WO2019232467A1 - Biomarqueurs pharmacodynamiques pour le traitement du cancer avec un inhibiteur de cdk8/19 - Google Patents

Biomarqueurs pharmacodynamiques pour le traitement du cancer avec un inhibiteur de cdk8/19 Download PDF

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WO2019232467A1
WO2019232467A1 PCT/US2019/035038 US2019035038W WO2019232467A1 WO 2019232467 A1 WO2019232467 A1 WO 2019232467A1 US 2019035038 W US2019035038 W US 2019035038W WO 2019232467 A1 WO2019232467 A1 WO 2019232467A1
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cdk8
optionally substituted
inhibitor
blood sample
antibody
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Matthew D. Shair
Henry E. Pelish
Hanna TUKACHINSKY
Stana K. NICKOLICH
Alexander Stuart
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President And Fellows Of Harvard College
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A61K31/4725—Non-condensed isoquinolines, e.g. papaverine containing further heterocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/08—Bridged systems
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • CDK8 and CDK19 proteins reversibly associate with the Mediator complex and form its key regulatory unit.
  • CDK8 and CDK19 oncogenes have been implicated in the proliferation of a variety of cancer cell lines via the modulation of various gene expression programs.
  • AML acute myeloid leukemia
  • Super enhancers in AML can be regulated by the use of CDK8 and CDK19 inhibitors such as cortistatin A.
  • CDK8 has a conserved function in transcription as described by Taatjes, D. L, Trends Biochem Sci 35, 315-322 (2010); and Conaway, R. C. and Conaway, J. W., Curr Opin Genet Dev 21, 225-230 (2011).
  • CDK8 has also been reported as an oncogene in both colon cancer (Firestein R. et ak, Nature 455:547-51 (2008); Morris E. J. et ah, Nature 455:552-6 (2008); Starr T. K. et ak, Science 323 : 1747-50 (2009)) and melanoma (Kapoor A. et ak, Nature 468: 1105-9 (2010)).
  • CDK8 is upregulated and amplified in a subset of human colon tumors, is known to transform immortalized cells and is required for colon cancer proliferation in vitro. Similarly, CDK8 has also been found to be overexpressed and essential for proliferation in melanoma. Kapoor, A. et al., Nature 468, 1105-1109 (2010). CDK8 has been shown to regulate several signaling pathways that are key regulators of both ES pluripotency and cancer. CDK8 activates the Wnt pathway by promoting expression of b-Catenin target genes (Firestein, R. et al., Nature 455, 547-551 (2008)) or by inhibiting E2F1, a potent inhibitor of b-Catenin transcriptional activity. Morris, E. J. et al., Nature 455, 552-556 (2008).
  • The‘019 patent discloses that such compounds are anti-angiogenic and can be used to treat proliferative diseases.
  • a hematopoietic cancer such as leukemia, multiple myeloma (MM), acute myelocytic leukemia (AML), a myeloproliferative neoplasm, acute lymphoblastic leukemia (ALL), chronic myelocytic leukemia (CML) and primary myelofibrosis (PMF).
  • MM multiple myeloma
  • AML acute myelocytic leukemia
  • ALL acute lymphoblastic leukemia
  • CML chronic myelocytic leukemia
  • PMF primary myelofibrosis
  • WO 2017/112815 titled“Cortistatin Analogues and Uses Thereof’ and WO 2017/142621 titled“Cortistatin Analogs” filed by Shair, et ah, and also assigned to the President and Fellows of Harvard College describes additional compounds that inhibit CDK8 and CDK19.
  • CDK8 and CDK19 inhibitors While a range of CDK8 and CDK19 inhibitors have now been identified, there is a need to know how best to use these compounds to treat patients with a medical disorder such as cancer or a tumor.
  • a pharmacodynamic diagnostic includes the use of a selection of biomarkers to measure the efficiency over time of CDK8 and/or CDK19 inhibitors in medical therapy.
  • This pharmacodynamic approach to assessing CDK8 and/or CDK19 drug efficacy over time is based on the observed loss of phosphorylation of predictive proteins that have been identified for this purpose for the first time using a range of parameters.
  • This advantageous real time diagnostic and kit allows the healthcare provider to monitor the patient during therapy to determine the level of response to treatment with the selected CDK8 and/or CDK19 inhibitor.
  • the CDK8 and/or CDK19 inhibitor may be administered to treat a tumor or cancer, or a non-tumor or cancerous disease that responds to CDK8 and/or CDK19 therapy, as described in more detail below.
  • the ability of these pharmacodynamic biomarkers to offer a real-time evaluation of efficacy is particularly important because cancer cells can develop resistance to treatment.
  • the tumor may acquire resistance by using an alternative kinase (not CDK8 and/or CDK19) to phosphorylate the same target.
  • the methods described herein may detect the resistance emerging as an increase (or less of a decrease) in the concentration of the pharmacokinetic biomarker.
  • a care provider may not discover that a patient’s cancer either is not sensitive to the drug or has developed resistance to therapy until after the cancer has progressed sufficiently to show (or not show) phenotypic signals.
  • biomarkers are not suitable for use in a pharmacodynamic diagnostic due to multiple requirements.
  • STAT1 and STAT5 are not useful kinase substrates when testing a patient’s whole blood, because they are not phosphorylated in sufficient quantities in whole blood and they are phosphorylated by a range of kinases, rendering the assay not meaningful. This is also the case with a number of other kinase substrates.
  • biomarkers for use in a pharmacodynamic diagnostic have now been discovered that can conveniently be used in a whole blood assay to monitor over time the effectiveness of the administered CDK8 and/or CDK19 inhibitor drug, such as one of the cortistatin derivatives described further below.
  • These biomarkers are selectively and directly phosphorylated by CDK8 and/or CDK19, and are present in whole blood in sufficient quantities for accurate analysis.
  • they are superior diagnostic agents for use to determine if CDK8 and/or CDK19 is being effectively inhibited in vivo by the drug in the tumor or cancer patient.
  • phosphoproteomic studies were conducted in two AML cell lines (MOLM-13 and MOLM-14) after CDK8 inhibitor exposure to identify candidate proteins that are not phosphorylated in the presence of the inhibitor. The same analysis was carried out in peripheral blood mononuclear cells from five healthy donors. A list of substrates reported in a previous phosphoproteomics study conducted in colorectal cancer cell line HCT116 using cortistatin A was also evaluated (Poss et ak, Cell Rep 2016 PMID 27050516).
  • specific monoclonal mouse antibodies were created against the top phosphorylated protein candidate biomarkers to determine if they are present in sufficient quantities in whole blood. These antibodies were used to quantify the amount of pharmacodynamic biomarker in the sample which can be indicative of treatment success, as described further herein.
  • This invention is advantageous in one respect in light of the observation that a CDK8 and/or CDK19 inhibitor may not suppress cancer proliferation in all tissues. It is known that tumors and cancer even within a narrow category can be heterogeneous. Due to the fact that individual tumors can be caused by a range of genetic abnormalities and as a result can express or suppress key proteins, resulting in a range of phenotypes, not all tumors or cancers within the narrow class will respond to the same drug therapy. Even with the most effective oncology drugs, it is expected that there will be responders and non-responders. And, patients who initially respond to a treatment regimen may develop resistance to that treatment.
  • this invention fills an unmet need to determine in real time over the course of therapy if a patient will or is responding to treatment with the selected CDK8 and/or CDK19 inhibitor.
  • the present invention also includes a method to determine if a patient’s cancer or other disease modulated by CDK8 and/or CDK19, has acquired drug resistance to the administered drug overtime.
  • the resulting pharmacodynamic diagnostic and therapeutic methods, materials and kits allow clinicians to monitor the responsiveness of cancer to CDK8 and/or CDK19 treatment in real-time.
  • the diagnostic is sensitive, does not require a biopsy, and allows for rapid feedback regarding treatment efficacy.
  • a method for treating a patient with a disorder that can be treated with a CDK8 and/or CDK19 inhibitor, such as a cancer or tumor comprising the following steps:
  • a diagnostic method or kit is provided to determine if a patient’s CDK8 and/or CDK19 mediated disorder is responding to treatment with a CDK8/19 inhibitor.
  • One non-limiting example comprises the following steps:
  • a“sufficient time for the CDK8/19 inhibitor to act” as used in the methods herein is at least about or not more than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or about 12 hours.
  • the sufficient period of time is at least about or not more than 0.2 half lives, 0.5 half lives, 0.7 half lives, 1 half-life, 1.5 half-lives, 2 half-lives, 2.5 half-lives, or 3 half-lives.
  • the sufficient period of time is at least about or not more than the compounds Tmax, 1.5 times its Tmax, 2 times its Tmax, 2.5 times its Tmax, or 3 times its Tmax.
  • the concentration of pharmacokinetic biomarker must decrease by at least about 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% after treatment with the CDK8/19 inhibitor to indicate that the CDK8 and/or CDK19 mediated disorder is responding to treatment.
  • the diagnostic includes a reference chart with standardized comparisons of the range of inhibition of phosphorylation in healthy people when given a range of doses of the CDK8 and or CDK19 inhibitor, as well as the range of inhibition of phosphorylation in patients when the drug is considered still effective, and optionally, when it has lost effectiveness.
  • the pharmacodynamic biomarkers are used to select patients for treatment with a CDK8/19 inhibitor.
  • blood samples bearing a cells from prospective patients can be removed and treated in vitro to determine if the patient will likely respond to therapy.
  • One non-limiting example of such a method comprises the following steps:
  • CDK8/19 inhibitor is added to the blood sample to make the sample have a concentration of at least 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 1 mM, 2 mM, or 10 mM of the CDK8/19 inhibitor.
  • This disclosure thus provides specific, non-limiting examples of methods to treat a patient that includes the administration of a CDK8 and/or CDK19 inhibitor along with a companion diagnostic assessment or kit that includes the use of pharmacodynamic biomarkers. Additionally, the disclosure provides specific, non-limiting methods for determining if a patient’s CDK8 and/or CDK19 mediated disorder is responding to treatment with a CDK8/19 inhibitor. The skilled artisan will recognize that other methods are in the alternative and are still encompassed within the scope of this invention.
  • the disclosure describes how to determine the concentration of phosphorylated CHD4 by contacting the sample with an antibody and then quantifying the amount of phosphorylated CHD4 bound to antibody by immunoblot, however the skilled artisan will recognize that other methods could be used, such as other spectrometric techniques or using alternative chemical reactions.
  • iii administer one or more dosages of a CDK8 and/or CDK19 inhibitor drug; iv. obtain a second blood sample from the patient after one or more treatments with the CDK8/19 inhibitor drug;
  • v. detect the concentration of the pharmacodynamic biomarker in the second blood sample by contacting the blood sample with one or more antibodies to pCHD4, pDPF2, or pDMAPl and determining the concentration of these proteins by immunoblot; and vi. determine if the patient’s CDK8 and/or CDK19 mediated disorder is responding to treatment, wherein less than a 10% decrease in the concentration of the one or more pharmacodynamic biomarkers in the second blood sample relative to the first blood sample indicates that the treatment regimen is not efficacious for treating the CDK8 and/or CDK19 mediated disorder in the subject.
  • a diagnostic kit for the determination of whether a patient is responding successfully to therapy that includes an antibody that binds to the phosphorylated pharmacodynamic biomarker.
  • the kit also includes a means to determine the concentration of the phosphorylated pharmacodynamic biomarker antibody complex, for example a colorimetric assay, a western blot assay, or an immunoblot assay.
  • the kit can include multiple antibodies for one or more different pharmacodynamic complexes.
  • the kit is a combined therapeutic and companion diagnostic that also includes dosage forms of the selected CDK8/19 inhibitor.
  • the kit provides specialized instructions, for example the kit may provide the healthcare provider instructions on how to check patient response in vitro as described herein.
  • kits comprising a drug with a cortistatin framework as described herein or a pharmaceutically acceptable salt, quaternary amine salt, or N- oxide thereof, for use as a medicament in the treatment of a CDK8 and/or CDK19 mediated disorder.
  • the kit includes an antibody for pCHD4, pDPF2, or pDMAPl .
  • the kit is for diagnosing the effectiveness of CDK 8/19 inhibition in the treatment of a hematological cancer.
  • the kit comprises a means for detecting or assaying phosphorylation of CHD4, DPF2, and/or DMAP1, or a combination thereof, both prior to administration of a CDK 8/19 inhibitor to treat a CDK8 and/or CDK19 mediated cancer (for example, a CDK8 and/or CDK19 mediated hematological cancerjand during continued administration of the CDK 8/19 inhibitor.
  • the kit is capable of analyzing the change in phosphorylation of CHD4, DPF2, and/or DMAP1 during treatment using a CDK 8/19 inhibitor, which is indicative of the effectiveness of the CDK 8/19 inhibitor treatment.
  • Non-limiting examples of hematopoietic lineage tumors or cancers that can be monitored include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphoblastic leukemia (CLL), B-cell acute lymphoblastic leukemia (B-ALL), childhood B-ALL, chronic myeloid leukemia, acute monocytic leukemia, acute megakaryoblastic leukemia, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, Burkitt’s lymphoma, AIDS-related lymphoma, chronic myeloproliferative disorder, primary central nervous system lymphoma, T-cell lymphoma, hairy cell leukemia, and multiple myeloma (MM).
  • ALL acute lymphoblastic leukemia
  • AML acute myeloid leukemia
  • CLL chronic lymphoblastic leukemia
  • B-ALL B-cell acute lymphoblastic leukemia
  • childhood B-ALL chronic mye
  • the invention also includes a diagnostic for and/or treatment of precursor cells to a hematopoietic tumor or cancer, such as found in myelodysplastic syndrome (MDS).
  • MDS myelodysplastic syndrome
  • the tumor or cancer may also be of a non-hematopoietic lineage, such as breast cancer, ovarian cancer, endometrioid carcinoma, squamous cell cancer, angiosarcoma, colon cancer, gastrointestinal tumors, metastasis-prone solid tumors, clear cell carcinoma, renal cell carcinoma, or esophageal cancer.
  • a non-hematopoietic lineage such as breast cancer, ovarian cancer, endometrioid carcinoma, squamous cell cancer, angiosarcoma, colon cancer, gastrointestinal tumors, metastasis-prone solid tumors, clear cell carcinoma, renal cell carcinoma, or esophageal cancer.
  • the CDK8/19 inhibitor is a cortistatin analogue, for example a cortistatin analogue described herein.
  • the pharmacodynamic biomarker is phosphorylated CHD4, DPF2, or DMAP1.
  • the pharmacodynamic biomarker is either CHD4 rT1553, DPF2 pT248, or DMAP1 pT445, wherein the pT# denotes which amino acid is phosphorylated.
  • any of the methods described herein utilize a different patient sample instead of blood.
  • the patient sample can be any sample from which sufficient quantities of cells can be isolated and analyzed because the detected biomarkers are not metabolites or secreted proteins/peptides but rather intracellular proteins.
  • the patient sample is a fluid sample. In another embodiment the patient sample is lymph fluid.
  • a method for determining the pharmacokinetic time course for a specific CDK8/19 inhibitor in a specific patient comprising the following steps:
  • iii. obtain a another fluid sample from the patient after waiting about .5 hours, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, or about 4 hours;
  • step iii 1, 2, 3, 4, 5, or 6 times;
  • v. detect the concentration of the one or more pharmacodynamic biomarkers selected from pCHD4, pDPF2, and pDMAPl in the blood samples collected in steps iii and iv wherein the concentration over time of the one or more pharmacodynamic biomarkers is the pharmacokinetic time course for the specific CDK8/19 inhibitor, wherein lower concentration of the one or more pharmacodynamic biomarkers indicates higher inhibition of CDK8 and/or CDK19;
  • the pharmacokinetic time course determined with the above method is used to establish the dose necessary to achieve full target inhibition, wherein once you see a plateau in pharmacokinetic biomarker concentration the kinase has been inhibited maximally. Combined with tolerated dose data, this data can establish the therapeutic index of a compound. Additionally the data can be used to determine the clearance rate out of tissue and blood.
  • one or more of the above methods is used to determine when the compound is exerting its effects and when it stops doing so. This data can be compared to pharmacokinetic time courses calculated through means known in the art (such as tracking concentration of the CDK8/19 inhibitor or a metabolite thereof).
  • any of the methods herein are used with samples from xenograph experiments.
  • the methods are used during clinical trials to determine effective dosage regimens.
  • FIG. l is a volcano plot of 5,784 phosphosites identified in TMT mass spectrometry from 5 different donor PBMC samples.
  • the y axis is the t-test value measured as p plotted in logarithmic base 10 units.
  • the x axis is the ratio of DMSO (vehicle) to Cortistatin A plotted in logarithmic base 2 units.
  • DPF2 pT248 and DMAP1 pT445 were top hits. The experimental preparation of these PBMC samples is provided in Example 5.
  • FIG. 2 is a volcano plot of 5,700 phosphosites identified in TMT mass spectrometry from heavy-labeled MOLM-13 cells treated with Cortistatin A.
  • the y axis is the t-test value measured as p plotted in logarithmic base 10 units.
  • the x axis is the ratio of DMSO (vehicle) to Cortistatin A plotted in logarithmic base 2 units. CHD4 was a top hit.
  • FIG. 3 is a volcano plot of 5,700 phosphosites identified in TMT mass spectrometry from heavy-labeled MOLM-13 cells treated with vehicle.
  • the y axis is the t-test value measured as p plotted in logarithmic base 10 units.
  • the x axis is the ratio of DMSO (vehicle) to Cortistatin A plotted in logarithmic base 2 units. CHD4 and DMAP1 were top hits.
  • FIG. 4 is a volcano plot of 5,700 phosphosites identified in TMT mass spectrometry from heavy-labeled MOLM-14 cells treated with Cortistatin A.
  • the y axis is the t-test value measured as p plotted in logarithmic base 10 units.
  • the x axis is the ratio of DMSO (vehicle) to Cortistatin A plotted in logarithmic base 2 units. CHD4 and DPF2 were top hits.
  • FIG. 5 is a volcano plot of 5,700 phosphosites identified in TMT mass spectrometry from heavy-labeled MOLM-14 cells treated with vehicle.
  • the y axis is the t-test value measured as p plotted in logarithmic base 10 units.
  • the x axis is the ratio of DMSO (vehicle) to Cortistatin A plotted in logarithmic base 2 units. CHD4 and DPF2 were top hits.
  • FIG. 6 is an immunoblot of freshly drawn whole blood treated with various concentrations of Compound 1 for 3 hours.
  • the PBMCs used were isolated using Lymphoprep and Sepmate tubes, washed, and lysed.
  • the concentration of wild type CHD4, DPF2, DMAP1 and their analogous phosphosites was assessed at various concentrations.
  • FIG. 7 is a dose response graph of pCHD4 at various concentrations of Compound 1. This graph was generated from the immunoblot in Figure 6. The y-axis is densitometry signal standardized to 100% vehicle. The x-axis is concentration of Compound 1 measured in micromolar units.
  • FIG. 8 is a dose response graph of pDPF2 at various concentrations of Compound 1. This graph was generated from the immunoblot in Figure 6. The y-axis is densitometry signal standardized to 100% vehicle. The x-axis is concentration of Compound 1 measured in micromolar units.
  • FIG. 9 is a dose response graph of pDMAPl at various concentrations of Compound 1. This graph was generated from the immunoblot in Figure 6.
  • the y-axis is densitometry signal standardized to 100% vehicle.
  • the x-axis is concentration of Compound 1 measured in micromolar units.
  • FIG. 10 is an immunoblot of freshly drawn whole blood treated with various concentrations of Compound 1 for 4 hours.
  • the blood sample used is different from that depicted in Figure 6.
  • the PBMCs were isolated using Lymphoprep and Sepmate tubes, washed, and lysed. The concentration of CHD4 and pCHD4 was assessed at various concentrations.
  • FIG. 11 is a dose response graph of pCHD4 at various concentrations of Compound 1. This graph was generated from the immunoblot in Figure 10.
  • the y-axis is densitometry signal standardized to 100% vehicle.
  • the x-axis is concentration of Compound 1 measured in micromolar units.
  • FIG. 12 is an immunoblot of freshly drawn whole blood treated with various concentrations of Compound 1 for 4 hours.
  • the blood sample used is different from that depicted in Figure 6.
  • the PBMCs were isolated using Lymphoprep and Sepmate tubes, washed, and lysed. The concentration of DPF2 and pDPF2 was assessed at various concentrations.
  • FIG. 13 is a dose response graph of pDPF2 at various concentrations of Compound 1. This graph was generated from the immunoblot in Figure 12.
  • the y-axis is densitometry signal standardized to 100% vehicle.
  • the x-axis is concentration of Compound 1 measured in micromolar units.
  • FIG. 14 is an immunoblot of freshly drawn whole blood treated with various concentrations of Compound 1 for 4 hours.
  • the blood sample used is the same as the one used in the immunoblots of Figure 10 and Figure 12.
  • the concentration of STAT1, STAT5, pSTATl, and pSTAT5 was assessed at various concentrations.
  • CDK8/19 targets neither pSTATl or pSTAT5 showed a robust Pharmacodynamic biomarker signal.
  • FIG. 15 is an immunoblot of freshly drawn whole blood treated with various concentrations of Compound 1 for 3 hours.
  • the PBMC’s used for this immunoblot were isolated from freshly drawn blood using Lymphoprep and Sepmate tubes and then treated in RPMI supplement with 10% serum for 3 hours.
  • the concentration of CHD4 and pCHD4 was assessed at various concentrations.
  • FIG. 16 is a dose response graph of pCHD4 at various concentrations of Compound 1. This graph was generated from the immunoblot in Figure 15. The y-axis is densitometry signal standardized to 100% vehicle. The x-axis is concentration of Compound 1 measured in nanomolar units. The IC50 determined in Figure 16 (with serum) is different from the IC50 determined in Figure 11 (without serum).
  • FIG. 17 is an immunoblot of freshly drawn whole blood treated with various concentrations of Compound 1 for 3 hours.
  • the PBMC’s used for this immunoblot were isolated from freshly drawn blood using Lymphoprep and Sepmate tubes and then treated in RPMI supplement with 10% serum for 3 hours.
  • the concentration of DPF2 and pDPF2 was assessed at various concentrations.
  • FIG. 18 is a dose response graph of pDPF2 at various concentrations of Compound 1. This graph was generated from the immunoblot in Figure 17.
  • the y-axis is densitometry signal standardized to 100% vehicle.
  • the x-axis is concentration of Compound 1 measured in nanomolar units.
  • the IC50 determined in Figure 18 (with serum) is different from the IC50 determined in Figure 13 (without serum).
  • FIG. 19 is an immunoblot of CHD4, pCHD4, DPF2, pDPF2, DMAP1, and pDMAPl taken from various cells cultured in the absence and presence of 100 nM Cortistatin A.
  • the cells used were EOL-l, HL-60, Kasumi-l, MEG-01, and MOLM-14. All five cell types show biomarker pCHD4, pDPF2, and pDMAPl as pharmacodynamic biomarkers.
  • FIG. 20 is an immunoblot of CHD4, pCHD4, DPF2, pDPF2, DMAP1, and pDMAPl taken from various cells cultured in the absence and presence of 100 nM Cortistatin A.
  • the cells used were NOMO-l, SET -2, SKNO-l, MV4-11, MOLM-16. All five cell types show biomarker pCHD4, pDPF2, and pDMAPl as pharmacodynamic biomarkers.
  • FIG. 21 is an immunoblot of CHD4 and pCHD4 taken from mouse xenographs. The mice were either treated with 1 milligram/kg (mpk) of Compound 1 or 2.5 milligram/kg (mpk) of Compound 1 PO. The xenograph model used is MV4-11.
  • FIG. 22 is an immunoblot of DPF2 and pDPF2 taken from mouse xenographs. The mice were either treated with 1 milligram/kg (mpk) of Compound 1 or 2.5 milligram/kg (mpk) of Compound 1 PO. The xenograph model used is MV4-11.
  • FIG. 23, FIG. 24, FIG. 25, FIG. 26, FIG. 27, FIG. 28, FIG. 29, FIG. 30, FIG. 31, FIG. 32, FIG. 33, FIG. 34, FIG. 35, FIG. 36, FIG. 37, and FIG. 38 provide non-limiting examples of specific cortistatin analogues that can be used in the present invention.
  • the present invention includes at least the following features:
  • a method for treating a patient with a CDK8 and/or CDK 19 mediated disorder comprising (i) obtaining a first blood sample from the patient; (ii) administering to the patient a first dose of a CDK8/19 inhibitor; (iii) obtaining a second blood sample from the patient after sufficient time for the CDK 8/19 inhibitor to act; (iv) detecting the concentration of the selected phosphorylated pharmacodynamic biomarker selected from the group consisting of CHD4 rT1553, DPF2 pT248, and/or DMAP1 pT445 in the first and second blood sample; (v) determining if the level of phosphorylated CHD4 pTl 553, DPF2 pT248, and/or DMAP1 pT445 is higher in the first blood sample than the second blood sample; (vi) if the decrease in the level of phosphorylated CHD4 rT1553, DPF2 pT248, and/or DMAP1 pT445 is higher in the
  • a method for selecting a patient with a CDK8 and/or CDK19 mediated disorder, for example cancer, for therapy with a cortistatin analogue comprising (i) obtaining a first blood sample from the patient; (ii) detecting the concentration of the selected phosphorylated pharmacodynamic biomarker selected from the group consisting of CHD4 rT1553, DPF2 pT248, and/or DMAP1 pT445 in the first and second blood sample; (iii) adding the CDK8/19 inhibitor to the blood sample to obtain a blood sample treated with the CDK8/19 inhibitor; (iv) detecting the concentration of the selected phosphorylated pharmacodynamic biomarker selected from the group consisting of CHD4 rT1553, DPF2 pT248, and/or DMAP1 pT445 in the first and second blood sample; and (v) determining if the level of phosphorylated CHD4 rT1553, DPF2 pT248, and/or DMAP1
  • step (v) is accomplished by comparing the concentration of the pharmacodynamic biomarker in the untreated and treated sample, wherein if the concentration is decreased by at least 10% in the treated sample as compared to the untreated sample, then the CDK8 and/or CDK19 mediated disorder responds to treatment.
  • biomarkers for use in a pharmacodynamic diagnostic have now been discovered that can conveniently be used in a whole blood assay to monitor over time the effectiveness of the administered CDK8 and/or CDK19 inhibitor drug, such as one of the cortistatin derivatives described further below.
  • These biomarkers are selectively and directly phosphorylated by CDK8 and/or CDK19, and are present in whole blood in sufficient quantities for accurate analysis.
  • they are superior diagnostic agents for use to determine if CDK8 and/or CDK19 is being effectively inhibited in vivo by the drug in the tumor or patient.
  • a patient’s CDK8 and/or CDK19 mediated disorder response to treatment with a CDK8/19 inhibitor can be assessed by determining the concentration of a pharmacodynamic biomarker before and after treatment with a CDK8/19 inhibitor.
  • a healthcare provider can determine whether the patient is responding to treatment with a CDK8/19 inhibitor.
  • pharmacodynamic biomarker refers to a protein whose concentration changes depending on the effectiveness of therapy with a CDK8/19 inhibitor.
  • the pharmacodynamic biomarker may serve as an indicator of a particular subtype of a disease or disorder (e.g., cancer) characterized by certain, molecular, pathological, histological, and/or clinical features.
  • the pharmacodynamic biomarker is a phosphorylated protein, for example, pCHD4, pDPF2, or pDMAPl .
  • multiple pharmacodynamic biomarkers are quantified.
  • the pharmacodynamic biomarker does not have to directly or even indirectly mediate the patient’s CDK8 and/or CDK19 mediated disorder. Even if the concentration of the pharmacodynamic biomarker does not predict a cell line’s sensitivity to CDK8/19 inhibition the method can still be used.
  • CHD4 rT1553, DPF2 pT248, and DMAP1 pT445 are particularly useful because they are found to be phosphorylated in relatively comparable levels in different AML cell lines and different donors’ whole blood unlike phospho-STATs which can often vary widely between different AML cell lines, and between the leukemic blasts and normal PBMCs.
  • the CDK8/19 inhibitor may act through epigenetic mechanisms. And thus, the inhibitor may cause undifferentiated leukemic blasts to enter a differentiation pathway. Healthy PBMCs are largely differentiated lymphocytes and monocytes, and should be far less sensitive to CDK8/19 inhibition than AML blasts.
  • a protein denoted with the letter“p” denotes the protein after one or more phosphorylations.
  • pCHD4 refers to the CHD4 protein with one or more phosphate groups added.
  • CHD4 pTl 553 refers to CHD4 that has specifically been phosphorylated at the 1443 amino acid.
  • the pharmacodynamic biomarker is selected from: pNELFA, pTP53BPl, pAFF4, pOGFR, pMSANTD2, and pBRD4.
  • pCHD4, pDPF2, or pDMAPl being used in a method described herein pNELFA, pTP53BPl, pAFF4, pOGFR, pMSANTD2, or pBRD4 is used.
  • the pharmacodynamic biomarker is selected from: NELFA pTS363, TP53BP1 pTS265, AFF4 pTS8l4, OGFR pTS349, MSANTD2 pTS27, and BRD4 pTS470.
  • NELFA pTS363, TP53BP1 pTS265, AFF4 pTS8l4, OGFR pTS349, MSANTD2 pTS27, or BRD4 pTS470 is used, or may be used independently in combination with any of the other biomarkers described herein.
  • STAT1 pTS727 and/or STAT5 pTS726 is used as the pharmacokinetic biomarker. As described herein STAT1 and STAT5 are not as sensitive to CDK8/19 inhibitors and thus the readouts are not as robust as those achieved for the other pharmacokinetic biomarkers described. However, if the method is sufficiently sensitive for the target method STAT1 and/or STAT5 may be used.
  • the pharmacodynamic biomarker selected, the particular sample’s level of phosphomarks, and the biomarkers dependence on CDK8/19 for phosphorylation will determine what percent reduction is indicative of treatment response.
  • CHD4 rT1553, DPF2 pT248, and DMAP1 pT445 each gave consistent results across multiple donors with CHD4 rT1553 concentration being reduced below 25% of the untreated sample, and DPF2 pT248 and DMAP1 pT445 below 50% of the untreated sample (as quantified by a Western blot set of titrations).
  • Even at maximal CDK8/19 inhibition there is typically not a 100% reduction in the concentration of the pharmacodynamic phosphorylated biomarker used because other kinases may also phosphorylate it.
  • capturing the pharmacodynamic biomarker with one or more antibodies capturing the pharmacodynamic biomarker with a solid support reagent, use of labelled antibodies, use of chemical reagents, or spectroscopic techniques.
  • a monoclonal antibody directed to pCHD4, pDPF2, or pDMAPl is used to capture the pCHD4, pDPF2, or pDMAPl from a test sample.
  • the concentration of this antibody protein complex can be determined with immunoassays, mass spectrometry, or any other desired detection technique.
  • the monoclonal antibody is tagged.
  • the monoclonal antibody is a mouse monoclonal antibody.
  • the monoclonal antibody is a rabbit monoclonal antibody.
  • an additional conjugate antibody is added to detect the presence of pCHD4, pDPF2, or pDMAPl that has been captured.
  • This additional antibody may possess a tag or utilize radiolabeling.
  • the additional antibody just serves as a means to more easily separate the mixture of proteins by SDS page or a related technique.
  • the immunoassay at least detects CHD4 rT1553, DPF2 pT248, and/or DMAP1 pT445 in the test sample, but does not detect non- phosphorylated CHD4, DPF2, and/or DMAP1.
  • the antibody in the immunodiagnostic reagent is comprised can be coated on a solid support such as for example, a microparticle, (e.g., magnetic particle), bead, test tube, microtiter plate, cuvette, membrane, scaffolding molecule, film, filter paper, disc or chip.
  • a microparticle e.g., magnetic particle
  • the immunodiagnostic reagent comprises one or more antibodies that will be used to capture one or more of pCHD4, pDPF2, or pDMAPl from the test sample
  • such antibody or antibodies can be co-coated on the same solid support or can be on separate solid supports.
  • the immunodiagnostic reagent antibody may be labeled with a detectable label or labeled with a specific partner that allows capture or detection.
  • the labels may be a detectable label, such as a fluorophore, radioactive moiety, enzyme, biotin/avidin label, chromophore, chemiluminescent label, or the like.
  • a chemiluminescent microparticle immunoassay is used.
  • the antibody can also incorporate a detectable label, such as a fluorophore, radioactive moiety, enzyme, biotin/avidin label, chromophore, chemiluminescent label, or the like.
  • a detectable label such as a fluorophore, radioactive moiety, enzyme, biotin/avidin label, chromophore, chemiluminescent label, or the like.
  • the antibody is an anti-IgG antibody or anti-IgM antibody, that can also incorporate a detectable label, such as a fluorophore, radioactive moiety, enzyme, biotin/avidin label, chromophore, chemiluminescent label, or the like.
  • a detectable label such as a fluorophore, radioactive moiety, enzyme, biotin/avidin label, chromophore, chemiluminescent label, or the like.
  • any suitable assay known in the art can be used for detecting pCHD4, pDPF2, and/or pDMAPl in the test sample.
  • assays include, but are not limited to, immunoassay, such as sandwich immunoassay (e.g., monoclonal -polyclonal sandwich immunoassays, including radioisotope detection (radioimmunoassay (RIA)) and enzyme detection (enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA) (e.g., Quantikine ELISA assays, R&D Systems, Minneapolis, Minn.)), competitive inhibition immunoassay (e.g., forward and reverse), fluorescence polarization immunoassay (FPIA), enzyme multiplied immunoassay technique (EMIT), bioluminescence resonance energy transfer (BRET), and homogeneous chemiluminescent assay, etc.
  • sandwich immunoassay e.g., monoclonal
  • immunoassays are performed in a l-step or 2-step format.
  • Solid phase reagents for capture of immune complexes formed in solution in the l-step assay include anti -biotin monoclonal antibody, streptavidin, or neutravidin to capture the biotinylated moiety, e.g., the biotinylated antibody for the capture of pCHD4, pDPF2, and/or pDMAPl in the test sample.
  • a capture reagent that specifically binds pCHD4, pDPF2, and/or pDMAPl is attached to the surface of a mass spectrometry probe, such as a pre-activated protein chip array.
  • pCHD4, pDPF2, and/or pDMAPl is then specifically captured on the biochip, and the captured moiety is detected by mass spectrometry.
  • pCHD4, pDPF2, and/or pDMAPl can be eluted from the capture reagent and detected by traditional MALDI (matrix- assisted laser desorption/ionization) or by SELDI.
  • a chemiluminescent microparticle immunoassay in particular one employing the ARCHITECT® automated analyzer (Abbott Laboratories, Abbott Park, Ill.), is an example of an immunoassay in which a combination of multiple anti-pCHD4, pDPF2, and/or pDMAPl antibodies may readily be employed.
  • test sample can comprise further moieties in addition to the polypeptide of interest, such as antibodies, antigens, haptens, hormones, drugs, enzymes, receptors, proteins, peptides, polypeptides, oligonucleotides or polynucleotides.
  • the sample can be a whole blood sample obtained from a subject. It can be necessary or desired that a test sample, particularly whole blood, be treated prior to immunoassay as described herein, e.g., with a pretreatment reagent. Even in cases where pretreatment is not necessary, pretreatment optionally can be done for mere convenience (e.g., as part of a regimen on a commercial platform).
  • the pretreatment reagent can be any reagent appropriate for use with the kit of the invention.
  • the pretreatment optionally comprises: (a) one or more solvents (e.g., methanol and ethylene glycol) and salt, (b) one or more solvents, salt and detergent, (c) detergent, or (d) detergent and salt.
  • Pretreatment reagents are known in the art, and such pretreatment can be employed, e.g., as used for assays on Abbott TDx, AxSYM®, and ARCHITECT® analyzers (Abbott Laboratories, Abbott Park, Ill.), as described in the literature (see, e.g., Yatscoff et ak, Abbott TDx Monoclonal Antibody Assay Evaluated for Measuring Cyclosporine in Whole Blood, Clin. Chem. 36: 1969- 1973 (1990), and Wallemacq et ak, Evaluation of the New AxSYM Cyclosporine Assay: Comparison with TDx Monoclonal Whole Blood and EMIT Cyclosporine Assays, Clin. Chem.
  • pretreatment can be done as described in Abbott's U.S. Pat. No. 5, 135,875, European Pat. Pub. No. 0 471 293, U.S. Provisional Pat. App. 60/878,017, filed Dec. 29, 2006, and U.S. Pat. App. Pub. No. 2008/0020401 (incorporated by reference in its entirety for its teachings regarding pretreatment).
  • the pretreatment reagent can be a heterogeneous agent or a homogeneous agent.
  • a mixture is prepared.
  • the mixture contains the test sample being assessed for pCHD4, pDPF2, and/or pDMAPl and an anti-pDMAPl, pDFP2, and/or pDMAPl antibody, wherein pCHD4, pDPF2, and/or pDMAPl, if present in the sample, and the respective antibody form a complex.
  • the anti-pCHD4, pDPF2, and/or pDMAPl capture antibody are immobilized on a solid phase.
  • the solid phase can be any solid phase known in the art, such as, but not limited to, a magnetic particle, a bead, a test tube, a microtiter plate, a cuvette, a membrane, a scaffolding molecule, a film, a filter paper, a disc and a chip.
  • Detection can be achieved by addition of a specific detection binding partner to the mixture to form a further complex.
  • the detection binding partner can be an anti-IgG antibody.
  • the detection binding partner can be labeled with or contains a detectable label.
  • the detectable label can be a radioactive label (such as 3H, 1251, 35S, 14C, 32P, and 33P), an enzymatic label (such as horseradish peroxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase, and the like), a chemiluminescent label (such as acridinium esters, thioesters, or sulfonamides; luminol, isoluminol, phenanthridinium esters, and the like), a fluorescent label (such as fluorescein (e.g., 5-fluorescein, 6-carboxyfluorescein, 3'6- carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachloro-fluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, and the
  • An acridinium compound can be used as a detectable label in a homogeneous chemiluminescent assay (see, e.g., Adamczyk et al., Bioorg. Med. Chem. Lett. 16: 1324-1328 (2006); Adamczyk et al., Bioorg. Med. Chem. Lett. 4: 2313-2317 (2004); Adamczyk et al., Biorg. Med. Chem. Lett. 14: 3917-3921 (2004); and Adamczyk et al., Org. Lett. 5: 3779-3782 (2003)).
  • the signal that is generated by the detectable label can be detected using routine techniques known to those skilled in the art. For example, when using a chemiluminescent based-label, based on the intensity of the signal generated, the amount of pCHD4, pDPF2, and/or pDMAPl in the sample can be quantified. For example, the amount of pCHD4, pDPF2, and/or pDMAPl in the sample is proportional to the intensity of the signal generated.
  • the amount pCHD4, pDPF2, and/or pDMAPl can be quantified by comparing the amount of light generated to a standard curve concentration of pCHD4, pDPF2, and/or pDMAPl or by comparison to a reference standard.
  • the standard curve can be generated using serial dilutions or solutions of known concentrations of pCHD4, pDPF2, and/or pDMAPl by mass spectroscopy, gravimetric methods, and other techniques known in the art.
  • pCHD4, pDPF2, and/or pDMAPl immunoassays can be conducted using any suitable format known in the art. Generally speaking, a sample being tested for pCHD4, pDPF2, and/or pDMAPl can be contacted with an antibody to pCHD4, pDPF2, or pDMAPl and a detection antibody, such as a labeled anti-IgG antibody, either simultaneously or sequentially and in any order.
  • a detection antibody such as a labeled anti-IgG antibody
  • the test sample can be first contacted with at least one capture antibody and then (sequentially) with at least one detection antibody.
  • the test sample can be first contacted with at least one detection antibody and then (sequentially) with at least one capture antibody.
  • the test sample can be contacted simultaneously with a capture antibody and a detection antibody.
  • the detectable label can be bound to the detection antibody either directly or through a coupling agent.
  • a coupling agent that can be used is EDAC (l-ethyl-3-(3- dimethylaminopropyl)carbodiimide, hydrochloride), which is commercially available from Sigma-Aldrich, St. Louis, Mo.
  • EDAC l-ethyl-3-(3- dimethylaminopropyl)carbodiimide, hydrochloride
  • Methods for binding a detectable label to an antibody are known in the art.
  • detectable labels can be purchased or synthesized that already contain end groups that facilitate the coupling of the detectable label to the antibody, such as CPSP-Acridinium Ester (i.e., 9-[N- tosyl-N-(3-carboxypropyl)]-l0-(3-sulfopropyl)acridinium carboxamide) or SPSP-Acridinium Ester (i.e., Nl0-(3-sulfopropyl)-N-(3-sulfopropyl)-acridinium-9-carboxamide).
  • CPSP-Acridinium Ester i.e., 9-[N- tosyl-N-(3-carboxypropyl)]-l0-(3-sulfopropyl)acridinium carboxamide
  • SPSP-Acridinium Ester i.e., Nl0-(3-sulfopropyl)-N-(3-sulfopropyl)
  • the amount of label in the complex can be quantified using techniques known in the art. For example, if an enzymatic label is used, the labeled complex is reacted with a substrate for the label that gives a quantifiable reaction such as the development of color. If the label is a radioactive label, the label is quantified using a scintillation counter. If the label is a fluorescent label, the label is quantified by stimulating the label with a light of one color (which is known as the “excitation wavelength”) and detecting another color (which is known as the “emission wavelength”) that is emitted by the label in response to the stimulation.
  • the label is quantified by detecting the light emitted either visually or by using luminometers, x-ray film, high speed photographic film, a CCD camera, etc.
  • the concentration of pCHD4, pDPF2, and/or pDMAPl in the test sample is determined by use of a standard curve that has been generated using serial dilutions of pCHD4, pDPF2, and/or pDMAPl or known concentrations of pCHD4, pDPF2, and/or pDMAPl .
  • the standard curve can be generated gravimetrically, by mass spectroscopy and by other techniques known in the art.
  • the detection technique is an adaptation of an existing assay.
  • the following configuration is exemplary.
  • a microfabricated silicon chip is manufactured with a pair of gold amperometric working electrodes and a silver-silver chloride reference electrode.
  • polystyrene beads (0.2 mm diameter) with immobilized capture antibody are adhered to a polymer coating of patterned polyvinyl alcohol over the electrode.
  • This chip is assembled into an I-STAT® cartridge with a fluidics format suitable for immunoassay.
  • a layer comprising the detection antibody labeled with alkaline phosphatase (or other label).
  • an aqueous reagent that includes p-aminophenol phosphate.
  • a sample is added to the holding chamber of the test cartridge and the cartridge is inserted into the I-STAT® reader.
  • a pump element within the cartridge forces the sample into a conduit containing the chip.
  • it is oscillated to promote formation of the sandwich between the capture antibody, pCHD4, pDPF2, and/or pDMAPl, and the labeled detection antibody.
  • fluid is forced out of the pouch and into the conduit to wash the sample off the chip and into a waste chamber.
  • the alkaline phosphatase label reacts with p-aminophenol phosphate to cleave the phosphate group and permit the liberated p-aminophenol to be electrochemically oxidized at the working electrode.
  • the reader is able to calculate the amount of pCHD4, pDPF2, and/or pDMAPlin the sample by means of an embedded algorithm and factory-determined calibration curve.
  • the methods provided herein can be used to monitor treatment in a subject receiving one or more CDK 8/19 inhibitors. Specifically, such methods involve obtaining a first test sample from a subject before the subject has been administered one or more CDK 8/19 inhibitors. The concentration or amount of pCHD4, pDPF2, and/or pDMAPl in a first test sample from a subject is determined (e.g., using the methods described herein or as known in the art). After the concentration or amount of pCHD4, pDPF2, and/or pDMAPl is determined, the subject is then administered one or more CDK 8/19 inhibitors in a therapeutic protocol for the treatment of a CDK8 and/or CDK19 mediated disorder, such as cancer.
  • a second test sample is drawn and the concentration or amount of pCHD4, pDPF2, and/or pDMAPl is determined and compared with the concentration of pCHD4, pDPF2, and/or pDMAPl in the first test sample.
  • the concentration or amount of pCHD4, pDPF2, and/or pDMAPl as determined in the first test sample is the same or lower than the concentration of amount of pCHD4, pDPF2, and/or pDMAPl in the second sample, then the subject is administered a higher dose of CDK 8/19 inhibitor, or administered an alternative therapeutic agent. However, If the concentration or amount of pCHD4, pDPF2, and/or pDMAPl as determined in the first test sample is higher than the concentration of amount of pCHD4, pDPF2, and/or pDMAPl in the second sample, then the subject is maintained on the administered dose of CDK 8/19 inhibitor.
  • samples may be drawn from the patient following a single dose of CDK8/19 inhibitor.
  • the second sample may be drawn at least about or not more than about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours after treatment.
  • concentration or amount of pCHD4, pDPF2, and/or pDMAPl is determined and compared with the concentration of pCHD4, pDPF2, and/or pDMAPl in the first test sample.
  • the concentration or amount of pCHD4, pDPF2, and/or pDMAPl as determined in the first test sample is the same or lower than the concentration of amount of pCHD4, pDPF2, and/or pDMAPl in the second sample, then the subject is administered a higher dose of CDK 8/19 inhibitor, or administered an alternative therapeutic agent. However, If the concentration or amount of pCHD4, pDPF2, and/or pDMAPl as determined in the first test sample is higher than the concentration of amount of pCHD4, pDPF2, and/or pDMAPl in the second sample, then the subject is maintained on the administered dose of CDK 8/19 inhibitor.
  • multiple samples are collected from the patient after receiving treatment.
  • concentration of pharmacodynamic biomarker in each sample is determined. These concentrations may be used to determine how that specific patient is metabolizing the CDK 8/19 inhibitor. For example, if the concentration of pharmacokinetic biomarker begins to increase more rapidly after administration of the CDK 8/19 inhibitor the caregiver may increase the frequency of dosing to ensure an effective dose is maintained.
  • subsequent test samples are then obtained from the subject to monitor the concentration or amount of pCHD4, pDPF2, and/or pDMAPl, and the administration of the CDK 8/19 inhibitor adjusted accordingly.
  • the number of test samples and the time in which said test samples are obtained from the subject are not critical. For example, a subsequent sample could be obtained 30 days after the subject’s second test sample.
  • a subsequent test sample is obtained at a period in time after the second test sample has been obtained from the subject.
  • a subsequent test sample from the subject can be obtained days, weeks or months after the second test sample has been obtained from the subject.
  • the subsequent test sample can be obtained from the subject at a time period of about about 7 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27 weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks, about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36 weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks, about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45 weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks, about 50 weeks, about 51 weeks, about 52 weeks, or more after the second test sample from the subject is obtained.
  • the present invention also provides a diagnostic kit adapted for carrying out any one of the methods of the present invention comprising means for the determination of the amount of a pharmacodynamic biomarker in a sample of a subject and means for comparing the amounts to reference amounts.
  • kit refers to a collection of the aforementioned means, preferably, provided in separately or within a single container.
  • the container also preferably, comprises instructions for carrying out the method of the present invention. Kits are described further below, and can be used with any of the methods described herein.
  • Diagnostic reagents in the field of specific binding assays are often best provided in the form of a kit, which comprises the specific binding agent and the auxiliary reagents required to perform the assay.
  • the present invention therefore also relates to an immunological kit comprising at least one specific binding agent for the pharmacodynamic biomarker and auxiliary reagents for measurement of the pharmacodynamic biomarker.
  • an immunological kit comprising at least one specific binding agent for the pharmacodynamic biomarker, at least one specific binding agent for a well characterized standard and auxiliary reagents for measurement of the pharmacodynamic biomarker and well characterized standard.
  • kits for diagnosing the effectiveness of CDK 8/19 inhibition in the treatment of a CDK8 and/or CDK19 mediated disorder is a cancer.
  • the cancer. is a hematological cancer.
  • the kit comprises a means for detecting or assaying phosphorylation of CHD4, DPF2, and/or DMAP1, or a combination thereof, both prior to administration of a CDK 8/19 inhibitor to treat a CDK8 and/or CDK19 mediated cancer (for example, CDK8 and/or CDK19 mediated hematological cancer) and during continued administration of the CDK 8/19 inhibitor.
  • the kit is capable of analyzing the change in phosphorylation of CHD4, DPF2, and/or DMAP1 during treatment using a CDK 8/19 inhibitor, which is indicative of the effectiveness of the CDK 8/19 inhibitor treatment.
  • the kit generally incorporates an immunoassay method for determining concentrations of one or more of the phosphorylated targets CHD4, DPF2, and DMAP1.
  • such methods include methods for isolating pCHD4, pDPF2, and/or pDMAPl present in the test sample.
  • a monoclonal antibody directed to pCHD4, pDPF2, or pDMAPl is used to capture the pCHD4, pDPF2, or pDMAPl from a test sample and then further conjugate antibodies are used to detect the presence of pCHD4, pDPF2, or pDMAPl that has been captured.
  • such antibodies preferably determine the presence of only phosphorylated CHD4, DPF2, and/or DMAP1 in the test sample.
  • Antibodies directed to only phosphorylated CHD4, DPF2, and/or DMAP1 are described herein, and methods of making such antibodies are generally known to those of skill in the art.
  • the antibodies used in the immunoassay are antibodies designed to detect CHD4 rT1553, DPF2 pT248, and/or DMAP1 pT445 in a test sample
  • the immunoassay at least detects CHD4 rT1553, DPF2 pT248, and/or DMAP1 pT445 in the test sample, but does not detect non-phosphorylated CHD4, DPF2, and/or DMAP1.
  • the pCHD4, pDPF2, or pDMAPl antibodies described above are contemplated for use as immunodiagnostic reagents in combination immunoassays designed for the detection of one of pCHD4, pDPF2, or pDMAPl found in a test sample from a subject prior to and after receiving a CDK 8/19 inhibitor for the treatment of a CDK8 and/or CDK 19 disorder (for example, a CDK8 and/or CDK19 mediated hematological cancer).
  • Immunodiagnostic reagents will be comprised of the above-described antibodies (typically in combination) such that they can be used in an immunoassay designed for the detection of pCHD4, pDPF2, or pDMAPl .
  • the kit may include antibodies coated on a solid support such as for example, a microparticle, (e.g., magnetic particle), bead, test tube, microtiter plate, cuvette, membrane, scaffolding molecule, film, filter paper, disc or chip.
  • a microparticle e.g., magnetic particle
  • the immunodiagnostic reagent comprises one or more antibodies that will be used to capture one or more of pCHD4, pDPF2, or pDMAPl from the test sample
  • such antibodies can be co- coated on the same solid support or can be on separate solid supports.
  • the immunodiagnostic reagent antibody may be labeled with a detectable label or labeled with a specific partner that allows capture or detection.
  • the labels may be a detectable label, such as a fluorophore, radioactive moiety, enzyme, biotin/avidin label, chromophore, chemiluminescent label, or the like.
  • the invention contemplates the preparation of a diagnostic kit comprising the immunodiagnostic reagents described herein and instructions for the use of the immunodiagnostic reagents in immunoassays for determining the change in phosphorylation patterns of CHD4, DPF2, or DMAP1 during treatment of a CDK8 and/or CDK19 mediated disorder (for example, a CDK8 and/or CDK19 mediated hematological cancer) before and after administration of a CDK 8/19 inhibitor in a test sample.
  • the kit can comprise instructions for assaying the test sample for pCHD4, pDPF2, or pDMAPl by immunoassay.
  • the antibodies used in the immunoassays of the present invention may be used in any immunoassay formats known to those of skill in the art for determining the presence of a protein in a test sample, including for example, but not limited to chemiluminescent microparticle immunoassays.
  • the instructions can be in paper form or computer-readable form, such as a disk, CD, DVD, or the like.
  • the kit can comprise a calibrator or control, e.g., purified, and optionally lyophilized pCHD4, pDPF2, and/or pDMAPl and/or at least one container (e.g., tube, microtiter plates or strips), which can be already coated with one or more antibodies for conducting the assay, and/or a buffer, such as an assay buffer or a wash buffer, either one of which can be provided as a concentrated solution, a substrate solution for the detectable label (e.g., an enzymatic label), or a stop solution.
  • the kit comprises all components, i.e., reagents, standards, buffers, diluents, etc., which are necessary to perform the assay.
  • any antibodies which are provided in the kit, such as anti-IgG antibodies and anti-IgM antibodies, can also incorporate a detectable label, such as a fluorophore, radioactive moiety, enzyme, biotin/avidin label, chromophore, chemiluminescent label, or the like, or the kit can include reagents for labeling the antibodies or reagents for detecting the antibodies (e.g., detection antibodies) and/or for labeling the analytes or reagents for detecting the analyte.
  • the antibodies, calibrators and/or controls can be provided in separate containers or pre-dispensed into an appropriate assay format, for example, into microtiter plates.
  • the kit includes quality control components (for example, sensitivity panels, calibrators, and positive controls).
  • quality control components for example, sensitivity panels, calibrators, and positive controls.
  • Preparation of quality control reagents is well-known in the art and is described on insert sheets for a variety of immunodiagnostic products.
  • Sensitivity panel members optionally are used to establish assay performance characteristics, and further optionally are useful indicators of the integrity of the immunoassay kit reagents, and the standardization of assays.
  • the kit can also optionally include other reagents required to conduct a diagnostic assay or facilitate quality control evaluations, such as buffers, salts, enzymes, enzyme co-factors, substrates, detection reagents, and the like.
  • Other components such as buffers and solutions for the isolation and/or treatment of a test sample (e.g., pretreatment reagents), also can be included in the kit.
  • the kit can additionally include one or more other controls.
  • One or more of the components of the kit can be lyophilized, in which case the kit can further comprise reagents suitable for the reconstitution of the lyophilized components.
  • kits for holding or storing a sample (e.g., a container or cartridge for a sample).
  • a sample e.g., a container or cartridge for a sample
  • the kit optionally also can contain reaction vessels, mixing vessels, and other components that facilitate the preparation of reagents or the test sample.
  • the kit can also include one or more instrument for assisting with obtaining a test sample, such as a syringe, pipette, forceps, measured spoon, or the like.
  • the kit can contain a solid support phase, such as a magnetic particle, bead, test tube, microtiter plate, cuvette, membrane, scaffolding molecule, film, filter paper, disc or chip.
  • a solid support phase such as a magnetic particle, bead, test tube, microtiter plate, cuvette, membrane, scaffolding molecule, film, filter paper, disc or chip.
  • immunoassay such as sandwich immunoassay (e.g., monoclonal-polyclonal sandwich immunoassays, including radioisotope detection (radioimmunoassay (RIA)) and enzyme detection (enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA) (e.g., Quantikine ELISA assays, R&D Systems, Minneapolis, Minn.)), competitive inhibition immunoassay (e.g., forward and reverse), fluorescence polarization immunoassay (FPIA), enzyme multiplied immunoassay technique (EMIT), bioluminescence resonance energy transfer (BRET), and homogeneous chemiluminescent assay, etc.
  • sandwich immunoassay e.g., monoclonal-polyclonal sandwich immunoassays, including radioisotope detection (radioimmunoassay (RIA)) and enzyme detection (enzyme immunoassay (EIA) or enzyme-
  • Pretreatment reagents may also be provided in this kit.
  • the test sample can comprise further moieties in addition to the polypeptide of interest, such as antibodies, antigens, haptens, hormones, drugs, enzymes, receptors, proteins, peptides, polypeptides, oligonucleotides or polynucleotides.
  • the sample can be a whole blood sample obtained from a subject. It can be necessary or desired that a test sample, particularly whole blood, be treated prior to immunoassay as described herein, e.g., with a pretreatment reagent. Even in cases where pretreatment is not necessary, pretreatment optionally can be done for mere convenience (e.g., as part of a regimen on a commercial platform).
  • the pretreatment reagent can be any reagent appropriate for use with the kit of the invention.
  • the pretreatment optionally comprises: (a) one or more solvents (e.g., methanol and ethylene glycol) and salt, (b) one or more solvents, salt and detergent, (c) detergent, or (d) detergent and salt.
  • Pretreatment reagents are known in the art, and such pretreatment can be employed, e.g., as used for assays on Abbott TDx, AxSYM®, and ARCHITECT® analyzers (Abbott Laboratories, Abbott Park, Ill.), as described in the literature (see, e.g., Yatscoff et ah, Abbott TDx Monoclonal Antibody Assay Evaluated for Measuring Cyclosporine in Whole Blood, Clin. Chem. 36: 1969- 1973 (1990), and Wallemacq et al., Evaluation of the New AxSYM Cyclosporine Assay: Comparison with TDx Monoclonal Whole Blood and EMIT Cyclosporine Assays, Clin. Chem.
  • pretreatment can be done as described in Abbott's U.S. Pat. No. 5, 135,875, European Pat. Pub. No. 0 471 293, U.S. Provisional Pat. App. 60/878,017, filed Dec. 29, 2006, and U.S. Pat. App. Pub. No. 2008/0020401 (incorporated by reference in its entirety for its teachings regarding pretreatment).
  • the pretreatment reagent can be a heterogeneous agent or a homogeneous agent.
  • a mixture is prepared.
  • the mixture contains the test sample being assessed for pCHD4, pDPF2, and/or pDMAPl and an anti-pDMAPl, pDFP2, and/or pDMAPl antibody, wherein pCHD4, pDPF2, and/or pDMAPl, if present in the sample, and the respective antibody form a complex.
  • the kit provides specific detection antibodies.
  • the detection binding partner can be an anti-IgG antibody.
  • the detection binding partner can be labeled with or contains a detectable label.
  • kits provided herein can be used to monitor treatment in a subject receiving treatment with one or more CDK 8/19 inhibitors. Specifically, such methods involve providing a first test sample from a subject before the subject has been administered one or more CDK 8/19 inhibitors. The concentration or amount of pCHD4, pDPF2, and/or pDMAPl in a first test sample from a subject is determined (e.g., using the methods described herein or as known in the art).
  • the subject is then administered one or more CDK 8/19 inhibitors in a therapeutic protocol for the treatment of CDK8 and/or CDK19 mediated disorder (for example, a CDK8 and/or CDK19 mediated hematological cancer).
  • a CDK8 and/or CDK19 mediated disorder for example, a CDK8 and/or CDK19 mediated hematological cancer.
  • a second test sample is drawn and the concentration or amount of pCHD4, pDPF2, and/or pDMAPl is determined and compared with the concentration of pCHD4, pDPF2, and/or pDMAPl in the first test sample.
  • the concentration or amount of pCHD4, pDPF2, and/or pDMAPl as determined in the first test sample is the same or lower than the concentration of amount of pCHD4, pDPF2, and/or pDMAPl in the second sample, then the subject is administered a higher dose of CDK 8/19 inhibitor, or administered an alternative therapeutic agent. However, If the concentration or amount of pCHD4, pDPF2, and/or pDMAPl as determined in the first test sample is higher than the concentration of amount of pCHD4, pDPF2, and/or pDMAPl in the second sample, then the subject is maintained on the administered dose of CDK 8/19 inhibitor.
  • subsequent test samples are then obtained from the subject to monitor the concentration or amount of pCHD4, pDPF2, and/or pDMAPl, and the administration of the CDK 8/19 inhibitor adjusted accordingly.
  • the number of test samples and the time in which said test samples are obtained from the subject are not critical. For example, a subsequent sample could be obtained 30 days after the subject’s second test sample.
  • a subsequent test sample is obtained at a period in time after the second test sample has been obtained from the subject.
  • a subsequent test sample from the subject can be obtained days, weeks or months after the second test sample has been obtained from the subject.
  • the subsequent test sample can be obtained from the subject at a time period of about about 7 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27 weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks, about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36 weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks, about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45 weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks, about 50 weeks, about 51 weeks, about 52 weeks, or more after the second test sample from the subject is obtained.
  • Preferred devices are those which can be applied without the particular knowledge of a specialized clinician, e.g., test strips or electronic devices which merely require loading with a sample.
  • the results may be given as output of raw data which need interpretation by the clinician.
  • the output of the device is, however, processed, i.e. evaluated, raw data the interpretation of which does not require a clinician.
  • Further preferred devices comprise the analyzing units/devices (e.g., biosensors, arrays, solid supports coupled to ligands specifically recognizing the natriuretic peptide, plasmon surface resonance devices, NMR spectrometers, mass-spectrometers etc.) and/or evaluation units/devices referred to above in accordance with the method of the invention.
  • the kit (or components thereof), as well as the method of determining the concentration of pCHD4, pDPF2, and/or pDMAPl can be adapted for use in a variety of automated and semi- automated systems (including those wherein the solid phase comprises a microparticle), as described, e.g., in U.S. Pat. Nos. 5,089,424 and 5,006,309, and as commercially marketed, e.g., by Abbott Laboratories (Abbott Park, Ill.) as ARCHITECT®.
  • Other platforms available from Abbott Laboratories include, but are not limited to, AxSYM®, IMx® (see, e.g., U.S. Pat. No.
  • the kit may include an adaptation of an existing assay.
  • an assay to the I-STAT® system
  • the following configuration is exemplary.
  • a microfabricated silicon chip is manufactured with a pair of gold amperometric working electrodes and a silver-silver chloride reference electrode.
  • polystyrene beads (0.2 mm diameter) with immobilized capture antibody are adhered to a polymer coating of patterned polyvinyl alcohol over the electrode.
  • This chip is assembled into an I-STAT® cartridge with a fluidics format suitable for immunoassay.
  • On a portion of the wall of the sample-holding chamber of the cartridge there is a layer comprising the detection antibody labeled with alkaline phosphatase (or other label).
  • an aqueous reagent that includes p-aminophenol phosphate.
  • the present invention relates to a diagnostic kit adapted for carrying out any one of the methods of the present invention comprising means for the determination of the amount of a pharmacodynamic biomarker in a sample of a subject and means for comparing the amounts to reference amounts.
  • the term “kit” as used herein refers to a collection of the aforementioned means, preferably, provided in separately or within a single container.
  • the container also preferably, comprises instructions for carrying out the method of the present invention.
  • kits may comprise a provided composition or antibody and a container (e.g., a vial, ampoule, bottle, syringe, and/or dispenser package, or other suitable container).
  • a container e.g., a vial, ampoule, bottle, syringe, and/or dispenser package, or other suitable container.
  • provided kits may optionally further include a second container comprising a suitable aqueous carrier for dilution or suspension of the provided composition for preparation to administer the compound to the subject.
  • the kit may include a second container comprising a suitable carrier for dilution of suspension of the provided antibody for ease in quantification.
  • contents of provided formulation container and solvent container combine to form at least one unit dosage form.
  • a single container may comprise one or more compartments for containing a provided composition or antibody, and/or appropriate aqueous carrier for suspension or dilution.
  • a single container can be appropriate for modification such that the container may receive a physical modification so as to allow combination of compartments and/or components of individual compartments.
  • a foil or plastic bag may comprise two or more compartments separated by a perforated seal which can be broken so as to allow combination of contents of two individual compartments once the signal to break the seal is generated.
  • the additional compartment is empty and can be filled with the patient’s fluid sample.
  • a pharmaceutical pack or kit may thus comprise such multi-compartment containers including a provided composition or antibody and appropriate solvent and/or appropriate aqueous carrier for suspension.
  • instructions for use are additionally provided in such kits of the invention.
  • Such instructions may provide, generally, for example, instructions for dosage and administration.
  • instructions may further provide additional detail relating to specialized instructions for particular containers and/or systems for administration.
  • instructions may provide specialized instructions for use in conjunction and/or in combination with additional therapy.
  • cortistatin or“cortistatin derivative” or“cortistatin analog” as used herein refers to a compound that has the general cortistatin framework of one of the known naturally occurring cortistatins (Cortistatins A, B, C, D, E, F, G, H, I, J, K or L) or is described in one of the Formulas below.
  • the cortistatin can be used if desired in the form of a pharmaceutically acceptable salt, including a quartemary ammonium salt, an N-oxide and/or in a pharmaceutically acceptable composition.
  • the CDK8/19 inhibitor for use in the present invention is a cortistatin analogue described in U.S. Patent Publication 2017/029435; PCT Patent Publication WO 2017/112815; PCT Patent Publication WO 2017/142621; each of which is incorporated by reference for all purposes.
  • Non-limiting examples of cortistatin based CDK8/19 inhibitors include compounds of Formula A, B, C, D, and E:
  • R 1 and R 2 are joined to form an optionally substituted heterocyclyl or optionally substituted heteroaryl;
  • R 3 is hydrogen or optionally substituted alkyl
  • R 4 is hydrogen, halogen, optionally substituted alkyl, or -Si(R A )3;
  • R 5 is hydrogen, halogen, or optionally substituted alkyl
  • each instance of designated as (a), ( «/), ( b ), and (c), represents a single or double bond, provided that when designated as (c) represents a double bond, then one of R B1 and R B2 is absent, and provided that when designated as (c) represents a single bond, then both R B1 and R B2 are present;
  • each instance of R B1 and R B2 is, independently, hydrogen, -LI-R B3 , or -X A R A wherein X A is -0-, -S-, or -N(R a )-; or R B1 and R B2 are joined to form an oxo group, provided that at least one of R B1 and R B2 is not hydrogen;
  • R B3 is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl, provided that when Li is a bond, then R B3 is not hydrogen;
  • each instance of R A is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, carbonyl, silyl, an oxygen protecting group when attached to oxygen, a sulfur protecting group when attached to sulfur, or a nitrogen protecting group when attached to nitrogen, optionally when attached to N the two R A groups may be joined to form an optionally substituted heterocyclyl or optionally substituted heteroaryl ring, and optionally when R B1 and R B2 are each -X A R A then two R A groups may be joined to form an optionally substituted heterocyclyl ring; and
  • cortistatin for use in the present invention is selected from the structures of Figure 23-38.
  • the present invention utilizes compounds of Formula A, B, C, D, or E, and additional active compounds described herein, with at least one desired isotopic substitution of an atom, at an amount above the natural abundance of the isotope, i.e., enriched.
  • Isotopes are atoms having the same atomic number but different mass numbers, i.e., the same number of protons but a different number of neutrons.
  • isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as 2 H, 3 H, U C, 13 C, 14 C, 15 N, 18 F 31 P, 32 P, 35 S, 36 CI, 125 I respectively.
  • the invention includes various isotopically labeled compounds as defined herein, for example those into which radioactive isotopes, such as 3 H, 13 C, and 14 C, are present.
  • Such isotopically labelled compounds are useful in metabolic studies (with 14 C), reaction kinetic studies (with, for example 2 H or 3 H), detection or imaging techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays, or in radioactive treatment of patients.
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • an 18 F labeled compound may be particularly desirable for PET or SPECT studies.
  • Isotopically labeled compounds of this invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.
  • R 1 and R 2 are joined to form an optionally substituted heterocyclyl, e.g., an optionally substituted 3-6 membered heterocyclyl. In certain embodiments, R 1 and R 2 are joined to form an optionally substituted 3-membered heterocyclyl, an optionally substituted 4-membered heterocyclyl, optionally substituted 5-membered heterocyclyl, or an optionally substituted 6-membered heterocyclyl. In certain embodiments, R 1 and R 2 are joined to form an optionally substituted 3-membered heterocyclyl, i.e., an optionally substituted aziridinyl.
  • R 1 and R 2 are joined to form an optionally substituted 4-membered heterocyclyl, e.g, an optionally substituted azetidinyl. In certain embodiments, R 1 and R 2 are joined to form an optionally substituted 5-membered heterocyclyl, e.g, an optionally substituted pyrrolidinyl or optionally substituted imidazolidine-2,4-dione.
  • R 1 and R 2 are joined to form an optionally substituted 6-membered heterocyclyl, e.g, an optionally substituted piperidinyl, optionally substituted tetrahydropyranyl, optionally substituted dihydropyridinyl, optionally substituted thianyl, optionally substituted piperazinyl, optionally substituted morpholinyl, optionally substituted dithianyl, optionally substituted dioxanyl, or optionally substituted triazinanyl.
  • an optionally substituted 6-membered heterocyclyl e.g, an optionally substituted piperidinyl, optionally substituted tetrahydropyranyl, optionally substituted dihydropyridinyl, optionally substituted thianyl, optionally substituted piperazinyl, optionally substituted morpholinyl, optionally substituted dithianyl, optionally substituted dioxanyl, or optionally substituted triazinanyl.
  • R 1 and R 2 are joined to form a group of formula: , e.g, to provide a compound of formula:
  • R 3 , R 4 , R B1 , and R B2 are as defined herein;
  • G is -0-, -S-, -NH-, -NR 7 -, -CFh-, -CH(R 7 )-, or -C(R 7 ) 2 -;
  • R 5A is selected from hydrogen, halogen, and optionally substituted alkyl
  • each instance of R 7 is independently halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, amino, substituted amino, hydroxyl, substituted hydroxyl, thiol, substituted thiol, carbonyl, sulfonyl, sulfmyl, or a nitrogen protecting group when attached to a nitrogen atom;
  • n 0, 1, 2, 3, or 4.
  • R 1 and R 2 are joined to form a group of formula: provide a compound of formula
  • R 3 , R 4 , R 5A , R b1 , and R B2 are as defined herein;
  • G is -0-, -S-, -NH-, -NR 7 -, -CH 2 -, -CH(R 7 )-, or -C(R 7 ) 2 -;
  • each instance of R 7 is independently halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, amino, substituted amino, hydroxyl, substituted hydroxyl, thiol, substituted thiol, carbonyl, sulfonyl, sulfmyl, or a nitrogen protecting group when attached to a nitrogen atom;
  • n 0, 1, 2, 3, or 4.
  • R 1 and R 2 are joined to form a group of formula: provi SdEeI aii compound of formula
  • R 3 , R 4 , R 5A , R b1 , and R B2 are as defined herein;
  • G is -0-, -S-, -NH-, -NR 7 -, -CH2-, -CH(R 7 )-, or -C(R 7 ) 2 -;
  • each instance of R 7 is independently halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, amino, substituted amino, hydroxyl, substituted hydroxyl, thiol, substituted thiol, carbonyl, sulfonyl, sulfmyl, or a nitrogen protecting group when attached to a nitrogen atom;
  • n 0, 1, 2, 3, or 4.
  • n is 0, and the ring system formed by the joining of R 1 and R 2 is not substituted with an R 7 group as defined herein.
  • n is 1, 2, 3, or 4, and the ring system is substituted with 1, 2, 3, or 4 R 7 groups as defined herein.
  • n is 1. In certain embodiments, n is 2. In certain embodiments, n is 3. In certain embodiments, n is 4.
  • n is not 0 (i.e ., n is 1, 2, 3, or 4) and at least one R 7 is attached to a carbon atom
  • the R 7 is halogen (e.g ., fluoro), hydroxyl, substituted hydroxyl, or carbonyl (e.g., -CO2H).
  • n is not 0 (i.e., n is 1, 2, 3, or 4) and two R 7 groups are attached to the same carbon atom
  • the two R 7 groups are each halogen, e.g, fluoro.
  • n is not 0 (i.e., n is 1, 2, 3, or 4) and two R 7 groups are attached to the same carbon atom, the two R 7 groups are joined to form an optionally substituted carbocyclyl ring or optionally substituted heterocyclyl ring (e.g, optionally substituted oxetanyl ring).
  • n is not 0 (i.e., n is 1, 2, 3, or 4) and two R 7 groups are attached to a different carbon atom
  • the two R 7 groups are joined to form an optionally substituted carbocyclyl ring or optionally substituted heterocyclyl ring.
  • G is -0-. In certain embodiments, G is -NR 7 -, e.g, wherein R 7 is optionally substituted alkyl (e.g, -CH3). In certain embodiments, G is -CH(R 7 )- or -C(R 7 )2- wherein at least one R 7 is hydroxyl, substituted hydroxyl, or carbonyl (e.g, -CO2H).
  • the group In certain embodiments, the group
  • group -LI-R B3 is a group of formula:
  • R n , R 1 , R 2 , R 3 , R 4 , R 5A , and m are as defined herein;
  • each instance of R 6C is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to oxygen, a sulfur protecting group when attached to sulfur, or a nitrogen protecting group when attached to nitrogen, optionally when attached to N the two R 6C groups may be joined to form an optionally substituted heterocyclyl or optionally substituted heteroaryl ring.
  • G is O. In certain embodiments, G is N- CFb. In certain embodiments, m is 0. In certain embodiments, m is 1. In certain embodiments, n is 0. In certain embodiments, n is 1.
  • G is not CH 2 .
  • m is 0. In certain embodiments, m is 1. In certain embodiments, n is 0. In certain embodiments, n is 1
  • G is -CH2-.
  • m is 0.
  • n is 0.
  • n is 1.
  • Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various stereoisomeric forms, e.g. , enantiomers and/or diastereomers. Also contemplated are stereoisomers featuring either a Z or E configuration, or mixture thereof, about a double bond.
  • the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer.
  • Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al ., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al. , Tetrahedron 33 :2725 (1977); Eliel, E.L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, S.H. Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The invention additionally encompasses compounds as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.
  • HPLC high pressure liquid chromatography
  • Isomeric mixtures containing any of a variety of isomer ratios may be utilized in accordance with the present invention. For example, where only two isomers are combined, mixtures containing 50:50, 60:40, 70:30, 80:20, 90: 10, 95:5, 96:4, 97:3, 98:2, 99: 1, or 100:0 isomer ratios are all contemplated by the present invention. Those of ordinary skill in the art will readily appreciate that analogous ratios are contemplated for more complex isomer mixtures.
  • the mixture may contain two enantiomers, two diastereomers, or a mixture of diastereomers and enantiomers.
  • a particular enantiomer of a compound described herein may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers.
  • a compound described herein is prepared by asymmetric synthesis with an enzyme. Enantiomers and diastereomers may be separated by means of fractional crystallization or chromatography (e.g., HPLC with a chiral column).
  • diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.
  • structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms.
  • compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, replacement of 19 F with 18 F, or the replacement of a carbon by a 13 C- or 14 C-enriched carbon are within the scope of the disclosure.
  • Such compounds are useful, for example, as analytical tools or probes in biological assays.
  • Ci-6 alkyl is intended to encompass, Ci, C2, C3, C 4 , C5, C 6 , Ci- 6, Ci-5, Ci-4, Ci— 3, Ci-2, C2-6, C2-5, C2-4, C2-3, C3-6, C3-5, C3-4, C4-6, C4-5, and C5-6 alkyl.
  • aliphatic refers to alkyl, alkenyl, alkynyl, and carbocyclic groups.
  • alkyl refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 10 carbon atoms (“Ci-io alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms (“C1-9 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“Ci- 7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“C1-6 alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C1-5 alkyl”).
  • an alkyl group has 1 to 4 carbon atoms (“Ci-4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C1-3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“Ci- 2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“Ci alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C2-6 alkyl”).
  • C1-6 alkyl groups include methyl (Ci), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec- butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl- 2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C 6 ).
  • Additional examples of alkyl groups include n-heptyl (C7), n-octyl (Cx) and the like.
  • each instance of an alkyl group is independently unsubstituted (an“unsubstituted alkyl”) or substituted (a“substituted alkyl”) with one or more substituents.
  • the alkyl group is an unsubstituted Ci-10 alkyl (e.g, -CFb).
  • the alkyl group is a substituted C1-10 alkyl.
  • “haloalkyl” is a substituted alkyl group as defined herein wherein one or more of the hydrogen atoms are independently replaced by a halogen, e.g., fluoro, bromo, chloro, or iodo.
  • Perhaloalkyl is a subset of haloalkyl, and refers to an alkyl group wherein all of the hydrogen atoms are independently replaced by a halogen, e.g, fluoro, bromo, chloro, or iodo.
  • the haloalkyl moiety has 1 to 8 carbon atoms (“Ci-s haloalkyl”).
  • the haloalkyl moiety has 1 to 6 carbon atoms (“Ci-6 haloalkyl”).
  • the haloalkyl moiety has 1 to 4 carbon atoms (“Ci-4 haloalkyl”).
  • the haloalkyl moiety has 1 to 3 carbon atoms (“C1-3 haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 2 carbon atoms (“C1-2 haloalkyl”). In some embodiments, all of the haloalkyl hydrogen atoms are replaced with fluoro to provide a perfluoroalkyl group. In some embodiments, all of the haloalkyl hydrogen atoms are replaced with chloro to provide a“perchloroalkyl” group. Examples of haloalkyl groups include -CF3, - CF 2 CF 3 , -CF 2 CF 2 CF 3 , -CCh, -CFCh, -CF 2 Cl, and the like.
  • alkenyl refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 10 carbon atoms and one or more carbon-carbon double bonds (e.g, 1, 2, 3, or 4 double bonds).
  • an alkenyl group has 2 to 9 carbon atoms (“C 2-9 alkenyl”).
  • an alkenyl group has 2 to 8 carbon atoms (“C 2- s alkenyl”).
  • an alkenyl group has 2 to 7 carbon atoms (“C 2-7 alkenyl”).
  • an alkenyl group has 2 to 6 carbon atoms (“C 2-6 alkenyl”).
  • an alkenyl group has 2 to 5 carbon atoms (“C 2-5 alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C 2-4 alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C 2-3 alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C 2 alkenyl”).
  • the one or more carbon-carbon double bonds can be internal (such as in 2- butenyl) or terminal (such as in l-butenyl).
  • Examples of C2-4 alkenyl groups include ethenyl (C 2 ), l-propenyl (C3), 2-propenyl (C3), l-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like.
  • Examples of C 2-6 alkenyl groups include the aforementioned C 2-4 alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C 6 ), and the like.
  • Additional examples of alkenyl include heptenyl (C7), octenyl (Cx), octatrienyl (Cx), and the like.
  • each instance of an alkenyl group is independently unsubstituted (an“unsubstituted alkenyl”) or substituted (a“substituted alkenyl”) with one or more substituents.
  • the alkenyl group is an unsubstituted C2-10 alkenyl.
  • the alkenyl group is a substituted C2-10 alkenyl.
  • alkynyl refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 10 carbon atoms and one or more carbon-carbon triple bonds ( e.g 1, 2, 3, or 4 triple bonds) (“C2-10 alkynyl”).
  • an alkynyl group has 2 to 9 carbon atoms (“C2-9 alkynyl”).
  • an alkynyl group has 2 to 8 carbon atoms (“C2-8 alkynyl”).
  • an alkynyl group has 2 to 7 carbon atoms (“C2-7 alkynyl”).
  • an alkynyl group has 2 to 6 carbon atoms (“C2-6 alkynyl”).
  • an alkynyl group has 2 to 5 carbon atoms (“C2-5 alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C2-4 alkynyl”). In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C2-3 alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C2 alkynyl”).
  • the one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in l-butynyl).
  • C2-4 alkynyl groups include, without limitation, ethynyl (C2), l-propynyl (C3), 2-propynyl (C3), l-butynyl (C 4 ), 2-butynyl (C 4 ), and the like.
  • Examples of C2-6 alkenyl groups include the aforementioned C2-4 alkynyl groups as well as pentynyl (C5), hexynyl (C 6 ), and the like. Additional examples of alkynyl include heptynyl (C7), octynyl (Cx), and the like.
  • each instance of an alkynyl group is independently unsubstituted (an“unsubstituted alkynyl”) or substituted (a“substituted alkynyl”) with one or more substituents.
  • the alkynyl group is an unsubstituted C2- 10 alkynyl.
  • the alkynyl group is a substituted C2-10 alkynyl.
  • “carbocyclyl” or“carbocyclic” refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 14 ring carbon atoms (“C3-14 carbocyclyl”) and zero heteroatoms in the non-aromatic ring system.
  • a carbocyclyl group has 3 to 10 ring carbon atoms (“C3-10 carbocyclyl”).
  • a carbocyclyl group has 3 to 9 ring carbon atoms (“C3-9 carbocyclyl”).
  • a carbocyclyl group has 3 to 8 ring carbon atoms (“C3-8 carbocyclyl”).
  • a carbocyclyl group has 3 to 7 ring carbon atoms (“C3-7 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms (“C3-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 4 to 6 ring carbon atoms (“C4-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 6 ring carbon atoms (“C5-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms (“C5-10 carbocyclyl”).
  • Exemplary C3-6 carbocyclyl groups include, without limitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C 4 ), cyclobutenyl (C 4 ), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like.
  • Exemplary C3-8 carbocyclyl groups include, without limitation, the aforementioned C3-6 carbocyclyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (Cx), cyclooctenyl (Cx), bicyclo[2.2. l]heptanyl (C7), bicyclo[2.2.2]octanyl (Cs), and the like.
  • C7 cycloheptyl
  • C7 cycloheptenyl
  • C7 cycloheptadienyl
  • C7 cycloheptatrienyl
  • Cx cyclooctyl
  • Cx cyclooctenyl
  • bicyclo[2.2. l]heptanyl (C7) bicyclo[2.2.2]octanyl (Cs), and the like.
  • Exemplary C3-10 carbocyclyl groups include, without limitation, the aforementioned C3-8 carbocyclyl groups as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C10), cyclodecenyl (C10), octahydro- 1 //-indenyl (C9), decahydronaphthalenyl (C10), spiro[4.5]decanyl (C10), and the like.
  • the carbocyclyl group is either monocyclic (“monocyclic carbocyclyl”) or polycyclic (e.g ., containing a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic carbocyclyl”) or tricyclic system (“tricyclic carbocyclyl”)) and can be saturated or can contain one or more carbon-carbon double or triple bonds.
  • “Carbocyclyl” also includes ring systems wherein the carbocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups wherein the point of attachment is on the carbocyclyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the carbocyclic ring system.
  • each instance of a carbocyclyl group is independently unsubstituted (an“unsubstituted carbocyclyl”) or substituted (a“substituted carbocyclyl”) with one or more substituents.
  • the carbocyclyl group is an unsubstituted C3-14 carbocyclyl.
  • the carbocyclyl group is a substituted C3-14 carbocyclyl.
  • heterocyclyl or“heterocyclic” refers to a radical of a 3- to l4-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“3-14 membered heterocyclyl”).
  • the point of attachment can be a carbon or nitrogen atom, as valency permits.
  • a heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or polycyclic (e.g., a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”) or tricyclic system (“tricyclic heterocyclyl”)), and can be saturated or can contain one or more carbon-carbon double or triple bonds.
  • Heterocyclyl polycyclic ring systems can include one or more heteroatoms in one or both rings.
  • Heterocyclyl also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system.
  • each instance of heterocyclyl is independently unsubstituted (an“unsubstituted heterocyclyl”) or substituted (a“substituted heterocyclyl”) with one or more substituents.
  • the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3-14 membered heterocyclyl.
  • aryl refers to a radical of a monocyclic or polycyclic (e.g ., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6-14 aryl”).
  • an aryl group has 6 ring carbon atoms (“C 6 aryl”; e.g, phenyl).
  • an aryl group has 10 ring carbon atoms (“C10 aryl”; e.g, naphthyl such as 1- naphthyl and 2-naphthyl).
  • an aryl group has 14 ring carbon atoms (“Ci 4 aryl”; e.g, anthracyl).
  • “Aryl” also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system.
  • each instance of an aryl group is independently unsubstituted (an“unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents.
  • the aryl group is an unsubstituted C6-14 aryl.
  • the aryl group is a substituted C6-14 aryl.
  • heteroaryl refers to a radical of a 5-14 membered monocyclic or polycyclic (e.g, bicyclic, tricyclic) 4n+2 aromatic ring system (e.g, having 6, 10, or 14 p electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-14 membered heteroaryl”).
  • the point of attachment can be a carbon or nitrogen atom, as valency permits.
  • Heteroaryl polycyclic ring systems can include one or more heteroatoms in one or both rings.
  • Heteroaryl includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system.“Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused polycyclic (aryl/heteroaryl) ring system.
  • Polycyclic heteroaryl groups wherein one ring does not contain a heteroatom e.g ., indolyl, quinolinyl, carbazolyl, and the like
  • the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g, 5-indolyl).
  • a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”).
  • a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”).
  • a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”).
  • the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur.
  • the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur.
  • the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.
  • each instance of a heteroaryl group is independently unsubstituted (an “unsubstituted heteroaryl”) or substituted (a “substituted heteroaryl”) with one or more substituents.
  • the heteroaryl group is an unsubstituted 5-14 membered heteroaryl.
  • the heteroaryl group is a substituted 5-14 membered heteroaryl.
  • Exemplary 5-membered heteroaryl groups containing 1 heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl.
  • Exemplary 5-membered heteroaryl groups containing 2 heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl.
  • Exemplary 5-membered heteroaryl groups containing 3 heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl.
  • Exemplary 5-membered heteroaryl groups containing 4 heteroatoms include, without limitation, tetrazolyl.
  • Exemplary 6-membered heteroaryl groups containing 1 heteroatom include, without limitation, pyridinyl.
  • Exemplary 6- membered heteroaryl groups containing 2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl.
  • Exemplary 6-membered heteroaryl groups containing 3 or 4 heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively.
  • Exemplary 7- membered heteroaryl groups containing 1 heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl.
  • Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl.
  • Exemplary 6,6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.
  • Exemplary tricyclic heteroaryl groups include, without limitation, phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl and phenazinyl.
  • the term“partially unsaturated” refers to a ring moiety that includes at least one double or triple bond.
  • the term“partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aromatic groups ( e.g ., aryl or heteroaryl moieties) as herein defined.
  • the term“saturated” refers to a ring moiety that does not contain a double or triple bond, i.e., the ring contains all single bonds.
  • alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups, as defined herein, are, in certain embodiments, optionally substituted.
  • Optionally substituted refers to a group which may be substituted or unsubstituted (e.g., “substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl,“substituted” or“unsubstituted” alkynyl,“substituted” or“unsubstituted” heteroalkyl, “substituted” or “unsubstituted” heteroalkenyl, “substituted” or “unsubstituted” “substituted” or “unsubstituted” carbocyclyl, “substituted” or “unsubstituted” heterocyclyl, “substituted” or“unsubstituted” aryl or“substituted” or“unsubstituted” heteroaryl group).
  • substituted or unsubstituted e.g., “substi
  • the term“substituted” means that at least one hydrogen present on a group is replaced with a permissible substituent, e.g, a substituent which upon substitution results in a stable compound, e.g, a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction.
  • a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position.
  • the present invention contemplates any and all such combinations in order to arrive at a stable compound.
  • heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety.
  • substituents include, but are not limited to, halogen, -CN, -NCte, -N3, -SO2H,
  • Ci-10 alkyl C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, heteroCi-10 alkyl, heteroC2-io alkenyl, heteroC2-io alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl;
  • R 33 is, independently, selected from C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, heteroCi-10 alkyl, heteroC2-ioalkenyl, heteroC2-ioalkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two R 33 groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring; each instance of R bb is, independently, independently,
  • halo or“halogen” refers to fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), or iodine (iodo, -I).
  • a“counterion” is a negatively charged group associated with a positively charged quartemary amine in order to maintain electronic neutrality.
  • exemplary counterions include halide ions (e.g., F , Cl , Br , G), ME-, C104-, OH , H2P04-, HS04-, sulfonate ions (e.g, methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate, naphthalene-l-sulfonic acid-5-sulfonate, ethan-l-sulfonic acid-2-sulfonate, and the like), and carboxylate ions (e.g, acetate, ethanoate, propanoate, benzoate, glycerate, lactate, tartrate
  • a“leaving group” is an art-understood term referring to a molecular fragment that departs with a pair of electrons in heterolytic bond cleavage, wherein the molecular fragment is an anion or neutral molecule. See, for example, Smith, March Advanced Organic Chemistry 6th ed. (501-502).
  • Exemplary leaving groups include, but are not limited to, halo (e.g, chloro, bromo, iodo) and -OS02R aa , wherein R aa as defined herein.
  • the group -OS02R aa encompasses leaving groups such as tosyl, mesyl, and besyl, wherein R aa is optionally substituted alkyl (e.g, -CH3) or optionally substituted aryl (e.g, phenyl, tolyl).
  • the term“hydroxyl” or“hydroxy” refers to the group -OH.
  • the term“thiol” or“thio” refers to the group -SH.
  • the term,“amino” refers to the group -NH2.
  • the term“substituted amino,” by extension, refers to a monosub stituted amino or a disubstituted amino, as defined herein.
  • the“substituted amino” is a monosub stituted amino or a disubstituted amino group.
  • sulfonyl refers to a group selected from -S02N(R bb )2, -S02R 33 , and -S020R 33 , wherein R aa and R bb are as defined herein.
  • sil refers to the group -Si(R aa )3, wherein R aa is as defined herein.
  • Nitrogen atoms can be substituted or unsubstituted as valency permits, and include primary, secondary, tertiary, and quarternary nitrogen atoms.
  • the substituent present on the nitrogen atom is a nitrogen protecting group (also referred to herein as an“amino protecting group”).
  • Nitrogen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis , T. W. Greene and P. G. M. Wuts, 3 rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
  • the substituent present on an oxygen atom is an oxygen protecting group (also referred to herein as an“hydroxyl protecting group”).
  • Oxygen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis , T. W. Greene and P. G. M. Wuts, 3 rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
  • Non-limiting examples of synthetic methods to prepare the cortistatin analogues described herein for use with the present invention include the procedures listed in: WO 2010/024930; WO 2015/100420; WO 2016/182904; WO 2016/182932; WO 2017/004411; WO 2017/112815; WO 2017/112823; and WO 2017/142621; each of which is incorporated by reference.
  • a CDK8/19 inhibitor other than cortistatin can be used in combination with the method of targeted selection of patients for therapy using the biomarkers identified herein.
  • a range of CDK8/19 inhibitors are known in the art, including but not limited to those described in the following publications: Schiemann, K. et al. Discovery of potent and selective CDK8 inhibitors from an HSP90 pharmacophore. Bioorg. Med. Chem. Lett. 26, 1443-1451 (2016); Mallinger, A. etal. Discovery of Potent, Selective, and Orally Bioavailable Small-Molecule Modulators of the Mediator Complex- Associated Kinases CDK8 and CDK19. J. Med. Chem.
  • CDK8/19 inhibitors are provided in the following U.S. Patent Applications: US2013/0217014; US2015/027953; US2004/0180848;
  • CDK8/19 inhibitor is an analog of Senexin. In another embodiment the CDK8/19 inhibitor is an analog of Selvita.
  • the cortistatin or other CDK8/19 inhibitor can be administered as the neat chemical, but are more typically administered as a pharmaceutical composition, that includes an effective amount for a host, typically a human, in need of such treatment of the selected cortistatin or the CDK8/19 inhibitor, as described herein. Accordingly, the disclosure provides pharmaceutical compositions comprising an effective amount of cortistatin or its pharmaceutically acceptable salt together with at least one pharmaceutically acceptable carrier for all of the uses described herein.
  • the pharmaceutical composition may contain the cortistatin as the only active agent, or, in an alternative embodiment, the compound and at least one additional active agent.
  • the pharmaceutical composition is in a dosage form that contains from about 0.1 mg to about 2000 mg, from about 10 mg to about 1000 mg, from about 5 mg to about 200 mg, or from about 5 mg to about 100 mg of the active compound and optionally an appropriate dosage amount of an additional active agent, in a unit dosage form.
  • dosage forms with at least 5, 10, 15, 25, 50, 75, 100, 200, 250, 300, 400, 500, 600, 700, or 750 mg of active compound.
  • the cortistatin or CDK8/19 inhibitor may be administered orally, topically, parenterally, by inhalation or spray, sublingually, via implant, including ocular implant, transdermally, via buccal administration, rectally, as an ophthalmic solution, injection, intravenous, intra-aortal, intracranial, subdermal, intraperitoneal, subcutaneous, transnasal, sublingual, or rectal or by other means, in dosage unit formulations containing conventional pharmaceutically acceptable carriers.
  • the present disclosure provides compositions comprising a cortistatin or CDK8/19 inhibitor, such as a cortistatin described herein or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxide thereof, for administration to a subject having a cancer or tumor that exhibits impaired RUNX1 activity.
  • a cortistatin or CDK8/19 inhibitor such as a cortistatin described herein or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxide thereof
  • the composition comprises a CDK8/19 inhibitor.
  • the composition further comprises a Jakl/2 inhibitor.
  • compositions are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally also suitable for administration to animals. If required, modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled person in the art will be able to design and perform such modification with merely ordinary, if any, experimentation.
  • Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, e.g., cattle, pigs, horses, sheep, cats, dogs, rodents, mice, hamsters, and/or rats; birds, e.g., chickens, ducks, geese, and turkeys.
  • mammals e.g., cattle, pigs, horses, sheep, cats, dogs, rodents, mice, hamsters, and/or rats
  • birds e.g., chickens, ducks, geese, and turkeys.
  • Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.
  • a pharmaceutical composition in accordance with the invention may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a“unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • Salts can be prepared from inorganic acids sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydriodic, phosphorus, and the like.
  • Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate, lactobionate, laurylsulphonate and isethionate salts, and the like.
  • Salts can also be prepared from organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl -substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. and the like.
  • organic acids such as aliphatic mono- and dicarboxylic acids, phenyl -substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. and the like.
  • Representative salts include acetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, methanesulfonate, and the like.
  • Pharmaceutically acceptable salts can include cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Also contemplated are the salts of amino acids such as arginate, gluconate, galacturonate, and the like. See, for example, Berge et ah, J. Pharm. Sci., 1977, 66, 1-19, which is incorporated herein by reference.
  • compositions may comprise between 0.1% and 100% (w/w) of active ingredient, e.g. a CDK8/19 inhibitor, or a cortistatin or cortistatin analog thereof, such as a compound described herein, or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxide thereof, and, optionally, any additional active ingredients, such as, for example, a JAK1/2 inhibitor.
  • active ingredient e.g. a CDK8/19 inhibitor, or a cortistatin or cortistatin analog thereof, such as a compound described herein, or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxide thereof
  • active ingredient e.g. a CDK8/19 inhibitor, or a cortistatin or cortistatin analog thereof, such as a compound described herein, or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxide thereof
  • any additional active ingredients such as, for example, a JAK1/2 inhibitor.
  • the composition comprises between 0.1% and 1%, between 1% and 10%, between 10% and 20%, between 20% and 30%, between 30% and 40%, between 40% and 50%, between 50% and 60%, between 60% and 70%, between 70% and 80%, between 80% and 90%, or between 90% and 100% (w/w) of active ingredient, and more generally, between 0.1 and 100% (w/w) of active ingredient.
  • compositions as provided herein may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • a pharmaceutically acceptable excipient includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • Remington s The Science and Practice of Pharmacy, 2lst Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference) discloses various excip
  • the excipient is one already approved for use in humans and for veterinary use, for example, by ETnited States Food and Drug Administration.
  • an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
  • compositions used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical formulations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator.
  • Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and/or combinations thereof.
  • Exemplary granulating and/or dispersing agents include, but are not limited to, potato starch, com starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, etc., and/or combinations thereof.
  • crospovidone cross-linked poly(vinyl-pyrrolidone)
  • Exemplary surface active agents and/or emulsifiers include, but are not limited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and Veegum® [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g.
  • natural emulsifiers e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin
  • colloidal clays e.g. bentonite [aluminum silicate
  • stearyl alcohol cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g.
  • polyoxyethylene monostearate [Myrj®45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. Cremophor®), polyoxyethylene ethers, (e.g.
  • polyoxyethylene lauryl ether [Brij®30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic®F 68, Poloxamer®l88, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations thereof.
  • Exemplary binding agents include, but are not limited to, starch (e.g. cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol,); natural and synthetic gums (e.g.
  • acacia sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; etc.; and combinations thereof.
  • Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and/or other preservatives.
  • Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabi sulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabi sulfite, and/or sodium sulfite.
  • Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate.
  • EDTA ethylenediaminetetraacetic acid
  • citric acid monohydrate disodium edetate
  • dipotassium edetate dipotassium edetate
  • edetic acid fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate.
  • antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/or thimerosal.
  • Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and/or sorbic acid.
  • Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol.
  • Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and/or phytic acid.
  • preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabi sulfite, Glydant Plus®, Phenonip®, methylparaben, Germall®l l5, Germaben®II, NeoloneTM, KathonTM, and/or Euxyl®.
  • Exemplary buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isot
  • Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.
  • oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl my ri state, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savour
  • oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and/or combinations thereof.
  • Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs.
  • liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art such as, for example,
  • oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents.
  • compositions are mixed with solubilizing agents such as Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
  • Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and/or suspending agents.
  • Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, as a solution in l,3-butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution.
  • Sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • Fatty acids such as oleic acid can be used in the preparation of injectables.
  • Injectable formulations can be sterilized, for example, by filtration through a bacterial- retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • the rate of drug release can be controlled.
  • biodegradable polymers include poly(orthoesters) and poly(anhydrides).
  • Depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.
  • compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing compositions with suitable non-irritating excipients such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.
  • suitable non-irritating excipients such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • an active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate and/or fillers or extenders (e.g. starches, lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g. carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g. glycerol), disintegrating agents (e.g.
  • the dosage form may comprise buffering agents.
  • solution retarding agents e.g. paraffin
  • absorption accelerators e.g. quaternary ammonium compounds
  • wetting agents e.g. cetyl alcohol and glycerol monostearate
  • absorbents e.g. kaolin and bentonite clay
  • lubricants e.g. talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate
  • the dosage form may comprise buffering agents.
  • Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.
  • Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • Dosage forms for topical and/or transdermal administration of a composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and/or patches.
  • an active ingredient is admixed under sterile conditions with a pharmaceutically acceptable excipient and/or any needed preservatives and/or buffers as may be required.
  • the present invention contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of a compound to the body.
  • dosage forms may be prepared, for example, by dissolving and/or dispensing the compound in the proper medium.
  • rate may be controlled by either providing a rate controlling membrane and/or by dispersing the compound in a polymer matrix and/or gel.
  • Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices such as those described in U.S. Patents 4,886,499; 5,190,521; 5,328,483; 5,527,288; 4,270,537; 5,015,235; 5, 141,496; and 5,417,662.
  • Intradermal compositions may be administered by devices which limit the effective penetration length of a needle into the skin, such as those described in PCT publication WO 99/34850 and functional equivalents thereof.
  • Jet injection devices which deliver liquid compositions to the dermis via a liquid jet injector and/or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis are suitable.
  • Jet injection devices are described, for example, in U.S. Patents 5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569, 189; 5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335; 5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880; 4,940,460; and PCT Publications WO 97/37705 and WO 97/13537.
  • Ballistic powder/particle delivery devices which use compressed gas to accelerate vaccine in powder form through the outer layers of the skin to the dermis are suitable.
  • conventional syringes may be used in the classical mantoux method of intradermal administration.
  • Formulations suitable for topical administration include, but are not limited to, liquid and/or semi liquid preparations such as liniments, lotions, oil in water and/or water in oil emulsions such as creams, ointments and/or pastes, and/or solutions and/or suspensions.
  • Topically- administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of active ingredient may be as high as the solubility limit of the active ingredient in the solvent.
  • Formulations for topical administration may further comprise one or more of the additional ingredients described herein.
  • a pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity.
  • a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 nm to about 7 nm or from about 1 nm to about 6 nm.
  • Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder and/or using a self-propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container.
  • Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nm and at least 95% of the particles by number have a diameter less than 7 nm. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nm and at least 90% of the particles by number have a diameter less than 6 nm.
  • Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.
  • Low boiling propellants generally include liquid propellants having a boiling point of below 65 °F at atmospheric pressure.
  • the propellant may constitute 50% to 99.9% (w/w) of the composition, and active ingredient may constitute 0.1% to 20% (w/w) of the composition.
  • a propellant may further comprise additional ingredients such as a liquid non-ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).
  • compositions formulated for pulmonary delivery may provide an active ingredient in the form of droplets of a solution and/or suspension.
  • Such formulations may be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising active ingredient, and may conveniently be administered using any nebulization and/or atomization device.
  • Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and/or a preservative such as methylhydroxybenzoate.
  • Droplets provided by this route of administration may have an average diameter in the range from about 0.1 nm to about 200 nm.
  • Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition.
  • Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 mih to 500 mih. Such a formulation is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nose.
  • Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of active ingredient, and may comprise one or more of the additional ingredients described herein.
  • the formulation suitable for nasal administration comprises between 0.1% and 1%, between 1% and 10%, between 10% and 20%, between 20% and 30%, between 30% and 40%, between 40% and 50%, between 50% and 60%, between 60% and 70%, between 70% and 80%, between 80% and 90%, or between 90% and 100% (w/w) of active ingredient.
  • the formulation suitable for nasal administration comprises between 0.1 and 100% (w/w) of active ingredient.
  • a pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for buccal administration.
  • formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may, for example, 0.1% to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein.
  • formulations suitable for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising active ingredient.
  • Such powdered, aerosolized, and/or atomized formulations when dispersed, may have an average particle and/or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein.
  • a pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for ophthalmic administration.
  • Such formulations may, for example, be in the form of eye drops including, for example, a 0.1/1.0% (w/w) solution and/or suspension of the active ingredient in an aqueous or oily liquid excipient.
  • Such drops may further comprise buffering agents, salts, and/or one or more other of any additional ingredients described herein.
  • Other opthalmically- administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are contemplated as being within the scope of this invention.
  • the methods described herein may be used to monitor or predict treatment outcomes for any disorder mediated by CDK8 and/or CDK19.
  • disorders mediated by CDK8 and CDK19 include tumors, cancers, disorders related to abnormal cellular proliferation, inflammatory disorders, immune disorders, and autoimmune disorders.
  • the method is an in vitro method. In certain embodiments, the method is an in vivo method.
  • a method of treating a condition associated with CDK8 and/or CDK19 kinase activity comprising administering to a subject in need thereof a compound of the present invention or a pharmaceutically acceptable composition, salt, isotopic analog such as a deuterated derivative, or prodrug thereof, wherein a method utilizing a pharmacodynamics biomarker described herein is utilized during treatment.
  • condition associated with CDK8 and/or CDK19 kinase activity is a disorder related to abnormal cellular proliferation.
  • Abnormal cellular proliferation notably hyperproliferation
  • Psoriasis is a benign disease of human skin generally characterized by plaques covered by thickened scales. The disease is caused by increased proliferation of epidermal cells of unknown cause. Chronic eczema is also associated with significant hyperproliferation of the epidermis.
  • Other diseases caused by hyperproliferation of skin cells include atopic dermatitis, lichen planus, warts, pemphigus vulgaris, actinic keratosis, basal cell carcinoma and squamous cell carcinoma.
  • hyperproliferative cell disorders include blood vessel proliferation disorders, fibrotic disorders, autoimmune disorders, graft-versus-host rejection, tumors and cancers.
  • Blood vessel proliferative disorders include angiogenic and vasculogenic disorders. Proliferation of smooth muscle cells in the course of development of plaques in vascular tissue cause, for example, restenosis, retinopathies and atherosclerosis. Both cell migration and cell proliferation play a role in the formation of atherosclerotic lesions.
  • Fibrotic disorders are often due to the abnormal formation of an extracellular matrix.
  • fibrotic disorders include hepatic cirrhosis and mesangial proliferative cell disorders.
  • Hepatic cirrhosis is characterized by the increase in extracellular matrix constituents resulting in the formation of a hepatic scar.
  • Hepatic cirrhosis can cause diseases such as cirrhosis of the liver.
  • An increased extracellular matrix resulting in a hepatic scar can also be caused by viral infection such as hepatitis. Lipocytes appear to play a major role in hepatic cirrhosis.
  • Mesangial disorders are brought about by abnormal proliferation of mesangial cells.
  • Mesangial hyperproliferative cell disorders include various human renal diseases, such as glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombotic micro- angiopathy syndromes, transplant rejection, and glomerulopathies.
  • Rheumatoid arthritis is generally considered an autoimmune disease that is thought to be associated with activity of autoreactive T cells, and to be caused by autoantibodies produced against collagen and IgE.
  • disorders that can include an abnormal cellular proliferative component include Bechet’s syndrome, acute respiratory distress syndrome (ARDS), ischemic heart disease, post- dialysis syndrome, leukemia, acquired immune deficiency syndrome, vasculitis, lipid histiocytosis, septic shock and inflammation in general.
  • ARDS acute respiratory distress syndrome
  • ischemic heart disease post- dialysis syndrome
  • leukemia CAD
  • acquired immune deficiency syndrome CAD
  • vasculitis lipid histiocytosis
  • septic shock and inflammation in general.
  • the condition associated with CDK8 and/or CDK19 kinase activity is a diabetic condition.
  • condition associated with CDK8 and/or CDK19 kinase activity is a viral disease.
  • CDK8 activity plays a role in interferon response, which is also important in cancer cell survival.
  • Treatment with Cortistatin A increases expression of genes in MOLM-14 AML cells that have been identified as interferon gamma signalling genes and interferon responsive genes. Viruses such as HIV block interferon induction to allow more effective replication. Further, Cortistatin A has been shown to inhibit the HIV virus as well as the HIV viral protein TAT-l .
  • the condition associated with CDK8 and/or CDK19 kinase activity is an infection.
  • the infection is a bacterial infection.
  • the infection is a fungal infection.
  • the infection is a protozoal infection.
  • the infection is a viral infection.
  • the viral infection is a retroviral infection, and the virus is a retrovirus, i.e., of the family Retroviridae .
  • the viral infection is a retroviral infection, and the virus is of the family Retroviridae and subfamily Orthoretrovirinae , Alpharetrovirus , Betaretrovirus, Deltaretrovirus , Epsilonretrovirus , Gammaretrovirus , or Lentivirus.
  • the viral infection is a retroviral infection, and the virus is of the family Retroviridae and subfamily Lentivirus.
  • Exemplary virus of the subfamily Lentivirus include human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), and Visna virus are all examples of lentiviruses.
  • the viral infection is a human immunodeficiency virus (HIV) infection.
  • HSV herpes simplex virus
  • HAV human immunodeficiency virus
  • HCMV human cytomegalovirus
  • the virus is an oncovirus, i.e., a virus which is associated with oncogenesis and/or causes cancer.
  • treatment of the viral infection is associated with inhibition of CDK8 and/or CDK19 kinase activity.
  • the methods provided herein are used to predict outcomes related to the treatment of HIV infections and other related conditions such as AIDS-related complex (ARC), persistent generalized lymphadenopathy (PGL), AIDS-related neurological conditions, anti-HIV antibody positive and HIV-positive conditions, Kaposi's sarcoma, thrombocytopenia purpura and opportunistic infections.
  • these compounds or formulations can be used prophylactically to prevent or retard the progression of clinical illness in individuals who are anti- HIV antibody or HIV-antigen positive or who have been exposed to HIV.
  • the methods provided herein are used to predict outcomes related to the treatment of HB V infections and other related conditions such as anti-HBV antibody positive and HBV-positive conditions, chronic liver inflammation caused by HBV, cirrhosis, acute hepatitis, fulminant hepatitis, chronic persistent hepatitis, and fatigue.
  • anti-HBV antibody positive and HBV-positive conditions chronic liver inflammation caused by HBV, cirrhosis, acute hepatitis, fulminant hepatitis, chronic persistent hepatitis, and fatigue.
  • These compounds or formulations can also be used prophylactically to prevent or retard the progression of clinical illness in individuals who are anti-HBV antibody or HBV-antigen positive or who have been exposed to HBV.
  • the condition is associated with an immune response.
  • Cutaneous contact hypersensitivity and asthma are just two examples of immune responses that can be associated with significant morbidity.
  • Others include atopic dermatitis, eczema, Sjogren's Syndrome, including keratoconjunctivitis sicca secondary to Sjogren's Syndrome, alopecia areata, allergic responses due to arthropod bite reactions, Crohn's disease, aphthous ulcer, ulceris, conjunctivitis, keratoconjunctivitis, ulcerative colitis, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, and drug eruptions.
  • immunologically mediated leukocyte infiltration In atopic dermatitis, and eczema in general, immunologically mediated leukocyte infiltration (particularly infiltration of mononuclear cells, lymphocytes, neutrophils, and eosinophils) into the skin importantly contributes to the pathogenesis of these diseases. Chronic eczema also is associated with significant hyperproliferation of the epidermis. Immunologically mediated leukocyte infiltration also occurs at sites other than the skin, such as in the airways in asthma and in the tear producing gland of the eye in keratoconjunctivitis sicca.
  • condition associated with CDK8 and/or CDK19 kinase activity is a degenerative disorder, e.g., Alzheimer's disease (AD) or Parkinson’s Disease.
  • AD Alzheimer's disease
  • Parkinson Parkinson
  • condition is mediated by the b-catenin pathway.
  • condition is mediated by the JAK-STAT pathway.
  • condition is mediated by STAT1 activity in a cell (e.g., the disorder may be treated by inhibiting phosphorylation of STAT1 S727 in the JAK-STAT pathway, leading to up- or down-regulation of specific STAT1 -associated genes).
  • the disorder is mediated by the TGF-beta/BMP pathway.
  • HIF-l-A HIF-l-alpha
  • CDK8 has been linked to regulation of hypoxic response, playing a role in induction of HIF-l-A (HIF-l-alpha) target genes. These genes are involved in angiogenesis, glycolysis, metabolic adaption, and cell survival, processes critical to tumor maintenance and growth. See, e.g., Galbraith, et al., Cell 153 : 1327-1339.
  • the monitored condition is related to hypoxia comprising.
  • the monitored condition is related to modulating HIF-l-A (HIF-l-alpha) activity (e.g., by inhibiting the expression HIF-l-alpha associated genes) in a cell.
  • the method is an in vitro method. In certain embodiments, the method is an in vivo method.
  • the monitored condition is associated with angiogenesis, such as, for example, a diabetic condition (e.g., diabetic retinopathy), an inflammatory condition (e.g., rheumatoid arthritis), macular degeneration, obesity, atherosclerosis, or a proliferative disorder, comprising administering to a subject in need thereof a compound of the present invention or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.
  • angiogenesis such as, for example, a diabetic condition (e.g., diabetic retinopathy), an inflammatory condition (e.g., rheumatoid arthritis), macular degeneration, obesity, atherosclerosis, or a proliferative disorder
  • a“diabetic condition” refers to diabetes and pre-diabetes. Diabetes refers to a group of metabolic diseases in which a person has high blood sugar, either because the body does not produce enough insulin, or because cells do not respond to the insulin that is produced. This high blood sugar produces the classical symptoms of polyuria (frequent urination), polydipsia (increased thirst) and polyphagia (increased hunger). There are several types of diabetes. Type I diabetes results from the body's failure to produce insulin, and presently requires the person to inject insulin or wear an insulin pump. Type 2 diabetes results from insulin resistance a condition in which cells fail to use insulin properly, sometimes combined with an absolute insulin deficiency.
  • Gestational diabetes occurs when pregnant women without a previous diagnosis of diabetes develop a high blood glucose level.
  • Other forms of diabetes include congenital diabetes, which is due to genetic defects of insulin secretion, cystic fibrosis-related diabetes, steroid diabetes induced by high doses of glucocorticoids, and several forms of monogenic diabetes, e.g., mature onset diabetes of the young (e.g., MODY 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).
  • Pre-diabetes indicates a condition that occurs when a person's blood glucose levels are higher than normal but not high enough for a diagnosis of diabetes.
  • the condition associated with angiogenesis is macular degeneration.
  • a method of treating macular degeneration comprising administering to a subject in need thereof a compound of the present invention or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.
  • the condition associated with angiogenesis is obesity.
  • “obesity” and“obese” as used herein refers to class I obesity, class II obesity, class III obesity and pre-obesity (e.g., being“over-weight”) as defined by the World Health Organization.
  • a method of treating obesity comprising administering to a subject in need thereof a compound of the present invention or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.
  • the condition associated with angiogenesis is atherosclerosis.
  • a method of treating atherosclerosis comprising administering to a subject in need thereof a compound of the present invention or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.
  • the condition associated with angiogenesis is a proliferative disorder.
  • a method of treating a proliferative disorder comprising administering to a subject in need thereof a compound of the present invention or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.
  • Exemplary proliferative disorders include, but are not limited to, tumors (e.g., solid tumors), benign neoplasms, pre-malignant neoplasms (carcinoma in situ), and malignant neoplasms (cancers).
  • tumors e.g., solid tumors
  • benign neoplasms e.g., pre-malignant neoplasms (carcinoma in situ)
  • pre-malignant neoplasms e.g., pre-malignant neoplasms (carcinoma in situ)
  • malignant neoplasms cancers.
  • Exemplary cancers include, but are not limited to, acoustic neuroma, adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma), appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g., cholangiocarcinoma), bladder cancer, breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast), brain cancer (e.g., meningioma; glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), bronchus cancer, carcinoid tumor, cervical cancer (e.g., cervical adenocarcinoma), choriocarcinoma, chordo
  • Wilms tumor, renal cell carcinoma), liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma), lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis (e.g., systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma, myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a.
  • HCC hepatocellular cancer
  • lung cancer e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung
  • myelofibrosis MF
  • chronic idiopathic myelofibrosis chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)
  • neuroblastoma e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis
  • neuroendocrine cancer e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor
  • osteosarcoma ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors), penile cancer (e.g., Paget’s disease of the pen
  • the disorder is myelodysplastic syndrome (MDS).
  • MDS myelodysplastic syndrome
  • the cancer or tumor is associated with CDK8 and/or CDK19 kinase activity. In certain embodiments, the cancer or tumor is associated with CDK8 kinase activity. In certain embodiments, the cancer or tumor is associated with CDK19 kinase activity. In certain embodiments, the cancer or tumor is associated with aberrant CDK8 kinase activity. In certain embodiments, the cancer or tumor is associated with aberrant CDK19 kinase activity. In certain embodiments, the cancer or tumor is associated with increased CDK8 kinase activity. In certain embodiments, the cancer is associated with increased CDK19 kinase activity.
  • the cancer is a hematopoietic cancer.
  • the hematopoietic cancer is a lymphoma.
  • the hematopoietic cancer is a leukemia.
  • the leukemia is acute myelocytic leukemia (AML).
  • the proliferative disorder is a myeloproliferative neoplasm.
  • the myeloproliferative neoplasm MPN
  • PMF primary myelofibrosis
  • the cancer is a solid tumor.
  • a solid tumor refers to an abnormal mass of tissue that usually does not contain cysts or liquid areas. Different types of solid tumors are named for the type of cells that form them. Examples of classes of solid tumors include, but are not limited to, sarcomas, carcinomas, and lymphomas, as described above herein. Additional examples of solid tumors include, but are not limited to, squamous cell carcinoma, colon cancer, breast cancer, prostate cancer, lung cancer, liver cancer, pancreatic cancer, and melanoma.
  • the patient is being treated with a CDK8/19 inhibitor and an additional active agent.
  • an additional active agent is added after determining if the cancer or tumor is responding to treatment.
  • a treatment regimen comprising the administration of a compound of this disclosure or a pharmaceutically acceptable composition, salt, isotopic analog (such as a deuterated derivative), or prodrug thereof in combination or in alternation with at least one additional therapeutic agent.
  • a compound of this disclosure or a pharmaceutically acceptable composition, salt, isotopic analog (such as a deuterated derivative), or prodrug thereof in combination or in alternation with at least one additional therapeutic agent.
  • the combinations and/or alternations disclosed herein can be administered for beneficial, additive, or synergistic effect in the treatment of abnormal cellular proliferative disorders.
  • the treatment regimen includes the administration of a compound of this disclosure or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof in combination or alternation with at least one additional kinase inhibitor.
  • the at least one additional kinase inhibitor is selected from a phosphoinositide 3- kinase (PI3K) inhibitor, a Bruton’s tyrosine kinase (BTK) inhibitor, another cyclin-dependent kinase inhibitor, or a spleen tyrosine kinase (Syk) inhibitor, or a combination thereof.
  • a compound of this disclosure or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof is combined in a dosage form with the PIk3 inhibitor.
  • PI3k inhibitors that may be used in the present invention are well known.
  • PI3 kinase inhibitors include but are not limited to Wortmannin, demethoxyviridin, perifosine, idelalisib, Pictilisib, Palomid 529, ZSTK474, PWT33597, CUDC-907, and AEZS-136, duvelisib, GS-9820, GDC-0032 (2-[4-[2-(2-Isopropyl-5-methyl-l,2,4-triazol-3-yl)-5,6-dihydroimidazo[l,2- d][l,4]benzoxazepin-9-yl]pyrazol-l-yl]-2-methylpropanamide), MLN-l 117 ((2R)-l-Phenoxy-2- butanyl hydrogen (S)-methylphosphonate; or Methyl(oxo) ⁇ [(2R)-l-phenoxy
  • BTK inhibitors for use in the present invention are well known.
  • BTK inhibitors include ibrutinib (also known as PCI-32765)(ImbruvicaTM)(l-[(3R)-3-[4-amino-3-(4- phenoxy-phenyl)pyrazolo[3 ,4-d]pyrimidin- 1 -yljpiperidin- 1 -yl]prop-2-en- 1 -one),
  • dianilinopyrimidine-based inhibitors such as AVL-101 and AVL-291/292 (N-(3-((5-fluoro-2-((4- (2-methoxyethoxy)phenyl)amino)pyrimidin-4-yl)amino)phenyl)acrylamide) (Avila Therapeutics) (see ETS Patent Publication No 2011/0117073, incorporated herein in its entirety), Dasatinib ([N- (2-chloro-6-methylphenyl)-2-(6-(4-(2-hydroxyethyl)piperazin-l-yl)-2-methylpyrimidin-4- ylamino)thiazole-5-carboxamide], LFM-A13 (alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5- ibromophenyl) propenamide), GDC-0834 ([R-N-(3-(6-(4-(l,4-dimethyl-3-o
  • the additional cyclin-dependent kinase inhibitor is a CDK7 inhibitor such as THZ1 (N-[3-[[5-chloro-4-(lH-indol-3-yl)pyrimidin-2-yl]amino]phenyl]-4-[[(E)-4- (dimethylamino)but-2-enoyl]amino]benzamide).
  • the additional cyclin-dependent kinase inhibitor is a CDK9 inhibitor such as flavopiridol (alvocidib).
  • a method of treating a tumor or cancer comprising administration of an effective amount of a compound described herein or a pharmaceutically acceptable salt thereof in combination or alternation with an effective amount of a Syk inhibitor to a host in need thereof.
  • a method of treating a tumor or cancer comprising administration of an effective amount of an analog of a compound described herein or a pharmaceutically acceptable salt thereof as provided herein in combination or alternation with an effective amount of a Syk inhibitor to a host in need thereof.
  • a method of treating a tumor or cancer comprising administration of an effective amount of a compound described herein or a pharmaceutically acceptable salt thereof in combination or alternation with imatinib (Gleevec) to a host in need thereof.
  • a method of treating a tumor or cancer comprising administration of an effective amount of an analog of a compound described herein or a pharmaceutically acceptable salt thereof as provided herein in combination or alternation with imatinib (Gleevec) to a host in need thereof.
  • Syk inhibitors for use in the present invention are well known, and include, for example, Cerdulatinib (4-(cyclopropylamino)-2-((4-(4-(ethylsulfonyl)piperazin-l- yl)phenyl)amino)pyrimidine-5-carboxamide), entospletinib (6-(lH-indazol-6-yl)-N-(4- morpholinophenyl)imidazo[l,2-a]pyrazin-8-amine), fostamatinib ([6-( ⁇ 5-Fluoro-2-[(3,4,5- trimethoxyphenyl)amino]-4-pyrimidinyl ⁇ amino)-2,2-dimethyl-3-oxo-2,3-dihydro-4H- pyrido[3,2-b][l,4]oxazin-4-yl]methyl dihydrogen phosphate), fostamatinib disodium salt (s
  • a compound of the present invention or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof is combined in a dosage form with the Syk inhibitor.
  • the method of treatment provided includes the administration of a compound of the present invention or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof in combination or alternation with at least one additional chemotherapeutic agent.
  • the at least one additional chemotherapeutic agent combined or alternated with a compound of the present invention is a protein cell death-l (PD-l) inhibitor.
  • PD- 1 inhibitors are known in the art, and include, for example, nivolumab (BMS), pembrolizumab (Merck), pidilizumab (CureTech/Teva), AMP -244 (Amplimmune/GSK), BMS-936559 (BMS), and MEDI4736 (Roche/Genentech).
  • BMS protein cell death-l
  • PD- 1 inhibitors include, for example, nivolumab (BMS), pembrolizumab (Merck), pidilizumab (CureTech/Teva), AMP -244 (Amplimmune/GSK), BMS-936559 (BMS), and MEDI4736 (Roche/Genentech).
  • a method of treating a tumor or cancer comprising administration of an effective amount of a compound described herein or a pharmaceutically acceptable salt thereof in combination or alternation with an effective amount of a PD-l inhibitor to a host in need thereof.
  • a method of treating a tumor or cancer comprising administration of an effective amount of an analog of a compound described herein or a pharmaceutically acceptable salt thereof as provided herein in combination or alternation with an effective amount of a PD-l inhibitor to a host in need thereof.
  • a method of treating a tumor or cancer comprising administration of an effective amount of a compound described herein or a pharmaceutically acceptable salt thereof in combination or alternation with pembrolizumab (Keytruda).
  • a method of treating a tumor or cancer comprising administration of an effective amount of an analog of a compound described herein or a pharmaceutically acceptable salt thereof as provided herein in combination or alternation with pembrolizumab (Keytruda).
  • the at least one additional chemotherapeutic agent combined or alternated with a compound of the present invention is a CTLA-4 inhibitor.
  • CTLA-4 inhibitors are known in the art, and include, for example, ipilimumab (Yervoy) marketed by Bristol-Myers Squibb and tremelimumab marketed by Pfizer.
  • the at least one additional chemotherapeutic agent combined or alternated with the compound of the present invention is a BET inhibitor.
  • BET inhibitors are known in the art, and include, for example, JQ1, I-BET 151 (a.k.a. GSK1210151A), I-BET 762 (a.k.a.
  • the BET inhibitor used in combination or alternation with a compound of the present invention for treatment of a tumor or cancer is JQ1 ((S)-tert-butyl 2-(4-(4-chlorophenyl)-2,3,9- trimethyl-6H-thieno[3,2-f [l,2,4]triazolo[4,3-a][l,4]diazepin-6-yl)acetate).
  • a method of treating a tumor or cancer comprising administration of an effective amount of a compound described herein or a pharmaceutically acceptable salt thereof in combination or alternation with an effective amount of a BET inhibitor to a host in need thereof.
  • a method of treating a tumor or cancer comprising administration of an effective amount of an analog of a compound described herein or a pharmaceutically acceptable salt thereof as provided herein in combination or alternation with an effective amount of a BET inhibitor to a host in need thereof.
  • a method of treating a tumor or cancer comprising administration of an effective amount of a compound described herein or a pharmaceutically acceptable salt thereof in combination or alternation with JQ1.
  • a method of treating a tumor or cancer comprising administration of an effective amount of an analog of a compound described herein or a pharmaceutically acceptable salt thereof as provided herein in combination or alternation with JQ1.
  • a method of treating a tumor or cancer comprising administration of an effective amount of a compound described herein or a pharmaceutically acceptable salt thereof in combination or alternation with I-BET 151.
  • a method of treating a tumor or cancer comprising administration of an effective amount of an analog of a compound described herein or a pharmaceutically acceptable salt thereof as provided herein in combination or alternation with I-BET 151.
  • the at least one additional chemotherapeutic agent combined or alternated with the compound of the present invention is a MEK inhibitor.
  • MEK inhibitors for use in the present invention are well known, and include, for example, tametinib/GSKl 120212 (N-(3- ⁇ 3-Cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-3,4,6,7- tetrahydropyrido[4,3-d]pyrimidin-l(2H-yl ⁇ phenyl)acetamide), selumetinob (6-(4-bromo-2- chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide), pimasertib/AS703026/MSC 1935369 ((S)-N-(2,3-dihydroxypropyl)-3-((2-
  • the at least one additional chemotherapeutic agent combined or alternated with the compound of the present invention is a Raf inhibitor.
  • Raf inhibitors for use in the present invention are well known, and include, for example, Vemurafmib (N-[3-[[5-(4- Chlorophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]carbonyl]-2,4-difluorophenyl]-l- propanesulfonamide), sorafenib tosylate (4-[4-[[4-chloro-3-
  • a compound of the present invention or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof is combined in a dosage form with the Raf inhibitor.
  • the at least one additional chemotherapeutic agent combined or alternated with the compound of the present invention is a B-cell lymphoma 2 (Bcl-2) protein inhibitor.
  • BCL-2 inhibitors are known in the art, and include, for example, ABT-199 (4-[4-[[2-(4- Chlorophenyl)-4,4-dimethylcyclohex-l-en-l-yl]methyl]piperazin-l-yl]-N-[[3-nitro-4- [[(tetrahydro-2H-pyran-4-yl)methyl]amino]phenyl]sulfonyl]-2-[(lH- pyrrolo[2,3-b]pyridin-5- yl)oxy]benzamide), ABT-737 (4-[4-[[2-(4-chlorophenyl)phenyl]methyl]piperazin-l-yl]-N-[4- [[(2R)-4-(dimethylamino)-l-
  • a compound of the present invention or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof is combined in a dosage form with the at least one BCL-2 inhibitor.
  • the at least one BCL-2 inhibitor is ABT-199 (Venetoclax).
  • a method of treating a tumor or cancer comprising administration of an effective amount of a compound described herein or a pharmaceutically acceptable salt thereof in combination or alternation with an effective amount of a BCL-2 inhibitor to a host in need thereof.
  • a method of treating a tumor or cancer comprising administration of an effective amount of an analog of a compound described herein or a pharmaceutically acceptable salt thereof as provided herein in combination or alternation with an effective amount of a BCL-2 inhibitor to a host in need thereof.
  • a method of treating a tumor or cancer comprising administration of an effective amount of a compound described herein or a pharmaceutically acceptable salt thereof in combination or alternation with ABT-199 to a host in need thereof.
  • a method of treating a tumor or cancer comprising administration of an effective amount of an analog of a compound described herein or a pharmaceutically acceptable salt thereof as provided herein in combination or alternation with ABT-199 to a host in need thereof.
  • the treatment regimen includes the administration of a compound of the present invention or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof in combination or alternation with at least one additional chemotherapeutic agent selected from, but are not limited to, Imatinib mesylate (Gleevac), Dasatinib (Sprycel), Nilotinib (Tasigna), Bosutinib (Bosulif), Trastuzumab (Herceptin), Pertuzumab (PeijetaTM), Lapatinib (Tykerb), Gefitinib (Iressa), Erlotinib (Tarceva), Cetuximab (Erbitux), Panitumumab (Vectibix), Vandetanib (Caprelsa), Vemurafenib (Zelboraf), Vorinostat (Zolinza), Romidepsin (Istodax), Bexarotene (Tagretin), A
  • the pharmaceutical combination or composition described herein can be administered to the subject in combination or further combination with other chemotherapeutic agents for the treatment of a tumor or cancer. If convenient, the pharmaceutical combination or composition described herein can be administered at the same time as another chemotherapeutic agent, in order to simplify the treatment regimen. In some embodiments, the pharmaceutical combination or composition and the other chemotherapeutic can be provided in a single formulation. In one embodiment, the use of the pharmaceutical combination or composition described herein is combined in a therapeutic regime with other agents.
  • Such agents may include, but are not limited to, tamoxifen, midazolam, letrozole, bortezomib, anastrozole, goserelin, an mTOR inhibitor, a PI3 kinase inhibitor as described above, a dual mTOR-PI3K inhibitor, a MEK inhibitor as described above, a RAS inhibitor, ALK inhibitor, an HSP inhibitor (for example, HSP70 and HSP 90 inhibitor, or a combination thereof), a BCL-2 inhibitor as described above, apopototic inducing compounds, an ART inhibitor, including but not limited to, Perifosine, (KRX- 0401), GDC-0068, Triciribine, AZD5363, Honokiol, PF-04691502, and Miltefosine, a PD-l inhibitor as described above including but not limited to, Nivolumab, CT-011, MK-3475, BMS936558, and AMP-514 or a FLT-3 inhibitor
  • mTOR inhibitors include but are not limited to rapamycin and its analogs, everolimus (Afmitor), temsirolimus, ridaforolimus, sirolimus, and deforolimus.
  • RAS inhibitors include but are not limited to Reolysin and siGl2D LODER.
  • ALK inhibitors include but are not limited to Crizotinib, AP26113, and LDK378.
  • HSP inhibitors include but are not limited to Geldanamycin or l7-N-Allylamino-l7- demethoxygeldanamycin (17AAG), and Radicicol.
  • a compound described herein is administered in combination with letrozole and/or tamoxifen.
  • Other chemotherapeutic agents that can be used in combination with the compounds described herein include, but are not limited to, chemotherapeutic agents that do not require cell cycle activity for their anti-neoplastic effect.
  • the treatment regimen includes the administration of a compound of the present invention or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof in combination or alternation with at least one additional therapy, wherein the second therapy is an immunotherapy.
  • the combination agent can be conjugated to an antibody, radioactive agent, or other targeting agent that directs the active compound as described herein to the diseased or abnormally proliferating cell.
  • the pharmaceutical combination or composition is used in combination with another pharmaceutical or a biologic agent (for example an antibody) to increase the efficacy of treatment with a combined or a synergistic approach.
  • the pharmaceutical combination or composition can be used with T-cell vaccination, which typically involves immunization with inactivated autoreactive T cells to eliminate a cancer cell population as described herein.
  • the pharmaceutical combination or composition is used in combination with a bispecific T-cell Engager (BiTE), which is an antibody designed to simultaneously bind to specific antigens on endogenous T cells and cancer cells as described herein, linking the two types of cells.
  • BiTE bispecific T-cell Engager
  • the additional therapy is a monoclonal antibody (MAb).
  • MAbs stimulate an immune response that destroys cancer cells. Similar to the antibodies produced naturally by B cells, these MAbs“coat” the cancer cell surface, triggering its destruction by the immune system.
  • bevacizumab targets vascular endothelial growth factor(VEGF), a protein secreted by tumor cells and other cells in the tumor’s microenvironment that promotes the development of tumor blood vessels. When bound to bevacizumab, VEGF cannot interact with its cellular receptor, preventing the signaling that leads to the growth of new blood vessels.
  • VEGF vascular endothelial growth factor
  • cetuximab and panitumumab target the epidermal growth factor receptor (EGFR), and trastuzumab targets the human epidermal growth factor receptor 2 (HER-2).
  • MAbs that bind to cell surface growth factor receptors prevent the targeted receptors from sending their normal growth-promoting signals. They may also trigger apoptosis and activate the immune system to destroy tumor cells.
  • MAbs are the immunoconjugates. These MAbs, which are sometimes called immunotoxins or antibody-drug conjugates, consist of an antibody attached to a cell-killing substance, such as a plant or bacterial toxin, a chemotherapy drug, or a radioactive molecule. The antibody latches onto its specific antigen on the surface of a cancer cell, and the cell-killing substance is taken up by the cell. FDA-approved conjugated MAbs that work this way include ado-trastuzumab emtansine, which targets the HER-2 molecule to deliver the drug DM1, which inhibits cell proliferation, to HER-2 expressing metastatic breast cancer cells.
  • FDA-approved conjugated MAbs that work this way include ado-trastuzumab emtansine, which targets the HER-2 molecule to deliver the drug DM1, which inhibits cell proliferation, to HER-2 expressing metastatic breast cancer cells.
  • Immunotherapies with T cells engineered to recognize cancer cells via bispecific antibodies (bsAbs) or chimeric antigen receptors (CARs) are approaches with potential to ablate both dividing and non/slow-dividing subpopulations of cancer cells.
  • Bispecific antibodies by simultaneously recognizing target antigen and an activating receptor on the surface of an immune effector cell, offer an opportunity to redirect immune effector cells to kill cancer cells.
  • Another approach is the generation of chimeric antigen receptors by fusing extracellular antibodies to intracellular signaling domains. Chimeric antigen receptor-engineered T cells are able to specifically kill tumor cells in a MHC-independent way.
  • the additional therapy is another therapeutic agent, for example, an anti inflammatory agent, a chemotherapeutic agent, a radiotherapeutic agent, or an immunosuppressive agent.
  • Suitable chemotherapeutic agents include, but are not limited to, a radioactive molecule, a toxin, also referred to as cytotoxin or cytotoxic agent, which includes any agent that is detrimental to the viability of cells, and liposomes or other vesicles containing chemotherapeutic compounds.
  • General anticancer pharmaceutical agents for administration as additional agents include: Vincristine (Oncovin) or liposomal vincristine (Marqibo), Daunorubicin (daunomycin or Cerubidine) or doxorubicin (Adriamycin), Cytarabine (cytosine arabinoside, ara-C, or Cytosar), L-asparaginase (Elspar) or PEG-L-asparaginase (pegaspargase or Oncaspar), Etoposide (VP- 16), Teniposide (Vumon), 6-mercaptopurine (6-MP or Purinethol), Methotrexate, Cyclophosphamide (Cytoxan), Prednisone, Dexamethasone (Decadron), imatinib (Gleevec marketed by Novartis), dasatinib (Sprycel), nilotinib (Tasigna), bosutinib (Bosulif
  • chemotherapeutic agents include but are not limited to 1- dehydrotestosterone, 5-fluorouracil decarbazine, 6-mercaptopurine, 6-thioguanine, actinomycin D, adriamycin, aldesleukin, an alkylating agent, allopurinol sodium, altretamine, amifostine, anastrozole, anthramycin (AMC)), an anti-mitotic agent, cis-dichlorodiamine platinum (II) (DDP) cisplatin), diamino dichloro platinum, anthracycline, an antibiotic, an antimetabolite, asparaginase, BCG live (intravesical), betamethasone sodium phosphate and betamethasone acetate, bicalutamide, bleomycin sulfate, busulfan, calcium leucouorin, calicheamicin, capecitabine, carboplatin, lomustine (CCNU), carmustine (BSNU)
  • Suitable immunosuppressive agents include, but are not limited to: calcineurin inhibitors, e.g. a cyclosporin or an ascomycin, e.g. Cyclosporin A (NEORAL), FK506 (tacrolimus), pimecrolimus, a mTOR inhibitor, e.g. rapamycin or a derivative thereof, e.g. Sirolimus (RAPAMUNE), Everolimus (Certican), temsirolimus, zotarolimus, biolimus-7, biolimus-9, a rapalog, e.g.ridaforolimus, azathioprine, campath 1H, a S1P receptor modulator, e.g.
  • fmgolimod or an analog thereof, an anti IL-8 antibody, mycophenolic acid or a salt thereof, e.g. sodium salt, or a prodrug thereof, e.g. Mycophenolate Mofetil (CELLCEPT), OKT3 (ORTHOCLONE OKT3), Prednisone, ATGAM, THYMOGLOBULIN, Brequinar Sodium, OKT4, T10B9.A-3A, 33B3.1, l5-deoxyspergualin, tresperimus, Leflunomide ARAVA, CTLAI-Ig, anti-CD25, anti-IL2R, Basiliximab (SIMULECT), Daclizumab (ZENAPAX), mizorbine, methotrexate, dexamethasone, ISAtx-247, SDZ ASM 981 (pimecrolimus, Elidel), CTLA4lg (Abatacept), belatacept, LFA3lg contend etanercept (sold
  • a pharmaceutical combination or composition described herein is administered to the subject prior to treatment with another chemotherapeutic agent, during treatment with another chemotherapeutic agent, after administration of another chemotherapeutic agent, or a combination thereof.
  • the selective pharmaceutical combination or composition can be administered to the subject such that the other chemotherapeutic agent can be administered either at higher doses (increased chemotherapeutic dose intensity) or more frequently (increased chemotherapeutic dose density).
  • Dose-dense chemotherapy is a chemotherapy treatment plan in which drugs are given with less time between treatments than in a standard chemotherapy treatment plan.
  • Chemotherapy dose intensity represents unit dose of chemotherapy administered per unit time. Dose intensity can be increased or decreased through altering dose administered, time interval of administration, or both.
  • the pharmaceutical combination or composition described herein can be administered in a concerted regimen with another agent such as a non- DNA-damaging, targeted anti -neoplastic agent or a hematopoietic growth factor agent.
  • another agent such as a non- DNA-damaging, targeted anti -neoplastic agent or a hematopoietic growth factor agent.
  • hematopoietic growth factors can have serious side effects.
  • the use of the EPO family of growth factors has been associated with arterial hypertension, cerebral convulsions, hypertensive encephalopathy, thromboembolism, iron deficiency, influenza like syndromes and venous thrombosis.
  • the G-CSF family of growth factors has been associated with spleen enlargement and rupture, respiratory distress syndrome, allergic reactions and sickle cell complications.
  • G-CSF granulocyte colony stimulating factor
  • Neupogen filamentgrastin
  • Neulasta peg-filgrastin
  • lenograstin granulocyte-macrophage colony stimulating factor
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • M-CSF macrophage colony stimulating factor
  • thrombopoietin megakaryocyte growth development factor (MGDF), for example sold as Romiplostim and Eltrombopag
  • interleukin IL
  • interleukin-3 interleukin- 11
  • SCF stem cell factor, steel factor, kit-ligand, or KL
  • the pharmaceutical combination or composition is administered prior to administration of the hematopoietic growth factor.
  • the hematopoietic growth factor administration is timed so that the pharmaceutical combination or composition’s effect on HSPCs has dissipated.
  • the growth factor is administered at least 20 hours after the administration of a pharmaceutical combination or composition described herein.
  • multiple doses of a pharmaceutical combination or composition described herein can be administered to the subject.
  • the subject can be given a single dose of a pharmaceutical combination or composition described herein.
  • the activity of an active compound for a purpose described herein can be augmented through conjugation to an agent that targets the diseased or abnormally proliferating cell or otherwise enhances activity, delivery, pharmacokinetics or other beneficial property.
  • Fv fragments are the smallest fragment made from enzymatic cleavage of IgG and IgM class antibodies. Fv fragments have the antigen-binding site made of the VH and VC regions, but they lack the CH1 and CL regions. The VH and VL chains are held together in Fv fragments by non-covalent interactions.
  • a selected compound as described herein can be administered in combination with an antibody fragment selected from the group consisting of an ScFv, domain antibody, diabody, triabody, tetrabody, Bis-scFv, minibody, Fab2, or Fab3 antibody fragment.
  • the antibody fragment is a ScFv.
  • ScFv single chain variable fragments
  • the linker is at least 12 residues long, the ScFv fragments are primarily monomeric.
  • the antibody fragment administered in combination with a selected compound described herein is a bivalent diabody. If the linker length is less than three residues, scFv molecules associate into triabodies or tetrabodies. In one embodiment, the antibody fragment is a triabody. In one embodiment, the antibody fragment is a tetrabody.
  • Multivalent scFvs possess greater functional binding affinity to their target antigens than their monovalent counterparts by having binding to two more target antigens, which reduces the off-rate of the antibody fragment.
  • the antibody fragment is a minibody. Minibodies are scFv-CH3 fusion proteins that assemble into bivalent dimers.
  • the antibody fragment is a Bis-scFv fragment. Bis-scFv fragments are bispecific. Miniaturized ScFv fragments can be generated that have two different variable domains, allowing these Bis-scFv molecules to concurrently bind to two different epitopes.
  • a selected compound described herein is administered in conjugation or combination with a bispecific dimer (Fab2) or trispecific dimer (Fab3). Genetic methods are also used to create bispecific Fab dimers (Fab2) and trispecific Fab trimers (Fab3). These antibody fragments are able to bind 2 (Fab2) or 3 (Fab3) different antigens at once.
  • Fab2 bispecific dimer
  • Fab3 trispecific dimer
  • a selected compound described herein is administered in conjugation or combination with an rlgG antibody fragment.
  • rlgG antibody fragments refers to reduced IgG (75,000 daltons) or half-IgG. It is the product of selectively reducing just the hinge-region disulfide bonds. Although several disulfide bonds occur in IgG, those in the hinge-region are most accessible and easiest to reduce, especially with mild reducing agents like 2-mercaptoethylamine (2-MEA).
  • Half-IgG are frequently prepared for the purpose of targeting the exposing hinge-region sulfhydryl groups that can be targeted for conjugation, either antibody immobilization or enzyme labeling.
  • a selected active compound described herein can be linked to a radioisotope to increase efficacy, using methods well known in the art.
  • Any radioisotope that is useful against cancer cells can be incorporated into the conjugate, for example, but not limited to, 131 1, 123 I, 192 Ir, 32 P , 90 Sr, 198 Au, 226 Ra, 90 Y, 241 Am, 252 Cf , 60 Co, or 137 Cs.
  • the linker chemistry can be important to efficacy and tolerability of the drug conjugates.
  • the thio-ether linked T-DM1 increases the serum stability relative to a disulfide linker version and appears to undergo endosomal degradation, resulting in intra-cellular release of the cytotoxic agent, thereby improving efficacy and tolerability, See , Barginear, M.F. and Budman, D.R., Trastuzumab-DMl : A review of the novel immune-conjugate for HER2-overexpressing breast cancer, The Open Breast Cancer Journal, 1 : 25-30, (2009).
  • composition or combination as described herein can be used to treat any disorder described herein.
  • a compound of the present invention is dosed in a combination or composition with an effective amount of a nucleoside or nucleoside analog.
  • nucleosides include: azacitidine, decitabine, didanosine, vidarabine, BCX4430, cytarabine, emtricitabine, lamivudine, zalcitabine, abacavir, aciclovir, entecavir, stavudine, telbivudine, zidovudine, idoxuridine, trifluridine, apricitabine, elvucitabine, amdoxovir, and racivir.
  • the compound of present invention is used in a combination or composition with an effective amount of a nucleoside or nucleoside analog to treat a viral infection.
  • the compound of present invention is used in a combination or composition with an effective amount of a nucleoside or nucleoside analog to treat a tumor or cancer.
  • the nucleoside analog is azacitidine and the disorder is tumor or cancer.
  • a method of treating tumor or cancer in a subject comprising administration of an analog of a compound described herein or a pharmaceutically acceptable salt thereof as provided herein in combination or alternation with an effective amount of a nucleoside analog to a host in need thereof.
  • a method of treating tumor or cancer in a subject comprising administration of an analog of a compound described herein or a pharmaceutically acceptable salt thereof as provided herein in combination or alternation with azacitidine to a host in need thereof.
  • Example 1 Exemplary Preparative Technique to Prepare Antigens to form Antibodies to the Pharmacodynamic Biomarkers.
  • recombinant expression of the protein is performed to obtain the immunogen.
  • the expression is done by applying a combination of the RTS 100 expression system and bacteria that expresses the pharmacodynamic biomarker.
  • the DNA sequence is analyzed and recommendations for high yield cDNA silent mutational variants and respective PCR-primer sequences are obtained using the“ProteoExpert RTS E. coli HY” system. This is a commercial web-based service (www.proteoexpert.com).
  • the“RTS 100 E. coli Linear Template Generation Set, His-tag” (Roche Diagnostics GmbH, Mannheim, Germany, Cat. No.
  • His-tag fusion protein Purification of His-tag fusion protein is done following standard procedures on aNi-chelate column. Briefly, 1 1 of bacteria culture containing the expression vector for the His-tag fusion protein is pelleted by centrifugation. The cell pellet is suspended in lysis buffer, containing phosphate, pH 8.0, 7 M guanidium chloride, imidazole and thioglycerole, followed by homogenization using a ETltra-Turrax®. Insoluble material is pelleted by high speed centrifugation and the supernatant is applied to a Ni-chelate chromatographic column. The column is washed with several bed volumes of lysis buffer followed by washes with buffer, containing phosphate, pH 8.0 and urea. Finally, bound antigen is eluted using a phosphate buffer containing SDS under acid conditions.
  • Example 2 Exemplary Preparative Technique to form Antibodies to the Pharmacodynamic Biomarkers.
  • mice 12 week old mice are initially immunized intraperitoneally with 100 pg of antigen (see above). This is followed after 6 weeks by two further intraperitoneal immunizations at monthly intervals. In this process each mouse is administered 100 pg antigen or antigen adsorbed to aluminium hydroxide and 10 9 germs of Bordetella pertussis. Subsequently the last two immunizations are carried out intravenously on the 3rd and 2nd day before fusion using 100 pg of antigen for each.
  • Spleen cells of the mice are fused with myeloma cells according to Galfre, G., and Milstein, C., Methods in Enzymology 73 (1981) 346.
  • ca. 1 x 10 8 spleen cells of the immunized mouse are mixed with 2> ⁇ l0 7 myeloma cells (P3X63-Ag8-653, ATCC CRL1580) and centrifuged (10 min at 300xg and 4° C.). The cells are then washed once with RPMI 1640 medium and centrifuged again at 400xg in a 50 ml conical tube.
  • the sedimented cells are taken up in RPMI 1640 medium containing 10% FCS and sown in hypoxanthine-azaserine selection medium (100 mmol/l hypoxanthine, 1 pg/ml azaserine in RPMI 1640+10% FCS).
  • Interleukin 6 at 100 U/ml is added to the medium as a growth factor.
  • Antibody positive primary cultures are cloned in 96-well cell culture plates by means of a fluorescence activated cell sorter. In this process again interleukin 6 at 100 U/ml is added to the medium as a growth additive.
  • the hybridoma cells obtained are sown at a density of U lO 5 cells per ml in RPMI 1640 medium containing 10% FCS and proliferated for 7 days in a fermenter (Thermodux Co., Wertheim/Main, Model MCS-104XL, Order No. 144-050). Purification of this antibody from the culture supernatant is carried out by conventional methods in protein chemistry (e.g. according to Bruck, C., et ak, Methods in Enzymology 121 (1986) 587-695).
  • Example 3 Exemplary Western Blot Technique to Detect the Concentration of Pharmacodynamic Biomarker in Human Plasma Samples
  • the membranes are washed 3 times in PBS/0.05% Tween-20 and blocked with SuperBlock Blocking Buffer (Pierce Biotechnology, Inc., Rockford, Ill., USA).
  • the biotinylated primary antibody is diluted in SuperBlock Blocking Buffer (0.01-0.2 pg/ml) and incubated with the membrane for 1 h.
  • the membranes are washed 3 times in PBS/0.05% Tween-20.
  • the specifically bound biotinylated primary antibody is labelled with a streptavidin-HRP-conjugate (20 mUABTs/ml in SuperBlock Blocking Buffer). After incubation for 1 h, the membranes are washed 3 times in PBS/0.05% Tween-20.
  • the bound streptavidin-HRP- conjugate is detected using a chemiluminescent substrate (Super Signal West Femto Substrate, Pierce Biotechnology, Inc., Rockford, Ill., USA) and autoradiographic film. Exposure times varies from 10 min to overnight.
  • chemiluminescent substrate Super Signal West Femto Substrate, Pierce Biotechnology, Inc., Rockford, Ill., USA
  • a 20 pl aliquot of a human serum or plasma sample or a serial dilution of the recombinant standard antigen were incubated with 100 m ⁇ biotinylated polyclonal antibody (1 pg/ml) and with digoxygenylated polyclonal antibody (1 pg/ml) in 10 mM phosphate, pH 7.4, 1% BSA, 0.9% NaCl and 0.1% Tween-20. After incubation for 90 min at 30° C., the plates were washed three times with 0.9% NaCl, 0.1% Tween-20.
  • PBMCs Cryogenically frozen PBMCs from 5 healthy donors (provided by StemCell Technologies) were thawed and maintained in the incubator in RPMI supplemented with 10% heat inactivated FBS in low attachment plates. After 1 hour recovery, 100 nM Cortistatin A or 0.1% DMSO was added to each sample and left in the incubator for 1 hour. PBMCs were collected and flash-frozen. Cells were lysed in 50 mM Tris pH 8.5, 8M Urea, 1% SDS, and protease and phosphatase inhibitors (Roche), reduced with DTT and alkylated with iodoacetamide.
  • Proteins were precipitated by methanol/chloroform, and digested sequentially using LysC (1 :50) and trypsin (1 : 100) protease/protein ratio. Peptides were quantified by micro-BCA (Pierce), and tandem mass tagged for multiplexing. TMT label incorporation was 99.7%. 3278 total peptides, 2254 unique peptides representing 1147 proteins were identified. Phosphopeptide enrichment was 83.9% (2752 total phospho peptides, 1968 unique phosphopeptides). Peptides were separated using a 3 to 25% acetonitrile gradient in 0.125% formic acid over 180 minutes. Peptides were detected (MS1) and quantified (MS3) in the Orbitrap.
  • AML cell lines MOLM-13 and MOLM-14 were split and grown in heavy-labeled and light labeled SILAC medium for weeks.
  • Cells were treated with vehicle or CDK8/19 inhibitor for one hour, and lysed. Lysates were mixed and analyzed by mass spectrometry. Both reciprocal iterations were performed (vehicle or inhibitor-treated cells heavy labeled).
  • CDK8/19 substrates were from phosphopeptides with a high ratio in vehicle: CDK8 inhibitor- treated samples. Confidence was assessed by ztest, calculating number of heavy and light labeled peptides detected in each sample. The resultant volcano plots are shown in Figures 2, 3, 4, and 5 which correspond to MOLM-13 CA treated, MOLM-13 vehicle treated, MOLM-14 CA treated, and MOLM-14 vehicle treated samples respectively.
  • Example 7 Preparation of Antibodies for Immunoblot Studies.
  • phosphorylated and unphosphorylated version of each peptide was synthesized for immunization, screening, and antibody purification.
  • Phosphorylated peptide was conjugated to keyhole limpet hemocyanin (KLH) and used for immunization.
  • KLH keyhole limpet hemocyanin
  • the phosphorylated peptides used in the generation of the antibodies were: CHD4 rT1553 : TQPN ⁇ pThr ⁇ PAPVPPAEDC, DPF2 pT248: DSQPP ⁇ pThr ⁇ PVSQRSEEC, DMAP1 pT445: V GAPL ⁇ pThr ⁇ PN SRKRREC .
  • the C- terminal cysteine was used for conjugation to KLH.
  • mice Three Balb/c and three C57BL/6 mice were injected with the immunogen on the following schedule: Primary immunization: 50 ug/animal with complete Freund’s adjuvant (CFA) subcutaneously, 2 boosts of 25 ug/animal on days 14, 28 after primary immunization with incomplete Freund’s adjuvant (IF A) subcutaneously. Total sera were screened by Western blotting using lysates of SET-2 cells treated with vehicle or 100 nM Cortistatin A for 3 hours. Two animals mounting the best phosphospecific response were selected for a final intravenous boost at day ⁇ 50, using 25 ug/animal and IFA.
  • CFA complete Freund’s adjuvant
  • IF A incomplete Freund’s adjuvant
  • Anti-CHD4 rT1553 clone 7F12 (BALB/c mouse, isotype IgGl), clones 4D8, 7F7, 8B6, and 11B6 (BALB/c mouse, isotype IgG2b);
  • Anti-DPF2 pT248 clones 1D4, 2D1 (BALB/c mouse, isotype IgG2a), clones 11B8, 12C8, 12F1 (BALB/c mouse, isotype IgG2b);
  • Anti-DMAPl pT445 clone 5G9 (BALB/c mouse, IgG2a), clones 6G4, 14F12 (BALB/c mouse, IgG2b)
  • SL164 titration stocks were prepared in DMSO and added to blood in T25 flasks that were incubated at 37 °C 5% CO2 for 3-4 hours.
  • PBMCs were then collected using Lymphoprep and Sepmate-50 tubes (StemCell Technologies) according to standard protocols.
  • PBMCs were transferred to microtubes, washed with PBS, and lysed in buffer containing 20 mM Tris pH 7.5, l50 mMNaCl, 1% Triton X-100, l mM Na2EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3V04, 1 pg/ml, IX HALT protease/phosphatase inhibitor cocktail, 2 mM PMSF and 1 mM 3,4- dichloroisocoumarin on ice. Samples were quantified by BCA (Thermo Fisher) and run on SDS-PAGE gels at 30 ug total protein per lane. Immunoblotting was performed using the monoclonal antibodies and anti-mouse HRP conjugated antibody.
  • PBMCs were transferred to microtubes, washed with PBS, and lysed in buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM Na2EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3V04, 1 pg/ml, IX HALT protease/phosphatase inhibitor cocktail, 2 mM PMSF and 1 mM 3,4-dichloroisocoumarin on ice. Samples were quantified by BCA (Thermo Fisher) and run on SDS-PAGE gels at 30 ug total protein per lane. Immunoblotting was performed using the monoclonal antibodies and anti-mouse HRP conjugated antibody.

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Abstract

L'invention concerne un diagnostic pharmacodynamique qui comprend l'utilisation d'une sélection de biomarqueurs pour mesurer l'efficacité dans le temps des inhibiteurs de CDK8 et/ou de CDK19 en thérapie médicale. Cette approche pharmacodynamique pour évaluer l'efficacité thérapeutique de CDK8 et/ou CDK19 au fil du temps est basée sur la perte observée de phosphorylation de protéines prédictives qui ont été identifiées à cet effet pour la première fois à l'aide d'une plage de paramètres. Ce diagnostic en temps réel et ce kit avantageux permettent au fournisseur de soins de santé de surveiller le patient pendant une thérapie pour déterminer le niveau de réponse à un traitement avec l'inhibiteur de CDK8 et/ou de CDK19 sélectionné.
PCT/US2019/035038 2018-06-01 2019-05-31 Biomarqueurs pharmacodynamiques pour le traitement du cancer avec un inhibiteur de cdk8/19 WO2019232467A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2754441C2 (ru) * 2019-12-30 2021-09-02 Закрытое Акционерное Общество "Биокад" Новые ингибиторы cdk8/19
WO2023044041A1 (fr) * 2021-09-16 2023-03-23 University Of South Carolina Inhibiteurs de cdk8/19 pour la prévention de la résistance aux médicaments
WO2023215235A3 (fr) * 2022-05-03 2024-01-25 The Regents Of The University Of California Inhibiteurs peptidiques de la protéine 4 de liaison à l'adn hélicase de chromodomaine (chd4)

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US20090304594A1 (en) * 2005-12-05 2009-12-10 Fantin Valeria R Methods for Predicting Treatment Response Based On the Expression Profiles of Protein and Transcription Biomarkers
US20120270233A1 (en) * 2008-02-11 2012-10-25 Historx, Inc. Association of biomarkers with patient outcome
WO2013148775A1 (fr) * 2012-03-30 2013-10-03 Merck Sharp & Dohme Corp. Biomarqueur prédictif utile pour thérapie anticancéreuse médiée par un inhibiteur de cdk
WO2016182904A1 (fr) * 2015-05-08 2016-11-17 President And Fellows Of Harvard College Sélection ciblée de patients pour un traitement par dérivés de cortistatine
US20180087114A1 (en) * 2015-03-05 2018-03-29 Trovagene, Inc. Early assessment of mechanism of action and efficacy of anti-cancer therapies using molecular markers in bodily fluid

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090304594A1 (en) * 2005-12-05 2009-12-10 Fantin Valeria R Methods for Predicting Treatment Response Based On the Expression Profiles of Protein and Transcription Biomarkers
US20120270233A1 (en) * 2008-02-11 2012-10-25 Historx, Inc. Association of biomarkers with patient outcome
WO2013148775A1 (fr) * 2012-03-30 2013-10-03 Merck Sharp & Dohme Corp. Biomarqueur prédictif utile pour thérapie anticancéreuse médiée par un inhibiteur de cdk
US20180087114A1 (en) * 2015-03-05 2018-03-29 Trovagene, Inc. Early assessment of mechanism of action and efficacy of anti-cancer therapies using molecular markers in bodily fluid
WO2016182904A1 (fr) * 2015-05-08 2016-11-17 President And Fellows Of Harvard College Sélection ciblée de patients pour un traitement par dérivés de cortistatine

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2754441C2 (ru) * 2019-12-30 2021-09-02 Закрытое Акционерное Общество "Биокад" Новые ингибиторы cdk8/19
WO2023044041A1 (fr) * 2021-09-16 2023-03-23 University Of South Carolina Inhibiteurs de cdk8/19 pour la prévention de la résistance aux médicaments
WO2023215235A3 (fr) * 2022-05-03 2024-01-25 The Regents Of The University Of California Inhibiteurs peptidiques de la protéine 4 de liaison à l'adn hélicase de chromodomaine (chd4)

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