Patent Description:
Within cellular signaling networks, RAS and RAF play significant roles in the regulation of various biological processes including cell growth, proliferation, differentiation, inflammatory responses, and programmed cell death. Notably, mutations in RAS genes were the first genetic alterations identified in human cancer. Activating mutations of HRAS, NRAS, and KRAS ('RAS'), as well as BRAF are found frequently in several types of cancer.

ERK inhibitors for inhibiting cancer cell growth have been described (<CIT> and<NPL>). Combined inhibition of the Ral pathway (via blockade of the CDK5 activity) with inhibition of either the RAF/MEK/ERK pathway (via the MEK inhibitor U0126 or the Raf inhibitor sorafenib) or PI3k/Akt pathway (via the PI3K inhibitor LY294002) has been described to inhibit pancreatic cancer growth (<NPL>). Likewise, the combination of the CDK inhibitor Dinaciclib with inhibitors od either the PI3k/Akt pathway (via the pan-AKT inhibitor MK2206) or the RAF/MEK/ERK pathway (via the ERK inhibitor SCH772984) has been described to inhibit pancreatic cancer growth and metastases (<NPL>).

To date, progress has been slow in developing effective, longer term treatment options for patients suffering from cancer in which one or more mutations of RAS and/or RAF are present. For example, drug resistance is a common problem with many current MAPK inhibitors used today.

In view of the foregoing, there is, inter alia, a need for new methods for treating malignancies associated with the MAPK signaling pathway of which RAS and RAF are members. The present application is directed to meeting these and other needs.

Accordingly the invention relates to a first anti-cancer agent (i), which is BVD-<NUM> or a pharmaceutically acceptable salt thereof and a second anti-cancer agent (ii), which is a cyclin dependent kinase (CDK) inhibitor or a pharmaceutically acceptable salt thereof, for use in treating or ameliorating the effects of cancer in a subject, wherein the CDK inhibitor is selected from the group consisting of palbociclib, LEE-<NUM>, pharmaceutically acceptable salts thereof, and combinations thereof. The invention further relates to an in vitro method of effecting cancer cell death comprising contacting the cancer cell with an effective amount of (i) a first anti-cancer agent, which is BVD-<NUM> or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent the first and second anti-cancer agent which is a cyclin dependent kinase (CDK) inhibitor or a pharmaceutically acceptable salt thereof, wherein the CDK inhibitor is selected from the group consisting of palbociclib, LEE-<NUM>, pharmaceutically acceptable salts thereof, and combinations thereof. Furthermore, the invention relates to a kit for use in treating or ameliorating the effects of a cancer in a subject comprising an effective amount of (i) a first anti-cancer agent, which is BVD-<NUM> or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent the first and second anti-cancer agent which is a cyclin dependent kinase (CDK) inhibitor or a pharmaceutically acceptable salt thereof, packaged together with instructions for their use, wherein the CDK inhibitor is selected from the group consisting of palbociclib, LEE-<NUM>, pharmaceutically acceptable salts thereof, and combinations thereof. In addition, the invention also relates to a pharmaceutical composition for use in treating or ameliorating the effects of a cancer in a subject, the pharmaceutical composition comprising a pharmaceutically acceptable diluent or carrier and an effective amount of (i) a first anti-cancer agent, which is BVD-<NUM> or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent the first and second anti-cancer agent which is a cyclin dependent kinase (CDK) inhibitor or a pharmaceutically acceptable salt thereof, wherein the CDK inhibitor is selected from the group consisting of palbociclib, LEE-<NUM>, pharmaceutically acceptable salts thereof, and combinations thereof, and wherein administration of the first and second anti-cancer agents provides a synergistic effect compared to administration of either anti-cancer agent alone. The invention further relates to a composition comprising BVD-<NUM> or a pharmaceutically acceptable salt thereof for use in combination with a CDK inhibitor or a pharmaceutically acceptable salt thereof for use in treating or ameliorating the effects of cancer in a subject, wherein the CDK inhibitor is selected from the group consisting of palbociclib, LEE-<NUM>, pharmaceutically acceptable salts thereof, and combinations thereof. Furthermore the invention relates to a composition comprising a CDK inhibitor or a pharmaceutically acceptable salt thereof for use in combination with BVD-<NUM> or a pharmaceutically acceptable salt thereof for use in treating or ameliorating the effects of cancer in a subject, wherein the CDK inhibitor is selected from the group consisting of palbociclib, LEE-<NUM>, pharmaceutically acceptable salts thereof, and combinations thereof.

Further disclosed herein is a method of treating or ameliorating the effects of a cancer in a subject in need thereof. The method comprises administering to the subject an effective amount of (i) a first anti-cancer agent, which is BVD-<NUM> or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent, which is a CDK inhibitor or a pharmaceutically acceptable salt thereof, to treat or ameliorate the effects of the cancer.

Also disclosed is a method of treating or ameliorating the effects of a cancer in a subject in need thereof. The method comprises administering to the subject an effective amount of (i) a first anti-cancer agent, which is BVD-<NUM> or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent, which is selected from the group consisting of palbociclib, LEE-<NUM>, pharmaceutically acceptable salts thereof and combinations thereof, to treat or ameliorate the effects of the cancer.

Further disclosed herein is an in vitro method of effecting cancer cell death. The method comprises contacting the cancer cell with an effective amount of (i) a first anti-cancer agent, which is BVD-<NUM> or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent, which is a CDK inhibitor or a pharmaceutically acceptable salt thereof.

Also disclosed is a kit for use in treating or ameliorating the effects of a cancer in a subject in need thereof. The kit comprises an effective amount of (i) a first anti-cancer agent, which is BVD-<NUM> or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent, which is a CDK inhibitor or a pharmaceutically acceptable salt thereof, packaged together with instructions for their use.

Further disclosed herein is a pharmaceutical composition for treating or ameliorating the effects of a cancer in a subject in need thereof. The pharmaceutical composition comprises a pharmaceutically acceptable diluent or carrier and an effective amount of (i) a first anti-cancer agent, which is BVD-<NUM> or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent, which is a CDK inhibitor or a pharmaceutically acceptable salt thereof, wherein administration of the first and second anti-cancer agents provides a synergistic effect compared to administration of either anti-cancer agent alone.

Disclosed herein is a method of treating or ameliorating the effects of a cancer in a subject in need thereof. The method comprises administering to the subject an effective amount of (i) a first anti-cancer agent, which is BVD-<NUM> or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent, which is a CDK inhibitor or a pharmaceutically acceptable salt thereof, to treat or ameliorate the effects of the cancer.

As used herein, the terms "treat," "treating," "treatment" and grammatical variations thereof mean subjecting an individual subject to a protocol, regimen, process or remedy, in which it is desired to obtain a physiologic response or outcome in that subject, e.g., a patient. In particular, the methods and compositions of the present disclosure may be used to slow the development of disease symptoms or delay the onset of the disease or condition, or halt the progression of disease development. However, because every treated subject may not respond to a particular treatment protocol, regimen, process or remedy, treating does not require that the desired physiologic response or outcome be achieved in each and every subject or subject population, e.g., patient population. Accordingly, a given subject or subject population, e.g., patient population may fail to respond or respond inadequately to treatment.

As used herein, the terms "ameliorate", "ameliorating" and grammatical variations thereof mean to decrease the severity of the symptoms of a disease in a subject.

As used herein, a "subject" is a mammal, preferably, a human. In addition to humans, categories of mammals include, for example, farm animals, domestic animals, laboratory animals, etc. Some examples of farm animals include cows, pigs, horses, goats, etc. Some examples of domestic animals include dogs, cats, etc. Some examples of laboratory animals include primates, rats, mice, rabbits, guinea pigs, etc..

Cancers include both solid and hemotologic cancers. Non-limiting examples of solid cancers include adrenocortical carcinoma, anal cancer, bladder cancer, bone cancer (such as osteosarcoma), brain cancer, breast cancer, carcinoid cancer, carcinoma, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, Ewing family of cancers, extracranial germ cell cancer, eye cancer, gallbladder cancer, gastric cancer, germ cell tumor, gestational trophoblastic tumor, head and neck cancer, hypopharyngeal cancer, islet cell carcinoma, kidney cancer, large intestine cancer, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer, lymphoma, malignant mesothelioma, Merkel cell carcinoma, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian epithelial cancer, ovarian germ cell cancer, pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pituitary cancer, plasma cell neoplasm, prostate cancer, rhabdomyosarcoma, rectal cancer, renal cell cancer, transitional cell cancer of the renal pelvis and ureter, salivary gland cancer, Sezary syndrome, skin cancers (such as cutaneous t-cell lymphoma, Kaposi's sarcoma, mast cell tumor,and melanoma), small intestine cancer, soft tissue sarcoma, stomach cancer, testicular cancer, thymoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms' tumor.

Examples of hematologic cancers include, but are not limited to, leukemias, such as adult/childhood acute lymphoblastic leukemia, adult/childhood acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia, lymphomas, such as AIDS-related lymphoma, cutaneous T-cell lymphoma, adult/childhood Hodgkin lymphoma, mycosis fungoides, adult/childhood non-Hodgkin lymphoma, primary central nervous system lymphoma, Sézary syndrome, cutaneous T-cell lymphoma, and Waldenstrom macroglobulinemia, as well as other proliferative disorders such as chronic myeloproliferative disorders, Langerhans cell histiocytosis, multiple myeloma/plasma cell neoplasm, myelodysplastic syndromes, and myelodysplastic/myeloproliferative neoplasms. A preferred set of cancers that may be treated according to the present invention include melanoma, neuroblastoma, leukemia, lymphoma, liver cancer, lung cancer, testicular cancer, and thyroid cancer. Preferably, the cancer is melanoma.

In the present invention, BVD-<NUM> is an ERK1/<NUM> inhibitor. BVD-<NUM> is a compound according to formula (I):
<CHM>
and pharmaceutically acceptable salts thereof. BVD-<NUM> may be synthesized according to the methods disclosed in, e.g., <CIT>. BVD-<NUM>'s mechanism of action is believed to be, inter alia, unique and distinct from certain other ERK1/<NUM> inhibitors, such as SCH772984. For example, SCH772984 inhibits autophosphorylation of ERK (Morris et al. , <NUM>), whereas BVD-<NUM> allows for the autophosphorylation of ERK while still inhibiting ERK. (See, e.g., <FIG>). This is important, inter alia, because it is believed that the properties of BVD-<NUM> allows for dissociation of multiple signaling pathways, for example, by controlling cell proliferation without substantially affecting cell death.

As used herein, "CDK" means a family of protein kinases that regulate the cell cycle. Known CDKs include cdk1, cdk2, ckd3, ckd4, cdk5, cdk6, cdk7, cdk8, cdk9, cdk10, and cdk11. A "CDK inhibitor" means those substances that (i) directly interact with CDK, e.g. by binding to CDK and (ii) decrease the expression or the activity of CDK.

Non-limiting examples of CDK inhibitors include <NUM>-Hydroxybohemine, <NUM>-ATA, <NUM>-Iodo-indirubin-<NUM>'-monoxime, <NUM>-Cyanopaullone, Aloisine A, Alsterpaullone <NUM>-Cyanoethyl, alvocidib (Sanofi), AM-<NUM> (Amgen), Aminopurvalanol A, Arcyriaflavin A, AT-<NUM> (Astex Pharmaceuticals), AZD <NUM> (<NPL>), BMS-<NUM> (<NPL>), BS-<NUM> (<NPL>), Butyrolactone I (<NPL>), Cdk/Crk Inhibitor (<NPL>), Cdk1/<NUM> Inhibitor (<NPL>), Cdk2 Inhibitor II (<NPL>), Cdk2 Inhibitor IV, NU6140 (<NPL>), Cdk4 Inhibitor (<NPL>), Cdk4 Inhibitor III (<NPL>), Cdk4/<NUM> Inhibitor IV (<NPL>), Cdk9 Inhibitor II (<NPL>), CGP 74514A, CR8, CYC-<NUM> (Cyclacel), dinaciclib (Ligand), (R)-DRF053 dihydrochloride (<NPL>), Fascaplysin, Flavopiridol, Hygrolidin, Indirubin, LEE-<NUM> (Astex Pharmaceuticals), LY-<NUM> (Eli Lilly), milciclib maleate (Nerviano Medical Sciences), MM-D37K (Maxwell Biotech), N9-Isopropyl-olomoucine, NSC <NUM> (<NPL>), NU2058 (<NPL>), NU6102 (<NPL>), Olomoucine, ON-<NUM> (Onconova), ON-<NUM> (Onconova), Oxindole I, P-<NUM>-<NUM> (Piramal), P-<NUM>-<NUM> (Piramal), palbociclib (Pfizer), PHA-<NUM> (<NPL>), PHA-<NUM> (<NPL>), PHA-<NUM> (<NPL>), Purvalanol A, Purvalanol B, R547 (<NPL>), RO-<NUM> (<NPL>), Roscovitine, SB-<NUM> (SBIO), SCH <NUM> (<NPL>), SEL-<NUM> (Selvita), seliciclib (Cyclacel), SNS-<NUM> (<NPL>), SU9516 (<NPL>), WHI-P180 (<NPL>), pharmaceutically acceptable salts thereof, and combinations thereof. Preferably, the CDK inhibitor is selected from the group consisting of dinaciclib, palbociclib, pharmaceutically acceptable salts thereof, and combinations thereof.

In another aspect of this embodiment, the subject with cancer has a somatic mutation in a MAPK pathway node, including RAS, RAF, MEK and ERK. As used herein, "somatic mutation" means a change occurring in any cell that is not destined to become a germ cell. The mutation may be a substitution, deletion, insertion, or a fusion. Preferably, the somatic mutation is a mutation in H-RAS, N-RAS, or K-RAS. More preferably, the cancer has a somatic N-RAS mutation. Table <NUM> shows the SEQ ID Nos. of representative nucleic acid and amino acid sequences of wild type N-RAS from various animals. These sequences may be used in methods for identifying subjects with a mutant N-RAS genotype (such as in the methods set forth below).

Methods for identifying mutations in nucleic acids, such as the above identified RAS genes, are known in the art. Nucleic acids may be obtained from biological samples. Biological samples include, but are not limited to, blood, plasma, urine, skin, saliva, and biopsies. Biological samples are obtained from a subject by routine procedures and methods which are known in the art.

Non-limiting examples of methods for identifying mutations include PCR, sequencing, hybrid capture, in-solution capture, molecular inversion probes, fluorescent in situ hybridization (FISH) assays, and combinations thereof.

Various sequencing methods are known in the art. These include, but are not limited to, Sanger sequencing (also referred to as dideoxy sequencing) and various sequencing-by-synthesis (SBS) methods as disclosed in, e.g., Metzker <NUM>, sequencing by hybridization, by ligation (for example, <CIT>), by degradation (for example, <CIT> and <CIT>) and nanopore sequencing (which is commercially available from Oxford Nanopore Technologies, UK). In deep sequencing techniques, a given nucleotide in the sequence is read more than once during the sequencing process. Deep sequencing techniques are disclosed in e.g., <CIT> and International Patent Publication No. <CIT>.

PCR-based methods for detecting mutations are known in the art and employ PCR amplification, where each target sequence in the sample has a corresponding pair of unique, sequence-specific primers. For example, the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method allows for rapid detection of mutations after the genomic sequences are amplified by PCR. The mutation is discriminated by digestion with specific restriction endonucleases and is identified by electrophoresis. See, e.g., Ota et al. Mutations may also be detected using real time PCR. See, e.g., International Application publication No. <CIT>.

Hybrid capture methods are known in the art and are disclosed in e.g., <CIT> and <CIT> and <CIT>. These methods are based on the selective hybridization of the target genomic regions to user-designed oligonucleotides. The hybridization can be to oligonucleotides immobilized on high or low density microarrays (on-array capture), or solution-phase hybridization to oligonucleotides modified with a ligand (e.g. biotin) which can subsequently be immobilized to a solid surface, such as a bead (in-solution capture).

Molecular Inversion Probe (MIP) techniques are known in the art and are disclosed in e.g., Absalan et al. This method uses MIP molecules, which are special "padlock" probes (Nilsson et al, <NUM>) for genotyping. A MIP molecule is a linear oligonucleotide that contains specific regions, universal sequences, restriction sites and a Tag (index) sequence (<NUM>-<NUM> bp). A MIP hybridizes directly around the genetic marker/SNP of interest. The MIP method may also use a number of "padlock" probe sets that hybridize to genomic DNA in parallel (Hardenbol et al. In case of a perfect match, genomic homology regions are ligated by undergoing an inversion in configuration (as suggested by the name of the technique) and creating a circular molecule. After the first restriction, all molecules are amplified with universal primers. Amplicons are restricted again to ensure short fragments for hybridization on a microarray. Generated short fragments are labeled and, through a Tag sequence, hybridized to a cTag (complementary strand for index) on an array. After the formation of Tag-cTag duplex, a signal is detected.

In another aspect of this embodiment, at least one additional therapeutic agent effective for treating or ameliorating the effects of the cancer is to be administered to the subject. The additional therapeutic agent may be selected from the group consisting of an antibody or fragment thereof, a cytotoxic agent, a toxin, a radionuclide, an immunomodulator, a photoactive therapeutic agent, a radiosensitizing agent, a hormone, an anti-angiogenesis agent, and combinations thereof.

As used herein, an "antibody" encompasses naturally occurring immunoglobulins as well as non-naturally occurring immunoglobulins, including, for example, single chain antibodies, chimeric antibodies (e.g., humanized murine antibodies), and heteroconjugate antibodies (e.g., bispecific antibodies). Fragments of antibodies include those that bind antigen, (e.g., Fab', F(ab')<NUM>, Fab, Fv, and rlgG). See also, e.g., <NPL>. The term antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. The term "antibody" further includes both polyclonal and monoclonal antibodies.

Examples of therapeutic antibodies that may be used include rituximab (Rituxan), Cetuximab (Erbitux), bevacizumab (Avastin), and Ibritumomab (Zevalin).

Cytotoxic agents include DNA damaging agents, antimetabolites, anti-microtubule agents, antibiotic agents, etc. DNA damaging agents include alkylating agents, platinum-based agents, intercalating agents, and inhibitors of DNA replication. Non-limiting examples of DNA alkylating agents include cyclophosphamide, mechlorethamine, uramustine, melphalan, chlorambucil, ifosfamide, carmustine, lomustine, streptozocin, busulfan, temozolomide, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof. Non-limiting examples of platinum-based agents include cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, triplatin tetranitrate, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof. Non-limiting examples of intercalating agents include doxorubicin, daunorubicin, idarubicin, mitoxantrone, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof. Non-limiting examples of inhibitors of DNA replication include irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof. Antimetabolites include folate antagonists such as methotrexate and premetrexed, purine antagonists such as <NUM>-mercaptopurine, dacarbazine, and fludarabine, and pyrimidine antagonists such as <NUM>-fluorouracil, arabinosylcytosine, capecitabine, gemcitabine, decitabine, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof. Anti-microtubule agents include without limitation vinca alkaloids, paclitaxel (Taxol®), docetaxel (Taxotere®), and ixabepilone (Ixempra®). Antibiotic agents include without limitation actinomycin, anthracyclines, valrubicin, epirubicin, bleomycin, plicamycin, mitomycin, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof.

Cytotoxic agents also include an inhibitor of the PI3K/Akt pathway. Non-limiting examples of an inhibitor of the PI3K/Akt pathway include A-<NUM> (<NPL>), AGL <NUM>, AMG-<NUM> (Amgen, Thousand Oaks, CA), AS-<NUM> (<NUM>-benzo[<NUM>,<NUM>]dioxol-<NUM>-ylmethylene-thiazolidine-<NUM>,<NUM>-dione), AS-<NUM> (<NUM>-(<NUM>,<NUM>-Difluoro-benzo[<NUM>,<NUM>]dioxol-<NUM>-ylmethylene)-thiazolidine-<NUM>,<NUM>-dione), AS-<NUM> (<NUM>-quinoxilin-<NUM>-methylene-<NUM>,<NUM>-thiazolidine-<NUM>,<NUM>-dione), AT7867 (<NPL>), benzimidazole series, Genentech (Roche Holdings Inc. , South San Francisco, CA), BML-<NUM> (<NPL>), CAL-<NUM> (Gilead Sciences, Foster City, CA), CAL-<NUM> (Gilead Sciences), CAL-<NUM> (Gilead Sciences), CAL-<NUM> (Gilead Sciences), CAL-<NUM> (Gilead Sciences),<NPL>,<NPL>, <NPL>, <NPL>, <NPL>, CCT128930 (<NPL>), CH5132799 (<NPL>), CHR-<NUM> (Chroma Therapeutics, Ltd. , Abingdon, UK), FPA <NUM> (<NPL>), GS-<NUM> (CAL-<NUM>) (Gilead Sciences), GSK <NUM> (<NPL>), H-<NUM> (<NPL>), Honokiol, IC87114 (Gilead Science), IPI-<NUM> (Intellikine Inc. ), KAR-<NUM> (Karus Therapeutics, Chilworth, UK), KAR-<NUM> (Karus Therapeutics), KIN-<NUM> (Karus Therapeutics), KT <NUM> (<NPL>), Miltefosine, MK-<NUM> dihydrochloride (<NPL>), ML-<NUM> (<NPL>), Naltrindole Hydrochloride, OXY-111A (NormOxys Inc. , Brighton, MA), perifosine, PHT-<NUM> (<NPL>), PI3 kinase delta inhibitor, Merck KGaA (Merck & Co. , Whitehouse Station, NJ), PI3 kinase delta inhibitors, Genentech (Roche Holdings Inc. ), PI3 kinase delta inhibitors, Incozen (Incozen Therapeutics, Pvt. , Hydrabad, India), PI3 kinase delta inhibitors-<NUM>, Incozen (Incozen Therapeutics), PI3 kinase inhibitor, Roche-<NUM> (Roche Holdings Inc. ), PI3 kinase inhibitors, Roche (Roche Holdings Inc. ), PI3 kinase inhibitors, Roche-<NUM> (Roche Holdings Inc. ), PI3-alpha/delta inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd. , South San Francisco, CA), PI3-delta inhibitors, Cellzome (Cellzome AG, Heidelberg, Germany), PI3-delta inhibitors, Intellikine (Intellikine Inc. , La Jolla, CA), PI3-delta inhibitors, Pathway Therapeutics-<NUM> (Pathway Therapeutics Ltd. ), PI3-delta inhibitors, Pathway Therapeutics-<NUM> (Pathway Therapeutics Ltd. ), PI3-delta/gamma inhibitors, Cellzome (Cellzome AG), PI3-delta/gamma inhibitors, Cellzome (Cellzome AG), PI3-delta/gamma inhibitors, Intellikine (Intellikine Inc. ), PI3-delta/gamma inhibitors, Intellikine (Intellikine Inc. ), PI3-delta/gamma inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd. ), PI3-delta/gamma inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd. ), PI3-gamma inhibitor Evotec (Evotec), PI3-gamma inhibitor, Cellzome (Cellzome AG), PI3-gamma inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd. ), PI3K delta/gamma inhibitors, Intellikine-<NUM> (Intellikine Inc. ), PI3K delta/gamma inhibitors, Intellikine-<NUM> (Intellikine Inc. ), pictilisib (Roche Holdings Inc. ), PIK-<NUM> (<NPL>), SC-<NUM> (Pfizer, New York, NY), SF-<NUM> (Semafore Pharmaceuticals, Indianapolis, IN), SH-<NUM>, SH-<NUM>, Tetrahydro Curcumin, TG100-<NUM> (Targegen Inc. , San Diego, CA), Triciribine, X-<NUM> (Xcovery, West Palm Beach, FL), XL-<NUM> (Evotech, Hamburg, Germany), pharmaceutically acceptable salts thereof, and combinations thereof.

As used herein, the term "toxin" means an antigenic poison or venom of plant or animal origin. An example is diphtheria toxin or portions thereof.

As used herein, the term "radionuclide" means a radioactive substance administered to the patient, e.g., intravenously or orally, after which it penetrates via the patient's normal metabolism into the target organ or tissue, where it delivers local radiation for a short time. Examples of radionuclides include, but are not limited to, I-<NUM>, At-<NUM>, Lu-<NUM>, Cu-<NUM>, I-<NUM>, Sm-<NUM>, Re-<NUM>, P-<NUM>, Re-<NUM>, In-<NUM>, and Y-<NUM>.

As used herein, the term "immunomodulator" means a substance that alters the immune response by augmenting or reducing the ability of the immune system to produce antibodies or sensitized cells that recognize and react with the antigen that initiated their production. Immunomodulators may be recombinant, synthetic, or natural preparations and include cytokines, corticosteroids, cytotoxic agents, thymosin, and immunoglobulins. Some immunomodulators are naturally present in the body, and certain of these are available in pharmacologic preparations. Examples of immunomodulators include, but are not limited to, granulocyte colony-stimulating factor (G-CSF), interferons, imiquimod and cellular membrane fractions from bacteria, IL-<NUM>, IL-<NUM>, IL-<NUM>, CCL3, CCL26, CXCL7, and synthetic cytosine phosphate-guanosine (CpG).

As used herein, the term "photoactive therapeutic agent" means compounds and compositions that become active upon exposure to light. Certain examples of photoactive therapeutic agents are disclosed, e.g., in <CIT>, "Photoactive Metal Nitrosyls For Blood Pressure Regulation And Cancer Therapy.

As used herein, the term "radiosensitizing agent" means a compound that makes tumor cells more sensitive to radiation therapy. Examples of radiosensitizing agents include misonidazole, metronidazole, tirapazamine, and trans sodium crocetinate.

As used herein, the term "hormone" means a substance released by cells in one part of a body that affects cells in another part of the body. Examples of hormones include, but are not limited to, prostaglandins, leukotrienes, prostacyclin, thromboxane, amylin, antimullerian hormone, adiponectin, adrenocorticotropic hormone, angiotensinogen, angiotensin, vasopressin, atriopeptin, brain natriuretic peptide, calcitonin, cholecystokinin, corticotropin-releasing hormone, encephalin, endothelin, erythropoietin, follicle-stimulating hormone, galanin, gastrin, ghrelin, glucagon, gonadotropin-releasing hormone, growth hormone-releasing hormone, human chorionic gonadotropin, human placental lactogen, growth hormone, inhibin, insulin, somatomedin, leptin, liptropin, luteinizing hormone, melanocyte stimulating hormone, motilin, orexin, oxytocin, pancreatic polypeptide, parathyroid hormone, prolactin, prolactin releasing hormone, relaxin, renin, secretin, somatostain, thrombopoietin, thyroid-stimulating hormone, testosterone, dehydroepiandrosterone, androstenedione, dihydrotestosterone, aldosterone, estradiol, estrone, estriol, cortisol, progesterone, calcitriol, and calcidiol.

Some compounds interfere with the activity of certain hormones or stop the production of certain hormones. These hormone-interfering compounds include, but are not limited to, tamoxifen (Nolvadex®), anastrozole (Arimidex®), letrozole (Femara®), and fulvestrant (FasIodex®). Such compounds are also within the meaning of hormone as used herein.

As used herein, an "anti-angiogenesis" agent means a substance that reduces or inhibits the growth of new blood vessels, such as, e.g., an inhibitor of vascular endothelial growth factor (VEGF) and an inhibitor of endothelial cell migration. Anti-angiogenesis agents include without limitation <NUM>-methoxyestradiol, angiostatin, bevacizumab, cartilage-derived angiogenesis inhibitory factor, endostatin, IFN-α, IL-<NUM>, itraconazole, linomide, platelet factor-<NUM>, prolactin, SU5416, suramin, tasquinimod, tecogalan, tetrathiomolybdate, thalidomide, thrombospondin, thrombospondin, TNP-<NUM>, ziv-aflibercept, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof.

In an additional aspect of this embodiment, administration of the first and second anti-cancer agents provides a synergistic effect compared to administration of either anti-cancer agent alone. As used herein, "synergistic" means more than additive. Synergistic effects may be measured by various assays known in the art, including but not limited to those disclosed herein, such as the excess over bliss assay.

Also disclosed is a method of treating or ameliorating the effects of a cancer in a subject in need thereof. The method comprises administering to the subject an effective amount of (i) a first anti-cancer agent, which is BVD-<NUM> or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent, which is selected from the group consisting of dinaciclib, palbociclib, and pharmaceutically acceptable salts thereof, to treat or ameliorate the effects of the cancer.

Suitable and preferred subjects are as disclosed herein. In this embodiment, the methods may be used to treat the cancers disclosed above, including those cancers with the mutational backgrounds identified above. Methods of identifying such mutations are also as set forth above.

In one aspect of this embodiment, the BVD-<NUM> or a pharmaceutically acceptable salt thereof is administered in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier or diluent.

In an additional aspect of this embodiment, the dinaciclib, palbociclib or a pharmaceutically acceptable salt thereof is administered in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier or diluent.

In another aspect of this embodiment, at least one additional therapeutic agent, preferably an inhibitor of the PI3K/Akt pathway is to be administered to the subject, as disclosed herein.

In another aspect of this embodiment, administration of the first and second anti-cancer agents provides a synergistic effect compared to administration of either anti-cancer agent alone.

Also disclosed is a method of effecting cancer cell death. The method comprises contacting the cancer cell with an effective amount of (i) a first anti-cancer agent, which is BVD-<NUM> or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent, which is a CDK inhibitor or a pharmaceutically acceptable salt thereof. In this embodiment, "contacting" means bringing BVD-<NUM>, the CDK inhibitors, and optionally one or more additional therapeutic agents into close proximity to the cancer cells. This may be accomplished using conventional techniques of drug delivery to mammals or in the in vitro situation by, e.g., providing BVD-<NUM>, the CDK inhibitors, and optionally other therapeutic agents to a culture media in which the cancer cells are located.

Suitable and preferred CDK inhibitors are as disclosed herein. In this embodiment, effecting cancer cell death may be accomplished in cancer cells having various mutational backgrounds and/or that are characterized as disclosed above. Methods of identifying such mutations are also as set forth above.

The methods of this embodiment, which may be carried out in vitro or in vivo, may be used to effect cancer cell death, by e.g., killing cancer cells, in cells of the types of cancer disclosed herein.

In one aspect of this embodiment, the cancer cell is a mammalian cancer cell. Preferably, the mammalian cancer cell is obtained from a mammal selected from the group consisting of humans, primates, farm animals, and domestic animals. More preferably, the mammalian cancer cell is a human cancer cell.

In another aspect of this embodiment, the method further comprises contacting the cancer cell with at least one additional therapeutic agent, preferably an inhibitor of the PI3K/Akt pathway, as disclosed herein.

In a further aspect of this embodiment, contacting the cancer cell with the first and second anti-cancer agents provides a synergistic effect compared to contacting the cancer cell with either anti-cancer agent alone.

Also disclosed is a kit for treating or ameliorating the effects of a cancer in a subject in need thereof. The kit comprises an effective amount of (i) a first anti-cancer agent, which is BVD-<NUM> or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent, which is a CDK inhibitor or a pharmaceutically acceptable salt thereof, packaged together with instructions for their use.

The kits may also include suitable storage containers, e.g., ampules, vials, tubes, etc., for each pharmaceutical composition and other reagents, e.g., buffers, balanced salt solutions, etc., for use in administering the pharmaceutical compositions to subjects. The pharmaceutical compositions and other reagents may be present in the kits in any convenient form, such as, e.g., in a solution or in a powder form. The kits may further include instructions for use of the pharmaceutical compositions. The kits may further include a packaging container, optionally having one or more partitions for housing the pharmaceutical composition and other optional reagents.

For use in the kits of the invention, suitable and preferred CDK inhibitors and subjects are as disclosed herein. In this embodiment, the kit may be used to treat the cancers disclosed above, including those cancers with the mutational backgrounds identified herein. Methods of identifying such mutations are as set forth above.

In an additional aspect of this embodiment, the kit further comprises at least one additional therapeutic agent, preferably an inhibitor of the PI3K/Akt pathway, as disclosed herein.

Also disclosed is a pharmaceutical composition for treating or ameliorating the effects of a cancer in a subject in need thereof. The pharmaceutical composition comprises a pharmaceutically acceptable diluent or carrier and an effective amount of (i) a first anti-cancer agent, which is BVD-<NUM> or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent, which is a CDK inhibitor or a pharmaceutically acceptable salt thereof, wherein administration of the first and second anti-cancer agents provides a synergistic effect compared to administration of either anti-cancer agent alone.

Suitable and preferred CDK inhibitors and subjects are as disclosed herein. The pharmaceutical compositions disclosed herein may be used to treat the cancers disclosed above, including those cancers with the mutational backgrounds identified herein. Methods of identifying such mutations are also as set forth above.

In another aspect of this embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent, preferably an inhibitor of the PI3K/Akt pathway, as disclosed herein.

The pharmaceutical compositions disclosed herein may be in a unit dosage form comprising both anti-cancer agents. In another aspect of this embodiment, the first anti-cancer agent is in a first unit dosage form and the second anti-cancer agent is in a second unit dosage form, separate from the first.

The first and second anti-cancer agents may be co-administered to the subject, either simultaneously or at different times, as deemed most appropriate by a physician. If the first and second anti-cancer agents are administered at different times, for example, by serial administration, the first anti-cancer agent may be administered to the subject before the second anti-cancer agent. Alternatively, the second anti-cancer agent may be administered to the subject before the first anti-cancer agent.

As used herein, an "effective amount" or a "therapeutically effective amount" of an anti-cancer agent disclosed herein, including the pharmaceutical compositions containing same, is an amount of such agent or composition that is sufficient to effect beneficial or desired results as described herein when administered to a subject. Effective dosage forms, modes of administration, and dosage amounts may be determined empirically, and making such determinations is within the skill of the art. It is understood by those skilled in the art that the dosage amount will vary with the route of administration, the rate of excretion, the duration of the treatment, the identity of any other drugs being administered, the age, size, and species of mammal, e.g., human patient, and like factors well known in the arts of medicine and veterinary medicine. In general, a suitable dose of an agent or composition disclosed herein will be that amount of the agent or composition, which is the lowest dose effective to produce the desired effect. The effective dose of an agent or composition of the present invention may be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day.

A suitable, non-limiting example of a dosage of an anti-cancer agent disclosed herein is from about <NUM>/kg to about <NUM>/kg per day, such as from about <NUM>/kg to about <NUM>/kg per day, <NUM>/kg per day to about <NUM>/kg per day, including from about <NUM>/kg to about <NUM>/kg per day. Other representative dosages of such agents include about <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, and <NUM>/kg per day. The effective dose of anti-cancer agents disclosed herein, e.g., BVD-<NUM> and CDK inhibitors, may be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day.

The anti-cancer agents or pharmaceutical compositions containing same disclosed herein may be administered in any desired and effective manner: for oral ingestion, or as an ointment or drop for local administration to the eyes, or for parenteral or other administration in any appropriate manner such as intraperitoneal, subcutaneous, topical, intradermal, inhalation, intrapulmonary, rectal, vaginal, sublingual, intramuscular, intravenous, intraarterial, intrathecal, or intralymphatic. Further, the anti-cancer agents or pharmaceutical compositions containing same disclosed herein may be administered in conjunction with other treatments. The anti-cancer agents or the pharmaceutical compositions disclosed herein may be encapsulated or otherwise protected against gastric or other secretions, if desired.

The pharmaceutical compositions disclosed herein may comprise one or more active ingredients, e.g. anti-cancer agents, in admixture with one or more pharmaceutically-acceptable diluents or carriers and, optionally, one or more other compounds, drugs, ingredients and/or materials. Regardless of the route of administration selected, the agents/compounds disclosed herein are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. See, e.g., <NPL>.

Pharmaceutically acceptable diluents or carriers are well known in the art (see, e.g., <NPL>. ) and<NPL>. )) and include sugars (e.g., lactose, sucrose, mannitol, and sorbitol), starches, cellulose preparations, calcium phosphates (e.g., dicalcium phosphate, tricalcium phosphate and calcium hydrogen phosphate), sodium citrate, water, aqueous solutions (e.g., saline, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection), alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol), polyols (e.g., glycerol, propylene glycol, and polyethylene glycol), organic esters (e.g., ethyl oleate and tryglycerides), biodegradable polymers (e.g., polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides)), elastomeric matrices, liposomes, microspheres, oils (e.g., corn, germ, olive, castor, sesame, cottonseed, and groundnut), cocoa butter, waxes (e.g., suppository waxes), paraffins, silicones, talc, silicylate, etc. Each pharmaceutically acceptable diluent or carrier used in a pharmaceutical composition disclosed herein must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Diluents or carriers suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable diluents or carriers for a chosen dosage form and method of administration can be determined using ordinary skill in the art.

The pharmaceutical compositions disclosed herein may, optionally, contain additional ingredients and/or materials commonly used in pharmaceutical compositions. These ingredients and materials are well known in the art and include (<NUM>) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (<NUM>) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose, sucrose and acacia; (<NUM>) humectants, such as glycerol; (<NUM>) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium starch glycolate, cross-linked sodium carboxymethyl cellulose and sodium carbonate; (<NUM>) solution retarding agents, such as paraffin; (<NUM>) absorption accelerators, such as quaternary ammonium compounds; (<NUM>) wetting agents, such as cetyl alcohol and glycerol monostearate; (<NUM>) absorbents, such as kaolin and bentonite clay; (<NUM>) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, and sodium lauryl sulfate; (<NUM>) suspending agents, such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth; (<NUM>) buffering agents; (<NUM>) excipients, such as lactose, milk sugars, polyethylene glycols, animal and vegetable fats, oils, waxes, paraffins, cocoa butter, starches, tragacanth, cellulose derivatives, polyethylene glycol, silicones, bentonites, silicic acid, talc, salicylate, zinc oxide, aluminum hydroxide, calcium silicates, and polyamide powder; (<NUM>) inert diluents, such as water or other solvents; (<NUM>) preservatives; (<NUM>) surface-active agents; (<NUM>) dispersing agents; (<NUM>) control-release or absorption-delaying agents, such as hydroxypropylmethyl cellulose, other polymer matrices, biodegradable polymers, liposomes, microspheres, aluminum monostearate, gelatin, and waxes; (<NUM>) opacifying agents; (<NUM>) adjuvants; (<NUM>) wetting agents; (<NUM>) emulsifying and suspending agents; (<NUM>), solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, <NUM>,<NUM>-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan; (<NUM>) propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane; (<NUM>) antioxidants; (<NUM>) agents which render the formulation isotonic with the blood of the intended recipient, such as sugars and sodium chloride; (<NUM>) thickening agents; (<NUM>) coating materials, such as lecithin; and (<NUM>) sweetening, flavoring, coloring, perfuming and preservative agents. Each such ingredient or material must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Ingredients and materials suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable ingredients and materials for a chosen dosage form and method of administration may be determined using ordinary skill in the art.

The pharmaceutical compositions disclosed herein suitable for oral administration may be in the form of capsules, cachets, pills, tablets, powders, granules, a solution or a suspension in an aqueous or non-aqueous liquid, an oil-in-water or water-in-oil liquid emulsion, an elixir or syrup, a pastille, a bolus, an electuary or a paste. These formulations may be prepared by methods known in the art, e.g., by means of conventional pan-coating, mixing, granulation or lyophilization processes.

Solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like) may be prepared, e.g., by mixing the active ingredient(s) with one or more pharmaceutically-acceptable diluents or carriers and, optionally, one or more fillers, extenders, binders, humectants, disintegrating agents, solution retarding agents, absorption accelerators, wetting agents, absorbents, lubricants, and/or coloring agents. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using a suitable excipient. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using a suitable binder, lubricant, inert diluent, preservative, disintegrant, surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine. The tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein. They may be sterilized by, for example, filtration through a bacteria-retaining filter. These compositions may also optionally contain opacifying agents and may be of a composition such that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. The active ingredient can also be in microencapsulated form.

Liquid dosage forms for oral administration include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. The liquid dosage forms may contain suitable inert diluents commonly used in the art. Besides inert diluents, the oral compositions may also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions may contain suspending agents.

The pharmaceutical compositions disclosed herein for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more active ingredient(s) with one or more suitable nonirritating diluents or carriers which are solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound. The pharmaceutical compositions disclosed herein which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such pharmaceutically-acceptable diluents or carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops and inhalants. The active agent(s)/compound(s) may be mixed under sterile conditions with a suitable pharmaceutically-acceptable diluent or carrier. The ointments, pastes, creams and gels may contain excipients. Powders and sprays may contain excipients and propellants.

The pharmaceutical compositions disclosed herein suitable for parenteral administrations may comprise one or more agent(s)/compound(s) in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Proper fluidity can be maintained, for example, by the use of coating materials, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These pharmaceutical compositions may also contain suitable adjuvants, such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption.

In some cases, in order to prolong the effect of a drug (e.g., pharmaceutical formulation), it is desirable to slow its absorption from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility.

The rate of absorption of the active agent/drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered agent/drug may be accomplished by dissolving or suspending the active agent/drug in an oil vehicle. Injectable depot forms may be made by forming microencapsule matrices of the active ingredient in biodegradable polymers. Depending on the ratio of the active ingredient to polymer, and the nature of the particular polymer employed, the rate of active ingredient release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.

The formulations may be present in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid diluent or carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.

The present invention provides combinations shown to enhance the effects of BVD-<NUM>. Herein, applicants have also shown that the combination of BVD-<NUM> with different CDK inhibitors (i.e. palbociclib, LEE-<NUM>, pharmaceutically acceptable salts thereof, and combinations thereof) is synergistic.

The following examples are provided to further illustrate the present invention.

For Western blot studies, HCT116 cells (<NUM> x <NUM><NUM>) were seeded into <NUM> dishes in McCoy's 5A plus <NUM>% FBS. A375 cells (<NUM> x <NUM><NUM>) were seeded into <NUM> dishes in DMEM plus <NUM>% FBS. Cells were allowed to adhere overnight prior to addition of the indicated amount of test compound (BVD-<NUM>) or vehicle control. Cells were treated for either <NUM> or <NUM> hours before isolation of whole-cell protein lysates, as specified below. Cells were harvested by trypsinisation, pelleted and snap frozen. Lysates were prepared with RIPA (Radio-Immunoprecipitation Assay) buffer, clarified by centrifugation and quantitated by bicinchoninic acid assay (BCA) assay. <NUM>-<NUM>µg of protein was resolved by SDS-PAGE electrophoresis, blotted onto PVDF membrane and probed using the antibodies detailed in Table <NUM> (for the <NUM>-hour treatment) and Table <NUM> (for the <NUM>-hour treatment) below.

<FIG> shows Western blot analyses of cells treated with BVD-<NUM> at various concentrations for the following: <NUM>) MAPK signaling components in A375 cells after <NUM> hours; <NUM>) cell cycle and apoptosis signaling in A375 <NUM> hours treatment with various amounts of BVD-<NUM>; and <NUM>) MAPK signaling in HCT-<NUM> cells treated for <NUM> hours. The results show that acute and prolonged treatment with BVD-<NUM> in RAF and RAS mutant cancer cells in-vitro affects both substrate phosphorylation and effector targets of ERK kinases. The concentrations of BVD-<NUM> required to induce these changes is typically in the low micromolar range.

Changes in several specific activity markers are noteworthy. First, the abundance of slowly migrating isoforms of ERK kinase increase following BVD-<NUM> treatment; modest changes can be observed acutely, and increase following prolonged treatment. While this could indicate an increase in enzymatically active, phosphorylated forms of ERK, it remains noteworthy that multiple proteins subject to both direct and indirect regulation by ERK remain "off" following BVD-<NUM> treatment. First, RSK1/<NUM> proteins exhibit reduced phosphorylation at residues that are strictly dependent on ERK for protein modification (T359/S363). Second, BVD-<NUM> treatment induces complex changes in the MAPK feedback phosphatase, DUSP6: slowly migrating protein isoforms are reduced following acute treatment, while total protein levels are greatly reduced following prolonged BVD-<NUM> treatment. Both of these findings are consistent with reduced activity of ERK kinases, which control DUSP6 function through both post-translational and transcriptional mechanisms. Overall, despite increases in cellular forms of ERK that are typically thought to be active, it appears likely that cellular ERK enzyme activity is fully inhibited following either acute or prolonged treatment with BVD-<NUM>.

Consistent with these observations, effector genes that require MAPK pathway signaling are altered following treatment with BVD-<NUM>. The G1/S cell-cycle apparatus is regulated at both post-translational and transcriptional levels by MAPK signaling, and cyclin-D1 protein levels are greatly reduced following prolonged BVD-<NUM> treatment. Similarly, gene expression and protein abundance of apoptosis effectors often require intact MAPK signaling, and total levels of Bim-EL increase following prolonged BVD-<NUM> treatment. As noted above, however, PARP protein cleavage and increased apoptosis were not noted in the A375 cell background; this suggests that additional factors may influence whether changes in BVD-<NUM>/ERK-dependent effector signaling are translated into definitive events such as cell death and cell cycle arrest.

Consistent with the cellular activity of BVD-<NUM>, marker analysis suggests that ERK inhibition alters a variety of molecular signaling events in cancer cells, making them susceptible to both decreased cell proliferation and survival.

In sum, <FIG> shows that BVD-<NUM> inhibits the MAPK signaling pathway and may be more favorable compared to RAF or MEK inhibition in this setting.

Finally, properties of BVD-<NUM> may make this a preferred agent for use as an ERK inhibitor, compared to other agents with a similar activity. It is known that kinase inhibitor drugs display unique and specific interactions with their enzyme targets, and that drug efficacy is strongly influenced by both the mode of direct inhibition, as well as susceptibility to adaptive changes that occur following treatment. For example, inhibitors of ABL, KIT, EGFR and ALK kinases are effective only when their cognate target is found in active or inactive configurations. Likewise, certain of these inhibitors are uniquely sensitive to either secondary genetic mutation, or post-translational adaptive changes, of the protein target. Finally, RAF inhibitors show differential potency to RAF kinases present in certain protein complexes and/or subcellular localizations. In summary, as ERK kinases are similarly known to exist in diverse, variable, and complex biochemical states, it appears likely that BVD-<NUM> may interact with and inhibit these targets in a fashion that is distinct and highly preferable to other agents.

Cancer cell lines are maintained in cell culture under standard media and serum conditions.

For all combination studies, MM415 cells (N-RAS mutant human melanoma cells) are seeded into triplicate <NUM>-well plates at a cell density of <NUM> cells/well in RPMI <NUM> media supplemented with <NUM>% (vol/vol) fetal bovine serum (FBS). HCT <NUM> cells (K-RAS mutant human colorectal carcinoma cells) are seeded into triplicate <NUM>-well plates at a cell density of <NUM> cells/well in McCoy's 5A medium plus <NUM>% FBS. A375 cells (BRAF V600 E human malignant melanoma) are seeded at a density of <NUM> cells/well in Dulbecco's Modified Eagle Medium (DMEM) plus <NUM>% FBS. Cells are allowed to adhere overnight prior to addition of test compound or vehicle control.

For dinaciclib studies, the following combinations are tested using a <NUM> x <NUM> dose matrix: dinaciclib (ranging from <NUM> - <NUM>) with BVD-<NUM> (ranging from <NUM> to <NUM>), dinaciclib (ranging from <NUM> - <NUM>) with dabrafenib (ranging from <NUM> to <NUM>), and dinaciclib (ranging from <NUM> - <NUM>) with trametinib (ranging from <NUM> to <NUM>). The final concentration of DMSO is <NUM>%. The compounds are incubated with the cells for <NUM> hours.

For palbociclib studies, the following combinations are tested using a <NUM> x <NUM> dose matrix: palbociclib (ranging from <NUM>-<NUM>) with BVD-<NUM> (<NUM> to <NUM>), palbociclib (ranging from <NUM>-<NUM>) with dabrafenib (ranging from <NUM> to <NUM>), and palbociclib (ranging from <NUM>-<NUM>) with trametinib (ranging from <NUM> to <NUM>). The final concentration of DMSO is <NUM>%. The compounds are incubated with the cells for <NUM> hours.

Next, Alamar Blue <NUM>% (v/v) is added and incubated with the cells for <NUM> hours prior to reading on a fluorescent plate reader. After reading Alamar Blue, the medium/Alamar Blue mix is flicked off, <NUM>µl of CellTiter-Glo/PBS (<NUM>:<NUM>) is added, and the plates are processed as per the manufacturer's instructions (Promega, Madison, WI). Media only background values are subtracted before the data is analyzed.

In brief, MM415 cells are seeded in triplicate in white <NUM>-well plates at a cell density of <NUM> cells/well in RPMI <NUM> plus <NUM>% FBS. A375 cells are seeded at a density of <NUM> cells/well in DMEM plus <NUM>% FBS. HCT <NUM> cells are seeded at a cell density of <NUM> cells/well in McCoy's 5A medium plus <NUM>% FBS. Cells are allowed to adhere overnight prior to addition of test compound or vehicle control. The final concentration of DMSO is <NUM>%, and <NUM> staurosporine is included as a positive control. <NUM> and <NUM> hour assay incubation periods are used. Then, Caspase-Glo® <NUM>/<NUM><NUM>% (v/v) is added, plates are mixed for <NUM> minutes on an orbital shaker and incubated for <NUM> hour at room temperature prior to reading on a luminescent plate reader. Media only background values are subtracted before the data is analysed.

The combination data may be presented as dose-response curves generated in GraphPad Prism (plotted using % viability relative to DMSO only treated controls).

Predicted fractional inhibition values for combined inhibition are calculated using the equation Cbliss =A + B - (A x B) where A and B are the fractional inhibitions obtained by drug A alone or drug B alone at specific concentrations. Cbliss is the fractional inhibition that would be expected if the combination of the two drugs is exactly additive. Cbliss values are subtracted from the experimentally observed fractional inhibition values to give an 'excess over Bliss' value. Excess over Bliss values greater than <NUM> indicate synergy, whereas values less than <NUM> indicate antagonism. Excess over Bliss values may be plotted as heat maps ± SD.

It is expected that the combinations of dinaciclib or palbociclib with BVD-<NUM> will be effective in inhibiting the growth of A375, MM415, and HCT116 cells. Dose response curves will be obtained. It is expected that the IC<NUM> of BVD-<NUM> in these cell lines will be approximately <NUM>. It is also expected that the IC<NUM> of dinaciclib and palbociclib in these cell lines will be approximately <NUM> (Parry et al. , <NUM>) and <NUM> (Fry et al. , <NUM>), respectively.

Female athymic nude mice (Crl:NU(Ncr)-Foxn/nu, Charles River) are nine weeks old with a body weight (BW) range of about <NUM> to about <NUM> grams on Day <NUM> of the study. The animals are fed ad libitum water (reverse osmosis, <NUM> ppm C!), and NIH <NUM> Modified and Irradiated Lab Diet® consisting of <NUM>% crude protein, <NUM>% crude fat, and <NUM>% crude fiber. The mice are housed on irradiated Enrich-o'cobsTM Laboratory Animal Bedding in static microisolators on a <NUM>-hour light cycle at <NUM>-<NUM> (<NUM>-<NUM>°F) and <NUM>-<NUM>% humidity. The recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care are complied with.

MM415 N-RAS mutant human melanoma cells are cultured in RPMI-<NUM> medium supplemented with <NUM>% fetal bovine serum, <NUM> glutamine, <NUM> units/mL penicillin G sodium, <NUM>µg/mL streptomycin sulfate, and <NUM>µg/mL gentamicin. The tumor cells are grown in tissue culture flasks in a humidified incubator at <NUM>, in an atmosphere of <NUM>% CO<NUM> and <NUM>% air.

The MM415 cells used for implantation are harvested during exponential growth and resuspended in <NUM>% Matrigel (BD Biosciences): <NUM>% phosphate buffered saline at a concentration of <NUM> x <NUM><NUM> cells/mL. On the day of tumor implant, each test mouse is injected subcutaneously in the right flank with <NUM> x <NUM><NUM> cells (<NUM> cell suspension), and tumor growth is monitored as the average size approaches the target range of <NUM> to <NUM><NUM>. Tumors are measured in two dimensions using calipers, and volume is calculated using the formula: <MAT> where w = width and l = length, in mm, of the tumor. Tumor weight may be estimated with the assumption that <NUM> is equivalent to <NUM><NUM> of tumor volume.

Ten days after tumor implantation, designated as Day <NUM> of the study, the animals are sorted into sixteen groups, each described below.

On Day <NUM> of the study, mice are sorted into groups each consisting of fifteen mice and one group consisting of ten mice, and dosing is initiated. All doses are given by oral gavage (p. ) except dacarbazine (DTIC), which is given intravenously (i. For each agent, the dosing volume of <NUM>/kg (<NUM> per <NUM> grams of BW) is scaled to the BW of the individual animal. The dinaciclib/palbociclib doses are to be given once daily (qd) until study end (qd to end), whereas the vehicle and BVD-<NUM> doses are to be given twice daily (bid) until study end (bid to end). For bid dosing, dosing is initiated in the afternoon of Day <NUM>, so that one dose is given on the first day ("first day <NUM> dose").

One group receives <NUM>% CMC vehicle p. bid to end, and serves as the control group for calculation of %TGD. Another group receives DTIC i. at <NUM>/kg once every other day (qod) for five doses (qod x <NUM>), and serves as the positive control for the model.

Four groups receive either dinaciclib at <NUM> or <NUM>/kg or palbociclib at <NUM> or <NUM>/kg. Two groups receive <NUM> or <NUM>/kg BVD-<NUM> p. bid to end.

Each one of two groups receives a combination of <NUM>/kg BVD-<NUM> with <NUM> or <NUM>/kg of dinaciclib. Two other groups receive <NUM>/kg BVD-<NUM> with <NUM> or <NUM>/kg of dinaciclib. Two additional groups will receive <NUM>/kg BVD-<NUM> with <NUM> or <NUM>/kg palbociclib, and another two groups will receive <NUM>/kg BVD-<NUM> with <NUM> or <NUM>/kg palbociclib.

Tumors are measured using calipers twice per week, and each animal is euthanized when its tumor reaches the pre-determined tumor volume endpoint of <NUM><NUM> or on the final day, whichever comes first. Animals that exit the study for tumor volume endpoint are documented as euthanized for tumor progression (TP), with the date of euthanasia. The time to endpoint (TTE) for analysis is calculated for each mouse by the following equation: <MAT> where TTE is expressed in days, endpoint volume is expressed in mm<NUM>, b is the intercept, and m is the slope of the line obtained by linear regression of a log-transformed tumor growth data set. The data set consists of the first observation that exceeds the endpoint volume used in analysis and the three consecutive observations that immediately precede the attainment of this endpoint volume. The calculated TTE is usually less than the TP date, the day on which the animal is euthanized for tumor size. Animals with tumors that do not reach the endpoint volume are assigned a TTE value equal to the last day of the study. Any animal classified as having died from NTR (non-treatment-related) causes due to accident (NTRa) or due to unknown etiology (NTRu) are excluded from TTE calculations (and all further analyses). Animals classified as TR (treatment-related) deaths or NTRm (non-treatment-related death due to metastasis) are assigned a TTE value equal to the day of death.

Treatment outcome is evaluated from TGD, defined as the increase in the median TTE in a treatment group compared to the control group: <MAT> expressed in days, or as a percentage of the median TTE of the control group: <MAT> where:.

Treatment efficacy may be determined from the incidence and magnitude of regression responses observed during the study. Treatment may cause partial regression (PR) or complete regression (CR) of the tumor in an animal. In a PR response, the tumor volume is <NUM>% or less of its Day <NUM> volume for three consecutive measurements during the course of the study, and equal to or greater than <NUM><NUM> for one or more of these three measurements. In a CR response, the tumor volume is less than <NUM><NUM> for three consecutive measurements during the course of the study. An animal with a CR response at the termination of the study is additionally classified as a tumor-free survivor (TFS). Animals are monitored for regression responses.

Animals are weighed daily on Days <NUM>-<NUM>, then twice per week until completion of the study. The mice are observed frequently for overt signs of any adverse, TR side effects, and clinical signs are recorded when observed. Individual BW loss is monitored as per protocol, and any animal whose weight exceeds the limits for acceptable BW loss is euthanized. Group mean BW loss also is monitored as per protocol. Dosing is to be suspended in any group that exceeds the limits for acceptable mean BW loss. If mean BW recovers, then dosing is to be resumed in that group, but at a lower dosage or less frequent dosing schedule. Acceptable toxicity for the maximum tolerated dose (MTD) is defined as a group mean BW loss of less than <NUM>% during the study and not more than <NUM>% TR deaths. A death is classified as TR if attributable to treatment side effects as evidenced by clinical signs and/or necropsy, or may also be classified as TR if due to unknown causes during the dosing period or within <NUM> days of the last dose. A death is classified as NTR if there is no evidence that death is related to treatment side effects. NTR deaths may be further characterized based on cause of death. A death is classified as NTRa if it results from an accident or human error. A death is classified as NTRm if necropsy indicates that it may result from tumor dissemination by invasion and/or metastasis. A death is classified as NTRu if the cause of death is unknown and there is no available evidence of death related to treatment side effects, metastasis, accident or human error, although death due to treatment side effects cannot be excluded.

Prism (GraphPad) for Windows <NUM> is used for graphical presentations and statistical analyses.

The logrank test, which evaluates overall survival experience, is used to analyze the significance of the differences between the TTE values of two groups. Logrank analysis includes the data for all animals in a group except those assessed as NTR deaths. Two-tailed statistical analyses are conducted at significance level P = <NUM>. The statistical tests are not adjusted for multiple comparisons. Prism summarizes test results as not significant (ns) at P > <NUM>, significant (symbolized by "*") at <NUM> < P < <NUM>, very significant ("**") at <NUM> < P ≤ <NUM>, and extremely significant ("***") at P ≤ <NUM>. Groups with regimens above the MTD are not evaluated statistically.

A scatter plot is constructed to show TTE values for individual mice, by group. Group mean tumor volumes are plotted as a function of time. When an animal exits the study due to tumor size, the final tumor volume recorded for the animal is included with the data used to calculate the mean volume at subsequent time points. Error bars (when present) indicate one standard error of the mean (SEM). Tumor growth plots exclude the data for NTR deaths, and are truncated after <NUM>% of the assessable animals in a group exit the study or after the second TR death in a group, whichever comes first. Kaplan-Meier plots show the percentage of animals in each group remaining in the study versus time. The Kaplan-Meier plot and logrank test share the same TTE data sets. Percent mean BW changes from Day <NUM> are calculated for each group for each day of BW measurement, and are plotted as a function of time. BW plots exclude the data for NTR deaths, and are truncated after <NUM>% of the assessable animals in a group exit the study.

It is expected that the combinations of dinaciclib or palbociclib with BVD-<NUM> will be effective against MM415 cell-derived tumors and that the results will be statistically significant. It is also expected that the side effects associated with the BVD-<NUM>/CDK inhibitor treatment will be minimal.

Cells were seeded in <NUM>-well plates at the densities indicated in Table <NUM> in RPMI containing <NUM>% FBS and allowed to adhere overnight prior to addition of compound or vehicle control. Compounds were prepared from DMSO stocks to give the desired final concentrations. The final DMSO concentration was constant at <NUM>%. Test compounds were incubated with the cells for <NUM> at <NUM>, <NUM>% CO<NUM> in a humidified atmosphere. CellTiter-Glo® reagent (Promega, Madison, WI) was added according to manufacturer's instructions and luminescence detected using the BMG FLUOstar plate reader (BMG Labtech, Ortenberg, Germany). A duplicate set of assay plates was incubated with 10µg/ml Hoechst <NUM> stain (Invitrogen, Grant Island, NY) in complete growth medium for <NUM> at <NUM>, <NUM>% CO<NUM> in a humidified atmosphere. The medium was then removed and replaced with PBS and fluorescence detected using a BMG FLUOstar Omega plate reader (BMG labtech, Ortenberg, Germany). The average media only background value was deducted and the data analysed using a <NUM>-parameter logistic equation in GraphPad Prism (GraphPad Software, La Jolla, CA).

Cells were seeded into triplicate <NUM>-well plates at the densities indicated in Table <NUM> in RPMI media containing <NUM>% FBS and allowed to adhere overnight prior to addition of test compound or vehicle control. Combinations were tested using a 10x8 dose matrix. The final DMSO concentration was constant at <NUM>%.

Test compounds were incubated with the cells for <NUM> at <NUM>, <NUM>% CO<NUM> in a humidified atmosphere. Cells were stained with Hoechst stain and fluorescence detected as described above. The average media only background value was deducted and the data analysed.

Combination interactions across the dose matrix were determined by the Loewe Additivity and Bliss independence models using Chalice™ Combination Analysis Software (Horizon Discovery Group, Cambridge, MA) as outlined in the user manual (available at chalice. horizondiscovery. com/chalice-portal/documentation/analyzer/home. Synergy is determined by comparing the experimentally observed level of inhibition at each combination point with the value expected for additivity, which is derived from the single-agent responses along the edges of the matrix. Potential synergistic interactions were identified by displaying the calculated excess inhibition over that predicted as being additive across the dose matrix as a heat map, and by reporting a quantitative 'Synergy Score' based on the Loewe model. The single agent data derived from the combination assay plates were presented as dose-response curves generated in Chalice™.

This study assessed the effects of combining the ERK inhibitors BVD-<NUM> and SCH772984 with two different CDK4/<NUM> inhibitors (Palbociclib and LEE-<NUM>) across a panel of four lung cancer cell lines, two mutant for KRas and two wild type.

The effects of BVD-<NUM>, the CDK4/<NUM> inhibitors, another ERK inhibitor (SCH772984), and a reference MEK inhibitor (Trametinib), as single agents on cell viability was assessed after <NUM> using two methods (<FIG>). The first method was by quantitating cellular ATP levels using CellTiter-Glo® (Promega, Madison, Wl). The second method was by quantitating total amount of DNA in an assay well after staining the DNA with Hoechst stain.

The single agent IC<NUM> values are shown in Table <NUM>. The two cell lines carrying a KRas mutation are more sensitive to BVD-<NUM> relative to the wild type cell lines. This may indicate that the presence of a KRas mutation may be a predictive biomarker for sensitivity to BVD-<NUM> as a single agent. The pattern of response to the ERK inhibitor SCH772984 was broadly similar to that of BVD-<NUM>.

The single agent results for the CDK4/<NUM> inhibitors were dependent on the readout for cell viability used, with cells appearing to be markedly more sensitive to inhibition when assessed using Hoechst staining. This suggests that measurement of ATP levels is not a suitable proxy for the number of viable cells in response to CDK4/<NUM> inhibition and, therefore, only the Hoechst stain readout was used in the combination assays.

Combination interactions between two compounds were assessed across a matrix of concentrations using the Loewe Additivity and Bliss Independence Models with Chalice™ Bioinformatics Software (Horizon Discovery Group, Cambridge, MA). Chalice™ enables potential synergistic interactions to be identified by displaying the calculated excess inhibition over that predicted as being additive across the dose matrix as a heat map, and by reporting a quantitative 'Synergy Score' based on the Loewe model.

Combination interactions between BVD-<NUM> and the two CDK4/<NUM> inhibitors are shown in <FIG> and <FIG>, respectively. Combination interactions between SCH772984 and the two CDK4/<NUM> inhibitors are shown in <FIG> and <FIG>, respectively. Combination interactions between Trametinib and the two CDK4/<NUM> inhibitors are shown in <FIG> and <FIG>, respectively.

Visualization of the Loewe 'excess inhibition' heat maps suggested that the combination of BVD-<NUM> with either of the two CDK4/<NUM> inhibitors was mainly additive in A549 and H226 cells, and additive with windows of potential synergy in H1437 and H2122. These windows of synergy appeared broader and stronger in H1437 relative to H2122 cells. Similar results were obtained with the ERK inhibitor SCH772984.

Activity over Loewe additivity can be quantified in Chalice™ using a simple volume score, which effectively calculates a volume between the measured and Loewe additive response surfaces, and emphasizes the overall synergistic (positive values) or antagonistic (negative values) effect of the combination. Volume scores for the combinations of BVD-<NUM> and SCH772984 with either of the two CDK4/<NUM> inhibitors are shown in <FIG> and Tables <NUM>-<NUM> and are consistent with the conclusions drawn from the heat maps.

In summary, these results suggest that interactions between BVD-<NUM> and CDK4/<NUM> inhibitors are at least additive, and in some cases synergistic, in lung cancer cell lines wild type or mutated for KRas.

RAF mutant melanoma cell line A375 cells were cultured in DMEM with <NUM>% FBS and seeded into triplicate <NUM>-well plates at an initial density of <NUM> cells per well. Combination interactions between ERK inhibitors BVD-<NUM> and SCH772984 were analized after <NUM> hours as described above in Example <NUM> and viability was determined using CellTiter-Glo® (Promega) reagent as described above in Example <NUM>.

Visualization of the Loewe and Bliss 'excess inhibition' heat maps suggested that the combination of BVD-<NUM> and SCH772984 was mainly additive with windows of potential synergy in mid-range doses (<FIG>).

Claim 1:
A first anti-cancer agent (i), which is BVD-<NUM> or a pharmaceutically acceptable salt thereof and a second anti-cancer agent (ii), which is a cyclin dependent kinase (CDK) inhibitor or a pharmaceutically acceptable salt thereof, for use in treating or ameliorating the effects of cancer in a subject, wherein the CDK inhibitor is selected from the group consisting of palbociclib, LEE-<NUM>, pharmaceutically acceptable salts thereof, and combinations thereof.