WO2012118599A1 - Inhibiteurs de la tyrosine kinase c-abl utiles pour inhibition de réplication de filovirus - Google Patents

Inhibiteurs de la tyrosine kinase c-abl utiles pour inhibition de réplication de filovirus Download PDF

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WO2012118599A1
WO2012118599A1 PCT/US2012/023953 US2012023953W WO2012118599A1 WO 2012118599 A1 WO2012118599 A1 WO 2012118599A1 US 2012023953 W US2012023953 W US 2012023953W WO 2012118599 A1 WO2012118599 A1 WO 2012118599A1
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tyrosine kinase
abll
patient
abl tyrosine
lanes
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Daniel Kalman
Gary J. Nabel
Mayra GARCIA
Arik Arie COOPER
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Emory University
The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/137—Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/4162—1,2-Diazoles condensed with heterocyclic ring systems
    • 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
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/5025—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/7056—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/212—IFN-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals

Definitions

  • c-Abl tyrosine kinase antagonists inhibit virus growth through their effect on VP-40 induced budding, and c-Abl tyrosine kinase antagonists inhibit virus growth.
  • c-Abl specific siRNAs and c-Abl tyrosine kinase inhibitors are provided herein as Ebola and Marburg viral replication inhibitors.
  • Compositions and methods for treating and preventing viral infection, including Ebola and Marburg viral infections, are also provided by this disclosure.
  • Ebolavirus EBOV
  • Marburg virus MARV
  • EBOV Ebolavirus
  • MAV Marburg virus
  • TKs tyrosine kinases
  • the present disclosure provides inhibitors of filovirus c-Abl tyrosine kinase pathway useful for inhibiting viral replication.
  • the disclosure fulfills the need for additional prophylactic and therapeutic treatments for Ebola virus and Marburg virus and provides additional advantages discussed below.
  • c-Abl specific siRNAs and c-Abl tyrosine kinase inhibitors are provided herein as Ebola and Marburg viral replication inhibitors.
  • a method of treating a Filoviridae viral infection comprising providing an effective amount of a c-Abl tyrosine kinase inhibitor to a patient in need thereof is provided by the disclosure.
  • the c-Abl tyrosine kinase inhibitor may be a biological inhibitor that decreases expression of the c-Abl tyrosine kinase, such as a c-Abl tyrosine kinase specific siRNA.
  • the c-Abl tyrosine kinase inhibitor is a small molecule c-Abl tyrosine kinase antagonist.
  • Suitable c-Abl tyrosine kinase antagonists include dasatinib, imatinib, tivozanib, ponatinib, bafetinib, saracatinib, fingolimod, AT 9283, KW 2449, danusertib, and nilotinib and the pharmaceutically acceptable salts thereof.
  • the c-Abl tyrosine kinase antagonist is dasatinib, imatinib, or nilotinib or a pharmaceutically acceptable salt.
  • the disclosure includes methods of treating a Filoviridae viral infection in which the c-Abl tyrosine kinase inhibitor is provided together with an additional active agent.
  • additional active agents include ribavirin and interferon, such as interferon alpha- 2b.
  • Methods of treatment include both therapeutic methods in which the patient is known to be infected with a Filoviridae virus, such as Ebola virus or Marburg virus, and prophylactic methods. Because Ebola virus and Marburg virus are highly contagious, treating health care workers who will be exposed to these viruses prophylactically to reduce their chance of becoming infected is particularly included as an aspect of this disclosure.
  • a Filoviridae virus such as Ebola virus or Marburg virus
  • the c-Abl tyrosine kinase inhibitor may be provided by any pharmaceutically acceptable method, such as administration as an oral dosage form or intravenously.
  • the c-Abl tyrosine kinase inhibitor is provided to an infected patient together with supportive treatment such as intravenous fluids given to prevent or reverse dehydration and/ or transfusions of platelets.
  • FIGURE 1 Transient transfection of NP, VP40, VP35, VP24 and GP gives rise to Ebola VLPs in 293T cells.
  • A Western blot analysis of EBOV VLP
  • FIGURE 2 Effect of c-Abll knockdown and kinase inhibition on Ebola VLP release in transfected 293T cells.
  • A Knockdown of c-Abll (lanes 1-3) or c-Abl2 (lanes 4-6) using non-targeting siRNA control or siRNA targeting c-Abll or c-Abl2 confirmed by Western blotting in cell lysates with antibodies specific to either c-Abll (lanes 1-3) or c-Abl2 (lanes 4-6). ⁇ -actin was used as a loading control. The results are representative of 5 independent experiments.
  • 293T cells were transfected with plasmids encoding VP24, VP35, VP40, NP, and GP.
  • EBOV VLPs were analyzed by immunoprecipitation with GP followed by Western blotting for NP and VP40 (lanes 10-12).
  • Cell lysates are shown in lanes 7-9. Data represent the mean + SEM of individual measures with cells from 4 independent experiments.
  • NP and VP40 present in cell lysates were determined (lanes 17-20), and EBOV VLPs in supernatants were measured as in (A) after knockdown with c-Abll individual siRNAs (lanes 21-24). Data represent the mean + SEM of individual measures with cells from 3 independent experiments. For A and B, quantitation of NP and VP40 protein bands is expressed as a percentage of the intensity of the siRNA control band (lower panels).
  • C Western blot analysis of EBOV VLP release for NP and VP40 content with imatinib (lanes 25-27) or nilotinib (lanes 28-30).
  • FIGURE 3 Toxicity analysis, morphology of Ebola intracellular
  • nucleocapsids and nucleocapsid formation.
  • A 293T cells viability analysis. Toxicity of 10 or 20 ⁇ imatinib or nilotinib or vehicle control was measured by 7-AAD exclusion in untransfected cells 36-48 hours after the drug was added. Data are presented as mean + SEM of 3 independent experiments.
  • B Morphology of Ebola virus nucleocapsids in 293T cells transfected with empty vector (left panel) or the five EBOV plasmids in combination with water or DMSO controls (middle panels) or drug treatments at 20 ⁇ (right panels) seen intracellularly (arrows) by electron microscopy. Size standards are shown.
  • FIGURE 4 GP levels in 293T cell lysates after drug treatment.
  • FIGURE 5 c-Abll- specific siRNA and nilotinib effects on VP40 VLP egress.
  • 293T cells were transfected with plasmid encoding VP40.
  • VP40 VLPs were analyzed by immunoprecipitation with monoclonal antibody anti-FLAG followed by Western blotting for VP40 after transfection with a pool (A, lanes 4-6) or individual (B, lanes 11-14) siRNAs specific to cAbll . Cell lysates are shown in lanes 1-3 and 7-10.
  • VP40 levels in VLPs were normalized to inputs. Quantitation of VP40 protein bands is expressed as a percentage of the intensity of the siRNA control band.
  • FIGURE 6 Tyrosine phosphorylation of c-Abll and VP40 after expression of c-Abll in 293T cells.
  • A Western blotting analysis of c-Abll activation in 293T cells transfected with empty vector control (lanes 1 and 4) or wild type c-Abll expression vector (lanes 2, 3, 5 and 63) 24 hours before EBOV plasmid transfection. Autophosphorylation on Tyr 412 and 245 was measured by Western blotting for phospho tyro sine in cell lysates after c-Abll immunoprecipitation (lanes 2 and 5).
  • FIGURE 7 Effect of c-Abll expression on VP40 phosphorylation and sensitivity to tyrosine kinase antagonist inhibition and siRNA.
  • A Western blotting analysis of transfected cell lysates (lanes 1-4) or VP40 immunoprecipitates (lanes 5-8) of cells transfected with empty vector control (lanes 1, 3, 5, and 7) or wild type c-Abll (lanes 2, 4, 6 and 8) in the presence of VP40 expression vector.
  • DMSO control (lanes 1, 2 and 5, 6) or 20 ⁇ of nilotinib (lanes 3, 4 and 7, 8) was added 12-18 hours after transfection.
  • kinase activity on VP40 was measured by Western blotting of inputs using an anti-phosphotyrosine antibody (upper panel). Western blotting was also performed for c-Abll and flag-tagged VP40 (middle panels). eIF4E was used as a loading control (lower panel). Analyses are representative of 3 independent experiments.
  • NP and VP40 were cotransfected with NP and VP40 and then treated with DMSO (lanes 39 and 44) or 20 ⁇ nilotinib (lanes 40 and 45).
  • NP and flag-tagged VP40 in cell lysates (lanes 36-40) and VP40 immunoprecipitates (lanes 41-45) were detected by Western blotting using rabbit anti-NP antiserum and anti-FLAG monoclonal antibody, ⁇ -actin was used as a loading control.
  • NP levels in VLPs were normalized to NP levels in lysates and then expressed as a ratio of the DMSO control sample (lower panel). Data are presented as mean + SEM from 4 independent experiments and significance analyzed by paired Student's t test.
  • FIGURE 8 Localization of VP40 c-Abll -mediated tyrosine phosphorylation and effect of Tyr mutation on VLP release.
  • A Expanded region of the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectrum of the VP40 (amino acids 3-21) acquired for the non-phosphorylated (left) vs. the phosphorylated (right) peptides from 293T cells co-transfected with VP40 and c-Abll. Analysis was performed on a gel slice of a flag-tagged VP40 immunoprecipitate. Arrow indicates site of Y13 phosphorylation of VP40.
  • MALDI-TOF MS matrix-assisted laser desorption ionization time-of-flight mass spectrometry
  • FIGURE 9 Mass spectrometry analysis of VP40 modification in the absence of c-Abll tyrosine kinase expression vectors in transfected 293T cells.
  • A Expanded region of the MALDI-TOF MS spectrum of the VP40 (amino acids 292-326) acquired for the non- phosphorylated (left) vs. the phosphorylated (right) peptides from 293T cells co-transfected with VP40 and c-Abll . Arrow indicates site of Y292 phosphorylation of VP40.
  • B Expanded region of the MALDI-TOF MS spectrum of the VP40 (amino acids 4-21) acquired for the non-phosphorylated (left) vs.
  • Cells were transfected with empty vector control (lanes 1, 3, 5, 7, 9, 11, 13, and 15) or wild type c-Abll vector (lanes 2, 4, 6, 8, 10, 12, 14, and 16) in the presence of WT (lanes 1, 2, 9, and 10) or Y13A (lanes 3, 4, 11, and 12) or Y292A (lanes 5, 6, 13, and 14) or Y13A+Y292A (lanes 7, 8, 15, and 16) VP40 labeled with a flag tag.
  • WT VP40 was used as a reference in all cases.
  • Phosphorylation on Tyr was measured by Western blotting (upper panel) in cell lysates (lanes 1-8) or in VP40 immunoprecipitates (lanes 9-16). Western blotting analysis was also carried for VP40 by flag labeling (middle panel), ⁇ -actin was used as a loading control (lower panel). The results are representative of 3 independent experiments.
  • FIGURE 10 Nedd4 and c-Abll interaction with VP40.
  • A Co- immunoprecipitation of Nedd4 (top panel) with VP40 in the absence (lane 3) or presence (lane 4) of 20 ⁇ nilotinib. Inputs are shown on lanes 1 and 2.
  • Western blotting analysis was also carried out for VP40 by flag labeling (middle panel).
  • eIF4E was used as a loading control (lower panel). Data are presented as mean + SEM of 3 independent experiments.
  • FIGURE 11 Effect of siRNA knockdown and c-Abll tyrosine kinase inhibition on EBOV replication.
  • A Effect of a non-targeted siRNA control or individual siRNAs targeting c-Abll (S9, S10, SI 1) on Zaire EBOV release from Vero cells on day 7 after infection. Cells were infected at multiplicity of infection of 1. Background viral load for day 1 was subtracted. Data are presented as mean + SEM of individual measures with cells from 2 independent experiments.
  • FIGURE 12 Infection assays.
  • A Vero cells viability analysis. Toxicity of 20 ⁇ nilotinib or DMSO compared to untreated cells was measured by 7-AAD exclusion in uninfected Vero cells on day 1, 2, 3, and 7 after the drug was added. Data are presented as mean + SEM of 3 independent experiments.
  • B Infection viability analysis. Toxicity of 20 ⁇ nilotinib or DMSO was measured by an approximation of the number of floating cells in Zaire EBOV-infected Vero cells on days 0, 1, 2, 5, 7 and 8 after the drug was added. The results are representative of 2 independent experiments.
  • C Adenovirus infection assay.
  • Isotopes include those atoms having the same atomic number but different mass numbers.
  • isotopes of hydrogen include tritium and deuterium and isotopes of carbon include U C, 13 C, and 14 C.
  • an “active agent” is any compound, element, or mixture, that when administered to a patient alone or in combination with one or more other agents confers a therapeutic benefit on the patient.
  • the active agent is a compound, solvates (including hydrates) of the free compound or salt, crystalline and non-crystalline forms, as well as various polymorphs or the compound are included.
  • an active agent can include optical isomers of the compound and pharmaceutically acceptable salts thereof either alone or in combination.
  • oral dosage form denotes a form of a pharmaceutical composition that contains an amount sufficient to achieve a therapeutic effect with a single administration.
  • oral dosage form is meant to include a unit dosage form prescribed or intended for oral administration.
  • An oral dosage form may or may not comprise a plurality of subunits such as, for example, microcapsules or microtablets, packaged for administration in a single dose.
  • the term "effective amount” means an amount effective, when administered to a human or non-human patient, to provide any therapeutic benefit such as an amelioration of symptoms, e.g., an amount effective to decrease the symptoms of viral infection, and preferably an amount sufficient to decrease the symptoms of Ebola virus or Marburg virus infection.
  • An "effective amount” may also be an amount sufficient to decrease viral load or viral antibodies in the patient's blood tissues and symptoms. In certain circumstances a patient suffering from a virus may not present symptoms of being affected.
  • a therapeutically effective amount of a compound is also an amount sufficient to provide a positive effect on any indicia of disease, e.g.
  • a significant increase or reduction in the detectable level of viral infection markers is any detectable change that is statistically significant in a standard parametric test of statistical significance such as Student's T-test, where p ⁇ 0.05.
  • Lymphocytic leukemia markers are discussed in more detail in the "method of treatment” section.
  • a "patient” is any human or non-human animal in need of medical treatment.
  • the patient is a human patient determined to have a filovirus infection, such as an Ebola virus infection or a Marburg virus infection.
  • Medical treatment can include treatment of an existing condition, such as a disease or disorder, or prophylactic or preventative treatment.
  • treatment can be treatment of a patient infected with a filovirus, such as an Ebola virus or a Marburg virus.
  • treatment can be prophylactic treatment of a patient, such as a health care worker, who does not yet show symptoms of infection, wherein the treatment additionally comprises determining the patient has been in contact with a human or non-human animal infected with Ebola virus or Marburg virus with the past 0 to 14 days or will be in contact with a human or non-human animal infected with Ebola virus or Marburg virus in the following 0 to 14 days.
  • prophylactic treatment will be continued for 1 to 10 days after the patient is no longer in contact with a human or non-human animal infected with Ebola virus or Marburg virus.
  • “Pharmaceutically acceptable salts” includes derivatives of the disclosed compounds, wherein the parent compound is modified by making non-toxic acid or base addition salts thereof, and further refers to pharmaceutically acceptable solvates, including hydrates, of such compounds and such salts.
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid addition salts of basic residues such as amines; alkali or organic addition salts of acidic residues such as carboxylic acids; and the like, and combinations comprising one or more of the foregoing salts.
  • the pharmaceutically acceptable salts include non-toxic salts and the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • non-toxic acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; other acceptable inorganic salts include metal salts such as sodium salt, potassium salt, cesium salt, and the like; and alkaline earth metal salts, such as calcium salt, magnesium salt, and the like, and
  • organic salts include salts prepared from organic acids such as acetic, trifluoroacetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, HOOC-(CH 2 ) n -COOH where n is 0-4, and the like; organic amine salts such as triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt,
  • ⁇ , ⁇ '-dibenzylethylenediamine salt and the like; and amino acid salts such as arginate, asparginate, glutamate, and the like, and combinations comprising one or more of the foregoing salts.
  • c-Abl tyrosine kinase inhibitors include both biological molecules and small molecules that reduce the activity of c-Abl tyrosine kinase whether by reducing the levels of c-Abl protein as in the case of c-Abl tyrosine kinase specific siRNAs or by interacting directly with the c-Abl tyrosine kinase protein and thereby reducing its enzymatic activity (c- Abl tyrosine kinase antagonists).
  • Effective c-Abl tyrosine kinase antagonists for use in this method include dasatinib (CAS Reg. No. 302962-49-8), imatinib (CAS Reg. No. 152459-95-5), imatinib mesylate (CAS Reg. No. 220127-57-1), and nilotinib (CAS Reg. No. 641571-10-0). These molecules have the following chemical structures:
  • c-Abl tyrosine kinase inhibitors useful in the methods of treatment described herein include tivozanib (CAS Reg. No. 475108-18-0), ponatinib (CAS Reg. No. 1114544-31-8), bafetinib (CAS Reg. No. 887650-05-7), saracatinib (CAS Reg. No. 379231- 04-6), fingolimod (CAS Reg. No. 162359-55-9), AT9283 (CAS Reg. No. 896466-04-9), KW-2449 (CAS Reg. No. 1000669-72-6), and danusertib (CAS Reg. No. 827318-97-8).
  • Ebola virus replication is regulated by the c- Abl tyrosine kinase.
  • VLPs Ebola virus-like particles
  • c-Abl-specific siRNA or kinase inhibitors This effect is mediated by the Ebola matrix protein, VP40.
  • Phosphorylation of tyrosine (Y) 13 on VP40 is stimulated by c-Abl expression, and a VP40 Y13A mutation inhibited release of Ebola VLPs.
  • Inventors have also identified an unexpected modification by c-Abl of an essential Ebola virus gene product, VP40. EBOV VLP egress is inhibited by c-Abl siRNAs.
  • Inventors examined the effect of c-Abl on Ebola virus assembly and release initially by using a cell transfection system previously shown to support virus-like particle (VLP) generation in vitro.
  • VLP virus-like particle
  • Inventors knocked doTwn c-Abll or the related c-Abl2 with specific siRNAs (FIG. 2A, lanes 1-6).
  • c-Abl2 siRNA had no effect on c-Abll levels (FIG. 2A, lane 1 vs. 3) or vice versa (FIG. 2A, lane 4 vs. 5).
  • transfection of c-Abll siRNA decreased the quantity of VLPs by ⁇ 5-fold as measured by NP, and by ⁇ 2.5-fold as measured by VP40 protein levels following immunoprecipitation with GP (FIG. 2A, lane 11).
  • No effect was observed on intracellular levels of Ebola NP or VP40 proteins (FIG. 2A, lanes 7-9). The effect was specific; similar effects were evident with three individual siRNAs for c-Abll (FIG.
  • c-Abl2 siRNA or a control siRNA had no effect (FIG. 2A, lanes 10 and 12), and c-Abll siRNAs did not alter expression of an unrelated control protein eIF4E (FIG 2B, compare lane 13 with 14-16).
  • c-Abll siRNAs had no effect on intracellular levels of Ebola NP or VP40 proteins (FIG. 2B, lanes 17-20). At the same time, they decreased VLP release (FIG 2B, lanes 21-24) as measured by NP and VP40 protein levels, suggesting that they affected egress of preassembled VLPs from the cell. Release of EBOV VLPs is decreased by c-Abll tyrosine kinase inhibitors
  • Imatinib and nilotinib inhibit c-Abll kinase activity and are used clinically for treatment of chronic myelogenous leukemia; a disease caused by translocations or mutations that dysregulate c-Abll.
  • Incubation of 293T cells with imatinib (FIG. 2C, left panel) or nilotinib (FIG. 2C, right panel) 12-18 hours after transfection of EBOV plasmids resulted in a dose-dependent reduction in VLP release measured by NP and VP40 protein levels. No toxicity was observed at the selected concentrations (FIG. 3A).
  • Inventors assessed the effect of c-Abll on VP40-induced VLPs lacking nucleocapsid. VP40 VLPs were reduced by c-Abll - specific siRNAs at levels similar to those observed in complete VLPs (FIG. 5A and B).
  • VP40 contains a sequence surrounding Y18 that is homologous to a sequence in the Tir protein of enteropathogenic E. coli, which serves as an Abl/Tec phosphorylation site and an SH2 binding site.
  • Inventors transfected 293T cells with a c-Abll expression vector in conjunction with an EBOV VP40 plasmid. Transfected c-Abll was catalytically active as evidenced by autophosphorylation on Y412 (FIG.
  • Endogenous c-Abll also phosphorylates VLP-associated VP40
  • Y13 is a site of phosphorylation in VP40 by c-Abll
  • Inventors determined the sites of tyrosine phosphorylation in VP40 by mass spectrometry after cotransfection of VP40 and c-Abll. Tryptic peptides containing phosphorylated Y residues were predicted to increase the m/z ratio by 40 compared to unphosphorylated peptides, and matrix-assisted laser desorption ionization (MALDI) analysis detected several peptides with such m/z deviations, including Y13 (FIG. 8A, right vs. left) and Y292 (FIG. 9A, right vs. left panels). A similar modification of VP40 Y13 was evident with endogenous c-Abll, in the absence of co-transfected c-Abll (FIG. 9B, right vs. left panels).
  • MALDI matrix-assisted laser desorption ionization
  • c-Abll tyrosine kinase inhibitors reduce productive replication of wild type Ebola virus.
  • Inventors next determined whether c-Abll regulates productive replication of EBOV Zaire, a strain that is lethal in humans and NHPs, which was identified after an outbreak in 1976 in the Ebola River valley of Zaire, now the Democratic Republic of the Congo.
  • Treatment of Vero E6 cells with the S9 or S10 c-Abll siRNAs reduced Zaire EBOV production by 8- to 10-fold seven days after infection (FIG. 11 A), measured by relative levels that generated 50 percent of a tissue culture infectious dose (TCID 50 ).
  • TCID 50 tissue culture infectious dose
  • EBOV infectious virus production decreased by up to ⁇ 4 logs eight days after infection (FIG. 11B).
  • FIG. 12A and B there was no detectable cytopathic effect of the drug alone (FIG. 12A and B), and nilotinib had no effect on infection by an unrelated virus, adenovirus 5 (FIG. 12C), confirming the specificity of this inhibition.
  • Inventors used a luciferase-pseudotyped HIV virus with EBOV envelope to infect 293A cells. EBOV GP-mediated entry was not affected by nilotinib in concentrations up to 20 ⁇ (FIG. 12D).
  • the greater magnitude of drug inhibition (FIG.1 IB), compared to c-Abll siRNA treatment (FIG.
  • c-Abll tyrosine kinase antagonists disclosed herein are useful for treating viral infections in patients, including filovirus infections, such as Ebola virus infection or Marburg virus infection.
  • This disclosure provides methods of treating viral infections, including filovirus infections, such as Ebola virus infections and Marburg virus infections, by providing a therapeutically effective amount of a c-Abll tyrosine kinase antagonist or pharmaceutically acceptable salt of thereof to patient infected with a virus.
  • the c-Abll tyrosine kinase antagonist or salt thereof may be provided as the only active agent or may be provided together with one or more additional active agents.
  • the c-Abll tyrosine kinase antagonist is administered together with interferon alpha 2b or ribavirin.
  • An effective amount of a c-Abll tyrosine kinase antagonist may be an amount sufficient to (a) inhibit the progression of the viral infection (b) cause a regression of the viral infection; or (c) cure of a viral infection such that the virus or viral antibodies can no longer be detected in a previously infected patient's blood or plasma.
  • An amount of a c-Abll tyrosine kinase antagonist effective to inhibit the progress or cause a regression of viral infection includes an amount effective to stop the worsening of symptoms of viral infection or reduce the symptoms experienced by a patient infected with the virus. Alternatively a halt in progression or regression of viral infection may be indicated by any of several markers for the disease.
  • a lack of increase or reduction in the viral load or a lack of increase or reduction in the number of circulating viral antibodies in a patient' s blood are markers of a halt in progression or regression of viral infection.
  • Other filovirus markers include elevated TNF-a levels, elevated interleukin levels, including IL-6, -10, -11, and -1 ⁇ .
  • Disease regression is usually marked by the return of interleukin and TNF levels to the normal range.
  • Symptoms of filovirus infection that may be affected by an effective amount of a c-Abll tyrosine kinase antagonist include fever, sore throat, weakness, severe headache, joint and muscle aches, diarrhea, vomiting, dehydration, cough, stomach pain, internal and external bleeding, and rash.
  • a c-Abll tyrosine kinase antagonist is provided together with one or more additional active agents.
  • additional active agents include interferon alpha- 2b and ribavirin.
  • Certain drugs increase the amount of c-Abll tyrosine kinase antagonists in the bloodstream and may increase the efficacy of c- Abll tyrosine kinase antagonists.
  • Methods of treatment include administering on or more of these compounds in combination with a c-Abll tyrosine kinase antagonist.
  • a c- Abll tyrosine kinase antagonist may be administered together with another antiviral compound such as but not limited to ritonavir, atazanavir sulfate, indinavir, nelfinavir, saquinavir; an antibiotic such as but not limited to telithromycin, erythromycin, or clarithromycin; an antifungal such as but not limited to ketoconazole or itraconazole; or nefadar.
  • another antiviral compound such as but not limited to ritonavir, atazanavir sulfate, indinavir, nelfinavir, saquinavir
  • an antibiotic such as but not limited to telithromycin, erythromycin, or clarithromycin
  • an antifungal such as but not limited to ketoconazole or itraconazole
  • nefadar nefadar
  • the compound c-Abll tyrosine kinase antagonist and additional active agent may be: (1) co-formulated and administered or delivered simultaneously in a combined formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by any other combination therapy regimen known in the art.
  • the methods of the invention may comprise administering or delivering the c-Abll tyrosine kinase antagonist and an additional active agent sequentially, e.g., in separate solution, emulsion, suspension, tablets, pills or capsules, or by different injections in separate syringes.
  • an effective dosage of each active ingredient is administered sequentially, i.e., serially
  • simultaneous therapy effective dosages of two or more active ingredients are administered together.
  • Various sequences of intermittent combination therapy may also be used.
  • Methods of treatment include providing certain dosage amounts of a c-Abll tyrosine kinase antagonist to a patient.
  • Dosage levels of each active agent of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per patient per day).
  • the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the patient treated and the particular mode of administration.
  • Dosage unit a compound of the invention. In certain embodiments 25 mg to 500 mg, or 25 mg to 200 mg of a compound of the invention are provided daily to a patient. Frequency of dosage may also vary depending on the compound used and the particular disease treated.
  • a dosage regimen of 4 times daily or less is preferred and a dosage regimen of 1 or 2 times daily is particularly preferred.
  • Preferred dosages include dasatinib, about 10 to about 250 mg per day, or about 50 to 150 mg per day, or about 100 mg per day or about 140 mg per day; nilotinib, about 20 to 1500 mg per day, or about 150 to 1000 mg per day, or about 400 to 800 mg per day, or about 150 mg per day, or about 800 mg per day, or about 400 mg administerd twice per day; imatinib, about 50 to 500 mg per day, or about 100 to about 400 mg per day; tivozanib, about 0.1 to 20 mg per day, or about 0.5 to 10 mg per day, or about 1.5 mg per day; ponatinib, about 10 to 150 mg per day, or about 20 to 100 mg per day or about 45 mg per day; bafetinib, about 100 to 1200 mg per day, or about 200 to 500 mg per day, or about 480 mg per day;
  • NP VP35
  • VP24 Ebola Viral Protein necessary for nucleocapsid assembly
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • P/S penicillin and 100 ⁇ g/mL streptomycin
  • Virus Ebola Zaire was kindly provided by Peter Gonzling. The filovirus stock was grown in Vero E6 cells within 150 mm tissue culture flasks with DMEM (Invitrogen) containing 4,500 mg/L d-glucose, 1-glutamine and supplemented with 2% heat-inactivated FBS (DMEM-2) at 37°C, 5% C0 2 , and 85% humidity. At 8 days post-infection, supernatant was collected and aliquots of the virus were stored at -80°C.
  • DMEM Invitrogen
  • FBS heat-inactivated FBS
  • Imatinib mesylate and nilotinib were synthesized by Bill Bornmann at MD Anderson Cancer Center.
  • Plasmids Plasmids. Expression vectors pGP(Z), pNP, pVP24, pVP35, and
  • pFLAG_VP40 (altogether pEBOV x5) contain a cytomegalovirus enhancer and promoter as previously described. Plasmid DNAs were purified using double cesium chloride
  • the cells were transfected using 0.5 ⁇ g of pNP, pVP24, pVP35, WT, or mutant pFLAG_VP40 and 0.00625 ⁇ g of pGP(Z) with LipofectamineTM 2000 (Invitrogen) transfection reagents according to the protocol supplied by the manufacturer.
  • the backbone plasmid was used as a negative control.
  • VP40 VLPs the cells were transfected only with 0.5 ⁇ g of pFLAG VP40.
  • 0.5 ⁇ g of FLAG_VP40 was transfected in combination with 1 ⁇ g of c-Abll WT.
  • VP40 with NP or Nedd5 cells were co-transfected with 1 ⁇ g of each plasmid.
  • VP40 with c-Abll 0.5 ⁇ g of WT FLAG_VP40 or FLAG_VP40 mutants were transfected alone or in combination with 2 ⁇ g of c-Abl WT.
  • nucleocapsid formation the cells were cotransfected using 0.5 ⁇ g of pNP, pVP24, and pVP35.
  • HEK 293T cells For endogenous c-Abll association or phosphorylation of VP40, 2.5 x 10 6 HEK 293T cells seeded in 10 cm plates were transfected with 5 ⁇ g of pFLAG_VP40.
  • 2.5 x 10 6 human embryonic kidney 293T cells were seeded in 10 cm plates in DMEM + 10% FBS + P/S.
  • the cells were transfected using 2 ⁇ g of pNP, pVP24, pVP35, WT or mutant pFLAG_VP40 and 0.025 ⁇ g of pGP(Z) with LipofectamineTM 2000 (Invitrogen).
  • LipofectamineTM 2000 (Invitrogen). When used, drugs were added 12-18 hours after transfection. Cells were harvested 36-48 hours later. Supernatant fluids were kept at 4°C and cell pellets frozen at
  • siRNA For siRNA experiments, 1.25 x 10 5 293T or Vero E6 cells per well were seeded in 6-well plates in DMEM + 10% FBS + P/S. On the following morning, a total of 100 pmol of smart pools of siRNA for c-Abll (ON-TARGETplus SMARTpool L-003100- 00-0005, Human ABL1, NM_005157) or c-Abl2 (ON-TARGETplus SMARTpool L-003101- 00-0005, Human ABL2, NM_005158) (Dharmacon, Lafayette, CO) were transfected using Lipofectamine 2000 (Invitrogen).
  • c-Abll ON-TARGETplus SMARTpool L-003100- 00-0005, Human ABL1, NM_005157
  • c-Abl2 ON-TARGETplus SMARTpool L-003101- 00-0005, Human ABL2, NM_005158
  • siRNAs against c-Abll ON-TARGETplus J-003100-09, called S9-, ON-TARGETplus J-003100-10, called S10-, and ON-TARGETplus J-003100-11, called S11-, Human ABLl, NM_007313; NM_005157) (Dharmacon) were used at the same concentration.
  • 293T cells were transfected for a second time with pGP(Z), pNP, pVP24, pVP35, and pFLAG_VP40. 36-48 hours later, cells were harvested. Supernatant fluids were kept at 4°C and cell pellets frozen at -80°C before immunoprecipitation.
  • the transfection mixture was removed and the cells were infected at a multiplicity of infection of 1 with Zaire EBOV for two hours. The inoculum was then removed, fresh media added and supernatant fluids were collected on day 7 after infection. A non-targeting control siRNA was used as a reference.
  • pseudotyped lentiviral viruses Production of pseudotyped lentiviral viruses.
  • the pseudotyped lentiviral vectors expressing a luciferase reporter gene were produced by cotransfecting 293T cells with expression vectors ⁇ 8.2, luciferase, and pGP(Z) by Ca as previously reported. After 24 hours, the transfection mixture was removed and fresh media added. 72 hours later, supernatant fluid was collected and filtered.
  • Membranes were probed with 1:1,000 dilution of primary antibodies against c-Abll (Bethyl, Montgomery, TX), 1 :1000 antibody against phospho-c-Abl (Tyr412) or anti-Nedd4 (Cell Signaling Technology), 2.5 g/mL dilution of c-Abl2 (Abeam, Cambridge, MA), 1:5,000 antibody against EBOV-NP or EBOV- GP from DNA-immunized mice or rabbits, 1:3,000 antibody against EBOV-VP35 from DNA-immunized guinea pigs, 1:100 of anti-P-Tyr (PY20) sc-508 (Santa Cruz Biotechnology, Inc.) or 4G10 (Millipore, Temecula, CA), or O.lng/ L of VP40 (IBT Bioservices,
  • Blots were washed three times with lx TBS + 0.1% Tween-20 at room temperature for 10 min and developed in 12 mL of ECL detection reagent (GE Healthcare, Piscataway, NJ) for 1 min. Semi-quantitative analysis was performed using ImageQuant TL 7.0 image analysis software (GE Healthcare).
  • DMEM-2 nilotinib prior to infection with Ebola Zaire for two additional hours at a multiplicity of infection of 1.
  • the inoculum was then removed and fresh media with the drug at the same concentration was added.
  • DMSO vehicle was used as a negative control. Aliquots of supernatant fluids were taken at days 0, 1, 2, 5, 7 or 8 after infection for TCID 50 determination.
  • adenovirus 5 infection we used the Adeno-XTM rapid titer kit and followed the protocol according to the manufacturer's indications (Clontech, Mountain View, CA).
  • TCID 0 assay The quantity of Ebola virus in samples was estimated by calculating the 50% Tissue Culture Infectious Dose (TCID 50 ) as originally described by Reed and Muench (Am. J. Hygiene (1938) 27: 493-497.). Briefly, a 96-well tissue culture plate was seeded with Vero E6 cells and then incubated at 37 °C and 5% CO 2 for 24 hours. Serial 1-log dilutions of the virus in DMEM-2 were added to five wells per dilution of a 96-well tissue culture plate (BD, Franklin Lakes, NJ). After 10 days, the monolayers were observed with phase contrast microscopy to identify CPE. Cells were then stained by adding 0.2 ml of a solution of crystal violet (PML Microbiologicals, WilsonviUe, OR) to each well. Wells that remained unstained were identified as having CPE and thus counted as infected.
  • TCID 50 50% Tissue Culture Infectious Dose
  • ESI-LC-MS/MS LC-MS/MS analysis was done on a Waters Ultima Q-Tof hybrid quadrupole/time-of-flight mass spectrometer with a nanoelectro spray source.
  • Capillary voltage was set at 1.8 kV and cone voltage 32 V; collision energy was set according to mass and charge of the ion, from 18 eV to 50 eV.
  • Chromatography was performed on an LC Packings HPLC with a CI 8 PepMap column using a two-hour linear acetonitrile gradient with flow rate of 200 nl/ min. Raw data files were processed using the MassLynx
  • ProteinLynx software and .pkl files were submitted for searching at www.matrixscience.com using the Mascot algorithm.
  • Vero cells were harvested 1, 2, 3 and 7 days and 293T cells 36-48 hours after the addition of 20 ⁇ nilotinib or DMSO control.
  • Cell viability was determined by 7-AAD exclusion in a BD LSR cell analyzer (BD Biosciences) using FlowJo version 9.1 (Tri Star Inc., Ashland, OR). Cytopathic effect was estimated by visual examination of Vero E6 cell monolayers infected with Ebola virus several days after infection.

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Abstract

Cette invention concerne une méthode de traitement d'une infection virale par des Filoviridae, telle qu'une infection par le virus d'Ebola ou une infection par le virus de Marburg, comportant l'utilisation d'une quantité efficace d'un inhibiteur de tyrosine kinase c-Abl à un patient en ayant besoin. L'inhibiteur de tyrosine kinase c-Abl peut être un inhibiteur biologique qui diminue l'expression de la tyrosine kinase c-Abl, tel qu'un ARNsi spécifique de la tyrosine kinase c-Abl. Cependant, il est préférable que l'inhibiteur de tyrosine kinase c-Abl soit un antagoniste à petite molécule de tyrosine kinase c-Abl. Des antagonistes appropriés de tyrosine kinase c-Abl comprennent le dasatinib, l'imatinib et le nilotinib et les sels de qualité pharmaceutique de ceux-ci.
PCT/US2012/023953 2011-02-28 2012-02-06 Inhibiteurs de la tyrosine kinase c-abl utiles pour inhibition de réplication de filovirus WO2012118599A1 (fr)

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WO2015069711A1 (fr) * 2013-11-06 2015-05-14 Celgene Corporation Compositions et procédés de traitement de maladies virales à l'aide de modulateurs pde4
WO2015103026A3 (fr) * 2014-01-03 2015-08-27 The Regents Of The University Of Michigan Traitement des troubles neurologiques
WO2016054468A1 (fr) * 2014-10-03 2016-04-07 The Board Of Trustees Of The Leland Stanford Junior University Méthodes et compositions pour le traitement de virus à enveloppe
CN109700823A (zh) * 2014-08-18 2019-05-03 中国医学科学院药物研究所 泰利霉素在抗埃博拉病毒感染中的应用
WO2020237909A1 (fr) * 2019-05-28 2020-12-03 中国人民解放军军事科学院军事医学研究院 Cible applicable au traitement d'une maladie à virus ebola

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WO2015069711A1 (fr) * 2013-11-06 2015-05-14 Celgene Corporation Compositions et procédés de traitement de maladies virales à l'aide de modulateurs pde4
WO2015103026A3 (fr) * 2014-01-03 2015-08-27 The Regents Of The University Of Michigan Traitement des troubles neurologiques
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CN109700823A (zh) * 2014-08-18 2019-05-03 中国医学科学院药物研究所 泰利霉素在抗埃博拉病毒感染中的应用
WO2016054468A1 (fr) * 2014-10-03 2016-04-07 The Board Of Trustees Of The Leland Stanford Junior University Méthodes et compositions pour le traitement de virus à enveloppe
WO2020237909A1 (fr) * 2019-05-28 2020-12-03 中国人民解放军军事科学院军事医学研究院 Cible applicable au traitement d'une maladie à virus ebola

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