formulations comprising the therapeutic compound and one or more chemotherapeutic agents can be any pharmaceutically acceptable dosage forms. In some embodiments, pharmaceutically acceptable dosage forms are selected from an oral dosage form, a parenteral dosage form, a peritoneal wash, or a topical dosage form. Oral dosage forms can be discrete units, such as hard or soft capsules, tablets, pills, or lozenges; or a liquid form such as emulsions, solutions, suspensions, syrups, and elixirs. Parenteral dosage forms can be a liquid form such as emulsions, solutions, and suspensions or a solid form packaged in a single-dose or multidose containers that is reconstituted prior to administration. In some embodiments, parenteral dosage form is a ready-to-use (RTU) liquid form. Topical dosage forms can be solutions, collodion, suspensions, emulsions (e.g., lotions), and semisolids (e.g., foams, ointments, pastes, creams, and gels), etc. A peritoneal wash can be any sterile physiological solution (e.g., physiological saline, a phosphate buffer, and the like) comprising the therapeutic compound and one or more chemotherapeutic agents as a single formulation or a separate formulation.

In some embodiments, the present disclosure provides a combination comprising one or more therapeutic compounds selected from calcimycin, romidepsin, panobinostat, and A-1331852 and one or more chemotherapeutic agents selected from a platinum compound and a taxane compound for use in treating cancer. In some embodiments, the present disclosure provides a combination comprising one or more therapeutic compounds selected from calcimycin, romidepsin, panobinostat, and A-1331852 and one or more chemotherapeutic agents selected fromcarboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel for use in treating cancer. In some embodiments, the present disclosure provides a combination selected from Table A for use in treating cancer. In some embodiments, said combination for use in treating cancer is a single formulation comprising the therapeutic compound and the one or more chemotherapeutic agents. In some embodiments, said combination for use in treating cancer is a kit comprising the therapeutic compound and the one or more chemotherapeutic agent as separate formulations. In some embodiments, the present disclosure provides a combination comprising one or more therapeutic compounds selected from calcimycin, romidepsin, panobinostat, and A-1331852 and one or more chemotherapeutic agents selected fromcarboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel for use in treating cancer selected from an ovarian cancer, a uterine cancer, a cervical cancer, a colorectal cancer, and a gastric cancer. The dosing amounts and the routes of administration for the four therapeutic compounds (calcimycin, romidepsin, panobinostat, and A-1331852) and for the chemotherapeutic agents (carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel) are as disclosed herein.
In some embodiments, the present disclosure provides use of a combination comprising one or more therapeutic compounds selected from calcimycin, romidepsin, panobinostat, and A-1331852 and one or more chemotherapeutic agents selected from a platinum compound and a taxane compound for treating cancer. In some embodiments, the present disclosure provides use of a combination comprising one or more therapeutic compounds selected from calcimycin, romidepsin, panobinostat, and A-1331852 and one or more chemotherapeutic agents selected fromcarboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel for treating cancer. In some embodiments, the present disclosure provides use of a combination selected from Table A for treating cancer. In some embodiments, the present disclosure provides use of a therapeutic compound selected from calcimycin, romidepsin, panobinostat, and A-1331852 as a medicament in combination with one or more chemotherapeutic agents selected from a platinum compound and a taxane compound for treating cancer. In some embodiments, said combination in said use is a single formulation comprising the therapeutic compound and the one or more chemotherapeutic agents. In some embodiments, said combination in said use is a kit comprising the therapeutic compound and the one or more chemotherapeutic agent as separate formulations. In some embodiments, the present disclosure provides use of a combination comprising one or more therapeutic compounds selected from calcimycin, romidepsin, panobinostat, and A-1331852 and one or more chemotherapeutic agents selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel for treating cancer selected from an ovarian cancer, a uterine cancer, a cervical cancer, a colorectal cancer, and a gastric cancer. The dosing amounts and the routes of administration for the four therapeutic compounds (calcimycin, romidepsin, panobinostat, and A-1331852) and for the chemotherapeutic agents (carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel) are as disclosed herein.

In some embodiments, the present disclosure provides a single formulation comprising one or more therapeutic compounds selected from calcimycin, romidepsin, panobinostat, and A-1331852 and one or more chemotherapeutic agents selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel. In some embodiments, the single formulation comprises calcimycin and one or more chemotherapeutic agents selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel. In some embodiments, the single formulation comprises romidepsin and one or more chemotherapeutic agents selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel. In some embodiments, the single formulation comprises panobinostat and one or more chemotherapeutic agents selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel. In some embodiments, the single formulation comprises A-1331852 and one or more chemotherapeutic agents selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel.

The present disclosure provides a kit comprising one or more therapeutic compounds selected from calcimycin, romidepsin, panobinostat, and A-1331852 and one or more chemotherapeutic agents selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel as separate formulations. In some embodiments, the kit comprises calcimycin and one or more chemotherapeutic agents selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel as separate formulations. In some embodiments, the kit comprises romidepsin and one or more chemotherapeutic agents selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel as separate formulations. In some embodiments, the kit comprises panobinostat and one or more chemotherapeutic agents selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel as separate formulations. In some embodiments, the kit comprises A-1331852 and one or more chemotherapeutic agents selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel as separate formulations.

It is to be understood that the foregoing descriptive matter is illustrative of the disclosure and not a limitation. While considerable emphasis has been placed herein on the particular features of this disclosure, it will be appreciated that various modifications can be made, and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. Those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein. Similarly, additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon description provided herein.

Descriptions of well-known/conventional methods/steps and techniques are omitted so as to not unnecessarily obscure the embodiments herein. Further, the disclosure herein provides for examples illustrating the above-described embodiments, and in order to illustrate the embodiments of the present disclosure certain aspects have been employed. The examples used herein for such illustration are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the following examples should not be construed as limiting the scope of the embodiments herein.
INCORPORATION BY REFERENCE
All references, articles, publications, patents, patent publications, and patent applications (if any) cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

EXAMPLES

Example 1: Identification of therapeutic compounds effective against ovarian cancer cells
To identify compounds that can be re-purposed for treatment of spheroid-forming cancer, the inventors established a spheroid-based phenotypic screening platform for high-throughput screening. The inventors employed an efficient method for generating the spheroids based on the methods described by Friedrich J et al., in the article entitled “Spheroid-based drug screen: considerations and practical approach” (Nat Protoc. 2009;4:309–324) and Kelm JM et al., in the article entitled “Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types” (BiotechnolBioeng. 2003;83(2):173-180. doi:10.1002/bit.10655). FIG. 1, panel “a” shows a schematic for generating 3D spheroids. FIG. 1, panel “b” shows the spheroids obtained on day 7 after seeding of cells.

The inventors employed cell viability assays- resazurin assay and acid phosphatase assays (Friedrich J, Eder W, Castaneda J, et al. “A reliable tool to determine cell viability in complex 3-d culture: the acid phosphatase assay.” J Biomol Screen. 2007 Dec;12(8):1115-9. doi:10.1177/1087057107306839; Ivanov DP, Parker TL, Walker DA, Alexander C, Ashford MB, et al. (2014) “Multiplexing Spheroid Volume, Resazurin and Acid Phosphatase Viability Assays for High-Throughput Screening of Tumour Spheroids and Stem Cell Neurospheres.” PLoS ONE 9(8): e103817. doi:10.1371/journal.pone.0103817) to determine the cytotoxic effects of drugs on OVCAR3 spheroids. FIG. 1, panel “c” shows a schematic and timeline of generating spheroids, screening of the library of compounds against the spheroids, followed by cytotoxicity/viability assays. FIG. 1, panel “d” shows Z-scores calculated for the screening assays with different conditions. A Z-score of >0.5 indicated that the assay was reliable (the assay conditions shown in FIG. 1, panel “c” provided the Z-score of > 0.5).

A library of 1427 compounds was assembled from Sigma (LOPAC) and NIH (Oncology set and Mechanistic set) and screened in the OVCAR3- MCTS model at a final concentration of 10?µM in 0.5% DMSO (v/v). To produce spheroids, OVCAR3 cells (obtained from ATCC (American Type Culture Collection), Manassas, Virgina, USA) were seeded at a density of 105 cells/well into 96-well flat bottom low attachment microplates (96 well-plate coated with polyHEMA). The plates were then incubated at 37?°C in a humidified atmosphere of 5% CO2 for 4 days. For testing of compounds, a 4-µL sample of each compound was transferred into an intermediate 96-well plate. The compounds were mixed with 16?µL defined medium per well. Subsequently, a 10-µL sample of each compound was dispensed into each well of a 96-well assay plate in duplicates. The plates were then incubated at 37?°C in a humidified atmosphere of 5% CO2 for another 3 days. Controls were added to each assay plate with puromycin at 10µg/ml as a positive control, 0.5% DMSO (v/v) as a vehicle control and an untreated control. After 3 days, the therapeutic potential of the compounds on ovarian carcinoma cell line OVCAR-3 was investigated using resazurin and acid phosphatase activity-based assays. The resazurin assay is a fluorometric assay used to measure the metabolic capacity of live cells and is based on the reduction of oxidized non-fluorescent blue resazurin to a red fluorescent dye (resorufin) by the mitochondrial respiratory chain in live cells. Acid phosphatase assay is used for determination of cell growth based on quantification of cytosolic acid phosphatase activity. The assay is based on the hydrolysis of the 4-methylumbelliferyl phosphate by intracellular acid phosphatases in viable cells to produce soluble fluorescent substance - methylumbelliferone. In addition, morphological assessment was also performed prior to and after treatment.

FIG. 2 shows the screening of compounds from the LOPAC library. Panel “a” shows cell viability of cells from the 3D-spheroids using resazurin (left graph) and APH assay (right graph) plotted for 1280 drugs. The ‘hit’ threshold was set to 0.75 for cell death. Panel “b” shows a correlation plot for resazurin and APH assay. Panel “c” shows compounds showing effects in resazurin and APH assay and also affecting the spheroid morphology. These compounds were selected for further studies. Panel “d” shows the inverted pyramid scheme showing the screening process to arrive at the lead compounds.

FIG. 3 shows the screening of compounds from the NIH library. Panel “a” shows cell viability of cells from the 3D-spheroids using resazurin (top graph) and APH assay (bottom graph) plotted for 147 drugs. The ‘hit’ threshold was set to 0.5 for cell death. Panel “b” shows compounds showing effects in resazurin and APH assay and also affecting the spheroid morphology. These compounds were selected for further studies. Panel “c” shows the inverted pyramid scheme showing the screening process to arrive at the lead compounds.

48 compounds from LOPAC (out of a total of 1280 compounds) and 11 compounds from the NIH library (out of a total of 147 compounds) were identified that show a decrease in cell proliferation. Out of the 48 actives identified from the LOPAC library screen, 10 compounds were chosen for further studies, based on an in-house microarray database of the target gene expression in cancer spheroids; some interesting targets include the ion transport system and the mitochondrial oxidative phosphorylation. From the NIH library screen, 10 drugs out of the 11 actives that also showed change in spheroid morphology were chosen for further studies.

Example 2: Evaluation of selected compounds
To verify the efficacy of the selected compounds, the selected compounds were further evaluated using a more resistant ovarian cancer cell line SKOV-3 (ATCC (American Type Culture Collection), Manassas, Virgina, USA). The efficacy of the compounds was compared using the 2D and 3D cultures for each drug treatment. To assess the drug concentration-effect, spheroids were generated in 96-well plates and incubated with a concentration ranging from 1 nM to 30 µM for 72 hours. Cell viability was measured using using resazurin and acid phosphatase activity-based assays.

One of the selected compounds was Venetoclax. Venetoclax is a B-cell lymphoma-2 (BCL-2) inhibitor, it possesses specificity only for BCL-2. For validation, another compound, A-1331852, was procured. A-1331852 is a new BCL-XL inhibitor that is engineered to avoid interaction with BCL-2 and BCL-W but have selectivity for BCL- XL.

Dose response studies were conducted using the shortlisted 20 compounds. From the dose response study, four compounds - calcimycin, romidepsin, panobinostat, and A-1331852 - showed synergy in combination with carboplatin, paclitaxel, etc. Accordingly, these four compounds were employed for further studies.

All 4 compounds reduced the cell viability in a dose dependent manner with an EC50 measured in both OVCAR3 and SKOV3 spheroids (Table 1).

Table 1: EC50 values of the hits in OVCAR3 and SKOV3 spheroids

Drugs EC50(µM)
OVCAR3 Spheroids SKOV3 Spheroids
Calcimycin 3.86 1.84
Romidepsin 0.133 0.286
Panobinostat 1 5.4
A-1331852 0.83 0.127 * 10-3

The efficacy of the four therapeutic compounds was further evaluated using OVCAR3, SKOV3 spheroids and compared in the 2D and 3D cultures for each compound treatment. FIG. 4, panel “a” shows dose response curves for Calcimycin, Romidepsin, Panobinostat, and A-1331852 against 2D cultures and 3D spheroids of OVCAR3 and SKOV3. Panel “b” shows a morphology change in drug-treated spheroids and untreated control.

Drug concentration-effect was also assessed for Carboplatin, Paclitaxel, and Docetaxel against OVCAR3 and SKOV3 2D-culture and 3D-spheroids. FIG. 4, panel “c” shows dose response curves for the chemotherapeutic agents - Carboplatin, Paclitaxel, and Docetaxel - against 2D and 3D cultures of OVCAR3 and SKOV3.

Example 3: Evaluation of combinations comprising the identified therapeutic compounds and chemotherapeutic agents
Effective drug combination is a useful strategy for enhancing anticancer activity, as it provides powerful means of targeting multiple cellular anomalies with the potential to achieve synergistic therapeutic effects, overcome drug resistance and possibly reduce toxic side effects due to reduced dosage for each individual drug in a combination. With this in mind, the cytotoxic effects of the four identified therapeutic compounds (calcimycin, romidepsin, panobinostat, and A-1331852) were examined in combination with Carboplatin, Paclitaxel, and Docetaxel.

Drug combination studies were performed using the Combination Index (CI) method described above. OVCAR3 spheroids were grown for 4 days before addition of Calcimycin (0.1 µM - 3µM) /Romidepsin (0.01 µM - 1µM)/ Panobinostat (0.01 µM - 1µM)/ A-1331852 (0.1 µM –1.5µM) and Carboplatin (100µM-1000 µM) or Paclitaxel/Docetaxel (1 µM or 100 nM) and 3 days later cell viability was evaluated using resazurin assay followed by APH assay. The viability data were then analyzed using CompuSyn software that employs the method of Chou and Talalay described above to calculate the CI at each molar ratio evaluated. Drug combinations, which yielded CI values less than 1 were considered to be synergistic. When the combined effect is greater than the sum of effects provided by the agents singly, the combination is considered to be synergistic. Strong synergy was observed overall in both OVCAR3 and SKOV3 spheroids.
Synergistic interactions were observed as follows:
• In combinations with all tested concentrations (0.1 µM - 3µM) of Calcimycin and at concentrations higher than 100µM of Carboplatin.
• In combinations with all tested concentrations (0.01 µM - 1µM) of Romidepsin and at intermediate concentrations of Carboplatin (200 µM - 300µM).
• In combinations with higher concentrations of Panobinostat (0.62 µM, 1 µM) and at intermediate concentrations of Carboplatin (200 µM, 300 µM).
• Almost all combinations of concentrations of A-1331852 and Carboplatin except at lower (100 µM) concentrations of Carboplatin in some instance.
FIGs. 5A-5D show the results of the combination treatment studies carried out in this Example in OVCAR3 and SKOV3 spheroids. Combination index (CI) calculated is represented as heatmap. CI<1 is considered to be synergistic.

FIG. 5A shows the CI heat map based on the cell viability tested using resazurin assay for a combination of calcimycin with carboplatin, paclitaxel, and docetaxel.

FIG. 5B shows the CI heat map based on the cell viability tested using resazurin and APH assays for a combination of romidepsin with carboplatin, paclitaxel, and docetaxel.

FIG. 5C shows the CI heat map based on the cell viability tested using resazurin and APH assays for a combination of Panobinostat with carboplatin.

FIG. 5D shows the CI heat map based on the cell viability tested using resazurin and APH assays for a combination of A-1331852 with carboplatin.

These results demonstrate that 4 single agents- Calcimycin, Romidepsin, Panobinostat, and A-1331852 - identified through a quantitative drug screen of libraries in OVCAR3 cells (Examples 1 and 2), when combined with standard chemotherapeutic drugs exhibit higher potency. Thus, these combinations can be used in the targeted treatment of ovarian cancer including the aggressive metastatic form of the ovarian cancer.

Example 4: Evaluation of a combination comprising calcimycin and carboplatin
In this experiment, the viability of OVCAR-3 cells grown in 2D and in 3D spheroids and the viability of non-cancerous fallopian epithelia hTERT FT282 cells (ATCC (American Type Culture Collection), Manassas, Virgina, USA) in the presence of calcimycin individually, and in combination with carboplatin was measured. To assess the drug concentration-effect, monolayers and spheroids were generated in 96-well plates and incubated with concentrations of the drugs individually (100 nM to 3000 nM of calcimycin) or in combination (100 nM to 3000 nM of calcimycin and 100000 nM (i.e., 100 µM) carboplatin) for 72 hours. Cell viability was measured using using resazurin.

The viability of OVCAR-3 cells in monolayers was very poor in the presence of calcimycin - both when carboplatin was present and absent (˜20%). On the other hand, the viability of OVCAR3 cells was high in lower concentrations of calcimycin (100 nM), when used alone but decreases upon higher concentrations (3000 nM). In the presence of carboplatin, their viability is very low at a high concentration of both carboplatin (100 µM) and calcimyin (3000 nM). The viability of non-cancerous cells was higher than that for OVACR3 cells (2D and 3D) for all concentrations of calcimycin and carboplatin used. This shows the specificity of the combination of calcimycin and carboplatin in targeting ovarian cancer cells.
,CLAIMS:
1. A combination comprising one or more therapeutic compounds selected from calcimycin, romidepsin, panobinostat, and A-1331852 and one or more chemotherapeutic agents selected from a platinum compound and a taxane.
2. The combination as claimed in claim 1, wherein the platinum compound is selected from carboplatin, cisplatin, and oxaliplatin.
3. The combination as claimed in claim 1 or 2, wherein the taxane is selected from paclitaxel, docetaxel, and cabazitaxel.
4. The combination as claimed in any one of claims 1-3, wherein the therapeutic compound is calcimycin and the one or more chemotherapeutic agents are selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel.
5. The combination as claimed in any one of claims 1-3, wherein the therapeutic compound is romidepsin and the one or more chemotherapeutic agents are selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel.
6. The combination as claimed in any one of claims 1-3, wherein the therapeutic compound is panobinostat and the one or more chemotherapeutic agents are selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel.
7. The combination as claimed in any one of claims 1-3, wherein the therapeutic compound is A-1331852 and the one or more chemotherapeutic agents are selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel.
8. The combination as claimed in claim 1, wherein the combination is selected from Table A.
9. The combination as claimed in any one of claims 1-8, wherein the combination is a single formulation comprising the one or more therapeutic compound and the one or more chemotherapeutic agent.
10. The combination as claimed in any one of claims 1-8, wherein the combination is a kit comprising the one or more therapeutic compound and the one or more chemotherapeutic agent as separate formulations.
11. The combination as claimed in claim 9, wherein the formulation is an oral or a parenteral dosage form.
12. The combination as claimed in claim 10, wherein the separate formulations are selected from an oral dosage form and a parenteral dosage form.
13. A method for treating a cancer in a subject in need thereof, comprising administering to the subject one or more therapeutic compounds selected from calcimycin, romidepsin, panobinostat, and A-1331852 and one or more chemotherapeutic agents selected from a platinum compound and a taxane.
14. The method of claim13, wherein the platinum compound is selected from carboplatin, cisplatin, and oxaliplatin.
15. The method of claim 13 or 14, wherein the taxane is selected from paclitaxel, docetaxel, and cabazitaxel.
16. The method of claim 13, wherein the therapeutic compound is calcimycin and the one or more chemotherapeutic agents are selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel.
17. The method of claim 13, wherein the therapeutic compound is romidepsin and the one or more chemotherapeutic agents are selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel.
18. The method of claim 13, wherein the therapeutic compound is panobinostat and the one or more chemotherapeutic agents are selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel.
19. The method of claim 13, wherein the therapeutic compound is A-1331852 and the one or more chemotherapeutic agents are selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel.
20. The method as claimed in any one of claims 13-19, wherein the cancer is an ovarian cancer, a uterine cancer, a cervical cancer, a colorectal cancer, or a gastric cancer.
21. The method as claimed in any one of claims 13-20, wherein the cancer comprises cells that form spheroids.
22. The method as claimed in any one of claims 13-21, wherein the one or more therapeutic compounds and the one or more chemotherapeutic agents are administered to the subject as a single formulation.
23. The method as claimed in claim 22, wherein the formulation is administered to the subject orally or parenterally.
24. The method as claimed in any one of claim 13-21, wherein the one or more therapeutic compounds and the one or more chemotherapeutic agents are administered to the subject as separate formulations.
25. The method as claimed in claim 24, wherein the one or more therapeutic compounds are administered to the subject orally or parenterally and the one or more chemotherapeutic agents are administered to the subject parenterally.
26. The combination as claimed in any one of claims 1-12 for use in treating a cancer.
27. The combination for use as claimed in claim 26, wherein the cancer is an ovarian cancer, a uterine cancer, a cervical cancer, a colorectal cancer, or a gastric cancer.
28. The combination for use as claimed in claim 26, wherein the cancer comprises cells that form spheroids.
29. The combination for use as claimed in any one of claims 26-28, wherein the combination is a single formulation.
30. The combination for use as claimed in claim 29, wherein the formulation is an oral or a parenteral dosage form.
31. The combination for use as claimed in any one of claims 26-28, wherein the combination is a kit comprising the therapeutic compound and the chemotherapeutic agent as separate formulations.
32. The combination for use as claimed in claim 31, wherein the separate formulations are selected from an oral dosage form and a parenteral dosage form.
33. A kit comprising one or more therapeutic compounds selected from calcimycin, romidepsin, panobinostat, and A-1331852 and one or more chemotherapeutic agents selected from carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and cabazitaxel.