leukemia compositions and treatment methods

专利摘要:
PHARMACEUTICAL COMPOSITION AND CASE UNDERSTANDING AN AGENT THAT INHIBITS BRD4, USES OF THAT AGENT, METHOD OF DETECTION OF THE LEUKEMIC CELL RESPONSE RESPONSE, AND METHOD OF TREATMENT REGIME SELECTION.The present invention provides compositions, methods, and kits for the treatment of acute myeloid leukemia in a subject.
公开号:BR112012029057A2
申请号:R112012029057-6
申请日:2011-05-16
公开日:2020-10-13
发明作者:Johannes Zuber；Junwei Shi；Christopher R. Vakoc
申请人:Dana-Farber Cancer Institute, Inc.；Cold Spring Harbor Laboratory；
IPC主号:

专利说明:

: Invention Patent Descriptive Report for "COMPOSITIONS AND METHODS OF TREATING LEUKEMIA",
CROSS REFERENCE TO RELATED REQUESTS This claim claims the benefit of U.S. Provisional Orders No. 61 / 334,991, filed on May 14, 2010; 61 / 370,745, registered on August 4, 2010; 61 / 375,863, registered on August 22, 2010; 61 / 467,376, registered on March 24, 2011; and 61 / 467,342, registered on March 24, 2011. The content of these requests is incorporated herein by reference in its entirety.
AFFIRMATION OF RIGHTS TO INVENTIONS - UNDER RESEARCH SPONSORED BY THE FEDERAL VIA 7 This work was sponsored by the following grant from the National Institutes of Health, Grant No.: KO8CA128972. The government has certain rights in the invention.
BACKGROUND OF THE INVENTION Acute Myeloid Leukemia (AML) represents a paradigm for understanding how complex patterns of cooperative genetic and epigenetic changes lead to tumorigenesis. This complexity poses a challenge to the development of targeted therapy, and several genetic mutations of AML generally converge in a functional way in the deregulation of similar nucleus cellular processes. A key event in the initiation of AML is the corruption of programs of cellular fate, so that they generate Leukemic Stem Cells (LSCs) that openly self-renew and thus maintain and spread the disease. Despite being incompletely understood, this process was linked to changes in regulatory chromatin modifications, whose impact on gene expression is well characterized. Thus, common oncogenes in AML, such as the A-ML1-ETO and MLL fusion proteins, induce self-renewal programs, at least in part, through the reprogramming of epigenetic pathways. Several epigenetic regulators are targets of somatic mutation. Since the changes ER
: epigenetics induced by oncogenic stimuli are potentially reversible, chromatin regulators have been explored as targets for candidate drugs.
SUMMARY OF THE INVENTION The invention provides compositions, methods, and kits for the detection and treatment of leukemia and related disorders (e.g., acute myeloid leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL) , Chronic Myeloid Leukemia (CML), Chronic Myelomonocytic Leukemia (CMML), Eosinophilic Leukemia, Hairy Cell Leukemia, Hodgkin's Lymphoma, Multiple Myeloma, Non-Hodgkin's Lymphoma, - Myeloproliferative or Syndromesmal Syndrome. f In one aspect, the invention generally provides a method of treating a leukemia or related disorder (for example, acute myeloid leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), Chronic Myeloid Leukemia (CML), Chronic Myelomococcal Leukemia (CMML), Eosinophilic Leukemia, Hair Cell Leukemia, Hodgkin's Lymphoma, Multiple Myeloma, Non-Hodgkin's Lymphoma, Myeloproliferative Disorders or Syndromes in syndromes; wherein the method involves administering to the subject an effective amount of an agent that inhibits Brd4 (e.g., an inhibitory nucleic acid that selectively addresses Brd4, JQ1) or a derivative thereof.
In another aspect, the invention provides a method of reducing the growth, proliferation or survival of a leukemic cell, in which the method involves contacting the cell with an effective amount of an agent that inhibits Brd4 or a derivative thereof, thereby reducing the growth, proliferation or survival of a leukemic cell.
In yet another aspect, the invention provides a method of inducing cell death or terminal differentiation in a leukemic cell, wherein the method comprises contacting the cell with an effective amount of an agent that inhibits Brd4 or a derivative thereof, thereby inducing cell death or terminal differentiation in the leukemic cell.
In yet another aspect, the invention provides a method of treating PE
; acute myeloid leukemia in a subject, where the method involves administering to a needy subject an effective amount of an agent that inhibits Brd4, thereby treating acute myeloid leukemia in a subject.
In yet another aspect, the invention provides a pharmaceutical composition containing a therapeutically effective amount of an agent that inhibits Brd4 or a derivative thereof in a pharmaceutically effective excipient.
In yet another aspect, the invention provides a kit for the treatment of leukemia, in which the kit contains a therapeutically effective amount of an agent that inhibits Brd4, and written instructions for administering the compound for use in the method as defined in claim 8.
In yet another aspect, the invention provides a method of detecting the clinical response capacity of a leukemic cell, in which the method involves contacting a leukemic cell with a Brd4 inhibitory agent, or its derivative, and detecting the expression in the cell of a specific differentiation marker for macrophages, where an increase in the expression of the specific differentiation marker for macrophages indicates that a - cell responds to the agent.
In yet another aspect, the invention provides a method of selecting a treatment regimen for a subject identified as suffering from leukemia, in which the method involves contacting a subject's leukemic cell with a Brd4 inhibitory agent, or its derivative, and detecting in the cell the expression of a specific differentiation marker for macrophages, in which an increase in the expression of the specific differentiation marker for macrophages indicates that a treatment regimen including this agent should be selected for the subject.
In yet another aspect, the invention provides a method of detecting the clinical response capacity of a leukemic cell, in which the method comprises contacting a leukemic cell with a Brd4 inhibitory agent, or its derivative, and detecting the expression in the cell of myc, where
NE a decrease in the expression of myc indicates that the cell responds to the agent.
In yet another aspect, the invention provides a method of selecting a treatment regimen for a subject, in which the method comprises contacting a leukemic cell with a Brd4 inhibitory agent, or its derivative, and detecting the expression of myc, in which a decrease in the expression of myc indicates that a treatment regimen including this agent should be selected for the subject. In various embodiments of any of the above or any other aspect of the invention outlined here, the agent is a compound - small (for example, JQ1 or a derivative thereof) or an inhibitory nucleic acid molecule (for example, siRNA, shRNA or nucleic acid molecule It is antisense). In other embodiments of the above aspects, the subject is a mammal (for example, a human patient). In other embodiments, the subject is an adult mammal (for example, an adult human patient). In other embodiments, the subject is a young mammal (for example, a young human patient). In other modalities of the above aspects, the method reduces the growth, proliferation or survival of a leukemic cell in a subject. In various embodiments of any of the above, the agent is a compound of any of Formulas | I-XXIIl or any other formula described herein. In particular modalities of the above aspects, the cell is in a subject. In other modalities of the above aspects, leukemia is acute myeloid leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), Chronic Myeloid Leukemia (CML), Chronic Myelomonocytic Leukemia (CMML), Eosinophilic Leukemia, Leukemia Hair Cell Cells, Hodgkin's Lymphoma, Multiple Myeloma, Non-Hodgkin's Lymphoma, Myelodysplasia or Myeloproliferative Disorders. In other modalities of the above aspects, the leukemic cell is derived from acute myeloid leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Leukemia —Acute Lymphocytic (ALL), Chronic Myeloid Leukemia (CML), Myeloid Leukemia Chronic lomonocytic (CMML), Eosinophilic Leukemia, Hairy Cell Leukemia, Hodgkin's Lymphoma, Multiple Myeloma, Non-Hodgkin's Lymphoma, V——
i Myelodysplasia or Myeloproliferative Disorders. In another aspect, the invention generally provides a method of treating a leukemia or related disorder (for example, acute myeloid leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), Myeloid Leukemia Chronic (CML), Chronic Myelomonocytic Leukemia (CMML), Eosinophilic Leukemia, Hairy Cell Leukemia, Hodgkin's Lymphoma, Multiple Myeloma, Non-Hodgkin's Lymphoma, Myeloproliferative Disorders or Myelodysplastic Syndrome in an individual subject, in an individual subject - providing the subject with an effective amount of an agent that inhibits Brd4 (for example, an inhibitory nucleic acid that selectively addresses Brd4, - JQ1) or a derivative thereof.
In another aspect, the invention provides a method of reducing the: growth, proliferation or survival of a leukemic cell, wherein the method comprises contacting the cell with an effective amount of an agent that inhibits Brd4 or a derivative thereof, thereby reducing the growth, proliferation or survival of a leukemic cell.
In yet another aspect, the invention provides a method of inducing cell death or terminal differentiation in a leukemic cell, wherein the method comprises contacting the cell with an effective amount of an agent that inhibits Brd4 or a derivative thereof, thereby inducing cell death or terminal differentiation in the leukemic cell.
In yet another aspect, the invention provides a method of treating acute myeloid leukemia in a subject, wherein the method comprises administering to an individual in need an effective amount of an agent that inhibits Brd4, thereby treating acute myeloid leukemia on a subject.
In yet another aspect, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of an agent that inhibits Brd4 or a derivative thereof in a pharmaceutically effective excipient.
In yet another aspect, the invention provides a kit for the treatment of leukemia, in which the kit comprises a therapeutic amount
Tically effective CRER of an agent that inhibits Brd4, and written instructions for administering the compound for use in the method as defined in claim 8. In yet another aspect, the invention provides a method of detecting the clinical responsiveness of a cell leukemic, in which the method comprises contacting a leukemic cell with a Brd4 inhibitory agent, or its derivative, and detecting in the cell the expression of a specific differentiation marker for macrophages, in which an increase in the expression of the specific differentiation marker for macrophages it indicates which cell corresponds to the agent. - In yet another aspect, the invention provides a method of selecting a treatment regimen for a subject identified as suffering from leukemia, in which the method comprises contacting a subject's leukemic cell with a Brd4 inhibitory agent, or its derivative , and detecting in the cell the expression of a specific differentiation marker for macrophages, in which an increase in the expression of the specific differentiation marker for macrophages indicates that a treatment regimen including this agent should be selected for the subject.
In yet another aspect, the invention provides a method of detecting the clinical response capacity of a leukemic cell, in which the method comprises contacting a leukemic cell with a Brd4 inhibitory agent, or its derivative, and detecting the expression in the cell of myc, in which a decrease in the expression of myc indicates that the cell responds to the agent.
In yet another aspect, the invention provides a method of selecting a treatment regimen for a subject, in which the method comprises contacting a leukemic cell with a Brd4 inhibitory agent, or its derivative, and detecting the expression of myc, in which a decrease in the expression of myc indicates that a treatment regimen including this agent should be selected for the subject.
In various embodiments of any of the above or any other aspect of the invention outlined here, the agent is an EREaa ———
small (for example, JQO1 or a derivative thereof) or an inhibitory nucleic acid molecule (for example, siRNA, shRNA or antisense nucleic acid molecule). In other embodiments of the above aspects, the subject is a mammal (for example, a human patient). In other modalities of the above aspects, the method reduces the growth, proliferation or survival of a leukemic cell in a subject. In various embodiments of any of the above, the agent is a compound of any of Formulas I-XXII or any other formula described herein. In particular modalities of the above aspects, the cell is in a subject. In other modalities of the above aspects, leukemia is acute myeloid leukemia - (AML), Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), Chronic Myeloid Leukemia (CML), Chronic Myelomonocytic Leukemia Is (CMML), Eosinophilic Leukemia, Hair Cell Leukemia, Hodgkin's Lymphoma, Multiple Myeloma, Non-Hodgkin's Lymphoma, Myelodysplasia or Myeloproliferative Disorders. In other modalities of the above aspects, the leukemic cell is derived from acute myeloid leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), Chronic Myeloid Leukemia (CML), Chronic Myelomonocytic Leukemia ( CMML), Eosinophilic Leukemia, Hairy Cell Leukemia, Hodgkin's Lymphoma, Multiple Myeloma, Non-Hodgkin's Lymphoma, Myelodysplasia or Myeloproliferative Disorders.
Other advantages and new features of the present invention will become clear from the following detailed description of various non-limiting modalities of the invention when considered in conjunction with adjunct aspheres. In cases where the present specification and a document incorporated by reference include disclosure that conflicts and / or inconsistent, the present specification controls.
Compositions and articles defined by the invention have been isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be clear from the detailed description, and through the claims. NEED BRIEF DESCRIPTION OF THE FIGURES
Figures 1A and 1B show chromatin regulators sensitive to Brd4 inhibition. Figure 1A includes a circular sector diagram showing the distribution of genes involved in chromatin modification and a graph showing screening for negative selection assembled in MLL-AF9 / Nrasº * leukemia illustrating changes in the representation of 1072 informative shRNAs during fourteen days in culture (Figure 1B). The numbers indicate the number of genes in each category.
For each gene, six ShRNAs were designed using the BIOPREDsi algorithm (Huesken et a /., Nat Biotech2005; 23: 995-1001) and adapted to the miR30 context. The library - was built using synthesis of oligonucleotides in chips on a large scale, followed by cloning by assembled PCR and verification of the sequence of individual clones, which resulted in a total of 1095 shRNAs (three to six per gene). Figure 1B includes a graph showing changes in the representation of the 1072 informative shRNAs during 14 days of culture.
Screening for pooled negative selection was performed on MLL-AF9 / Nrasº * Leukemic cells, and shRNA abundance ratios were calculated as the number of readings after fourteen days of doxycycline administration (T14) divided by readings before administration doxycycline (To). The results were represented graphically as the average of two replicates in ascending order.
The completely depleted shRNAs (zero Ti readings, n = 71) were plotted as a ratio of 10 *; the shRNAs highlighted in this group are shown with uniform spacing in alphabetical order.
ShRNAs with a positive score (with a depletion greater than twenty times in both replicates, n = 177) are marked in dark gray.
Positive controls include shRNAs that target Rpa1, Rpa3, Pena, or Polr2b.
The control shRNAs needed
They selectively address Renilla (Ren) or Braf luciferase.
Figures 2A-2D show the screening by means of RNAi in an AML model competent for Tet-On.
Figure 2A is a schematic diagram that describes the RNAi screening strategy.
The screening was performed on a model of Acute Myeloid Leukemia (AML) competent for
Da e
Tet-On generated by retroviral vector cotransduction encoding MTA3-IRES-MLL-AF9 and Luciferase-IRES-Nrasº * in stem cells and hemotopoietic progenitors (HSPC). Leukemic cells collected from terminally ill mice were cultured and used for screening. A custom shRNA library targeting selectively chromatin regulatory genes was synthesized using synthesis of chip oligonucleotides, and was cloned into a pooled format. A library matching of 1095 shRNAs verified for the sequence was subcloned in TRMPV-Neo (Zuber et al., Nat Biotechnol 2011; 29: 79-83) and was transduced into leukemic cells, followed by selection with G418. The cells were then treated with doxycycline for fourteen days (equivalent to twelve cell passages), followed by fluorescence activated cells (FACS) screening to isolate the reds-positive cells / expressing shR-NA. Genomic DNA was prepared from leukemic cells subjected to screening (Tu), as well as pre-treated (To), and used as a model for PCR amplification of ShRNA guide strips, which were subjected to in-depth sequencing to quantify the relative abundance of each ShRNA in the library. The main hits were defined in the screening as genes for which at least two shRNA exhibited a depletion greater than twenty times. Thirty-eight genes met these criteria and were subjected to one-to-one validation using a different MLL-AF9 / NrasS * Induced AML cell line and a constitutive shRNA expression vector (LMN). Figure 2B is a scatter plot illustrating the correlation of normalized readings by shRNA between the plasmid assembly and two replicates of leukemia cells transduced from the library after drug selection (To). The correlation finds that the representation of the library is mostly unaffected by retroviral transduction and drug selection. Figure 2C is a scatter plot of readings normalized by ShRNA in T, compared to T., in an assay. The low correlation suggests substantial changes in the representation of sShRNA. Figure 2D is a dispersion graph illustrating the correlation of normalized readings by sShRNA in Ti in two independent replicates. The high correlation indicates that changes in
NEERNCCC sShRNA abundance are due to specific effects. r, Pearson's correlation coefficient.
Figures 3A and 3B validate the screening strategy. Figure 3A is a schematic diagram that describes a strategy for validating RNAi screening. Each gene that scored positively on the primary pooled screen (criteria: at least two shRNAs depleted more than seven times in two independent replicates) was subjected to validation one by one. The shRNAs designed to target this gene have been subcloned into the LMN vector, which expresses sShRNAs miR30 under the control of the constitutive LTR promoter and features GFP and NeoR reporters. LMN-shRNAs were trans-. produced in a leukemia cell line MLL-AF9 / Nrasº ' derived independently with an average infection efficiency of 20%. The relative change in% GFP was monitored over ten days using flow cytometry and was used as a reading of cell growth inhibition, represented graphically as depletion times [GFP% (d2) divided by GFP% ( d12)]. Figure 3B is a bar diagram showing depletion times for all LMN-shRNAs addressing the thirty-eight assumptions identified in the primary screen. The times of depletion of all LMN-shRNAs addressing the thirty-eight hits identified in the primary screening. It was not possible to validate several genes, which may be due to (i) true false positives in primary screening, (ii) variable effects in the independent leukemia lineage, or (iii) differences between the expression systems. shRNA pressure. Based on the total number of shRNAs identified showing maximum depletion (twenty-five times), Brd4 was identified as the main success in the screening.
Figures 44-4E show comparisons of sShnRNAs effects for Brd4 in leukemia, MEF, and G1E cells. In each of the experiments shown, doxycycline-inducible shRNAs in the TITMPV vector were transduced into cells competent for Tet-On, followed by selection with G418. Figure 4A includes diagrams showing the RT-QPCR results of Brd4 MRNA levels after 48 hours of dox treatment. (n = 4). Figure 4B includes diagrams showing the results of PE proliferation assays—
competitive education. The selected cells were mixed with cells not transduced at an 8: 1 ratio, and were subsequently cultured with doxycycline. The relative percentage of Venus-positive / TurboRFP-positive cells (ie, expressing shRNA) was determined at the indicated times, and changes were used to read growth-inhibiting effects (n = 3). Error bars represent m.e.p. Figure 4C includes flow cytometry graphs of cell cycle analyzes (double staining with BrdU / 7-AAD) of cells evaluated in Figure 4B, after five days of doxycycline administration. Figure 4D includes graphs showing apoptosis measurements using double staining, with Appendix V / DAPI, of cells evaluated in Figure 4A, after five days of doxycycline administration. The cluster was first applied to live cells (FSC / SSC), followed by clustering of RFP + / sShRNA + cells. This is responsible for the absence of accumulated dead cells (Annexin V + / DAPI +). Figure 4E includes diagrams showing the degree of GFP depletion of LMN-shRNAs performed on G1E as illustrated in Figure 3A. (n = 3). Error bars represent m.e.p. Figures 5A-5D show that ShRNA silencing of BRDA4 is sufficient to inhibit the growth of human AML cell lines THP-1 and MOLM-13. shnRNAs targeting human BRD4 selectively were cloned into the TRMPV-Neo vector, followed by retro-viral transduction of the human AML cell lines Eco-receptor + / competent for Tet-On THP-1 and MOLM-13. The cells were selected with G418 for one week. Figure 5A includes a graph showing the efficiency of BRDA4 silencing by suppression by conditional RNAi. RT-qPCR was performed on TRMPV-MOLM-13 lines after 48 hours of dox treatment (n = 3). Error bars represent m.e.p. Figures 5B and 5C include graphs showing the results of competitive proliferation assays for MOLM-13 and THP-1. The selected cells were mixed with non-transduced cells and were subsequently cultured in —dox The relative percentage of dsRed + / shRNA + cells was determined at the indicated times, and changes were used to measure growth inhibitory effects. The results are the average of two experiments NERD
: dependents. All results were normalized to a control shRNA (shRen.713). Error bars represent m.e.p. Figure 5D includes flow cytometry for cell cycle analysis (double staining with BrU / DAPI) of cells in Figures 5B and 5C after 5 days of dox treatment. The events were grouped into dsRed + / shRNA + cells.
Figures 6A-6BE show that the growth of AML is sensitive to inhibition of Brd4. Figure 6A (top panel) includes a "Western" staining representative of complete cell lysates prepared from cultures of murine embryonic fibroblasts (MEF) transduced with the indicated TtTMPV-shRNAs and induced with doxycycline for five days. Figure 6A (bottom panel) shows the relative change of% GFP after transduction of leukemia cultures MLL-AF9 / Nrasº * with LMN-shRNAs.
Figures 6B-6E show the inhibition of cell proliferation in murine (Figures 6B and 6D) and human (Figures 6C and 6E) cells by treatment with JO1. Figures 6B and 6C include graphs showing the proliferation rates of cells treated with JQ1. Curves were generated measuring the increase in the number of viable cells after three days in culture and adjusting the data to an exponential growth curve. The results were plotted against the proliferation rate of control cells, set at 1 (n = 3). The results were normalized to the proliferation rate of cells treated with vehicle / DMSO, fixed at 1. (n = 3).
The term CML-BC designates a blast crisis of chronic myeloid leukemia. The term T-ALL designates acute T-cell lymphoblastic leukemia. Figures 6D and 6E include diagrams showing percentages (BrdU-positive) of the quantified phase after treatment with JQO1 for forty-eight hours at the indicated concentrations (n = 3). BrdU was pulsed for thirty minutes in all experiments shown. All error bars represent m.e.p.
Figures 7A and 7B show that JQ1 exhibits broad anti-leukemia activity in several human leukemia cell lines. Figures 7A and 7B include graphs showing the proliferation rates of cell lines treated with JQ1. Curves were generated measuring the DU——
increasing the number of viable cells after 3 days in culture and adjusting the data to an exponential growth curve. The results are shown in relation to the proliferation rate of control cells (treated with DMSO), fixed at 1. (n = 3). Error bars represent m.e.p. Most human myeloid leukemia cell lines exhibit an IC 50 <500 nM.
Figures 8A-8D show the sensitivity to JQ1 of adult AML samples derived from patients. Figure 8A includes a table of clinical and pathological information about the analyzed AML specimens.
Figure 8B includes a table that summarizes the impact of JQ1 on proliferation - (3 H-thymidine uptake), apoptosis (Giemsa stain), and cell maturation (Wright-Giemsa stain). Since the proliferation assay is different from those used in Figure 7, strains HL-60 and MOLM-13 were included to ensure that the IC50 measurements were consistent with the other findings. Figure 8C includes graphs showing the proliferation curves of AML specimens treated with JQ1, in the presence of cytokines. (n = 3). Error bars represent m.e.p. Figure 8D includes an image of a Wright-Giemsa cytocentrifuge from the AML 4 sample, showing morphological characteristics of macrophage differentiation.
Figures 9A-9C show the sensitivity to JQ1 of pediatric leukemia samples derived from patients. Figure 9A includes a table summarizing information from leukemia samples from patients and sensitivity data from the JQ1 experiments. The MV4-11 cell line was included as a control to ensure that proliferation measurements with the WST1 assay were comparable to the results shown in Figure 7. The samples were treated with JQ1 for 72 hours, followed by analysis with WST-1 reagent. or analysis with Anexin V staining. Wright-Giemsa staining of cytocentrifuges was performed on specimens treated with 250 nM JO1 for 48 hours. Figure 9B includes a graph showing the proliferation curves. The results were normalized to control cells treated with DMSO. (n = 3). Error bars represent m.e.p. Figure 9C includes an image of a Wright-Giemsa cytocentrifuge from the Upp sample——
PEDO025, demonstrating lymphoid differentiation characteristics.
Figures 10A-10C show that treatment with JQO1 leads to apoptosis of leukemic cells.
Figures 10A and 10B include graphs showing the quantification of cell death for murine cells (Figure 10A) and human cells (Figure 10B). The cells were treated with 250 nM JQ1 for forty-eight hours, followed by staining with propidium iodide (IP). Positive cells for IP staining were quantified using FACS; n = 3. All error bars represent m.e.p.
Figure 10C includes graphs showing apoptosis measurements for MLL-AF9 / Nrasº * P leukemia cells treated with JQ1 for forty-eight hours. (n
- = 3). The results of representative experiments are presented.
Figures 11A-11F show that TRMPV-Neo clonal leukemia strains exhibit robust disease inhibition by inducing, with doxycycline, sShRNA expression.
TRMPV-Neo clones were generated by performing series limiting dilutions.
Figure 11A includes a schematic diagram that describes the in vivo RNAi and JQO1 experiments. Leukemia cells competent for Tet-On were transduced with TRMPV-Neo-shRNAs, followed by selection with G418, and subsequently were transplanted into recipient mice irradiated sublethally.
Due to the onset of the disease (determined using bioluminescent imaging, typically after five or six days), the expression of shRNA was induced by supplementation with doxycycline in drinking water and feed.
An animal's disease burden was then assessed using bioluminescent imaging, overall survival, and quantification of dsRed-positive cells.
Figure 11 FACSinclusion charts of doxycycline-treated leukemia clones.
The results verify the high percentage of Venus + / dsRed + cells in these cell populations.
The identified clones are> 99.9% positive, although TRMPV-Neo meetings are typically -85% Venus + / dsRed + (see Figure 12). Figure 11C includes bioluminescent images of leukemia burden.
Doxycycline was administered after the onset of the disease (day 5-6 post-transplant). Figure 11D includes a graph showing quantification of bioluminescent imaging responses after dox treatment.
This is--
: 15/140 the number of mice in each treatment arm is indicated and the error bars represent m.e.p. Figure 11E includes a graph showing Kaplan-Meier survival curves from recipient mice transplanted with the indicated TRMPV-shRNA leukemia clones. The dox treatment interval is indicated by an arrow. The overall survival benefit of clonal shBrd4 disease is 9-10 days, whereas with non-clonal meetings the median survival is 4 days. Figure 11F includes flow cytometry plots of bone marrow cells (CD45.2 +) derived from donors in dox-treated mice with terminal illness. The grouping shown includes dsRed + / sShRNA + cells.
- Figures 12A-121 show that Brd4 is required for leukemia progression in vivo. Figure 12A includes bioluminescent images of mice administered doxycycline due to the onset of the disease, that is, six days post-transplant. Day zero is the first day of doxycycline administration. Figure 12B includes a graph showing the quantification of bioluminescent imaging responses after doxycycline administration. The average values of four replicated mice are shown. Figure 12C includes a graph showing Kaplan-Meier survival curves of recipient mice transplanted with the indicated leukemia cell line TRMPV-shRNA. The period of doxycycline administration is indicated by an arrow. Statistical significance for sSshRNAs that selectively address Renilla's luciferase (s-hRen) was calculated using a Log-Rank test; * p = 0.0001, ** p <0.0001. Figure 12D includes flow cytometry of bone marrow cells (CD45.2-positive) derived from donors in terminally ill mice administered doxycycline. The cluster shown includes dsRed-positive / shRNA-positive cells. Figure 12E includes a graph showing the quantification of the percentage of dsRed-positive / shRNA-positive in CDA45.2-positive terminal leukemia burden. Figure 12F includes bioluminescent images of recipient mice with MLL-AF9 / Nrasº * leukemia treated with JQ1 (50 mg / kg / day) or DMSO carrier. Figure 12G includes a graph showing quantification of image responses AND
; 16/140 bioluminescent geniology to treatment with JQ1. The average values of 6 mice treated with DMSO and 7 treated with JQ1 are shown. The p-values were calculated using a two-tailed Student's paired t-test. Figure 12H includes a graph showing Kaplan-Meier survival curves from control mice treated with JQ1. Statistical significance was calculated using a Log-Rank test. At 12F, 12G, and 12H, treatment with JQ1 was started on day 1 after transplantation of 50,000 leukemia cells. Figure 12! includes a graph showing quantification of bioluminescent imaging responses to treatment with JQ1 in established disease. The mice were transplan-. with 500,000 leukemia cells, followed by initiation of treatment 6 days post-transplant, when it was possible to obtain the first images of the disease. The average values of 6 mice treated with DMSO and 7 treated with JQ1 are shown. The p-values were calculated using a two-tailed Student's paired t-test. All error bars shown represent m.e.p.
Figures 13A-13E show that treatments with 100 mg / kg / day and 50 mg / kg / day of JO1 exhibit single agent activity in established MLL-AF9 / NrasG12D leukemia. Figure 13A includes bioluminescent images of leukemic mice treated with 100 mg / kg / day of JQ1. The mice were transplanted with 1 million leukemia cells, followed by initiation of treatment on day 4 (when the disease becomes visible by imaging). Figure 13B includes a graph showing quantification of the bioluminescent images. (n = 8 in each group). Error bars represent m.e.p. Figure 13C includes a graph showing Kaplan-Meier survival curves from control mice and treated with JQ1. Treatment was started on day 4 post-transplant (indicated by the horizontal line). Statistical significance was calculated using a Log-Rank test. Figure 13D includes bioluminescent images of leukemic mice treated with 50 mg / kg / day of JQ1. The mice were transplanted with 500,000 leukemia cells, followed by initiation of treatment on day 6 (when the disease became visible by imaging). RR
Quantification is shown in Figure 121. Figure 13E includes a graph showing Kaplan-Meier survival curves from control and JQ1-treated mice shown in Figure 13D. Treatment was started on day 6 post-transplant (indicated by the horizontal line). Statistical significance was calculated using a Log-Rank test.
Figures 14A-14C show that JQ1 exhibits single agent antileukemia activity in the AML mouse model AML1- ETO9a / Nrasº " P / p53". Figure 14A is a schematic diagram showing the experimental strategy. HSPCs p53 "were cotransduced with AML1I-ETOS9a and Luciferase-I | RES-NrasG12D constructions, followed by cell transplantation to a sublethally irradiated recipient mouse, with high penetration capacity, the mice succumb to AML as previously described (Dick, JE, Blood 2008; 112: 4793-807). Splenic leukemia material derived from dying mice was transplanted to secondary recipient animals. Treatment with 50 mg / kg / day of JQ1 was started 5 days after the onset of the disease, confirmed by bioluminescent ology Figure 14B includes bioluminescent images of leukemic mice at indicated times Figure 14C includes a graph showing quantification of bioluminescent imaging responses to treatment c om JQ1. The average values of 8 mice are shown in each treatment group, the error bars represent m.e.p., the p-values were calculated using a two-tailed Student t-test.
Figure 15 includes graphs showing the effects of treatment with JQ1 on peripheral hematopoietic cell counts. Healthy C57BI / 6 mice were treated with JQ1 (50 or 100 mg / kg media) or DMSO transporter (400 uL / day), both administered by intraperitoneal injection for 20 days. Peripheral blood was collected through submandibular bleeding, and was analyzed using a Hemavet 950 analyzer (Drew Scientific). The values represent average values of 3 replicated mice; error bars indicate m.e.p. ERR
: Figure 16 includes cell stains showing that 20 days of JQO1 administration have minimal impact on normal bone marrow hematopoiesis. Healthy C57BL / 6 mice were treated with daily intraperitoneal injections of 50 mg / kg or 100 mg / kg of JQO1 for 20 days prior to bone marrow analysis. Histopathology stained with H&E of bone marrow of the sternum of mice treated with vehicle or with JQ1 revealed normal cellularity and normal mixed hematopoiesis. n = 3-5 mice for each treatment group. Representative images are displayed.
Figures 17A and 17B show that daily administration of JQ1 has minimal impact on normal hematopoiesis. Healthy C57BL / 6 mice were treated with daily injections of 50 mg / kg or 100 É mg / kg of JQ1 for 20 days before the bone marrow FACS analysis. Figure 17A includes FACS plots representative of bone marrow cells demonstrating the grouping used to distinguish and quantify percentages of Lin-, ckit + (LK parents) and Lin-Sca1 + ckit + (LSK stem cells) percentages. ). Figure 17B includes graphs showing the percentage of total bone marrow cells staining for the indicated antibodies. (n = 3). Error bars indicate m.e.p.
Figures 18A-181l show that inhibition of Brd4 leads to myeloid differentiation and leukemia stem cell depletion. Figures 18A and 18B include light microscopy images of MLL-AF9 / NrasG12D leukemia cells with May-Grunwald / Giemsa stain after 2 days of dox-induced sShRNA expression or 2 days of treatment with 100 nM OJ. The expression of shRNA was induced in leukemia cells transduced with TRMPV. Imaging was performed with a 40X objective. Figures 18C and 18D include FACS graphs of Mac-1 and c-kit surface expression after 4 days of shRNA expression or after 2 days of treatment with 100 nM JQ1. Figures 18E-18H include graphs of - Gene Enrichment Analysis (GSEA) that evaluate changes in genetic signatures of macrophages and LSC by inhibition of Brd4. In Figures 18E and 18G, RNA for expression series was obtained from BELIEVE
| dsRed + / sShRNA + cells subjected to screening (Ren versus three different shRNAs for Brd4) after 2 days of dox induction. In Figures 18F and 18H, microseries data were obtained from leukemia cells treated for 2 days with DMSO or 100 nM JQ1. NES = normalized enrichment classification. FDR q-val = q Value of the False Discovery Ratio, which is the probability that a set of genes with a given NES represents a false positive finding. Figure 181 includes graphs showing RT-QPCR results. RT-qPCR was performed to analyze the genes involved in macrophage functions after 2 days of dox-induced shRNA expression or 2 days of treatment with 100 nM JQ1. - ShRNA expression was induced using the TRMPV vector. For sShRNA experiments, dsRed + / shRNA + cells were screened by FACS to prepare RNA. The data shown from sShRNA to Brd4 are an average of samples from shRNA Brd4,552, 1448, and 2097. The signals were normalized to GAPDH, with control samples set to 1. (n = 3). Error bars indicate m.e.p.
Figure 19 includes graphs of GSEA showing that JQ1 triggers a similar pattern of changes in gene expression in human AML THP-1 cells, as seen in the murine AML model MLL- —AF9 / Nrasº * . THP-1 cells were treated with 250 nM JQ1 for 48 hours before RNA collection. Expression series were performed using Affymetrix ST 1.0 human gene series. GSEA was performed to assess changes in genetic signatures of macrophages, LSC, and Myc by inhibiting Brd4, as shown.
Figures 20A-20H show that JQ1 suppresses the Myc pathway in leukemia cells. Figures 20A and 20B include graphs showing RT-qPCR results of relative levels of Myc RNA in mouse (Figure 20A) or human cells (Figure 20B) after treatment for 48 hours with JQ1. The results were normalized to GAPDH, with levels of RNA in untreated cells set to 1 (n = 3). Figure 20C includes a "Western" stain of whole cell lysates prepared from MLL-AF9 / Nrasº leukemia cells treated for 48 hours with PES CC
DMSO or 250 nM JQ1. Figure 20D includes a graph showing RT-PCR results.
RT-qPCR was performed at the indicated times after treatment of MLL-AF9 / Nrasº * P leukemia cells with 250 nM JQ1. The results were normalized to GAPDH, with MRNA levels in untreated cells set to 1 (n = 3). Figure 20E includes a graph showing ChiP-gPCR results.
ChIP-qPCR was performed on leukemia cells MLL-AF9 / Nrasº * with antibodies and indicated "primer" locations (n = 6 for DMSO; n = 4 for JQ1 treated). TSS = transcription start site.
Figure 20F includes a "Western" staining of whole cell lysates prepared from MLL-AF9 / NrasSº O leukemia cells - transduced with empty vector or MSCV retrovirus containing Myc cDNA.
The cells were treated for 48 hours with DMSO or 250 nM JQ1. Figure 20G includes a graph showing the quantification of BrdU incorporation after a 30 minute pulse in MLL-AF9 / Nrasº ' transduced with the empty control vector or the Myc cDNA.
The cells were treated with JQ1 for 5 days at the indicated concentrations. (n = 3) Does Figure 20H include light microscopy images of MLL-AF9 / Nrasº * leukemia cells with May-Grunwald / Giemsa stains transduced with an empty vector or containing the Myc cDNA.
The cells were treated for 5 days with 50 nM JQO1. Representative images taken with a 40X objective are shown.
All error bars shown represent
also m.e.p.
Figures 21A-21D show that Brd4 silencing via ShRNA leads to negative regulation of Myc levels and negative regulation of Myc target gene expression.
Figures 21A and 21B include graphs showing the results of RT-QPCR analysis of MRNA levels of Brd4 (Figures 21A) and Myc (Figures 21B) prepared from TurboRFP + leukemia cells (expressing sShRNA) undergoing transduced screening with the indicated TtTMPV-sShRNA constructs.
The cells were treated with dox for 3 days.
The results were normalized to GAP-DH.
Figure 21C includes a "Western" stain of extracts prepared from cells that express shRNA for Brd4. Clones of
NA—— "nÂ" "
leukemia MLL-AF9 / Nras transduced with TRMPV. The cells were treated with dox for 3 days. Figure 21D includes graphs of GSEA assessing changes in gene expression of targets downstream from Myc. Mini-series data were obtained from RNA samples described in Figure 21A. Myc target gene sets have been previously described (Kim et al., Cell 2010; 143: 313-24; and Schuhmacher et al., Nucleic Acids Res 2001; 29: 397- 406). Figure 22 shows that JQ1 triggers downregulation of Myc target gene expression. Figure 22 includes graphs of GSEA assessing the alteration induced by JQ1 in downstream genetic signatures. of Myc. Microseries data were obtained from MLL-AF9 / Nrasº * P leukemia cells treated for 48 hours with DMSO or 100 nM JO1.
Figures 23A and 23B show that 48 hours of treatment with JQ1 suppress Myc expression selectively in leukemia cells.
Figures 23A and 23B include graphs showing RT-gPCR results. RT-PCR was performed to determine Myc RNA levels in mouse (Figures 23A) or human (Figures 23B) cell lines. The results were normalized to GAPDH, with RNA levels in untreated cells set to 1 (n = 3). Error bars indicate m.e.p.
Figures 24A-24D show the impact of retroviral Myc overexpression on the sensitivity of leukemia cells to JQ1. Figure 24A includes a schematic diagram of the retroviral vectors used for Myc overexpression. Figure 24B includes a graph showing results for RT-qPCR. RT-QPCR was performed to evaluate genes related to ma- —crophages by treatment with JQ1 for 5 days of leukemia cells overexpressing Myc or empty vector control. n = 3. The error bars represent m.e.p. Figure 24C includes a graph showing the cumulative number of cells in MLL-AF9 / Nrasº ' P leukemia cells of control and transduced with Myc in the presence of 50 nM JQ1 or transporter control of —DMSO. Figure 24D includes a graph showing the quantification of cell death of cells treated with JQ1 on day 4. IP + cells were quantified using FACS (n = 3). Error bars represent m.e.p.
NW ——— DnR—
Figures 25A-25D show that Myc overexpression prevents cell cycle arrest and ShRNA-induced macrophage differentiation to Brd4. Figure 25A includes representative flow cytometry graphs showing an analysis of the cell cycle (double staining with —BrdU / DAPI) of leukemia cultures MLL-AF9 / Nrasº * P co-translated with MSCV-Myc or empty vector together with conditional sShRNA vector TtTMPV, and subsequently selected with puromycin and G418. The cells were treated with dox for 3 days to induce expression of sShRNA. The events were grouped into dsRed + / shRNA + cells. Figure 25B includes a graph showing the quantification of BrdU incorporation in one. population shRNA + / dsRed +. n = 3. The error bars represent m.e.p. Figure 25C includes light microscopy images of leukemia cells MLL-AF9 / Nrasº * with May-Grunwald / Giemsa stain. Dox treatment was administered for 2 days. The images were taken with a 40X objective. Figure 25D includes a graph showing results of RT-qPCR. RT-gPCR was performed to evaluate macrophage-related genes after 2.5 dox-induced shdNA expression for Brd4 in leukemia cells competent for Tet-On transduced with MSCV-Myc or empty MSCV vector. The shRNAs were expressed using the TtTMPV vector. n =
3.The error bars represent m.e.p.
Figures 26A-26C show that most of the changes in gene expression induced by JQ1 consist of side effects of Myc inhibition. MLL-AF9 / Nrasº 'leukemia cells transduced with MSCV-Myc or empty vector control were treated with 100 nM JO1 for 48 hours, followed by RNA collection for analysis of expression micro series. Figure 26A includes a normalized inline thermal map representation of the relative abundance of MRNAs encoding selected genes based on whether they are up-regulated (left) or down-regulated (right) 2 times in leukemia cells in empty vector control after treatment with JQ1. The modest level of overexpression of Myc used here influences gene expression prior to treatment with JQ1. Figure 26B includes representations of thermal maps demonstrating pnnp——. ")
. 23/140 influence of Myc overexpression on alterations in the gene expression of indicated sets of genes. The color scale in Figures 26A and 26B indicates normalized expression values in line. Figure 26C includes diagrams showing the classification of genetic expression changes in- —duced by JQ1 based on the relationship with Myc expression. Genes whose expression is altered 2 times after treatment with control cell JQ1 have been classified as being independent of Myc if their expression can still be altered 2 times in leukemia cells transduced with MSCV-Myc. Genes were classified as being Myc dependent if their expression has not been altered twice in cells - MSCV-Myc treated with JQ1. Figures 27A-27D show that ShcNA silencing of Myc inhibits the growth of MLL-AF9 / Nrasº * P leukemia and triggers terminal myeloid differentiation. Figure 27A includes a graph showing the inhibition of cell growth when LMN-shRNAs were transduced into an MLL-AF9 / Nrasº ' leukemia cell line. The relative change in% GFP was monitored over 6 days by flow cytometry and was used as a measure of cell growth inhibition. Figure 27B includes FACS plots showing c-kit and Mac-1 surface expression of LMN-transduced leukemia cells on day 4 post-infection. All events were grouped into GFP + / ShRNA + cells. Figure 27C includes light microscopy images of MLL-AF9 / Nrasº ' clonal with May-Grunwald / Giemsa stain after 2 days of doxycycline-induced TRMPV-shRNA expression. Figure 28D includes a graph showing RT-QPCR results. RT-gPCR was performed to analyze the genes involved in macrophage functions after 2 days of dox-induced ShRNA expression. The expression of shRNA was induced using the TRMPV vector. The signals were normalized to GAPDH, with control samples set to 1. (n = 3). Error bars represent m.e.p.
Figures 28A and 28B show that Brd4 is not consistently overexpressed in AML over other cell types. Figures 28A and 28B include graphs showing RT-QPCR results. RT- N —— "W) bp qPCR was performed on the indicated lines of mouse cells (Figures 28A) or human (Figures 28B). The results were normalized to GAPDH. N = 3. The error bars represent mep As Figures 29A and 29B show the results of the pharmacokinetic study of (+) - JO1 in mice. Figure 29A includes a table of pharmacokinetic data and measured parameters. Plasma drug concentrations were measured using quadrupole LCMS-MS. (API-2000) after a single intraperitoneal injection of (+) - JQ1 (50 mg / kg) in adult male C1 mice, at pre-specified times, as shown. (+) - JQ1 administration at this dose - results in an excellent maximum plasma concentration (Cmax> 20 µM) and total drug exposure (ADC> 20,000 h * ng / mL). ALQ indicates samples where (+) - JQ1 was beyond the quantifiable limit of the detection assay - pharmacokinetic tion (1.00 ng / mL). Figure 29B includes a graph showing the concentration profile in plasma-time for (+) - JQ1 using data listed in Figure 29A. The data represent mean measurements and the error bars indicate the standard deviation, both from independent measurements in triplicate. Plasma concentrations of the drug above the biologically active concentration observed in vitro (100 nM; red horizontal line) are observed for more than 10 hours by means of extrapolation.
Figures 30A-30C show the largely overlapping effects on transcription induced by suppression of Brd4, Myb, and MLL-AF9, with negative regulation of Myc by suppression of any of the three factors. Figure 30A includes graphs of GSEA evaluating the downstream transcription signatures of MLL-AF9 and Myb. MLL-AF9 500 and Myb 500 were defined using RMA as the main 500 negatively regulated genes based on the change in number of times by Tet-Off-mediated MLL-AF9 negative regulation or Myb sSshRNA silencing, respectively. Does the 500 gene limit correspond to a change in the number of times of Log de-1.17 for Myb and -1.77 for MLL-AF9. Figure 30B includes a representation of the thermal map of Myc expression in the replicas of the indicated microseries. Changes in the number of times of Log, and adj.P.Val were HUH
: ram calculated using the Limma algorithm, implemented using Bio-conductor.
Figure 30C includes a graph showing results of RT-qPCR.
RT-qgPCR was performed to validate that the treatment with JQ1 does not influence the expression of Hoxa7, Hoxa9, and Meis1 expression, which are well-established direct targets of MLL-AF9. This indicates that inhibition of Brd4 does not neutralize the global function of MLL-AF9; instead, it suppresses a large subset of other downstream targets, for example, Myc. n = 3. The error bars represent m.e.p.
Definitions By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof. is As used herein, the term "alkyl" means a straight or branched chain saturated non-cyclic hydrocarbon typically having from 1 to 10 carbon atoms.
Representative straight chain saturated alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl and n-decyl; whereas branched saturated alkyls include isopropyl, sec-butyl, isobutyl, ferc-butyl, isopentyl, 2-methylbutyl, 3-methylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-methylhexyl, 3-methylhexyl , 4-methylhexyl, 5-methylhexyl, 2,3-dimethylbutyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylpentyl , 2,2-dimethylhexyl, 3,3-dimethylpentyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylpentyl, 3-ethylpentyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2 -ethylpentyl, 2-methyl-3-ethylpentyl, 2-methyl-4-ethylpentyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2-methyl-4-ethylhexyl, 2,2-diethylpentyl, 3 , 3-diethylhexyl, 2,2-diethylhexyl, 3,3-diethylhexyl and the like.
Alkyl groups included in compounds of this invention may be unsubstituted, or may be optionally substituted with one or more substituents, such as amino, alkylamino, arylamino, heteroarylamino, alkoxy, alkylthio, oxo, halo, acyl, nitro, hydroxyl , cyano, aryl, heteroaryl, alkylaryl, alkylarroaryl, aryloxy, heteroaryloxy, arylthio, heteroarylthio, arylamino, heteroarylamino, carbocyclyl, carbocyclyloxy, carbocyclylthio, carbocyclicamino, heterocyclyl, hetero -—— ""
rocyclyloxy, heterocyclylamino, heterocyclylthio, and the like. Lower alkyl groups are typically preferred for the compounds of that invention.
By "alteration" is meant a modification (increase or decrease) in the levels of expression or activity of a gene or polypeptide, detected by methods known in the common art, such as those described here. As used here, a change includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or more change. expression levels.
By "improving" we mean to decrease, to suppress, to attenuate. air, decrease, halt, or stabilize the development or progression of a disease. Ú By "analogue" is meant a molecule that is not identical, but that has similar functional or structural characteristics. For example, a polypeptide analog retains at least some of the biological activity of a corresponding naturally occurring polypeptide, but having certain biochemical modifications that enhance the function of the analog with respect to a naturally occurring polypeptide. These biochemical changes can increase resistance to proteases, membrane permeability, or analog half-life, without altering, for example, the binding of ligands. An analogue can include an unnatural amino acid.
As used here, the term an "aromatic ring" or "aryl" means a ring or radical in a monocyclic or polycyclic aromatic ring comprising carbon and hydrogen atoms. Examples of suitable aryl groups —which include, but are not limited to, phenyl, tolyl, anthracenyl, fluorine, indenyl, azulenyl, and naphthyl, as well as benzo-fused carbocyclic fractions, such as 5,6,7,8-tetra- hydronaftyl. An aryl group may be unsubstituted or optionally substituted with one or more substituents, for example, substituents as described herein for alkyl groups (including, without limitation, alkyl (preferably, lower alkyl or alkyl substituted with one or plus halo), hydroxy, alkoxy (preferably lower alkoxy), alkylthio, cyano, halo, amino, boronic acid (-B (OH)>, and nitro). In certain monpn ——— "Â Â p;
: 27/140 dalities, the aryl group is a monocyclic ring, in which the ring comprises 6 carbon atoms. By "bromodomain" is meant a portion of a polypeptide that recognizes acetylated lysine residues. In one embodiment, a bromodomain of a polypeptide member of the BET family comprises approximately 110 amino acids and shares a conserved fold comprising a left-handed bundle of four alpha helices linked by different loop regions that interact with chromatin. By "polypeptide of the BET family" is meant a polypeptide comprising two bromodomains and an extraterminal domain "(ET), or a fragment thereof, having transcriptional regulatory activity or acetylated lysine binding activity. Exemplary members of the family. BET include BRD2, BRD3, BRD4 and BRDT. By "BRD2 polypeptide" is meant a protein, or fragment thereof, having at least 85% identity with NP. 005095 that is capable of binding to chromatin or regulating The sequence of an exemplary BRD2 polypeptide is as follows: MLONVTPHNKLPGEGNAGLLGLGPEAAAPGKRIRKPSLLYEGF ESPTMASVPALQLTPANPPPPEVSNPK KPGRVTNQLOYLHKVVMKALWKHQFAWPFRQPVDAVKLGLP DYHKIIKQPMDMGTIKRRLENNYYWAASE CMQDFNTMFTNCYIYNKPTDDIVLMAQTLEKIFLOKVASMPQE EQELVVTIPKNSHKKGAKLAALQGSVT SAHQVPAVSSVSHTALYTPPPEIPTTVLNIPHPSVISSPLLKSLH
SAGPPLLAVTAAPPAQPLAKKKGVK RKADTTTPTPTAILAPGSPASPPGSLEPKAARLPPMRRESGRP | KPPRKDLPDSQQAQHASSKKGKLSEQL KHCNGILKELLSKKHAAYAWPFYKPVDASALGLHDYHDIIKHPM DLSTVKRKMENRDYRDAQEFAADVRL MFSNCYKYNPPDHDVVAMARKLQDVFEFRYAKMPDEPLEPGP LPVSTAMPPGLAKSSSESSSEESSSESS HUH SEEEEEEDEEDEEEEESESSDSEEERAHRLAELOQEQLRAVHE QLAALSQGPISKPKRKREKKEKKKKRKA EKHRGRAGADEDDKGPRAPRPPQPKKSKKASGSGGGSAALG PSGFGPSGGSGTKLPKKATKTAPPALPTG YDSEEEEESRPMSYDEKRQLSLDINKLPGEKLGRVVHIIQAREP SLRDSNPEEIEIDFETLKPSTLRELE RYVLSCLRKKPRKPYTIKKPVGKTKEELALEKKRELEKRLQDVS GQLNSTKKPPKKANEKTESSSAQQVA
VSRLSASSSSSDSSSSSSSSSSSDTSDSDSG By "BRD2 nucleic acid molecule" we mean one: polynucleotide that encodes a BRD2 polypeptide, or its fragment. By "BRD3 polypeptide" is meant a protein, and or its fragment, having at least 85% identity with NP 031397.1 which is capable of binding to chromatin or regulating transcription. The sequence of one exemplary BRD3 polypeptide if- it is the fore: 1 mstattvapa gipatpgpvn ppppevsnps kpgrktnglga ymgnvvvktl 61 w- khafawpfy qpvdaikinl pdyhkiiknp mdmgtikkrl ennyywsase cmadfntmft NC- vyiynkptd 121 divimagale kiflgkvagm pgaeevellpp apkgkgrkpa agaqsagtaq vaavssvspa 181 tpfasvpptv satpviaatp vptitanvis vpvppaaapp ppatpivpvyv Pptppvvkkk 241 gvkrkadttt pttsaitasr sesppplsdp Kkgakvvarre sggrpikppk k- diedgevpqg 301 hagkkgklse hirycdsitr emiskkhaay awpfykpvda ealelhdyhd ii- khpmdlst 361 vkrkmdgrey pdaqggfaadv rimísncyky nppdhevvam arklgdvfem —rfakmpdepaa
: 481 saapvnkpkk kkekkekekk kkdkekekek hkvkaeeekk akvappak- qa qgkkapakka 541 nstttagra! kKkKggkgasas ydseeeeegl pmsydekral sidinripge kl- grvvhiiq 601 srepslrdsn pdeieidfet Ikpttirele ryvksclgkk qrkpfsasgk kga- akskeel 661 agekkkelek riqgdsssgsssgssgsssgsssssgsssgssssgsssgssssgsssgsssssgsssgsssssgsssgssssgsssgssssgssssgssssgsssgssssgsssgssssgsssgssssgsssgssssgssssgssssgsssgssssgsssg polynucleotide that encodes a BRD3 polypeptide. By "BRD4 polypeptide" is meant a protein, or fragment thereof, having at least 85% identity with NP 055114 which is capable of binding to chromatin or regulating transcription. 1 msaesgpgtr Irnilpvmgdg letsqmsttq agaqpapana astnppppet snpnkpkrqt 61 nalayllrvv Iktiwkhafa wpfggpvdav kInlpdyyki iktpmdmgti kkr- lennyyw 121 nagecigdfn tmftncyiyn kpgddivima ealeklflgk inelpteete imiv- gqakgrg 181 rorketgtak pgavstvpntt gastppatat papnpppvga tphpfpavtp dlivgtovmt 241 vvppaplato ppvyppapapp papapapvas hppiiaatpq pvktkkgvkr kadtttptti 301 dpiheppsip pepkttklgg rressrpvkp pkkdvpdsag hpapeksskv seqlkcesgi 361 lkemfakkha ayawpfykpv dvealglhdy cdiikhbpmadm stiksklear eyrdagefga 421 dvrimfsncy kynppdhevv amarklgdvf emrfakmpde peepvvavss —pavppptkwv 481 appsssdsss dsssdsdsst ddseeeragr laelgeglka vheglaalsa paqnkkk
: 30/140 541 kdkkekkkek hkrkeeveen kkskakeppp kktkknnssn snvskkepap mkskppptye 601 seeedkckpm syeekrqls | dinkIpgekl grvvhiigsr epslknsnpd ei- eidfetlk 661 pstlrelery viscirkkrk pgaekvdvia gsskmkgfss sesesssess ssdsedsetg 721 pa By "Brd4 nucleic acid molecule" it is intended to designate a polynucleotide that encodes a BR4 polypeptide. By "BRDT polypeptide" is meant a protein, or "its fragment, having at least 85% identity with NP 001717 which is able to bind to chromatin or regulate transcription.: 1 mslpsrqtai ivnppppeyi ntkkngrltn qglgylgkvvl kdlwkhsfsw pd davk 61 Iglpdyytii kKnpmdintik krlenkyyak aseciedfnt mfsncylynk pgaddi- vimaq 121 aleklimqgkl sampgeeqvv gvkerikkgt gqniavssak eksspsatek vfkageipsy 181 fpkisispin vvagasvnss sqataagvtkg vkrkadtttp atsavkasse fsptíteksv 241 alppikenmp knvipdsagq ynvvktvkvt eglrheseil kemlakkhfs yawpfynpvd 301 vnalglhnyy dvwvknpmdig tikekmdnge ykdaykfaad vrimfmncyk ynppdhevvt 361 marmigdvfe thfskipiep vesmplcyik tditettgre ntneassegn ssddsederv 421 krlakigeqg! kavhaqglavl sqavpírkInk kkekskkekk kekvnnsnen prkmceqmr! 481 kekskrnapk krkgafiglk sedednakpm nydekralsl ninklIpgdkl —grvvhiigsr 541 epslsnsnpd eieidfetlk astireleky vsacilrkrpl kppaqimk ke- PEER
601 elekrildvn nqinsrkrgt ksdktgpska venvsrises sssssssses essssdisss 661 dssdsesemf pkftevkpnd spskenvkkm knecilpegr tgvtgigycv qdttsantt! 721 vhattpshvm ppnhhqglafn ygelehlatv knisplgilp psgdsealsn gitumhpsgd 781 sdttimlesec gapvakdiki knadswkslg kpvkpsgvmk ssdelfnagfr kaaiekevka 841 rtgelirkhlqggggkngggkgnkggkggkgnkggkgggkggkggkggkgnk 901 ksklwllkdr dlargkeger rrreamvagti dmtlgsdimt mfennfd By "BRDT nucleic acid molecule" we mean a polynucleotide that encodes a BRDT polypeptide. Regarding the nomenclature of a chiral center, the terms "d" and "l" configuration are as defined by the IUPAC Recommendations. As for the use of the terms diastereomer, racemate, epimer and enantiomer, these will be used in their normal context to describe the stereochemistry of preparations.
By "compound" is meant any chemical compound, small molecule, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.
In such disclosure, "comprises", "comprising", "containing" and "having" and the like may have the meaning given in U.S. Patent Law and may mean "includes", "including", and the like; "consisting essentially of" or "consists essentially of" also has the meaning given in US Patent Law and the term is open, allowing for the presence of more than is recited, provided that basic or new features of what is recited are not altered by the presence of more than is recited, but excludes modalities of the previous
By "computational modeling" we mean the application of a computer program to determine one or more of the following: the location and proximity of a ligand to a fraction of N——
bond, the space occupied by a bonded bond, the amount of complementary contact surface between a bond fraction and a bond, the bond deformation energy of a given bond to a bond fraction, and some estimate of the bond strength hydrogen bonds, van der Waals interaction energies, hydrophobic interaction, and / or electrostatic interaction between ligand and bond fraction. Computational modeling can also provide comparisons between the characteristics of a model system and a candidate compound. For example, a computer modeling experiment can compare a pharmacophore model of the invention with a candidate compound to assess the fit of the candidate compound to the model. - By "means capable of being read by computer" we mean any means that can be read and accessed directly by a computer, for example, so that the means are suitable for use in the aforementioned computer system. The media includes, but is not limited to, the following: magnetic storage media, such as floppy disks, hard disk storage media and magnetic tape; optical storage media, such as optical discs or CD-ROM; electrical storage media, such as RAM and ROM; and hybrids of these categories, as magnetic / optical storage media.
By a "computer system" it is intended to designate the hardware means, software means and data storage means used to analyze atomic coordinate data. The minimal hardware means of the computer based systems of the present invention comprise a central processing unit (CPU), input means, output means and data storage means. Desirably, a monitor is provided to view data on structures. The means for storing data can be RAM or means for accessing means capable of being read by the computer of the invention. Examples of such systems are microcomputational workstations available from Silicon Gra- phics Incorporated and Sun Microsystems that work with Unix, Windows NT or IBM OS / 2 based operating systems. N ——— ÓDÕDR
Ê 33/140 "Detect" refers to the identification of the presence, absence or quantity of the analyte to be detected.
By "detectable tag" is meant a composition that, when attached to a molecule of interest, makes the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron density reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.
The term "diastereomers" refers to stereoisomers with two - or more centers of dissymmetry and whose molecules are not images in mirror with each other. By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases susceptible to treatment with compounds outlined here include leukemias and related disorders (for example, example, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), eosinophilic leukemia, hair cell leukemia, lymphoma Hodgkin's, Multiple Myeloma, Non-Hodgkin's Lymphoma, Myeloproliferative Disorders or Myelodysplastic Syndromes).
By "effective amount" is meant the amount of an agent required to ameliorate the symptoms of a disease in relation to an untreated patient. The effective amount of active compound (s) used to practice the present invention for the therapeutic treatment of a disease varies, depending on the mode of administration, age, body weight, and general health. of the subject. The attending physician or veterinarian will finally decide which amount and dosage regimen are appropriate. This quantity is called an "effective" quantity.
The term "enantiomers" refers to two stereoisomers of a compound that are mirror images not overlapping one another. An equimolar mixture of two enantiomers is called a "racemic mixture" TVR Cet
: 34/140 or "racemate". By "adjustment" we mean the determination, by automatic or semi-automatic means, of interactions between one or more atoms of an agent molecule and one or more atoms or binding sites of a member of the BET family (for example, a bromodomain of BRD2, BRD3, BRD4 and BRDT), and the determination of the extent to which these interactions are stable. Various computationally based methods for adjustment are also described here. By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the nucleic acid or polypeptide molecule A fragment can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
The term "haloalkyl" is intended to include alkyl groups as defined above that are mono-, di- or polysubstituted with halogen, for example, fluoromethyl and trifluoromethyl.
The term "halogen" means -F, -CI, -Br or —l.
The term "heteroaryl" refers to a 5-8 membered monocyclic aromatic, 8-12 membered, bicyclic or 11-14 membered tricyclic ring system having 1-4 ring heteroatoms if it is monocyclic, 1- 6 heteroatoms if it is bicyclic, or 1-9 heteroatoms if it is tricyclic, where said heteroatoms are selected from O, N, or S, and the remaining ring atoms! are carbon. Heteroaryl groups can optionally be substituted with one or more substituents, for example, substituents as described here for aryl groups. Examples of heteroaryl groups include but are not limited to pyridyl, furanyl, benzodioxolyl, thienyl, pyrrole, oxazolyl, oxadiazolyl, imidazolyl, thiazolyl, isoxazolyl, quinolinyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinin, pyrimidinin, pyrimidinin, pyrimidin, triazolyl, thiadiazolyl, isoquinolinyl, indazolyl, benzoxazolyl, benzofuryl, indolizinyl, imidazo-pyridyl, tetrazolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, benzo- E—
xadiazolyl, and indolyl.
The term "heteroatom", as used here, designates an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus. The term "isomers" or "stereoisomers" refers to compounds that have an identical chemical constitution, but that differ with respect to the arrangement of atoms or groups in space.
The term "heterocyclic", as used here, refers to organic compounds that contain at least one atom other than carbon (for example, S, O, N) in a ring structure. Organic compounds can be aromatic or, in certain embodiments, non-aromatic.Some examples of heterocyclic fractions include but are not limited to pyridine, pyrimidine, pyrrolidine, furan, tetrahydrofuran, tetrahydrothiophene, and dioxane.
"Hybridization" means hydrogen bonding, which can be reverse Watson-Crick, Hoogsteen or Hoogsteen hydrogen bonding between complementary nucleobases. For example, adenine and thymine are complementary nucleotides that pair through the formation of hydrogen bonds.
The term "hydroxyl" means -OH.
By "inhibitory nucleic acid" is meant a double-stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic, which, when administered to a mammalian cell, results in a decrease (for example, 10%, 25%, 50%, 75%, or even —mo90-100%) of the expression of a target gene. Typically, a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule, or an ortholog thereof, or comprises at least a portion of the complementary strand of a target nucleic acid molecule. For example, an inhibitory nucleic acid molecule comprises at least a portion of any or all of the nucleic acids outlined here.
By "isolated polynucleotide" is meant a nucleic acid (for example, a DNA) that is devoid of the genes that, in ge- NEREERS —————
õ 36/140 the naturally occurring name of the organism from which the nucleic acid molecule of the invention derives, flanks the gene. Consequently, the term includes, for example, recombinant DNA that is incorporated into a vector; on a plasmid or autonomously replicating virus; or in the genomic DNA of a prokaryote or eukaryote; or that it exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or digestion with restriction endonucleases) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as recombinant DNA that is part of a hybrid gene that encodes a polypeptide sequence. additional information.
By an "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally adhere to it. Typically, the polypeptide is isolated when it is at least 60% by weight, devoid of the naturally occurring organic proteins and molecules to which it is naturally associated. Preferably, the preparation consists of at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, of a polypeptide of the invention. An isolated polypeptide of the invention can be obtained, for example, by extracting a natural source, by expressing a recombinant nucleic acid that encodes that polypeptide; or through chemical synthesis of the protein. Purity can be measured by any appropriate method, for example, column chromatography, electrophoresis on polyacrylamide gel, or by HPLC analysis.
The term "isomers" or "stereoisomers" refers to compounds that have an identical chemical constitution, but that differ with respect to the arrangement of atoms or groups in space. The term "isotopic derivatives" includes derivatives of compounds in which one or more atoms of compounds are replaced by isotopes of the corresponding atoms. For example, an isotopic derivative of a compound containing a carbon atom (0 ) Is such that the carbon atom of the compound is replaced by the C " Isotope. ESSES SC WC
By "leukemic cell" is meant a cell derived from a leukemia.
By "marker" is meant any protein or polynucleotide having a change in the level of expression or activity that is associated with a disease or disorder.
The language "inhibiting the growth" of a cancer cell includes slowing, stopping, halting or stopping its growth and metastasis, and does not necessarily indicate a complete elimination of growth.
Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide. of the invention, or a fragment thereof. These nucleic acid molecules are not required to be 100% identical to an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" with an endogenous sequence are typically capable of hybridizing to at least one strand of a double stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule encoding a polypeptide of the invention, or a fragment thereof. It is not necessary for these nucleic acid molecules to be 100% identical to an endogenous nucleic acid sequence, but they will typically exhibit substantial identity. Polynucleotides having "substantial identity" with an endogenous sequence are typically capable of hybridizing to at least one strand of a double stranded nucleic acid molecule. By "hybridizing" is meant to pair to form a double-stranded molecule between complementary polynucleotide sequences (for example, a gene described here), or their portions, under various stringent conditions. (See, for example, Wahl, GM and SL Berger (1987) Methods Enzymol. 152: 399; Kimmel, AR (1987) Methods Enzymol. 152: 507.) The term "optical isomers", as used here, includes molecules, also called chiral molecules, which are images in the exact mirror not overlapping each other.
The phrases "parenteral administration" and "administered parenterally", as used here, mean different modes of administration from enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular injection and infusion, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intra-peritoneal, transtraqueal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinatus and intrasternum. to the radical of two or more cyclic rings (for example, cycloalkyls, cycloalkenyls, cycloalkylyls, aryls and / or heterocyclyl) in which two or more carbons are common. to two adjacent rings, for example, the rings are "fused rings ". Rings that are joined through non-adjacent atoms are called" bridged "rings. Each of the polycyclic rings can be replaced with substituents like those described above, for example o, halogen, hydroxyl, alkoxycarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonate, alkylate, amino, dialkyl, amino , arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonate, sulfamoyl, sulfonyl, trifluorohydride, nitro- , cyano, azido, heterocyclyl, alkyl, alkylaryl, or an aromatic or heteroaromatic moiety. The term "polymorph", as used herein, refers to solid crystalline forms of a compound of the present invention or its complex. Different polymorphs of the same compound can exhibit different physical, chemical and / or spectroscopic properties. Different physical properties include but are not limited to stability (for example, heat or light), compressibility and density (important in the formulation and manufacture of products), and dissolution rates (which can affect bioavailability). Differences in stability can result from changes in chemical reactivity (for example, differential oxidation, so that a dosage form undergoes discoloration more quickly when understood from a well.
Limorphic ERR than when comprised of another polymorph) or mechanical characteristics (for example, tablets disintegrate on storage when a polymorph favored by kinetics is converted into a thermodynamically more stable polymorph) or both (for example, polymorphic tablets more susceptible to breakdown at high humidity). Different
These physical properties of polymorphs can affect their processing.
The term "prodrug" includes compounds with fractions that can be metabolized in vivo.
In general, prodrugs are metabolized in vivo, by esterases or by other mechanisms, to active drugs.
Examples of prodrugs and their uses are well known in the art - (see, for example, Berge et al. (1977) "Pharmaceutical Salts", J.
Pharm.
Sci. 66: 1-19). The prodrugs can be prepared in situ during the final isolation and purification of the compounds, or by reacting the purified compound separately, in its free acid or hydroxyl form, with a suitable esterifying agent.
Hydroxyl groups can be converted to esters via treatment with a carboxylic acid.
Examples of prodrug fractions include substituted and unsubstituted, branched or unbranched fractions of lower alkyl esters (eg, propionic acid esters), lower alkenyl esters, di-lower alkyl-amino lower alkyl esters (for example, dimethylaminoethyl ester), acylamino lower alkyl esters (eg acetyloxymethyl ester), acyloxy lower alkyl esters (eg pivaloyloxymethyl ester), aryl esters (phenyl esters), aryl esters lower alkyl (e.g., benzyl ester), substituted aryl and lower aryl-alkyl esters (e.g., with methyl, halo, or methoxy substitutes), amides, lower alkyl amides, di-lower alkyl amides, and hydroxy amides.
Preferred prodrug fractions are propionic acid esters and acyl esters.
Also included are prodrugs that are converted into active forms by other mechanisms in vivo.
In addition, the indication of stereochemistry in a carbon-carbon double bond is also opposed to the general chemical domain, since "Z" refers to what is often called the "cis" conformation (even side), to the whereas "E" refers to what is often called compliance
——
. 40/140 "trans" (opposite side). Both configurations, cis / trans and / or Z / E, are covered by the compounds of the present invention.
By "reduce" or "increase" is meant a negative or positive change, respectively, of at least about 10%, 25%, 50%, 75%, or 100% relative to a reference.
By "reducing cell survival" is meant to inhibit the viability of a cell or induce cell death relative to a reference cell.
By "reference" we mean a standard or control condition. . A "reference sequence" is a defined sequence used as a basis for comparing sequences.
A reference sequence É can be a subset or the whole of a specified sequence; for example, a segment of a complete cCDNA or genetics sequence, or the complete CDNA or genetics sequence.
For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids.
For nucleic acids, the reference nucleic acid sequence length will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides — or any integer close to or between them By "mean square deviation" is meant the square root of the arithmetic mean of the squares of the mean deviations.
Sequence identity is typically measured using sequence analysis software (for example, the Genetics Computer Group's —Sequence Analysis Software Package, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST programs , BESTFIT, GAP, or PILEUP / PRETTYBOX). That softwa- and—.
: 41/140 re matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions in the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determine the degree of identity, a BLAST program can be used, with a probability score between e.sup.-3 and e.sup.-100 indicating a closely related sequence.
By "siRNA" is meant a double-stranded RNA. In . optimally, a siRNA is 18, 19, 20, 21, 22, 23 or 24 nucleotides in length and has a 2-base protuberance at its 3 end. These dsRNAs can be introduced into an individual cell or a complete animal; for example, they can be introduced systemically via the bloodstream. These siRNAs are used to down-regulate MRNA levels or promoter activity.
By "specifically binds" is meant a compound or antibody that recognizes and binds to a polypeptide of the invention, but that does not recognize or substantially bind to other molecules in a sample, for example, a biological sample , which naturally includes a polypeptide of the invention.
By "subject" is meant a mammal, including but not limited to a human or non-human mammal, such as a bovine, equine, canine, sheep, or feline.
By "substantially identical" is meant a polypeptide or nucleic acid molecule that exhibits at least 85% identity with a reference amino acid sequence (for example, any of the amino acid sequences described here) or sequence reference nucleic acid (for example, any of the nucleic acid sequences described here). Preferably, that sequence is at least 85%, 90%, 95%, 99% or even 100% identical, at the level of amino acids or nucleic acid, to the sequence used for comparison. NR
: 42/140 The term "sulfhydryl" or "thiol" means -SH.
As used here, the term "tautomers" refers to isomers of organic molecules that are easily interconverted through tauterization, in which a hydrogen atom or proton migrates in the reaction, sometimes accompanied by an exchange of a single bond and an adjacent double bond.
The invention provides some targets that are useful for the development of highly specific drugs for the treatment of a disorder, characterized by the methods outlined here. In addition, invention methods provide an easy way to identify therapies that are safe. for use on subjects. In addition, the methods of the invention provide a way to analyze virtually any number of compounds for effects on a disease described here, with high volume productivity, high sensitivity, and low complexity.
As used here, the terms "prevent", "preventing", "prevention", "prophylactic treatment" and the like refer to reducing the likelihood of developing a disorder or condition in a subject, who does not suffer but is at risk or is susceptible to developing a disorder or condition.
"An effective amount" refers to an amount of a compound that imparts a therapeutic effect to the treated subject. The therapeutic effect can be objective (that is, measurable by some test or marker) or subjective (that is, the subject gives an indication of an effect or feels an effect). An effective amount of a compound described herein can range from about 1 mg / kg to about 5000 mg / kg of body weight. Effective doses will also vary, depending on the route of administration, as well as the possibility of coutilization with other agents.
It is understood that the ranges provided here are an abbreviation for all values within the range. For example, it is understood that a range from 1 to 50 includes any number, combination of numbers, or —subvalue of the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15,16, 17,18, 19,20,21, 22, 29,24 25,26, 27, 26, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42.43, 44, 45, 46, 47, 48, 49, or 50. RRR —————
à: 43/140 As used here, the terms "treat", "treating", "treatment" and the like refer to the reduction or improvement of a disorder and / or symptoms associated with it. By "improving" is meant to decrease, suppress, attenuate, decrease, halt, or stabilize the development or progression of a disease. It will be appreciated that, while not being excluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated with them be completely eliminated.
Unless specifically stated or obvious from the context, as used here, the term "or" is understood to be inclusive. Unless it is specifically stated or is obvious from the context,. as used here, the terms "one", "one", and "o" and "a" are understood to be singular or plural. ! Unless specifically stated or obvious from the context, as used here, the term "about" is understood to mean that it is within a normal tolerance range in the art, for example, within 2 standard deviations from the average. About can be understood as being within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1% , 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided here are modified by the term about.
Reciting a listing of chemical groups in any definition of a variable here includes definitions of that variable as any isolated group or combination of groups listed. The recitation of a modality for a variable or aspect here includes that modality as - any modality alone or in combination with any other modality or its portions.
Any compositions or methods provided here can be combined with one or more of any of the other compositions and methods provided here.
DETAILED DESCRIPTION OF THE INVENTION The invention features compositions and methods useful for the treatment of leukemia and related disorders (e.g., leukemia mie- ERR>
. 44/140 acute loide (AML), Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), Chronic Myeloid Leukemia (CML), Chronic Myelomonocytic Leukemia (CMML), Eosinophilic Leukemia, Hair Cell Leukemia, Hodg's Linma , Multiple Myeloma, Non-Hodgkin's Lymphoma, Myeloproliferative Disorders or Myelodysplastic Syndromes).
The invention is based, at least in part, on the discovery that agents that inhibit Brd4 are useful to inhibit the growth or progression of acute myeloid leukemia. This inhibition may involve suppression of Myc activity. These findings also highlight the usefulness of RNAi screening as a discovery platform for revealing vulne- +. epigenetic abilities for direct pharmacological intervention in cancer.
As reported in detail below, the discovery that the introduction of Brd4 is useful for the treatment of leukemia was made using an undistorted approach to probe epigenetic vulnerabilities in acute myeloid leukemia (AML) - a malignancy aggressive hematopoietic that is associated with aberrant chromatin. By screening a personalized ShRNA library directed to known chromatin regulators in genetically defined leukemias, the protein containing Brd4 bromodomain has been identified as a critical requirement for the maintenance of AML disease. Suppression of Brd4 using sShRNAs or the small molecule inhibitor JQ1 led to robust anti-leukemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation and elimination of leukemic stem cells (LSCs). These effects were due to the requirement of Brd4 in maintaining Myc expression and promoting open self-renewal Proteins containing bromodomains Genetic regulation is fundamentally governed by a reversible and non-covalent assembly of macromolecules. Transduction of the signal to RNA polymerase requires protein complexes of a higher order, spatially regulated by assembly factors capable of interpreting the post-translational chromatin modification states. Epigenetic readers are structurally diverse proteins, each having one or more -
: 45/140 effector modules evolutionarily conserved, which recognize covalent modifications of proteins or histone DNA.
E-N-acetylation of lysine residues (Kac) in histone tails is associated with an open chromatin architecture and transcription activation (Marushige Proc Natl Acad Sci USA 7T3, 3937-3941, (1976)). The context-specific molecular recognition of acetyl-lysine is mainly mediated by bromodomains.
Proteins containing bromodomains have substantial biological interest, as components of transcription factor complexes (TAF1, PCAF, Gcn5 and CBP) and determinants of epigenetic memory (Dey etal, Mol Biol Ceil 20, 4899-4909, (2009)). There are 41 human proteins con-. having a total of 57 different bromodomains.
Despite great variations in the sequences, all bromodomains share a conservative fold comprising a left-handed bundle of four alpha helices (az, da, de, 0- c), connected by several regions of loops (loops ZA and BC) that determine the specificity for the substrate.
Cocrystalline structures with peptide substrates showed that acetyl lysine is recognized by a central hydrophobic cavity and is anchored by a hydrogen bond with an asparagine residue present in most bromodomains (Owen, D.
J. et al. "The structural basis for the recognition of acetylated histone H4 by the bromodomain of histone acetyltransferase gen5p". Embo J 19, 6141-6149, (2000)). The bromodomain family and extra-terminal domain (BET) (BRD2, BRD3, BRD4 and BRDT) share a common domain architecture comprising two N-terminal bromodomains, which exhibit a high level of sequence conservation, and a domain of more divergent C-terminal recruitment (Zeng et al., FEBS Lett 513, 124-128, (2002). The invention features compositions and methods that are useful for inhibiting human bromodomain proteins.
Compounds of the Invention The invention provides compounds (for example, JQ1 and compounds of formulas outlined here) that bind to the binding bag of the apo crystal structure of the first bromodomain of a member of the BET family (for example, BRD2, BRD3, BRD4 ). Without claiming to be restricted by theory, these ——
: 46/140 compounds can be particularly effective in inhibiting leukemias, including but not limited to acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML) ), Chronic Myelomonocytic Leukemia (CMML), Leukemia — Eosinophilic, Hair Cell Leukemia, Hodgkin's Lymphoma, Multiple Myeloma, Non-Hodgkin's Lymphoma, Myeloproliferative Disorders or Myelodysplastic Syndrome. In one approach, compounds useful for treating leukemias and related disorders are selected using a molecular anchoring program to identify compounds that are expected to bind to a bromodomain's structural binding pouch. In . certain embodiments, a compound of the invention can prevent, inhibit, or dismantle, or reduce by at least 10%, 25%, 50%, 75%, or 100% the biological activity of a member of the BET family (for example, BRD 2, BRD3, BRD4, BRDT) and / or dismantle the subcellular location of these proteins, for example, by means of binding to a binding site in a bromodomain apo binding pouch.
In certain embodiments, a compound of the invention is a small molecule with a molecular weight less than about 1000 Dalton, less than 800, less than 600, less than 500, less than 400, or less than about 300 Dalton. Examples of compounds of the invention include JQ1 and other compounds that bind to the ligation pouch of the apo crystal structure of the first bromodomain of a member of the BET family (for example, BRD4 (hereinafter called BRD4 (1); PDB ID 20SS) JO1 is a new thieno-triazole-1,4-diazepine The invention also provides pharmaceutically acceptable salts of these compounds.
In certain embodiments, a compound of the invention is a small molecule with a molecular weight less than about 1000 Dalton, less than 800, less than 600, less than 500, less than 400, or less than about 300 Dalton. Examples of compounds of the invention include JQ1 and other compounds that bind to the ligation pouch of the apo crystal structure of the first bromodomain of a member of the BET family (for example, BRD4 (hereinafter called BRD4 (1); PDB ID E—
. 47/140 20SS). JO1 is a new thieno-triazole-1,4-diazepine. The invention also provides pharmaceutically acceptable salts of these compounds. In one aspect, the compound is a compound of Formula |
R ema NOR, (Rad A CX ve (Ro
RE EX o) where - XéNouCRs ;, Rs; is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroari- É la, each of which is optionally substituted; Rg is H, alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or -COO-R; 3, each of which is optionally substituted; ring A is aryl or heteroaryl; each Ra, is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or any two Ra, together with the atoms to which each is attached, can form a fused aryl or heteroaryl group; R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; each of which is optionally substituted; R1 is - (CH> 2), - L, where n is 0-3 and L is H, -COO-R3, -CO-R; 3, -CO- N (R3R4), -S (O) 2 -R3, -S (O) aN (R3Ra), N (RsR4), N (Ra) C (OJRs, optionally substituted aryl, or optionally substituted heteroaryl; R7 is H, D (deuterium), halogen, or optionally alkyl each R; is independently selected from the group consisting of (i) H, aryl, substituted aryl, heteroaryl, or substituted heteroaryl;
: 48/140 (ii) heterocycloalkyl or substituted heterocycloalkyl; (iii) -C1-Cg alkyl, -C7-Cg alkenyl or -C2-C; g alkynyl, each containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N; -C3-C12 cycloalkyl, -C3-C12 substituted cycloalkyl, -C3-C12 cycloalkenyl, or -C3-C12 cycloalkenyl substituted, each of which may be optionally substituted; and (iv) NHo, N = CRaR6; each R is independently H, alkyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; . or R; and R, are taken together with the nitrogen atom to which they are attached to form a 4-10 membered ring; : Re is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; orR, eRçss are taken together with the carbon atom to which they are attached to form a 4-10 membered ring; mo, 12,, 0u3, provided that (a) if ring A is thienyl, Xé N, R is phenyl or substituted phenyl R-éH, Rs is methyl, and R; is - (CH>)) - L, where ne 1 and L is -CO-N (R3R1), so R; 3 and R are not taken together with the nitrogen atom to which they are attached to form a ring morpholino; (b) if ring A is thienyl, X is N, R is substituted phenyl, R2 is H, Rg is methyl, and R; is - (CH>), - L, where n is 1 and L is -CO-N (R3R4), and one of R; eR, éH, then the other of R; and R, is not methyl, hydroxyethyl, alkoxy, phenyl, substituted phenyl, pyridyl or substituted pyridyl; and (c) if ring A is thienyl, X is N, R is substituted phenyl, R; is H, Rg is methyl, and R, is - (CH>), - L, where n is 1 and L is -COO-R ;, then R; 3 is not methyl or ethyl; or a salt, solvate or hydrate thereof.
In certain embodiments, R is aryl, or heteroaryl, each of which is optionally substituted.
: 49/140 In certain embodiments, L is H, -COO-R3, -CO-N (R3R4), -S (O) 2- Ra, -S (O)> - N (R3Ra), N (R3R4) , N (Ra) C (OJ) R3 or optionally substituted aryl. In certain embodiments, each R3 is independently selected from the group consisting of: H, -C1-C; alkyl, containing O, 1, 2, or 3 heteroatoms selected from O, S, or N; or NH2, N = CRaRs. In certain embodiments, R; is H, D, halogen or methyl. In certain embodiments, Rg is alkyl, hydroxyalkyl, haloalkyl, or alkoxy; each of which is optionally replaced. In certain embodiments, Rg is methyl, ethyl, hydroxy methyl, methoxymethyl, trifluoromethyl, COOH, COOMe, COOEt, or COOCH2OC (O) CH ;. - In certain embodiments, ring A is a 5- or 6-membered aryl or heteroaryl. In certain embodiments, ring A is tiofuranila, phenyl, naphthyl, biphenyl Õ, tetrahydrofuran hidronaftila, indanila, pyridyl, furanyl, indolyl, pyrimidinyl, dizinila piri-, pyrazinyl, imidazolyl, oxazolyl, thienyl, thiazolyl, triazolila, isoxazolyl, quinolinila , pyrrolyl, pyrazolyl, or 5,6,7,8-tetrahydroisoquinolinyl. In certain embodiments, ring A is phenyl or thienyl. In certain modalities, m is 1 or 2, and at least one occurrence of Ra is methyl. In certain embodiments, each Ra is independently H, an optionally substituted alkyl, or any two Ra, together with the atoms to which each is attached, can form an aryl. In another aspect, the compound is a compound of Formula | 1:
R E XY 'SON e
RX (1) in which XéNouCRs ;, Rs is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted;
. 50/140 Rg is H, alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or -COO-R; 3, each of which is optionally substituted; each R is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or any two Ra, together with the atoms to which each is attached, can form a fused aryl or heteroaryl group; R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; R ', is H, -COO-R3, -CO-R ;, optionally substituted aryl, or * "optionally substituted heteroaryl; each R; is independently selected from the group consisting of: (i) H, aryl, substituted aryl, heteroaryl, substituted heteroaryl; (ii) heterocycloalkyl or substituted heterocycloalkyl; (iii) -C1-Cg alkyl, -C27-Cg alkenyl or -C3-C; g alkynyl, each containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N; -C3-C; 2 cycloalkyl, -C3-C12 substituted cycloalkyl; -C3-C12 cycloalkenyl, or -C3-C12 cycloalkenyl substituted; each of which may optionally be subs- titrated; mean, 1,2,0u3; provided that if R 'is -COO-R3, X is N, R is substituted phenyl, and Rg is methyl, then R; 3 is not methyl or ethyl; or one of its salt, solvate or hydrate.
In certain embodiments, R is aryl, or heteroaryl, each of which is optionally substituted. In certain embodiments, R is phenyl or pyridyl, each of which is optionally substituted. In certain modalities, R is p-Cl-phenyl, o-Cl-phenyl, m-Cl-phenyl, p-F-phenyl, o-F-phenyl, m-F-phenyl or pyridinyl.
In certain embodiments, R, is -COO-R ;, optionally substituted aryl, or optionally substituted heteroaryl; and R3 is -C1-C; g alkyl, which contains 0, 1, 2, or 3 heteroatoms selected from O, S, or N, and which
- 51/140, can be optionally replaced. In certain embodiments, R ', is -CO- O-R; 3, and R3 is methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, or t-butyl; or R ', is H or optionally substituted phenyl. In certain embodiments, Rg is methyl, ethyl, hydroxy methyl, methoxymethyl, trifluoromethyl, COOH, COOMe, COOEt, COOCH; OC (O0) CHs3. In certain embodiments, Rg is methyl, ethyl, hydroxy methyl, methoxymethyl, trifluoromethyl, COOH, COOMe, COOEt, or COOCH2OC (O0) CH ;. In certain embodiments, each Ra is independently an optionally substituted alkyl, or any two Ra, together with the atoms to which each is attached, can form a fused aryl. . In certain embodiments, each R is methyl. In another aspect, the compound is a compound of the formula Ill:: R% Ra 2 / SN N (Ram A CI Ra
NR Rx (1) where XéNouCRs, Rs; is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; Rg is H, alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or -COO-R3, each of which is optionally substituted; ring A is aryl or heteroaryl; each R is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or any two Ra, together with the atoms to which each one - attached, can form a fused aryl or heteroaryl group; R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; each R; 3 is independently selected from the group consisting of: (i) H, aryl, substituted aryl, heteroaryl, or substituted heteroaryl; (ii) heterocycloalkyl or substituted heterocycloalkyl; (iii) -C1-Cg alkyl, -C7-Cg alkenyl or -C2-Cg alkynyl, each containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N; -C3-C17 cycloalkyl, -C3-C12 substituted cycloalkyl, -C3-C12 cycloalkenyl, or -C3-C12 cycloalkenyl substituted, each of which may optionally be substituted (iv) NH2, N = CR4Re; each R is independently H, alkyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or R; 3 and Ra are taken together with the nitrogen atom to which they are attached to form a 4-10 membered ring; Rs is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or R, and Rg are taken together with the carbon atom to which they are attached to form a 4-10 membered ring; mé 0oO, 1,2,0u3, provided that: (a) if ring A is thienyl, X is N, R is phenyl or substituted phenyl, Rg is methyl, then R; and R, 4 are not taken together with the nitrogen atom to which they are attached to form a morpholino ring; and (b) if ring A is thienyl, X is N, R is substituted phenyl, R2 is H, Rg is methyl, and one of R3 and R, is H, then the other of R3 and R, is not methyl, hydroxyethyl, alkoxy, phenyl, substituted phenyl, pyridyl or substituted pyridyl; and or a salt, solvate or hydrate thereof.
In certain embodiments, R is aryl, or heteroaryl, each of which is optionally substituted. In certain embodiments, R is phenyl or pyridyl, each of which is optionally substituted.
In certain embodiments, R is p-Ci-phenyl, o-Cl-phenyl, m-Cl-phenyl, p-F-phenyl, o-F-phenyl, m-F-phenyl or pyridinyl. In certain embodiments, R3 is H, NH> 72, or N = CR, Reg.
In certain embodiments, each R is independently H, alkyl, cycloalkyl, heterocycloalkite, aryl, heteroaryl; each of which is optionally replaced.
In certain embodiments, R; is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted.
In another aspect, gomoneio is a compound of formula IV: =. / N R1 (Rad e ey SÁ Ro
RSX (V) where XéNouCRs ;, Rs; is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; Rg is H, alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or -COO-R3, each of which is optionally substituted; ring A is aryl or heteroaryl; each Ra is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or any two Ra, together with the atoms to which each is attached, can form a fused aryl or heteroaryl group; R1 is - (CH2)) - L, where n is 0-3 and L is H, -COO-R3, -CO-R ;, -CO-N (R3R4), -S (0) 2-R3, -S (O) 2-N (R3R4), N (R3R4), N (Ra) C (OJR; 3, optional aryl-
nally substituted, or optionally substituted heteroaryl; R> is optionally substituted H, D, halogen, or alkyl; each R; it is independently selected from the group consisting of: (i) H, aryl, substituted aryl, heteroaryl, or substituted heteroaryl; (ii) heterocycloalkyl or substituted heterocycloalkyl; (iii) -C1-Cg alkyl, -C2-Cg alkenyl or -C2-C; g alkynyl, each containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N; -C3-C12 cycloalkyl, -C3-C12 substituted cycloalkyl, -C3-C12 cycloalkenyl, or -C3-C12. substituted cycloalkenyl, each of which may optionally be replaced; and (iv) NHo, N = CRaRs; each R is independently H, alkyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or R3 and Ra, are taken together with the nitrogen atom to which they are attached to form a 4-10 membered ring; Rs is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or R, and R; they are taken together with the carbon atom to which they are attached to form a 4-10 membered ring; mean, 1.2.0u3; provided that (a) if ring A is thienyl, Xé N, R is H, Rg is methyl and R; is - (CH>)) - L, where n is 0 and L is -CO-N (R3R4), so R3 and Ra are not taken together with the nitrogen atom to which they are attached to form a morpholino ring; (b) if ring A is thienyl, Xé N, Rz is H, Rg is methyl and R is - (CH>)) - L, where né0Oelé-CO-N (R3Rs1), and one of R; and R, is H, then the other for R3 and Ra is not methyl, hydroxyethyl, alkoxy, phenyl, substituted phenyl, pyridyl or substituted pyridyl; and
(c) if ring A is thienyl, Xé N, RR is H, Rg is methyl, and R is - (CH2), - L, where n is 0 and L is -COO-R ;, then R; 3 it is not methyl or ethyl; or a salt, solvate or hydrate thereof. In certain embodiments, R, is - (CH>), - L, where n is 0-3 and Lé -COO-R; 3, optionally substituted aryl, or optionally substituted heteroaryl; and R3 is -C1-Cg alkyl, which contains 0, 1, 2, or 3 heteroatoms selected from O, S, or N, and which can be optionally substituted. In certain embodiments, n is 1 or 2 and L is alkyl or -COO-R ;, and R3 is methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, or t-butyl; or right 1 or 2e Lé H or optionally substituted phenyl. O. In certain embodiments, R; is H or methyl. In certain embodiments, Rg is methyl, ethyl, hydroxy methyl, metho- ximethyl, trifluoromethyl, COOH, COOMe, COOEt, COOCH; OC (O0) CH ;. In certain embodiments, ring A is phenyl, naphthyl, biphenyl, tetrahydronaphile, indanyl, pyridyl, furanyl, indolyl, pyrimidinyl, pyridizinyl, pyrazine, imidazolyl, oxazolyl, thienyl, thiazolyl, triazolyl, isoxazolyl, isoxazole, pyridine, isoxazole rolila, pyrazolyl, or 5,6,7,8-tetrahydroisoquinolinyl. In certain embodiments, each R is independently an optionally substituted alkyl, or any two Ra, together with the atoms to which each is attached, can form an aryl. The methods of the invention also refer to compounds of Formulas V-XXII, and to any compound described herein. In another aspect, the compound is a compound represented by the formula: cl - a o Se s
JA or a salt, solvate or hydrate thereof.
In certain embodiments, the compound is (+) - JQ1: cl 1 N = N. O. E are only NONO XxX Ds or a salt, solvate or hydrate thereof. In another aspect, the compound is a compound represented by the formula: cl == N H: it is TO "m so NONNO Ds es
N s NA | "+ = N SeNH, ie. NONE
NZ cl õ
The QC =! gives. No e! TA q ”N
HN o,
or
CI —N H No / N Ss NONO IN oH. or a salt, solvate or hydrate thereof. In another aspect, the compound is a compound represented by the formula:
CI OH = N, nu. O . and É | X9FA, OY are EIA TO IN 7 o or cl the node Na = NHH "| Y n N OO NO FA Pre mo mo mer NT in neo 6 Ss NONO o = or a salt, solvate or hydrate thereof. In another aspect, the compound is a compound represented by any of the following formulas:
ee e. N N
N NH FX FE SCENE A NR that are Ss FS TO Es fS JN | “=> EN YAN: the o JOIS RX JU
Ó Ç ci F Cc cl
IF N E No Y Ss a CAR Y a NA a. Do (= N Xen A E) = "JOIR au Y. E
1. 10 O oa
NR cr R É e) Do Y cl (NO "s O) o EXX Y. E E. ES e = N mo o $ + L. bone O 4E Q Jo2o ei cr Cc Ns io 2 N Se do -. and Y. E 3 FF NO are 8 | SIA NH "4 AA A. FN sos F ks! cr cl cr or a salt, solvate or hydrate thereof. In another aspect, the compound is a compound represented by any of the following formulas: Cl B (OH), 7 á —N A Nr Ss NONO = or
So B (OH), is —N nude
They are not only NONO dA or a salt, solvate or hydrate thereof. In another aspect, the compound is a compound represented by any of the following structures: s. : IF the A Oo is X) Cc Es Ss
SW IS EN O
RS Cc oa, Ss = UU "= N NH * to NH Cr Em oH
MO O N =
SEE cr
N and N s = "= N NH and Fa po Cc
> <eN s-
ALMA ST Sas cl near
A Nm O) VW ec
PAC Ss. AU Á .. Y. é y * ci C. e: s “& N Y o Cc a. A: The Di d nm Do [ei] —N H | JP: s and o a
The es Q
E: Er [e or Do & e Ds. Er AIR
Ci “E
O + fe “E FF Tv O Sa e 'e:
N Ss Ç x N-
ELO 22N NH> Ó
And CC
N Ss TO No uJ> E = N HN- o cr E »s: UU SN No” Õ is S N
We are. o Na <O eN = N No ó ci De E Sp - 1º = N HN o ci Te,
O "= N No IA act ci ci = N: É Mame à) De ame o = N HN à. Ci ci:
or a salt, solvate or hydrate thereof. In certain embodiments, a compound of the invention can be represented by one of the following structures:
N E x A o à. Yes, Ss "laughs.
AE - N fs o Cc: N o> N gs Nà e | mo Y = N Na o Cc à If N =: e = UU "= N NH" 2 cl;
A and <NON | o Sd NR = N NH o
SN cl
N É »E S o No FásraR | ““
UNITE the Sã ci “EP s
UU NE = N o o - N a, "o - s NA NE UU and ÍA = N yo ó ci; TE, RR s = - |." YNN Ho o f;
At the
AND NON S Nx was];
EN Va s “4 No N =" S MAO = N Ho ci ci
A Ss = N yo
Õ: 7: 2N, -: p Cr S e ci; No: And Co a ci a N, by Ci /
S NO | ;
We are So 2 cl A., Sm Is: Act sº NONO LN IN:
CI, —N 3 "N" N Lo Do, or cl = N Ds Ss "N q: No or a salt, solvate or hydrate thereof. In one embodiment, the compound is represented by the structure: NN = S ps E SW" ”= N NH &> cl or a salt, solvate or hydrate thereof. In another modality, the compound is represented by the structure:
N Fx ss NA Is ds Y = N Xena the NH 2
N ci; or a salt, solvate or hydrate thereof. In another modality, the compound is represented by the structure:
N o A and ss NA SE | ST YAN No Cl or a salt, solvate or hydrate thereof. In certain embodiments, a compound of the invention can have the opposite chirality of any compound shown here. In certain embodiments, the compound is a representative compound. 5 of Formula (V), (VI), or (VII):
R —N. : - O Ve NONO X no
JD Ré VV),
N A: - = N, R2 * to Ji do (VD), ci - = N. O.
RE Ss NONO K / N Re (VI) where R, R ;, and Rz and Rg have the same meaning as Formula (1); YéoO, N, S, or CRs, where Rs has the same meaning as Formula (1); right O or 1; and the dashed circle in Formula (VII) indicates an aromatic or non-aromatic ring; or a salt, solvate, or hydrate thereof. In certain embodiments of any of Formulas | -IV and VI (or any formula presented here), Rs represents the non-carbonyl portion of an aldehyde shown in Table A, below (that is, for an aldehyde of the formula R & ãCHO, R; is the non-carbonyl portion of the aldehyde). In certain modalities, Ra and Rg together represent the non-carbonyl portion of a ketone shown in Table A (that is, for a ketone of the formula RgC (OJRa, Ra and Rk are the non-carbonyl portion of the ketone). A: Plate 1 01 02 03 O4 O5 OG O7 OB O9 10 11 12 à CAE O RO A to ad À, g u bo a ton of REG a nm bm: cd au AO mano o! DOE 6 MA SEE OO OF ZAP Sh E SIA AR at MARA OO O00o Frio LEO o sd in tos OR MO o GuougAa Mere beat Rom dl HE io »* +. Nl LO: CO Deo: e SO
Fduca 01 02 03 04 O5 OG O7 OB OS 10 11 12 A donates to the Se É BL AÇO oo EP Ena DP Finger Go S QT gt TO RAS a DE ok STE 2 e LU E oe OR Wo ça a tr FAAO nu TO ÚCOO A as DT O É ghana fo AO Ao o O und OR Con Dn O MD DO O3 DA OS OB 07 08 09 10 11 (2 AA AO O NBR AE ONLY IN BE O dL STEEL or PR TO o At 6 Sage and DA OO 0 ”ol So DO QoS RIBBON 0% OA FROM TT
FTA IO LO AE HD o CEL O. Be DO 4 EPI
Plate 4 01 02 03 O4 O5 OG O7 O8 O9 10 11 12 AO QT a Of ne of Pon a BR Os EO E SO A On a E c Am SA, 7 AA O na, SO pot a TO been been so E - THE DTONT CUBOS Fo Nx | Fargo tas odado ÃO | GG SEE Qt OD To EE
H In one embodiment, the compound is a compound represented by the formula: Cc = N o,: PR: * PP (VIII), or a salt, solvate, or hydrate thereof. In certain embodiments, the compound is JQ1 (racemic); in certain modalities, the compound is (+) - JQ1. In certain modalities, the compound is a compound selected from the group consisting of: cl 7 N = N2N H - N. 2 (6) and
[oil —N. H No
FT - Ss N po Oo / N oH a), or a salt, solvate, or hydrate thereof. Additional examples of compounds include compounds according to any of the following formulas: ss. > Ss' + = AN de = FN NODE 7 cl PN NO / o NAN No. 2) CI: NNNN o tro AR NO PNR o) n = 1,2,3 RR n = 1,2,3 os n = 1, 2.3x
SN ba Á nO OX IAD d NO N N N 1, ci Nn. A 7 (Os NNNN NH OX) (Og om (OR om = o R op. R R '= H, D, Me R' = H, D, Me ea ne 123 n = 123 "SN es = NO and INE NEN HN / Cc!
R N N XV) R R R XVI) R "= OMe, CH, OH, CH, NH7, CHzOMe
Ss Ss ATA Ja À TN F. = = N N N We YZ nº> yº N ATT o, N N (XVI x XVI O, o õ à 20 Also 2- and 4-pyridyl
SN SON deg o sq Nº. / s Nº / e! ENNNNN— / cl (XXI) n N to SI (XXI) - No. 0%, nR & R í In Formulas IX-XXII, R and R 'can be, for example, H, aryl, substituted aryl, heteroaryl, heteroaryl, heterocycloalkyl, -C1-Cs alkyl, -C2-Cg alkenyl, -C2-C; g alkynyl, -C3-C12 cycloalkyl, -C3-C12 cycloalkyl substituted, -C3-Ci> cycloalkenyl, or -C3- Substituted C 1 cycloalkenyl, each of which may be optionally substituted. In Formulas XIV, X can be any substituent for an aryl group as described here. The compounds of the invention can be prepared by a variety of methods, some of which are known in the art. For example, the Chemical Examples provided below provide synthetic schemes for the preparation of compound JQ1 (in the form of the racemate) and enantiomers (+) - JQ1 and (-) - JO1 (see Schemes S1 and S2). A variety of compounds of Formulas (1) - (VIII) can be prepared by analogous methods with replacement of appropriate starting materials. For example, starting from JQ1, the analogous amine can be prepared as shown in Scheme 1, below.
Ss SN - Qd a of ”Cl 1) DPPA, NEt Io p— LL. NEts WA 7 AZ Frcoo N A PN = 2B0H N | and! o o o If he NHCbz
OJ | BBr3 sq SS Ss ho NaH, Mel did 1) RoHO == and q 2) NaBH (OAc) s eb - NH NH. - x KR 2 & Scheme 1 As shown in Scheme 1, the hydrolysis of the t-butyl ester of JQ1 gives rise to the carboxylic acid, which is treated with diphenylphosphoryl azide (DPPA) and subjected to Curtius rearrangement conditions to provide the amine protected with Cbz, which is then unprotected to give rise to the amine. The subsequent elaboration of the amine group, for example, by means of reductive amination, gives rise to secondary amines, which can be further alkylated to provide tertiary amines.
. 76/140
SO Ss VS = MN = N / F HoN F N N and o o SN -, and Ss ——- = N == 7 N HN = N> IT
E N N o 3rd o x A q HaN OR de! faith R
ES IS - N ° o Scheme 2 Scheme 2 shows the synthesis of other examples of the compounds of the invention, for example, Formula |, in which the fused ring core is modified (for example, by replacing a different aromatic ring as Ring A in Formula |). The use of aminodiaryl ketones having appropriate functionality (for example, instead of aminodiaryl ketone in Scheme S1, infra) provides new compounds having a variety of fused ring nuclei and / or appendages of aryl groups (corresponding to the R group in the Formula |). These aminodiaryl ketones are available on the market or can be prepared by a variety of methods, some of which are known in the art. Scheme 3 provides additional exemplary synthetic schemes for the preparation of other compounds of the invention.
- s Y sÀ Ss nn DAM DAM, (=) cy nO 1) Base 2 Cc o N re P OT re o N LDA. DAMBr O N 2) D; 0.0u Me! A O o o Y & Y | Acid hs À in ft = S q E ci
N N o N. R
O - . Scheme 3 As shown in Scheme 3, a fused bicyclic precursor (see scheme S1, below, for the synthesis of this compound) is functionalized with an R fraction (DAM protecting group = dimethylaminomethylene) and is then made by reaction with a hydrazide to form the fused tricyclic core. The substituent R, can be varied by selecting a suitable hydrazide.
Additional examples of compounds of the invention (which can be prepared by the methods described here) include: Amides: Amides can be prepared, for example, by preparing a corresponding carboxylic acid or ester, followed by amidation with an appropriate amine using standard conditions. In certain embodiments, an amide provides a two-carbon "linker" with a ring containing terminal nitrogen (for example, pyridyl, piperidyl, piperazinyl, imidazolyl (including N-methylimidazolyl), morpholinyl, and the like). Structures of exemplary amides include:
> 78/140 SN to SON Q / e NO c Nº N N N
N NHESINOO, MO o W7 o =
YN Ss SN Mei e MEN A a ta D N JA DO NO ”cl Cs Ex es a —N NH À o Ô * Ss SN NO ec NOS c Ru V N
NH NH PFN ADO AO) o [o] N It is preferred to use a two-carbon linker between the amide fraction and the ring containing terminal nitrogen.
- 79/140 "Reverse amides": Y TS Ss and NA er E - and NAN oO, and N N Position of N can be different p LT |
There is PP. N
SON Ss S o = no. E f NO) Cc nº fo N 7 fe N N o N 8 o - A OS fo o ON =: O à SN à. SN e - NA ci NON (=) o o o o n N "E NnaNT
. 80/140 Secondary amines:
SN sq 4 EN AUEet nN N) c NO / e! It is
N N>. "THAT H A"
E O Ca br 2 NU = cl o n No. NS N cl O and Nx N | Dt aa Es and SN NOT = N c Les A N GIVES
A Boronic acids: cl B (OH) z and O Cr ”and in di o B (OH), —N H 3 e and NÃ O N
FT à 81/140 In certain modalities, a compound having at least one chiral center is present in racemic form. In certain embodiments, a compound having at least one chiral center is enantiomerically enriched, that is, it has an enantiomeric excess (ee) of at least about 10%, 20%, 30%, 40%, 50%, 60 %, 70%, 80%, 85%, 90%, 90%, 95%, 99%, 99% or 100%. In certain embodiments, a compound has the same absolute configuration as the compound (+) - JQ1 (2- (4- (4-Chlorophenyl) - 2,3,9-trimethyl-6H-thieno [3,2-f] [1 , 2, A] triazole [4,3-a] [1 AJdiazepin-B-yl) (S) -tert-butyl acetate) described herein.
In certain modalities of any of the Formulas disclosed. here, the compound is not represented by the following structure: 3! N RA, | a = N R1 R3 where: R'1 is C1-C, alkyl; R'2 is hydrogen, halogen, or C1-C, alkyl optionally substituted with a halogen atom or a hydroxyl group; R '; is a halogen atom, phenyl optionally substituted with a halogen atom, C1-C, alkyl, C1-C, alkoxy, or cyano; -NR5- (CH2) 0m-Rs where Rs is a hydrogen atom or C1-C, alkyl, m is an integer from 0-4, and Rg is phenyl or pyridyl optionally substituted with a halogen atom; or -NR; -CO - (CH>)) - Rs where R; is a hydrogen atom or C; -C, alkyl, n is an integer from 0-2, and Rg; is phenyl or pyridyl optionally substituted with a halogen atom; and R'1a is - (CH2) a-CO-NH-Rg where a is an integer 1-4, and Rg is C1-C, alkyl; Hydroxyalkyl C1-Ca; Cy1-C, alkoxy; or phenyl or pyridyl optionally — optionally substituted with C1-C, alkyl, C1-C, alkoxy, amino or a hydroxyl group or - (CH2), - COOR 15 where b is an integer 1-4, and Rio It is
82/140 C1-C, alkyl.
The term "pharmaceutically acceptable salt" also refers to a salt prepared from a compound disclosed herein (for example, JO1, a compound of Formulas | -XXII) or any other compound outlined here, having an acidic functional group, as a carboxylic acid functional group, and a pharmaceutically acceptable inorganic or organic base. Suitable bases include but are not limited to alkali metal hydroxides, such as sodium, potassium, and lithium; hydroxides of alkaline earth metals, such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, and organic amines, such as mono-, di-, or trialkylamines unsubstituted or substituted with hydroxy; dicyclohexylamine; tributyl amine; pyridine; N-methyl-N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris- (2-hydroxy-lower alkyl amines), such as mono-, bis-, or tris- (2-hydroxyethyl) -amine, 2-hydroxy-tert-butylamine, or tris- ( hydroxymethyl) methylamine, NN-di-lower alkyl-N- (hydroxy lower alkyl) -amines, such as N N-dimethyl-N- (2-hydroxyethyl) -amine, or tri- (2-hydroxyethyl) amine; N-methyl-D-glucamine; and amino acids, such as arginine, lysine, and the like. The term "pharmaceutically acceptable salt" also refers to a salt prepared from a compound disclosed herein, or any other compound outlined here, having a basic functional group, such as an amino functional group, and an inorganic acid or pharmaceutically acceptable organic. Suitable acids include but are not limited to hydrogen sulfate, citric acid, acetic acid, oxalic acid, hydrochloric acid, hydrogen bromide, hydrogen iodide, nitric acid, phosphoric acid, isonicotinic acid, lactic acid, salicylic acid, tartaric acid, ascorbic acid, succinic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucaronic acid, saccharic acid, formic acid, benzoic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and p- toluenesulfonic.
In addition to small compounds that inhibit Brd4, the invention also provides other agents that inhibit the expression or biological activity of Brd4.
à 83/140 Nucleic Acids Inhibitors The invention also provides molecules of inhibitory nucleic acids that inhibit the expression or activity of Brd4, and the use of these agents for the treatment of leukemias (for example, acute myeloid leukemia ( AML), Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), Chronic Myeloid Leukemia (CML), Chronic Myelomonocytic Leukemia (CMML), Eosinophilic Leukemia, Hairy Cell Leukemia, Hodgkin's Lymphoma, Non-Multiple Heloid Myeloma , Myeloproliferative Disorders, Myelodysplasia). These oligonucleotides include single- and double-stranded nucleic acid molecules (for example, DNA, RNA, and their analogs) that bind to a nucleic acid molecule that encodes Brd4 (for example, antisense molecules, siRNA , shRNA) as well as nucleic acid molecules that bind directly to Brd4 to modulate its biological activity (eg aptamers).
Ribozymes Catalytic RNA molecules or ribozymes that include an antisense Brd4 sequence of the present invention can be used to inhibit the expression of a Brd4 nucleic acid molecule in vivo. The inclusion of ribozyme sequences in antisense RNAs gives them the same RNA cleavage activity, thereby increasing the activity of the constructs. The design and use of RNA-specific ribozymes is described in Haseloff et al., Nature 1988; 334: 585-591 and U.S. Patent Application Publication No. 2003/0003469 A1, each of which is incorporated by reference.
Accordingly, the invention also features a catalytic RNA molecule that includes, in the binding arm, an antisense RNA having between eight and nineteen consecutive nucleobases. In preferred embodiments of this invention, the catalytic nucleic acid molecule is formed into a hammerhead or hairpin motif. Examples of such hammerhead motifs are described by Rossi et a /., Aids Research and Human Retroviruses 1992; 8: 183. Examples of hairpin motifs are described by Hampel et a /., "RNA Catalyst for Cleaving Specific
It's 84/140 RNA Sequences ”, registered on September 20, 1989, which is a continuation-in-parts of U.S.
Series No. 07 / 247,100 registered on September 20, 1988, Hampel and Tritz, Biochemistry 1989; 28: 4929 and Hampel et al, Nucleic Acids Research 1990; 18: 299. These specific reasons are not —limiting in the invention, and practitioners will recognize that all that is important in an enzyme nucleic acid molecule of that invention is that it has a specific substrate binding site that is complementary to one or more of the RNA regions of target genes, and which has nucleotide sequences, within or around that substrate binding site, which give the molecule RNA cleavage activity. . Small hairpin RNAs consist of a stem-loop structure with optional 3 'UU protrusions.
Although there may be variations, the rods can vary from 21 to 31 bp (desirably 25 to 29 bp), and the handles can range from 4 to 30 bp (desirably 4 to 23 bp). For the expression of sShRNAs in cells, plasmid vectors containing the RNA polymerase promoter Ill H1 or U6 can be employed, a cloning site for the rod-loop RNA insert, and a 4-5- transcription termination signal. thymidine.
In general, Polymerase ll promoters have well-defined initiation and termination sites and their transcripts do not have poly (A) tails. The termination signal for these promoters is defined by the polythymidine tract, and the transcript is typically cleaved after the second uridine.
Cleavage in this position generates a 3 'UU protrusion in the expressed shR-NA, which is similar to the 3' protuberances of synthetic siRNAs.
Additional methods for expressing ShRNA in mammalian cells are described in the references cited above. siRNA small double-stranded RNAs with twenty-one to twenty-five nucleotides are effective in downregulating gene expression (Za- more et al., Cell 101: 25-33; Elbashir et al, Nature 2001; 411: 494 -498 incorporated- —ported here by reference). The therapeutic efficacy of a SiRNA approach in mammals has been demonstrated in vivo by McCaffrey et al.
Nature 2002; 418: 38-39.
- 85/140 Given the sequence of a target gene, siRNAs can be designed to inactivate that gene. For example, these siRNAs can be administered directly to an affected tissue, or they can be administered systemically. The nucleic acid sequence of a Brd4 gene can be used to design small interfering RNAs (siRNAs). 21 to 25 nucleotide siRNAs can be used, for example, as therapeutic agents to treat a vascular disease or disorder. The inhibitory nucleic acid molecules of the present invention can be used as double-stranded RNAs for silencing, mediated by RNA interference (RNAi), of Brd4 expression. In a . modality, the expression of Brd4 is reduced in a hematopoietic cell or a leukemic cell. RNAi is a method to decrease expression | cell of specific proteins of interest (reviewed in Tuschl, Chembio-chem 2001; 2: 239-245; Sharp, Genes & Devel. 2000; 15: 485-490; Hutvagner and Zamore, Curr. Opin. Genet. Devel. 2002; 12: 225-232; and Hannon, Nature 2002; 418: 244-251). The introduction of siRNAs into cells, through dsRNA transfection or through siRNA expression using a plasmid-based expression system, is increasingly being used to create loss-of-function phenotypes in mammalian cells.
In one embodiment of the invention, a double-stranded RNA (AsRNA) molecule is prepared that includes between eight and nineteen consecutive nucleobases of an invention nucleobase oligomer. The dsRNA can consist of two distinct strands of RNA that have formed a duplex, or a single strand of RNA that has autoformed a duplex (small hairpin RNA (sSh) RNA)). Typically, dsRNAs have about 21 or 22 pairs of bases, but can be smaller or larger (up to about 29 nucleobases), if desired. dsRNA can be prepared using common techniques (for example, chemical synthesis or in vitro transcription). Cases are available, for example, from Ambion (Austin, TX) and Epicenter (Madison, WI). Methods of expressing dsRNA in mammalian cells are described in Brummelkamp et al., Science 2002; 296: 550-553; Paddison et al, Genes & Devel. 2002; 16: 948-958; Paul et al., Nature Biotechnol. 2002; 20: 505-508; Sui et and 86/140 al., Proc. Natl. Acad. Sci. USA 2002; 99: 5515-5520; Yu et al., Proc. Natl. Acad. Sci. USA 2002; 99: 6047-6052; Miyagishi et al., Nature Biotechnol. 2002; 20: 497-500; and Lee et al., Nature Biotechnol. 2002; 20: 500-505, each of which is incorporated herein by reference.
Small hairpin RNAs (ShRNAs) comprise an RNA sequence having a stem-loop structure. A "stem-loop structure" refers to a nucleic acid having a secondary structure that includes a region of nucleotides that is known or predicted to form a double or duplex strand (portion of the stem) that is attached, in one side, through a region of nucleotides predominantly single-stranded (por- ”of the loop). The term "hairpin" is also used here to designate stem-handle structures. Such structures are well known in the art, and the term is used in a manner consistent with their meaning known in the art. As is known in the art, the secondary structure does not require an exact base pairing. Thus, the stem may include one or more defective mating or protrusions. Alternatively, the base pairing can be accurate, that is, without including any faulty pairing. The multiple stem-loop structures can be linked together via a linker, such as, for example, a nucleic acid linker, a flanking sequence of mRNA, another molecule, or some combination thereof.
As used here, the term "small hairpin RNA" includes a conventional rod-loop sShRNA, which forms a pre-cursor miRNA (pre-miRNA). Although there may be some variation in the range, a conventional rod-loop shRNA can comprise a rod ranging from 19 to 29 bp, and a loop ranging from 4 to 30 bp. "shRNA" also includes ShRNAs with embedded micro-RNA (mMiRNA-based sShRNAs), in which the guide strand and the passing strand of the miRNA duplex are embedded in an existing (or natural) or modified miRNA synthetic (designed). In some cases, the precursor miRNA molecule may include more than one stem-loop structure. MicroRNAs are endogenously encoded RNA molecules that have about 22 nucleotides
. 87/140 tids in length and are generally expressed in a highly specific manner for tissues or stages of development and that regulate, in the post-transcription, target genes. More than 200 different miRNAs have been identified in plants and animals. These small regulatory RNAs - are believed to perform important biological functions through two prevalent modes of action: (1) through the repression of the translation of target MRNAs, and (2) through RNA interference (RNAi), this that is, cleavage and degradation of MRNAs. In the latter case, miRNAs function similarly to small interference RNAs (siRNAs). Thus, it is possible to design and express artificial miRNAs based on the characteristics of existing miRNA genes.
In this regard, small hairpin RNAs can be designed to mimic endogenous miRNAs. Many MIiRNA intermediates can be used as templates for ShRNA or shRNAmir, including, without limitation, a miRNA comprising a main chain design of miR-15a, -16, -19b, -20, -23a, -27b, - 29a, -30b, -30c, -104, - 132s, -181, -191, -223 (see US Publication No. 2005/0075492). In some embodiments, SshnRNA molecules are designed based on the human miR-30 sequence, re-designed to allow the expression of artificial shR-NAs by replacing the pri-miR-30 stem sequences with base sequences unrelated pairs (Siolas et al, 2005, Nat. Biotech. 23: 227-231; Silva et a /., 2005, Nat. Genet. 37: 1281-1288); Zeng et al. (2002), Molec. Cell 9: 1327-1333). The miR-30 natural stem sequence can be replaced by a stem sequence having — from about 16 to about 29 nucleotides in length, in particular from about 19 to 29 nucleotides in length. The loop sequence can be changed so that the length ranges from about 4 to about 23 nucleotides. In one embodiment, the stem of the s-hRNA molecule is about 22 nucleotides in length. In another embodiment, the rod is about 29 nucleotides in length. Thus, the invention can be practiced using shRNAs that are produced synthetically, as well as microRNA (mIRNA) molecules that are found in nature
* 88/140 and that can be remodeled to function as synthetic, small silencing RNAs in hair clips. ShRNAs can be expressed from DNA vectors to provide sustained silencing and high-throughput distribution in almost all cell types. In some modalities, the vector is a viral vector. Exemplary viral vectors include retroviral vectors, including lentiviral, adenoviral, baculoviral and avian viral, and the inclusion of these vectors allows for stable single-copy genomic integrations. Retroviruses from which retroviral plasmid vectors can be derived include but are not limited to Moloney's Murine Leukemia Virus, spleen neurosis virus, Rous sarcoma virus, Harvey's sarcoma virus, viruses avian leukosis, gibbon monkey leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus. A retroviral plasmid vector can be used to transduce packaging cell lines in order to form producer cell lines. Examples of packaging cells that can be transfected include, but are not limited to, the PESOI, PA3I7, R-2, R-AM, PA12, T19-14x, VT-19-17-H2, RCRE, RCRIP, GP + E cell lines. -86, GP + envAm12, and DAN, as described in Miller, Human Gene Therapy 1: 5-14 (1990), which is incorporated herein by reference in its entirety. The vector can transduce the packaging cells by any means known in the art. A producer cell line generates infectious retroviral vector particles that include a polynucleotide that encodes a DNA replicating protein. These retroviral vector particles can then be used to transduce eukaryotic cells, in vitro or in vivo. The transduced eukaryotic cells will express a DNA replicating protein.
Essentially any method of introducing a nucleic acid construct into cells can be employed. Physical methods of introducing nucleic acids include injecting a solution containing the construct, bombarding particles covered with the construct, imbibing a cell, tissue or organism sample into a solution
- 89/140 nucleic acid, or electroporation of cell membranes in the presence of the construct.
A viral construct packaged in a viral particle can be used to effectively introduce an expression construct into the cell and transcribe the encoded sShRNA.
Other methods known in the art can be used to introduce nucleic acids into cells, such as transport by means of a lipid-mediated transporter, chemically mediated transport, such as calcium phosphate, and the like.
Thus, the shRNA-encoding nucleic acid construct can be introduced together with components that perform one or more of the following activities: increase RNA uptake by the cell, promote hybridization of the + duplex strands, stabilization of the hybridized strands , or increased inhibition of the target gene.
For expression in cells, DNA vectors can be used, for example, plasmid vectors, comprising a polymerase RNA promoter or RNA polymerase Ill.
The expression of endogenous miRNAs is controlled by promoters of RNA polymerase | (Pol II) and, in some cases, ShRNAs are very efficiently driven by promoters of Pol Il, compared to promoters of RNA polymerase | ll (Dickins et a /., 2005, Nat.
Genet. 39: 914-921). In some embodiments, sShRNA expression can be controlled by an inducible promoter or a conditional expression system, including, without limitation, type 1 RNA polymerase promoters.
Examples of promoters useful in the context of the invention are tetracycline-inducible promoters (including TRE-Tight), IPTG-inducible promoters, tetracycline transactivator systems, and tetracycline reverse (tTA) transactivator systems. Constitutive promoters can also be used, as well as specific promoters for cells or tissues.
Many promoters will be ubiquitous, so they are expressed in all types of cells and tissues.
A certain modality uses promoters that respond to tetracycline, one of the most effective conditional gene expression systems in in vitro and in vivo studies.
See International Patent Application PCT / US2003 / 030901 (Publication No. WO 2004-029219 A2) and Fewell et al., 2006, Drug Discovery Today 11: 975-982, for a description of shR-
is: 90/140 NA inducible.
Small hairpin RNAs (ShRNAs) comprise an RNA sequence having a stem-loop structure. A "stem-loop structure" refers to a nucleic acid having a secondary structure that includes a region of nucleotides that is known or predicted to form a double or duplex strand (portion of the stem) that is attached, in one side, through a region of nucleotides predominantly single-stranded (portion of the loop). The term "hairpin" is also used here to designate rod-handle structures. Such structures are well known in the art, and eotherm is used in a manner consistent with their known meaning in the art. As is known in the art, the secondary structure does not require an exact base match. Thus, the stem may include one or more defective mating or protrusions. Alternatively, the base pairing can be accurate, that is, without including any faulty pairing. The multiple rod-loop structures can be linked together via a linker, such as, for example, a nucleic acid linker, a flanking sequence of mMIRNA, another molecule, or some combination thereof. As used herein, the term "small hairpin RNA" includes conventional stem-loop shRNA, which forms a pre-cursor mRNA (pre-miRNA). Although there may be some variation in the range, a conventional rod-loop shRNA can comprise a rod ranging from 19 to 29 bp, and a loop ranging from 4 to 30 bp. "shRNA" also includes ShRNAs with embedded micro-RNA (ShRNAs based on —miRNA), where the guide strand and the passing strand of the miRNA duplex are embedded in an existing (or natural) or modified miRNA or synthetic (designed). In some cases, the precursor miRNA molecule may include more than one stem-loop structure. MicroRNAs are endogenously encoded RNA molecules that are about 22 nucleotides in length and are generally expressed in a highly specific way for tissues or stages of development and that regulate, in the post-transcription, target genes. More than 200 distinct miRNAs have been identified
- 91/140 of those in plants and animals. These small regulatory RNAs are believed to perform important biological functions through two prevalent modes of action: (1) by suppressing the translation of target mRNAs, and (2) by RNA interference (RNAi), ie , cleavage and degradation of —mRNAs. In the latter case, miRNAs function similarly to small interference RNAs (siRNAs). Thus, it is possible to design and express artificial MIRNAs based on the characteristics of existing miRNA genes.
In this regard, small hairpin RNAs can be designed to mimic endogenous miRNAs. Many MIRNA intermediates can be used as templates for ShRNA or shRNArmir, including, without limitation, a miRNA comprising a main chain design of miR-15a, -16, -19b, -20, -23a, -27b, -29a, -30b, -30c, -104, - 132s, -181, -191, -223 (see US Publication No. 2005/0075492). In some embodiments, shRNA molecules are designed based on the human miR-30 sequence, reconceived to allow the expression of artificial shR-NAs by replacing the pri-miR-30 stem sequences with base sequences unrelated pairs (Siolas et al., 2005, Nat. Biotech. 23: 227-231; Silva et al., 2005, Nat. Genet. 37: 1281-1288); Zeng et al. (2002), Molec. Cell 9: 1327-1333). The miR-30 natural stem sequence can be replaced by a stem sequence having from about 16 to about 29 nucleotides in length, in particular from about 19 to 29 nucleotides in length. The loop sequence can be changed so that the length ranges from about 4 to about 23 nucleotides. In one embodiment, the stem of the s-hRNA molecule is about 22 nucleotides in length. In another embodiment, the stem is about 29 nucleotides in length. Thus, the invention can be practiced using sShRNAs that are produced in a synthetic way, as well as microRNA molecules (miRNA) that are found in nature and that can be remodeled to function as synthetic silencing RNAs in a hairpin. sShRNAs can be expressed from DNA vectors to
- 92/140 provide sustained silencing and high-performance distribution in almost all cell types. In some modalities, the vector is a viral vector. Exemplary viral vectors include retroviral vectors, including lentiviral, adenoviral, baculoviral and avian viral, and the inclusion of these vectors allows for stable single-copy genomic integrations. Retroviruses from which retroviral plasmid vectors can be derived include but are not limited to Moloney's Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma virus, Harvey's sarcoma virus, avian leukosis virus, gibbon monkey leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus. A retroviral plasmid vector can be employed to effect the transduction of packaging cell lines in order to form producer cell lines. Examples of packaging cells that can be transfected include, but are not limited to, the PESOI, PA3I7, R-2, R-AM, PA12, T19-14x, VT-19-17-H2, RCORE, RCRIP, GP + E cell lines. -86, GP + envAm12, and DAN, as described in Miller, Human Gene Therapy 1: 5-14 (1990), which is incorporated herein by reference in its entirety. The vector can transduce the packaging cells by any means known in the art. A producer cell line generates infectious retroviral vector particles that include a polynucleotide that encodes a DNA replicating protein. These retroviral vector particles can then be used to transduce eukaryotic cells, in vitro or in vivo. The transduced eukaryotic cells will express a DNA replicating protein.
Essentially any method of introducing a nucleic acid construct into cells can be employed. Physical methods of introducing nucleic acids include injecting a solution containing the construct, bombarding particles covered with the construct, imbibing a cell, tissue or organism sample into a solution of the nucleic acid, or electroporating cell membranes in the presence of the construct. A viral construct packaged in a viral particle can be used to effectively introduce an expression construct
- 93/140 in the cell and transcription of the encoded sShRNA.
Other methods known in the art can be used to introduce nucleic acids into cells, such as transport by means of a lipid-mediated transporter, chemically mediated transport, such as calcium phosphate, and the like.
Thus, the ShRNA-encoding nucleic acid construct can be introduced together with components that perform one or more of the following activities: increased RNA uptake by the cell, promoting hybridization of duplex strands, stabilization of hybridized strands, or else increased inhibition of the target gene. mo For expression in cells, DNA vectors can be used, for example, plasmid vectors comprising a polymerase RNA promoter or RNA polymerase III.
The expression of endogenous miRNAs is controlled by promoters of RNA polymerase |! (Pol II) and, in some cases, ShRNAs are very efficiently driven by Pol | 1 promoters, compared to RNA polymerase II promoters (Dickins et a / l., 2005, Nat.
Genet. 39: 914-921). In some embodiments, ShRNA expression can be controlled by an inducible promoter or a conditional expression system, including, without limitation, type II RNA polymerase promoters.
Examples of promoters useful in the context of the invention are tetracycline-inducible promoters (including TRE-Tight), IPTG-inducible promoters, tetracycline transactivator systems, and tetracycline reverse transactivator (rtTA) systems. Constitutive promoters can also be used, as well as specific promoters for cells or tissues.
Many promoters will be ubiquitous, so they are expressed in all types of cells and tissues.
A certain modality uses promoters that respond to tetracycline, one of the most effective conditional gene expression systems in in vitro and in vivo studies.
See International Patent Application PCT / US2003 / 030901 (Publication No. WO 2004-029219 A2) and Fewell et al., 2006, Drug Discovery Today 11: 975-982, for a description of shR-
Inducible NA
”94/140 Distribution of Nucleobase Oligomers Naked inhibitor nucleic acid molecules, or their analogs, are able to enter mammalian cells and inhibit the expression of a gene of interest, for example, Brd4. Still, it may be desirable to use a formulation that aids in the delivery of oligonucleotides, or other nucleobase oligomers, in cells (see, for example, U.S. Patents.
Nos. 5,656,611, 5,753,613, 5,785,992, 6,120,798, 6,221,959, 6,346,613, and 6,353,055, each of which is incorporated herein by reference). Pharmaceutical Therapeutic Agents no In other embodiments, agents that have been found to have medicinal value (for example, JQ1 or a compound of a formula outlined a-: qui) using the methods described here are useful as a drug or as information for structural modification of existing compounds, for example, through rational drug design.
For therapeutic uses, the compositions or agents identified using the methods disclosed herein can be administered systemically, for example, formulated in a pharmaceutically acceptable buffer, such as saline.
Preferred routes of administration include, for example, subcutaneous, intravenous, interperitoneal, intramuscular, or intradermal injections, which provide continuous and sustained levels of the drug in the patient.
Treatment of human or other animals will be carried out using a therapeutically effective amount of a therapeutic agent identified here in a physiologically acceptable carrier.
Suitable carriers and their formulation are described, for example, in "Remington's Pharmaceutical Sciences" by E. W.
Martin.
The amount of the therapeutic agent to be administered varies, depending on the mode of administration, the age and weight of the patient's body, and the clinical symptoms of leukemia (for example, acute myeloid leukemia (AML), Chronic Lymphocytic Leukemia ( CLL), Acute Lymphocytic Leukemia (ALL), Chronic Myeloid Leukemia (CML), Chronic Myeloid Leukemia (CMML), Eosinophilic Leukemia, Hairy Cell Leukemia, Hodgkin's Lymphoma, Multiple Myeloma, Non-Hodgkin's Lymphoma, Myeloproliferative Disorders). In general, the quantities
- 95/140 will be in the range of those used for other agents used in the treatment of other diseases associated with leukemias, although, in certain cases, smaller quantities are required due to the increased specificity of the compound. A compound is administered at a dosage that reduces the proliferation, growth or survival of a cancer cell, determined by a method known to the practitioner, or using any of these assays that measure cell proliferation or viability.
Formulation of Pharmaceutical Compositions The administration of a compound for the treatment of a leukemia can be carried out by any suitable means that results in a concentration of the therapeutic agent that, combined with other components, is effective in reducing proliferation. or survival of a leukemic cell. The compound can be contained in any suitable amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition can be provided in a dosage form that is suitable for the parenteral route of administration (for example, subcutaneous, intravenous, intramuscular, or intraperitoneal). Pharmaceutical compositions can be formulated in accordance with conventional pharmaceutical practice (see, for example, "Remington: The Science and Practice of Pharmacy" (20th edition), editor AR Gennaro, Lippincott Williams & Wilkins, 2000, and "Encyclopedia of Pharmaceutical Technology ", editors J. Swarbrick and JC Boylan, 1988-1999, Marcel Dekker, New York). In a particular embodiment, a person of the invention is directly administered to a subject in a systematic way.
25. doctor.
Quantities of human dosage can be initially determined by extrapolating the amount of compound used in mice, since, as recognized by the professional, it is routine in the area to modify the dosage for humans compared to animal models. In one embodiment, an agent of the invention is administered orally or systemically at 50 mg / kg. In certain different embodiments, it is considered that the dosage can vary from about 1 µg of
- 96/140 to / Kg of body weight up to about 5000 mg of compound / Kg of body weight; or from about 5 mg / kg of body weight to about 4000 mg / kg of body weight, or from about 10 mg / kg of body weight to about 3000 mg / kg of body weight; or from about 50 mg / kg of body weight to about 2000 mg / kg of body weight; or from about 100 mg / kg of body weight to about 1000 mg / kg of body weight; or from about 150 mg / kg of body weight to about 500 mg / kg of body weight. In other embodiments, this dose may be about 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, - 10 750,800,850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, Á 4500, or 5000 mg / kg of body weight. In other modalities, it is considered that the doses may be in the range of about 5 mg of compound / kg of the body to about 100 mg of compound / kg of the body. In other embodiments, doses can be about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg / Kg of body weight. Obviously, this dosage amount can be adjusted upwards or downwards, as is done routinely in these treatment protocols, depending on the results of the initial clinical trials and the needs of a particular patient.
The pharmaceutical compositions according to the invention can be formulated to release the active compound substantially immediately upon administration, or at any predetermined time or period after administration. The latter types of compositions are generally known as controlled-release formulations, which include (i) formulations that create a substantially constant concentration of the drug in the body over an extended period of time; (ii) formulations that, after a predetermined delay time, create a substantially constant concentration of the drug in the body for an extended period of time; (iii) formulations that sustain action for a predetermined period of time by maintaining a relatively constant effective level in the body,
- 97/140 mitigation of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that locate the action, for example, through the spatial placement of an adjacent controlled release composition or in contact with the thymus; (v) formulations that allow for convenient dosing, so that doses are administered, for example, once every one or two weeks; and (vi) formulations that selectively address leukemia, including but not limited to acute myeloid leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), Leu- - 10 Chronic Myeloid Leukemia (CML) , Chronic Myelomonocytic Leukemia (CMML), Eosinophilic Leukemia, Hair Cell Leukemia, Hodgkin's Lymphoma, 'Multiple Myeloma, Non-Hodgkin's Lymphoma, Myelodysplasia, and Myeloproliferative Disorders. For some applications, controlled-release formulations obviate the need for frequent dosing during the day to sustain the plasma level at a therapeutic level.
Any of some strategies can be followed to achieve controlled release, in which the rate of release exceeds the rate of metabolism of the compound in question. In one example, controlled release is achieved through the appropriate selection of various parameters and ingredients of the formulation, including, for example, various types of controlled release compositions and coatings. Thus, the therapeutic agent is formulated, with appropriate excipients, into a pharmaceutical composition that, upon administration, releases the therapeutic agent in a controlled manner. Examples include compositions of a single or multiple units of tablets or capsules, oily solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, plasters, and liposomes.
Parenteral Compositions The pharmaceutical composition can be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, or the like) in dosage forms, formulations, or via delivery devices or suitable implants containing
.: 98/140 conventional, non-toxic and pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of these compositions are well known to those in the field of pharmaceutical formulation. Formulations can be found in "Remington: The Science and Practice of Pharmacy", supra.
Compositions for parenteral use can be provided in unit dosage forms (for example, in single-dose ampoules), or in vials containing several doses and to which a suitable preservative can be added (see below). The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented in the form of a dry powder to be reconstituted with water or another suitable vehicle before use. In addition to the active agent, which reduces the growth, proliferation or survival of a leukemic cell, the composition may include suitable parenterally acceptable carriers and / or excipients. The active therapeutic agent (s) can be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. In addition, the composition may include suspending, solubilizing, stabilizing, pH adjusting agents, tonicity adjusting agents, and / or dispersing agents.
As indicated above, the pharmaceutical compositions according to the invention can be in a form suitable for sterile injection. To prepare this composition, the appropriate active therapeutic agent (s) —which (s) is (are) dissolved or suspended in a liquid vehicle acceptable from the point of view of parenteral. Among acceptable vehicles and solvents that can be employed are water, water adjusted to a suitable pH by adding an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution. The aqueous formulation can also contain one or more preservatives (for example, methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the
: 99/140 compounds is only sparse or slightly soluble in water, a dissolution enhancing agent or solubilizer may be added, or the solvent may include 10-60% w / w propylene glycol or the like.
Controlled Release Parenteral Compositions Controlled release parenteral compositions can be in the form of aqueous suspensions, microspheres, microcapsules, magnetic microspheres, oily solutions, oily suspensions, or emulsions.
Alternatively, the active drug can be incorporated into bio-compatible carriers, liposomes, nanoparticles, implants, or infusion devices. 10 Materials for use in the preparation of microspheres and / or Microcapsules are, for example, biodegradable / bioerodible polymers , such as polygalactin, poly- (isobutyl cyanoacrylate), poly (2-hydroxyethyl-L-glutaminin) and poly (lactic acid). Biocompatible carriers that can be used in the formulation of a controlled-release parenteral composition are carbohydrates (eg, dextrans), proteins (eg, albumin), lipoproteins, or antibodies.
Materials for use in implants can be non-biodegradable (for example, polydimethyl siloxane) or biodegradable (for example, poly (caprolactone), lactic polyphacid), glycolic polysaccharide) or poly (ortho esters) or combinations thereof. Solid Dosage Forms for Oral Use Formulations for oral use include tablets containing the active ingredient (s) in a mixture with pharmaceutically acceptable non-toxic excipients.
These formulations are known to the professional.
Excipients can be, for example, inert diluents or fillers (for example, sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (for example, cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (eg, sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin,
- 100/140 starch, pregelatinized starch, microcrystalline cellulose, aluminum and magnesium silicate, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating, gliding, and anti-adhesive agents (for example, magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients can be dyes, flavoring agents, plasticizers, humectants, buffering agents, and the like.
The tablets may be uncoated or may be coated using known techniques, optionally to delay disintegration and absorption in the gastrointestinal tract and thereby provide sustained action for a longer period. The coating can be adapted to release the active drug in a predetermined pattern (for example, to achieve a controlled release formulation), or it can be adapted to not release the active drug until after it has passed through the stage. stomach (enteric lining). The coating can be a sugar coating, a film coating (for example, based on hydroxypropyl methylcellulose, methylcellulose, methyl hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers, polyethylene glycols and / or polyvinylpyrrolidone), or an enteric coating (for example, based on methacrylic acid cup-polymer, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose succinate, polyvinyl acetate phthalate, shellac, and / or ethyl cellulose). In addition, a time-delaying material, such as, for example, glyceryl monostearate or glyceryl distearate can be employed.
Solid tablet compositions may include a coating adapted to protect the composition from unwanted chemical changes (for example, chemical degradation prior to the release of the active therapeutic substance). The coating can be applied in the form of solid dosage in a similar manner to that described in "Encyclopedia of Pharmaceutical Technology", supra.
At least two therapeutic agents can be mixed in
. 101/140 set in the tablet, or can be partitioned. In one example, the first active therapeutic agent is contained within the tablet, and the second active therapeutic agent is on the outside, so that a substantial portion of the second therapeutic agent is released prior to the release of the first therapeutic agent.
Formulations for oral use can also be presented in the form of chewable tablets, or as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (eg potato starch, lactose, microcrystalline cellulose, 10 calcium bonate, calcium phosphate or kaolin), or in the form of soft gelatin capsules in which the active ingredient is mixed with water or an oily medium, for example, peanut oil, liquid paraffin, or olive oil.
Powders and granules can be prepared using the ingredients mentioned above for tablets and capsules in a conventional manner, using, for example, a mixer, a fluidized bed apparatus or a spray drying equipment.
Oral Controlled Release Dosage Forms Controlled release compositions for oral use can be constructed, for example, to release the active therapeutic agent by controlling the dissolution and / or diffusion of the active substance. Controlled release by dissolution or diffusion can be achieved through the appropriate coating of a tablet, capsule, pellet, or granular formulation of compounds, or by incorporating the compound into an appropriate matrix. A controlled release coating may include one or more of the aforementioned coating substances and / or, for example, shellac, beeswax, glucose wax, castor wax, carnauba wax, stearyl alcohol, monostearate glycerol, glyceryl distearate, glycerol palmitostearate, ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate, methylmethacrylate, 2-hydroxy methacrylate, 1,3-butylene glycol, ethylene glycol methacrylate, and / or polyethylene glycols. In a release matrix formulation
- 102/140 controlled ration, the matrix material can also include, for example, hydrated methylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate, methyl methacrylate, polyvinyl chloride , polyethylene, and / or halogenated fluorocarbons.
A controlled release composition containing one or more therapeutic compounds can also be in the form of a floating tablet or capsule (i.e., a tablet or capsule that, by oral administration, floats on the gastric contents for a certain period of time). A floating tablet formulation of the compound (s) can be stopped by granulating a mixture of the compound (s) with excipients and 20-75% w / w hydrocolloids, as hydroxyethylcellulose, hydroxypropylcellulose, or hydroxypropylmethylcellulose. The granules obtained can then be compressed to form tablets. Upon contact with gastric juice, the tablet forms a substantially impermeable gel barrier around its surface. This gel barrier has a role in maintaining a density less than one, thereby allowing the tablet to remain floating in the gastric juice.
Combination Therapies Optionally, a therapeutic agent for the treatment of leukemia, including but not limited to acute myeloid leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), Chronic Myeloid Leukemia (CML), Chronic Myelomonocytic Leukemia (CMML), Eosinophilic Leukemia, Hairy Cell Leukemia, Hodgkin's Lymphoma, Multiple Myeloma, Non-Hodgkin's Lymphoma, Myelodysplasia, and Myeloproliferative Disorders, are administered alone or in combination with other cancer treatments for common therapies ; these methods are known to the practitioner and are described in E. W. Martin's "Remington's Pharmaceutical Sciences". If desired, agents of the invention (for example, JQ1, compounds of the formulas outlined here, and their derivatives) are administered in combination with any conventional chemotherapeutic agent useful for the treatment of cancer.
Pharmaceutical Cases or Systems
- 103/140 The present compositions can be assembled in cases or pharmaceutical systems for use in the treatment of leukemia (for example, acute myeloid leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), Myeloid Leukemia Chronic (CML), Chronic Myelomonocytic Leukemia (CMML), Eosinophilic Leukemia, Hair Cell Leukemia, Hodgkin's Lymphoma, Multiple Myeloma, Non-Hodgkin's Lymphoma, Myelodysplasia, and Myeloproliferative Disorders). Pharmaceutical cases or systems according to that aspect of the invention comprise a carrier medium, such as a box, cardboard, tube or the like, containing, in close confinement, one or more containers, such as vials, tubes, ampoules, bottles and the like.
The pharmaceutical kits or systems of the invention may also comprise associated instructions for using the agents of the invention.
Therapy
Therapy can be provided wherever cancer therapy is performed: at home, in the doctor's office, in a clinic, in a hospital's outpatient care department, or in a hospital.
Treatment usually begins in a hospital, so that the doctor can closely observe the effects of the therapy and make any necessary adjustments.
The duration of therapy depends on the type of cancer to be treated, the age and condition of the patient, the stage and type of disease of the patient, and how the patient's body responds to treatment.
Drug administration can be performed at different intervals (for example, daily, weekly, or monthly). The therapy can be administered in cycles — with-and-without that include rest periods, so that the patient's body has a chance to build new healthy cells and regain its resistance.
As described above, if desired, treatment with a compound of the invention (eg, JQ1), an inhibitory nucleic acid molecule that selectively addresses Brd4, can be combined with therapies for the treatment of disease proliferative (for example, radiotherapy, surgery, or chemotherapy). The practice of the present invention employs, unless
- 104/140 unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which belong to the professional. These techniques are fully explained in the literature, such as "Molecular Cloning: A Labo- —ratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Syntheisis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzyme" "Handbook of Experimental Immunology" (Weir, 1996), "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain - 10 Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). * These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, can be considered in the implementation and practice of the invention. Techniques particularly useful for particular modalities will be discussed in the following sections.
The following examples are presented in order to provide professionals with a full disclosure and description of how to prepare and use the trial, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors consider to be their own. - convention.
EXAMPLES |. CHEMICAL EXAMPLES - SYNTHESIS AND METHODS OF PRE-
PARACTION The compounds of the invention can be synthesized by methods described here, and / or according to methods known to the person skilled in the art, considering the description presented here
& 105/140 Diagram S1. Synthesis of the racemic bromodomain inhibitor (+ *) - JQ1. à f re Fmoc-Asp (OrBurCH O tia NC.
Eid O S morpholine SÁ HOT FauEt sd alo dh À gor 7070 xo DNF 230 Rb s1 s2 s3 e o o meraee o are AÊ caneco - ss prt reentato ORA and nt sa e ss *: o | ec CE and her, "WA ue lRiaiaiaiaiaSiRiDiataaar oo rá Me ss Cc BEX (Z steps) tros | (2-amino-4,5-dimethylthiofen-3-yl) (4-chlorophenyl) netanone (S2) according to the scheme shown above.
Sulfur (220 mg, 6.9 mmol, 1.00 equivalent) was added, as a solid, to a solution of 4-chlorobenzoyl acetonitrile S1 (1.24 g, 6.9 mmol, 1 equivalent), 2- butanone (0.62 mL, 6.9 mmol, 1.00 equivalent), and morpholine (0.60 mL, 6.9 mmol, 1.00 equivalent) in ethanol (20 mL, 0.35 M) at 23º ºC ” . The mixture was then heated to 70 ° C.
After 12 hours, the reaction mixture was cooled to 23 ° C and was poured into saline (100 ml). The aqueous layer was extracted with ethyl acetate (3 x 50 ml). The combined organic layers were washed with saline (50 ml), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure.
The residue was purified by means of flash column chromatography (Combiflash RF system, 40 grams of silica gel, gradient O to 100% ethyl acetate-hexanes), providing S2 (1.28 g, 70 %) as a yellow solid. 3- (1 [(9H-Fluoren-9-yl)] methoxycarbonylamino) -4 - [[3- (4-
. 106/140 chlorobenzoyl) -4,5-dimethylthiophen-2-yl (amino) -4 (S) -ferc-butyl (S3) oxobutanoate
(2- (6-chloro-1H-benzotriazole-1-11) -1,1,3,3-tetramethylammonium hexafluorophosphate (HCTU) (827 mg, 2.0 mmol, 2.00 equivalents), and N, N -diisopropylethylamine (0.72 mL, 4.0 mmol, 4.00 equivalents) was added sequentially to a solution of 9-fluorenylmethoxycarbonyl-aspartic acid B-ferc-butyl ester [Fmoc-Asp (Ot-Bu) -OH] (864 mg, 2.1 mmol, 2.10 equivalents) in N, N-dimethylformamide (1.5 mL, 1.0 M) The mixture was then stirred at 23 ° C for 5 minutes.
Then S2 (266 mg, 1.0 mmol, 1 equivalent - 10 valent) was added as a solid.
The reaction mixture was stirred at 23 ° C.
After 16 hours, ethyl acetate (20 mL) and saline solution (20 mL) were added. The two layers were separated, and the aqueous layer was extracted with ethyl acetate (2 x 20 ml). The combined organic layers were washed with saline (30 ml), dried over anhydrous sodium sulfate, filtered, and were concentrated under reduced pressure.
The residue was purified by means of flash column chromatography (Combi-flash RF, 40 grams of silica gel, gradient 0 to 100% ethyl acetate-hexanes), providing S3 (625 mg, 90%) as a brown oil. (S) -fert-butyl 3-Amino-4 - ((3- (4-chlorobenzoyl) -4,5-dimethylthiophen-2-i) amino) -4-oxobutanoate Compound S3 (560 mg, 0.85 mmol, 1 equivalent) was dissolved in 20% piperidine in DMF solution (4.0 mL, 0.22 M) at 23 ° C.
After 30 minutes, ethyl acetate (20 ml) and saline solution (20 ml) were added to the reaction mixture. The two layers were separated, and the aqueous layer was extracted with ethyl acetate (2 x 20 mL). The combined organic layers were washed with saline (3 x 25 ml), dried over anhydrous sodium sulfate, filtered, and were concentrated under reduced pressure.
The residue was purified by flash column chromatography (Combiflash RF system, 24 grams of silica gel, gradient 0 to 100% ethyl acetate-hexanes), providing the free amine S4 (370 mg, 90% ) in the form of a yellow solid.
Enantiomeric purity dropped to 75% (determined by Berger Supercritical Fluid Chromatography (SFC) using a
"107/140 AS-H column). 2- (5- (4-Chlorophenyl) -6,7-dimethyl-2-0x0-2,3-dihydro-1H-thieno [2,3- e] [ 1 AJdiazepin-3-yl) (S) -ferc-butyl acetate (S5) Amino ketone (S4) (280 mg, 0.83 mmol) was dissolved in 10% acetic acid ethanol solution (21 mL, 0.03 The reaction mixture was heated to 85 ° C. After 30 minutes, all solvents were removed under reduced pressure. The residue was purified by means of flash column chromatography (Combiflash RF system, 12 grams of silica). gel, gradient O to 100% ethyl acetate-hexanes), providing compound S5 (241 mg, 95%) - 10 in the form of a white solid.The S5 enantiomeric purity was 67% (determined by Supercritical Fluid Chromatography ( SFC) Berger using an AS-H). 2- (5- (4-Chlorophenyl) -6,7-dimethyl-2-thioxo-2,3-dihydro-1H-thieno [2,3- el [ 1 AJdiazepin-3-yl) tert-butyl acetate (S6) Phosphorus pentasulfide (222 mg, 1.0 mmol, 2.00 equivalents), sodium bicarbonate (168 mg, 2.0 mmol, 4.00 equivalent) have been added sequentially added to a solution of S5 (210 mg, 0.5 mmol, 1 equivalent) in diglyme (1.25 mL, 0.4 M). The reaction mixture was heated to 90 “ºC. After 16 hours, saline solution (20 ml) and ethyl acetate (35 ml) were added. The two layers were separated, and the aqueous layer was extracted with ethyl acetate (3 x 30 ml). The combined organic layers were washed with saline (2 x 15 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by means of flash column chromatography (Combiflash RF system, 24 grams of silica gel, gradient O to 100% ethyl acetate-hexanes), providing S6 (141 mg, 65%) in the form of a brown solid with recovered S5 (73 mg, 34%).
2- (4- (4-Chlorophenyl) -2,3,9-trimethyl-SH-thieno [3,2-f [1,2, A] triazole [4,3-al [1 AJdiazepin-6-yl) tert-butyl acetate [((+) JQ1] Hydrazine (0.015 mL, 0.45 mmol, 1.25 equivalents) was added to a solution of S6 (158 mg, 0.36 mmol, 1 equivalent) in THF (2.6 mL, 0.14 M) at 0 ° C. The reaction mixture was heated to 23 ° C, and was stirred
* 108/140 da at 23 ºC for 1 hour.
All solvents were removed under reduced pressure.
The resulting hydrazine was used directly without purification.
The hydrazine was then dissolved in a 2: 3 mixture of trimethyl orthoacetate and toluene (6 mL, 0.06 M). The reaction mixture was heated to 120 ° C.
After 2 hours, all solvents were removed under reduced pressure.
The residue was purified by means of flash column chromatography (Combiflash system, 4 g silica gel, gradient 0 to 100% ethyl acetate-hexanes), providing JQ1 (140 mg, 85% in 2 steps) in the form of a white solid.
Reaction conditions further epimerized the stereogenic center, resulting in the racemate, JQ1 (determined by Berger Supercritical Fluid Chromatography (SFC) with an AS-H column). It is Scheme S2. Synthesis of (+) - JQ1 enantiomerically enriched. % scoore: À core: F Bu FTCoor8u No Fmoc-AsplOr-Bur-OH im Eneas Piperidine aa, sd PyBOP, i-Pr, NE:. EMF, 23 * C to SA IS and Q & FR am A O 72% Me nte Ns.
Me Ma e ct s2 s3 sa Pp No So, Toluene med E RISEANTIAS a ANE meta, Soda, EASAiNd ana RA and E EA re Mme 98% à CH; CONHNH ;, n-BuOH, 60 * C "sa m Me Me .. Me Me ão. jx: ss 4Jo1 3- (1I (9H-Fluoren-9-yl) methoxy] lcarbonyljamino) -4 - [[3- (4- — chlorobenzoyl) -4,5-dimethylthiofen-2-ylJamino ) (S) -fert-butyl (S3) -4-oxobutanoate (Benzotriazol-1-yloxy) tripyrrolidinophosphonium (PYBOP) (494 mg, 0.95 mmol, 0.95 equivalents), N N-diisopropylethylamine (0 , 50 mL, 2.8 mmol, 2.75 equivalents) were added sequentially to a 9-fluorenylmethoxycarbonyl-aspartic acid [Fmoc-Asp (Ot-Bu) -OH] B-tert-butyl ester solution (411 mg , 1.00 mmol, 1.0 equivalent) in N, N-dimethylformamide (1.0 mL, 1.0 M) The mixture was then stirred at 23 ° C for 5 minutes.
Then S2 (266 mg, 1.0 mmol, 1 equivalent) was added as a solid.
The reaction mixture was stirred at 23 ° C.
After 4 hours,
. 109/140 ethyl tact (20 mL) and saline (20 mL). The two layers were separated, and the aqueous layer was extracted with ethyl acetate (2 x 20 mL). The combined organic layers were washed with saline, dried over anhydrous sodium sulfate, filtered, and were concentrated under reduced pressure. The residue was purified by means of flash column chromatography (Combiflash RF system, 40 grams of silica gel, gradient 0 to 100% ethyl acetate-hexanes), providing S3 (452 mg, 72%) in the form of a brown oil. 3-Amino-4 - ((3- (4-chlorobenzoyl) -4,5-dimethylthiophen-2-yl) amino) -4- - 10 (S) -fert-butyl oxobutanoate (S4) The compound S3 (310 mg, 0.47 mmol, 1 equivalent) was dissolved in 20% piperidine in DMF solution (2.2 mL, 0.22 M) at 23 ° C. After 30 minutes, ethyl acetate (20 ml) and saline solution (20 ml) were added to the reaction mixture. The two layers were separated, and the aqueous layer was extracted with ethyl acetate (2 x 20 mL). The combined organic layers were washed with saline (3 x 25 ml), dried over anhydrous sodium sulfate, filtered, and were concentrated under reduced pressure. The residue was purified by flash column chromatography (Combiflash RF system, 24 grams of silica gel, gradient O to 100% ethyl acetate-hexane), providing the free amine S4 (184 mg, 90% ) as a yellow solid. The enantiomeric purity was 91% (checked by Chromatography with Supercritical Fluid (SFC) Berger using an AS-H column).
2- (5- (4-Chlorophenyl) -6,7-dimethyl-2-0x0-2,3-dihydro-1H-thieno [2,3- e] [1 4Jdiazepin-3-yl) ( S) -fert-butyl (S5) Amino ketone (S4) (184 mg, 0.42 mmol) was dissolved in toluene (10 mL, 0.04 M). Silica gel (300 mg) was added, and the reaction mixture was heated to 90 ° C. After 3 hours, the reaction mixture was cooled to 23 ° C. The silica gel was filtered, and was washed with ethyl acetate. The combined filtrates were concentrated. The residue was purified by means of flash column chromatography (Combiflash RF system, 12 grams of silica gel, gradient O to 100% ethyl acetate-hexanes), providing compound S5 (168 mg, 95%) in the form of a white solid. The enantiomeric purity of
. 110/140 S5 was 90% (determined by Berger Supercritical Fluid Chromatography (SFC) using an AS-H column) 2- (4- (4-Chlorophenyl) -2,3,9-trimethyl-SH-thienof3,2- f] [1,2,4Jtriazole [4,3- al [1 AJdiazepin-6-yl) (S) -tert-butyl acetate [(+) JQ1] Ss potassium tert-Butoxide (1.0 M solution in THF, 0.3 mL, 0.30 mmol, 1.10 equivalents) was added to a solution of S5 (114 mg, 0.27 mmol, 1 equivalent) in THF (1.8 mL, 0.15 M) at -78 ºC. The reaction mixture was heated to -10 ° C, and was stirred at 23 ° C for 30 minutes. The reaction mixture was cooled to -78 ° C. Diethyl chlorophosphate - 10 (0.047 mL, 0.32 mmol, 1.20 equivalents) was added to the reaction mixture. The resulting mixture was heated to -10 ° C over 45 minutes. Acetic hydrazide (30 mg, 0.40 mmol, 1.50 equivalents) was added to the regional mixture. The reaction mixture was stirred at 23 ° C. After 1 hour, 1-butanol (2.25 mL) was added to the reaction mixture, which was heated to 90 ° C. After 1 hour, all solvents were removed under reduced pressure. The residue was purified by flash column chromatography (Combiflash system, 4 g silica gel, gradient O to 100% ethyl acetate-hexanes), providing (+) - JQ1 (114 mg, 92%) at as a white solid with 90% enantiomeric purity (determined by Supercritical Fluid Chromatography (SFC) Berger using an AS-H column, 85% hexanes-methanol, 210 nm, tr (R enantiomer) = 1.59 minutes , tr (S enantiomer) = 3.67 minutes). The product was further purified by means of chiral preparative HPLC (Agilent High Pressure Liquid Chromatography using an OD-H column) to provide the S enantiomer by more than 99% ee.
1H NMR (600 MHz, CDCl3, 25 "C) 5 7.39 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 4.54 (t , J = 6.6 MHz, 1H), 3.54-3.52 (m, 2H), 2.66 (s, 3H), 2.39 (s, 3H), 1.67 (s, 3H) , 1.48 (s, 9H) * 3C NMR (150 MHz, CDCl3, 25 ° C) 5 171.0, 163.8, 155.7, 150.0, 136.9, 131.1, 130.9, 130.6, 130.3, 128.9, 81.2, 54.1, 38.1, 28.4, 14.6, 13.5, 121 HRMS (ESI) calculated for C2: H2.CIN203S [M + H] ": 457.1460, experimental 457.1451 m / z.
"111/140 TLC (EtOAc), Rf: 0.32 (UV) [a] éo = + 75 (c 0.5, CHCI;) (-) - JQ1 was synthesized in a similar way, using Fmoc-D- Asp (Ot-Bu) -OH as starting material, and was further purified by means of chiral preparative HPLC (Agilent High Pressure Liquid Chromatography using an OD-H column) to provide the R enantiomer by more than 99% ee. [A] p = - 72 (c 0.5, CHCk) Synthesis of Additional Compounds Additional compounds of the invention were prepared as illus- - 10 in Scheme S3. Scheme S3. Synthesis of hydrazine derivatives. cl cl CI 3 and —no mm —N o —N. H
OH S o to KR and O NE NH
JN AN JN (1), (+) - JO1 2) (3) cl a - o No is CX only NONO IN oH (4) As shown in Scheme S3, the (+) - JQ1 t-butyl ester (1) was cleaved to give free acid (2), which was coupled to hydrazine to provide the hydrazide (3). The reaction with 4-hydroxybenzaldehyde provided the hydrazone (4). Hydrazide (3) and hydrazone (4) exhibited activity in at least one biological assay. A compound library was prepared by reacting hydrazide (3) with a variety of carbonyl-containing compounds (see
- 112/140 Table A, above). Additional compounds have been prepared for use, for example, as probes for the development of assays.
An exemplary synthesis is shown in Scheme S4, below.
Scheme S4. Synthesis of derivatives useful as probes.
Cl CI, Cl, HCOOH, 23 C MeOococ !; je et O, B5% =, goto EN Ee dao o A dao = SO NOÔWO nO SK PP 7 “mi - CI, oH FITC, EtOH. 23 / C D Das se == N Ho Ss o o o N, ss FCOXDDPHLOS SS NONO A RR) = CcoH 5 N - o For FITC test and el ——— and TAN HO Sm own NHTA 2! EDC biotin.
HOB1 23'C Ji Pi CI, o un = n, H H "SS" FF JOANA ”NOW O o si: For Alpha test
Additional compounds were prepared as presented in RSA E DI OO oo eee and Com- Structure Name MS [M + H] "post m / z (Obtained EOA DE served) (S) -JQO1 EX, 457.1 ss E 2. Y
Õ E a a a a aa act R) -JO1 N 457.1 À (R) O - = N o o): cl VQs SE 415.1
SN 1 au Y Sen The N
A to aaa postpones and! ->. DIO. VQ4 Ev or pd 19.1 s. A Am o N = cr E OA JQ6 E 493.1 s NA Cs. = N NH MAs
EA and and A Tao vo7 SE, 579.0 s-. <
AAA / ó No O "o SO, Na SS iSSiDDS ç Ses cl
. 114/140 Name of Com- “o Estutira - MS [IMHHY rank m / z (Ob- EO E to EE AEE served) E. 494 1:. to
It is pe & Ps> »ss Ç) N o e O. Jo1o - Eh 501.1 Ss
E Y = N o ee is aa. = Do - —— FzC. vo 7 In 511.1 Ss E o. It is the e A a a he VQ1-FITC. 804.1 = N ns E * Ji VJQ1-Biotin bs o 829.3 3 so n ue 7 d red = vo13 Cc! 526.2 -) H N
HIGH s - LON = N
- 115/140 Name of the Com- Structure MS [M + H] 'post m / z (Observed) ra eee meme ee Tete eeeTO KS1 "429.1 Ss UU dy Y = N ro O o) cl RENO to VQO18 o 487.1 Lx
N 4 with the Chemical Formula: "UU -. And CH>: CINÇO, S IS Exact Mass: 486.14924 Molecular Weight: 487.01418 ci DA vQ19 Lx 471.1 F NgN are NA T e. Y Chemical Formula: NEN Yo Cr4H7 / CINÇO, S IS Mass kExata: 470.15432 Molecular Weight: 471.01478 aa. C == -
N JQ20 Fu 370.1 s NA From leader A X HW Exact Mass: 370.10190 Molecular Weight: 370.89896 JQI-1-023 er
N vJQa21 Pv 443.1
NZ ne YV sanroz. | ee: = O Chemical Formula: C ,, H7; CIN, O0; S of Exact Mass: 442.12302% J Molecular Weight: 442.96162 od anna: NO24A F 456.1 Q:: BE »05 V = N o Is Chemical Formula: CayH, CIN; O: S Exact Mass: 4551424 Molecular Weight: 456,0001 Cc Aeee
. 116/140 Name of Com- Structure MS [M + H] * post m / z (Observed) NJQO24B fi e O 456.1 sn NA A - No 4-0 O Chemical Formula: C;, H; sCINHO; S Hassa Exact: 455.14% 4 Molecular Weight: 456,0001 ci and SE 506.1 so NA in SA = NA Chemical Formula: C, yH;, CINO; SO MassExact: 5) 51339 fa: Ã Molecular Weight: 506 , 0191 Cc JQB 2,389.2 and “
O NS O Chemical Formula: C ,, l, NO, Exact Mass: 388.1899 Molecular Weight: 3684623 NQO30 is 456.2, “à s' YY É Chemical Formula: CyyH, eClNcOS o Exact Mass: 455.155 FE Weight Molecular: 456,0034 E and JO31 oil. 456.2 Ds So
S) A 1:: NA link, Chemical Formula: CoHCNsOS "Exact Mass: 455/1547 Molecular Weight: 456.0034 As - E to JQO32 E". 468.1 NA FF
E YAN AN Chemical Formula: CHiCIFANSOS H O nassa Exact: d67.0704 FE Molecular Weight: 467.8951
The Mae Siad
. 117/140 Name of the Com- Structure MS [M + H] 'post m / z (Observed) JO33 e. À 512.2 and AO So N e | A eN dd E) »Chemical Form: C; sHxCINKOS From Hassa Exact: 512,176 ci Molecular Weight: 5130548 N In JQO34 s and 505.1
NA YZ S-., And | rr = N NH is 3 Ó = Chemical Formula: CH, CINÇOS Y Exact Mass: 504/1469 ci Molecular Weight: SO5,0343 NO35 2N 540.2 * —x P NEN K | = eN NH o Chemical Formula: C.; HUyCIN; OS Exact Mass: 5392234 cy Molecular Weight: 540.1232 Jo36 ES 540.2
NZ EN N N— À Decceca
SAN À o Chemical Formula: CyHyCIN; OS Exact Mass: 539.2234 cy Molecular Weight: 5401232 NvVO37 = N. 424.2: and AN v Y 22 Chemical Formula: CzH3sN; O; S o Exact Mass: 423/1729 C y Molecular Weight: 423.5312 - SN and No38 Ta) 508.2 NA nO | NEN sN) NH
Õ ”) Chemical Formula: Ca, rt: CIN; OS% Exact Mass: 507.1808 ci Molecular Weight: 598.0382 à: 118/140 Name of Com- Structure MS [M + H]" post m / z (Ob - served) NQ39 E. TA 505.1
E SAS If f- = 1 A O = N HNA Í o D D Chemical formula: CafH,: CINHOS
SO Exact Mass: S04,1468 ci Molecular Weight: 595/1473 JQ40 se, 512,2 4 AA Md VN NA As * AO Chemical formula: Ca Ha CIN; OS “/ Exact Mass: 511,15921 Molecular Weight: 512,0700 - SEO ci REA SH JO41 es [540.2 a ”7 S / A NON 7 Ns
XY FT Í = N HN a O Chemical formula: Co; HyCIN; C LC E Exact Mass: 539.22 Molecular Weight: 540.1232 A nndnndanataa) cr - ai JQo42 and 41 2 A NL a ao 2N) Chemical roma: CasHisFN.O: S o Exact Mass: 440.1682 Molecular Weight: 440.5336
F Jo43 se Ps 494.1 NA o Bo N Q go = N NH A O Chemical Formula: CaHaCIN; OS £) Exact Mass: 493/1452 Molecular Weight: 454.0117 O. DO NQ44 aê. R 513.2 seo Sn e N Í o = N HN o Is it Chemical Formula: C, .H ;; CIN; C, S x Exact Mass: 512/1761 ei Molecular Weight: 5130548
. 119/140 NomedoCom- | | Structure MS IM + H] "station m / z (Ob- E served) JQ45 DA Es 494.1 A Na = =
A NA = Chemical Formula: CH CIN; OS &) Exact Mass: 493/1452 Molecular Weight: 494.0117 EE LEA A nana a need N o VJQ46 steel and> 499.2 Ss and Nº
N = N a f Chemical Formula: CASHs; CINsOS Of the Exact Mass: 4981969: ci Molecular Weight: 4869.0712 vo4s7 2. G 626.3 NM. Te = WeZ Chemical Formula: CaHyCINC: S Hassa Exact: 625,206 7 y n Molecular Weight: 626,2555
SS NO A AA er AE E) Jo48 N-N P 471.2
E A and Ann Exata: 470/1543 RA Molecular weight: 471.0148 = »dean VQO49 + 429.1 -. n Exact hash: 428/1074 in N Molecular Weight: Á26.935] SO NN O
AN NQ50 is 540.2 and KE Exact: 539/2234 (as accurate: ”H Pro Molecular Weight: S40.1232“ to Ns. = N dk 120/140 Name of Com- - Structure NS MS [M + H] " ] station m / z (Ob- the EEE HH RH NH served) JOS E 667.2 AA QN
AAA O - JOHHAIA4 Exact Mass: 666,: 816 | NolecuLar weight: 66 /, 1/84 ia JQs2 513.2 jatis Exact Mass: 5122125% NE - lecular: S 097: II A Paso Mo. S13.0978
IN Vos3 and A 400.1 = N, JIN A NT Me mA calming acid: SN
TAN The spectral data for each compound was consistent with the assigned structure.
"n BIOLOGICAL ACTIVITY AND METHODS OF TREATMENT
MENT Example 1: Brd4 is critical and specifically required for the proliferation of Acute Myeloid Leukemia cells.
To systematically scan for epigenetic pathways required for the maintenance of Acute Myeloid Leukemia (AML), a ShRNA screening was performed. For this purpose, a personalized shRNA library was built for the 243 known chromatin regulators. This library mainly included "writers", "readers", and 'erasers' of epigenetic marks (Figure 1A). This library of 1,095 shRNAs (three to six per gene) was built in TRMPV, a vector optimized for screening by negative selection RNAi. In a primary screen, the library was transduced, like a pool, into a cell line from a Tet-On competent AML mouse model, which included an MLL-AF9 and Nrasº * P fusion gene. (Zuber et al., Nat Biotechnol 2011; 29: 79-83). After
. 121/140 drug selection, shRNA expression was induced by the addition of doxycycline (dox). Changes in library representation after fourteen days of culture were monitored using depth sequencing of ShRNA guide strips amplified from genomic DNA (Figures 1Be2A-2D). In each of two independent replicates, 177 shR-NAs exhibited depletion greater than twenty times, which was used as a scoring criterion. Positive scores were achieved for all eight positive control sShRNAs that address essential genes (Rpa1, Rpa3, Pcna, Polr2b), as well as several shRNAs that address two known MLL-AF9 - 10 cofactors (Meni and Psip1). The genes having at least two independent sShRNAs that met the scoring criteria in the primary screen were subjected to extensive one-to-one validation using an AML MLL-AF9 / Nrasº ' º independent strain and vector system (Figure 3A) ( for additional details see PCT Publication No. WO / 2010/111712).
In both primary screens and validation stages, the ShRNAs that addressed the Brd4 transcription factor were among the most heavily depleted. Globally, Brd4 was identified as the gene most responsive to the experimental conditions of this ShRNA screening (Figures 1B and 3B).
Brd4 is a member of the BET family of proteins containing bromodomains that bind to acetylated histones to influence transcription. BRD4 is also a proto-oncogene that is mutated, via chromosomal translocation, into a rare form of squamous cell carcinoma. A role for Brd4 in leukemia has not been described. The recent development of small molecule BET bromodomain inhibitors (Filippakopoulos et al., Nature 2010; 468: 1067-73), together with the identification of Brd4 as the most responsive gene in the ShRNA screening mentioned above , suggested that Brd4 is a new drug target for the treatment of AML. Five independent Brd4 shRNAs showed a close correspondence between silencing efficiency and growth inhibition, indicating effects on the target (Figures 6A and 6B). Suppression of Brd4 led to cell cycle arrest and leukemic cell apoptosis, whereas
* 122/140 equivalent silencing in immortalized murine embryonic fibroblasts (MEF) led only to a modest inhibition of the cell cycle without cytotoxicity (Figures 4A-4D). Brd4 silencing was also not able to influence the growth of unprocessed G1E erythroblasts (Figure 4E). In addition, ShRNAs addressing BRDA4 were also sufficient to induce cell cycle arrest in two human AML MLL-AF9 + strains (Figure 5A-5D). Together, these results indicated that Brd4 is a critical requirement in AML MLL-AF9 +. Example 2: The proliferation of acute myeloid leukemia - 10 (AML) cells is specifically blocked by the bromodomain protein inhibitor JQ1.
The effects of JQ1, a small molecule inhibitor, first in class, of BET bromodomains with the highest affinity for the first Brd4 bromodomain (Filippakopoulos et a /., 2010), were tested on a variety of cell types. leukemia. The proliferation of MLL fused mouse leukemia cells was remarkably sensitive to submicromolar concentrations of JQO1, compared to fibroblasts and G1E (Figure 6B), in agreement with the relative impact of ShRNAs to Brd4 on the proliferation of these different cell types. The growth inhibitory effects of JO1 in a series of human leukemia cell images established in adult and pediatric primary leukemia samples were also examined. A large growth suppressing activity of JQ1 (IC50 <500 nM) was observed in 13/14 AML cell lines (Figures 6C and 7A) and 12/15 primary AMLs through various genetic subtypes (Figures 8 and 9). In addition, 3/3 primary pediatric leukemias with rearranged MLL tested were highly sensitive to JQ1 (Figures 9A and 9B), whereas other non-AML leukemia cell lines and tested solid tumors exhibited minimal sensitivity to the compound ( Figures 6C and 7B). In all tested AML strains, treatment with JO1 universally triggered cell cycle arrest and apoptosis, similar to the effects observed after shRNA-mediated Brd4 silencing (Figures 6D, 6E, 8A-8D, 9A- 9C, 10A-10C). In
"123/140 together, these data indicate that Brd4 is important for the growth of AML in vitro, which can be effectively addressed selectively using the bromodomain inhibitor JQ1.
Example 3: Leukemia progression in vivo is inhibited by suppression of Brd4.
The in vivo relevance of Brd4 for AML progression was investigated. To suppress Brd4 in AML established in mice, leukemia cells MLL-AF9 / Nrasº * competent for Tet-On were transduced with TRMPV constructs containing anti-Brd4 shRNAs or containing control shRNAs. These cells were then transplanted into secondary recipient mice that had previously been irradiated sublethally. After the onset of the disease, which was confirmed by means of bioluminescent imaging, the expression of shRNA was induced by administering doxycycline (Figures 11A-11F). Subsequent monitoring revealed that Brd4 suppression resulted in a marked delay in the progression of leukemia and provided a significant survival benefit (Figures 12A-12C). Taking advantage of the dsRed reporter linked to shRNA expression in the TRMPV vector (Zuber ef al., Nat Biotechnol 2011; 29: 79-83), flow cytometry analysis found that cells positive for shRNA for Brd4 were depleted in the load terminal leukemia compared to controls. These data indicate that the lethality in the mice studied was a consequence of an expansion of cells negative for ShRNA to Brd4 (Figures 12D and 12E). Taken together, these data indicate that suppression, mediated by RNAi, of Brd4 inhibits the expansion of leukemia in vivo.
Example 4: Treatment with JQ1 inhibits established AML in vivo.
To examine whether JQ1 has activity as the sole agent in AML, mice transplanted with MLL- —AF9 / Nras ** leukemia cells were treated with daily injections of JQ1 (50 mg / kg) or vehicle. Administration of JQ1 led to a marked retardation of disease progression and significantly prolonged survival (Fig.
F 124/140 ras 12F-12H). JQ1 also exhibited single agent activity in the established disease scenario, as observed in AML models MLL-AF9 / Nrasº and in AML1-ETO9a / Nrasº " P / p53” (Figures 121, 13A-13E, and 14A-14C), both of which are known to be insensitive to conventional chemotherapy (Zuber et al, Genes Dev 2009; 23: Consistent with previous findings (Filippakopoulos et al., Nature 2010; 468: 1067-73), treatment with JQ1 was well tolerated in mice, with little, if any, impact on normal hematopoiesis (Figures 15 , 16, 17A and 17B). These findings demonstrate that JQ1 has potent and specific effects * 10 paraleukemia as the only agent in vivo. Example 5: Inhibition of Brd4, by ShRNA or JQ1, reduces the stem cell potential of leukemia cells and induces their differentiation.
AML is characterized by an expanded capacity for self-renewal linked to an inability to complete terminal myeloid differentiation. Thus, it was then considered whether the presence of Brd4 influences the state of differentiation of leukemia cells. Both the expression of shRNA for Brd4 and the treatment with JQ1 altered the morphology of MLL-AF9 / Nrasº * Leukemic cells, from myelomonocytic blasts to cells with a macrophage-like appearance (Figures 18A and 18B). By inhibiting Brd4, through treatment with sShRNA or JQ1, regulated genes positively involved in macrophage functions and Mac-1, a myeloid differentiation marker. The inhibition of Brd4 negatively regulated c-kit, whose levels are correlated with cell frequencies - leukemic stem cells (LSC) in leukemia with MLL rearrangement (Figures 18C and 18D). In addition, treatment with JQ1 induced morphological signs of maturation phenotypes in most of the tested samples of primary leukemia, although to varying degrees (Figures 8 and 9). In order to further validate whether Brd4 suppression eradicates the LSC compartment, Gene Set Enrichment Analysis (GSEA) was performed on expression microarrays obtained from leukemia cells treated with ShRNA for Brd4 and JQ1 (Subramanian ef al ., Proc
* 125/140 Natl Acad Sci USA 2005; 102: 15545-50). GSEA revealed significant positive regulation of macrophage-specific gene expression after inhibition of Brd4 (Figures 18E and 18F), as well as global loss of a gene expression signature that has previously been shown to distinguish LSCs from —setsets of leukemia cells that do not exhibit self-renewal (Figures 18G and 18H) (Somervaille et al., Cell Stem Cell 2009; 4: 129-40). Figure 181 includes graphs showing RT-qPCR results. A similar profile of alterations in gene expression was observed in a human AML cell line treated with JQ1 THP-1 (Figure 19). It is important to note that the strong phenotypic similarity between Brd4 silencing via shRNA and pharmacological inhibition of BET bromodomains between these assays establishes that Brd4 is a target for JO1. Accordingly, these results reveal that Brd4 is essential for the maintenance of leukemic stem cell populations and for the prevention of their terminal differentiation.
Example 6: In murine and human leukemia cells, JQ1 suppresses the Myc pathway, a pathway associated with self-renewing leukemic stem cells.
Since the Myc pathway is associated with self-renewal of leukemic stem cells and Myc appears to be a downstream target of —Brd4, the effects of Brd4 inhibition on Myc levels have been studied. In leukemia cells MLL-AF9 / Nrasº ** in mice, inhibition of Brd4 via treatment with sShRNAs or JQ1 led to a dramatic reduction in Myc MRNA levels and Myc protein levels; in contrast, inhibition of Brd4 had minimal effects on MEF or G1E cells (Figures 20A-20C, 21A, e21B) The negative regulation of Myc MRNA levels occurred within a 60-minute period of exposure to JQ1, qualitatively preceding the expression increased number of genes related to macrophage differentiation, such as Cd74 (Figure 20D). In addition to supporting direct transcription regulation, chromatin immunoprecipitation experiments identified a focal region of Brd4 focal -2 kilobases upstream of the Myc promoter, which was eliminated after exposure to JQ1 (Figure 20E). As expected, the suppression, induced by RNAi or JQ1, of inhibition of
"126/140 Brd4 with sShRNA or JQ1 also led to an overall reduction in Myc target gene expression (Figures 21C and 22) (see also, Kim et al, Cell 2010; 143: 313-24; and Schuhmacher et al ., Nucleic Acids Res 2001; 29: 397-406. Notably, treatment with JQ1 triggered regulation - Mycegative negative - a wide range of mouse and human leukemia cell lines examined (Figures 20A-20C, Figures 23A and 23B ), indicating that JQ1 provides a means to suppress the Myc pathway in a range of leukemia subtypes.
Figure 21D includes GSEA plots that assess changes in expression of target genes downstream of Myc.
Example 7: Brd4 regulates cell survival via an independent Myc E pathway. Next, experiments were conducted to further assess whether the antiproliferative effects of treatment with JQ1 occur via suppression of Myc activity.
Here, leukemia cultures were generated MLL-AF9 / Nrasº ' so that Myc cDNA was expressed ectopically from a retroviral promoter, which resulted in mild but constitutive Myc overexpression that was totally resistant to JQ1-induced transcription suppression (Figures 20F, 24A and 24B). Notably, ectopic Myc conferred almost complete resistance to JQO1, sShRNA-induced cell cycle arrest for Brd4, and macrophage differentiation (Figures 20G, 20H, and 25A-D). In addition, the establishment of the global expression profile revealed that the vast majority of transcription changes induced by JO1 are, in fact, side effects of Myc negative regulation (Figures 26A-26C). Myc's own shRNA silencing also triggered a pattern of growth arrest and myeloid differentiation resembling Brd4 inhibition (Figures 27A-D), which further supports Myc as an important mediator of effects induced by Myc JQ1. It is important to note that ectopic expression of Myc was unable to prevent cell death induced by JQ1, suggesting additional roles, independent of Myc, for Brd4 in the regulation of cell survival (Figures 24C and 24D). These findings indicate that Brd4 plays an important role in maintaining Myc activation to preserve a
undifferentiated cellular status in leukemia.
Following an undistorted screening approach aimed at epigenetic regulators, Brd4 was identified as a critical factor required for the maintenance of AML disease. Since Brd4 is not evidently mutated or overexpressed in AML (Figures 28A and 28B), the fine sensitivity of leukemia cells to the inhibition of Brd4 would not have been revealed simply through the genetic characterization or transcription of this disease . In addition, the results described here demonstrate that the JQ1 bromodomain inhibitor has broad activity in different contexts - 10 deAMLe, by comparing its effects with those induced by ShRNAs for Brd4, provide evidence that Brd4 is the relevant target para It is the anti-leukemic activity of JQ1. JQO1 is a robust anti-leukemic molecule with a half-life in rodents of about one hour (Figure 29).
These effects are also seen in vivo with sShRNAs for Brd4, highlighting unequivocally the usefulness of RNAi screening in revealing new targets for cancer drugs.
As a competitive inhibitor of the acetyl-lysine binding domain, JQ1 interferes with Brd4's ability to “read histone acetylation marks that facilitate transcription activation (Filippakopoulos et a /., 2010).
When applied to leukemia cells, JQ1 interferes with transcription circuits that support self-renewal; thus, JQ1 induces terminal differentiation in leukemic stem cells (LSCs). Myb is a central mediator of transcription programs induced by MLL-AF9 and is important for aberrant self-renewal states, so that Mybé inhibition is sufficient to eradicate the disease (Zuber et al, submitted). It is interesting to note that alterations in gene expression, generated after genetic or pharmacological inhibition of Brd4, are remarkably similar to those observed by suppression of MLL-AF9 or Myb (Figures 30A-30C). However, treatment with JQ1 does not influence the expression of Hoxa7, Hoxa9, or Meis1, which are well-established direct targets of MLL-AF9. This indicates that the inhibition of Brd4 does not neutralize the global function of MLL-AFS9; instead, it suppresses a large subset of other downstream targets, for example, via an
P 128/140 Myc lesson.
Taken together, it appears that MLL-AF9, Myb, and Brd4 exhibit a functional intersection in a common transcription circuit essential for malignant self-renewal.
A key effector of this program is the Myco oncoprotein (Zuber et al., Submitted), which has been validated as an attractive therapeutic target but which is not prone to traditional pharmacological inhibition.
The examples mentioned above demonstrate decisively that the selective Brd4 approach extinguishes Myc expression and limits self-renewal with selectivity for the leukemic context, thereby avoiding hematopoietic toxicities potentially associated with systemic Myc inhibition.
As a result, inhibition of Brd4 via silencing by means of RNAi or treatments with JQ1 defines a specific and effective strategy for disarming elusive oncogenic pathways related to murine and human leukemias through direct modulation of the epigenetic machinery.
The results reported here in the Examples above were obtained using the following materials and methods.
Plasmids For conditional RNAi experiments, SshRNAs were expressed from the TRMPV-Neo vector or TITMPV-Neo vector, which were previously described (Zuber et al., Nat Biotechnol 2011; 29: 79-83). For screening validation, ShRNAs were cloned into LMN (MSCV-miR30-PGK-NeoR-IRES-GFP), which was generated based on LMP3 by replacing the PuroR transgene with a NeoR cassette.
For Myc rescue experiments, the wild type mouse Myc cDNA was subcloned into MSCV-PGK-Puro-IRES-GFP (MSCV-PIG) (Hemann et al., Nat Genet 2003; 33: 396-400). Pooled negative selection RNAi screening A custom shRNA library was designed to selectively target 243 mouse genes regulating chromatin using BIOPREDsi predictions adapted to miR30 (Huesken ef al., Nature Biotechnology 2005; 23: 995-1001) (6 shRNAs / gene), and was constructed by PCR cloning of a pool of oligonucleotides synthesized in customized 55k series (Agilent Technologies, Lexington, MA)
'129/140 as previously described (Zuber et al., 2011). After checking the sequences, 1095 shRNAs (3-6 / gene) were combined with several positive and negative control SshRNAs, at equal concentrations, in one meeting. This meeting was subcloned in TRMPV-Neo and transduced into MLL-AF9 / Nrasº 'leukemia cells Tet-On using conditions that predominantly led to a single retroviral integration and represent each ShRNA in an estimated number of> 500 cells (total of 30 million cells in infection, 2% transduction efficiency). The transduced cells were selected for 5 days using 1 mg / mL of G418 (Invitrogen, Carls- - 10 bad, CA); at each pass,> 20 million cells were maintained to preserve the representation of the library throughout the experiment. After drug selection, TO samples (-20 million cells per replicate) were obtained and the cells were subsequently cultured by adding 0.5 mg / ml of G418 and 1 µg / ml of doxycycline to induce sSshRNA expression. .
After 14 days (= 12 passages, T14), each replicate was screened — 15 million cells expressing shRNA (dsRed + / Venus +) using a FACSAriall "M (BD Biosciences, Sparks, MD). TO and T14 samples were isolated by means of two rounds of extraction with phenol using PhaseLock "tubes" (5Prime, Gaithersburg, MD), followed by precipitation with isopropanol. Libraries of in-depth sequencing models were generated by PCR amplification of sSshRNA guide tapes as previously described (Zuber et a /., 2011). The libraries were analyzed on an Illuminaº Genome Analyzer (San Diego, CA) at a final concentration of 8 pM; 18 nucleotides were sequenced using a '' primer 'that reads in reverse for the guide strip (miR30EcoRISeg, TAGCCCCTTGAATTCCGAGGCAGTAGGCA. To provide sufficient reference to detect shRNA depletion in experimental samples, it was desirable to acquire> 500 readings per ShRNA in the sample TO, which required> 10 million readings per sample to compensate for disparities in the representation of shRNA inherent in the assembled plasmid preparation or introduced by PCR distortions.These conditions acquired TO references of> 500 readings for 1072 (97% of the total) ShRNAs.
Invention Patent Descriptive Report for "COMPOSITE
PHARMACEUTICAL TION AND CASE UNDERSTANDING AN AGENT THAT INHIBITS BRDA4, USES OF THAT AGENT, METHOD OF DETECTION OF THE LEUKEMIC CELL RESPONSE CLINICAL RESPONSE, AND METHOD OF TREATMENT REGIME SELECTION ",
CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Applications No. 61 / 334,991, filed on May 14, 2010; 61 / 370,745, registered on August 4, 2010; 61 / 375,863, registered on August 22, 2010; * t 10 61 / 467,376, registered on March 24, 2011; and 61 / 467,342, registered on March 24, 2011. The content of these requests is incorporated here | by reference in its entirety AFFIRMATION OF RIGHTS TO INVENTIONS CARRIED OUT
UNDER RESEARCH SPONSORED BY THE FEDERAL VIA This work was sponsored by the following grant from the National Institutes of Health, Grant No. KO8CA128972. The government has certain rights in the invention.
BACKGROUND OF THE INVENTION Acute Myeloid Leukemia (AML) represents a paradigm for understanding how complex patterns of cooperating genetic and epigenetic changes lead to tumorigenesis. This complexity poses a challenge to the development of targeted therapy, and several genetic mutations of AML usually converge in a functional way in the deregulation of similar nucleus cellular processes. A key event in the initiation of AML is the corruption of programs of cellular fate, so that they generate Leukemic Stem Cells (LSCs) that openly self-renew and thus maintain and spread the disease. Despite being incompletely understood, this process was linked to changes in regulatory chromatin modifications, whose impact on gene expression is well characterized. Thus, common oncogenes in AML, such as the A-ML1I-ETO and MLL fusion proteins, induce self-renewal programs, at least in part, through the reprogramming of epigenetic pathways. Several epigenetic regulators are targets of somatic mutation. Since the changes

权利要求:
Claims (24)
[1]
1. Method of treating leukemia or a related disorder in a subject, characterized by the fact that it comprises administering to the subject an effective amount of an agent that inhibits Brd4, or a derivative thereof.
[2]
2. Method according to claim 1, characterized by the fact that the agent is a compound with any of Formulas I-XXII, or any compound disclosed herein, or a derivative thereof.
[3]
3. Method according to claim 1, characterized by * 10 fatodequea leukemia is acute myeloid leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), Chronic Myeloid Leukemia (CML), Myelomonocytic Leukemia Chronic (CMML), Eosinophilic Leukemia, Hairy Cell Leukemia, Hodgkin's Lymphoma, Multiple Myeloma, Non-Hodgkin's Lymphoma, Myeloproliferative Disorders or —myelodysplastic Syndromes.
[4]
4. A method of reducing the growth, proliferation or survival of a leukemic cell, characterized by the fact that it comprises contacting the cell with an effective amount of an agent that inhibits Brd4 or a derivative thereof, thereby reducing growth, proliferation or survival of a leukemic cell.
[5]
5. Method of inducing cell death or terminal differentiation in a leukemic cell, characterized by the fact that it comprises contacting the cell with an effective amount of an agent that inhibits Brd4 or a derivative thereof, thereby inducing cell death or terminal differentiation —Nuclear cell.
[6]
Method according to any one of claims 1 to 4, characterized by the fact that the cell is in a subject.
[7]
Method according to any one of claims 1 to 4, characterized in that the cell is derived from acute myeloid leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), Myeloid Leukemia Chronic (CML), Chronic Myelomonocytic Leukemia (CMML), Eosinophilic Leukemia, Hairy Cell Leukemia, Lin-
Hodgkin's form, Multiple Myeloma, Non-Hodgkin's Lymphoma, Myelodysplasia or Myeloproliferative Disorders.
[8]
8. Method of treating acute myeloid leukemia in a subject, characterized by the fact that it comprises administering to an individual in need an effective amount of an agent that inhibits Brd4, thereby treating acute myeloid leukemia in a subject.
[9]
Method according to any one of claims 1 to 8, characterized in that the agent is a small compound or molecule of inhibitory nucleic acid.
[10]
Oo 10. Method according to claim 8, characterized by the healthy fact that the small compound is JQ1 or a derivative thereof.
[11]
Method according to claim 8, characterized in that the inhibitory nucleic acid molecule is a siRNA, shRNA or antisense nucleic acid molecule.
[12]
12. Method according to claim 8, characterized by the fact that the subject is a mammal.
[13]
13. Method according to claim 12, characterized by the fact that the subject is a human patient.
[14]
Method according to claim 13, characterized by the fact that the human patient is an adult.
[15]
15. Method according to claim 13, characterized by the fact that the human patient is a child.
[16]
16. Method according to claim 8, characterized by the fact that it reduces the growth, proliferation or survival of a leukemic cell in a subject.
[17]
17. Pharmaceutical composition characterized by the fact that it comprises a therapeutically effective amount of an agent that inhibits Brd4 or a derivative thereof in a pharmaceutically effective excipient.
[18]
18. Kit for the treatment of leukemia, characterized by the fact that it comprises a therapeutically effective amount of an agent that inhibits Brd4, and written instructions for administering the compound for use in the method as defined in claim 8
[19]
19. Method for detecting the clinical response capacity of a leukemic cell, characterized by the fact that the method comprises contacting a leukemic cell with a Brd4 inhibitory agent, or its derivative, and detecting the expression of a specific differentiation for macrophages, in which an increase in the expression of the specific differentiation marker for macrophages indicates that the cell responds to the agent.
[20]
20. Method of selecting a treatment regimen for a subject identified as suffering from leukemia, characterized by the fact that> 10 the method involves contacting a subject's leukemic cell with a Brd4 inhibitor, or its derivative, and detecting in the cell the expression of a specific differentiation marker for macrophages, in which an increase in the expression of the specific differentiation marker for macrophages indicates that a treatment regimen including this agent should be selected for the subject.
[21]
21. Method of detecting the clinical response capacity of a leukemic cell, characterized by the fact that it comprises contacting a leukemic cell with a Brd4 inhibitory agent, or its derivative, and detecting in the cell the expression or biological activity of myc, in which a decrease in myc expression or biological activity indicates that the cell responds to the agent.
[22]
22. Method of selecting a treatment regimen for a subject, characterized by the fact that it comprises contacting a leukemic cell with a Brd4 inhibitory agent, or its derivative, and detecting the expression or biological activity of myc , in which a decrease in the expression or biological activity of myc indicates that a treatment regimen including this agent should be selected for the subject.
[23]
23. Use of an agent that inhibits Brd4 or its derivative, characterized by the fact that it is in the preparation of a pharmaceutical composition or kit to treat leukemia or a related disorder in a subject, reduce the growth, proliferation or survival of a leukemic cell, induce cell death or terminal differentiation in a leukemic cell,
treat acute myeloid leukemia in a subject, detect the clinical response capacity of a leukemic cell, and / or select a treatment regimen for a subject identified as suffering from leukemia
[24]
24. Invention, in any form of its embodiments or in - any applicable category of claim, for example, product, process or use encompassed by the material initially described, revealed or illustrated in the present patent application.
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AREA PALLAS auKS2 txmAT Dlastocytes in AML = acute myeloid leukemia; CML = chronic myeloid leukemia * obtained from "Guide to Leukemia-Lymphoma Cel) Lines", Hans G. Drexler, Rlenã Collection of Wicrorganisnos and Cell Cultures, Braunschwveig Rlemanha, 2nd edition, 2010 R —— m——
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Tso É 1 NAOE E A Asç, a and RITO ço! ã E ar nan ATA ne Db ad. q o & a: z ú 8 Ma gn OM TBMO BO 1000 concentration JQ1 (NM) Cell line - cell description GOIMR-O primary fibroblasts human lung DOBI6-FIO murine metastatic melanoma uses human osteosaccharide of HeLa human cervical adenocarcinoma -
Figure 8A who gs cream pays ES fears, SAGE AA Auade BABI ANS ATA IF MD Cariólapa o erations ORA AR ERRRO ERRÓ ERAR E RARO aseotio TETTA £ O Eco Go MO aMenatáda 36 9 7mo cv mus Pro MO eso 8a6 alice cu INPMAMO MEO 6% 00% dTaVWMihzA MULAPS FLTIDOSS NPIMTM) Pa mM Se MO ARLGGENPMEMO 1945 25% 58% sexy FLISTO KIT DOE.
NPedimo js om mM O AME NPEMOO PELA PURE Bh ABA Fira MD.
NetIM already or Mo aMmentameneetticao 98 6h 60 ana FI TO [rom So Nm love pens 88 Bh AMO cambio Ee FG MS AML In nidiateada MLP TR 7% 4SNOGTTA then By cm MEO AM postpones 60 (ho ah araxamaidas j% FO 48 MO Amenioviao 082 ATO 69 Asnuavie ATO janom SN Mo amceiBato (65 6% 80% aRaXfaA7) fe AAA Mo AMAR AA AA AR RO o RSRCNARI ra tania ne THESE patients were analyzed at recidivism.
Abbreviations: COB, convagen white blood cells; F, female; M, male; FAB, French-. American-British Cooperative Study Croup; WHO, World Health Organization; SP, peripheral blood: HO, bone marrow; NPHIM, MPII mutated Figure $ 8B SE Fundraising EN-tarnddias TIRATION A To NmanbaraSs TITO scitoquinas * -cytoquinas Apoptotic cells (Ciensa) |. AML; ICSQ (M) ICSQIM) SOGNMJOT 1O0CAMIAO! Naturation à TERES o ní T AS gm oo 7 u 22 + tnacrotago) | to the Pan nt nt nt * se 420 ta 18 ++ tmacrófago) It is to the nt 30 so + beginning) hand am + aa1oido) so ne as 2 <inieloido) .. so 38 od - i sm so so o EE oo mo cc and Ss ++ immacrophage) a no so nt ne nd nt Í 2 so no nt E are Ha Foo MOLMI3 + er vs - TC cells are incubated with 701 in the presence of C-CSF (100 ng / ml) , SCF (100 ng / ml), and TL-8 (100 ng / 2L) 77 The percentage of apoptotic cells was determined in civocentrifuge slides by Wright-Ciensa staining; percentages of apoptotic cells measured in a control medium (usually <10% cells) were subtracted in each case
Figure $ C 205 ao e REALE b au ARE .. AA Am TS g E A .. & AMLSID Sá: au O aa EE RA oa nm AML 12 o HO WAY HO AGA est) à Es:: ao SE são ELptan ao SAAENA At . It is what presents nun = -. Aa co asna ENO RE ENE TÁ bore concentration JOL (nt) JoL tnMi concentration Fiqura 8D for Dwarf o,
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Figure 94 - Aptosis Induction ** 1 m Geo Molecular / - WwsTI "Apoptotic Cells (AVB) sample Ages - Phenotype - Cytogenotic ICSO (nt SOONM.JO! 100ONMJAI Naturation! TED TactenceMPAL fimioioid) MICAR Jia à WLinfosde) PEDNS Lactente MPAL E 'myeloide) - MLLAFA o 3 1 “ruMieloide) PEDOSI Criença - AML FETO so8 a =: PEDOOS Child - AM menossomía RGRARI | 4s1 io» + (Myeloid) Ani Pale infant, MULENL AND us iihinfoide) PEDIGA Crisnça AML GBFB -MYHOO, 1 21 as Masi UA Aa HELAFA FUTATOO TIS o Fa i Abbreviations: HPAL: Acute Phenotype Mixed Leukemia nt: not tested 7h percentage of apoptotic cells was determined by 13-pin flow cytometry to Annex V (AVE); Apoptoric cells measured in control Uçio were subtracted in each case Figure 9B e. sm PEDOSS É Fr -— PEDOS1 3> PEDOOS Es 601345 Sb x »* PEDO63 a. q ne MASI 4th dreguiao Section PEDO2S 4 kills. 3 and naaaad ! LN SAS E g.
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引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

US3709898A|1971-02-09|1973-01-09|Upjohn Co|Process for the production of triazolobenzodiazepines and intermediates|

---

US3681343A|1971-05-11|1972-08-01|Upjohn Co|6-phenyl-s-triazolo{8 4,3-a{9 {8 1,4{9 {0 benzodiazepines|

---

US3812259A|1971-08-09|1974-05-21|Upjohn Co|Animal feed and process|

---

CH622019A5|1975-10-23|1981-03-13|Upjohn Co|Process for the preparation of aminotriazolobenzodiazepines|

---

FR2329668B1|1975-10-27|1979-07-27|Upjohn Co|

---

EP0087850A1|1982-01-04|1983-09-07|The Upjohn Company|Benzodiazepines for anti-hypertensive use|

---

DE3435973A1|1984-10-01|1986-04-10|Boehringer Ingelheim KG, 6507 Ingelheim|PHARMACEUTICAL COMPOSITIONS CONTAINING DIAZEPINE WITH PAF-ANTAGONISTIC EFFECT|

---

DE3724164A1|1986-07-25|1988-01-28|Boehringer Ingelheim Kg|Novel 1,4-benzodiazepines, their preparation and use|

---

EP0315698B1|1987-05-28|1993-03-03|Yoshitomi Pharmaceutical Industries, Ltd.|Thienodiazepine compounds and medicinal application of the same|

---

JPH0676326B2|1988-05-24|1994-09-28|吉富製薬株式会社|Cardiovascular drug|

---

EP0368175A1|1988-11-06|1990-05-16|Boehringer Ingelheim Kg|3S,7S-3--5--7,10-dimethyl-3,4-dihydro-2H,7H-cyclopenta[4,5]thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine.|

---

EP0387613A1|1989-03-03|1990-09-19|Boehringer Ingelheim Kg|Thienodiazepines|

---

YU133090A|1989-07-12|1993-10-20|Boehringer Ingelheim Kg.|NEW HETEROASEPINES WITH PAF-ANTAGANOSTIC EFFECT AND PROCEDURE FOR THEIR PREPARATION|

---

DE4010528A1|1990-04-02|1991-10-17|Boehringer Ingelheim Kg|PRECARATIVE CHROMATOGRAPHIC SEPARATION METHOD FOR THE PRODUCTION OF ENANTIOMERALLY PURE HETRAZEPINE|

---

US5593988A|1991-10-11|1997-01-14|Yoshitomi Pharmaceutical Industries, Ltd.|Therapeutic agent for osteoporosis and diazepine compound|

---

WO1994006802A1|1992-09-18|1994-03-31|Yoshitomi Pharmaceutical Industries, Ltd.|Thienodiazepine compound and medicinal use thereof|

---

US5658780A|1992-12-07|1997-08-19|Ribozyme Pharmaceuticals, Inc.|Rel a targeted ribozymes|

---

EP0692483A1|1993-03-30|1996-01-17|Yoshitomi Pharmaceutical Industries, Ltd.|Cell adhesion inhibitor and thienotriazolodiazepine compound|

---

WO1995032963A1|1994-06-01|1995-12-07|Yoshitomi Pharmaceutical Industries, Ltd.|Thienylazole compound and thienotriazolodiazepine compound|

---

AT219660T|1994-09-30|2002-07-15|Inex Pharmaceuticals Corp|AGENT FOR INSERTING POLYANIONIC MATERIALS IN CELLS|

---

US5753613A|1994-09-30|1998-05-19|Inex Pharmaceuticals Corporation|Compositions for the introduction of polyanionic materials into cells|

---

US5656611A|1994-11-18|1997-08-12|Supratek Pharma Inc.|Polynucleotide compositions|

---

US6221959B1|1994-11-18|2001-04-24|Supratek Pharma, Inc.|Polynucleotide compositions|

---

US6353055B1|1994-11-18|2002-03-05|Supratek Pharma Inc.|Polynucleotide compositions|

---

AT203026T|1995-01-06|2001-07-15|Hoffmann La Roche|HYDROXYMETHYL IMIDAZODIAZEPINE AND ITS ESTER|

---

TR199800406T1|1995-09-09|1998-05-21|F.Hoffmann-La Roche Ag|Use of a thienotriazolodiazepine to increase Alipoprotein A-I levels.|

---

PL330635A1|1996-06-12|1999-05-24|Japan Tobacco Inc|Inhibitor of cytokin production, triazepin compound and intermediate compounds used for obtaining the last mentioned one|

---

KR100338144B1|1996-09-13|2002-05-24|가마쿠라 아키오|Tienotriazolodiazepine compounds and medicinal uses thereof|

---

US6444664B1|1997-04-18|2002-09-03|Nederlandse Organisatie Voor Toegepast - Natuurweten Schappelijk Onderzoek |Method for controlling the plasma level of lipoproteins to treat alzheimeris disease|

---

AU7983798A|1997-06-23|1999-01-04|Sequus Pharmaceuticals, Inc.|Liposome-entrapped polynucleotide composition and method|

---

IE970794A1|1997-09-24|2000-08-23|Elan Corp Plc|Composition and method for enhancing paracellular transport across cell layers|

---

JPH11228576A|1997-12-10|1999-08-24|Japan Tobacco Inc|Apoptosis inhibitor|

---

FR2779652B1|1998-06-15|2001-06-08|Sod Conseils Rech Applic|USE OF DIAZEPINES FOR THE PREPARATION OF MEDICINES FOR THE TREATMENT OF CONDITIONS OR DISEASES IN WHICH ONE OF THE SOMATOSTATIN RECEPTORS IS INVOLVED|

---

KR20000016732A|1998-12-12|2000-03-25|Japan Tobacco Inc|Cytokine production inhibitors, triazepine compounds, and intermediates thereof|

---

MXPA01011632A|1999-05-14|2002-11-07|Imclone Systems Inc|Treatment of refractory human tumors with epidermal growth factor receptor antagonists.|

---

US20060142257A1|2001-01-19|2006-06-29|Eberhard Nieschlag|Male contraceptive formulation comprising norethisterone|

---

US7589167B2|2000-02-22|2009-09-15|J. David Gladstone Institutes|ZA loops of bromodomains|

---

KR100838686B1|2000-06-16|2008-06-16|미쯔비시 웰 파마 가부시키가이샤|Compositions controlling release pH range and/or speed|

---

WO2002002557A2|2000-06-30|2002-01-10|Neurogen Corporation|2-phenylimidazo[1,2-a]pyridine derivatives: a new class of gaba brain receptor ligands|

---

US6979682B2|2001-02-23|2005-12-27|Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College|Platelet-activating factor antagonist inhibition of angiogenesis and tumor growth induced by basic fibroblast|

---

US20030216758A1|2001-12-28|2003-11-20|Angiotech Pharmaceuticals, Inc.|Coated surgical patches|

---

GB0206215D0|2002-03-15|2002-05-01|Novartis Ag|Organic compounds|

---

AU2003283976B2|2002-09-27|2009-12-10|Cold Spring Harbor Laboratory|Cell-based RNA interference and related methods and compositions|

---

US6861422B2|2003-02-26|2005-03-01|Boehringer Ingelheim Pharma Gmbh & Co. Kg|Dihydropteridinones, processes for preparing them and their use as pharmaceutical compositions|

---

AU2004215359A1|2003-02-27|2004-09-10|Abbott Laboratories|Heterocyclic kinase inhibitors|

---

GB0305929D0|2003-03-14|2003-04-23|Novartis Ag|Organic compounds|

---

AU2004251654A1|2003-06-02|2005-01-06|Icoria, Inc.|Use of genes differentially expressed during aging of liver for treatment and diagnosis|

---

WO2005002526A2|2003-07-01|2005-01-13|President And Fellows Of Harvard College|Method and compositions for treatment of viral infections|

---

US8106180B2|2003-08-07|2012-01-31|Whitehead Institute For Biomedical Research|Methods and products for expression of micro RNAs|

---

WO2005026130A1|2003-09-18|2005-03-24|Novartis Ag|2,4-di pyrimidines useful in the treatment of proliferative disorders|

---

EP1528056A1|2003-10-29|2005-05-04|Academisch Ziekenhuis bij de Universiteit van Amsterdam|Deoxynojirimycin analoguesand their usesas glucosylceramidase inhibitors|

---

US7759485B2|2004-08-14|2010-07-20|Boehringer Ingelheim International Gmbh|Process for the manufacture of dihydropteridinones|

---

US20060058311A1|2004-08-14|2006-03-16|Boehringer Ingelheim International Gmbh|Combinations for the treatment of diseases involving cell proliferation|

---

US20060035903A1|2004-08-14|2006-02-16|Boehringer Ingelheim International Gmbh|Storage stable perfusion solution for dihydropteridinones|

---

US20060074088A1|2004-08-14|2006-04-06|Boehringer Ingelheim International Gmbh|Dihydropteridinones for the treatment of cancer diseases|

---

CN1759834B|2004-09-17|2010-06-23|中国医学科学院医药生物技术研究所|Application of berberine or associated with Simvastatin in preparing product for preventing or curing disease or symptom related to blood fat|

---

US8624027B2|2005-05-12|2014-01-07|Abbvie Inc.|Combination therapy for treating cancer and diagnostic assays for use therein|

---

TWI447120B|2005-05-30|2014-08-01|Mitsubishi Tanabe Pharma Corp|Thienotriazolodiazepine compound and a medicinal use thereof|

---

EP1915155A1|2005-08-03|2008-04-30|Boehringer Ingelheim International GmbH|Dihydropteridinones in the treatment of respiratory diseases|

---

NZ592990A|2005-11-01|2013-01-25|Targegen Inc|Bi-aryl meta-pyrimidine inhibitors of kinases|

---

US8133900B2|2005-11-01|2012-03-13|Targegen, Inc.|Use of bi-aryl meta-pyrimidine inhibitors of kinases|

---

US8604042B2|2005-11-01|2013-12-10|Targegen, Inc.|Bi-aryl meta-pyrimidine inhibitors of kinases|

---

PE20071042A1|2005-11-04|2007-10-12|Wyeth Corp|PHARMACEUTICAL PRODUCT INCLUDING TEMSIROLIMUS AND SUNITINIB MALATE|

---

KR20080083680A|2005-12-23|2008-09-18|스미스클라인 비참 코포레이션|Azaindole inhibitors of aurora kinases|

---

BRPI0706560A2|2006-01-17|2011-03-29|Hoffmann La Roche|aryl-isoxazol-4-yl-imidazo [1,2-a] pyridine derivatives useful for the treatment of alzheimer's disease via gaba receptors|

---

TW200804389A|2006-02-14|2008-01-16|Vertex Pharma|Dihydrodiazepines useful as inhibitors of protein kinases|

---

US20070218135A1|2006-03-14|2007-09-20|Glenmark Pharmaceuticals Limited|Sustained release matrix pharmaceutical composition|

---

JP2009535425A|2006-05-03|2009-10-01|ノバルティスアクチエンゲゼルシャフト|Use of organic compounds|

---

MX2009004077A|2006-10-19|2009-05-05|Signal Pharm Llc|Heteroaryl compounds, compositions thereof, and their use as protein kinase inhibitors.|

---

WO2008066887A2|2006-11-30|2008-06-05|Albert Einstein College Of Medicine Of Yeshiva University|Small molecule inhibitors of bcl6|

---

JP2008156311A|2006-12-26|2008-07-10|Institute Of Physical & Chemical Research|Brd2 bromodomain binder|

---

CN101687780B|2006-12-26|2016-01-20|兰休斯医疗成像公司|For making the part of imaging cardiac innervation|

---

WO2008137081A1|2007-05-02|2008-11-13|The Trustees Of Columbia University In The City Of New York|Substituted -dihydronaphraalenyl compounds as reversible male contraceptives|

---

KR20080107050A|2007-06-05|2008-12-10|울산대학교 산학협력단|Pharmaceutical composition for preventing or treating chronic graft-versus-host disease comprising anti-cd137 monoclonal antibody|

---

US20110028405A1|2007-12-20|2011-02-03|Richard John Harrison|Sulfamides as zap-70 inhibitors|

---

JP5478262B2|2007-12-28|2014-04-23|田辺三菱製薬株式会社|Anticancer drug|

---

US20110098288A1|2008-03-11|2011-04-28|Jeremy Major|Sulfonamides as zap-70 inhibitors|

---

WO2010015340A1|2008-08-06|2010-02-11|Bayer Schering Pharma Aktiengesellschaft|Daa-pyridine as peripheral benzodiazepine receptor ligand for diagnostic imaging and pharmaceutical treatment|

---

EA018620B1|2008-10-30|2013-09-30|Сёркомед Ллк|THIENOTRIAZOLODIAZEPINE DERIVATIVES ACTIVE ON Apo A1|

---

EP3828185A3|2009-01-06|2021-09-01|Dana Farber Cancer Institute, Inc.|Pyrimido-diazepinone kinase scaffold compounds and methods of treating disorders|

---

KR101779137B1|2009-01-23|2017-09-18|다케다 야쿠힌 고교 가부시키가이샤|Polypolymerase inhibitors|

---

WO2010111712A2|2009-03-27|2010-09-30|Cold Spring Harbor Laboratory|Identification of rnai targets and use of rnai for rational therapy of chemotherapy-resistant leukemia and other cancers|

---

GB0919423D0|2009-11-05|2009-12-23|Glaxosmithkline Llc|Novel compounds|

---

GB0919432D0|2009-11-05|2009-12-23|Glaxosmithkline Llc|Use|

---

GB0919426D0|2009-11-05|2009-12-23|Glaxosmithkline Llc|Novel compounds|

---

GB0919431D0|2009-11-05|2009-12-23|Glaxosmithkline Llc|Novel compounds|

---

EA023441B1|2009-11-05|2016-06-30|ГЛАКСОСМИТКЛАЙН ЭлЭлСи|Benzodiazepine bromodomain inhibitor|

---

GB0919434D0|2009-11-05|2009-12-23|Glaxosmithkline Llc|Novel compounds|

---

HUE026421T2|2009-11-05|2016-05-30|Glaxosmithkline Llc|Benzodiazepine bromodomain inhibitor|

---

EP2536725B1|2010-02-17|2015-10-28|Boehringer Ingelheim International GmbH|Dihydropteridinones, method for production and use thereof|

---

EP2571875A4|2010-05-14|2013-10-30|Dana Farber Cancer Inst Inc|Male contraceptive compositions and methods of use|

---

JP5913292B2|2010-05-14|2016-04-27|ダナ−ファーバー キャンサー インスティテュート， インコーポレイテッド|Compositions and methods for modulating metabolism|

---

KR101857599B1|2010-05-14|2018-05-14|다나-파버 캔서 인스티튜트 인크.|Compositions and methods for treating neoplasia, inflammatory disease and other disorders|

---

AU2011252799B2|2010-05-14|2015-05-14|Cold Spring Harbor Laboratory|Compositions and methods for treating leukemia|

---

ES2526671T3|2010-06-22|2015-01-14|Glaxosmithkline Llc|Compounds of benzotriazoldiazepine bromodomain inhibitors|

---

CA2805413A1|2010-06-25|2011-12-29|Shire Human Genetic Therapies, Inc.|Methods and compositions for cns delivery of heparan n-sulfatase|

---

US20120014979A1|2010-07-16|2012-01-19|Alexander Dent|Use of bcl6 inhibitors for treating autoimmune diseases|

---

TWI583689B|2010-08-04|2017-05-21|達納 法柏癌症學院有限公司|Compositions and methods for treating neoplasia, inflammatory disease and other disorders|

---

WO2012050907A2|2010-09-28|2012-04-19|The Regents Of The University Of California|Gaba agonists in the treatment of disorders associated with metabolic syndrome and gaba combinations in treatment or prophylaxis of type i diabetes|

---

WO2012075456A1|2010-12-02|2012-06-07|Constellation Pharmaceuticals|Bromodomain inhibitors and uses thereof|

---

AR084070A1|2010-12-02|2013-04-17|Constellation Pharmaceuticals Inc|BROMODOMINIUM INHIBITORS AND USES OF THE SAME|

---

US20130004481A1|2011-01-12|2013-01-03|Boehringer Ingelheim International Gmbh|Anticancer therapy|

---

EP2677865A4|2011-02-23|2015-04-22|Icahn School Med Mount Sinai|Inhibitors of bromodomains as modulators of gene expression|

---

EP3323820A1|2011-02-28|2018-05-23|Epizyme, Inc.|Substituted 6,5-fused bicyclic heteroaryl compounds|

---

WO2012119095A1|2011-03-02|2012-09-07|Board Of Regents, The University Of Texas System|Fus1/tusc2 therapies|

---

WO2013019710A1|2011-07-29|2013-02-07|The Children's Hospital Of Philadelphia|Compositions and methods for the treatment of hiv|

---

WO2013033270A2|2011-08-29|2013-03-07|Coferon, Inc.|Bromodomain ligands capable of dimerizing in an aqueous solution, and methods of using same|

---

WO2013033269A1|2011-08-29|2013-03-07|Coferon, Inc.|Bioorthogonal monomers capable of dimerizing and targeting bromodomains and methods of using same|

---

WO2013033420A1|2011-08-30|2013-03-07|Whitehead Institute For Biomedical Research|Methods of downregulating translocated oncogene expression using bromodomain inhibitors|

---

WO2013097052A1|2011-12-30|2013-07-04|Abbott Laboratories|Bromodomain inhibitors|

---

CN104203267A|2012-02-17|2014-12-10|安迅生物制药公司|Methods, compounds and compositions for treatment of parainfluenza virus in immunocompromised patients|

---

EP2830629A4|2012-03-28|2016-01-20|Icahn School Of Medicine Mount Sinai|Compositions and methods for reactivating latent immunodeficiency virus|

---

WO2013192274A2|2012-06-19|2013-12-27|The Broad Institute, Inc.|Diagnostic and treatment methods in subjects having or at risk of developing resistance to cancer therapy|

---

CN104968334B|2012-09-28|2018-09-14|翁科埃斯克斯有限公司|Include the pharmaceutical preparation of thieno triazol diazepine compound|

---

EP2917203B1|2012-11-02|2019-04-03|Dana-Farber Cancer Institute, Inc.|Method for identifying myc inhibitors|

---

EP2958923A1|2013-02-22|2015-12-30|Bayer Pharma Aktiengesellschaft|4-substituted pyrrolo- and pyrazolo-diazepines|

---

CN105189514A|2013-02-22|2015-12-23|拜耳医药股份有限公司|Pyrrolo- and pyrazolo-triazolodiazepines as bet-protein inhibitors for treating hyperproliferative diseases|

---

AU2014223990A1|2013-02-28|2015-09-10|Washington University|Methods of treatment of human cytomegalovirus infection and diseases with bromodomain inhibitors|

---

WO2014159392A1|2013-03-14|2014-10-02|Dana-Farber Cancer Institute, Inc.|Bromodomain binding reagents and uses thereof|

---

EP2968263A2|2013-03-15|2016-01-20|Genentech, Inc.|Treating th2-mediated diseases by inhibition of bromodomain-comprising proteins brd7 and brd9|

---

CA2913741A1|2013-05-28|2014-12-04|The Brigham And Women's Hospital, Inc.|Bet inhibition therapy for heart disease|

---

JP2016531113A|2013-07-25|2016-10-06|ダナ−ファーバー キャンサー インスティテュート， インコーポレイテッド|Inhibitors of transcription factors and uses thereof|

---

WO2015018522A1|2013-08-06|2015-02-12|Oncoethix Sa|Bet-bromodomain inhibitor shows synergism with several anti-cancer agents in pre-clinical models of diffuse large b-cell lymphoma |

---

JP2016529246A|2013-08-06|2016-09-23|オンコエシックス ゲーエムベーハー|Method for treating diffuse large B-cell lymphoma using BET bromodomain inhibitor|

---

EP3033423A4|2013-08-16|2017-04-26|Rana Therapeutics Inc.|Epigenetic regulators of frataxin|

---

WO2015042249A1|2013-09-19|2015-03-26|Dana-Farber Cancer Institute, Inc.|Methods of bh3 profiling|

---

CN105979958A|2013-10-11|2016-09-28|基因泰克公司|Use of cbp/ep300 bromodomain inhibitors for cancer immunotherapy|

---

US20160279141A1|2013-11-08|2016-09-29|Dana-Farber Cancer Institute, Inc.|Combination therapy for cancer using bromodomain and extra-terminal protein inhibitors|

---

WO2015081284A1|2013-11-26|2015-06-04|Coferon, Inc.|Bivalent bromodomain ligands, and methods of using same|

---

MX2016009976A|2014-01-31|2016-11-15|Dana-Farber Cancer Inst Inc|Diazepane derivatives and uses thereof.|

---

US10793571B2|2014-01-31|2020-10-06|Dana-Farber Cancer Institute, Inc.|Uses of diazepane derivatives|

---

MX2016009974A|2014-01-31|2016-10-31|Dana-Farber Cancer Inst Inc|Diaminopyrimidine benzenesulfone derivatives and uses thereof.|

---

CA2940472A1|2014-02-28|2015-09-03|Tensha Therapeutics, Inc.|Treatment of conditions associated with hyperinsulinaemia|

---

CN107635979A|2014-10-27|2018-01-26|腾沙治疗公司|Bromine domain inhibitor|

---

CA2969417A1|2014-12-05|2016-06-09|H. Lee Moffitt Cancer Center And Research Institute, Inc.|Bromodomain inhibitor as adjuvant in cancer immunotherapy|

---

JP2018520124A|2015-06-26|2018-07-26|テンシャ セラピューティクス，インコーポレイテッド|Treatment of NUT midline cancer|

---

CA2999523A1|2015-10-02|2017-04-06|Dana-Farber Cancer Institute, Inc.|Combination therapy of bromodomain inhibitors and checkpoint blockade|KR101857599B1|2010-05-14|2018-05-14|다나-파버 캔서 인스티튜트 인크.|Compositions and methods for treating neoplasia, inflammatory disease and other disorders|

---

EP2571875A4|2010-05-14|2013-10-30|Dana Farber Cancer Inst Inc|Male contraceptive compositions and methods of use|

---

AU2011252799B2|2010-05-14|2015-05-14|Cold Spring Harbor Laboratory|Compositions and methods for treating leukemia|

---

JP5913292B2|2010-05-14|2016-04-27|ダナ−ファーバー キャンサー インスティテュート， インコーポレイテッド|Compositions and methods for modulating metabolism|

---

WO2012075456A1|2010-12-02|2012-06-07|Constellation Pharmaceuticals|Bromodomain inhibitors and uses thereof|

---

AR084070A1|2010-12-02|2013-04-17|Constellation Pharmaceuticals Inc|BROMODOMINIUM INHIBITORS AND USES OF THE SAME|

---

US9422292B2|2011-05-04|2016-08-23|Constellation Pharmaceuticals, Inc.|Bromodomain inhibitors and uses thereof|

---

US9328117B2|2011-06-17|2016-05-03|Constellation Pharmaceuticals, Inc.|Bromodomain inhibitors and uses thereof|

---

KR20200063135A|2012-01-12|2020-06-04|예일 유니버시티|Compounds and Methods for the Enhanced Degradation of Targeted Proteins and Other Polypeptides by an E3 Ubiquitin Ligase|

---

TWI602820B|2012-06-06|2017-10-21|星宿藥物公司|Bromodomain inhibitors and uses thereof|

---

US9624244B2|2012-06-06|2017-04-18|Constellation Pharmaceuticals, Inc.|Benzo [B] isoxazoloazepine bromodomain inhibitors and uses thereof|

---

WO2013192274A2|2012-06-19|2013-12-27|The Broad Institute, Inc.|Diagnostic and treatment methods in subjects having or at risk of developing resistance to cancer therapy|

---

US10426799B2|2012-12-21|2019-10-01|Astellas Institute For Regenerative Medicine|Methods for production of platelets from pluripotent stem cells and compositions thereof|

---

CA2903463A1|2013-03-11|2014-10-09|The Regents Of The University Of Michigan|Bet bromodomain inhibitors and therapeutic methods using the same|

---

KR102216288B1|2013-03-15|2021-02-18|인사이트 홀딩스 코포레이션|Tricyclic heterocycles as bet protein inhibitors|

---

GB201311891D0|2013-07-03|2013-08-14|Glaxosmithkline Ip Dev Ltd|Novel compound|

---

GB201311888D0|2013-07-03|2013-08-14|Glaxosmithkline Ip Dev Ltd|Novel compounds|

---

WO2015006193A1|2013-07-08|2015-01-15|Incyte Corporation|Tricyclic heterocycles as bet protein inhibitors|

---

JP2016531113A|2013-07-25|2016-10-06|ダナ−ファーバー キャンサー インスティテュート， インコーポレイテッド|Inhibitors of transcription factors and uses thereof|

---

WO2015014998A1|2013-08-01|2015-02-05|Oncoethix Sa|Pharmaceutical formulation containing thienotriazolodiazepine compounds|

---

US20160279141A1|2013-11-08|2016-09-29|Dana-Farber Cancer Institute, Inc.|Combination therapy for cancer using bromodomain and extra-terminalprotein inhibitors|

---

US9399640B2|2013-11-26|2016-07-26|Incyte Corporation|Substituted pyrrolo[2,3-c]pyridines and pyrazolo[3,4-c]pyridines as BET protein inhibitors|

---

US9315501B2|2013-11-26|2016-04-19|Incyte Corporation|Bicyclic heterocycles as BET protein inhibitors|

---

US9757385B2|2013-11-27|2017-09-12|Merck Sharp & Dohme Corp.|Method of treating leukemia using pharmaceutical formulation containing thienotriazolodiazepine compounds|

---

WO2015095492A1|2013-12-19|2015-06-25|Incyte Corporation|Tricyclic heterocycles as bet protein inhibitors|

---

MX2016009974A|2014-01-31|2016-10-31|Dana-Farber Cancer Inst Inc|Diaminopyrimidine benzenesulfone derivatives and uses thereof.|

---

US10793571B2|2014-01-31|2020-10-06|Dana-Farber Cancer Institute, Inc.|Uses of diazepane derivatives|

---

EP3104845A4|2014-02-10|2017-09-27|Salk Institute for Biological Studies|Increasing storage of vitamin a, vitamin d and/or lipids|

---

EP3110818B1|2014-02-28|2019-10-23|The Regents of The University of Michigan|9h-pyrimido[4,5-b]indoles and related analogs as bet bromodomain inhibitors|

---

CA2940472A1|2014-02-28|2015-09-03|Tensha Therapeutics, Inc.|Treatment of conditions associated with hyperinsulinaemia|

---

WO2015144636A1|2014-03-24|2015-10-01|INSERM |Methods for the treatment of t-cell acute lymphoblastic leukemias|

---

SG10201809353TA|2014-04-23|2018-11-29|Incyte Corp|1H-PYRROLO[2,3-c]PYRIDIN-7-ONES AND PYRAZOLO[3,4-c]PYRIDIN-7-ONES AS INHIBITORS OF BET PROTEINS|

---

JP2017514909A|2014-05-02|2017-06-08|オンコエシックス ゲーエムベーハー|Method for treating acute myeloid leukemia and / or acute lymphoblastic leukemia using thienotriazolodiazepine compound|

---

SI3157928T1|2014-06-20|2019-06-28|Constellation Pharmaceuticals, Inc.|Crystalline forms of 2--6--1-methyl-4h-benzoisoxazoloazepin-4-yl)acetamide|

---

CN106793775B|2014-08-08|2020-06-02|达纳-法伯癌症研究所股份有限公司|Dihydropyrazinone derivatives and uses thereof|

---

RU2017104897A|2014-08-08|2018-09-10|Дана-Фарбер Кэнсер Инститьют, Инк.|DIAZEPANE DERIVATIVES AND THEIR APPLICATIONS|

---

US10071164B2|2014-08-11|2018-09-11|Yale University|Estrogen-related receptor alpha based protac compounds and associated methods of use|

---

CN107427524A|2014-08-28|2017-12-01|翁科埃斯克斯有限公司|Utilize the method for the medicine composite for curing acute myelocytic leukemia comprising thieno triazol diazacompounds or acute lymphatic leukemia|

---

EP3194406B8|2014-09-15|2021-03-31|Incyte Corporation|Tricyclic heterocycles for use as bet protein inhibitors|

---

CN107635979A|2014-10-27|2018-01-26|腾沙治疗公司|Bromine domain inhibitor|

---

WO2016070037A2|2014-10-31|2016-05-06|Massachusetts Institute Of Technology|Massively parallel combinatorial genetics for crispr|

---

US10307407B2|2015-02-27|2019-06-04|The Regents Of The University Of Michigan|9H-pyrimido [4,5-B] indoles as BET bromodomain inhibitors|

---

JP2018512449A|2015-03-18|2018-05-17|アルビナス インコーポレイテッド|Compounds and methods for improved degradation of target proteins|

---

JP6851978B2|2015-04-20|2021-03-31|スミトモ ダイニッポン ファーマ オンコロジー， インコーポレイテッド|Prediction of alvocidib response by mitochondrial profiling|

---

CN111349118A|2015-05-18|2020-06-30|特雷罗药物股份有限公司|Avasidil prodrugs with increased bioavailability|

---

WO2016196065A1|2015-05-29|2016-12-08|Genentech, Inc.|Methods and compositions for assessing responsiveness of cancers to bet inhibitors|

---

US10702527B2|2015-06-12|2020-07-07|Dana-Farber Cancer Institute, Inc.|Combination therapy of transcription inhibitors and kinase inhibitors|

---

KR20180034538A|2015-08-03|2018-04-04|톨레로 파마수티컬스, 인크.|Concurrent therapy for the treatment of cancer|

---

WO2017030814A1|2015-08-19|2017-02-23|Arvinas, Inc.|Compounds and methods for the targeted degradation of bromodomain-containing proteins|

---

WO2017044792A1|2015-09-11|2017-03-16|Dana-Farber Cancer Institute, Inc.|Acetamide thienotriazoldiazepines and uses thereof|

---

US20170121347A1|2015-10-29|2017-05-04|Incyte Corporation|Amorphous solid form of a bet protein inhibitor|

---

BR112018008918A8|2015-11-02|2019-02-26|Univ Yale|directed proteolysis chimera compounds and methods for their preparation and use|

---

SG10201913450PA|2015-11-25|2020-03-30|Dana Farber Cancer Inst Inc|Bivalent bromodomain inhibitors and uses thereof|

---

ES2882066T3|2016-02-15|2021-12-01|Univ Michigan Regents|Fused 1,4-oxazepines and related analogs as BET bromodomain inhibitors|

---

EP3436006A1|2016-03-30|2019-02-06|Wisconsin Alumni Research Foundation|Methods and compositions for modulating frataxin expression|

---

CN113788818A|2016-04-06|2021-12-14|密执安大学评议会|MDM2 protein degrading agent|

---

WO2017176958A1|2016-04-06|2017-10-12|The Regents Of The University Of Michigan|Monofunctional intermediates for ligand-dependent target protein degradation|

---

AU2017250076B2|2016-04-12|2021-07-22|The Regents Of The University Of Michigan|Bet protein degraders|

---

EP3472157A1|2016-06-20|2019-04-24|Incyte Corporation|Crystalline solid forms of a bet inhibitor|

---

CN110062759A|2016-09-13|2019-07-26|密执安大学评议会|Condensed 1,4- oxygen azepineas BET protein degradation agent|

---

EP3512853B1|2016-09-13|2020-12-23|The Regents of The University of Michigan|Fused 1,4-diazepines as bet protein degraders|

---

JP2020502148A|2016-12-16|2020-01-23|エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト|Process for the production of diazepine derivatives|

---

JP6619519B2|2016-12-19|2019-12-11|トレロ ファーマシューティカルズ， インコーポレイテッド|Profiling peptides and methods for sensitivity profiling|

---

US11046709B2|2017-02-03|2021-06-29|The Regents Of The University Of Michigan|Fused 1,4-diazepines as BET bromodomain inhibitors|

---

US11267822B2|2017-09-13|2022-03-08|The Regents Of The University Of Michigan|BET bromodomain protein degraders with cleavable linkers|

---

WO2019148055A1|2018-01-26|2019-08-01|Yale University|Imide-based modulators of proteolysis and methods of use|

---

AU2019284971A1|2018-06-13|2020-12-03|Dybly Ag|Preparation of condensed triazepine derivatives and their use as BET inhibitors|

---

EP3890749A1|2018-12-04|2021-10-13|Sumitomo Dainippon Pharma Oncology, Inc.|Cdk9 inhibitors and polymorphs thereof for use as agents for treatment of cancer|

---

WO2021074338A1|2019-10-16|2021-04-22|INSERM |Methods of determining whether patients suffering from acute myeloid leukemia will achieve a response to an myc-targeting therapy|

---

WO2021092464A2|2019-11-08|2021-05-14|Phio Pharmaceuticals Corp.|Chemically modified oligonucleotides targeting bromodomain containing protein 4for immunotherapy|

法律状态:
2020-10-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2020-10-27| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2021-04-13| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2021-05-04| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-08-17| B11B| Dismissal acc. art. 36, par 1 of ipl - no reply within 90 days to fullfil the necessary requirements|

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2021-11-23| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US33499110P| true| 2010-05-14|2010-05-14|

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US61/334,991|2010-05-14|

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US37074510P| true| 2010-08-04|2010-08-04|

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US61/370,745|2010-08-04|

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US37586310P| true| 2010-08-22|2010-08-22|

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US61/375,863|2010-08-22|

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US201161467342P| true| 2011-03-24|2011-03-24|

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US201161467376P| true| 2011-03-24|2011-03-24|

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US61/467,376|2011-03-24|

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US61/467,342|2011-03-24|

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PCT/US2011/036672|WO2011143660A2|2010-05-14|2011-05-16|Compositions and methods for treating leukemia|

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