WO2015095833A1 - Treatment of hematologic cancers - Google Patents

Treatment of hematologic cancers Download PDF

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Publication number
WO2015095833A1
WO2015095833A1 PCT/US2014/071733 US2014071733W WO2015095833A1 WO 2015095833 A1 WO2015095833 A1 WO 2015095833A1 US 2014071733 W US2014071733 W US 2014071733W WO 2015095833 A1 WO2015095833 A1 WO 2015095833A1
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Prior art keywords
adult acute
leukemia
agent
adult
myeloid leukemia
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PCT/US2014/071733
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French (fr)
Inventor
Saurabh Saha
Dean WELSCH
Gary Decrescenzo
Jeffrey James ROIX
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Biomed Valley Discoveries, Inc.
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Publication of WO2015095833A1 publication Critical patent/WO2015095833A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

Definitions

  • the present invention provides, inter alia, methods, pharmaceutical compositions, and kits for treating or ameliorating the effects of a hematologic cancer in a subject in need of such treatment. Also provided are methods of modulating ribosomal S6 kinase (RSK) phosphorylation in the blood of a subject that has a hematologic cancer.
  • RSK ribosomal S6 kinase
  • sequence listing text file "0375607.txt”, file size of 468 KB, created on December 15, 2014.
  • sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. ⁇ 1.52(e)(5).
  • Ras plays a role in the regulation of various biological processes including cell growth, proliferation, differentiation, inflammatory responses and programmed cell death. Mutations in ras genes were the first genetic alterations identified in human cancer. Activating mutations of HRAS, NRAS, and KRAS ('RAS') are found in 10-30% of myeloid malignancies. To date, however, treatments for myeloid malignancies have had limited success.
  • One embodiment of the present invention is a method of treating or ameliorating the effects of a hematologic cancer in a subject in need thereof. This method comprises administering to the subject an effective amount of an ERK1/2 inhibitor to treat or ameliorate the effects of the hematologic cancer.
  • Another embodiment of the present invention is a method of treating or ameliorating the effects of a hematologic cancer in a subject in need thereof.
  • This method comprises administering to the subject an effective amount of BVD-523 or a pharmaceutically acceptable salt thereof to treat or ameliorate the effects of the hematologic cancer.
  • a further embodiment of the present invention is a method of modulating ribosomal S6 kinase (RSK) phosphorylation in the blood of a subject that has a hematologic cancer.
  • This method comprises administering to the subject an effective amount of an ERK1/2 inhibitor to modulate RSK phosphorylation in the blood of the subject.
  • RSK ribosomal S6 kinase
  • An additional embodiment of the present invention is a pharmaceutical composition for treating or ameliorating the effects of a hematologic cancer in a subject in need thereof.
  • This pharmaceutical composition comprises a pharmaceutically acceptable carrier and an effective amount of an ERK1/2 inhibitor or a pharmaceutically acceptable salt thereof.
  • Another embodiment of the present invention is a pharmaceutical composition for treating or ameliorating the effects of a hematologic cancer in a subject in need thereof.
  • This pharmaceutical composition comprises a pharmaceutically acceptable carrier and an effective amount of BVD-523 or a pharmaceutically acceptable salt thereof.
  • a further embodiment of the present invention is a kit for treating or ameliorating the effects of a hematologic cancer.
  • This kit comprises any pharmaceutical composition disclosed herein, packaged together with instructions for its use.
  • Figure 1A shows that BVD-523 inhibited ERK 1 and 2 kinases at near-nanomolar potencies. Kinase assays using fluorogenic activity readouts were performed at various inhibitor concentrations.
  • Figure 1 B shows that both direct ERK substrate phosphorylation and known effector pathways are modulated following acute and prolonged treatment with BVD-523 in vitro.
  • Western blots were performed using a variety of antibodies to detect changes in whole-cell lysates of cancer lines exposed to BVD-523.
  • A375 BRAF mutant cell line a human melanoma cell line
  • HCT1 16 KRAS mutant cell line a human colorectal carcinoma cell line
  • Figures 2A and 2B show pharmacokinetic and pharmacodynamic findings observed in patients administered BVD-523 twice- a-day for 15 days, at various dose levels ranging 10 mg to 600 milligrams.
  • Figure 2A shows the maximum and trough exposures of BVD-523 in plasma across multiple subjects and dose levels.
  • Figure 2B shows that, after repeated clinical administration of BVD-523, reductions in an ERK-dependent biomarker (phosphorylation of RSK1/2 serine 380 detected using standard antibody-based ELISA methods) was observed in patient-derived peripheral blood mononuclear cells that were isolated and then stimulated ex vivo.
  • an ERK-dependent biomarker phosphorylation of RSK1/2 serine 380 detected using standard antibody-based ELISA methods
  • Specimens from BVD-523-treated subjects were compared to negative and positive control specimens that were, respectively, obtained from healthy volunteers and left untreated, or treated ex-vivo with 10 ⁇ BVD-523.
  • Near- complete inhibition of ERK-dependent RSK phosphorylation was seen at maximal exposures for several dose levels, and likewise trough exposures and correlative biomarker inhibition data suggest that complete, durable target inhibition of ERK kinases can occur in the peripheral blood tissue compartments following administration of BVD-523 at various dose levels.
  • FIG. 3A shows that BVD-523 can treat acquired resistance to targeted drugs in vitro.
  • Cell line variants of A375 melanoma were selected for 3 months following passage in increasing amounts of dabrafenib, trametinib, BVD-523, or combinations of these agents at various fixed ratios.
  • Cell line variants were obtained that could grow in the presence of dabrafenib or trametinib at concentrations greater than 100 times the IC 50 of these agents in parental A375 cell.
  • cell lines resistant to BVD-523 could only be maintained in less than 10X of parental IC 50 concentration.
  • ST052C A patient-derived line, ST052C, was isolated from a BRAFV600E melanoma patient that progressed following 10 months of therapy with MAPK-pathway directed therapies. Treated ex vivo, ST052C exhibited acquired cross-resistance to dabrafenib at 50 mg/kg BID. Meanwhile, BVD-523 was effective in ST052C as a single-agent at 100 mg/kg BID.
  • Figure 4 shows that BVD-523 inhibits cell growth in a variety of solid tumor cell lines, and that oncogenic mutations relevant to MAPK pathway activation (BRAF and RAS gain-of-function mutations, NF1 inactivation) may confer high drug sensitivity.
  • Cell viability assays (Alamar Blue) suggest a strong cytostatic response occurs following 96 hours of treatment with BVD-523, with cellular IC 50 potencies typically in the low micromolar range.
  • Relevant genetic factors appear to dictate the cytostatic activity of BVD-523: 2 of 15 cell lines not harboring likely MAPK activating mutations appeared to be sensitive at relatively low concentrations, while 1 1 of 19 lines harboring changes in BRAF, KRAS or NF1 genes appeared to be sensitive.
  • FIG. 5A-5OO show the dose response curve of various compounds as specified in the figure legends, including BVD-523, in 40 different cell lines.
  • BVD-523 and ERK inhibition are effective in a variety of malignancies, particularly in cancers where aberrant activation of the MAPK pathway has occurred following oncogenic mutation of various genes, including those in the RAF and RAS oncogene families.
  • both pharmacologically relevant drug exposures and reduced cellular levels of the RSK biomarker are observed after administration of BVD-523 in human subjects.
  • BVD-523 and therapeutic ERK inhibition may exhibit novel utility as an anti-cancer strategy in human lymphoid and myeloid malignancies, when used either as a first-line therapy in treatment- naive subjects, as well as in patients with disease that presents following acquired drug resistance.
  • one embodiment of the present invention is a method of treating or ameliorating the effects of a hematologic cancer in a subject in need thereof.
  • This method comprises administering to the subject an effective amount of an ERK1/2 inhibitor to treat or ameliorate the effects of the hematologic cancer.
  • 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.
  • the methods and compositions of the present invention 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.
  • 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.
  • ameliorate means to decrease the severity of the symptoms of a disease in a subject.
  • a "subject" is a mammal, preferably, a human.
  • other categories of mammals within the scope of the present invention include, for example, farm animals, domestic animals, laboratory animals, etc.
  • farm animals include cows, pigs, horses, goats, etc.
  • domestic animals include dogs, cats, etc.
  • laboratory animals include primates, rats, mice, rabbits, guinea pigs, etc.
  • an ERK1/2 "inhibitor” means those substances that (i) directly interact with ERK1 and/or ERK2, e.g. , by binding to ERK1/2 and (ii) decrease the expression or the activity of ERK1 and/or ERK2 protein kinases. Therefore, inhibitors that act upstream of ERK1/2, such as MEK inhibitors and RAF inhibitors, are not ERK1/2 inhibitors according to the present invention. Preferred ERK1/2 inhibitors of the present invention do not decrease the amount of phosphorylated ERK1 and/or ERK2 but decrease the activity of phosphorylated ERK1 and/or ERK2.
  • Non-limiting examples of ERK1/2 inhibitors according to the present invention include AEZS-131 (Aeterna Zentaris), AEZS-136 (Aeterna Zentaris), BVD-523 (BioMed Valley Discoveries, Inc.), SCH-722984 (Merck & Co.), SCH-772984 (Merck & Co.), SCH-900353 (MK-8353, Merck & Co.), pharmaceutically acceptable salts thereof, and combinations thereof.
  • the ERK1/2 inhibitor is BVD- 523 or a pharmaceutically acceptable salt thereof.
  • BVD-523 a preferred ERK1/2 inhibitor
  • ERK1/2 inhibitor corresponds to a compound according to formula (I):
  • BVD-523 may be synthesized according to the methods disclosed in, e.g. , U.S. Patent No. 7,354,939. Enantiomers and racemic mixtures of both enantiomers of BVD-523 are also contemplated within the scope of the present invention.
  • BVD-523 is a preferred ERK1/2 inhibitor because its mechanism of action is believed to be, e.g. , unique and distinct from certain other ERK1/2 inhibitors, such as SCH772984.
  • ERK1/2 inhibitors such as SCH772984, inhibit autophosphorylation of ERK (Morris et al., 2013), whereas BVD-523 allows for the autophosphorylation of ERK while still inhibiting ERK ( Figure 1 B).
  • the hematologic cancer may be selected from RAS mutant myelodysplastic syndromes (MDS), including refractory anemia with excess blast (RAEB), RAEB in transformation (RAEBt), and Acute myeloid leukemia (AML) following MDS, lymphoid cancers, or myeloid cancers.
  • MDS RAS mutant myelodysplastic syndromes
  • RAEB refractory anemia with excess blast
  • RAEBt RAEB in transformation
  • AML Acute myeloid leukemia
  • Additional non-limiting examples of hematologic cancer within the scope of the present invention include Adult Acute Megakaryoblastic Leukemia (M7), Adult Acute Minimally Differentiated Myeloid Leukemia (MO), Adult Acute Monoblastic Leukemia (M5a), Adult Acute Monocytic Leukemia (M5b), Adult Acute Myeloblasts Leukemia With Maturation (M2), Adult Acute Myeloblasts Leukemia Without Maturation (M1 ), Adult Acute Myeloid Leukemia With 1 1 q23 (MLL) Abnormalities, Adult Acute Myeloid Leukemia With Del(5q), Adult Acute Myeloid Leukemia With Inv(16)(p13;q22), Adult Acute Myeloid Leukemia With t(16;16)(p13;q22), Adult Acute Myeloid Leukemia With t(8;21 )(q22;q22), Adult Acute Myelomonocytic Leukemia (M4), Adult Erythrole
  • the subject with cancer has a somatic RAS and/or BRAF mutation.
  • a "mutation" means a change, e.g., in the nucleic acid sequence of a cell.
  • the RAS mutation is a mutation in H-RAS, N-RAS, or K-RAS.
  • Tables 1 , 2, 3, and 4 show the SEQ ID Nos. of representative nucleic acid and amino acid sequences of wild type H-RAS, K-RAS, N-RAS, and BRAF from various mammalian sources, respectively. These sequences may be used in methods for identifying subjects with a mutant -RAS, K-RAS, N-RAS, and/or BRAF genotype (such as in the methods set forth below).
  • Bos taurus variant X1 33 nucleic acid cow, Bos taurus variant X1
  • 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 2005, sequencing by hybridization, by ligation (for example, WO 2005021786), by degradation (for example, U.S. Patent Nos. 5,622,824 and 6, 140,053) and nanopore sequencing (which is commercially available from Oxford Nanopore Technologies, UK).
  • 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., U.S. Patent Publication No. 20120264632 and International Patent Publication No. WO2012125848.
  • 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.
  • PCR-RFLP polymerase chain reaction-restriction fragment length polymorphism
  • the mutation is discriminated by digestion with specific restriction endonucleases and is identified by electrophoresis. See, e.g., Ota et al., 2007. Mutations may also be detected using real time PCR. See, e.g., International Application publication No. WO2012046981.
  • Hybrid capture methods are known in the art and are disclosed in e.g., U.S. Patent Publication No. 20130203632 and U.S. Patent Nos. 8,389,219 and 8,288,520. 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).
  • a ligand e.g. biotin
  • MIP Molecular Inversion Probe
  • 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 a Tag- cTag duplex, a signal is detected.
  • the method further comprises treating the subject with a combination therapy.
  • the combination therapy may include administering at least one additional therapeutic agent 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.
  • 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. , multi- specific antibodies such as bispecific antibodies). Fragments of antibodies include those that bind antigen, (e.g. , Fab', F(ab') 2 , Fab, Fv, and rlgG). See also, e.g., Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, III.); Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., New York (1998). The term antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. The term "antibody” further includes both polyclonal and monoclonal antibodies.
  • therapeutic antibodies examples 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 6-mercaptopurine, dacarbazine, and fludarabine, and pyrimidine antagonists such as 5-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 according to the present invention also include an inhibitor of the PI3K/Akt pathway.
  • an inhibitor of the PI3K/Akt pathway include A-674563 (CAS # 552325-73-2), AGL 2263, AMG-319 (Amgen, Thousand Oaks, CA), AS-041 164 (5-benzo[1 ,3]dioxol-5- ylmethylene-thiazolidine-2,4-dione), AS-604850 (5-(2,2-Difluoro- benzo[1 ,3]dioxol-5-ylmethylene)-thiazolidine-2,4-dione), AS-605240 (5- quinoxilin-6-methylene-1 ,3-thiazolidine-2,4-dione), AT7867 (CAS # 857531- 00-1 ), benzimidazole series, Genentech (Roche Holdings Inc., South San Francisco, CA), BML-257 (CAS # 32387-96-5), CAL-120
  • PI3 kinase delta inhibitors-2 Incozen (Incozen Therapeutics), PI3 kinase inhibitor, Roche-4 (Roche Holdings Inc.), PI3 kinase inhibitors, Roche (Roche Holdings Inc.), PI3 kinase inhibitors, Roche-5 (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-1 (Pathway Therapeutics Ltd.), PI3-delta inhibitors, Pathway Therapeutics-2 (Pathway Therapeutics Ltd.), PI3-delta/gamma inhibitors, Cellzome (Cellzome AG), PI3-delta
  • toxin means an antigenic poison or venom of plant or animal origin.
  • An example is diphtheria toxin or portions thereof.
  • 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.
  • radionuclides include, but are not limited to, 1-125, At-21 1 , Lu-177, Cu-67, I- 131 , Sm-153, Re-186, P-32, Re-188, ln-1 14m, and Y-90.
  • 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.
  • immunomodulators include, but are not limited to, granulocyte colony- stimulating factor (G-CSF), interferons, imiquimod and cellular membrane fractions from bacteria, IL-2, IL-7, IL-12, CCL3, CCL26, CXCL7, and synthetic cytosine phosphate-guanosine (CpG).
  • G-CSF granulocyte colony- stimulating factor
  • interferons imiquimod and cellular membrane fractions from bacteria
  • IL-2, IL-7, IL-12, CCL3, CCL26, CXCL7 and synthetic cytosine phosphate-guanosine (CpG).
  • photoactive therapeutic agent means compounds and compositions that become active upon exposure to light. Certain examples of photoactive therapeutic agents are described in U.S. Patent Application Serial No. 201 1/0152230 A1 , "Photoactive Metal Nitrosyls For Blood Pressure Regulation And Cancer Therapy.”
  • radiosensitizing agent means a compound that makes tumor cells more sensitive to radiation therapy.
  • radiosensitizing agents include misonidazole, metronidazole, tirapazamine, and trans sodium crocetinate.
  • hormone means a substance released by cells in one part of a body that affects cells in another part of the body.
  • 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, in
  • hormone-interfering compounds include, but are not limited to, tamoxifen (Nolvadex®), anastrozole (Arimidex®), letrozole (Femara®), and fulvestrant (Faslodex®). Such compounds are also within the meaning of hormone in the present invention.
  • 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.
  • VEGF vascular endothelial growth factor
  • Anti-angiogenesis agents include without limitation 2-methoxyestradiol, angiostatin, bevacizumab, cartilage-derived angiogenesis inhibitory factor, endostatin, IFN-a, IL-12, itraconazole, linomide, platelet factor-4, prolactin, SU5416, suramin, tasquinimod, tecogalan, tetrathiomolybdate, thalidomide, thrombospondin, thrombospondin, TNP-470, ziv-aflibercept, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof.
  • the ERK1/2 inhibitor is administered in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier or diluent.
  • Another embodiment of the present invention is a method of treating or ameliorating the effects of a hematologic cancer in a subject in need thereof.
  • This method comprises administering to the subject an effective amount of BVD-523 or a pharmaceutically acceptable salt thereof to treat or ameliorate the effects of the hematologic cancer.
  • Suitable and preferred subjects and hematologic cancers are as disclosed herein.
  • the BVD-523 or a pharmaceutically acceptable salt thereof is administered in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier or diluent.
  • the method further comprises treating the subject with a combination therapy.
  • Suitable and preferred additional therapeutic agents for the combination therapy are as disclosed herein.
  • a further embodiment of the present invention is a method of modulating ribosomal S6 kinase (RSK) phosphorylation in the blood of a subject that has a hematologic cancer.
  • This method comprises administering to the subject an effective amount of an ERK1/2 inhibitor to modulate RSK phosphorylation in the blood of the subject.
  • RSK ribosomal S6 kinase
  • the terms “modulate,” “modulating,” and grammatical variations thereof mean to change, i.e., the phosphorylation level of RSK.
  • the modulation may be a decrease in RSK phosphorylation.
  • the decrease comprises substantially inhibiting RSK phosphorylation by ERK in the blood of the subject.
  • ERK1/2 inhibitors Suitable and preferred subjects, ERK1/2 inhibitors, and hematologic cancers are as disclosed herein.
  • the ERK1/2 inhibitor is administered in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier or diluent.
  • An additional embodiment of the present invention is a pharmaceutical composition for treating or ameliorating the effects of a hematologic cancer in a subject in need thereof.
  • This pharmaceutical composition comprises a pharmaceutically acceptable carrier and an effective amount of an ERK1/2 inhibitor or a pharmaceutically acceptable salt thereof.
  • ERK1/2 inhibitors Suitable and preferred subjects, ERK1/2 inhibitors, and hematologic cancers are as disclosed herein.
  • the pharmaceutical composition further comprises at least one additional therapeutic agent effective for treating or ameliorating the effects of the hematologic cancer. Suitable and preferred additional therapeutic agents are as disclosed herein.
  • Another embodiment of the present invention is a pharmaceutical composition for treating or ameliorating the effects of a hematologic cancer in a subject in need thereof.
  • This pharmaceutical composition comprises a pharmaceutically acceptable carrier and an effective amount of BVD-523 or a pharmaceutically acceptable salt thereof.
  • Suitable and preferred subjects and hematologic cancers are as disclosed herein.
  • the pharmaceutical composition further comprises at least one additional therapeutic agent effective for treating or ameliorating the effects of the hematologic cancer.
  • additional therapeutic agents are as disclosed herein.
  • a further embodiment of the present invention is a kit for treating or ameliorating the effects of a hematologic cancer.
  • This kit comprises any pharmaceutical composition disclosed herein, packaged together with instructions for its use.
  • kits of this invention suitable and preferred subjects having hematologic cancer and types of hematologic cancers are as disclosed herein.
  • the kit further comprises at least one additional therapeutic agent effective for treating or ameliorating the effects of the hematologic cancer.
  • additional therapeutic agents are as disclosed herein.
  • kits may also include suitable storage containers, e.g., ampules, vials, tubes, etc., for each inhibitor (which may, e.g., be in the form of pharmaceutical compositions) and other reagents, e.g. , buffers, balanced salt solutions, etc., for use in administering the pharmaceutical compositions to subjects.
  • the inhibitor 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 a packaging container, optionally having one or more partitions for housing the pharmaceutical composition(s) and other optional reagents.
  • an "effective amount” or a "therapeutically effective amount” of a compound or composition, including pharmaceutical compositions, disclosed herein is an amount of such compound 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.
  • a suitable dose of a composition according to the invention will be that amount of the composition, which is the lowest dose effective to produce the desired effect.
  • the effective dose of a compound 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 ERK1/2 inhibitor disclosed herein is from about 1 mg/kg to about 2400 mg/kg per day, such as from about 1 mg/kg to about 1200 mg/kg per day, 75 mg/kg per day to about 300 mg/kg per day, including from about 1 mg/kg to about 100 mg/kg per day.
  • Other representative dosages of such agents include about 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, 1000 mg/kg, 1 100 mg/kg, 1200 mg/kg, 1300 mg/kg, 1400 mg/kg, 1500 mg/kg, 1600 mg/kg, 1700 mg/kg, 1800 mg/kg, 1900 mg/kg, 2000 mg/kg, 2100 mg/kg, 2200 mg/kg, and 2300 mg/kg per day.
  • the effective dose of ERK1/2 inhibitors e.g
  • An ERK1/2 inhibitor or a pharmaceutical composition of the present invention 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 ERK1/2 inhibitor or the pharmaceutical composition of the present invention may be administered in conjunction with other treatments.
  • the ERK1/2 inhibitor or the pharmaceutical composition of the present invention may be encapsulated or otherwise protected against gastric or other secretions, if desired.
  • compositions of the invention may comprise one or more active ingredients in admixture with one or more pharmaceutically-acceptable carriers and/or diluents and, optionally, one or more other compounds, drugs, ingredients and/or materials. Regardless of the route of administration selected, the agents/compounds of the present invention are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. See, e.g. , Remington, The Science and Practice of Pharmacy (21 st Edition, Lippincott Williams and Wilkins, Philadelphia, PA.).
  • Pharmaceutically acceptable diluents or carriers are well known in the art (see, e.g. , Remington, The Science and Practice of Pharmacy (21 st Edition, Lippincott Williams and Wilkins, Philadelphia, PA.) and The National Formulary (American Pharmaceutical Association, Washington, D.C.)) 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 e.g., liposomes, microspheres, oils (e.g.
  • Each pharmaceutically acceptable diluent or carrier used in a pharmaceutical composition of the invention 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.
  • compositions of the invention may, optionally, contain additional ingredients and/or materials commonly used in pharmaceutical compositions.
  • ingredients and materials are well known in the art and include (1 ) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, such as glycerol; (4) 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; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glycerol monostearate; (8)
  • compositions of the present invention 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 nonaqueous 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 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.
  • 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.
  • 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.
  • compositions of the present invention 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions of the present invention 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.
  • suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents 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.
  • 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.
  • a drug e.g. , pharmaceutical formulation
  • 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.
  • 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 presented 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.
  • sterile liquid diluent or carrier for example water for injection
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.
  • BVD-523 is an ERK1/2 inhibitor
  • BVD-523 The ability of BVD-523 to inhibit ERK1 and EKR2 was assayed using Z'-LYTETM Kinase Assay Kits (Life Technologies, Invitrogen Corp., Madison, Wl) according to the manufacturer's instructions.
  • Z'-LYTETM Kinase Assay Kits (Life Technologies, Invitrogen Corp., Madison, Wl) according to the manufacturer's instructions.
  • the 2X MAPK3 (ERK1 )/Ser/Thr 03 mixture is prepared in 50 mM HEPES pH 7.5, 0.01 % BRIJ-35, 10 mM MgCI2, 1 mM EGTA.
  • the final 10 ⁇ _ Kinase Reaction consists of 5.94-94.5 ng MAPK3 (ERK1 ) and 2 ⁇ Ser/Thr 03 in 50 mM HEPES pH 7.5, 0.01 % BRIJ-35, 10 mM MgCI2, 1 mM EGTA.
  • MAPK3 MAPK3
  • 2 ⁇ Ser/Thr 03 in 50 mM HEPES pH 7.5, 0.01 % BRIJ-35, 10 mM MgCI2, 1 mM EGTA.
  • the 2X MAPK1 (ERK2)/Ser/Thr 03 mixture is prepared in 50 mM HEPES pH 7.5, 0.01 % BRIJ-35, 10 mM MgCI2, 1 mM EGTA.
  • the final 10 ⁇ _ Kinase Reaction consists of 2-45.5 ng MAPK1 (ERK2) and 2 ⁇ Ser/Thr 03 in 50 mM HEPES pH 7.5, 0.01 % BRIJ-35, 10 mM MgCI2, 1 mM EGTA.
  • ERK2 MAPK1
  • Ser/Thr 03 in 50 mM HEPES pH 7.5, 0.01 % BRIJ-35, 10 mM MgCI2, 1 mM EGTA.
  • 5 ⁇ _ of a 1 : 1024 dilution of Development Reagent A was added.
  • BVD-523 and ERK inhibition exhibited anti-cancer activity, particularly following oncogenic activation of MAPK-pathwav signaling
  • Each cell line was treated with BVD-523, another ERK2 inhibitor (TCS ERK 1 1 e, or 4-[2-[(2-Chloro-4-fluorophenyl)amino]-5-methyl-4- pyrimidinyl]-N-[(1 S)-1-(3-chlorophenyl)-2-hydroxyethyl]-1 H-pyrrole-2- carboxamide), a standard chemotherapy drug as positive control, vehicles, and culture medium.
  • TCS ERK 1 1 e 4-[2-[(2-Chloro-4-fluorophenyl)amino]-5-methyl-4- pyrimidinyl]-N-[(1 S)-1-(3-chlorophenyl)-2-hydroxyethyl]-1 H-pyrrole-2- carboxamide
  • Both BVD-523 and TCS ERK 1 1 e were dissolved in DMSO and stored at room temperature.
  • doxorubicin was purchased from Zhejiang (Haizheng, China) in the form of a powder and stored at 4°C; cisplatin was purchased from Nanjing Zhiyao (China) in the form a powder and stored at room temperature; and mitomycin was purchased from Zhejiang (Haizheng, China) in the form of a powder and stored at room temperature.
  • the 40 cell lines used for this Example are listed below in Table 5.
  • the positive controls used were cisplatin, doxorubicin, or mitomycin, and the incubation time was 96 hours.
  • CellTiter-Glo® Luminescent Cell Viability Assay (Cat. No.: G7572, Promeg) was stored at -20°C. To prepare the reagent, the CellTiter- Glo Buffer was thawed and equilibrated to room temperature prior to use for up to 48 hours prior to use.
  • Cells were harvested respectively during the logarithmic growth period and count cell number using Countstar. The cell concentrations were adjusted to 5.56x10 4 cells/ml with respective culture medium. Then 90 ⁇ of cell suspensions were added to two 96-well plates (A and B) with the final cell density of approximately 2*10 3 cells/well.
  • test plates were incubated for 72 hours in the humidified incubator at 37°C with 5% CO 2 , and then measured by means of a CellTiter-Glo® Luminescent Cell Viability Assay as follows.
  • the plate and its contents were equilibrated at room temperature for approximately 30 minutes.
  • a volume of CellTiter-Glo® Reagent equal to the volume of cell culture medium present in each well was added to each well (e.g., add 100 ⁇ of reagent to 100 ⁇ of medium containing cells for a 96-well plate).
  • the contents were mixed for 2 minutes on an orbital shaker to induce cell lysis.
  • the plates were allowed to incubate at room temperature for 10 minutes to stabilize the luminescent signal.
  • the luminescence signals were recorded using an EnVision Multi Label Reader.
  • the Surviving rate (%) (ODTest article-ODMedium control)/ (ODNone treated-ODMedium control)* 100%.
  • Table 7 Summary of cell growth normalized by TO data (set TO data at 1 .0)
  • Sensitive cell lines generally exhibit reduced cell growth following treatment with BVD-523 at concentrations in the low micromolar range over 96 hours.
  • BVD-523 sensitivity is observed in several different cancer histologies, including a variety of lines originating from colorectal and hepatocellular carcinomas.
  • Cell lines with, inter alia, RAS/BRAF mutations show enhanced sensitivity in the presence of the ERK1/2 inhibitor, BVD-523.
  • the liver cancer cell line HepG2 which has a mutation in N-RAS, was sensitive to BVD-523.
  • the colon cancer cell line, SW620 (contains both K-RAS and MAP2K4 mutation) also showed high sensitivity to BVD-523.
  • BVD-523 altered markers of MAPK kinase activity and effector function
  • HCT1 16 cells (5 x 10 6 ) were seeded into 10 cm dishes in McCoy's 5A plus 10% FBS.
  • A375 cells (2.5 x 10 6 ) were seeded into 10 cm dishes in DMEM plus 10% FBS. Cells were allowed to adhere overnight prior to addition of the indicated amount of test compound (BVD-523) or vehicle control. Cells were treated for either 4 or 24 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-lmmunoprecipitation Assay) buffer, clarified by centrifugation and quantitated by bicinchoninic acid assay (BCA) assay. 20-50 ⁇ g of protein was resolved by SDS-PAGE electrophoresis, blotted onto PVDF membrane and probed using the antibodies detailed in Table 9 (for the 4-hour treatment) and Table 10 (for the 24-hour treatment) below.
  • RIPA Radio-lmmunoprecipitation Assay
  • BCA bicinchoninic acid assay
  • Figure 1 B shows Western blot analyses of cells treated with BVD- 523 at various concentrations for the following: 1 ) MAPK signaling components in A375 cells after 4 hours; 2) cell cycle and apoptosis signaling in A375 24 hours treatment with various amounts of BVD-523; and 3) MAPK signaling in HCT-1 16 cells treated for 4 hours.
  • the results show that acute and prolonged treatment with BVD-523 in RAF and RAS mutant cancer cells in-vitro affects both substrate phosphorylation and effector targets of ERK kinases.
  • the concentrations of BVD-523 required to induce these changes is typically in the low micromolar range.
  • BVD-523 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-523 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-523.
  • effector genes that require MAPK pathway signaling are altered following treatment with BVD-523.
  • 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-523 treatment.
  • gene expression and protein abundance of apoptosis effectors often require intact MAPK signaling, and total levels of Bim-EL increase following prolonged BVD-523 treatment.
  • BVD-523 can treat acquired resistance to targeted drugs in vitro
  • Cancer cell lines were maintained in cell culture under standard media and serum conditions. For dose escalation studies, A375 cells were split, grown to about 40-60% confluence, and then treated with the initial dose of the specified drug. Table 1 1 shows a summary of drug treatments that were escalated.
  • Proliferation assays were used to track changes in sensitivity to the escalated agent(s) at appropriate time intervals (e.g. each month, although the timing is dependent on adequate cell numbers being available).
  • proliferation assays cells were seeded in 96-well plates at 3000 cells per well in drug-free DMEM medium containing 10% FBS and allowed to adhere overnight prior to addition of compound or vehicle control.
  • Compounds were prepared from DMSO stocks. The final DMSO concentration was constant at 0.1 %.
  • Test compounds were incubated with the cells for 96 hours at 37°C and 5% CO 2 in a humidified atmosphere. Alamar Blue 10% (v/v) was then added and incubated for 4 hours and fluorescent product was detected using a BMG FLUOstar plate reader. The average media only background value was deducted and the data analyzed using a 4-parameter logistic equation in GraphPad Prism. Paclitaxel was used as a positive control.
  • T 30 nM
  • T 16 nM
  • A375 cells were seeded into triplicate 96-well plates at a cell density of 3000 cells/well in DMEM plus 10% FBS and allowed to adhere overnight prior to addition of test compound or vehicle control. Combinations were tested using a 10x8 dose matrix with a final DMSO concentration of 0.2%. A 96 hour assay incubation period followed, with subsequent addition of Alamar Blue 10% (v/v) and 4 hours incubation prior to reading on a fluorescent plate reader. After reading Alamar Blue, the medium/Alamar Blue mix was flicked off and 100 ⁇ of CellTiter-Glo/PBS (1 :1 ) added and the plates processed as per the manufacturer's instructions (Promega). Media only background values were subtracted before the data was analysed. The Bliss additivity model was then applied.
  • HCT1 16 cells were seeded in triplicate in white 96-well plates at a cell density of 5000 cells/well in McCoy's 5A plus 10% FBS.
  • A375 cells were seeded at a density of 5000 cells/well in DMEM plus 10% FBS. Cells were allowed to adhere overnight prior to addition of the indicated amount of test compound or vehicle control. The final concentration of DMSO was 0.2%, and 800 nM staurosporine was included as a positive control. 24 and 48 hour assay incubation periods were used.
  • Figure 3A shows single and combination agent escalation for month 3 of the studies.
  • Cell line variants were obtained that could grow in the presence of dabrafenib or trametinib at concentrations greater than 100 times the IC 5 o of these agents in parental A375 cell.
  • cell lines resistant to BVD-523 could only be maintained in less than 10X of parental IC50 concentration.
  • Sensitivity testing suggested dabrafenib and trametinib-resistant cell lines remained relatively sensitive to BVD-523; the increased IC50 "shift" for BVD-523 in resistant cell lines was more modest than those corresponding IC 50 increases following dabrafenib or trametinib treatment.
  • BVD-523 can treat acquired resistance to targeted drugs in vivo
  • mice between 5-8 weeks of age were housed on irradiated corncob bedding (Teklad) in individual HEPA ventilated cages (Sealsafe ® Plus, Techniplast USA) on a 12-hour light-dark cycle at 70-74 (21- 23°C) and 40-60% humidity. Animals were fed water ad libitum (reverse osmosis, 2 ppm Cl 2 ) and an irradiated standard rodent diet (Teklad 2919) consisting of 19% protein, 9% fat, and 4% fiber.
  • Tumor Growth Inhibition (TGI) study was typically completed once all groups reached a predetermined mean tumor volume or a predetermined study day was reached; treatment groups may be taken out past this timepoint and collected measurements on that date used for analysis.
  • %TGI percent tumor growth inhibition
  • mice lacking palpable tumors were classified as complete responders (CR); a CR that persisted until study completion was considered a tumor-free survivor (TFS); TFS animals were excluded from TGI calculations.
  • CR complete responders
  • TFS tumor-free survivor
  • Statistical differences in tumor volume were determined using a two-tailed One-Way Analysis of Variance (ANOVA) followed by the Dunnett's multiple comparisons test comparing treated single agent groups with control and combinations with standard agent when possible.
  • ANOVA One-Way Analysis of Variance
  • T d tumor doubling time
  • BVD-523 was administered p.o. at 100 mg/kg on a bidx31 schedule. Dabrafenib was also evaluated as a single agent on the same treatment regimen. Weight loss was reported in all groups; one technical related death was reported in the single agent BVD-523 group on Day 21 (Table 18). Two partial responders (PR) were reported in the combination group. TGI values of 78%, 76% and 22% were calculated, respectively; activity was found statistically significant (p ⁇ 0.05) in single agent BVD-523 with vehicle control (Table 19).
  • BVD-523 Human plasma was treated with K 2 EDTA anticoagulant (Bioreclamation Inc., East Meadow, NY). BVD-523 was extracted from human plasma by protein precipitation and analyzed using liquid chromatography (LC) with tandem mass spectrometric detection (MS/MS). The standard curve range is from 1.00 to 1000 ng/mL for BVD-523 using a plasma sample volume of 0.0500 mL.
  • the chromatographic conditions were as follows.
  • the column used was a Supeico, Ascentis® Express C18, 50 x 3.0 mm, 2.7 pm particle size.
  • the pre-filter was 0.5 gin, Upchurch.
  • the column temperature was 40°C.
  • solution A was Type 1 Water
  • solution B was acetonitrile.
  • the gradient program was set with initial conditions of 0.800 mL/min; 30% solution B. The rest of the conditions are detailed in Table 20 below.
  • the flow rate was set to be 0.800 ml/min, and the typical back pressure was 170 bars.
  • the sample tray was refrigerated (2 to 8°C).
  • the injection volume was typically 3-5 ⁇ , but not to exceed 10 ⁇ total.
  • Acetonitrile was used for both the rinse port injector wash solution and the rinse pump injector wash solution. Needle stroke was set to be 50 mm. Rinse pump setting was from rinse pump to rinse port. The rinse volume was 450 ⁇ . The rinse mode was set to be before and after sampling. The rinse dip time was 5 seconds, and the rinse time was 1 second. The acquisition time was approximately 3.2 minutes, and the cycle time was approximately 5.0 minutes (from one injection start to the next injection start).
  • the regression type and weighting were linear, 1/x 2 , respectively.
  • the units of concentration were ng/mL, and the response was measured using peak area ratios (BVD-523/ISTD1 ).
  • Target biomarkers biomarkers that lie directly downstream of a protein of interest, are key resources in assessing target coverage when modulating the activity of the protein of interest with a small molecule inhibitor in preclinical and clinical in vivo studies.
  • RSK1 a member of the RSK serine/threonine kinase family, is a direct substrate of the MAP Kinases ERK1 and ERK2 (Romeo, Zhang and Roux, 2012).
  • RSK1 and ERK1/2 form an inactive complex in unstimulated cells.
  • ERK1/2 phosphorylates Thr573, Thr359 and Ser363 on RSK1.
  • Thr573 resides in the activation loop of the carboxy terminal kinase domain of RSK1 and once phosphorylated, enables RSK1 to autophosphorylate Ser380 (Cargnello and Roux, 201 1 ). Phosphorylation of Ser380 on RSK1 can then be used as a target biomarker for ERK1 and ERK2 activity.
  • phosphorylation of RSK1 on Ser380 was used as a target biomarker for assessment of ERK inhibition by BVD-523 in ex vivo treated human whole blood samples from ten healthy individuals.
  • Efforts consist of the determination of IC 50 values for the inhibition of phorbol 12-myristate 13-acetate (PMA) stimulated RSK1 phosphorylation by BVD-523. An eight point concentration curve ranging from 10 ⁇ to 5 nM BVD-523 was used for each IC 50 determination. Controls consisted of three unstimulated samples and three PMA stimulated samples for each donor. Two donors were analyzed in each experiment. A total of ten different volunteers were evaluated with an age range of 22 to 61 years.
  • PMA phorbol 12-myristate 13-acetate
  • PBMC peripheral blood mononucleated cell
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS Fetal Bovine Serum
  • Dulbecco's Phosphate Buffered Saline (DPBS) (Ca ++ , Mg-HE free) from Gibco Catalog Number: 14190;
  • Phosphatase Inhibitor Cocktail 3 (100X) from Sigma-Aldrich Catalog Number: P0044;
  • Phosphatase Inhibitor Cocktail 2 (100X) from Sigma-Aldrich Catalog Number: P5726;
  • BVD-523 was stored at room temperature in the dark.
  • DMSO was added to all tubes in groups "A” and "C” to a final concentration of 0.1 % (10 ⁇ per ml of a 10% DMSO solution in DMEM containing 10% FBS and penicillin/streptomycin/glutamine). Samples were then rocked gently at room temperature.
  • BVD-523 (10 mM in 100% DMSO) was serially diluted with 3- fold dilutions into 100% DMSO. These serially diluted BVD-523 samples in 100% DMSO were then diluted 10-fold in DMEM containing 10% FBS and penicillin/streptomycin/glutamine, and 10 ⁇ of each of these working solutions was added per ml of blood for each designated BVD-523 concentration. Each concentration of BVD-523 was run in duplicate, two 1 ml blood samples each, yielding sixteen total samples for the full eight point concentration curve. Samples were then rocked gently at room temperature for a minimum of two hours, but no longer than three hours.
  • PBMCs were isolated from the human whole blood.
  • One ml of blood from each sample was gently layered onto 0.75 ml of Histopaque 1077 in a 2 ml microcentrifuge tube. Histopaque 1077 was maintained at room temperature.
  • the samples were centrifuged for two minutes at 16,000 x g in an Eppendorf microcentrifuge.
  • the interface and upper layers were removed and added to tubes containing 1 ml cold Dulbecco's Phosphate-Buffered Saline (DPBS). These samples were then centrifuged for 30 seconds at 16,000 x g in an Eppendorf microcentrifuge to pellet the cells.
  • DPBS cold Dulbecco's Phosphate-Buffered Saline
  • the buffer supernatant was removed by aspiration and the pellets were re-suspended in 1 ml of cold DPBS. The pellets from each sample were then re-pelleted as above. The buffer was removed by aspiration and the pellets were lysed as indicated below.
  • complete lysis buffer consisted of MSD Tris lysis buffer, 1X Halt Protease inhibitor cocktail, 1X Phosphatase inhibitor cocktail 2, 1X Phosphatase inhibitor cocktail 3, 2mM PMSF and 0.1 % SDS. Lysis buffer was kept on ice and made fresh for each sample group. Final cell pellets were lysed by the addition of 120 ⁇ of complete lysis buffer. Samples were vortexed until the cell pellet disappeared and then flash frozen on dry ice. Samples were stored at -20°C prior to measurement of pRSK and total RSK by ELISA.
  • Both ELISAs were developed using the follow steps. Gently remove the tape and wash wells: Discard liquid contents of each plate into a receptacle. Wash 4 times with 1X Wash Buffer (provided in ELISA kit), 200 ⁇ each time for each well. For each wash, strike plates on fresh towels hard enough to remove the residual solution in each well, but do not allow wells to completely dry at any time. Add 100 ⁇ of Detection Antibody (provided in ELISA kit) to each well. Seal with tape and incubate the plate for 1 hour at 37°C. Repeat wash procedure as noted above. Add 100 ⁇ of HRP-Linked secondary antibody (provided in ELISA kit) to each well.
  • SoftMax Pro Version 4.6 (Molecular Devices), Microsoft Excel 2010 (Microsoft) and GraFit Data Analysis Software (Version 5.0.12) were used for data analysis.
  • BVD-523 The preclinical activity of BVD-523 on enzymes and cells, as well as several favorable pharmaceutical properties of the molecule (e.g. absorption, metabolism and distribution characteristics not shown here), supported the use of BVD-523 as an investigational anti-cancer agent in a human clinical trial.
  • BVD-523 exhibits oral bioavailability in humans: patients exhibit systemic exposures of BVD-523 within hours following administration of a single dose presented as a powder in standard gelatin capsules, and systemic BVD-523 exposures increase as oral dose increases (not shown). After repeated, twice-daily administration for up to 15 days, systemic exposures of BVD-523 at steady-state also appear to increase with increases in dose level (Figure 2A). The absolute concentration of BVD-523 observed at various dose levels appears consistent with concentrations required for ERK target enzyme inhibition and cellular bioactivity (above, Figure 2A).
  • ERK inhibition in hematologic malignancies takes advantage of oncogenic driver mutations in genetic components coupled to the MAPK signaling cascade, which would likely provide an enhanced therapeutic index. It is possible that particular hematologic malignancies may exhibitaki intrinsic sensitivity to BVD- 523, while simultaneously displaying insensitivity to either RAF or MEK inhibitors. In a subset of hairy-cell leukemias, for example, oncogenic MAP2K1 gene mutations confer MEK inhibitor insensitivity— variants of this type may remain sensitive to ERK inhibition and BVD-523.
  • BVD-523 and ERK inhibition may exhibit unique anti-cancer activity in malignancies that have acquired resistance to other targeted agents, including RAF and MEK inhibitors.
  • MAP2K1 activating mutations have been identified in subjects with melanoma that exhibited disease progression following therapy with RAF and MEK inhibitors, either alone or in combination. It is possible that these variants, as well as other adaptive changes that occur during cancer therapy, may evoke drug resistance to a variety of agents, while not altering sensitivity to BVD-523 or ERK inhibition.
  • BVD-523 properties of BVD-523 may make this a preferred agent for use as an ERK inhibitor, compared to other agents with a similar activity.
  • 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.
  • inhibitors of ABL, KIT, EGFR and ALK kinases are effective only when their cognate target is found in active or inactive configurations.
  • certain of these inhibitors are uniquely sensitive to either secondary genetic mutation, or post-translational adaptive changes, of the protein target.
  • RAF inhibitors show differential potency to RAF kinases present in certain protein complexes and/or subcellular localizations.
  • ERK kinases are similarly known to exist in diverse, variable, and complex biochemical states, it appears likely that BVD-523 may interact with and inhibit these targets in a fashion that is distinct and highly preferable to other agents.
  • BVD-523 Given the evidence of pharmacologically relevant exposures and potential bioactivity of BVD-523 in humans, coupled with the fact that many hematologic cancers have mutations in the RAS oncogene, which is activated upstream of ERK in the MAPK pathway (Li et al., 201 1 ; Lauchle et al., 2009; Borthakur ei al., 2012), BVD-523 is expected to have clinically relevant potency in human lymphoid and myeloid malignancies as well as preleukemic myelodysplastic syndromes (MDS) and lymphomas.
  • MDS preleukemic myelodysplastic syndromes
  • BVD-523 is effective in inhibiting the growth of hematologic cancer cell lines in vitro
  • Cancer cell lines are maintained in cell culture under standard media and serum conditions.
  • BVD-523 is effective in inhibiting the growth of the above hematologic cancer cell lines. Dose response curves will be obtained. It is expected that the IC 50 of BVD-523 in these cell lines will be approximately 150 nM. Example 8
  • BVD-523 is effective in treating hematologic cancers in vivo
  • mice Female athymic nude mice (Crl:NU(Ncr)-Foxn/ nu , Charles River) are nine weeks old with a body weight (BW) range of about 15 to about 30 grams on Day 1 of the study.
  • the animals are fed ad libitum water (reverse osmosis, 1 ppm CI), and NIH 31 Modified and Irradiated Lab Diet ® consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fiber.
  • the mice are housed on irradiated Enrich-o'cobsTM Laboratory Animal Bedding in static microisolators on a 12-hour light cycle at 20-22°C (68-72°F) and 40-60% 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.
  • Tumor xenografts (1-2x10 6 human myeloid leukaemia HL60 cells) are subcutaneously implanted into the flank of athymic nude mice. Tumors are measured in two dimensions using calipers, and volume is calculated using the formula:
  • Tumor weight may be estimated with the assumption that 1 mg is equivalent to 1 mm 3 of tumor volume.
  • BVD-523 is supplied as a dry powder and is stored at room temperature protected from light.
  • BVD-523 doses are prepared by suspending the required amount of BVD-523 powder in 1 % carboxymethyl cellulose in deionized water ("1 % CMC"). A 10 mg/mL BVD-523 stock is prepared, and is used to dose the 100 mg/kg BVD-523 group. Aliquots of the stock are diluted with the vehicle to a concentration of 5.0 mg/mL to provide the 50 mg/kg BVD-523 dosage in a dosing volume of 10 mL/kg. The BVD-523 doses are stored at 4°C protected from light for up to one week.
  • the 1 % CMC vehicle (“Vehicle”) is used to dose the control group.
  • Doxorubicin is approved to treat various cancers including Acute Lymphoblastic Leukemia (ALL) and Acute Myeloid Leukemia (AML). Doxorubicin is dissolved in distilled water at a concentration such that the dose could be given in a volume of 0.1 ml/10 g of body weight.
  • ALL Acute Lymphoblastic Leukemia
  • AML Acute Myeloid Leukemia
  • mice are sorted into five groups (Group 1-5) each consisting of fifteen mice, and dosing is initiated according to the treatment plan summarized in Table 24 below.
  • Each BVD-523 dose is given by oral gavage (p.o.) in a dosing volume of 10 mL/kg (0.2 mL per 20 grams of body weight), scaled to the body weight of each individual animal.
  • Each doxorubicin dose is given by intravenous injection (i.v.) in a dosing volume of 0.1 mL/10g of body weight, scaled to the body weight of each individual animal.
  • the vehicle and the BVD-523 doses are to be given twice daily until study end (bid to end), and the doxorubicin doses are to be given once a week until study end (qw to end).
  • bid dosing dosing is initiated in the afternoon on Day 1 , so that one dose is given on the first day ("first day 1 dose").
  • one dose is given in the afternoon on the first day and one dose in the morning on the last day.
  • Group 1 receives 1 % CMC vehicle, and serves as the control group for calculation of %TGD.
  • Group 5 receives doxorubicin at 100 mg/kg and serves as a reference group.
  • Groups 2 and 3 receive 50 and 100 mg/kg BVD-523, respectively. Tumor measurements
  • Tumors are measured using calipers twice per week, and each animal is euthanized when its tumor reaches the pre-determined tumor volume endpoint of 2000 mm 3 or on the final day (day 50), 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.
  • TTE time to endpoint
  • TTE loqn (endpoint volume) - b
  • TTE is expressed in days
  • endpoint volume is expressed in mm 3
  • b is the intercept
  • 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 exceeded the endpoint volume used in analysis and the three consecutive observations that immediately preceded 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 did not reach the endpoint volume are assigned a TTE value equal to the last day of the study.
  • TTE tumor growth delay
  • TGD T - C, expressed in days, or as a percentage of the median TTE of the control group:
  • T median TTE for a treatment group
  • 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.
  • PR partial regression
  • CR complete regression
  • the tumor volume is 50% or less of its Day 1 volume for three consecutive measurements during the course of the study, and equal to or greater than 135 mm 3 for one or more of these three measurements.
  • a CR response the tumor volume is less than 135 mm 3 for three consecutive measurements during the course of the study.
  • An animal with a CR response at the termination of a study is additionally classified as a tumor-free survivor (TFS). Animals are monitored for regression responses.
  • TFS tumor-free survivor
  • Acceptable toxicity for the maximum tolerated dose is defined as a group mean body-weight loss of less than 20% during the study and not more than 10% treatment-related (TR) deaths.
  • TR treatment-related
  • 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 14 days of the last dose.
  • a death is classified as non-treatment-related (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 resulted from an accident or human error.
  • a death is classified as NTRm if necropsy indicated that it may have resulted 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.
  • mice per group When available, five mice per group are euthanized by terminal cardiac puncture under carbon dioxide anesthesia at 3, 6 and 12 hours post final dose, and the full blood volume of each animal is collected. The serum is separated and stored frozen at -80°C until shipment. In addition, the tumors of these mice are harvested and divided into two parts. One part is snap frozen and stored at -80°C. The other part is fixed for 16-24 hours in 10% neutral buffered formalin, and then transferred to 70% ethanol. For groups with mice that had no detectable tumor, the implant site including full skin and muscle thickness is collected from three mice per group.
  • 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).
  • 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 body weight changes from Day 1 are calculated for each group for each day of body weight measurement, and are plotted as a function of time. Tumor growth and body weight plots will exclude the data for NTR deaths, and are truncated after 50% of the assessable animals in a group which has exited the study.
  • BVD-523 is effective against HL60 cancer cells at both 50 and 100 mg/kg doses and that the effects are statistically significant. It is also expected that the side effects associated with BVD-523 treatment are minimal.
  • GILMARTIN AG Bleam MR, Groy A, ei al. GSK1 120212 (JTP-74057) is an inhibitor of MEK activity and activation with favorable pharmacokinetic properties for sustained in vivo pathway inhibition.
  • Clinical cancer research an official journal of the American Association for Cancer
  • GSK21 18436 dabrafenib mediated by NRAS or MEK mutations.
  • MT (201 1 ). "Deep sequencing the transcriptome reveals seasonal adaptive mechanisms in a hibernating mammal”. PLoS One. 6 (10). HARDENBOL, P., et al. Multiplexed genotyping with sequence-tagged molecular inversion probes. Nat. Biotechnol. 2003, no.21 , p.673-678. HATZIVASSILIOU G, Liu B, O'Brien C, et al. ERK inhibition overcomes acquired resistance to MEK inhibitors. Molecular cancer therapeutics. May 2012; 1 1 (5): 1 143-1 154.
  • HODIS E Watson IR, Kryukov GV, et al. A landscape of driver mutations in melanoma. Cell. Jul 20 2012; 150(2):251-263.
  • RAF inhibitor PLX4032 inhibits ERK signaling and tumor cell proliferation in a V600E BRAF- selective manner. Proceedings of the National Academy of Sciences of the United States of America. Aug 17 2010; 107(33): 14903-14908.

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Abstract

The present invention provides, inter alia, methods of treating or ameliorating the effects of a hematologic cancer in a subject in need of such treatment. These methods include administering to the subject an effective amount of an ERK1/2 inhibitor, such as BVD-523, to treat or ameliorate the effects of the hematologic cancer. Also provided are methods of modulating ribosomal S6 kinase (RSK) phosphorylation in the blood of a subject that has a hematologic cancer. Pharmaceutical compositions and kits for treating or ameliorating the effects of a hematologic cancer are additionally provided.

Description

TREATMENT OF HEMATOLOGIC CANCERS CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Provisional Patent Application No. 61/919,293, filed December 20, 2013, which is incorporated herein by reference in its entirety.
FIELD OF INVENTION
[0002] The present invention provides, inter alia, methods, pharmaceutical compositions, and kits for treating or ameliorating the effects of a hematologic cancer in a subject in need of such treatment. Also provided are methods of modulating ribosomal S6 kinase (RSK) phosphorylation in the blood of a subject that has a hematologic cancer.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
[0003] This application contains references to amino acids and/or nucleic acid sequences that have been filed concurrently herewith as sequence listing text file "0375607.txt", file size of 468 KB, created on December 15, 2014. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. § 1.52(e)(5).
BACKGROUND OF THE INVENTION
[0004] Within cellular signaling networks, Ras plays a role in the regulation of various biological processes including cell growth, proliferation, differentiation, inflammatory responses and programmed cell death. Mutations in ras genes were the first genetic alterations identified in human cancer. Activating mutations of HRAS, NRAS, and KRAS ('RAS') are found in 10-30% of myeloid malignancies. To date, however, treatments for myeloid malignancies have had limited success.
[0005] In view of the foregoing, there is, inter alia, a need for new methods for treating myeloid malignancies. The present application is directed to meeting these and other needs.
SUMMARY OF THE INVENTION
[0006] One embodiment of the present invention is a method of treating or ameliorating the effects of a hematologic cancer in a subject in need thereof. This method comprises administering to the subject an effective amount of an ERK1/2 inhibitor to treat or ameliorate the effects of the hematologic cancer.
[0007] Another embodiment of the present invention is a method of treating or ameliorating the effects of a hematologic cancer in a subject in need thereof. This method comprises administering to the subject an effective amount of BVD-523 or a pharmaceutically acceptable salt thereof to treat or ameliorate the effects of the hematologic cancer.
[0008] A further embodiment of the present invention is a method of modulating ribosomal S6 kinase (RSK) phosphorylation in the blood of a subject that has a hematologic cancer. This method comprises administering to the subject an effective amount of an ERK1/2 inhibitor to modulate RSK phosphorylation in the blood of the subject.
[0009] An additional embodiment of the present invention is a pharmaceutical composition for treating or ameliorating the effects of a hematologic cancer in a subject in need thereof. This pharmaceutical composition comprises a pharmaceutically acceptable carrier and an effective amount of an ERK1/2 inhibitor or a pharmaceutically acceptable salt thereof.
[0010] Another embodiment of the present invention is a pharmaceutical composition for treating or ameliorating the effects of a hematologic cancer in a subject in need thereof. This pharmaceutical composition comprises a pharmaceutically acceptable carrier and an effective amount of BVD-523 or a pharmaceutically acceptable salt thereof.
[0011] A further embodiment of the present invention is a kit for treating or ameliorating the effects of a hematologic cancer. This kit comprises any pharmaceutical composition disclosed herein, packaged together with instructions for its use.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
[0013] Figure 1A shows that BVD-523 inhibited ERK 1 and 2 kinases at near-nanomolar potencies. Kinase assays using fluorogenic activity readouts were performed at various inhibitor concentrations.
[0014] Figure 1 B shows that both direct ERK substrate phosphorylation and known effector pathways are modulated following acute and prolonged treatment with BVD-523 in vitro. Western blots were performed using a variety of antibodies to detect changes in whole-cell lysates of cancer lines exposed to BVD-523. In the A375 BRAF mutant cell line (a human melanoma cell line) and in the HCT1 16 KRAS mutant cell line (a human colorectal carcinoma cell line), phosphorylation of ERK-dependent residues (T359/S363) in RSK 1 and 2 proteins was reduced after 4 hours of treatment with BVD-523 at micromolar concentrations. Following 24 hours of treatment, direct substrate inhibition was maintained in BRAF mutant cell lines, and the MAPK feedback phosphatase DUSP6 was greatly reduced, suggesting durable and nearly complete MAPK pathway inhibition. Lastly, consistent with cytostatic effects of BVD-523 across multiple cell line backgrounds, the MAPK effector and G1/S-cell-cycle determinant gene cyclin-D1 was greatly reduced after 24 hours of treatment. In the A375 cell line, while the apoptosis effector and ERK substrate Bim-EL was increased following prolonged treatment, increased apoptosis was not observed, consistent with a lack of PARP cleavage, as well as other observations (not shown) that additional factors influence the capacity for BVD-523 to induce cell death.
[0015] Figures 2A and 2B show pharmacokinetic and pharmacodynamic findings observed in patients administered BVD-523 twice- a-day for 15 days, at various dose levels ranging 10 mg to 600 milligrams. Figure 2A shows the maximum and trough exposures of BVD-523 in plasma across multiple subjects and dose levels. Figure 2B shows that, after repeated clinical administration of BVD-523, reductions in an ERK-dependent biomarker (phosphorylation of RSK1/2 serine 380 detected using standard antibody-based ELISA methods) was observed in patient-derived peripheral blood mononuclear cells that were isolated and then stimulated ex vivo. Specimens from BVD-523-treated subjects were compared to negative and positive control specimens that were, respectively, obtained from healthy volunteers and left untreated, or treated ex-vivo with 10 μΜ BVD-523. Near- complete inhibition of ERK-dependent RSK phosphorylation was seen at maximal exposures for several dose levels, and likewise trough exposures and correlative biomarker inhibition data suggest that complete, durable target inhibition of ERK kinases can occur in the peripheral blood tissue compartments following administration of BVD-523 at various dose levels.
[0016] Figure 3A shows that BVD-523 can treat acquired resistance to targeted drugs in vitro. Cell line variants of A375 melanoma were selected for 3 months following passage in increasing amounts of dabrafenib, trametinib, BVD-523, or combinations of these agents at various fixed ratios. Cell line variants were obtained that could grow in the presence of dabrafenib or trametinib at concentrations greater than 100 times the IC50 of these agents in parental A375 cell. In comparison, cell lines resistant to BVD-523 could only be maintained in less than 10X of parental IC50 concentration. Sensitivity testing suggested dabrafenib and trametinib-resistant cell lines remained relatively sensitive to BVD-523; the increased IC50 "shift" for BVD-523 in resistant cell lines was more modest than those corresponding IC50 increases following dabrafenib or trametinib treatment. Likewise, compared to dabrafenib or trametinib treatment, more complete inhibition of cell growth was observed when resistant cell lines were treated with BVD-523 at concentrations 10-fold above its IC50 in the parental A375 line. In total, patterns of resistance and cross-sensitivity suggest BVD-523 may remain effective in settings of acquired resistance. [0017] Figure 3B shows that BVD-523 can treat acquired resistance to targeted drugs in-vivo. A patient-derived line, ST052C, was isolated from a BRAFV600E melanoma patient that progressed following 10 months of therapy with MAPK-pathway directed therapies. Treated ex vivo, ST052C exhibited acquired cross-resistance to dabrafenib at 50 mg/kg BID. Meanwhile, BVD-523 was effective in ST052C as a single-agent at 100 mg/kg BID.
[0018] Figure 4 shows that BVD-523 inhibits cell growth in a variety of solid tumor cell lines, and that oncogenic mutations relevant to MAPK pathway activation (BRAF and RAS gain-of-function mutations, NF1 inactivation) may confer high drug sensitivity. Cell viability assays (Alamar Blue) suggest a strong cytostatic response occurs following 96 hours of treatment with BVD-523, with cellular IC50 potencies typically in the low micromolar range. Relevant genetic factors appear to dictate the cytostatic activity of BVD-523: 2 of 15 cell lines not harboring likely MAPK activating mutations appeared to be sensitive at relatively low concentrations, while 1 1 of 19 lines harboring changes in BRAF, KRAS or NF1 genes appeared to be sensitive. Alternatively, 1 1 of the 13 lines showing high BVD-523 sensitivity harbored mutations likely to activate the MAPK pathway. Additional factors may dictate whether BVD-523 induces cell death and cytotoxicity: while the majority of lines exhibiting cell death following BVD-523 are mutant in relevant MAPK pathway components, only a smaller subset of those lines exhibited cytotoxicity (not shown). In total, activation and subsequent addiction to MAPK pathway signaling appears to sensitize a variety of malignancies to the anti-cancer effects of BVD-523. [0019] Figures 5A-5OO show the dose response curve of various compounds as specified in the figure legends, including BVD-523, in 40 different cell lines.
DETAILED DESCRIPTION OF THE INVENTION
[0020] In the present invention, we demonstrate that BVD-523 and ERK inhibition is effective in a variety of malignancies, particularly in cancers where aberrant activation of the MAPK pathway has occurred following oncogenic mutation of various genes, including those in the RAF and RAS oncogene families. In clinical studies, both pharmacologically relevant drug exposures and reduced cellular levels of the RSK biomarker are observed after administration of BVD-523 in human subjects. Integrated preclinical and clinical pharmacology suggests that BVD-523 and therapeutic ERK inhibition may exhibit novel utility as an anti-cancer strategy in human lymphoid and myeloid malignancies, when used either as a first-line therapy in treatment- naive subjects, as well as in patients with disease that presents following acquired drug resistance.
[0021] Thus, one embodiment of the present invention is a method of treating or ameliorating the effects of a hematologic cancer in a subject in need thereof. This method comprises administering to the subject an effective amount of an ERK1/2 inhibitor to treat or ameliorate the effects of the hematologic cancer.
[0022] 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 invention 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.
[0023] 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.
[0024] As used herein, a "subject" is a mammal, preferably, a human. In addition to humans, which is a preferred mammal in the present invention, other categories of mammals within the scope of the present invention 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.
[0025] As used herein, an ERK1/2 "inhibitor" means those substances that (i) directly interact with ERK1 and/or ERK2, e.g. , by binding to ERK1/2 and (ii) decrease the expression or the activity of ERK1 and/or ERK2 protein kinases. Therefore, inhibitors that act upstream of ERK1/2, such as MEK inhibitors and RAF inhibitors, are not ERK1/2 inhibitors according to the present invention. Preferred ERK1/2 inhibitors of the present invention do not decrease the amount of phosphorylated ERK1 and/or ERK2 but decrease the activity of phosphorylated ERK1 and/or ERK2. Non-limiting examples of ERK1/2 inhibitors according to the present invention include AEZS-131 (Aeterna Zentaris), AEZS-136 (Aeterna Zentaris), BVD-523 (BioMed Valley Discoveries, Inc.), SCH-722984 (Merck & Co.), SCH-772984 (Merck & Co.), SCH-900353 (MK-8353, Merck & Co.), pharmaceutically acceptable salts thereof, and combinations thereof. Preferably, the ERK1/2 inhibitor is BVD- 523 or a pharmaceutically acceptable salt thereof.
[0026] In the present invention, BVD-523, a preferred ERK1/2 inhibitor, corresponds to a compound according to formula (I):
Figure imgf000010_0001
and pharmaceutically acceptable salts thereof. BVD-523 may be synthesized according to the methods disclosed in, e.g. , U.S. Patent No. 7,354,939. Enantiomers and racemic mixtures of both enantiomers of BVD-523 are also contemplated within the scope of the present invention. BVD-523 is a preferred ERK1/2 inhibitor because its mechanism of action is believed to be, e.g. , unique and distinct from certain other ERK1/2 inhibitors, such as SCH772984. In particular, other ERK1/2 inhibitors, such as SCH772984, inhibit autophosphorylation of ERK (Morris et al., 2013), whereas BVD-523 allows for the autophosphorylation of ERK while still inhibiting ERK (Figure 1 B).
[0027] In the present invention, the hematologic cancer may be selected from RAS mutant myelodysplastic syndromes (MDS), including refractory anemia with excess blast (RAEB), RAEB in transformation (RAEBt), and Acute myeloid leukemia (AML) following MDS, lymphoid cancers, or myeloid cancers. Additional non-limiting examples of hematologic cancer within the scope of the present invention include Adult Acute Megakaryoblastic Leukemia (M7), Adult Acute Minimally Differentiated Myeloid Leukemia (MO), Adult Acute Monoblastic Leukemia (M5a), Adult Acute Monocytic Leukemia (M5b), Adult Acute Myeloblasts Leukemia With Maturation (M2), Adult Acute Myeloblasts Leukemia Without Maturation (M1 ), Adult Acute Myeloid Leukemia With 1 1 q23 (MLL) Abnormalities, Adult Acute Myeloid Leukemia With Del(5q), Adult Acute Myeloid Leukemia With Inv(16)(p13;q22), Adult Acute Myeloid Leukemia With t(16;16)(p13;q22), Adult Acute Myeloid Leukemia With t(8;21 )(q22;q22), Adult Acute Myelomonocytic Leukemia (M4), Adult Erythroleukemia (M6a), Adult Pure Erythroid Leukemia (M6b), Recurrent Adult Acute Myeloid Leukemia, and Untreated Adult Acute Myeloid Leukemia.
[0028] In one aspect of this embodiment, the subject with cancer has a somatic RAS and/or BRAF mutation. As used herein, a "mutation" means a change, e.g., in the nucleic acid sequence of a cell. Preferably, the RAS mutation is a mutation in H-RAS, N-RAS, or K-RAS. The following Tables 1 , 2, 3, and 4 show the SEQ ID Nos. of representative nucleic acid and amino acid sequences of wild type H-RAS, K-RAS, N-RAS, and BRAF from various mammalian sources, respectively. These sequences may be used in methods for identifying subjects with a mutant -RAS, K-RAS, N-RAS, and/or BRAF genotype (such as in the methods set forth below).
Table 1 H-RAS sequences
Figure imgf000012_0001
SEQ ID polypeptide or nucleic Organism Other No. acid sequence Information
familiaris
24 polypeptide dog, Canis lupus variant 2 familiaris
25 nucleic acid cat, Felis catus variant 1
26 polypeptide cat, Felis catus variant 1
27 nucleic acid cat, Felis catus variant 2
28 polypeptide cat, Felis catus variant 2
29 nucleic acid cow, Bos taurus variant 1
30 polypeptide cow, Bos taurus variant 1
31 nucleic acid cow, Bos taurus variant 2
32 polypeptide cow, Bos taurus variant 2
33 nucleic acid cow, Bos taurus variant X1
34 polypeptide cow, Bos taurus variant X1
35 nucleic acid chicken, Gallus
gallus
36 polypeptide chicken, Gallus
gallus
Table 2 K-RAS sequences
SEQ ID polypeptide or nucleic Organism Other No. acid sequence Information
37 nucleic acid human isoform a
38 polypeptide human isoform a
39 nucleic acid human isoform b
40 polypeptide human isoform b
41 nucleic acid rat (Rattus
norvegicus)
42 polypeptide rat (Rattus
norvegicus)
43 nucleic acid mouse, Mus
musculus
44 polypeptide mouse, Mus
musculus
45 nucleic acid rabbit, Oryctolagus
cuniculus
46 polypeptide rabbit, Oryctolagus
cuniculus
47 nucleic acid guinea pig, Cavia variant 1 porcellus
48 polypeptide guinea pig, Cavia variant 1 porcellus
49 nucleic acid guinea pig, Cavia variant 2 porcellus
50 polypeptide guinea pig, Cavia variant 2 porcellus SEQ ID polypeptide or nucleic Organism Other
No. acid sequence Information
51 nucleic acid dog, Canis lupus variant 1 familiaris
52 polypeptide dog, Canis lupus variant 1 familiaris
53 nucleic acid dog, Canis lupus variant 2 familiaris
54 polypeptide dog, Canis lupus variant 2 familiaris
55 nucleic acid cat, Felis catus variant 1
56 polypeptide cat, Felis catus variant 1
57 nucleic acid cat, Felis catus variant 2
58 polypeptide cat, Felis catus variant 2
59 nucleic acid cow, Bos taurus
60 polypeptide cow, Bos taurus
61 nucleic acid cow, Bos taurus variant X2
62 polypeptide cow, Bos taurus variant X2
63 nucleic acid cow, Bos taurus variant X3
64 polypeptide cow, Bos taurus variant X3
65 nucleic acid chicken, Gallus
gallus
66 polypeptide chicken, Gallus
gallus
Table 3 N-RAS sequences
Figure imgf000014_0001
SEQ ID polypeptide or nucleic Organism Other No. acid sequence Information
77 nucleic acid dog, Canis lupus
familiaris
78 polypeptide dog, Canis lupus
familiaris
79 nucleic acid cat, Felis catus
80 polypeptide cat, Felis catus
81 nucleic acid cow, Bos taurus
82 polypeptide cow, Bos taurus
83 nucleic acid chicken, Gallus
gallus
84 polypeptide chicken, Gallus
gallus
Table 4 BRAF
Figure imgf000015_0001
SEQ ID Nucleic acid or Organism Other
NO polypeptide information
105 nucleic acid cow, Bos taurus variant X3
106 polypeptide cow, Bos taurus variant X3
107 nucleic acid cow, Bos taurus variant X4
108 polypeptide cow, Bos taurus variant X4
109 nucleic acid cow, Bos taurus variant X5
1 10 polypeptide cow, Bos taurus variant X5
1 1 1 nucleic acid cow, Bos taurus variant X6
1 12 polypeptide cow, Bos taurus variant X6
1 13 nucleic acid cow, Bos taurus variant X7
1 14 polypeptide cow, Bos taurus variant X7
1 15 nucleic acid cow, Bos taurus variant X8
1 16 polypeptide cow, Bos taurus variant X8
1 17 nucleic acid cow, Bos taurus variant X9
1 18 polypeptide cow, Bos taurus variant X9
1 19 nucleic acid cow, Bos taurus variant X10
120 polypeptide cow, Bos taurus variant X10
121 nucleic acid cow, Bos taurus variant X1 1
122 polypeptide cow, Bos taurus variant X1 1
123 nucleic acid cow, Bos taurus variant 2
124 polypeptide cow, Bos taurus variant 2
125 nucleic acid horse, Equus caballus
126 polypeptide horse, Equus caballus
127 nucleic acid chicken, Gallus gallus
128 polypeptide chicken, Gallus gallus
[0029] Methods for identifying mutations in nucleic acids, such as the above identified RAS and BRAF genes, are known in the art. Nucleic acids may be obtained from biological samples. In the present invention, 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.
[0030] 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. [0031] 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 2005, sequencing by hybridization, by ligation (for example, WO 2005021786), by degradation (for example, U.S. Patent Nos. 5,622,824 and 6, 140,053) 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., U.S. Patent Publication No. 20120264632 and International Patent Publication No. WO2012125848.
[0032] 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., 2007. Mutations may also be detected using real time PCR. See, e.g., International Application publication No. WO2012046981.
[0033] Hybrid capture methods are known in the art and are disclosed in e.g., U.S. Patent Publication No. 20130203632 and U.S. Patent Nos. 8,389,219 and 8,288,520. 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).
[0034] Molecular Inversion Probe (MIP) techniques are known in the art and are disclosed in e.g., Absalan et al., 2008. This method uses MIP molecules, which are special "padlock" probes (Nilsson et al, 1994) for genotyping. A MIP molecule is a linear oligonucleotide that contains specific regions, universal sequences, restriction sites and a Tag (index) sequence (16-22 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., 2003). 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 a Tag- cTag duplex, a signal is detected.
[0035] In another aspect of this embodiment, the method further comprises treating the subject with a combination therapy. The combination therapy may include administering at least one additional therapeutic agent 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.
[0036] 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. , multi- specific antibodies such as bispecific antibodies). Fragments of antibodies include those that bind antigen, (e.g. , Fab', F(ab')2, Fab, Fv, and rlgG). See also, e.g., Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, III.); Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., New York (1998). The term antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. The term "antibody" further includes both polyclonal and monoclonal antibodies.
[0037] Examples of therapeutic antibodies that may be used in the present invention include rituximab (Rituxan), Cetuximab (Erbitux), bevacizumab (Avastin), and Ibritumomab (Zevalin).
[0038] Cytotoxic agents according to the present invention 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 6-mercaptopurine, dacarbazine, and fludarabine, and pyrimidine antagonists such as 5-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.
[0039] Cytotoxic agents according to the present invention also include an inhibitor of the PI3K/Akt pathway. Non-limiting examples of an inhibitor of the PI3K/Akt pathway include A-674563 (CAS # 552325-73-2), AGL 2263, AMG-319 (Amgen, Thousand Oaks, CA), AS-041 164 (5-benzo[1 ,3]dioxol-5- ylmethylene-thiazolidine-2,4-dione), AS-604850 (5-(2,2-Difluoro- benzo[1 ,3]dioxol-5-ylmethylene)-thiazolidine-2,4-dione), AS-605240 (5- quinoxilin-6-methylene-1 ,3-thiazolidine-2,4-dione), AT7867 (CAS # 857531- 00-1 ), benzimidazole series, Genentech (Roche Holdings Inc., South San Francisco, CA), BML-257 (CAS # 32387-96-5), CAL-120 (Gilead Sciences, Foster City, CA), CAL-129 (Gilead Sciences), CAL-130 (Gilead Sciences), CAL-253 (Gilead Sciences), CAL-263 (Gilead Sciences), CAS # 612847-09-3, CAS # 681281-88-9, CAS # 75747-14-7, CAS # 925681-41-0, CAS # 98510- 80-6, CCT128930 (CAS # 885499-61-6), CH5132799 (CAS # 1007207-67-1 ), CHR-4432 (Chroma Therapeutics, Ltd., Abingdon, UK), FPA 124 (CAS # 902779-59-3), GS-1 101 (CAL-101 ) (Gilead Sciences), GSK 690693 (CAS # 937174-76-0), H-89 (CAS # 127243-85-0), Honokiol, IC871 14 (Gilead Science), IPI-145 (Intellikine Inc.), KAR-4139 (Karus Therapeutics, Chilworth, UK), KAR-4141 (Karus Therapeutics), KIN-1 (Karus Therapeutics), KT 5720 (CAS # 108068-98-0), Miltefosine, MK-2206 dihydrochloride (CAS # 1032350- 13-2), ML-9 (CAS # 105637-50-1 ), Naltrindole Hydrochloride, OXY-1 1 1A (NormOxys Inc., Brighton, MA), perifosine, PHT-427 (CAS # 1 191951-57-1 ), 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. Ltd., Hydrabad, India), PI3 kinase delta inhibitors-2, Incozen (Incozen Therapeutics), PI3 kinase inhibitor, Roche-4 (Roche Holdings Inc.), PI3 kinase inhibitors, Roche (Roche Holdings Inc.), PI3 kinase inhibitors, Roche-5 (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-1 (Pathway Therapeutics Ltd.), PI3-delta inhibitors, Pathway Therapeutics-2 (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, lntellikine-1 (Intellikine Inc.), PI3K delta/gamma inhibitors, lntellikine-1 (Intellikine Inc.), pictilisib (Roche Holdings Inc.), PIK-90 (CAS # 677338-12-4), SC-103980 (Pfizer, New York, NY), SF-1 126 (Semafore Pharmaceuticals, Indianapolis, IN), SH-5, SH-6, Tetrahydro Curcumin, TG100-1 15 (Targegen Inc., San Diego, CA), Triciribine, X-339 (Xcovery, West Palm Beach, FL), XL-499 (Evotech, Hamburg, Germany), pharmaceutically acceptable salts thereof, and combinations thereof.
[0040] In the present invention, the term "toxin" means an antigenic poison or venom of plant or animal origin. An example is diphtheria toxin or portions thereof.
[0041] In the present invention, 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, 1-125, At-21 1 , Lu-177, Cu-67, I- 131 , Sm-153, Re-186, P-32, Re-188, ln-1 14m, and Y-90.
[0042] In the present invention, 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-2, IL-7, IL-12, CCL3, CCL26, CXCL7, and synthetic cytosine phosphate-guanosine (CpG).
[0043] In the present invention, the term "photoactive therapeutic agent" means compounds and compositions that become active upon exposure to light. Certain examples of photoactive therapeutic agents are described in U.S. Patent Application Serial No. 201 1/0152230 A1 , "Photoactive Metal Nitrosyls For Blood Pressure Regulation And Cancer Therapy."
[0044] In the present invention, 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.
[0045] In the present invention, 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.
[0046] 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 (Faslodex®). Such compounds are also within the meaning of hormone in the present invention.
[0047] 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 2-methoxyestradiol, angiostatin, bevacizumab, cartilage-derived angiogenesis inhibitory factor, endostatin, IFN-a, IL-12, itraconazole, linomide, platelet factor-4, prolactin, SU5416, suramin, tasquinimod, tecogalan, tetrathiomolybdate, thalidomide, thrombospondin, thrombospondin, TNP-470, ziv-aflibercept, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof.
[0048] In another aspect of this embodiment, the ERK1/2 inhibitor is administered in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier or diluent.
[0049] Another embodiment of the present invention is a method of treating or ameliorating the effects of a hematologic cancer in a subject in need thereof. This method comprises administering to the subject an effective amount of BVD-523 or a pharmaceutically acceptable salt thereof to treat or ameliorate the effects of the hematologic cancer.
[0050] Suitable and preferred subjects and hematologic cancers are as disclosed herein.
[0051] In one aspect of this embodiment, the BVD-523 or a pharmaceutically acceptable salt thereof is administered in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier or diluent.
[0052] In another aspect of this embodiment, the method further comprises treating the subject with a combination therapy. Suitable and preferred additional therapeutic agents for the combination therapy are as disclosed herein.
[0053] A further embodiment of the present invention is a method of modulating ribosomal S6 kinase (RSK) phosphorylation in the blood of a subject that has a hematologic cancer. This method comprises administering to the subject an effective amount of an ERK1/2 inhibitor to modulate RSK phosphorylation in the blood of the subject.
[0054] As used herein, the terms "modulate," "modulating," and grammatical variations thereof mean to change, i.e., the phosphorylation level of RSK. For example, the modulation may be a decrease in RSK phosphorylation. Preferably, the decrease comprises substantially inhibiting RSK phosphorylation by ERK in the blood of the subject.
[0055] Suitable and preferred subjects, ERK1/2 inhibitors, and hematologic cancers are as disclosed herein.
[0056] In one aspect of this embodiment, the ERK1/2 inhibitor is administered in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier or diluent.
[0057] An additional embodiment of the present invention is a pharmaceutical composition for treating or ameliorating the effects of a hematologic cancer in a subject in need thereof. This pharmaceutical composition comprises a pharmaceutically acceptable carrier and an effective amount of an ERK1/2 inhibitor or a pharmaceutically acceptable salt thereof.
[0058] Suitable and preferred subjects, ERK1/2 inhibitors, and hematologic cancers are as disclosed herein.
[0059] In one aspect of this embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent effective for treating or ameliorating the effects of the hematologic cancer. Suitable and preferred additional therapeutic agents are as disclosed herein. [0060] Another embodiment of the present invention is a pharmaceutical composition for treating or ameliorating the effects of a hematologic cancer in a subject in need thereof. This pharmaceutical composition comprises a pharmaceutically acceptable carrier and an effective amount of BVD-523 or a pharmaceutically acceptable salt thereof.
[0061] Suitable and preferred subjects and hematologic cancers are as disclosed herein.
[0062] In one aspect of this embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent effective for treating or ameliorating the effects of the hematologic cancer. Suitable and preferred additional therapeutic agents are as disclosed herein.
[0063] A further embodiment of the present invention is a kit for treating or ameliorating the effects of a hematologic cancer. This kit comprises any pharmaceutical composition disclosed herein, packaged together with instructions for its use.
[0064] For use of the kits of this invention, suitable and preferred subjects having hematologic cancer and types of hematologic cancers are as disclosed herein.
[0065] In one aspect of this embodiment, the kit further comprises at least one additional therapeutic agent effective for treating or ameliorating the effects of the hematologic cancer. Suitable and preferred additional therapeutic agents are as disclosed herein.
[0066] The kits may also include suitable storage containers, e.g., ampules, vials, tubes, etc., for each inhibitor (which may, e.g., be in the form of pharmaceutical compositions) and other reagents, e.g. , buffers, balanced salt solutions, etc., for use in administering the pharmaceutical compositions to subjects. The inhibitor 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 a packaging container, optionally having one or more partitions for housing the pharmaceutical composition(s) and other optional reagents.
[0067] In the present invention, an "effective amount" or a "therapeutically effective amount" of a compound or composition, including pharmaceutical compositions, disclosed herein is an amount of such compound 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 a composition according to the invention will be that amount of the composition, which is the lowest dose effective to produce the desired effect. The effective dose of a compound 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. [0068] A suitable, non-limiting example of a dosage of an ERK1/2 inhibitor disclosed herein is from about 1 mg/kg to about 2400 mg/kg per day, such as from about 1 mg/kg to about 1200 mg/kg per day, 75 mg/kg per day to about 300 mg/kg per day, including from about 1 mg/kg to about 100 mg/kg per day. Other representative dosages of such agents include about 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, 1000 mg/kg, 1 100 mg/kg, 1200 mg/kg, 1300 mg/kg, 1400 mg/kg, 1500 mg/kg, 1600 mg/kg, 1700 mg/kg, 1800 mg/kg, 1900 mg/kg, 2000 mg/kg, 2100 mg/kg, 2200 mg/kg, and 2300 mg/kg per day. The effective dose of ERK1/2 inhibitors, e.g., BVD-523, disclosed herein may be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day.
[0069] An ERK1/2 inhibitor or a pharmaceutical composition of the present invention 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 ERK1/2 inhibitor or the pharmaceutical composition of the present invention may be administered in conjunction with other treatments. The ERK1/2 inhibitor or the pharmaceutical composition of the present invention may be encapsulated or otherwise protected against gastric or other secretions, if desired.
[0070] The pharmaceutical compositions of the invention may comprise one or more active ingredients in admixture with one or more pharmaceutically-acceptable carriers and/or diluents and, optionally, one or more other compounds, drugs, ingredients and/or materials. Regardless of the route of administration selected, the agents/compounds of the present invention are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. See, e.g. , Remington, The Science and Practice of Pharmacy (21st Edition, Lippincott Williams and Wilkins, Philadelphia, PA.).
[0071] Pharmaceutically acceptable diluents or carriers are well known in the art (see, e.g. , Remington, The Science and Practice of Pharmacy (21 st Edition, Lippincott Williams and Wilkins, Philadelphia, PA.) and The National Formulary (American Pharmaceutical Association, Washington, D.C.)) 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 of the invention 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.
[0072] The pharmaceutical compositions of the invention 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 (1 ) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, such as glycerol; (4) 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; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, and sodium lauryl sulfate; (10) suspending agents, such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth; (11 ) buffering agents; (12) 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; (13) inert diluents, such as water or other solvents; (14) preservatives; (15) surface-active agents; (16) dispersing agents; (17) control-release or absorption-delaying agents, such as hydroxypropylmethyl cellulose, other polymer matrices, biodegradable polymers, liposomes, microspheres, aluminum monostearate, gelatin, and waxes; (18) opacifying agents; (19) adjuvants; (20) wetting agents; (21 ) emulsifying and suspending agents; (22), solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-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; (23) propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane; (24) antioxidants; (25) agents which render the formulation isotonic with the blood of the intended recipient, such as sugars and sodium chloride; (26) thickening agents; (27) coating materials, such as lecithin; and (28) 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.
[0073] The pharmaceutical compositions of the present invention 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 nonaqueous 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.
[0074] 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.
[0075] 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.
[0076] The pharmaceutical compositions of the present invention 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 of the present invention 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. [0077] 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.
[0078] The pharmaceutical compositions of the present invention 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.
[0079] 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.
[0080] 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.
[0081] The formulations may be presented 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.
[0082] The following examples are provided to further illustrate the methods of the present invention. These examples are illustrative only and are not intended to limit the scope of the invention in any way. EXAMPLES
Example 1
BVD-523 is an ERK1/2 inhibitor
[0083] The ability of BVD-523 to inhibit ERK1 and EKR2 was assayed using Z'-LYTE™ Kinase Assay Kits (Life Technologies, Invitrogen Corp., Madison, Wl) according to the manufacturer's instructions. Specifically, for ERK1 , the 2X MAPK3 (ERK1 )/Ser/Thr 03 mixture is prepared in 50 mM HEPES pH 7.5, 0.01 % BRIJ-35, 10 mM MgCI2, 1 mM EGTA. The final 10 μΙ_ Kinase Reaction consists of 5.94-94.5 ng MAPK3 (ERK1 ) and 2 μΜ Ser/Thr 03 in 50 mM HEPES pH 7.5, 0.01 % BRIJ-35, 10 mM MgCI2, 1 mM EGTA. For ERK2, the 2X MAPK1 (ERK2)/Ser/Thr 03 mixture is prepared in 50 mM HEPES pH 7.5, 0.01 % BRIJ-35, 10 mM MgCI2, 1 mM EGTA. The final 10 μΙ_ Kinase Reaction consists of 2-45.5 ng MAPK1 (ERK2) and 2 μΜ Ser/Thr 03 in 50 mM HEPES pH 7.5, 0.01 % BRIJ-35, 10 mM MgCI2, 1 mM EGTA. For both kinases, after the 1 hour Kinase Reaction incubation, 5 μΙ_ of a 1 : 1024 dilution of Development Reagent A was added.
[0084] The results of the assays are presented in Figure 1A, which shows that BVD-523 inhibits ERK 1 and 2 kinases at near-nanomolar potencies.
Example 2
BVD-523 and ERK inhibition exhibited anti-cancer activity, particularly following oncogenic activation of MAPK-pathwav signaling
Materials and Methods
[0085] To measure the 50% inhibition concentration (IC50) of BVD-523 on a panel based on 40 cell lines, the CellTiter-Gloluminescent cell viability assay was used. The details of the experiments were as follows.
[0086] Each cell line was treated with BVD-523, another ERK2 inhibitor (TCS ERK 1 1 e, or 4-[2-[(2-Chloro-4-fluorophenyl)amino]-5-methyl-4- pyrimidinyl]-N-[(1 S)-1-(3-chlorophenyl)-2-hydroxyethyl]-1 H-pyrrole-2- carboxamide), a standard chemotherapy drug as positive control, vehicles, and culture medium. Both BVD-523 and TCS ERK 1 1 e were dissolved in DMSO and stored at room temperature. For the positive controls, doxorubicin was purchased from Zhejiang (Haizheng, China) in the form of a powder and stored at 4°C; cisplatin was purchased from Nanjing Zhiyao (China) in the form a powder and stored at room temperature; and mitomycin was purchased from Zhejiang (Haizheng, China) in the form of a powder and stored at room temperature.
Equipment
[0087] The following equipment were used in carrying out the experiments of this Example: Synergy2, Biotek (Gene Limited Company, Beijing, China); Countstar, Inno-Alliance Biotech (Wilmington, DE); Forma Series II Water Jacket CO2 Incubator, Thermo Scientific (Waltham, MA); Biological safety cabinet, Thermo Scientific; and inverted Microscope, Olympus CKX41 SF (Tokyo, Japan). Cell Lines
[0088] The 40 cell lines used for this Example are listed below in Table 5. The positive controls used were cisplatin, doxorubicin, or mitomycin, and the incubation time was 96 hours.
Table 5 List of cell lines tested
Figure imgf000039_0001
Human cancer No. Cell line Growth Condition
Oral Cancer 34 KB Adherent
CML 35 K562 Suspension
36 SK-OV-3 Adherent
Ovarian Cancer
37 OVCAR3 Adherent
Nasopharyngeal 38 CNE Adherent
39 A375 Adherent
Melanoma
40 SK-MEL-5 Adherent
[0089] All the cells were cultured in the media supplemented with 10% FBS unless otherwise labeled, in the temperature of 37°C, 5% CO2 and 95% humidity. Culture media were purchased from GIBCO or Sigma, USA.
CellTiter-Glo® Luminescent Cell Viability Assay
[0090] CellTiter-Glo® Luminescent Cell Viability Assay (Cat. No.: G7572, Promeg) was stored at -20°C. To prepare the reagent, the CellTiter- Glo Buffer was thawed and equilibrated to room temperature prior to use for up to 48 hours prior to use.
[0091] The appropriate volume (100 ml) of CellTiter-Glo Buffer was transferred into an amber bottle containing CellTiter-Glo Substrate to reconstitute the lyophilized enzyme/substrate mixture. This mixture formed the CellTiter-Glo Reagent. The mixture was achieved by gently vortexing, swirling or by inverting the contents to obtain a homogeneous solution.
ICm Determination
[0092] Cells were harvested respectively during the logarithmic growth period and count cell number using Countstar. The cell concentrations were adjusted to 5.56x104 cells/ml with respective culture medium. Then 90 μΙ of cell suspensions were added to two 96-well plates (A and B) with the final cell density of approximately 2*103 cells/well.
[0093] The next day, the following steps were taken to take the TO readings. 10 μΙ culture medium were added to each well of plate A. The plates were then incubated overnight in a humidified incubator at 37°C with 5% CO2. Subsequently, the plate and its contents were equilibrated at room temperature for approximately 30 minutes. A volume of CellTiter-Glo® Reagent equal to the volume of cell culture medium present in each well (e.g., add 100 μΙ of reagent to 100 μΙ of medium containing cells for a 96-well plate). The contents were mixed for 2 minutes on an orbital shaker to induce cell lysis. The plates were allowed to incubate at room temperature for 10 minutes to stabilize luminescent signal. The luminescence (at TO) were recorded using an EnVision Multi Label Reader (Perkin Elmer, Waltham, MA).
[0094] To determine the effect of various compounds on the cells, the following steps were taken. Plate B was incubated overnight in the humidified incubator at 37°C with 5% CO2. Various compounds (including positive controls) were dissolved with DMSO or PBS as stock solutions to first reach a concentration of 20 mM, then a 200* solution (or 4 mM) of compounds with DMSO. The DMSO solutions were then diluted with culture medium (20-fold) to form a 10* solution (or 200 μΜ). Subsequently, a 10 μΙ (10 X ) drug solution of various compounds were dispensed in each well (triplicate for each drug concentration) of plate B. (The final concentration of DMSO in culture medium was 0.5% [v/v]). The test plates were incubated for 72 hours in the humidified incubator at 37°C with 5% CO2, and then measured by means of a CellTiter-Glo® Luminescent Cell Viability Assay as follows. The plate and its contents were equilibrated at room temperature for approximately 30 minutes. A volume of CellTiter-Glo® Reagent equal to the volume of cell culture medium present in each well was added to each well (e.g., add 100 μΙ of reagent to 100 μΙ of medium containing cells for a 96-well plate). The contents were mixed for 2 minutes on an orbital shaker to induce cell lysis. The plates were allowed to incubate at room temperature for 10 minutes to stabilize the luminescent signal. The luminescence signals were recorded using an EnVision Multi Label Reader.
Data Analysis
[0095] The data are displayed graphically using GraphPad Prism 5.0. In order to calculate IC50, a dose-responsive curve was fitted using a nonlinear regression model with a sigmoidal dose response. The formula of surviving rate is shown below, and the IC50 was automatically produced by GraphPad Prism 5.0.
The Surviving rate (%)= (ODTest article-ODMedium control)/ (ODNone treated-ODMedium control)* 100%.
Results
[0096] The results are shown below in Tables 6 and 7 and in Figures 4 and 5.
Table 6. Summary of IC50 & Max inhibition determined by CTG assay
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Table 7. Summary of cell growth normalized by TO data (set TO data at 1 .0)
Figure imgf000046_0001
Cell No. Cell lines Cancer Type Non-treated control
21 HT-1080 Fibrosarcoma 9.3
22 786-0 Kindney 5.1
23 BxPC-3 6.0
24 MIA PaCa-2 Pancreatic 7.8
25 PANC-1 3.7
26 Hep 3B 2.6
27 Hep G2 Live 3.3
28 SK-HEP-1 5.6
29 MKN7 2.4
30 MKN1 Gastric 4.2
31 NCI-N87 2.9
32 U-2 OS Osteosarcoma 7.0
Multiple
33 RPMI-8226 1 .6
Myeloma
34 KB-WT Oral Cancer 3.2
35 K562 CML 7.4
36 SK-OV-3 2.9
Ovarian
37 OVCAR3 3.1
38 CNE Nasopharyngeal 3.8
39 A375 2.5
Melanoma
40 SK-MEL-5 4.0 [0097] We have identified a variety of cancer cell lines that were sensitive to BVD-523 by in vitro screening. Examples of sensitive cell lines are listed in Table 8, along with relevant genetic information.
Table 8 cell lines sensitive to BVD-523
Figure imgf000048_0001
[0098] Sensitive cell lines generally exhibit reduced cell growth following treatment with BVD-523 at concentrations in the low micromolar range over 96 hours. BVD-523 sensitivity is observed in several different cancer histologies, including a variety of lines originating from colorectal and hepatocellular carcinomas. Cell lines with, inter alia, RAS/BRAF mutations show enhanced sensitivity in the presence of the ERK1/2 inhibitor, BVD-523. For example, the liver cancer cell line HepG2, which has a mutation in N-RAS, was sensitive to BVD-523. In addition, the colon cancer cell line, SW620 (contains both K-RAS and MAP2K4 mutation) also showed high sensitivity to BVD-523. These data show that inhibition of the MAPK pathway, as exemplified by the inhibition of ERK by BVD-523, may result in blocking cell proliferation and/or enhancing cell death in cancers with mutations that are known typically to activate the MAPK signaling pathway.
Example 3
BVD-523 altered markers of MAPK kinase activity and effector function
[0099] For Western blot studies, HCT1 16 cells (5 x 106) were seeded into 10 cm dishes in McCoy's 5A plus 10% FBS. A375 cells (2.5 x 106) were seeded into 10 cm dishes in DMEM plus 10% FBS. Cells were allowed to adhere overnight prior to addition of the indicated amount of test compound (BVD-523) or vehicle control. Cells were treated for either 4 or 24 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-lmmunoprecipitation Assay) buffer, clarified by centrifugation and quantitated by bicinchoninic acid assay (BCA) assay. 20-50 μg of protein was resolved by SDS-PAGE electrophoresis, blotted onto PVDF membrane and probed using the antibodies detailed in Table 9 (for the 4-hour treatment) and Table 10 (for the 24-hour treatment) below.
Table 9 - Antibody Details
Figure imgf000049_0001
Incubation/
Size
Antigen Supplier Cat No Dilution Block Secondary
(kDa)
Conditions
Cell o/n 4°C 5%
pErk 1/2 42/44 9106S 1 :500 anti-mouse
Signaling milk
Cell o/n 4°C 5%
Total ERK 42/44 9102 1 :2000 anti-rabbit
Signaling milk
Cell o/n 4°C 5%
pMEK1/2 45 9154 1 :1000 anti-rabbit
Signaling BSA
Cell o/n 4°C 5%
Total MEK 45 9126 1 :1000 anti-rabbit
Signaling BSA
pS6- Cell o/n 4°C 5%
32 2211 S 1 :3000 anti-rabbit pS235 Signaling milk
Cell o/n 4°C 5%
Total S6 32 2217 1 :2000 anti-rabbit
Signaling milk
Cell o/n 4°C 5%
DUSP6 48 3058S 1 :1000 anti-rabbit
Signaling BSA
Total BD Bioo/n 4°C 5%
73 610152 1 :2000 anti-mouse CRAF sciences milk
pCRAF- Cell o/n 4°C 5%
73 9427 1 :1000 anti-rabbit Ser338 Signaling BSA
pRB Cell o/n 4°C 5%
105 9307 1 :2000 anti-rabbit (Ser780) Signaling BSA
o/n 4°C 5%
β-Actin 42 Sigma A5441 1 :500,000 anti-mouse milk
Table 10 - Antibody details
Figure imgf000050_0001
Incubation/
Size
Antigen Supplier Cat No Dilution Block Secondary
(kDa)
Conditions
Caspase 3 Signaling milk
Cell o/n 4°C 5%
DUSP6 48 3058S 1 :1000 anti-rabbit
Signaling BSA
pRSK1/2 Cell o/n 4°C 5%
90 9335 1 :1000 anti-rabbit pS380 Signaling BSA
pRSK1/2 Cell o/n 4°C 5%
90 1 1989 1 :2000 anti-rabbit pS380 Signaling BSA
pRSK- o/n 4°C 5%
90 Millipore 04-419 1 :40000 anti-rabbit T359/S363 BSA
Cell o/n 4°C 5%
Total RSK 90 9333 1 :1000 anti-rabbit
Signaling BSA
Cell o/n 4°C 5%
pErk 1/2 42/44 9106S 1 :500 anti-mouse
Signaling milk
Cell o/n 4°C 5%
Total ERK 42/44 9102 1 :2000 anti-rabbit
Signaling milk
o/n 4°C 5%
B-Actin 42 Sigma A5441 1 :500,000 anti-mouse milk
[0100] Figure 1 B shows Western blot analyses of cells treated with BVD- 523 at various concentrations for the following: 1 ) MAPK signaling components in A375 cells after 4 hours; 2) cell cycle and apoptosis signaling in A375 24 hours treatment with various amounts of BVD-523; and 3) MAPK signaling in HCT-1 16 cells treated for 4 hours. The results show that acute and prolonged treatment with BVD-523 in RAF and RAS mutant cancer cells in-vitro affects both substrate phosphorylation and effector targets of ERK kinases. The concentrations of BVD-523 required to induce these changes is typically in the low micromolar range.
[0101] Changes in several specific activity markers are noteworthy. First, the abundance of slowly migrating isoforms of ERK kinase increase following BVD-523 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-523 treatment. First, RSK1/2 proteins exhibit reduced phosphorylation at residues that are strictly dependent on ERK for protein modification (T359/S363). Second, BVD-523 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-523 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-523.
[0102] Consistent with these observations, effector genes that require MAPK pathway signaling are altered following treatment with BVD-523. 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-523 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-523 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-523/ERK-dependent effector signaling are translated into definitive events such as cell death and cell cycle arrest.
[0103] Consistent with the cellular activity of BVD-523, 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.
Example 4
BVD-523 can treat acquired resistance to targeted drugs in vitro
Materials and Methods
[0104] Cancer cell lines were maintained in cell culture under standard media and serum conditions. For dose escalation studies, A375 cells were split, grown to about 40-60% confluence, and then treated with the initial dose of the specified drug. Table 1 1 shows a summary of drug treatments that were escalated.
Table 11 - Summary of Treatments Being Escalated
Figure imgf000053_0001
[0105] Single agent dose escalations were performed based on Little et al. , 201 1 . Cells were allowed to grow until 70-90% confluence and split. Split ratios were kept as "normal" as possible and reasonably consistent between treatments (e.g. a minimum of 50% of the normal split ratio of the parentals). Medium was refreshed every 3-4 days. When cells again reached about 40-60% confluence, the dose was escalated. In the event that the 40-60% window was missed, the cells were split again and dosed once they reached 40-60% confluence. Again, medium was refreshed every 3-4 days. The process was repeated as required.
[0106] For single agent treatments, starting concentrations and dose increases were conducted by starting with the approximate IC5o, escalating in small increments or, gently, for the initial 4-5 doses, doubling the dose, increasing by the same increment for the next 4 doses, then moving to 1 .5-fold increases in concentration for subsequent doses.
[0107] For combination treatments, starting concentrations and dose increases were conducted by starting with half of the approximate IC5o of each compound (combination assay suggests this will result in about 40-70% inhibition range), escalating as per single agents (i.e. doing an initial doubling and then increasing by the same increment for the next 4 doses, then moving to 1 .5-fold increases in concentration). Table 12 shows the projected dose increases using these schemes. Table 12 - Dose Increases - Month 1
Figure imgf000055_0001
[0108] Clonal resistant cell populations were derived from resistant cell pools by limiting dilution.
[0109] Proliferation assays were used to track changes in sensitivity to the escalated agent(s) at appropriate time intervals (e.g. each month, although the timing is dependent on adequate cell numbers being available). For proliferation assays, cells were seeded in 96-well plates at 3000 cells per well in drug-free DMEM medium containing 10% FBS and allowed to adhere overnight prior to addition of compound or vehicle control. Compounds were prepared from DMSO stocks. The final DMSO concentration was constant at 0.1 %. Test compounds were incubated with the cells for 96 hours at 37°C and 5% CO2 in a humidified atmosphere. Alamar Blue 10% (v/v) was then added and incubated for 4 hours and fluorescent product was detected using a BMG FLUOstar plate reader. The average media only background value was deducted and the data analyzed using a 4-parameter logistic equation in GraphPad Prism. Paclitaxel was used as a positive control.
[0110] Proliferation assays for month 1 were initiated at day 28 using cells growing in the concentrations of each agent indicated in Table 13.
Table 13 - Initial Concentrations of Drugs Used in Proliferation Assays - Month 1
Figure imgf000056_0001
[0111] Proliferation assays for month 2 were initiated at day 56 using cells growing in the concentrations of each agent indicated in Table 14. Table 14 - Initial Concentrations of Drugs Used in Proliferation Assays - Month 2
Figure imgf000057_0001
[0112] At the end of the 3 month escalation period, cultures were maintained at the top concentration for 2 weeks prior to the final round of proliferation assays and potential single cell cloning. As the proliferation assays/single cell cloning required actively proliferating cells, for treatments where cells were proliferating very slowly at the top concentration or that were only recently escalated, a backup culture was also maintained at a lower concentration (Table 15). For the BVD-523 treatment, where cells appeared to have almost completely stopped growing and looked particularly fragile at the top concentration (1 .8 μΜ), cultures were maintained at a lower concentration for the 2 week period.
Table 15 - Details of Treatments Being Cultured at a Fixed Concentration for 2
Weeks
Figure imgf000057_0002
Treatment Inhibitor Culture 1 Backup Culture
D: 160 nM D: 80 nM
4 Dab + Tram
T: 30 nM T: 16 nM
D: 42 nM D: 28 nM
5 Dab + BVD-523
523: 1 .4 μΜ 523: 0.9 μΜ
T: 4 nM T: 2.5 nM
6 Tram + BVD-523
523: 0.6 μΜ 523: 0.4 μΜ
[0113] Proliferation assays for month 3 used cells growing in th concentrations of each agent indicated in Table 16.
Table 16 - Initial Concentrations of Drugs Used in Proliferation Assays - Month
Figure imgf000058_0001
[0114] For combination studies, A375 cells (ATCC) were seeded into triplicate 96-well plates at a cell density of 3000 cells/well in DMEM plus 10% FBS and allowed to adhere overnight prior to addition of test compound or vehicle control. Combinations were tested using a 10x8 dose matrix with a final DMSO concentration of 0.2%. A 96 hour assay incubation period followed, with subsequent addition of Alamar Blue 10% (v/v) and 4 hours incubation prior to reading on a fluorescent plate reader. After reading Alamar Blue, the medium/Alamar Blue mix was flicked off and 100 μΙ of CellTiter-Glo/PBS (1 :1 ) added and the plates processed as per the manufacturer's instructions (Promega). Media only background values were subtracted before the data was analysed. The Bliss additivity model was then applied.
[0115] In brief, predicted fractional inhibition values for combined inhibition were calculated using the equation CbMss =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. Cb|iss is the fractional inhibition that would be expected if the combination of the two drugs were exactly additive. Cbiiss values are subtracted from the experimentally observed fractional inhibition values to give an 'excess over Bliss' value. Excess over Bliss values greater than 0 indicate synergy, whereas values less than 0 indicate antagonism. Excess over Bliss values are plotted as heat maps ± SD.
[0116] The single and combination data are also presented as dose- response curves generated in GraphPad Prism (plotted using % viability relative to DMSO only treated controls).
[0117] For focused combination studies, the Alamar Blue viability assays were performed as described above for combination studies. Additionally, Caspase-Glo 3/7 assays were performed. In brief, HCT1 16 cells were seeded in triplicate in white 96-well plates at a cell density of 5000 cells/well in McCoy's 5A plus 10% FBS. A375 cells were seeded at a density of 5000 cells/well in DMEM plus 10% FBS. Cells were allowed to adhere overnight prior to addition of the indicated amount of test compound or vehicle control. The final concentration of DMSO was 0.2%, and 800 nM staurosporine was included as a positive control. 24 and 48 hour assay incubation periods were used. Then, Caspase-Glo® 3/7 50% (v/v) was added, plates were mixed for 5 minutes on an orbital shaker and incubated for 1 hour at room temperature prior to reading on a luminescent plate reader. Media only background values were subtracted before the data was analysed.
Results
[0118] Figure 3A shows single and combination agent escalation for month 3 of the studies. Cell line variants were obtained that could grow in the presence of dabrafenib or trametinib at concentrations greater than 100 times the IC5o of these agents in parental A375 cell. In comparison, cell lines resistant to BVD-523 could only be maintained in less than 10X of parental IC50 concentration. Sensitivity testing suggested dabrafenib and trametinib-resistant cell lines remained relatively sensitive to BVD-523; the increased IC50 "shift" for BVD-523 in resistant cell lines was more modest than those corresponding IC50 increases following dabrafenib or trametinib treatment. Likewise, compared to dabrafenib or trametinib treatment, more complete inhibition of cell growth was observed when resistant cell lines were treated with BVD-523 at concentrations 10-fold above its IC50 in the parental A375 line. In total, patterns of resistance and cross-sensitivity suggest BVD-523 may remain effective in settings of acquired resistance. Example 5
BVD-523 can treat acquired resistance to targeted drugs in vivo
Materials and Methods
[0119] Immunocompromised mice between 5-8 weeks of age were housed on irradiated corncob bedding (Teklad) in individual HEPA ventilated cages (Sealsafe® Plus, Techniplast USA) on a 12-hour light-dark cycle at 70-74 (21- 23°C) and 40-60% humidity. Animals were fed water ad libitum (reverse osmosis, 2 ppm Cl2) and an irradiated standard rodent diet (Teklad 2919) consisting of 19% protein, 9% fat, and 4% fiber.
[0120] Low passage patient-derived xenograft (START-PDX) models representing human cancer were from START (San Antonio, TX). In subcutaneous models, animals were implanted unilaterally on the flank with tumor fragments harvested from host animals each implanted from a specific passage lot. Pre-study tumor volumes were recorded for each experiment beginning approximately one week prior to its estimated start date. When tumors reached the appropriate Tumor Volume Initiation (TVI) range, animals were randomized into treatment and control groups and dosing initiated (Day 0); animals in all studies were tagged and followed individually throughout the experiment.
[0121] Initial dosing began at Day 0; animals in all groups were dosed by weight (0.01 ml per gram; 10 ml/kg). Dose concentration(s), route(s) of administration and schedule(s) for each agent are listed in the Experimental Design section. [0122] Beginning Day 0, animals were observed daily and weighed twice weekly using a digital scale; data including individual and mean gram weights (Mean We ± SD), mean percent weight change versus Day 0 (%vDo) and mean percent weight change versus prior measurement (%vD-x) were recorded for each group and %vD0 plotted at study completion. Animal deaths were recorded daily and designated as drug-related (D), technical (T), tumor related (B), or unknown (U) based on weight loss and gross observation; groups reporting a mean %vD0 >20% for a period of >7 days and/or >20% mortality or morbidity were considered above the maximum tolerated dose (MTD) for that treatment on the evaluated regimen. Maximum mean %vD0 (weight nadir) for each treatment group was reported at study completion.
[0123] For all studies, beginning Day 0, tumor dimensions were measured twice weekly by digital caliper and data including individual and mean estimated tumor volumes (Mean TV ± SEM) recorded for each group; tumor volume was calculated using the formula (Yasui et al., 1997): TV= width2 x length x 0.52. A Tumor Growth Inhibition (TGI) study was typically completed once all groups reached a predetermined mean tumor volume or a predetermined study day was reached; treatment groups may be taken out past this timepoint and collected measurements on that date used for analysis. For TGI analysis percent tumor growth inhibition (%TGI) values were calculated and reported for each treatment group (T) versus control (C) using initial (i) and final (f) tumor measurements by the formula: %TGI= 1- (Tf - TQ / ( Cf - CI). Individual mice reporting a tumor volume <50% of the Day 0 measurement for two consecutive measurements over a seven day period were considered partial responders (PR). If the PR persisted until study completion, percent tumor regression (%TR) was determined using the formula according to Corbette et al., 2004: %TR= 1- (Tf / T), x 100; a mean value was calculated if multiple PR mice occur in one group. Individual mice lacking palpable tumors (< 4x4 mm2 for two consecutive measurements over a seven day period) were classified as complete responders (CR); a CR that persisted until study completion was considered a tumor-free survivor (TFS); TFS animals were excluded from TGI calculations. Statistical differences in tumor volume were determined using a two-tailed One-Way Analysis of Variance (ANOVA) followed by the Dunnett's multiple comparisons test comparing treated single agent groups with control and combinations with standard agent when possible. The experimental design is summarized in Table 17 below.
Table 17: Experimental Design In Vivo Evaluation in a START-PDX
Group -n- Agent Dose (mq/kq/dose) ROA/ Schedule
1 10 VEHICLE - PO/ BID TO END
2 10 BVD-523 100 (200/DAY) PO/ BID TO END
3 10 DABRAFENIB 50 (100/DAY) PO/ BID TO END
Animal Information: Female nude mice (Crl:NU(NCr)-Foxn1 nu); 5-8 weeks old Study Type/ Endpoint(s): Efficacy/ TGI, Individual Group Mean TV= 2 cm3 or Day 60
Tumor Model Information ST052C Results
[0124] A vehicle of 1 % carboxymethylcellulose was administered per os
(p.o.) twice daily for 31 days (qdx31 ); minor weight loss was reported and regained by TGI study completion (Table 18). Final mean group weight was 23 ± 2 grams (+2% vs. Day 0). No deaths were reported. Based on vehicle control tumor volume, a tumor doubling time (Td) of 7.9 days was calculated for this model.
[0125] BVD-523 was administered p.o. at 100 mg/kg on a bidx31 schedule. Dabrafenib was also evaluated as a single agent on the same treatment regimen. Weight loss was reported in all groups; one technical related death was reported in the single agent BVD-523 group on Day 21 (Table 18). Two partial responders (PR) were reported in the combination group. TGI values of 78%, 76% and 22% were calculated, respectively; activity was found statistically significant (p<0.05) in single agent BVD-523 with vehicle control (Table 19).
Figure imgf000065_0001
Example 6
Clinical pharmacology of BVD-523 in humans
Materials and methods for the pharmacokinetic study
[0126] Human plasma was treated with K2EDTA anticoagulant (Bioreclamation Inc., East Meadow, NY). BVD-523 was extracted from human plasma by protein precipitation and analyzed using liquid chromatography (LC) with tandem mass spectrometric detection (MS/MS). The standard curve range is from 1.00 to 1000 ng/mL for BVD-523 using a plasma sample volume of 0.0500 mL.
[0127] The following equipment (or its equivalent) was used.
• Automated liquid handling system, Microlab Nimbus96, (Hamilton Robotics Inc. Reno, NV) or Tomtec (Munich, Germany) .
• Analytical column, Ascentis® Express C18, 50 x 3.0 mm, 2.7 [tm particle size, Supeico (Sigma-Aldrich, St. Louis, MO)
• Prefilter, 0.5 jam, Upchurch Scientific (Oak Harbor, WA)
• Solvent delivery system, LC-20AD, Shimadzu (Kyoto, Japan)
• Autoinjector, SIL-20AC, Prominence, Shimadzu
• Column heater, CTO-20AC, Shimadzu
• Triple quadrupole mass spectrometer, API 5000, Sciex Inc.
(Framingham, MA)
• Acetonitrile (ACN), HPLC grade, Sigma-Aldrich
• Dimethyl sulfoxide (DMSO), HPLC grade, Fisher (Pittsburg, PA)
• Water, Type 1 , ELGA system (ELGA Labwater, Quebec, Canada)
[0128] The chromatographic conditions were as follows. The column used was a Supeico, Ascentis® Express C18, 50 x 3.0 mm, 2.7 pm particle size. The pre-filter was 0.5 gin, Upchurch. The column temperature was 40°C.
[0129] For the mobile phase, solution A was Type 1 Water, and solution B was acetonitrile. The gradient program was set with initial conditions of 0.800 mL/min; 30% solution B. The rest of the conditions are detailed in Table 20 below.
Table 20 - chromatographic conditions
Figure imgf000067_0001
The flow rate was set to be 0.800 ml/min, and the typical back pressure was 170 bars.
[0130] The sample tray was refrigerated (2 to 8°C).
[0131] The injection volume was typically 3-5 μΙ, but not to exceed 10 μΙ total.
[0132] Acetonitrile was used for both the rinse port injector wash solution and the rinse pump injector wash solution. Needle stroke was set to be 50 mm. Rinse pump setting was from rinse pump to rinse port. The rinse volume was 450 μΙ. The rinse mode was set to be before and after sampling. The rinse dip time was 5 seconds, and the rinse time was 1 second. The acquisition time was approximately 3.2 minutes, and the cycle time was approximately 5.0 minutes (from one injection start to the next injection start).
[0133] The mass spectrometer parameters set forth in Tables 21 and 22 were used.
Table 21 - Mass spectrometer parameters
Figure imgf000068_0001
Table 22 - Mass spectrometer parameters
Figure imgf000068_0002
[0134] The column equilibrated within 10 minutes of starting flow. Four injections were used at the start of each batch to equilibrate the system.. [0135] The collision energy of BVD-523 is de-optimized from 50 eV. The de-optimized collision energy values may be adjusted to different instruments to make sure that there is no saturation of BVD-523 at the Upper Limit of Quantification (ULOQ).
[0136] For data analysis, the regression type and weighting were linear, 1/x2, respectively. The units of concentration were ng/mL, and the response was measured using peak area ratios (BVD-523/ISTD1 ).
Materials and methods for determining the effects of BVD-523 on RSK1 phosphorylation in human whole blood
[0137] Target biomarkers, biomarkers that lie directly downstream of a protein of interest, are key resources in assessing target coverage when modulating the activity of the protein of interest with a small molecule inhibitor in preclinical and clinical in vivo studies. RSK1 , a member of the RSK serine/threonine kinase family, is a direct substrate of the MAP Kinases ERK1 and ERK2 (Romeo, Zhang and Roux, 2012). RSK1 and ERK1/2 form an inactive complex in unstimulated cells. Upon activation of the mitogenic pathway, ERK1/2 phosphorylates Thr573, Thr359 and Ser363 on RSK1. Thr573 resides in the activation loop of the carboxy terminal kinase domain of RSK1 and once phosphorylated, enables RSK1 to autophosphorylate Ser380 (Cargnello and Roux, 201 1 ). Phosphorylation of Ser380 on RSK1 can then be used as a target biomarker for ERK1 and ERK2 activity. [0138] In this study, phosphorylation of RSK1 on Ser380 (pRSK) was used as a target biomarker for assessment of ERK inhibition by BVD-523 in ex vivo treated human whole blood samples from ten healthy individuals.
[0139] Efforts consist of the determination of IC50 values for the inhibition of phorbol 12-myristate 13-acetate (PMA) stimulated RSK1 phosphorylation by BVD-523. An eight point concentration curve ranging from 10 μΜ to 5 nM BVD-523 was used for each IC50 determination. Controls consisted of three unstimulated samples and three PMA stimulated samples for each donor. Two donors were analyzed in each experiment. A total of ten different volunteers were evaluated with an age range of 22 to 61 years.
[0140] Peripheral blood mononucleated cell (PBMC) preparation reagents used were as follows:
• Dulbecco's Modified Eagle Medium (DMEM) High Glucose from Gibco Catalog Number: 1 1965;
• Fetal Bovine Serum (FBS) from Gibco Catalog Number: 26140;
• Penicillin/Streptomycin/Glutamine from Gibco Catalog Number: 10378;
• Dimethyl Sulfoxide (DMSO) from Sigma-Aldrich Catalog Number:
D2438;
• Phorbol 12,Myristate 13 Acetate (PMA) from Sigma-Aldrich Catalog Number: P1585;
• Dulbecco's Phosphate Buffered Saline (DPBS) (Ca++, Mg-HE free) from Gibco Catalog Number: 14190;
• Histopaque 1077 from Sigma-Aldrich Catalog Number: 10771 ;
• 50 ml conical tubes from VWR International Catalog Number: 430829; and • 2 ml microtubes from VWR International Catalog Number: 89004-302.
[0141] The reagents for lysis buffer were as follows:
• MSD Tris Lysis Buffer from Meso Scale Discovery Catalog Number:
R6OTX-2;
• Phosphatase Inhibitor Cocktail 3 (100X) from Sigma-Aldrich Catalog Number: P0044;
• Phosphatase Inhibitor Cocktail 2 (100X) from Sigma-Aldrich Catalog Number: P5726;
• Halt Protease Inhibitor Solution (100X) from LabSource Catalog
Number: PI87785;
• Phenylmethylsulfonyl fluoride (PMSF) from Sigma-Aldrich Catalog
Number: P7626-5G; and
• Sodium Dodecyl Sulfate (SDS) from Calbiochem Catalog Number:
7990.
[0142] The reagents for ELISA were as follows:
• Phospho-RSK1 (Ser380) Sandwich ELISA kits from Cell Signaling
Catalog Number: 7965; and
• RSK1 Sandwich ELISA kits from Cell Signaling Catalog Number: 7966.
[0143] BVD-523 was stored at room temperature in the dark.
[0144] Using a sterile serological pipet, human whole blood was transferred and combined from three vacutainers per donor into a 50 ml conical tube. From the 50 ml conical tube, one ml of whole blood was added to each of twenty-two 2 ml microtubes per donor. A new serological pipet was used for each donor. The microtubes were labeled with the donor number (1- 10) and the subsequent treatment designation: "A" for PMA stimulation only (maximum), "B" for BVD-523 containing samples that received PMA stimulation; and "C" for the unstimulated samples (minimum).
[0145] DMSO was added to all tubes in groups "A" and "C" to a final concentration of 0.1 % (10 μΙ per ml of a 10% DMSO solution in DMEM containing 10% FBS and penicillin/streptomycin/glutamine). Samples were then rocked gently at room temperature.
[0146] BVD-523 (10 mM in 100% DMSO) was serially diluted with 3- fold dilutions into 100% DMSO. These serially diluted BVD-523 samples in 100% DMSO were then diluted 10-fold in DMEM containing 10% FBS and penicillin/streptomycin/glutamine, and 10 μΙ of each of these working solutions was added per ml of blood for each designated BVD-523 concentration. Each concentration of BVD-523 was run in duplicate, two 1 ml blood samples each, yielding sixteen total samples for the full eight point concentration curve. Samples were then rocked gently at room temperature for a minimum of two hours, but no longer than three hours.
[0147] For stimulation of human whole blood, samples in groups "A" and "B" for all donors were stimulated with PMA at a final concentration of 100 nM for twenty minutes at room temperature. A stock solution of PMA at 1 mM in 100% DMSO was diluted to 10 μΜ in DMEM containing 10% FBS and penicillin/streptomycin, and 10 μΙ of this working solution was added per ml of blood. Samples were gently rocked at room temperature for ten minutes then placed upright for 10 minutes prior to PBMC isolation. Samples in group "C" were not treated with PMA but were rocked and handled as all other samples.
[0148] Upon completion of PMA treatment for each sample, PBMCs were isolated from the human whole blood. One ml of blood from each sample was gently layered onto 0.75 ml of Histopaque 1077 in a 2 ml microcentrifuge tube. Histopaque 1077 was maintained at room temperature. The samples were centrifuged for two minutes at 16,000 x g in an Eppendorf microcentrifuge. The interface and upper layers were removed and added to tubes containing 1 ml cold Dulbecco's Phosphate-Buffered Saline (DPBS). These samples were then centrifuged for 30 seconds at 16,000 x g in an Eppendorf microcentrifuge to pellet the cells. The buffer supernatant was removed by aspiration and the pellets were re-suspended in 1 ml of cold DPBS. The pellets from each sample were then re-pelleted as above. The buffer was removed by aspiration and the pellets were lysed as indicated below.
[0149] To prepare the lysates, complete lysis buffer consisted of MSD Tris lysis buffer, 1X Halt Protease inhibitor cocktail, 1X Phosphatase inhibitor cocktail 2, 1X Phosphatase inhibitor cocktail 3, 2mM PMSF and 0.1 % SDS. Lysis buffer was kept on ice and made fresh for each sample group. Final cell pellets were lysed by the addition of 120 μΙ of complete lysis buffer. Samples were vortexed until the cell pellet disappeared and then flash frozen on dry ice. Samples were stored at -20°C prior to measurement of pRSK and total RSK by ELISA.
[0150] For the pRSK ELISA, thawed lysates were combined 1 :1 with Sample Diluent (provided in ELISA kit), 120 μΙ of lysate added to 120 μΙ of Sample Diluent in a Corning round bottom 96-well plate. This combination was then transferred to the pRSK microwells at 100 μΙ per well. For the total RSK ELISA, 20 μΙ of the lysate already diluted 1 : 1 in Sample Diluent was further diluted in 200 μΙ of Sample Diluent in a Corning round bottom 96-well plate. This combination was then transferred to the total RSK microwells at 100 μΙ per well. The plates were sealed with a plate seal and incubated 16-18 hours at 4°C, a time that was shown to yield the best detection of the target protein.
[0151] Both ELISAs were developed using the follow steps. Gently remove the tape and wash wells: Discard liquid contents of each plate into a receptacle. Wash 4 times with 1X Wash Buffer (provided in ELISA kit), 200 μΙ each time for each well. For each wash, strike plates on fresh towels hard enough to remove the residual solution in each well, but do not allow wells to completely dry at any time. Add 100 μΙ of Detection Antibody (provided in ELISA kit) to each well. Seal with tape and incubate the plate for 1 hour at 37°C. Repeat wash procedure as noted above. Add 100 μΙ of HRP-Linked secondary antibody (provided in ELISA kit) to each well. Seal with tape and incubate the plate for 30 minutes at 37°C. Repeat wash procedure as noted above. Add 100 μΙ of TMB Substrate (provided in ELISA kit) to each well. Seal with tape and incubate the plate for up to 30 minutes at 25°C. Add 100 μΙ of STOP Solution (provided in ELISA kit) to each well. Shake gently for a few seconds. Clean the underside of all wells with a lint-free tissue. Read absorbance at 450 nm within 30 minutes after adding STOP Solution. Plates were analyzed using a Spectramax-M2 plate reader (Molecular Devices).
[0152] SoftMax Pro, Version 4.6 (Molecular Devices), Microsoft Excel 2010 (Microsoft) and GraFit Data Analysis Software (Version 5.0.12) were used for data analysis.
[0153] The preclinical activity of BVD-523 on enzymes and cells, as well as several favorable pharmaceutical properties of the molecule (e.g. absorption, metabolism and distribution characteristics not shown here), supported the use of BVD-523 as an investigational anti-cancer agent in a human clinical trial.
[0154] The clinical pharmacology of BVD-523 administered in humans further suggests the potential utility of this agent for treatment of hematological disorders. BVD-523 exhibits oral bioavailability in humans: patients exhibit systemic exposures of BVD-523 within hours following administration of a single dose presented as a powder in standard gelatin capsules, and systemic BVD-523 exposures increase as oral dose increases (not shown). After repeated, twice-daily administration for up to 15 days, systemic exposures of BVD-523 at steady-state also appear to increase with increases in dose level (Figure 2A). The absolute concentration of BVD-523 observed at various dose levels appears consistent with concentrations required for ERK target enzyme inhibition and cellular bioactivity (above, Figure 2A). Consistent with this hypothesis, patient-derived, ex-vivo stimulated peripheral blood mononuclear cells exhibit reduced ERK- dependent RSK1/2 protein phosphorylation, in a fashion that appears overall to correlate well with both BVD-523 dose and exposure (Figure 2B). Lastly, a variety of additional clinical findings can be observed in subjects treated with BVD-523, ranging from drug-related adverse events, decreased tumor metabolic activity as indicated by FDG-PET, as well as changes in circulating and tumor cell biomarkers. In summary, a variety of evidence suggests that systemic exposure of BVD-523 following administration in humans can elicit primary pharmacology following inhibition of ERK 1 and 2 protein kinases. [0155] In the present invention, ERK inhibition in hematologic malignancies takes advantage of oncogenic driver mutations in genetic components coupled to the MAPK signaling cascade, which would likely provide an enhanced therapeutic index. It is possible that particular hematologic malignancies may exhibit exquisite intrinsic sensitivity to BVD- 523, while simultaneously displaying insensitivity to either RAF or MEK inhibitors. In a subset of hairy-cell leukemias, for example, oncogenic MAP2K1 gene mutations confer MEK inhibitor insensitivity— variants of this type may remain sensitive to ERK inhibition and BVD-523.
[0156] Additionally, it is possible that BVD-523 and ERK inhibition may exhibit unique anti-cancer activity in malignancies that have acquired resistance to other targeted agents, including RAF and MEK inhibitors. MAP2K1 activating mutations have been identified in subjects with melanoma that exhibited disease progression following therapy with RAF and MEK inhibitors, either alone or in combination. It is possible that these variants, as well as other adaptive changes that occur during cancer therapy, may evoke drug resistance to a variety of agents, while not altering sensitivity to BVD-523 or ERK inhibition.
[0157] Finally, properties of BVD-523 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-523 may interact with and inhibit these targets in a fashion that is distinct and highly preferable to other agents.
[0158] Given the evidence of pharmacologically relevant exposures and potential bioactivity of BVD-523 in humans, coupled with the fact that many hematologic cancers have mutations in the RAS oncogene, which is activated upstream of ERK in the MAPK pathway (Li et al., 201 1 ; Lauchle et al., 2009; Borthakur ei al., 2012), BVD-523 is expected to have clinically relevant potency in human lymphoid and myeloid malignancies as well as preleukemic myelodysplastic syndromes (MDS) and lymphomas.
Example 7
BVD-523 is effective in inhibiting the growth of hematologic cancer cell lines in vitro
[0159] Cancer cell lines are maintained in cell culture under standard media and serum conditions.
[0160] For this study, various cell lines listed in table 8 below are seeded into triplicate 96-well plates at a cell density of 3000 cells/well in DMEM plus 10% FBS and are allowed to adhere overnight prior to addition of BVD-523 or vehicle control. A 96 hour assay incubation period follows, with subsequent addition of Alamar Blue 10% (v/v) and 4 hours incubation prior to reading on a fluorescent plate reader. After reading Alamar Blue, the medium/Alamar Blue mix is flicked off and 100 μΙ of CellTiter-Glo/PBS (1 : 1 ) added and the plates processed as per the manufacturers instructions (Promega). Media only background values are subtracted before the data is analysed.
Table 23 - Hematologic cancer cell lines
Figure imgf000078_0001
[0161] It is expected that BVD-523 is effective in inhibiting the growth of the above hematologic cancer cell lines. Dose response curves will be obtained. It is expected that the IC50 of BVD-523 in these cell lines will be approximately 150 nM. Example 8
BVD-523 is effective in treating hematologic cancers in vivo
Mice
[0162] Female athymic nude mice (Crl:NU(Ncr)-Foxn/nu, Charles River) are nine weeks old with a body weight (BW) range of about 15 to about 30 grams on Day 1 of the study. The animals are fed ad libitum water (reverse osmosis, 1 ppm CI), and NIH 31 Modified and Irradiated Lab Diet® consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fiber. The mice are housed on irradiated Enrich-o'cobs™ Laboratory Animal Bedding in static microisolators on a 12-hour light cycle at 20-22°C (68-72°F) and 40-60% 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.
In Vivo Implantation and Tumor Growth
[0163] Tumor xenografts (1-2x106 human myeloid leukaemia HL60 cells) are subcutaneously implanted into the flank of athymic nude mice. Tumors are measured in two dimensions using calipers, and volume is calculated using the formula:
Tumor Volume (mm3) -w2 x \
2
where w = width and / = length, in mm, of the tumor. Tumor weight may be estimated with the assumption that 1 mg is equivalent to 1 mm3 of tumor volume.
[0164] Six days after tumor implantation, designated as Day 1 of the study, the animals are sorted into five groups (Groups 1-5), each consisting of fifteen mice. Therapeutic Agents
[0165] BVD-523 is supplied as a dry powder and is stored at room temperature protected from light.
[0166] BVD-523 doses are prepared by suspending the required amount of BVD-523 powder in 1 % carboxymethyl cellulose in deionized water ("1 % CMC"). A 10 mg/mL BVD-523 stock is prepared, and is used to dose the 100 mg/kg BVD-523 group. Aliquots of the stock are diluted with the vehicle to a concentration of 5.0 mg/mL to provide the 50 mg/kg BVD-523 dosage in a dosing volume of 10 mL/kg. The BVD-523 doses are stored at 4°C protected from light for up to one week.
[0167] The 1 % CMC vehicle ("Vehicle") is used to dose the control group.
[0168] Doxorubicin is approved to treat various cancers including Acute Lymphoblastic Leukemia (ALL) and Acute Myeloid Leukemia (AML). Doxorubicin is dissolved in distilled water at a concentration such that the dose could be given in a volume of 0.1 ml/10 g of body weight.
Treatment
[0169] On Day 1 of the study, mice are sorted into five groups (Group 1-5) each consisting of fifteen mice, and dosing is initiated according to the treatment plan summarized in Table 24 below. Each BVD-523 dose is given by oral gavage (p.o.) in a dosing volume of 10 mL/kg (0.2 mL per 20 grams of body weight), scaled to the body weight of each individual animal. Each doxorubicin dose is given by intravenous injection (i.v.) in a dosing volume of 0.1 mL/10g of body weight, scaled to the body weight of each individual animal. The vehicle and the BVD-523 doses are to be given twice daily until study end (bid to end), and the doxorubicin doses are to be given once a week until study end (qw to end). For bid dosing, dosing is initiated in the afternoon on Day 1 , so that one dose is given on the first day ("first day 1 dose").
Table 24 - Protocol Design for the HL60 in vivo Study
Figure imgf000081_0001
Vehicle = 1 % carboxymethylcellulose (CMC) in Dl water
For bid doses, one dose is given in the afternoon on the first day and one dose in the morning on the last day.
Controls
[0170] Group 1 receives 1 % CMC vehicle, and serves as the control group for calculation of %TGD. Group 5 receives doxorubicin at 100 mg/kg and serves as a reference group.
Monotherapy Treatments
[0171] Groups 2 and 3 receive 50 and 100 mg/kg BVD-523, respectively. Tumor measurements
[0172] Tumors are measured using calipers twice per week, and each animal is euthanized when its tumor reaches the pre-determined tumor volume endpoint of 2000 mm3 or on the final day (day 50), 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:
TTE = loqn (endpoint volume) - b
m
where TTE is expressed in days, endpoint volume is expressed in mm3, 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 exceeded the endpoint volume used in analysis and the three consecutive observations that immediately preceded 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 did 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. [0173] Treatment outcome is evaluated from tumor growth delay (TGD), defined as the increase in the median time to endpoint (TTE) in a treatment group compared to the control group:
TGD = T - C, expressed in days, or as a percentage of the median TTE of the control group:
%TGD = T - C x 100
C where:
T = median TTE for a treatment group, and
C = median TTE for the designated control group.
Criteria for Regression Responses
[0174] 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 50% or less of its Day 1 volume for three consecutive measurements during the course of the study, and equal to or greater than 135 mm3 for one or more of these three measurements. In a CR response, the tumor volume is less than 135 mm3 for three consecutive measurements during the course of the study. An animal with a CR response at the termination of a study is additionally classified as a tumor-free survivor (TFS). Animals are monitored for regression responses.
Toxicity
[0175] Animals are weighed daily on Days 1-5, then twice per week until completion of the study. The mice are observed frequently for overt signs of any adverse, treatment-related (TR) side effects, and clinical signs are recorded when observed. Individual body weight loss is monitored as per protocol, and any animal that exceeded the limits for acceptable body weight loss is euthanized. Group mean body weight loss also is monitored as per protocol. Dosing is to be suspended in any group that exceeded the limits for acceptable mean body weight loss. If mean body weight recovered, 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 body-weight loss of less than 20% during the study and not more than 10% treatment-related (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 14 days of the last dose. A death is classified as non-treatment-related (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 resulted from an accident or human error. A death is classified as NTRm if necropsy indicated that it may have resulted 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.
Sampling
[0176] When available, five mice per group are euthanized by terminal cardiac puncture under carbon dioxide anesthesia at 3, 6 and 12 hours post final dose, and the full blood volume of each animal is collected. The serum is separated and stored frozen at -80°C until shipment. In addition, the tumors of these mice are harvested and divided into two parts. One part is snap frozen and stored at -80°C. The other part is fixed for 16-24 hours in 10% neutral buffered formalin, and then transferred to 70% ethanol. For groups with mice that had no detectable tumor, the implant site including full skin and muscle thickness is collected from three mice per group.
Statistical and Graphical Analyses
[0177] Prism (GraphPad) for Windows 3.03 is used for graphical presentations and statistical analyses.
[0178] 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 = 0.05. The statistical tests are not adjusted for multiple comparisons. Prism summarizes test results as not significant (ns) at P > 0.05, significant (symbolized by "*") at 0.01 < P < 0.05, very significant ("**") at 0.001 < PO.01 , and extremely significant ("***") at PO.001.
[0179] 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). 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 body weight changes from Day 1 are calculated for each group for each day of body weight measurement, and are plotted as a function of time. Tumor growth and body weight plots will exclude the data for NTR deaths, and are truncated after 50% of the assessable animals in a group which has exited the study.
Results
[0180] It is expected that BVD-523 is effective against HL60 cancer cells at both 50 and 100 mg/kg doses and that the effects are statistically significant. It is also expected that the side effects associated with BVD-523 treatment are minimal.
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[0181] All documents cited in this application are hereby incorporated by reference as if recited in full herein.
[0182] Although illustrative embodiments of the present invention have been described herein, it should be understood that the invention is not limited to those described, and that various other changes or modifications may be made by one skilled in the art without departing from the scope or spirit of the invention.

Claims

What is claimed is:
1. A method of treating or ameliorating the effects of a hematologic cancer in a subject in need thereof comprising administering to the subject an effective amount of an ERK1/2 inhibitor to treat or ameliorate the effects of the hematologic cancer.
2. The method according to clam 1 , wherein the subject is a mammal.
3. The method according to claim 2, wherein the mammal is selected from the group consisting of humans, primates, farm animals, and domestic animals.
4. The method according to claim 2, wherein the mammal is a human.
5. The method according to claim 1 , wherein the ERK1/2 inhibitor is selected from the group consisting of AEZS-131 (Aeterna Zentaris), AEZS- 136 (Aeterna Zentaris), BVD-523 (BioMed Valley Discoveries, Inc.), SCH- 722984 (Merck & Co.), SCH-772984 (Merck & Co.), SCH-900353 (MK-8353, Merck & Co.), pharmaceutically acceptable salts thereof, and combinations thereof.
6. The method according to claim 1 , wherein the hematologic cancer is selected from the group consisting of RAS mutant myelodysplastic syndromes (MDS), lymphoid cancers, and myeloid cancers.
7. The method according to claim 1 , wherein the hematologic cancer is selected from the group consisting of Adult Acute Megakaryoblastic Leukemia (M7), Adult Acute Minimally Differentiated Myeloid Leukemia (MO), Adult Acute Monoblastic Leukemia (M5a), Adult Acute Monocytic Leukemia (M5b), Adult Acute Myeloblasts Leukemia With Maturation (M2), Adult Acute Myeloblasts Leukemia Without Maturation (M1 ), Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities, Adult Acute Myeloid Leukemia With Del(5q), Adult Acute Myeloid Leukemia With Inv(16)(p13;q22), Adult Acute Myeloid Leukemia With t(16;16)(p13;q22), Adult Acute Myeloid Leukemia With t(8;21 )(q22;q22), Adult Acute Myelomonocytic Leukemia (M4), Adult Erythroleukemia (M6a), Adult Pure Erythroid Leukemia (M6b), Recurrent Adult Acute Myeloid Leukemia, and Untreated Adult Acute Myeloid Leukemia.
8. The method according to claim 1 further comprising treating the subject with a combination therapy.
9. The method according to claim 8, wherein the combination therapy comprises administering at least one additional therapeutic agent selected from the group consisting of an antibody or fragment thereof, a cytotoxic agent, a drug, a toxin, a radionuclide, an immunomodulator, a photoactive therapeutic agent, a radiosensitizing agent, a hormone, an anti-angiogenesis agent, and combinations thereof.
10. The method according to claim 9, wherein the cytotoxic agent is selected from the group consisting of an alkylating agent, a platinum-based agent, an intercalating agent, an inhibitor of DNA replication, an antimetabolite, an anti-microtubule agent, an antibiotic agent, and combinations thereof.
11. The method according to claim 9, wherein the additional therapeutic agent is selected from the group consisting of cyclophosphamide, mechlorethamine, uramustine, melphalan, chlorambucil, ifosfamide, carmustine, lomustine, streptozocin, busulfan, temozolomide, cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, triplatin tetranitrate, doxorubicin, daunorubicin, idarubicin, mitoxantrone, irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, methotrexate premetrexed, 6-mercaptopurine, dacarbazine, fludarabine, 5-fluorouracil, arabinosylcytosine, capecitabine, gemcitabine, decitabine, vinca alkaloids, paclitaxel, docetaxel, ixabepilone, actinomycin, anthracyclines, valrubicin, epirubicin, bleomycin, plicamycin, mitomycin, 2-methoxyestradiol, angiostatin, bevacizumab, cartilage-derived angiogenesis inhibitory factor, endostatin, IFN-a, IL-12, itraconazole, linomide, platelet factor-4, prolactin, SU5416, suramin, tasquinimod, tecogalan, tetrathiomolybdate, thalidomide, thrombospondin, thrombospondin, TNP-470, ziv-aflibercept, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof.
12. A method of treating or ameliorating the effects of a hematologic cancer in a subject in need thereof comprising administering to the subject an effective amount of BVD-523 or a pharmaceutically acceptable salt thereof to treat or ameliorate the effects of the hematologic cancer.
13. The method according to claim 12, wherein the subject is a mammal.
14. The method according to claim 13, wherein the mammal is selected from the group consisting of humans, primates, farm animals, and domestic animals.
15. The method according to claim 13, wherein the mammal is a human.
16. The method according to claim 12, wherein the BVD-523 or a pharmaceutically acceptable salt thereof is administered in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier or diluent.
17. The method according to claim 13, wherein the hematologic cancer is selected from the group consisting of RAS mutant myelodysplastic syndromes (MDS), lymphoid cancers, and myeloid cancers.
18. The method according to claim 12, wherein the hematologic cancer is selected from the group consisting of Adult Acute Megakaryoblastic Leukemia (M7), Adult Acute Minimally Differentiated Myeloid Leukemia (MO), Adult Acute Monoblastic Leukemia (M5a), Adult Acute Monocytic Leukemia (M5b), Adult Acute Myeloblasts Leukemia With Maturation (M2), Adult Acute Myeloblasts Leukemia Without Maturation (M1 ), Adult Acute Myeloid Leukemia With 1 1 q23 (MLL) Abnormalities, Adult Acute Myeloid Leukemia With Del(5q), Adult Acute Myeloid Leukemia With Inv(16)(p13;q22), Adult Acute Myeloid Leukemia With t(16; 16)(p13;q22), Adult Acute Myeloid Leukemia With t(8;21 )(q22;q22), Adult Acute Myelomonocytic Leukemia (M4), Adult Erythroleukemia (M6a), Adult Pure Erythroid Leukemia (M6b), Recurrent Adult Acute Myeloid Leukemia, and Untreated Adult Acute Myeloid Leukemia.
19. The method according to claim 12 further comprising treating the subject with a combination therapy.
20. The method according to claim 19, wherein the combination therapy comprises administering at least one additional therapeutic agent selected from the group consisting of an antibody or fragment thereof, a cytotoxic agent, a drug, a toxin, a radionuclide, an immunomodulator, a photoactive therapeutic agent, a radiosensitizing agent, a hormone, an anti-angiogenesis agent, and combinations thereof.
21. The method according to claim 20, wherein the cytotoxic agent is selected from the group consisting of an alkylating agent, a platinum-based agent, an intercalating agent, an inhibitor of DNA replication, an antimetabolite, an anti-microtubule agent, an antibiotic agent, and combinations thereof.
22. The method according to claim 19, wherein the additional therapeutic agent is selected from the group consisting of cyclophosphamide, mechlorethamine, uramustine, melphalan, chlorambucil, ifosfamide, carmustine, lomustine, streptozocin, busulfan, temozolomide, cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, triplatin tetranitrate, doxorubicin, daunorubicin, idarubicin, mitoxantrone, irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, methotrexate premetrexed, 6-mercaptopurine, dacarbazine, fludarabine, 5-fluorouracil, arabinosylcytosine, capecitabine, gemcitabine, decitabine, vinca alkaloids, paclitaxel, docetaxel, ixabepilone, actinomycin, anthracyclines, valrubicin, epirubicin, bleomycin, plicamycin, mitomycin, 2-methoxyestradiol, angiostatin, bevacizumab, cartilage-derived angiogenesis inhibitory factor, endostatin, IFN-a, IL-12, itraconazole, linomide, platelet factor-4, prolactin, SU5416, suramin, tasquinimod, tecogalan, tetrathiomolybdate, thalidomide, thrombospondin, thrombospondin, TNP-470, ziv-aflibercept, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof.
23. A method of modulating ribosomal S6 kinase (RSK) phosphorylation in the blood of a subject that has a hematologic cancer comprising administering to the subject an effective amount of an ERK1/2 inhibitor to modulate RSK phosphorylation in the blood of the subject.
24. The method according to clam 23, wherein the subject is a mammal.
25. The method according to claim 24, wherein the mammal is selected from the group consisting of humans, primates, farm animals, and domestic animals.
26. The method according to claim 24, wherein the mammal is a human.
27. The method according to claim 23, wherein the ERK1/2 inhibitor is selected from the group consisting of AEZS-131 (Aeterna Zentaris), AEZS- 136 (Aeterna Zentaris), BVD-523 (BioMed Valley Discoveries, Inc.), SCH- 722984 (Merck & Co.), SCH-772984 (Merck & Co.), SCH-900353 (MK-8353, Merck & Co.), pharmaceutically acceptable salts thereof, and combinations thereof.
28. The method according to claim 23, wherein the modulation is a decrease in RSK phosphorylation.
29. The method according to claim 28, wherein the decrease comprises substantially inhibiting RSK phosphorylation by ERK in the blood of the subject.
30. The method according to claim 29, wherein the ERK1/2 inhibitor is BVD-523.
31. The method according to claim 23, wherein the hematologic cancer is selected from the group consisting of RAS mutant myelodysplastic syndromes (MDS), lymphoid cancers, and myeloid cancers.
32. The method according to claim 23, wherein the hematologic cancer is selected from the group consisting of Adult Acute Megakaryoblastic Leukemia (M7), Adult Acute Minimally Differentiated Myeloid Leukemia (MO), Adult Acute Monoblastic Leukemia (M5a), Adult Acute Monocytic Leukemia (M5b), Adult Acute Myeloblasts Leukemia With Maturation (M2), Adult Acute Myeloblasts Leukemia Without Maturation (M1 ), Adult Acute Myeloid Leukemia With 1 1 q23 (MLL) Abnormalities, Adult Acute Myeloid Leukemia With Del(5q), Adult Acute Myeloid Leukemia With Inv(16)(p13;q22), Adult Acute Myeloid Leukemia With t(16; 16)(p13;q22), Adult Acute Myeloid Leukemia With t(8;21 )(q22;q22), Adult Acute Myelomonocytic Leukemia (M4), Adult Erythroleukemia (M6a), Adult Pure Erythroid Leukemia (M6b), Recurrent Adult Acute Myeloid Leukemia, and Untreated Adult Acute Myeloid Leukemia.
33. A pharmaceutical composition for treating or ameliorating the effects of a hematologic cancer in a subject in need thereof comprising a pharmaceutically acceptable carrier and an effective amount of an ERK1/2 inhibitor or a pharmaceutically acceptable salt thereof.
34. The pharmaceutical composition according to claim 33, wherein the subject is a mammal.
35. The pharmaceutical composition according to claim 34, wherein the mammal is selected from the group consisting of humans, primates, farm animals, and domestic animals.
36. The pharmaceutical composition according to claim 34, wherein the mammal is a human.
37. The pharmaceutical composition according to claim 33, wherein the ERK1/2 inhibitor is selected from the group consisting of AEZS-131 (Aeterna Zentaris), AEZS-136 (Aeterna Zentaris), BVD-523 (BioMed Valley Discoveries, Inc.), SCH-722984 (Merck & Co.), SCH-772984 (Merck & Co.), SCH-900353 (MK-8353, Merck & Co.), pharmaceutically acceptable salts thereof, and combinations thereof.
38. The pharmaceutical composition according to claim 33, wherein the hematologic cancer is selected from the group consisting of RAS mutant myelodysplastic syndromes (MDS), lymphoid cancers, and myeloid cancers.
39. The pharmaceutical composition according to claim 33, wherein the hematologic cancer is selected from the group consisting of Adult Acute Megakaryoblastic Leukemia (M7), Adult Acute Minimally Differentiated Myeloid Leukemia (MO), Adult Acute Monoblastic Leukemia (M5a), Adult Acute Monocytic Leukemia (M5b), Adult Acute Myeloblastic Leukemia With Maturation (M2), Adult Acute Myeloblastic Leukemia Without Maturation (M1 ), Adult Acute Myeloid Leukemia With 1 1 q23 (MLL) Abnormalities, Adult Acute Myeloid Leukemia With Del(5q), Adult Acute Myeloid Leukemia With Inv(16)(p13;q22), Adult Acute Myeloid Leukemia With t(16;16)(p13;q22), Adult Acute Myeloid Leukemia With t(8;21 )(q22;q22), Adult Acute Myelomonocytic Leukemia (M4), Adult Erythroleukemia (M6a), Adult Pure Erythroid Leukemia (M6b), Recurrent Adult Acute Myeloid Leukemia, and Untreated Adult Acute Myeloid Leukemia.
40. The pharmaceutical composition according to claim 33 further comprising at least one additional therapeutic agent effective for treating or ameliorating the effects of the hematologic cancer.
41. The pharmaceutical composition according to claim 40, wherein the additional therapeutic agent is selected from the group consisting of an antibody or fragment thereof, a cytotoxic agent, a drug, a toxin, a radionuclide, an immunomodulator, a photoactive therapeutic agent, a radiosensitizing agent, a hormone, an anti-angiogenesis agent, and combinations thereof.
42. The pharmaceutical composition according to claim 41 , wherein the cytotoxic agent is selected from the group consisting of an alkylating agent, a platinum-based agent, an intercalating agent, an inhibitor of DNA replication, an antimetabolite, an anti-microtubule agent, an antibiotic agent, and combinations thereof.
43. The pharmaceutical composition according to claim 40, wherein the additional therapeutic agent is selected from the group consisting of cyclophosphamide, mechlorethamine, uramustine, melphalan, chlorambucil, ifosfamide, carmustine, lomustine, streptozocin, busulfan, temozolomide, cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, triplatin tetranitrate, doxorubicin, daunorubicin, idarubicin, mitoxantrone, irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, methotrexate premetrexed, 6-mercaptopurine, dacarbazine, fludarabine, 5-fluorouracil, arabinosylcytosine, capecitabine, gemcitabine, decitabine, vinca alkaloids, paclitaxel, docetaxel, ixabepilone, actinomycin, anthracyclines, valrubicin, epirubicin, bleomycin, plicamycin, mitomycin, 2-methoxyestradiol, angiostatin, bevacizumab, cartilage-derived angiogenesis inhibitory factor, endostatin, IFN-a, IL-12, itraconazole, linomide, platelet factor-4, prolactin, SU5416, suramin, tasquinimod, tecogalan, tetrathiomolybdate, thalidomide, thrombospondin, thrombospondin, TNP-470, ziv-aflibercept, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof.
44. A pharmaceutical composition for treating or ameliorating the effects of a hematologic cancer in a subject in need thereof comprising a pharmaceutically acceptable carrier and an effective amount of BVD-523 or a pharmaceutically acceptable salt thereof.
45. The pharmaceutical composition according to clam 44, wherein the subject is a mammal.
46. The pharmaceutical composition according to claim 45, wherein the mammal is selected from the group consisting of humans, primates, farm animals, and domestic animals.
47. The pharmaceutical composition according to claim 45, wherein the mammal is a human.
48. The pharmaceutical composition according to claim 44, wherein the hematologic cancer is selected from the group consisting of RAS mutant myelodysplastic syndromes (MDS), lymphoid cancers, and myeloid cancers.
49. The pharmaceutical composition according to claim 44, wherein the hematologic cancer is selected from the group consisting of Adult Acute Megakaryoblastic Leukemia (M7), Adult Acute Minimally Differentiated Myeloid Leukemia (MO), Adult Acute Monoblastic Leukemia (M5a), Adult Acute Monocytic Leukemia (M5b), Adult Acute Myeloblasts Leukemia With Maturation (M2), Adult Acute Myeloblasts Leukemia Without Maturation (M1 ), Adult Acute Myeloid Leukemia With 1 1 q23 (MLL) Abnormalities, Adult Acute Myeloid Leukemia With Del(5q), Adult Acute Myeloid Leukemia With Inv(16)(p13;q22), Adult Acute Myeloid Leukemia With t(16;16)(p13;q22), Adult Acute Myeloid Leukemia With t(8;21 )(q22;q22), Adult Acute Myelomonocytic Leukemia (M4), Adult Erythroleukemia (M6a), Adult Pure Erythroid Leukemia (M6b), Recurrent Adult Acute Myeloid Leukemia, and Untreated Adult Acute Myeloid Leukemia.
50. The pharmaceutical composition according to claim 44 further comprising at least one additional therapeutic agent effective for treating or ameliorating the effects of the hematologic cancer.
51. The pharmaceutical composition according to claim 50, wherein the additional therapeutic agent is selected from the group consisting of an antibody or fragment thereof, a cytotoxic agent, a drug, a toxin, a radionuclide, an immunomodulator, a photoactive therapeutic agent, a radiosensitizing agent, a hormone, an anti-angiogenesis agent, and combinations thereof.
52. The pharmaceutical composition according to claim 51 , wherein the cytotoxic agent is selected from the group consisting of an alkylating agent, a platinum-based agent, an intercalating agent, an inhibitor of DNA replication, an antimetabolite, an anti-microtubule agent, an antibiotic agent, and combinations thereof.
53. The pharmaceutical composition according to claim 51 , wherein the additional therapeutic agent is selected from the group consisting of cyclophosphamide, mechlorethamine, uramustine, melphalan, chlorambucil, ifosfamide, carmustine, lomustine, streptozocin, busulfan, temozolomide, cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, triplatin tetranitrate, doxorubicin, daunorubicin, idarubicin, mitoxantrone, irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, methotrexate premetrexed, 6-mercaptopurine, dacarbazine, fludarabine, 5-fluorouracil, arabinosylcytosine, capecitabine, gemcitabine, decitabine, vinca alkaloids, paclitaxel, docetaxel, ixabepilone, actinomycin, anthracyclines, valrubicin, epirubicin, bleomycin, plicamycin, mitomycin, 2-methoxyestradiol, angiostatin, bevacizumab, cartilage-derived angiogenesis inhibitory factor, endostatin, IFN-a, IL-12, itraconazole, linomide, platelet factor-4, prolactin, SU5416, suramin, tasquinimod, tecogalan, tetrathiomolybdate, thalidomide, thrombospondin, thrombospondin, TNP-470, ziv-aflibercept, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof.
54. A kit for treating or ameliorating the effects of a hematologic cancer comprising a pharmaceutical composition according to any one of claims 33 or 44 packaged together with instructions for its use.
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