WO2015134603A2 - Procédés de traitement du cancer - Google Patents

Procédés de traitement du cancer Download PDF

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WO2015134603A2
WO2015134603A2 PCT/US2015/018724 US2015018724W WO2015134603A2 WO 2015134603 A2 WO2015134603 A2 WO 2015134603A2 US 2015018724 W US2015018724 W US 2015018724W WO 2015134603 A2 WO2015134603 A2 WO 2015134603A2
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mll
nucleic acid
leukemia
cancer
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WO2015134603A3 (fr
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Roy Macfarlane Pollock
Scott Richard Daigle
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Epizyme Inc
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Epizyme Inc
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    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates generally to the field of cancer treatment, and in particular, the treatment of leukemia.
  • DOT1L Disease-associated chromatin-modifying enzymes
  • the disclosure relates to methods that include diagnosing if a subject has a partial tandem duplication (PTD) of the MLL gene (MLL-PTD) and, if the subject has MLL-PTD, administering a therapeutically effective amount of a DOT1L inhibitor to the subject.
  • PTD partial tandem duplication
  • MLL-PTD MLL gene
  • the present invention provides a method that includes steps of (i) providing a nucleic acid sample from a biological sample obtained from a subject; (ii) detecting the presence of a chromosomal rearrangement, wherein the chromosomal rearrangement is MLL-PTD; (iii) identifying the subject as a candidate for treatment; and selecting a therapy that includes the administration of a therapeutically effective amount of a DOT1L inhibitor to the subject identified in step (iii).
  • the present invention also provides a method that includes the steps of (i) providing a nucleic acid sample from a biological sample obtained from a subject; (ii) contacting the nucleic acid sample with at least one primer that specifically hybridizes to a nucleic acid sequence of the MLL gene SEQ ID NO:61 and/or SEQ ID NO:62, a complement or a fragment thereof; (iii) amplifying at least a portion of the nucleic acid sequence of the MLL gene, a complement or a fragment thereof; (iv) detecting the presence of the chromosomal rearrangement MLL-PTD by detecting the presence of an amplified nucleic acid with molecular weight larger than the molecular weight of an amplified nucleic acid of the corresponding wild type MLL gene; (v) identifying the subject as a candidate for treatment; and (vi) selecting a therapy that includes the administration of a therapeutically effective amount of a DOT1L inhibitor to the subject identified in step (v).
  • the method further comprises administering a therapeutically effective amount of a DOT1L inhibitor to the subject.
  • the detecting step is carried out by polymerase chain reaction (PCR), nested PCR, real-time PCR or multiplex ligation dependent probe amplification.
  • PCR polymerase chain reaction
  • the detecting step comprises contacting the nucleic acid sample with at least one primer that specifically hybridizes to a nucleic acid sequence of the MLL gene SEQ ID NO:61 and/or SEQ ID NO:62, a complement or a fragment thereof.
  • the detecting step further comprises amplifying at least a portion of the nucleic acid sequence of the MLL gene, a complement or a fragment thereof.
  • the amplification is carried out by polymerase chain reaction (PCR), nested polymerase chain reaction (PCR), real-time PCR or multiplex ligation dependent probe amplification.
  • the primer is any one of SEQ ID NOs: 1-60.
  • the subject has a hematological cancer.
  • the subject had at least one prior therapy to treat a hematological cancer.
  • the hematological cancer is refractory to the prior therapy.
  • the hematological cancer shows recurrence following remission.
  • the subject received and failed all known effective therapies for the hematological cancer.
  • the hematological cancer is selected from the group consisting of acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, a myeloproliferative disorder, and chronic myelogenous leukemia.
  • the hematological cancer is leukemia.
  • the subject has a leukemia characterized by MLL gene rearrangement.
  • MLL gene rearrangement is a partial tandem duplication of the
  • the subject is simultaneously being treated with another therapy to treat acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, a myeloproliferative disorder, or chronic myelogenous leukemia.
  • another therapy is standard of care for the treatment of acute myeloid leukemia.
  • another therapy is standard of care for the treatment of acute lymphoblastic leukemia.
  • the DOT1L inhibitor used in any methods described herein is Compound A2 (also called EPZ-5676) having the formula:
  • Figures 1A and IB are diagrams showing mixed-lineage leukemia (ML ) chromosome: Figure 1 A is wild type and Figure IB is rearranged chromosome indicating a partial tandem duplication (PTD) of the MLL gene. "Old” and “new” indicate old and new numbering of exons, respectively.
  • PTD partial tandem duplication
  • FIG. 2 is a graph showing that EOL-1 (MLL-PTD + ) cells are sensitive to DOT1L inhibitors. Inhibition of EOL-1 proliferation (graph to the left) correlates with DOT1L inhibitor biochemical potency (table to the right).
  • Figure 3 is a graph demonstrating that DOT1L inhibitor EPZ-5676 (i.e., Compound A2) inhibits EOL-1 tumor growth treated by vehicle or by EPZ-5676 at various dosages
  • Figures 4A-4D are multiple graphs showing results of each individual experiment of each group shown in Figure 3.
  • Figure 4A shows results of each individual experiment for vehicle treated animals.
  • Figure 4B shows results of each individual experiment for EPZ-5676 treated animals (70mg/kg/day continuous infusion for 21 days).
  • Figure 4C shows results of each individual experiment for EPZ-5676 treated animals (35mg/kg/day continuous infusion for 21 days).
  • Figure 4D shows results of each individual experiment for EPZ-5676 treated animals (17.5mg/kg/day continuous infusion for 21 days).
  • Figure 5 is a graph showing body weight change of animals treated by vehicle or EPZ- 5676 at various dosages.
  • Figure 6 includes tables showing mean or median EPZ-5676 plasma levels in the treated animals at different time points.
  • Figures 7A-7D are multiple graphs demonstrating inhibition of H3K79 methylation in EOL-1 surrogate tissue.
  • EOL-1 tumored nude rats were treated with vehicle or with EPZ-5676 for 7 days at 17.5, 35, and 70 mg/kg/day by continuous infusion.
  • PBMCs and bone marrow were harvested for analysis.
  • Figure 7A shows H3K79me2 quantitative ELISA results of tumor tissue.
  • Figure 7B shows H3K79me2 quantitative ELISA results of bone marrow.
  • Figure 7C shows H3K79me2 quantitative Western Blot results of PBMCs.
  • Figure 7D shows EPZ-5676 plasma concentration.
  • Figures 8A-8F are graphs showing tumor target gene expression in EOL-1 tumors.
  • Figure 8A shows expression of HOXA9.
  • Figure 8B shows expression of MEIS2.
  • Figure 8C shows expression of TBP.
  • Figure 8D shows expression of BCL.
  • Figure 8E shows expression of MEIS1.
  • Figure 8F shows expression of FLT3.
  • Figures 9A-9C show examples of the MLL-PTD variants including their relative occurrence frequency. Exemplary PCR primer landing sites to detect the MLL-PTD variants are indicated in Figures 9A-9C as well.
  • Figure 9A shows exemplary primer landing sites for MLL- PTD variant MLL-PTD 9-3.
  • Figure 9B shows exemplary primer landing sites for MLL-PTD variant MLL-PTD 10-3.
  • Figure 9C shows exemplary primer landing sites for MLL-PTD variant MLL-PTD 11-3.
  • the present invention is based upon the discovery that cells having a partial tandem duplication (PTD) chromosome rearrangement of the mixed-lineage leukemia ⁇ MLL) gene are sensitive to DOT1L inhibitors.
  • PTD partial tandem duplication
  • MLL Mixed lineage leukemia
  • AML adult acute myeloid leukemia
  • MLL represents a particularly aggressive form of leukemia and patients with this disease generally have poor prognoses; these patients often suffer from early relapse after treatment with current chemotherapies.
  • MLL can be characterized by the genetic lesions of the mixed-lineage leukemia ⁇ MLL) gene.
  • Such genetic lesions include chromosomal rearrangements, such as translocations, deletions, and/or duplications of the MLL gene.
  • MLL has been categorized or characterized as having a chimeric fusion of MLL, partial tandem duplication of the MLL gene (MLL-PTD), or nonrearranged MLL.
  • MLL genetic alterations of the MLL gene are commonly identified in acute leukemias.
  • AML acute myeloid leukemia
  • PTD partial tandem duplication
  • the mutation leads to an in-frame duplication of exons 5 to 11 resulting in the production of an aberrant MLL protein.
  • Myeloid leukemia cells with MLL-PTD are sensitive to pharmacological inhibition of the H3K79 methytransferase DOT1L. See e.g., Kuhn et al., ASH Annual Meeting 2013; Poster Abstract, incorporated herein by reference. There is thus a great and present need for diagnosis and treatment modalities for patient suffering with MLL-PTD.
  • the present invention provides methods for treating or alleviating a symptom of cancer in a subject in need of by first detecting the presence of chromosomal rearrangement MLL-PTD in the subject.
  • the present invention provides methods for identifying a subject who is responsive to DOT1L inhibitor treatment by (i) providing a nucleic acid sample from a biological sample obtained from the subject; (ii) detecting the presence of a chromosomal rearrangement, wherein the chromosomal rearrangement is MLL-PTD; and (iii) identifying the subject as a candidate for treatment if MLL-PTD is detected.
  • the present invention provides methods that include steps of (i) providing a nucleic acid sample from a biological sample obtained from a subject; (ii) detecting the presence of a chromosomal rearrangement, wherein the chromosomal rearrangement is MLL- PTD; (iii) identifying the subject as a candidate for treatment; and (iv) selecting a therapy that includes the administration of a therapeutically effective amount of a DOT1L inhibitor to the subject identified in step (iii).
  • the methods further comprise administering a therapeutically effective amount of a DOT1L inhibitor to the subject identified in step (iii).
  • the nucleic acid sequence of the MLL gene (wild type) is SEQ ID NO: 61 (NM 001197104.1). In some embodiments, the nucleic acid sequence of the MLL gene (wild type) is SEQ ID NO: 62 (NM_005933.3).
  • KMT2A Homo sapiens lysine (K)-specific methyltransferase 2A (KMT2A), transcript variant 1, mRNA (SEQ ID NO: 61)
  • KMT2A Homo sapiens lysine (K) -specific methyltransferase 2A (KMT2A) , transcript variant 2, mRNA (SEQ ID NO: 62)
  • Detection of a MLL-PTD chromosomal rearrangement can be performed by any method known in the art. For example, PCR, reverse transcriptase PCR, real-time PCR, nested PCR, multiplex ligation dependent probe amplification (DNA-MLPA), FISH (fluorescence in situ hybridization) or DNA sequencing methods known in the art such as Sanger sequencing, de novo sequencing, shotgun sequencing, or next generation sequencing methods or any standard method for detecting translocations (such as utilizing sequence specific probes) may be used.
  • MLL-PTD is detected by real-time PCR.
  • PCR has been described in Dohner et al. J. Clin Oncol.
  • MLL-PTD variants include any partial tandem duplication (PTD) of MLL gene, including, but are not limited to, Exon9/Exon 3 duplication, ExonlO/Exon 3 duplication, Exon 11/Exon 3 duplication, Exon 12/Exon 3, Exon 9/Exon 5, Exon 8/Exon 3 and Exon 8+ ⁇ See e.g., Balgobind et al, European J. of Cancer 2010; 46: 1892-199; Caligiuri et al, Cancer Res 1996; 56: 1418-1425 (with old numbering), Dohner et al.
  • PTD partial tandem duplication
  • Figures 9A-9C show examples of the MLL- PTD variants including their relative occurrence (See also the references cited in this paragraph). Exemplary PCR primer landing sites to detect the MLL-PTD variants are indicated in Figure 9 as well. As indicated in Figure 1, there are old and new two numbering systems for the exons of the MLL gene.
  • the detecting step may comprise contacting the nucleic acid sample with at least one primer that specifically hybridizes to a nucleic acid sequence of the MLL gene, a complement or a fragment thereof.
  • the hybridization step is followed by an amplification step where the nucleic acid sequence of the MLL gene, a complement or a fragment thereof is amplified. The presence of an amplified nucleic acid with molecular weight larger than the molecular weight of an amplified nucleic acid of the corresponding wild type MLL gene indicates the presence of the chromosomal rearrangement MLL-PTD.
  • the presence of an amplified nucleic acid that comprises a duplicate of at least a portion of one or more exons of the MLL gene indicates the presence of the chromosomal rearrangement MLL-PTD.
  • the presence of an amplified nucleic acid that includes (from 5' to 3') at least a portion of a higher numbered exon followed by at least a portion of a lower numbered exon of the MLL gene indicates the presence of the chromosomal rearrangement MLL-PTD.
  • the present invention provides a method that includes the steps of (i) providing a nucleic acid sample from a biological sample obtained from a subject; (ii) contacting the nucleic acid sample with at least one primer that specifically hybridizes to a nucleic acid sequence of MLL gene, a complement or a fragment thereof; (iii) amplifying the nucleic acid sequence of MLL gene, a complement or a fragment thereof; (iv) detecting the presence of the chromosomal rearrangement MLL-PTD by detecting the presence of an amplified nucleic acid with molecular weight larger than the molecular weight of an amplified nucleic acid of the corresponding wild type MLL gene; and (vi) selecting a therapy that includes the administration of a therapeutically effective amount of a DOT1L inhibitor to the subject identified in step (v).
  • the presence of an amplified nucleic acid that comprises a duplicate of at least a portion of one or more exons of the MLL gene indicates the presence of the chromosomal rearrangement MLL-PTD in step (iv).
  • the presence of an amplified nucleic acid that include (from 5' to 3') at least a portion of a higher numbered exon followed by at least a portion of a lower numbered exon of the MLL gene indicates the presence of the
  • the method further comprises administering a therapeutically effective amount of a DOT1L inhibitor to the subject identified in step (v).
  • a "subject" or “subject in need thereof is a subject having cancer, hematologic cancer or leukemia or a subject having an increased risk of developing such disorder relative to the population at large.
  • the leukemia is acute myeloid leukemia, acute lymphoblastic leukemia, acute mixed lineage leukemia, myelodysplasia syndrome, a myeloproliferative disorder, or chronic myelogenous leukemia.
  • the subject has a leukemia involving MLL-PTD.
  • the subject is the subject is unable to receive other therapy to treat the cancer due to age or intercurring illness.
  • the subject is at least 50 years old, or at least 60 years old, or at least 65 years old, or at least 70 years old or older.
  • the subject in need thereof had at least one prior therapy to treat acute myeloid leukemia, acute lymphoblastic leukemia, acute mixed lineage leukemia, myelodysplasia syndrome, a myeloproliferative disorder, or chronic myelogenous leukemia.
  • the subject has refractory cancer.
  • Refractory cancer is a malignancy for which surgery is ineffective, which is either initially unresponsive to chemo- or radiation therapy, or which becomes unresponsive over time.
  • the subject has hematological cancer recurrence following remission.
  • the subject is not a candidate for allogeneic stem cell
  • the subject is simultaneously being treated with another therapy to treat acute myeloid leukemia, acute lymphoblastic leukemia, acute mixed lineage leukemia, myelodysplasia syndrome, a myeloproliferative disorder, or chronic myelogenous leukemia.
  • Additional therapies and agents that can be used to treat these cancers are known to the skilled artisan; and are described in U.S. Provisional Application No. 61/785,446, the contents of which are incorporated herein by reference in their entireties.
  • the patient is treated with the standard of care treatment as described in the most current National
  • NCN Comprehensive Cancer Network
  • AML such therapy may include all-trans retinoic acid (ATRA), cytaribine (Ara-C), daunorubicin, idarubicine, arsenic trioxide (ATO, 6-mercaptopurine and/or methotrexate.
  • ATRA all-trans retinoic acid
  • Ara-C cytaribine
  • daunorubicin idarubicine
  • ATO arsenic trioxide
  • 6-mercaptopurine 6-mercaptopurine
  • methotrexate 6-mercaptopurine
  • the standard of care may include vincristine, corticosteroids such as prednisone, and/or methotrexate.
  • the subject received and failed all known effective therapies for the hematological cancer that the subject has or is suffering from.
  • a subject in need thereof may have a secondary cancer as a result of a previous therapy.
  • Secondary cancer means cancer that arises due to or as a result from previous carcinogenic therapies, such as chemotherapy.
  • the secondary cancer is a hematologic cancer, such as leukemia.
  • the subject has increased mR A, protein, and/or activity level of at least one protein selected from the group consisting of HOXA9, FLT3, MEIS1, MEIS2, TBP, BCL, and DOTIL.
  • a "subject" includes a mammal.
  • the mammal can be e.g., any mammal, e.g., a human, primate, bird, mouse, rat, fowl, dog, cat, cow, horse, goat, camel, sheep or a pig.
  • the mammal is a human.
  • responsiveness is interchangeable with terms “responsive”, “sensitive”, and “sensitivity”, and it is meant that a subject is showing therapeutic responses when administered an DOTIL inhibitor, e.g., tumor cells or tumor tissues of the subject undergo apoptosis and/or necrosis, and/or display reduced growing, dividing, or proliferation.
  • an DOTIL inhibitor e.g., tumor cells or tumor tissues of the subject undergo apoptosis and/or necrosis, and/or display reduced growing, dividing, or proliferation.
  • the term "increase in activity" refers to increased or a gain of function of a gene product/protein compared to the wild type.
  • increased activity can be caused by increased mRNA and/or increased protein levels.
  • Increased mRNA levels can be caused by gene amplification and increased transcription, for example.
  • Increased protein levels can be caused by increased stability, inhibition of degradation pathways, or increased transcription.
  • increased activity levels can be caused by a gain of function mutation resulting from a point mutation (e.g.
  • a substitution, a missense mutation, or a nonsense mutation an insertion, and/or a deletion, or a rearrangement in a polypeptide selected from the group consisting of HOXA9, FLT3, MEIS1, MEIS2, TBP, BCL, and DOTIL, or a nucleic acid sequence encoding a polypeptide selected from the group consisting of HOXA9, FLT3, MEIS1, MEIS2, TBP, BCL, and DOTIL.
  • the mutations referred herein are somatic mutations.
  • the term "somatic mutation" refers to a deleterious alteration in at least one gene allele that is not found in every cell of the body, but is found only in isolated cells.
  • wild-type refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source.
  • a wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designed the "normal” or "wild-type” form of the gene.
  • an increase in mRNA or protein expression and/or activity levels can be detected using any suitable method available in the art.
  • an increase in activity level can be detected by measuring the biological function of a gene product, such as the histone methyltransferase activity of DOT1L (i.e., methylation of histone substrates such as H3K79 by immunoblot); transcriptional activity of HOXA9 or MEIS1 (i.e., expression levels of HOXA9 or MEIS1 target genes by RT-PCR); or phosphorylation activity of FLT3 (i.e., phosphorylation status of FLT3 targets by immunoblot or radioimmunoassay).
  • a gene product such as the histone methyltransferase activity of DOT1L (i.e., methylation of histone substrates such as H3K79 by immunoblot); transcriptional activity of HOXA9 or MEIS1 (i.e., expression levels of HOXA9 or MEIS1 target genes by RT
  • a gain of function mutation can be determined by detecting any alternation in a nucleic acid sequence encoding a protein selected from the group consisting of HOXA9, FLT3, MEIS1 and DOT1L.
  • a nucleic acid sequence encoding HOXA9, FLT3, MEIS1 and DOT1L having a gain of function mutation can be detected by whole-genome resequencing or target region resequencing (the latter also known as targeted resequencing) using suitably selected sources of DNA and polymerase chain reaction (PCR) primers in accordance with methods well known in the art.
  • PCR polymerase chain reaction
  • the method typically and generally entails the steps of genomic DNA purification, PCR amplification to amplify the region of interest, cycle sequencing, sequencing reaction cleanup, capillary electrophoresis, and/or data analysis.
  • the method may include the use of microarray-based targeted region genomic DNA capture and/or sequencing. Kits, reagents, and methods for selecting appropriate PCR primers and performing resequencing are commercially available, for example, from Applied Biosystems, Agilent, and NimbleGen (Roche Diagnostics GmbH).
  • Detection of mRNA expression can be detected by methods known in the art, such as Northern blot, nucleic acid PCR, and quantitative RT-PCR. Detection of polypeptide expression (i.e., wild-type or mutant) can be carried out with any suitable immunoassay in the art, such as Western blot analysis.
  • sample it means any biological sample derived from the subject, includes but is not limited to, cells, tissues samples, body fluids (including, but not limited to, mucus, blood, plasma, serum, urine, saliva, and semen), tumor cells, and tumor tissues.
  • the sample is selected from bone marrow, peripheral blood cells, blood, plasma and serum. Samples can be provided by the subject under treatment or testing. Alternatively samples can be obtained by the physician according to routine practice in the art.
  • “Risk” in the context of the present invention relates to the probability that an event will occur over a specific time period and can mean a subject's "absolute" risk or "relative” risk.
  • Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period.
  • Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no-conversion.
  • the DOT1L inhibitor used in any method described herein is Compound A2 (also called EPZ-5676) having the formula:
  • the DOT1L inhibitor used in any method described herein is Compound T (i.e., Compound D 16 or EPZ004777) having the formula
  • monotherapy refers to the administration of a single active or therapeutic compound to a subject in need thereof.
  • monotherapy will involve administration of a therapeutically effective amount of a single active compound.
  • cancer monotherapy with one of the compound of the present invention, or a pharmaceutically acceptable salt, to a subject in need of treatment of cancer.
  • the single active compound is a compound of the present invention, or a pharmaceutically acceptable salt.
  • treating or “treat” describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of a compound of the present invention, or a pharmaceutically acceptable salt, to alleviate the symptoms or complications of a disease, condition or disorder, or to eliminate the disease, condition or disorder.
  • a compound of the present invention can also be used to prevent a disease, condition or disorder.
  • preventing or “prevent” describes reducing or eliminating the onset of the symptoms or complications of the disease, condition or disorder.
  • the term "alleviate” is meant to describe a process by which the severity of a sign or symptom of a disorder is decreased. Importantly, a sign or symptom can be alleviated without being eliminated. In a preferred embodiment, the administration of
  • compositions of the invention leads to the elimination of a sign or symptom, however, elimination is not required.
  • Effective dosages are expected to decrease the severity of a sign or symptom. For instance, a sign or symptom of a disorder such as cancer, which can occur in multiple locations, is alleviated if the severity of the cancer is decreased within at least one of multiple locations.
  • severity is meant to describe the potential of cancer to transform from a precancerous, or benign, state into a malignant state. Alternatively, or in addition, severity is meant to describe a cancer stage, for example, according to the TNM system
  • Cancer stage refers to the extent or severity of the cancer, based on factors such as the location of the primary tumor, tumor size, number of tumors, and lymph node involvement (spread of cancer into lymph nodes).
  • Tumor grade is a system used to classify cancer cells in terms of how abnormal they look under a microscope and how quickly the tumor is likely to grow and spread. Many factors are considered when determining tumor grade, including the structure and growth pattern of the cells. The specific factors used to determine tumor grade vary with each type of cancer. Severity also describes a histologic grade, also called differentiation, which refers to how much the tumor cells resemble normal cells of the same tissue type (see, National Cancer Institute, www.cancer.gov).
  • severity describes a nuclear grade, which refers to the size and shape of the nucleus in tumor cells and the percentage of tumor cells that are dividing (see, National Cancer Institute, www.cancer.gov).
  • severity describes the degree to which a tumor has secreted growth factors, degraded the extracellular matrix, become vascularized, lost adhesion to juxtaposed tissues, or metastasized.
  • severity describes the number of locations to which a primary tumor has metastasized.
  • severity includes the difficulty of treating tumors of varying types and locations. For example, inoperable tumors, those cancers which have greater access to multiple body systems (hematological and immunological tumors), and those which are the most resistant to traditional treatments are considered most severe.
  • symptom is defined as an indication of disease, illness, injury, or that something is not right in the body. Symptoms are felt or noticed by the individual experiencing the symptom, but may not easily be noticed by others. Others are defined as non- health-care professionals.
  • signs are also defined as an indication that something is not right in the body. But signs are defined as things that can be seen by a doctor, nurse, or other health care professional.
  • Treating cancer can result in a reduction in size of a tumor.
  • a reduction in size of a tumor may also be referred to as "tumor regression".
  • tumor size is reduced by 5% or greater relative to its size prior to treatment; more preferably, tumor size is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30%> or greater; more preferably, reduced by 40%> or greater; even more preferably, reduced by 50%> or greater; and most preferably, reduced by greater than 75% or greater.
  • Size of a tumor may be measured by any reproducible means of measurement. The size of a tumor may be measured as a diameter of the tumor.
  • Treating cancer can result in a reduction in tumor volume.
  • tumor volume is reduced by 5% or greater relative to its size prior to treatment; more preferably, tumor volume is reduced by 10%> or greater; more preferably, reduced by 20%> or greater; more preferably, reduced by 30%> or greater; more preferably, reduced by 40%> or greater; even more preferably, reduced by 50%> or greater; and most preferably, reduced by greater than 75% or greater.
  • Tumor volume may be measured by any reproducible means of measurement.
  • Treating cancer results in a decrease in number of tumors.
  • tumor number is reduced by 5% or greater relative to number prior to treatment; more preferably, tumor number is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30%> or greater; more preferably, reduced by 40%> or greater; even more preferably, reduced by 50%> or greater; and most preferably, reduced by greater than 75%.
  • Number of tumors may be measured by any reproducible means of measurement.
  • the number of tumors may be measured by counting tumors visible to the naked eye or at a specified magnification.
  • the specified magnification is 2x, 3x, 4x, 5x, lOx, or 50x.
  • Treating cancer can result in a decrease in number of metastatic lesions in other tissues or organs distant from the primary tumor site.
  • the number of metastatic lesions is reduced by 5% or greater relative to number prior to treatment; more preferably, the number of metastatic lesions is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40%> or greater; even more preferably, reduced by 50%> or greater; and most preferably, reduced by greater than 75%.
  • the number of metastatic lesions may be measured by any reproducible means of measurement.
  • the number of metastatic lesions may be measured by counting metastatic lesions visible to the naked eye or at a specified magnification.
  • the specified magnification is 2x, 3x, 4x, 5x, lOx, or 50x.
  • Treating cancer can result in an increase in average survival time of a population of treated subjects in comparison to a population receiving carrier alone.
  • the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days.
  • An increase in average survival time of a population may be measured by any reproducible means.
  • An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound.
  • An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.
  • Treating cancer can result in an increase in average survival time of a population of treated subjects in comparison to a population of untreated subjects.
  • the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days.
  • An increase in average survival time of a population may be measured by any reproducible means.
  • An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound.
  • An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.
  • Treating cancer can result in increase in average survival time of a population of treated subjects in comparison to a population receiving monotherapy with a drug that is not a compound of the present invention, or a pharmaceutically acceptable salt thereof.
  • the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days.
  • An increase in average survival time of a population may be measured by any reproducible means.
  • An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound.
  • An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.
  • Treating cancer can result in a decrease in the mortality rate of a population of treated subjects in comparison to a population receiving carrier alone. Treating cancer can result in a decrease in the mortality rate of a population of treated subjects in comparison to an untreated population. Treating cancer can result in a decrease in the mortality rate of a population of treated subjects in comparison to a population receiving monotherapy with a drug that is not a compound of the present invention, or a pharmaceutically acceptable salt thereof.
  • the mortality rate is decreased by more than 2%; more preferably, by more than 5%; more preferably, by more than 10%; and most preferably, by more than 25%.
  • a decrease in the mortality rate of a population of treated subjects may be measured by any reproducible means.
  • a decrease in the mortality rate of a population may be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following initiation of treatment with an active compound.
  • a decrease in the mortality rate of a population may also be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following completion of a first round of treatment with an active compound.
  • Treating cancer can result in a decrease in tumor growth rate.
  • tumor growth rate is reduced by at least 5% relative to number prior to treatment; more preferably, tumor growth rate is reduced by at least 10%; more preferably, reduced by at least 20%>; more preferably, reduced by at least 30%>; more preferably, reduced by at least 40%>; more preferably, reduced by at least 50%>; even more preferably, reduced by at least 50%>; and most preferably, reduced by at least 75%.
  • Tumor growth rate may be measured by any reproducible means of measurement. Tumor growth rate can be measured according to a change in tumor diameter per unit time.
  • Treating cancer can result in a decrease in tumor regrowth.
  • tumor regrowth is less than 5%; more preferably, tumor regrowth is less than 10%; more preferably, less than 20%; more preferably, less than 30%>; more preferably, less than 40%>; more preferably, less than 50%>; even more preferably, less than 50%>; and most preferably, less than 75%.
  • Tumor regrowth may be measured by any reproducible means of measurement. Tumor regrowth is measured, for example, by measuring an increase in the diameter of a tumor after a prior tumor shrinkage that followed treatment. A decrease in tumor regrowth is indicated by failure of tumors to reoccur after treatment has stopped.
  • Treating or preventing a cell proliferative disorder can result in a reduction in the rate of cellular proliferation.
  • the rate of cellular proliferation is reduced by at least 5%; more preferably, by at least 10%>; more preferably, by at least 20%>; more preferably, by at least 30%>; more preferably, by at least 40%>; more preferably, by at least 50%>; even more preferably, by at least 50%>; and most preferably, by at least 75%.
  • the rate of cellular proliferation may be measured by any reproducible means of measurement.
  • the rate of cellular proliferation is measured, for example, by measuring the number of dividing cells in a tissue sample per unit time.
  • Treating or preventing a cell proliferative disorder can result in a reduction in the proportion of proliferating cells.
  • the proportion of proliferating cells is reduced by at least 5%; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%.
  • the proportion of proliferating cells may be measured by any reproducible means of measurement.
  • the proportion of proliferating cells is measured, for example, by quantifying the number of dividing cells relative to the number of nondividing cells in a tissue sample.
  • the proportion of proliferating cells can be equivalent to the mitotic index.
  • Treating or preventing a cell proliferative disorder can result in a decrease in size of an area or zone of cellular proliferation.
  • size of an area or zone of cellular proliferation is reduced by at least 5% relative to its size prior to treatment; more preferably, reduced by at least 10%; more preferably, reduced by at least 20%>; more preferably, reduced by at least 30%>; more preferably, reduced by at least 40%>; more preferably, reduced by at least 50%>; even more preferably, reduced by at least 50%>; and most preferably, reduced by at least 75%.
  • Size of an area or zone of cellular proliferation may be measured by any reproducible means of measurement.
  • the size of an area or zone of cellular proliferation may be measured as a diameter or width of an area or zone of cellular proliferation.
  • Treating or preventing a cell proliferative disorder can result in a decrease in the number or proportion of cells having an abnormal appearance or morphology.
  • the number of cells having an abnormal morphology is reduced by at least 5% relative to its size prior to treatment; more preferably, reduced by at least 10%>; more preferably, reduced by at least 20%>; more preferably, reduced by at least 30%>; more preferably, reduced by at least 40%>; more preferably, reduced by at least 50%>; even more preferably, reduced by at least 50%>; and most preferably, reduced by at least 75%.
  • An abnormal cellular appearance or morphology may be measured by any reproducible means of measurement.
  • An abnormal cellular morphology can be measured by microscopy, e.g., using an inverted tissue culture microscope.
  • An abnormal cellular morphology can take the form of nuclear pleiomorphism.
  • the term "selectively" means tending to occur at a higher frequency in one population than in another population.
  • the compared populations can be cell populations.
  • a compound of the present invention, or a pharmaceutically acceptable salt thereof acts selectively on a cancer or precancerous cell but not on a normal cell.
  • a compound of the present invention, or a pharmaceutically acceptable salt thereof acts selectively to modulate one molecular target ⁇ e.g., a target protein methyltransferase) but does not significantly modulate another molecular target ⁇ e.g., a non-target protein methyltransferase).
  • the invention also provides a method for selectively inhibiting the activity of an enzyme, such as a protein methyltransferase.
  • an event occurs selectively in population A relative to population B if it occurs greater than two times more frequently in population A as compared to population B.
  • An event occurs selectively if it occurs greater than five times more frequently in population A.
  • An event occurs selectively if it occurs greater than ten times more frequently in population A; more preferably, greater than fifty times; even more preferably, greater than 100 times; and most preferably, greater than 1000 times more frequently in population A as compared to population B.
  • cell death would be said to occur selectively in cancer cells if it occurred greater than twice as frequently in cancer cells as compared to normal cells.
  • a compound of the present invention, or a pharmaceutically acceptable salt thereof can modulate the activity of a molecular target (e.g., a target protein methyltransferase). Modulating refers to stimulating or inhibiting an activity of a molecular target.
  • a compound of the present invention, or a pharmaceutically acceptable salt thereof modulates the activity of a molecular target if it stimulates or inhibits the activity of the molecular target by at least 2-fold relative to the activity of the molecular target under the same conditions but lacking only the presence of said compound.
  • a compound of the present invention modulates the activity of a molecular target if it stimulates or inhibits the activity of the molecular target by at least 5 -fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold relative to the activity of the molecular target under the same conditions but lacking only the presence of said compound.
  • the activity of a molecular target may be measured by any reproducible means.
  • the activity of a molecular target may be measured in vitro or in vivo.
  • the activity of a molecular target may be measured in vitro by an enzymatic activity assay or a DNA binding assay, or the activity of a molecular target may be measured in vivo by assaying for expression of a reporter gene.
  • a compound of the present invention does not significantly modulate the activity of a molecular target if the addition of the compound does not stimulate or inhibit the activity of the molecular target by greater than 10% relative to the activity of the molecular target under the same conditions but lacking only the presence of said compound.
  • the term "isozyme selective" means preferential inhibition or stimulation of a first isoform of an enzyme in comparison to a second isoform of an enzyme (e.g., preferential inhibition or stimulation of a protein methyltransferase isozyme alpha in comparison to a protein methyltransferase isozyme beta).
  • a compound of the present invention, or a pharmaceutically acceptable salt thereof demonstrates a minimum of a fourfold differential, preferably a tenfold differential, more preferably a fifty fold differential, in the dosage required to achieve a biological effect.
  • a compound of the present invention demonstrates this differential across the range of inhibition, and the differential is exemplified at the IC 50 , i.e., a 50% inhibition, for a molecular target of interest.
  • Treating cancer or a cell proliferative disorder can result in cell death, and preferably, cell death results in a decrease of at least 10% in number of cells in a population. More preferably, cell death means a decrease of at least 20%; more preferably, a decrease of at least 30%; more preferably, a decrease of at least 40%>; more preferably, a decrease of at least 50%>; most preferably, a decrease of at least 75%.
  • Number of cells in a population may be measured by any reproducible means. A number of cells in a population can be measured by fluorescence activated cell sorting (FACS), immunofluorescence microscopy and light microscopy. Methods of measuring cell death are as shown in Li et al., Proc Natl Acad Sci USA. 100(5): 2674-8, 2003. In an aspect, cell death occurs by apoptosis.
  • an effective amount of a compound of the present invention or a
  • a therapeutically effective amount of a compound is not significantly cytotoxic to normal cells if administration of the compound in a therapeutically effective amount does not induce cell death in greater than 10% of normal cells.
  • a therapeutically effective amount of a compound does not significantly affect the viability of normal cells if administration of the compound in a therapeutically effective amount does not induce cell death in greater than 10% of normal cells. In an aspect, cell death occurs by apoptosis.
  • a pharmaceutically acceptable salt thereof can induce or activate cell death selectively in cancer cells.
  • Contacting a cell with a compound of the present invention, or a pharmaceutically acceptable salt thereof can induce cell death selectively in one or more cells affected by a cell proliferative disorder.
  • administering to a subject in need thereof a compound of the present invention, or a pharmaceutically acceptable salt thereof induces cell death selectively in one or more cells affected by a cell proliferative disorder.
  • the present invention relates to a method of treating or preventing cancer by
  • a compound of the present invention or a pharmaceutically acceptable salt thereof, to a subject in need thereof, where administration of the compound of the present invention, or a pharmaceutically acceptable salt thereof, results in one or more of the following: accumulation of cells in Gl and/or S phase of the cell cycle, cytotoxicity via cell death in cancer cells without a significant amount of cell death in normal cells, antitumor activity in animals with a therapeutic index of at least 2, and activation of a cell cycle checkpoint.
  • therapeutic index is the maximum tolerated dose divided by the efficacious dose.
  • the present invention relates to use of the compounds disclosed herein in preparation of a medicament for treating or preventing cancer.
  • the cancer is leukemia. More preferably, the cancer is acute myeloid leukemia, acute lymphocytic leukemia or mixed lineage leukemia.
  • the present invention also provides pharmaceutical compositions comprising a compound disclosed herein in combination with at least one pharmaceutically acceptable excipient or carrier.
  • a "pharmaceutical composition” is a formulation containing the compounds of the present invention in a form suitable for administration to a subject.
  • the pharmaceutical composition is in bulk or in unit dosage form.
  • the unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler or a vial.
  • the quantity of active ingredient (e.g., a formulation of the disclosed compound or salt, hydrate, solvate or isomer thereof) in a unit dose of composition is an effective amount and is varied according to the particular treatment involved.
  • active ingredient e.g., a formulation of the disclosed compound or salt, hydrate, solvate or isomer thereof
  • the dosage will also depend on the route of administration.
  • routes of administration A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalational, buccal, sublingual, intrapleural, intrathecal, intranasal, and the like.
  • Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.
  • the phrase "pharmaceutically acceptable” refers to those compounds, materials, compositions, carriers, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use as well as human pharmaceutical use.
  • a “pharmaceutically acceptable excipient” as used in the specification and claims includes both one and more than one such excipient.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral ⁇ e.g., inhalation), transdermal (topical), and transmucosal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens;
  • antioxidants such as ascorbic acid or sodium bisulfite
  • chelating agents such as
  • ethylenediaminetetraacetic acid ethylenediaminetetraacetic acid
  • buffers such as acetates, citrates or phosphates
  • agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • a compound or pharmaceutical composition of the invention can be administered to a subject in many of the well-known methods currently used for chemotherapeutic treatment.
  • a compound of the invention may be injected directly into tumors, injected into the blood stream or body cavities or taken orally or applied through the skin with patches.
  • the dose chosen should be sufficient to constitute effective treatment but not as high as to cause unacceptable side effects.
  • the state of the disease condition e.g., cancer, precancer, and the like
  • the health of the patient should preferably be closely monitored during and for a reasonable period after treatment.
  • the term "therapeutically effective amount”, as used herein, refers to an amount of a pharmaceutical agent to treat, ameliorate, or prevent an identified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect.
  • the effect can be detected by any assay method known in the art.
  • the precise effective amount for a subject will depend upon the subject's body weight, size, and health; the nature and extent of the condition; and the therapeutic selected for administration.
  • Therapeutically effective amounts for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician.
  • the disease or condition to be treated is cancer.
  • the disease or condition to be treated is a cell proliferative disorder.
  • the therapeutically effective amount can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually rats, mice, rabbits, dogs, or pigs.
  • the animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • Therapeutic/prophylactic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED 50 (the dose therapeutically effective in 50% of the population) and LD 50 (the dose lethal to 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD 50 /ED 50 .
  • Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The dosage may vary within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • Dosage and administration are adjusted to provide sufficient levels of the active agent(s) or to maintain the desired effect.
  • Factors which may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug interaction(s), reaction sensitivities, and
  • Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.
  • compositions containing active compounds of the present invention may be manufactured in a manner that is generally known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • Pharmaceutical compositions may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and/or auxiliaries that facilitate processing of the active compounds into preparations that can be used pharmaceutically. Of course, the appropriate formulation is dependent upon the route of administration chosen.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol and sorbitol, and sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible pharmaceutically acceptable carrier.
  • compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser, which contains a suitable propellant, e.g. , a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g. , a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the active compounds can be prepared with pharmaceutically acceptable carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved.
  • the dosages of the pharmaceutical compositions used in accordance with the invention vary depending on the agent, the age, weight, and clinical condition of the recipient patient, and the experience and judgment of the clinician or practitioner administering the therapy, among other factors affecting the selected dosage. Generally, the dose should be sufficient to result in slowing, and preferably regressing, the growth of the tumors and also preferably causing complete regression of the cancer. Dosages can range from about 0.01 mg/kg per day to about 5000 mg/kg per day. In preferred aspects, dosages can range from about 1 mg/kg per day to about 1000 mg/kg per day.
  • the dose will be in the range of about 0.1 mg/day to about 50 g/day; about 0.1 mg/day to about 25 g/day; about 0.1 mg/day to about 10 g/day; about 0.1 mg to about 3 g/day; or about 0.1 mg to about 1 g/day, in single, divided, or continuous doses (which dose may be adjusted for the patient's weight in kg, body surface area in m 2 , and age in years).
  • An effective amount of a pharmaceutical agent is that which provides an objectively identifiable improvement as noted by the clinician or other qualified observer. For example, regression of a tumor in a patient may be measured with reference to the diameter of a tumor. Decrease in the diameter of a tumor indicates regression. Regression is also indicated by failure of tumors to reoccur after treatment has stopped.
  • the term "dosage effective manner" refers to amount of an active compound to produce the desired biological effect in a subject or cell.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • compositions of the present invention are capable of further forming salts. All of these forms are also contemplated within the scope of the claimed invention.
  • pharmaceutically acceptable salts refer to derivatives of the compounds of the present invention wherein the parent compound is modified by making acid or base salts thereof.
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like.
  • the pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • such conventional non-toxic salts include, but are not limited to, those derived from inorganic and organic acids selected from 2- acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic, benzene sulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethane disulfonic, 1,2-ethane sulfonic, fumaric,
  • amine acids
  • compositions include hexanoic acid, cyclopentane propionic acid, pyruvic acid, malonic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4- chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-l-carboxylic acid, 3-phenylpropionic acid,
  • the present invention also encompasses salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g. , an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.
  • a metal ion e.g. , an alkali metal ion, an alkaline earth ion, or an aluminum ion
  • coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.
  • the compounds, or pharmaceutically acceptable salts thereof are administered orally, nasally, transdermally, pulmonary, inhalationally, buccally, sublingually, intraperintoneally, subcutaneously, intramuscularly, intravenously, rectally, intrapleurally, intrathecally and parenterally.
  • the compound is administered orally.
  • One skilled in the art will recognize the advantages of certain routes of administration.
  • the dosage regimen utilizing the compounds is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed.
  • An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter, or arrest the progress of the condition.
  • Suitable pharmaceutically acceptable carriers include inert solid fillers or diluents and sterile aqueous or organic solutions.
  • the compounds will be present in such pharmaceutical compositions in amounts sufficient to provide the desired dosage amount in the range described herein.
  • EOL-1 Human leukemia cell line EOL-1 (Catalog # ACC-386) was purchased from DSMZ and were grown in Roswell Park Memorial Institute medium (RPMI) with 10% Fetal Bovine Serum (FBS). Cells were kept in log growth as outlined in the technical data sheet provided by the vendor.
  • RPMI Roswell Park Memorial Institute medium
  • FBS Fetal Bovine Serum
  • EOL-1 cells were plated in 96-well plates at a density of 3 X 10 4 viable cells/well. Each treatment was seeded in triplicate with a final well volume of 150 ⁇ ⁇ . Cells were incubated with increasing concentrations of DOT1L inhibitor up to50 ⁇ . Viable cell number was determined every 3 - 4 days for 11 days using the Guava Viacount assay (Millipore # 4000-0040) and analyzed on a Guava EasyCyte Plus instrument according to the manufacturer's protocol. On the days of cell counts, growth media and inhibitor were
  • Frozen pellets were allowed to thaw briefly on ice and then lysed by a 5 minute incubation on ice with 250 ⁇ nuclear extraction buffer (10 mM Tris-HCl, pH 7.6, 10 mM MgCl 2 , 25 mM KC1, 1% Triton X-100, 8.6%> Sucrose, plus a Roche protease inhibitor tablet
  • EOL-1 cells For immunoblot analysis of the H3K79me2 inhibition by EPZ-5676, exponentially growing EOL-1 cells were seeded at 2> ⁇ 10 5 cells/mL and incubated in the presence of increasing concentrations of EPZ-5676 for 4 days. Following incubation, cells (2-3 x lO 6 ) were harvested and histones extracted as described. Histones (400 ng) were fractionated on a 10-20% Tris HC1 gels (Bio-Rad) with Tris-Glycine SDS running buffer (Invitrogen) under denaturing conditions and transferred to a nitrocellulose filter.
  • Tris HC1 gels Bio-Rad
  • Tris-Glycine SDS running buffer Invitrogen
  • the filter was incubated for 1 hour in blocking buffer (Odyssey blocking buffer, Li-cor, 927-40000) at RT and then incubated overnight at 4°C in blocking buffer containing a antibody specific for H3K79me2 (1 :5000 dilution, abeam ab3594). Filters were washed 3 times for 5 minutes with wash buffer (PBST) and incubated with infrared tagged secondary antibody (Alexa Flour 680 goat anti-rabbit IgG (1 :20,000), Invitrogen A- 21076) at RT for 1 hour.
  • blocking buffer Odyssey blocking buffer, Li-cor, 927-40000
  • Filters were washed in PBST and reprobed for 1 hour at RT with the appropriate total histone antibody control (mouse anti-histone H3 (1 :20,000), CST 3638, or mouse anti-histone H4 (1 : 10,000), CST 2935). Filters were washed again in PBST and incubated with infrared tagged secondary antibody (IRDye 800Cw donkey- anti-mouse IgG (1 :20,000), Li- Cor 926-32212) at RT for 1 hour. After a final wash in PBST, filters were scanned using the Odyssey infared imager (Li-cor). Signal intensities specific for each methyl-specific antibody was quantified using Odyssey software and normalized to that of the appropriate total histone control signal on the same filter by dividing the methyl-specific antibody signal intensity by the total histone control signal intensity.
  • EOL-1 cells were plated in a 12 well plate at 2> ⁇ 10 5 cells/mL. Cells were incubated in the presence of increasing concentrations of EPZ-5676 up to 10 ⁇ . On day 4, cells were maintained in log phase culture by reseeding at 5x 10 5 cells/mL and compound was replenished. At day 6 cells were washed twice with PBS and pelleted by centrifugation at 200 X g. Cell pellets were lysed in 300 RLT buffer (Qiagen) and total RNA was isolated using the RNeasy total RNA isolation kit (Qiagen 74106).
  • RNA (1 ⁇ g) was reverse transcribed using a high capacity cDNA reverse transcription kit (Applied Biosystems 4368813). RNA isolation and cDNA synthesis were carried out according to the manufacturer's protocol. Predesigned labeled primer and probe sets ⁇ ⁇ 9 (Hs00365956), MEISI (HsOO 180020) and FLT3 (Hs00975659) were purchased from Applied Biosystems. Quantitative real-time PCR (qPCR) reactions contained 50 ng cDNA, IX labeled primer and probe set, and IX Taqman universal PCR master mix (Applied Biosystems 4304437).
  • EOL-1 (1 X 10 7 ) cells resuspended in 100% Matrigel (BD Biosciences) were implanted subcutaneously into female athymic nude rats (rnu/rnu, Harlan). Tumors were measured by calipers and rats were randomized according to tumor size into treatment groups before the initiation of dosing with EPZ-5676.
  • EPZ-5676 was delivered by IV infusion into the femoral vein continuously for 21 days in 5% hydroxypropyl-P-cyclodextrin (HPBCD) in saline.
  • HPBCD 5% hydroxypropyl-P-cyclodextrin
  • the dosing rate was fixed at 7 mL/animal/day in all groups, and dosing concentrations were adjusted for the last recorded weights of individual animals.
  • 10 animals were randomized into each treatment group when tumor volumes reached approximately 500 mm 3 . Rats were weighed and tumors measured with calipers twice weekly until the end of the study. Each test animal was euthanized when its neoplasm reached the predetermined endpoint volume or on the last day of the study, whichever came first. Optimal tumor volume endpoints were chosen based the growth characteristics of the EOL-1 xenograft model in pilot studies. Significance of treatment compared to control was measured using the repeated measures ANOVA with Dunnet post test versus the vehicle treated group.
  • blood samples (0.2 mL) were drawn from the saphaneous vein, flash frozen and subsequently analysed for EPZ-5676 levels by HPLC/MS/MS.
  • 5 animals were randomized in each treatment group when tumors volumes reached approximately 1000 mm 3 and then animals were dosed by continuous IV infusion for 7 days. On day 7 animals were euthanized and tumors, peripheral blood mononuclear cells and bone marrow cells were isolated and flash frozen.
  • tissue isolation and processing for PBMC whole cell lysates, tumor and bone marrow histones and tumor RNA can be found below.
  • Rats were euthanized at the end of infusion by terminal cardiac puncture under C0 2 anesthesia and sampled for full blood volume.
  • PBMCs were isolated from whole blood by centrifugation over Ficoll-Paque Plus (GE Healthcare) and treatment with ACK lysis buffer (Gibco).
  • PBMC pellets were flash frozen and stored at -80 °C.
  • PBMC whole cell lysates were prepared by thawing pellets and adding 50 ⁇ , Tris-Glycine SDS protein sample buffer
  • Rats were euthanized as described above and tumors were harvested, snap frozen in liquid nitrogen, pulverized using a mortar and pestle and stored at -80°C. 30 mg of tumor powder was lysed in 500 ⁇ . nuclear extraction buffer (10 mM Tris-HCl, pH 7.6, 10 mM MgCl 2 , 25 mM KC1, 1% Triton X-100, 8.6% Sucrose, plus a Roche protease inhibitor tablet 1836145). After 5 minutes in extraction buffer the samples were homogenized using a handheld homogenizer (Cole Parmer, R-04727-11).
  • Rats were euthanized as described above and, bone marrow cells were flushed from rat tibia, femur and hip bones using ice cold PBS. Cell pellets were flash frozen and stored at -80 °C. Histones were extracted from cell pellets following lysis in 500 ⁇ of nuclear extraction buffer (10 mM Tris-HCl, 10 mM MgC12, 25 mM KC1, 1% Triton X-100, 8.6% Sucrose, plus a Roche protease inhibitor tablet 1836145). Nuclei were collected by centrifugation at 600 g for 5 minutes at 4° C and washed once in TE buffer (pH 7.4).
  • Rats were euthanized and tumors were collected and powdered as described above. 10 mg of powdered tumor was lysed in 600 ⁇ , RLTlysis buffer (Qiagen) and homogenized in a TissueLyser (Qiagen, 85210). Supernatant was collected and total RNA was isolated using the RNeasy Total RNA isolation kit (Qiagen 74106) according to manufacturer's instructions.
  • Step 1 obtaining a biological sample from a subject.
  • Any biological sample derived from the subject can be used in the assay.
  • cells, tissues samples, body fluids including, but not limited to, mucus, blood, plasma, serum, urine, saliva, and semen
  • tumor cells and tumor tissues.
  • the sample is selected from bone marrow, peripheral blood cells, blood, plasma and serum. Samples can be provided by the subject under treatment or testing. Alternatively samples can be obtained by the physician according to routine practice in the art.
  • Step 2 isolating a nucleic acid sample (DNA or mRNA) from the biological sample obtained from a subject according to any known method in the art.
  • Step 3 carrying out PCR, real time PCR, nested PCR or multiplex ligation dependent probe amplification assay according to the standard protocol available in the art utilizing the isolated nucleic acid as the template and one or more primers (SEQ ID NO: 1-60) described herein.
  • Step 4 determining the molecular weight (size) of the amplified nucleic acid by, for example, running an electrophoresis gel. If the molecular weight of at least some of the amplified nucleic acid is larger than the molecular weight of an amplified nucleic acid of the corresponding wild type MLL gene, the tested subject has chromosomal rearrangement MLL- PTD and could be treated with a DOT1L inhibitor (e.g., Compound A2).
  • DOT1L inhibitor e.g., Compound A2

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Abstract

La présente invention concerne des procédés qui consistent à diagnostiquer si un sujet présente un MLL-PTD et, si le sujet présente un MLL-PTD, à lui administrer une quantité thérapeutiquement efficace d'un inhibiteur de DOT1L.
PCT/US2015/018724 2014-03-05 2015-03-04 Procédés de traitement du cancer Ceased WO2015134603A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016090271A1 (fr) * 2014-12-05 2016-06-09 Epizyme, Inc. Traitement de la leucémie par administration de l'inhibiteur de dot1l pinometostat
WO2017077326A1 (fr) * 2015-11-05 2017-05-11 King's College London Combinaison d'un inhibiteur de parp avec un inhibiteur de gsk-3 ou dot1l
US11633420B2 (en) 2012-09-06 2023-04-25 Epizyme, Inc. Method of treating leukemia

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* Cited by examiner, † Cited by third party
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US20030096255A1 (en) * 1997-02-19 2003-05-22 Felix Carolyn A. Methods and kits for analysis of chromosomal rearrangements associated with cancer
US7442685B2 (en) * 2003-06-13 2008-10-28 The University Of North Carolina At Chapel Hill DOT1 histone methyltransferases as a target for identifying therapeutic agents for leukemia
WO2010017515A2 (fr) * 2008-08-08 2010-02-11 Integrated Diagnostics Inc. Marqueurs spécifiques du cancer du sein et procédés d’utilisation
US8895526B2 (en) * 2009-03-27 2014-11-25 Cold Spring Harbor Laboratory Identification of RNAI targets and use of RNAI for rational therapy of chemotherapy-resistant leukemia and other cancers

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11633420B2 (en) 2012-09-06 2023-04-25 Epizyme, Inc. Method of treating leukemia
WO2016090271A1 (fr) * 2014-12-05 2016-06-09 Epizyme, Inc. Traitement de la leucémie par administration de l'inhibiteur de dot1l pinometostat
WO2017077326A1 (fr) * 2015-11-05 2017-05-11 King's College London Combinaison d'un inhibiteur de parp avec un inhibiteur de gsk-3 ou dot1l
US10799501B2 (en) 2015-11-05 2020-10-13 King's College Hospital Nhs Foundation Trust Combination of an inhibitor of PARP with an inhibitor of GSK-3 or DOT1L

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