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WO2018039581A1 - Utilisation d'inhibiteurs de hdac6 pour la prévention et le traitement de la cystogenèse rénale, d'un carcinome à cellules rénales et de ciliopathies rénales - Google Patents

Utilisation d'inhibiteurs de hdac6 pour la prévention et le traitement de la cystogenèse rénale, d'un carcinome à cellules rénales et de ciliopathies rénales Download PDF

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WO2018039581A1
WO2018039581A1 PCT/US2017/048646 US2017048646W WO2018039581A1 WO 2018039581 A1 WO2018039581 A1 WO 2018039581A1 US 2017048646 W US2017048646 W US 2017048646W WO 2018039581 A1 WO2018039581 A1 WO 2018039581A1
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aurka
vhl
hdac6
renal
inhibitor
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Ruhee DERE
Cheryl L. WALKER
Eric JONASCH
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Texas A&M University System
Texas A&M University
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Texas A&M University System
Texas A&M University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys

Definitions

  • sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification.
  • the name of the text file containing the sequence listing is 64311_ST25.txt.
  • the text file is 1 KB; was created on August 24, 2017; and is being submitted via EFS-Web with the filing of the specification.
  • HDAC6 signaling inhibitors for the treatment of renal diseases and disorders, including ciliopathy, Von Hippel-Lindau (VHL) disease, renal cystogenesis, renal cell carcinoma, and Tuberous Sclerosis Complex (TSC), and the like.
  • the HDAC6 signaling inhibitor is a direct inhibitor of HDAC6.
  • the HDAC6 signaling inhibitor is ACY- 1215 (ricolinostat) or a pharmaceutically acceptable salt thereof.
  • Renal disease manifestations include conditions that are associated with abnormal cilia development or function. Often, such aberrant development or function involves defects in the primary cilium, a microtubule-based organelle that projects from the apical surface of cells. Such disorders are commonly referred to as ciliopathies. However, the mechanism for loss of primary cilia is incompletely understood. Accordingly, there remains a need for therapeutic interventions to prevent abnormal cilia development, or restore cilia function, to address various maladies of the kidney. The present disclosure addresses these and related needs.
  • provided herein are methods of treating a renal disease in a subject, comprising administering a therapeutically effective amount of an HDAC6 inhibitor to the subject.
  • kits for treating a renal condition in a subject associated with high HDAC6 signaling comprising administering a therapeutically effective amount of an HDAC6 inhibitor to the subject.
  • provided herein are methods of treating a renal condition in a subject associated with high AURKA-HDAC6 signaling, comprising administering a therapeutically effective amount of a combination of an HDAC6 inhibitor and an AURKA inhibitor to the subject.
  • the renal disease is, or is associated with, ciliopathy, Von Hippel-Lindau (VHL) disease, renal cystogenesis, renal cell carcinoma, and Tuberous Sclerosis Complex (TSC), and the like.
  • VHL Von Hippel-Lindau
  • TSC Tuberous Sclerosis Complex
  • the HDAC6 signaling inhibitor directly inhibits the activity of HDAC6.
  • the HDAC6 inhibitor is ACY-1215 or a pharmaceutically effective salt thereof.
  • the subject is a mammal, such as a primate, rodent, rabbit, cat, dog, horse, cow, and the like.
  • the subject is a human, mouse, or rat.
  • HDAC6 histone deacetylase 6
  • ring B is aryl or heteroaryl
  • Ri is an aryl or heteroaryl, each of which may be optionally substituted by OH, halo, or Ci-6-alkyl;
  • R is H or Ci -6- alkyl.
  • the compound of Formula I is
  • HDAC6 histone deacetylase 6
  • ring B is aryl or heteroaryl
  • Ri is an aryl or heteroaryl, each of which may be optionally substituted by OH, halo, or Ci -6- alkyl;
  • R is H or Ci-6-alkyl.
  • the compound of Formula l is or a pharmaceutically acceptable salt thereof.
  • kits for treating a renal condition in a subject associated with high aurora kinase A (AURKA)- histone deacetylase 6 (HDAC6) signaling comprising administering a therapeutically effective amount of a combination of an HDAC6-specific inhibitor and an AURKA inhibitor to the subject, wherein the HDAC6-specific inhibitor is a compound of Formula I
  • ring B is aryl or heteroaryl
  • Ri is an aryl or heteroaryl, each of which may be optionally substituted by OH, halo, or Ci-6-alkyl;
  • R is H or Ci-6-alkyl.
  • the renal disease is, or is associated with, ciliopathy, Von Hippel-Lindau (VHL) disease, renal cystogenesis, renal cell carcinoma, and Tuberous Sclerosis Complex (TSC).
  • VHL Von Hippel-Lindau
  • TSC Tuberous Sclerosis Complex
  • the HDAC6-specific inhibitor directly inhibits the activity of HDAC6.
  • the subject is a mammal.
  • the subject is a human, mouse, or rat.
  • FIGURES 1 A-1D illustrate that VHL modulates AURKA protein levels.
  • FIGURE 1A Lysates from 786-0 and isogenic 786-0 cells overexpressing VHL (VHL24) cultured at sub-confluent (cycling) or confluent and serum starved conditions probed as indicated.
  • FIGURE IB Densitometric quantitation of the average ratio of AURKA to tubulin expression from 786-0 (black bars) and 786-0 VHL 2 4 (gray bars) cells. *p ⁇ 0.01.
  • FIGURE 1C Lysates harvested from 786-0 and 786-0 VHL 24 cells cultured to confluence and serum starved for 24 hours prior to treatment with cycloheximide (CHX) for the indicated time points probed as shown.
  • CHX cycloheximide
  • FIGURE ID Densitometric quantitation showing an average ratio of AURKA to tubulin in 786-0 (black bars) and 786-0 VHL24 (gray bars) cells treated with CHX. * p ⁇ 0.01. # denotes significant differences exclusively in the 786-0 VHL 24 cell line at the each of the indicated CHX treatment time points compared to the Oh time point (p ⁇ 0.05). Error bars denote standard error of the mean (SEM).
  • FIGURES 2A-2E illustrate that VHL E3 ligase directly ubiquitinates AURKA.
  • FIGURE 2B Densitometric quantitation from 786-0 (black bars) and 786-0 VHL 2 4 (gray bars) cells showing an averaged ratio of AURKA to tubulin in the absence and presence of MG132. *p ⁇ 0.00001.
  • FIGURE 2C in vivo ubiquitination assay using lysates from 786-0 and 786-0 VHL 24 cell lines in the absence or presence of MG132.
  • Immunoprecipitated AURKA probed using an anti-ubiquitin (P4D1) antibody, and pull down efficiency measured with an anti-AURKA antibody.
  • Arrowhead indicates the expected molecular weight of AURKA.
  • Input shows AURKA, VHL and tubulin.
  • FIGURE 2D Graphical representation of densitometric quantitation showing an average ratio of ubiquitinated AURKA (probed using the P4D1 anti-ubiquitin antibody) to the pull down efficiency (using an anti-AURKA antibody). Black and gray bars represent the ratio in 786-0 and 786-0 VHL24 cells, respectively. *p ⁇ 0.01.
  • FIGURE 2E In vitro ubiquitination assay. All error bars denote standard error of the mean (SEM).
  • FIGURES 3A-3H illustrate that VHL multi-monoubiquitinates AURKA independent of prolyl hydroxylation.
  • FIGURE 3A In vivo ubiquitination assay showing ubiquitinated AURKA from 786-0 and isogenic 786-0 cells overexpressing VHL (VHL24) probed using anti-P4Dl
  • FIGURE 3B In vivo ubiquitination assay showing ubiquitinated AURKA from cells overexpressing EGFP-AURKA and HAUb with and without overexpressed VHL 2 4, treated with defroxamine (DFX). Input lysates immunoblotted with the indicated antibodies.
  • DFX defroxamine
  • FIGURE 3C Densitometric quantitation showing an average ratio of EGFP- AURKA to GAPDH without (black bars) and with (gray bars) overexpressed VHL 2 4 treated with DFX. *p ⁇ 0.05. All error bars denote standard error of the mean (SEM).
  • FIGURE 3D In vivo ubiquitination assay showing ubiquitinated AURKA from cells overexpressing EGFP-AURKA and HA-Ub with and without overexpressed VHL 2 4, treated with dimethyloxaloylglycine (DMOG). Input lysates immunoblotted with the indicated antibodies.
  • DMOG dimethyloxaloylglycine
  • FIGURE 3E Densitometric quantitation showing an average ratio of EGFP AURKA to tubulin without (black bars) and with (gray bars) overexpressed VHL24 treated with DMOG. *p ⁇ 0.05. All error bars denote standard error of the mean (SEM).
  • FIGURE 3F In vitro ubiquitination assay performed with either wild type or mutant recombinant ubiquitin.
  • FIGURE 3G In vivo ubiquitination assay showing ubiquitinated AURKA from cells overexpressing EGFP-AURKA and wild type or mutant ubiquitin in the absence and presence of VHL24.
  • FIGURE 3H Densitometric quantitation showing an average ratio of EGFP AURKA to GAPDH in cells without (black bars) and with (gray bars) overexpressed VHL 2 4 in the presence of wild type or mutant ubiquitin. *p ⁇ 0.01
  • FIGURES 4A-4E illustrate that VHL R167Q mutant fails to ubiquitinate and degrade AURKA.
  • FIGURE 4A Schematic of VHL 24 (long isoform of VHL, VHLL) showing the domain (amino acid 63-204) and -domain (amino acid 155-193), and the R167Q mutation.
  • FIGURE 4B Lysates from 786-0 (parental) and isogenic 786-0 cells stably re- expressing WT (VHL24), or R167Q VHL mutant probed with the indicated antibodies.
  • FIGURE 4C Densitometric quantitation showing an average ratio of AURKA expression to tubulin.
  • Parental -black bar WT-gray bar, R167Q-black bar with white dots. *p ⁇ 0.0001.
  • FIGURE 4D In vivo ubiquitination assay using lysates from 786-0 and isogenic 786-0 VHL 24 (WT), and R167Q cell lines treated with MG132. Ubiquitinated AURKA probed using anti-ubiquitin P4D1 antibody. Arrowhead indicates the expected molecular weight of ubiquitinated AURKA. Input lysates probed with AURKA, VHL and tubulin.
  • FIGURE 4E Graphical representation of densitometric quantitation showing an averaged ratio of ubiquitinated AURKA to the pull down efficiency.
  • Black bar Parental
  • FIGURES 5A-5E illustrate that VHL regulates AURKA protein half-life and abundance.
  • FIGURES 5A and 5B Representative images of hTERT RPE-1 cells transfected with Dendra2C-AURKA, and HA-Ub without (5A) or with (5B) overexpressed VHL (VHL24) prior to photoconversion (green and red channels), immediately after photoconversion (Oh), and 2h post conversion.
  • Scale bar 10 ⁇
  • FIGURE 5C Quantitation of the corrected mean red fluorescent intensity
  • Dendra2C-AURKA measured every 15min from cells expressing Dendra2C-AURKA and HA-Ub with (gray line), and without (black line) overexpressed VHL 2 4 (averaged from 8 independent biological and technical replicates). *p ⁇ 0.02 (compared to the Oh time point).
  • FIGURE 5D Lysates from hTERT RRE-1 cells expressing Dendra2C-AURKA and HA-Ub in the absence or presence of VHL 2 4 probed for the indicated antibodies.
  • FIGURE 5E Densitometric quantitation showing an average ratio of Dendra2C-
  • FIGURES 6A-6E illustrate that VHL promotes AURKA degradation as cells enter quiescence.
  • FIGURE 6A In vivo ubiquitination assay using hTERT RPE-1 cells overexpressing EGFP-AURKA and HA-Ub with and without overexpressed VHL in the presence of MG132, harvested following serum withdrawal at the indicated time points. Ubiquitinated AURKA immunoprecipitated using an anti-HA antibody and probed with an anti-AURKA antibody. Input lysates immunoblotted for the indicated antibodies.
  • FIGURE 6B Lysates from hTERT RPE-1 cells overexpressing EGFP-AURKA and HA-Ub with and without overexpressed VHL 2 4 in the absence of MG132 probed for the indicated antibodies.
  • FIGURE 6C Densitometric quantitation showing an average ratio of EGFP- AURKA to tubulin in cells without (black bars) and with (gray bars) overexpressed VHL 2 4 at the indicated time points. *p ⁇ 0.01.
  • FIGURES 6D and 6E Densitometric quantitation of EGFP-AURKA to tubulin expression averaged from 3 independent experiments at each of the indicated time points normalized to the Oh time point in the absence of MG132 treatment (D) and the presence of MG132 treatment (E). *p ⁇ 0.01 (compared to the Oh time point). All error bars denote standard error of the mean (SEM).
  • FIGURES 7A-7F illustrate that AURKA and HDAC6 inhibition rescues ciliogenesis in VHL-deficient cells.
  • FIGURE 7A Representative images from hTERT RPE-1 cells transiently transfected with siControl (siC) or siVHL, treated with vehicle (DMSO), alisertib (MLN8237) or ricolinostat (ACY-1215) at the time of serum withdrawal for 48h. Ciliation monitored by immunofluorescent staining using acetylated a-tubulin (cilia marker) and pericentrin (basal body marker). Nuclei counterstained using DAPI. Highlighted boxes show magnified cilia. Scale bar, 3 ⁇ .
  • FIGURES 7B and 7D Percentage of cells that fail to ciliate (unciliated) normalized to 1, from siC or siVHL transfected cells treated with (B) alisertib or (D) ricolinostat. * and # statistical significance.
  • FIGURES 7C and 7E Cilia length ( ⁇ ) from siC or siVHL transfected cells treated with (C) alisertib or (E) ricolinostat. At least 100 cells were counted from each replicate. * and # statistical significance. All error bars indicate standard error of the mean (SEM).
  • FIGURE 7F Model depicting a role for VHL's ubiquitin ligase function in conditions of normoxia targeting HIFla and hypoxia targeting AURKA.
  • FIGURES 8A-8C illustrate that VHL regulates AURKA protein levels.
  • FIGURE 8A Lysates from A-498 and isogenic VHL L (VHL 24 ) and VHL S
  • VHLi 9 stable cell lines cultured under confluent and serum starved (for 48h) conditions probed with the indicated antibodies.
  • FIGURE 8B Densitometric quantitation of the ratio of AURKA to GAPDH expression from A-498, A-498 VHL L and VHL S cells, averaged from 6 independent experiments. *p ⁇ 0.05. Error bars denote standard error of the mean (SEM).
  • FIGURE 8C Lysates from hTERT RPE-1 cells overexpressing varying doses of VHL 2 4 ⁇ g) as denoted, probed for the indicated antibodies.
  • FIGURE 9 illustrates that VHL interacts with AURKA hTERT RPE-1 cells expressing EGFP-AURKA and HA-VHL30 used in coimmunoprecipitation assays to show binding between HA-VHL30 and EGFP-AURKA. Pulldown efficiencies are indicated for each immunoprecipitation.
  • FIGURES 10A and 10B illustrate that VHL R167Q mutant fails to ubiquitinate and degrade AURKA.
  • FIGURE 10A In vivo ubiquitination assay using lysates from 786-0 and isogenic
  • FIGURE 10B Graphical representation of densitometric quantitation from the representative experiment shown in (10A) denoting a ratio of ubiquitinated AURKA (probed using the P4D1 anti -ubiquitin antibody) to the pull down efficiency (using an anti-AURKA antibody).
  • Black bar parental 786-0
  • gray bar WT
  • black bar with white dots R167Q.
  • FIGURES 11A and 11B illustrate that VHL regulates AURKA protein half-life and abundance.
  • FIGURE 11 A In vivo ubiquitination assay performed using hTERT RPE-1 cells overexpressing Dendra2C-AURKA and HA-Ub with and without overexpressed VHL 2 4 in the absence and presence of MG132. Ubiquitinated AURKA immunoprecipitated under denaturing conditions using an anti-HA antibody and probed with an anti-AURKA antibody. Input lysates immunoblotted for the indicated antibodies.
  • FIGURE 11B In vivo ubiquitination assay performed using hTERT RPE-1 cells overexpressing EGFP-AURKA and HA-Ub with and without overexpressed VHL 2 4 in the absence of MG132. Cells were grown to confluence and harvested following serum withdrawal at the indicated time points. Ubiquitinated AURKA immunoprecipitated using an anti-HA antibody and probed with an anti-AURKA antibody. Input lysates immunoblotted for the indicated antibodies.
  • FIGURES 12A-12C illustrate the rescue of ciliogenesis in VHL-deficient cells with alisertib and ricolinostat.
  • FIGURE 12A Graph showing fold change in VHL mRNA transcript levels.
  • FIGURES 12B and 12C Lysates from siC and siVHL transfected cells treated with vehicle (DMSO) and ricolinostat (ACY-1215) (in 12B) or vehicle (DMSO) and alisertib (MLN8237) (in 12C) probed for the indicated antibodies.
  • VHL von Hippel Lindua
  • RRC renal cell carcinoma
  • ciliopathies This disclosure is based on an investigation into the mechanistic roles of loss of von Hippel Lindua (VHL) tumor suppressor in the development of renal cell carcinoma (RCC) and ciliopathies.
  • VHL is an E3 ubiquitin ligase that targets its most well- understood substrate, HIFla, for proteasome mediated degradation under conditions of normal oxygen tension. Loss of VHL expression or activity results in aberrant gene expression and leads to angiogenesis and tumor formation.
  • anti-angiogenic therapy is most commonly used in the clinic to address VHL deficiencies, limitations have promoted investigations into alternate functions and targets of VHL.
  • VHL was reported to regulate microtubule stability and consequentially loss of VHL resulted in loss of the primary cilia, a microtubule based organelle that projects from the apical surface of cells. This has led to the inclusion of VHL-deficiency as a "ciliopathy,” a group of disorders that result in defects of the primary cilium. The mechanism for this loss of the primary cilium upon loss of VHL is incompletely understood.
  • VHL targets Aurora kinase A (AURKA) in a hypoxia-independent manner for ubiquitination and degradation.
  • AURKA Aurora kinase A
  • HDAC6 Histone Deacetylase 6
  • the HDAC6-specific inhibitor, ACY-1215 (ricolinostat) was used in a murine model of renal cystogenesis, which demonstrated that application of the HDAC6 inhibitor significantly prevented the mice from developing cysts in the kidneys. This data demonstrates the utility of direct inhibitors of the HDAC6 signaling axis for the treatment of renal ciliopathy disorders.
  • VHL multi-monoubiquitinates AURKA in quiescent cells and targets it for proteasome- mediated degradation independent of oxygen-dependent prolyl hydroxylase activity.
  • VHL-mediated ubiquitination of AURKA provides a mechanism by which VHL protects the stability of axoneme microtubules to regulate the primary cilium.
  • the inability of the most common pathogenic variant of VHL which retains an intrinsic ability to degrade HIFa, to ubiquitinate and degrade AURKA, suggests an equally important role for this hypoxia-independent function of VHL in tumorigenesis.
  • Hydroxylation of proline residues by prolyl hydroxylases in normoxia is critical for VHL to recognize several of its substrates including HIFa (6,7), Sprouty2 (Spyr2) (10) and RNA polymerase subunit Rpbl (8,9), although it is not yet clear if this is the case for all VHL substrates. Hydroxylated substrates are recognized, and poly- ubiquitinated by VHL which targets them for proteasome-mediated degradation. K48 (lysine 48) linkages are the canonical signal for proteasomal degradation (31), and VHL mediated K48-linkage was specifically demonstrated for the VHL substrate EGFR (13), analogous to that demonstrated for HIFa.
  • VHL hypoxia-independent VHL activity was thought to be distinct from its function as an ubiquitin ligase, which was believed to be oxygen-dependent.
  • the data demonstrate VHL's ability to recognize and ubiquitinate AURKA independent of the activity of prolyl hydroxylases, revealing VHL's E3 ligase activity that can function under hypoxic conditions.
  • VHL has an established role in modulating microtubule stability during mitosis, and in the ciliary axoneme (18,19), the mechanism by which VHL promotes microtubule stability was unclear.
  • AURKA as a target for VHL ubiquitination identifies a VHL-AURKA-HDAC6 axis, and provides a mechanism whereby increased AURKA in VHL-deficient cells destabilizes axonemal microtubules of the primary cilium.
  • RRC renal cell carcinoma
  • AURKA is polyubiquitinated by the APC complex and targeted for degradation during mitosis (41-43). While the majority of AURKA is thus eliminated well before midbody formation and cytokinesis, a small pool of AURKA persists in interphase cells. The role of this non-mitotic AURKA is only beginning to emerge with the phosphorylation of HDAC6 its first reported activity in quiescent cells (30). However, heretofore it was not known how AURKA levels are regulated outside of mitosis. The finding that VHL targets AURKA for degradation in quiescent cells now provides a regulatory mechanism for non-mitotic AURKA activity.
  • AURKA can phosphorylate VHL at serine 72 (S72), a priming phosphorylation for GSK3P phosphorylation, which regulates VHL's role in microtubule stability (21). It would now be interesting to determine if phosphorylation of VHL by AURKA modulates VHL's hypoxia-independent E3 ligase activity during ciliogenesis.
  • VHL-mutants have also been shown to have differential and/or compromised ability to stabilize microtubules with the identification of two VHL domains spanning residues 1-53 and 95-123 thought to be important for maintaining primary cilia (46).
  • HIF-driven progression of tumors arising from defects in metabolism and increased tumor angiogenesis in patients with loss of VHL is clearly established, virtually nothing is known about the earliest events that result in tumorigenesis and the renal cystogenesis common in VHL patients.
  • the data establishing VHL's ability to regulate AURKA and modulate microtubule stability at the cilium could shed light on some of the early events in tumorigenesis.
  • AURKA as a substrate of VHL presents new opportunities for intervention, not only in RCC, but also in other cancers such as colorectal carcinoma, where genomic data from patients reveals the existence of VHL mutations (47,48), elevated AURKA (49) and ciliary defects (50).
  • VHL mutations 47,48
  • elevated AURKA 419
  • ciliary defects 50.
  • the demonstration that primary cilia can be rescued with AURKA and HDAC6 inhibitors provides proof-of- concept that targeting the VHL-AURKA-HDAC6 axis may have therapeutic efficacy.
  • Future studies using in vivo cancer models, as well as ciliopathy models such as polycystic kidney disease (PKD), in which AURKA signaling is dysregulated, now become candidates for therapeutic targeting of the VHL-AURKA-HDAC6 axis.
  • PPD polycystic kidney disease
  • the disclosure provides a method of treating defective ciliogenesis in a cell, wherein the cell is treated with a signaling inhibitor of HDAC6 or an inhibitor of HDAC6 combined with an inhibitor of AURKA, wherein the treated cell exhibits at least one cilium or a more-developed (e.g., longer) cilium than in a suitable control cell not treated with the signaling inhibitor.
  • the signaling inhibitor can be an inhibitor of HDAC6, such as one selected from the group consisting of ricolinostat, Tubacin, Tubastatin A, ST-3-06, ST-2-92, Nexturastat A, and Nexturastat B, Vorinostat, LBH589, ITF2357, PXD-101, Depsipeptide, MS-275, and MGCD0103.
  • the signaling inhibitor can be an AURKA inhibitor, such as alisertib.
  • the cell is treated with both an inhibitor of HDAC6 and an AURKA inhibitor.
  • the cell can be a renal cell, such as a renal epithelial cell.
  • the cell can be human.
  • the disclosure provides a method of treating a renal disease in a subject.
  • the method comprises administering a therapeutically effective amount of an HDAC6 specific inhibitor to the subject.
  • the disclosure provides a method of treating a renal condition associated with high HDAC6 specific in a subject, comprising administering a therapeutically effective amount of an HDAC6 signaling inhibitor to the subject.
  • the renal disease can be a ciliopathy, or ciliopathy associated condition.
  • a ciliopathy is a disease of the cilia or cilia anchoring structures, resulting in dysfunction thereof.
  • the condition is a dysfunction of cilia that occurs at least within the kidney, although the ciliopathy can also affect other tissues and organs as well.
  • Illustrative ciliopathies applicable in the present methods are described in more detail in Waters, A.M., and Beales, P.L., "Ciliopathies: an expanding disease spectrum," Pedriatr. Nephrol. 2(5: 1039-1056 (2011), incorporated herein by reference in its entirety.
  • the renal disease is, or is associated with, Von Hippel-Lindau (VHL) disease, renal cystogenesis, renal cell carcinoma, and Tuberous Sclerosis Complex (TSC), and the like.
  • VHL Von Hippel-Lindau
  • TSC Tuberous Sclerosis Complex
  • the renal disease is, or is associated with, renal cystogenesis.
  • the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
  • the terms “comprise,” “have,” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes,” and “including,” are also open-ended. For example, any method that "comprises,” “has,” or “includes” one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps.
  • composition refers to a mixture of at least one compound useful within the invention with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition facilitates administration of the compound to a patient or subject. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.
  • the term "pharmaceutically acceptable salt” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form.
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • the pharmaceutically acceptable salts of the present invention include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • the pharmaceutically acceptable salts of the present invention may be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods.
  • such salts may be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.
  • alkyl by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain hydrocarbon having the number of carbon atoms designated (i.e., Ci-C 6 -alkyl means an alkyl having one to six carbon atoms) and includes straight and branched chains. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, and hexyl. Other examples of Ci-C 6 -alkyl include ethyl, methyl, isopropyl, isobutyl, n-pentyl, and n-hexyl.
  • alkoxy refers to an -O-alkyl moiety.
  • alkylene refers to divalent aliphatic hydrocarbyl groups, for example, having from 1 to 4 carbon atoms that are either straight-chained or branched. This term includes, by way of example, methylene (-CH 2 -), ethylene (- CH 2 CH 2 -), n-propylene (-CH 2 CH 2 CH 2 -), iso-propylene (-CH 2 CH(CH 3 )-), and the like.
  • halo or halogen alone or as part of another substituent means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably, fluorine, chlorine, or bromine, more preferably, fluorine or chlorine.
  • cycloalkyl means a non-aromatic carbocyclic system that is partially or fully saturated having 1, 2 or 3 rings wherein such rings may be fused.
  • fused means that a second ring is present (i.e., attached or formed) by having two adjacent atoms in common (i.e., shared) with the first ring.
  • Cycloalkyl also includes bicyclic structures that may be bridged or spirocyclic in nature with each individual ring within the bicycle varying from 3-8 atoms.
  • cycloalkyl includes, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[3.1.0]hexyl, spiro[3.3]heptanyl, and bicyclo[l . l . l]pentyl.
  • cycloalkylene means a divalent cycloalkyl system, wherein cycloalkyl is defined above.
  • heterocycloalkyl means a non-aromatic carbocyclic system containing 1, 2, 3 or 4 heteroatoms selected independently from N, O, and S and having 1, 2 or 3 rings wherein such rings may be fused, wherein fused is defined above.
  • Heterocycloalkyl also includes bicyclic structures that may be bridged or spirocyclic in nature with each individual ring within the bicycle varying from 3-8 atoms, and containing 0, 1, or 2 N, O, or S atoms.
  • heterocycloalkyl includes cyclic esters (i.e., lactones) and cyclic amides (i.e., lactams) and also specifically includes, but is not limited to, epoxidyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl (i.e., oxanyl), pyranyl, dioxanyl, aziridinyl, azetidinyl, pyrrolidinyl, 2,5-dihydro-lH-pyrrolyl, oxazolidinyl, thiazolidinyl, piperidinyl, morpholinyl, piperazinyl, pyrrolidyl, thiomorpholinyl, 1,3-oxazinanyl, 1,3-thiazinanyl, 2-azabicyclo[2.1.1]hexanyl, 5- azabicyclo[2.1.1]hexanyl, 6-azabic
  • heterocycloalkylene means a divalent heterocycloalkyl system, wherein heterocycloalkyl is defined above.
  • aromatic refers to a carbocycle or heterocycle with one or more polyunsaturated rings and having aromatic character, i.e., having (4n + 2) delocalized ⁇ (pi) electrons, where n is an integer.
  • aryl means an aromatic carbocyclic system containing 1, 2 or 3 rings, wherein such rings may be fused, wherein fused is defined above. If the rings are fused, one of the rings must be fully unsaturated and the fused ring(s) may be fully saturated, partially unsaturated or fully unsaturated.
  • aryl includes, but is not limited to, phenyl, naphthyl, indanyl, and 1,2,3,4-tetrahydronaphthalenyl.
  • heteroaryl means an aromatic carbocyclic system containing 1, 2, 3, or 4 heteroatoms selected independently from N, O, and S and having 1, 2, or 3 rings wherein such rings may be fused, wherein fused is defined above.
  • heteroaryl includes, but is not limited to, furanyl, thiophenyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, imidazo[l,2-a]pyridinyl, pyrazolo[l,5-a]pyridinyl, 5,6,7,8-tetrahydroisoquinolinyl, 5,6,7,8-tetrahydroquinolinyl, 6,7-dihydro-5H-cyclopenta[b]pyridinyl, 6,7-dihydro-5H-cyclopenta[c]pyridinyl, 1,4,5,6- tetrahydrocyclopenta
  • aryl, heteroaryl, cycloalkyl, or heterocycloalkyl moiety may be bonded or otherwise attached to a designated moiety through differing ring atoms ⁇ i.e., shown or described without denotation of a specific point of attachment), then all possible points are intended, whether through a carbon atom or, for example, a trivalent nitrogen atom.
  • pyridinyl means 2-, 3- or 4-pyridinyl
  • thiophenyl means 2- or 3 -thiophenyl, and so forth.
  • substituted means that an atom or group of atoms has replaced hydrogen as the substituent attached to another group.
  • combination refers to two or more therapeutic agents to treat a therapeutic condition or disorder described in the present disclosure. Such combination of therapeutic agents may be in the form of a single pill, capsule, or intravenous solution. However, the term “combination” also encompasses the situation when the two or more therapeutic agents are in separate pills, capsules, or intravenous solutions.
  • combination therapy refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in the present disclosure. Such administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients or in multiple, or in separate containers (e.g., capsules) for each active ingredient. In addition, such administration also encompasses use of each type of therapeutic agent in a sequential manner, either at approximately the same time or at different times. In either case, the treatment regimen will provide beneficial effects of the drug combination in treating the conditions or disorders described herein.
  • HDAC histone deacetylases
  • HDAC 1 HDACs which include HDAC 1, HDAC2, HDAC 3, and HDAC 8 are related to the yeast RPD3 gene.
  • Class II HDACs which include HDAC4, HDAC 5, HDAC6, HDAC7, HDAC9, and HDAC 10, are related to the yeast Hdal gene.
  • Class III HDACs which are also known as the sirtuins are related to the Sir2 gene and include SIRT1-7.
  • Class IV HDACs which contains only HDAC1 1, has features of both Class I and II HDACs.
  • HDAC refers to any one or more of the 18 known histone deacetylases, unless otherwise specified.
  • HDAC6 specific means that the compound binds to HDAC6 to a substantially greater extent, such as 5X, 10X, 15X, 20X greater or more, than to any other type of HDAC enzyme, such as HDACl or HDAC2. That is, the compound is selective for HDAC6 over any other type of HDAC enzyme.
  • a compound that binds to HDAC 6 with an IC 50 of 10 nM and to HDACl with an IC 50 of 50 nM is HDAC 6 specific.
  • a compound that binds to HDAC6 with an IC 50 of 50 nM and to HDAC1 with an IC 50 of 60 nM is not HDAC6 specific
  • inhibitor is synonymous with the term antagonist.
  • VHL The hypoxia-regulated tumor suppressor von Hippel-Lindau
  • PHD oxygen-dependent prolyl hydroxylase
  • HIFa hypoxia-inducible factor a
  • VHL has an equally important function distinct from its hypoxia-regulated activity.
  • Aurora kinase A AURKA
  • AURKA Aurora kinase A
  • VHL mono-, rather than poly-ubiquitinates AURKA, in a PHD-independent reaction targeting AURKA for degradation in quiescent cells, where degradation of AURKA is required to maintain (via regulation of HDAC6) the primary cilium.
  • VHL Tumor-associated variants of VHL differentiate between these two functions, as a pathogenic VHL mutant that retains intrinsic ability to ubiquitinate HIFa is unable to ubiquitinate AURKA. Together, these data identify VHL as an E3 ligase with important cellular functions under both normoxic and hypoxic conditions.
  • VHL Von Hippel Lindau
  • ccRCC clear cell renal cell carcinoma
  • the PHD-family of prolyl hydroxylases requires oxygen to hydroxylate their targets (11).
  • the best-known VHL substrates are the hypoxia inducible factors 1 and 2 a (HIFla and HIF2a), which are hydroxylated at proline residues by PHD proteins under normoxic conditions.
  • hypoxia inducible factors 1 and 2 a HIFla and HIF2a
  • PHD-mediated proline hydroxylation target HIFla and HIF2a for VHL-mediated ubiquitination and proteasome-mediated degradation (6), inhibiting transcription of HIFa targets that increase glucose uptake and angiogenesis (12).
  • hypoxic conditions PHD-mediated hydroxylation and VHL-mediated ubiquitination are inhibited, resulting in HIFa stabilization.
  • VHL substrates include epidermal growth factor receptor (EGFR) (13), atypical protein kinase C (aPKC) (14), Sprouty2 (SPRY2) (10), ⁇ -adrenergic receptor II (ADRB2) (15), myb-binding protein 160 (MYBBP1A) (16), and RNA polymerase subunits Rpbl (8,9) and Rpb7 (17). More recently the differential and often compromised capability of pathogenic VHL mutants to stabilize microtubules has highlighted an additional function thought to contribute to VHL's function as a tumor suppressor (18, 19). However, the exact mechanism(s) by which VHL regulates microtubules is incompletely understood.
  • EGFR epidermal growth factor receptor
  • aPKC atypical protein kinase C
  • SPRY2 Sprouty2
  • ADRB2 ⁇ -adrenergic receptor II
  • MYBBP1A myb-binding protein 160
  • Rpbl RNA polymerase subunits
  • VHL has an equally important function in cells that is hypoxia-independent. VHL directly ubiquitinates AURKA independent of oxygen-dependent prolyl hydroxylase activity to regulate formation of the primary cilium in quiescent cells. Furthermore, in contrast to other known targets, VHL multi-monoubiquitinates AURKA to target this kinase for proteasome-mediated degradation. These data establish a new tumor suppressor activity for the VHL E3 ligase distinct from polyubiquitination of its oxygen- dependent targets such as HIFa, and points to AURKA as a potential target for therapy in VHL-deficient tumors.
  • the HDAC6 signaling inhibitor (also referred to herein as “inhibitor,” or “inhibitors” when addressed in a nonlimiting plural sense) directly inhibits the activity of HDAC6.
  • the term “directly inhibits” indicates that the inhibitor inhibits through physical contact with the HDAC6 protein to diminish the activity of the protein.
  • the HDAC6 signaling inhibitor inhibits expression of the genes encoding the HDAC6 protein, or otherwise inhibits the translation of the proteins from the gene transcript, to result in lower levels of functional protein.
  • the compounds, combinations, and methods of the invention comprise a histone deacetylase (HDAC) inhibitor.
  • HDAC histone deacetylase
  • the HDAC inhibitor may be any HDAC inhibitor.
  • the HDAC inhibitor may be selective or non-selective to a particular type of histone deacetylase enzyme.
  • the HDAC inhibitor is a selective HDAC inhibitor. More preferably, the HDAC inhibitor is an HDAC6 specific inhibitor.
  • the HDAC6 specific inhibitor is a compound of Formula I:
  • ring B is aryl or heteroaryl
  • Ri is an aryl or heteroaryl, each of which may be optionally substituted by OH, halo, or Ci-6-alkyl;
  • R is H or Ci-6-alkyl.
  • ring B is phenyl and Ri is phenyl, wherein phenyl may be optionally substituted by OH, halo, or Ci-C 6 -alkyl.
  • Representative compounds of Formula I include, but are not limited to:
  • the HDAC6 signaling inhibitor is a direct inhibitor of HDAC6.
  • HDAC6 is a 131 kDa protein considered to be a key regulator of cytoskeleton dynamics and cell-cell interactions, among other functions (Hubbert et al., Nature 417:455-458 (2002); Valenzuela-Fernandez, et al., Trends in Cell Biology 18:291-297 (2008), each incorporated herein by reference in its entirety).
  • the inhibitor selectively inhibits HDAC6, i.e., resulting in reduced or minimized off target modifications. This can be important for minimizing unintended side effects to the administration of the inhibitor.
  • the HDAC6 inhibitor is ACY-1215, also referred to as ricolinostat, which is represented by the following chemical structure:
  • HDAC6 inhibitors include ACY-241, ACY-738, ACY-775, ACY-1083, Tubacin, Tubastatin A, ST-3-06, ST-2-92, Nexturastat A, Nexturastat B, Vorinostat, LBH589, ITF2357, PXD-101, Depsipeptide, and CAY10603. Additional inhibitors of HDAC6 are known and applicable in the described methods. See, e.g., Sabrina Dallavalle, Claudio Pisano, and Franco Zunino, "Development and therapeutic impact of HDAC6-selective inhibitors," Biochemical Pharmacology, 84(6), 2012, pages 756-76, incorporated herein by reference in its entirety. The inhibitor can be administered in any pharmaceutically effective and appropriate salt.
  • the HDAC6 inhibitor can be applied in combination with an inhibitor of AURKA.
  • the HDAC6 specific inhibitor is administered in a therapeutically effective amount or dosage.
  • a “therapeutically effective amount” is an amount of an HDAC6 specific inhibitor (a compound of Formula I) that, when administered to a patient by itself, effectively treats a renal disease.
  • the amount of the compound that corresponds to a therapeutically effective amount is strongly dependent on the type of renal disease, the age of the patient being treated, and other facts. In general, therapeutically effective amounts of these compounds are well-known in the art, such as provided in the supporting references cited above.
  • the pharmaceutical compositions and methods include an HDAC6 specific inhibitor of Formula I.
  • the pharmaceutical compositions and methods include ACY-1215.
  • the pharmaceutical combinations and methods include an HDAC6 specific inhibitor (ACY-1215) and an AURKA inhibitor.
  • the pharmaceutical composition of compounds can inhibit cancer growth, inhibit cystogenesis, or even achieve treatment of von-Hippel Lindau (VHL) disease.
  • VHL von-Hippel Lindau
  • the amount of an HDAC6 specific inhibitor should result in the effective treatment of renal disease, the amount is preferably not excessively toxic to the patient (i.e., the amounts are preferably within toxicity limits as established by medical guidelines). In some embodiments, either to prevent excessive toxicity and/or provide a more efficacious treatment of renal disease, a limitation on the total administered dosage is provided. Typically, the amounts considered herein are per day; however, half-day and two-day or three-day cycles also are considered herein.
  • a daily dosage such as any of the exemplary dosages described above, is administered once, twice, three times, or four times a day for three, four, five, six, seven, eight, nine, or ten days.
  • a shorter treatment time e.g., up to five days
  • a longer treatment time e.g., ten or more days, or weeks, or months, or longer
  • the HDAC6 specific inhibitor can be dosed every week, every 21 days, every 28 days, every two months, every 16 weeks, or every 24 weeks.
  • a once- or twice-daily dosage is administered every other day.
  • the HDAC6 specific inhibitors, or their pharmaceutically acceptable salts or solvate forms, in pure form or in an appropriate pharmaceutical composition, can be administered via any of the accepted modes of administration or agents known in the art.
  • the compounds can be administered, for example, orally, nasally, parenterally (intravenous, intramuscular, or subcutaneous), topically, transdermally, intravaginally, intravesically, intracistemally, or rectally.
  • the dosage form can be, for example, a solid, semi-solid, lyophilized powder, or liquid dosage forms, such as for example, tablets, pills, soft elastic or hard gelatin capsules, powders, solutions, suspensions, suppositories, aerosols, or the like, preferably in unit dosage forms suitable for simple administration of precise dosages.
  • a particular route of administration is oral, particularly one in which a convenient daily dosage regimen can be adjusted according to the degree of severity of the disease to be treated.
  • pharmaceutical combination includes a combination of two drugs in either a single dosage form or separate dosage forms, i.e., the pharmaceutically acceptable carriers and excipients described throughout the application can be combined with an HDAC6 specific inhibitor and an AURKA inhibitor in a single unit dose, as well as individually combined with an HDAC6 specific inhibitor and an AURKA inhibitor when these compounds are administered separately.
  • Auxiliary and adjuvant agents may include, for example, preserving, wetting, suspending, sweetening, flavoring, perfuming, emulsifying, and dispensing agents. Prevention of the action of microorganisms is generally provided by various antibacterial and antifungal agents. Isotonic agents may also be included. Prolonged absorption of an injectable pharmaceutical form can be brought about by the use of agents delaying absorption.
  • the auxiliary agents also can include wetting agents, emulsifying agents, pH buffering agents, and antioxidants.
  • Solid dosage forms can be prepared with coatings and shells, such as enteric coatings and others well-known in the art. They can contain pacifying agents and can be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedded compositions that can be used are polymeric substances and waxes. The active compounds also can be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. Such dosage forms are prepared, for example, by dissolving, dispersing, etc., the HDAC6 specific inhibitors described herein, or a pharmaceutically acceptable salt thereof, and optional pharmaceutical adjuvants in a carrier to thereby form a solution or suspension.
  • the pharmaceutically acceptable compositions will contain about 1% to about 99% by weight of the compounds described herein, or a pharmaceutically acceptable salt thereof, and 99% to 1%) by weight of a pharmaceutically acceptable excipient.
  • the composition will be between about 5% and about 75% by weight of a compound described herein, or a pharmaceutically acceptable salt thereof, with the rest being suitable pharmaceutical excipients.
  • the disclosed compounds can be formulated in a physiologically- or pharmaceutically-acceptable form and administered by any suitable route known in the art including, for example, oral, nasal, rectal, topical, and parenteral routes of administration.
  • parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, and intrasternal administration, such as by injection.
  • Administration of the disclosed compounds or compositions can be a single administration, or at continuous or distinct intervals as can be readily determined by a person skilled in the art.
  • Effective amounts of the inhibitors can be readily determined by persons of ordinary skill in the art, with consideration given to the particular disorder, the formulation, administration, and the administration schedule (if multiple administrations). For example, in the experiments below, mice were administered 50 mg/kg once daily for a period of time. Amounts can be readily adjusted. Exemplary, non-limiting doses include about 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or more mg/kg (such as up to 500 mg/kg), or any value therein. Illustrative treatment regimens for humans can include about 10 mg to about 1000 mg per day in single or multiple doses. Doses vary depending one administration route, and co-administration of other therapeutic agents.
  • typical dosing of ACY-1215 has ranged from about 40 mg to 160 mg once or twice daily.
  • Typical dosing of ACY-1215 in clinical trials has been at about 180 mg up to about 480 mg. Accordingly, these are non-limiting, illustrative of potential doses in adult humans.
  • ACY-1215 is in an amount from 600 mg to 3000 mg (e.g., about 600, about 800, about 1000, about 1200, about 1400, about 1600, about 1800, about 2000 mg). In a further embodiment of the methods, ACY-1215 is in an amount from 600 mg to 2000 mg. In another embodiment of the methods, ACY-1215 is in an amount from 5 mg to 600 mg (e.g., about 5, about 25, about 50, about 100, about 200, about 300, about 400, about 500, about 600 mg). In yet another embodiment of the methods ACY-1215 is 10 mg to 200 mg.
  • the inhibitors disclosed herein, and compositions comprising them can also be administered utilizing liposome technology, slow-release capsules, implantable pumps, and biodegradable containers. These delivery methods can, advantageously, provide a uniform dosage over an extended period of time.
  • the inhibitors can be formulated for single administrations, with such administrations being administered one or more times in a course of treatment.
  • the compounds can also be administered in their salt derivative forms or crystalline forms.
  • the inhibitors disclosed herein for performance of the methods can be formulated according to known methods for preparing pharmaceutically acceptable compositions. Formulations are described in detail in a number of sources which are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Science by E.W. Martin (1995), incorporated herein by reference in its entirety, describes formulations that can be used in connection with the disclosed methods. In general, the compounds disclosed herein can be formulated such that an effective amount of the compound is combined with a suitable carrier in order to facilitate effective administration of the compound.
  • the compositions used can also be in a variety of forms.
  • compositions include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspension, suppositories, injectable and infusible solutions, and sprays.
  • the preferred form depends on the intended mode of administration and therapeutic application.
  • the compositions also preferably include conventional pharmaceutically-acceptable carriers and diluents which are known to those skilled in the art. Extemporaneous injection solutions and suspensions can be prepared from sterile powder, granules, tablets, etc. It should be understood that in addition to the ingredients particularly mentioned above, the compositions disclosed herein can include other agents conventional in the art with regard to the type of formulation in question.
  • the term "subject” refers to any individual who is the target of administration or treatment.
  • the subject can be a vertebrate, for example, a mammal.
  • the subject can be a human or veterinary patient.
  • the "subject” can include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds.
  • “Subject” can also include a mammal, such as a primate or a human. In one embodiment, the subject is a human.
  • prevent or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.
  • treat or other forms of the word, such as “treated” or “treatment,” is meant to administer a composition or to perform a method in order to reduce, prevent, inhibit, or eliminate a particular characteristic or event (e.g., tumor growth or survival or disease state).
  • control is used synonymously with the term “treat.”
  • treatment includes the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • provided herein are methods of treating a renal disease in a subject in need thereof comprising administering a therapeutically effective amount of a histone deacetylase 6 (HDAC6)-specific inhibitor, or a pharmaceutically acceptable salt thereof, to the subject.
  • HDAC6 histone deacetylase 6
  • methods of treating a renal disease in a subject in need thereof comprising administering a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof, to the subject.
  • methods of treating a renal disease in a subject in need thereof comprising administering a therapeutically effective amount of ACY-1215, or a pharmaceutically acceptable salt thereof, to the subject.
  • provided herein are methods of treating renal cystogenesis in a subject in need thereof comprising administering a therapeutically effective amount of a histone deacetylase 6 (HDAC6)-specific inhibitor, or a pharmaceutically acceptable salt thereof, to the subject.
  • HDAC6 histone deacetylase 6
  • methods of treating renal cystogenesis in a subject in need thereof comprising administering a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof, to the subject.
  • methods of treating renal cystogenesis in a subject in need thereof comprising administering a therapeutically effective amount of ACY-1215, or a pharmaceutically acceptable salt thereof, to the subject.
  • provided herein are methods of treating a renal disease in a subject associated with high histone deacetylase 6 (HDAC6) signaling, comprising administering a therapeutically effective amount of a histone deacetylase 6 (HDAC6)-specific inhibitor, or a pharmaceutically acceptable salt thereof, to the subject.
  • methods of treating a renal disease in a subject associated with high histone deacetylase 6 (HDAC6) signaling comprising administering a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof, to the subject.
  • HDAC6 histone deacetylase 6
  • provided herein are methods of treating renal cystogenesis in a subject associated with high histone deacetylase 6 (HDAC6) signaling, comprising administering a therapeutically effective amount of a histone deacetylase 6 (HDAC6)-specific inhibitor, or a pharmaceutically acceptable salt thereof, to the subject.
  • methods of treating renal cystogenesis in a subject associated with high histone deacetylase 6 (HDAC6) signaling comprising administering a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof, to the subject.
  • HDAC6 histone deacetylase 6
  • provided herein are methods of treating a renal disease in a subject associated with high aurora kinase A (AURKA)- histone deacetylase 6 (HDAC6) signaling, comprising administering a therapeutically effective amount of a histone deacetylase 6 (HDAC6)-specific inhibitor, or a pharmaceutically acceptable salt thereof, to the subject.
  • methods of treating a renal disease in a subject associated with high aurora kinase A (AURKA)- histone deacetylase 6 (HDAC6) signaling comprising administering a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof, to the subject.
  • kits for treating a renal disease in a subject associated with high aurora kinase A (AURKA)- histone deacetylase 6 (HDAC6) signaling comprising administering a therapeutically effective amount of ACY-1215, or a pharmaceutically acceptable salt thereof, to the subject.
  • AURKA high aurora kinase A
  • HDAC6 histone deacetylase 6
  • provided herein are methods of treating renal cystogenesis in a subject associated with high aurora kinase A (AURKA)- histone deacetylase 6 (HDAC6) signaling, comprising administering a therapeutically effective amount of a histone deacetylase 6 (HDAC6)-specific inhibitor, or a pharmaceutically acceptable salt thereof, to the subject.
  • methods of treating renal cystogenesis in a subject associated with high aurora kinase A (AURKA)- histone deacetylase 6 (HDAC6) signaling comprising administering a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof, to the subject.
  • kits for treating renal cystogenesis in a subject associated with high aurora kinase A (AURKA)- histone deacetylase 6 (HDAC6) signaling comprising administering a therapeutically effective amount of ACY-1215, or a pharmaceutically acceptable salt thereof, to the subject.
  • AURKA high aurora kinase A
  • HDAC6 histone deacetylase 6
  • VUL has a hypoxia-independent E3 ligase activity to ubiquitinate and regulate AURKA and, consequently, HDAC6 activity, to affect ciliogenesis.
  • the synthesis of the compounds of Formula I (e.g., ACY-1215) is provided in PCT/US2011/021982, which is incorporated herein by reference in its entirety.
  • Human recombinant VHL complex (cat # 23-044), UBE1 (cat # 23-021), UbcH4 (cat # 23-025), AURKA (cat # 14-511), and non-radioactive ATP (cat # 20-306) were purchased from EMD Millipore.
  • Human recombinant wild type ubiquitin (cat # U-100H), ubiquitin mutant K48 (cat # UM-K480), ubiquitin mutant K48R (cat # UM-K48R), and ubiquitin mutant K0 (cat # UM-NOK) were purchased from Boston Biochem.
  • the in vitro reaction was set up by incubating 10 nM UBE1, 1 ⁇ UbcH4, 1 ⁇ g of the VHL complex, 10 ⁇ ATP, and 500 ⁇ ubiquitin (or ubiquitin mutants) with either 50 nM or 100 nM AURKA in a buffer containing 25 mM MOPS [pH7.5], 0.01% Tween 20 and 5 mM MgC12.
  • the reaction was initiated by the addition of ubiquitin and following a 30 min incubation at room temperature, the reaction was terminated using a stop solution (25 mM MOPS [pH7.5] containing 125 mM EDTA, 150 mM NaCl, and 0.05% Tween 20) and loading dye.
  • Cells transfected with EGFP-AURKA/Dendra2C-AURKA, HA-Ub and VHL24 were cultured to 100% confluence and treated with 10 ⁇ MG132 16 hours prior to harvesting.
  • cell lysates were collected in cold modified IX RIPA buffer (10 mM Tris [pH 7.5], 150 mM NaCl, 5 mM EDTA, 1% P40, 1%) Sodium Deoxycholate, 0.025% SDS) containing IX complete protease inhibitor (Roche), 1 mM Na3V04, and 1 ⁇ N-ethylmaleimide (NEM, Sigma-Aldrich).
  • the lysates were immunoprecipitated overnight with anti-HA (1 :50) and anti-AURKA (1 :50) antibodies from Cell Signaling.
  • the immune complexes were incubated with prewashed magnetic beads (Pierce) for an hour, and washed 3 times with modified IX RIPA buffer prior to resuspension in loading dye.
  • Dendra2c-AURKA was visualized using the 488-laser, which was set to 5% power to prevent and minimize spontaneous conversion of green Dendra2C- AURKA to the red fluorescent state.
  • Single cells expressing high Dendra2C-AURKA levels were selected for photoconversion.
  • Photoconversion was achieved on a selected region of interest (ROI) using the 405 laser at 10% power, for 1 sec. Further details are provided in Supplementary Materials and Methods.
  • Human 786-0, VHL-deficient, renal cell carcinoma (RCC) cell line was maintained in RPMI-1640 media (Life Technologies) supplemented with 10% fetal bovine serum-FBS (Sigma-Aldrich).
  • Immortalized retinal epithelial (hTERT RPE-1) cells (a kind gift from Dr. Gregory Pazour, University of Massachusetts Medical School) were maintained in DMEM/F-12 media (Life Technologies), supplemented with 10% FBS.
  • Stable cells lines re-expressing VHL were generated by clonal selection of 786-0 (VHL- deficient) cells transfected with a wild type (WT) VHL24 construct (G57T3, previously described (1)), and maintained under antibiotic selection using 800 ⁇ g/ml G418 (Life Technologies).
  • VHL mutant and VHL24 (WT) expressing stables were generated in 786-0 as described previously (2).
  • A-498 (VHL-deficient) stables re-expressing VHL24 (pCR3-VHL24) or VHL19 (pCR3-VHL19) constructs (3) constructs were a gift from Dr. Alan Schoenfeld, Adelphi University, NY) were generated by transfection and selection using 800 ⁇ g/ml G418 (Life Technologies). All human cell lines were STR fingerprinted and validated using the Characterized Cell Line Core Facility (U.T. M.D. Anderson Cancer Center).
  • EGFP tagged wild type AURKA (EGFP-AURKA) (HsCD00036078) was purchased from DNASU Plasmid Repository (Arizona State University).
  • the pDendra2- C vector was purchased from Clontech.
  • Dendra2C-AURKA was generated by PCR amplification of AURKA cDNA from the EGFP-AURKA construct using primers (see below) designed to have BamHl and EcoRl sticky ends to enable cloning into the multiple cloning site (MCS) of the pDendra2-C vector.
  • PCR amplification (95°C - 2 min, 35 cycles of 95°C - 1 min, 44°C - 30 sec, 72°C - 2 min and, 72°C - 5 min) was performed using Pfu polymerase (Promega). Following PCR amplification the PCR product was gel purified using a QIA quick gel extraction kit (Qiagen). The purified PCR product was cloned into the BamHl/EcoRl digested pDendra2-C vector. The Dendra2C-AURKA construct was sequence verified using Genewiz Inc. In addition, localization of overexpressed Dendra2C-AURKA was confirmed at the centrosomes.
  • HA tagged VHL30 construct (HA-VHL30) was a kind gift from Dr. Wilhelm Krek, (ETH, Institute of Cell Biology, Zurich, Switzerland).
  • the pCR3 VHL24 construct was a kind gift from Dr. Alan Schoenfeld (Adelphi University, Garden City, NY).
  • pRK5-HA-Ubiquitin-WT (HA-Ub) (Addgene plasmid # 17608), pRK5- HAUbiquitin- K48 (HA-K48) (Addgene plasmid # 17605), pRK5-HA-Ubiquitin-K48R (HAK48R) (Addgene plasmid # 17604), pRK5-HA-Ubiquitin-KO (HA-K0) (Addgene plasmid # 17603) were gifts from Dr. Ted Dawson and purchased from Addgene.
  • constructs were transfected into cells using Lipofectamine2000 (Invitrogen) according to the manufacturer's protocol.
  • On-Target plus SMART pool siRNAs (nontargeting, and VHL specific siRNA) were purchased from Dharmacon (Thermo Fisher Scientific), and transfected per the manufacturer's protocol using DharmaFECTl .
  • DMOG Dimethyloxalylglycine
  • DFX deferoxamine
  • 250 ⁇ Dimethyloxalylglycine
  • MG132 10 ⁇
  • Bortezomib 10 ng/ml
  • Bafilomycin A 200 nM
  • All treatments using DMOG, DFX, MG132, and Bafilomycin A were performed overnight (16h).
  • Cycloheximide (Sigma- Aldrich) was solubilized in ethanol, and cells treated at a final concentration of 20 ng/ml for the indicated time points.
  • Alisertib (MLN8237, Millenium Pharmaceuticals Inc.), and ricolinostat (ACY- 1215, Selleck Chemicals) were solubilized in DMSO, and cells treated at a final concentration of 2 uM and 100 nM respectively, at the time of serum withdrawal for 48 hours.
  • Monoclonal anti-polyubiquitin (FKl, 1 : 1000) antibody was purchased from Enzo Life Sciences. Horseradish peroxidase-conjugated goat anti- mouse and goat anti-rabbit secondary antibodies were purchased from Santa Cruz Biotechnology. Immunoblots were visualized using LumiGLO (KPL), Pierce ECL (Thermo Fisher Scientific), or Amersham ECL Prime (GE Life Sciences) substrates.
  • Cells transfected with EGFP- AURKA and HA-VHL30 were grown to 100% confluence prior to harvesting in cold IX cell lysis buffer containing IX complete protease inhibitor (Roche), and 1 mM Na3V04.
  • the lysates were immunoprecipitated overnight using an anti-HA (1 :50) or anti-GFP (5 ⁇ g) antibodies.
  • the immune complexes were incubated with pre-washed magnetic beads (Pierce) for an hour, and washed 3 times with IX cell lysis buffer prior to resuspending the pellet in loading dye and boiling for 5 min at 95°C. Eluted proteins were immunoblotted with anti-HA (1 : 1000) and anti-GFP (1 : 1000) antibodies.
  • RT-PCR analysis to assess knockdown efficiency of VHL was performed as described previously(4). Briefly, mRNA was isolated from cells transfected with siControl/siVHL, and cDNA prepared by reverse-transcription. Gene expression was assessed by real-time qPCR using specific TaqMan probes (Thermo Fisher), and a TaqMan Fast Universal master mix on a Viia7 system (Thermo Fisher). mRNA expression was evaluated for VHL, AURKA and GAPDH, which was used as an endogenous control. The following set of conditions were used for each real-time reaction: 95°C for 20 minutes followed by 40 cycles of 1 second at 95°C and 20 seconds at 60°C. The real-time PCR reactions were all performed in triplicate and were quantified using the - ⁇ cycle threshold (CT) method. RT-PCR was performed for each replicate to ensure optimal knockdown of VHL.
  • CT - ⁇ cycle threshold
  • hTERT RPE-1 cells were plated on glass coverslips, transfected, starved and treated for 48 hours to induce cilia formation.
  • Cells were fixed using 4% paraformaldehyde (15 min), permeabilized with 0.05% Triton-X (10 min), followed by blocking in 3.75% BSA solution (lh).
  • Primary antibodies for acetylated a-tubulin (clone 6-11B-1, 1 :5000; Sigma-Aldrich) and pericentrin (1 :5000; Abeam) were applied in blocking buffer for 1 hour.
  • AlexaFluor 488 and 546 goat anti-mouse or anti-rabbit secondary antibodies (Life Technologies) were subsequently applied for another hour.
  • hTERT RPE-1 cells were co-transfected with the Dendra2C-AURKA construct and HAUb in the absence or presence of VHL24 and the cells cultured to confluence. Serum free media was added to the cells prior to live cell imaging. Live cell imaging was performed using a Nikon Al confocal microscope with an environmental chamber. Dendra2c8 AURKA can be visualized using the 488-laser, which was set to 5% power to prevent and minimize spontaneous conversion of green Dendra2C- AURKA to the red fluorescent state. Cells were scanned (60X) and single cells expressing high Dendra2C- AURKA levels selected for photoconversion. Photoconversion was achieved on a selected region of interest (ROI) using the 405 laser at 10% power, for 1 sec.
  • ROI region of interest
  • VHL modulates AURKA protein levels
  • VHL-null renal cell carcinoma (RCC) 786-0 cells re-expression of VHL significantly decreased AURKA in quiescent cells, while in cycling 786-0 cells re- expressing VHL AURKA levels remained unchanged (FIGURES 1A and IB).
  • a similar decrease in non-mitotic AURKA was observed in quiescent cultures of A-498 (VHL- deficient RCC cells) re-expressing the two known isoforms of VHL; VHL24, and VHL 19 (FIGURES 8A and 8B).
  • RPE-1 retinal pigmented epithelial
  • AURKA is a Target for the VHL E3 Ligase
  • VHL was recently reported to be phosphorylated by AURKA (21), and a VHL- AURKA interaction was confirmed using co- immunoprecipitation assays (FIGURE 9).
  • VHL Ubiquitination of AURKA is Hypoxia-independent In response to proline hydroxylation by PHDs, VHL is well documented to polyubiquitinate its substrates (22-25). However, endogenous AURKA immunoprecipitated from quiescent cells was recognized by the P4D1 anti -ubiquitin antibody, which detects both mono- and polyubiquitination, but not the FK1 antiubiquitin antibody, which exclusively recognizes only polyubiquitination (FIGURE 3A). This suggested that VHL was multi-monoubiquitinating AURKA rather than polyubiquitinating this kinase.
  • VHLR167Q mutant protein is unstable, if overexpressed or stabilized VHL-R167Q has been shown to retain its intrinsic PHD- and oxygen-dependent ability to ubiquitinate HIFa (27).
  • Generating stable lines of 786-0 cells re-expressing either wild type VHL-WT or a VHL-R167Q revealed that the VHL-R167Q mutant when overexpressed reduced HIF2a levels (FIGURE 4B), corroborating previous studies (27).
  • this mutant failed to even marginally reduce AURKA levels compared to VHL-WT expressing cells (FIGURES 4B and 4C).
  • this pathogenic mutant retained the ability to recognize HIFa, but had lost the ability to recognize/ubiquitinate AURKA, suggests ubiquitination of AURKA may participate in VHL's activity as a tumor suppressor.
  • VHL targets AURKA for degradation in quiescent cells
  • a photoswitchable AURKA Dendra2C-AURKA
  • Confluent hTERT RPE-1 cells expressing Dendra2C-AURKA and HA-Ub in the presence or absence of overexpressed VHL were imaged prior to, immediately after photoconversion, and every 15 mins thereafter for 2h (FIGURES 5 A and 5B).
  • VHL has been shown to play a role in stabilizing microtubules, including the microtubule axoneme of the primary cilium, with loss of this tumor suppressor severely attenuating the ability of cells to make a primary cilium (20,28,29).
  • the role of VHL in promoting ciliogenesis has been thought to be hypoxia-independent, although the mechanism by which VHL functioned in this hypoxia-independent capacity has not been elucidated.
  • AURKA ubiquitination by VHL was hypoxia- independent, the inability of VHL-deficient cells to target AURKA for degradation was responsible for defective ciliogenesis observed in VHL-deficient cells was assessed.
  • the ciliary axoneme is made up of highly acetylated a-tubulin, and AURKA is known to phosphorylate and activate HDAC6 (30), which promotes a-tubulin deacetylation. Therefore, a possible VHL-AURKA-HDAC6 signaling axis that regulated ciliogenesis was explored.
  • Direct inhibition of AURKA with alisertib (MLN8237) or HDAC6 using ricolinostat (ACY-1215) rescued deficient ciliogenesis (FIGURES 7A, 7B and 7D) and increased cilia length (FIGURES 7A, 7C and 7E) in VUL-deficient cells.
  • ACY-1215 treatment of cystogenesis in a Tsc2-null murine model The following is a description of a demonstration that the in vivo administration of an HDAC6-specific inhibitor, ACY-1215, significantly reduces the incidence of cystogenesis in a Tsc2-null murine model.
  • a goal of this project was to determine if HDAC6 inhibitor intervention prevents cyst development in vivo.
  • the strategy was to treat mice with an acute loss of tumor suppressor tuberin (Tsc2) with an HDAC6 inhibitor and determine whether HDAC6 inhibition prevents cystogenesis following loss of the cystoprotein.
  • Tsc2 -deficient mice are a preclinical model of cystogenesis obtained by administering tamoxofen at 1 month of age to CreER T2 ;Tsc2 f/" mice.
  • the HDAC inhibitor is administered at 2 months of age, which is prior to cyst formation.
  • This model was selected because of the lack of a Vhl deficient mouse model for renal cystogenesis or tumorigenesis, and preliminary data provides proof-of-concept of the value of this model of preclinical cystogenesis, and the expertise at performing the outlined studies in this model.
  • mice treated with ACY-1215 have a reduced incidence of cystogenesis.
  • One kidney was sagittally sectioned, with one half paraffin embedded for immunohistochemistry and the other fixed and frozen for immunofluorescence studies. The other kidney was flash frozen for protein and RNA extraction. Loss of Tsc2 was monitored using immunohistochemistry and immunoblotting for phospho-S6, a downstream target of mTOR signaling as well as RT- PCR analysis. Cellular inhibition of HDAC6 was monitored using acetylation of a- tubulin. Cystogenesis was evaluated using cystic indices and kidney weight/body weight ratios to determine the efficacy of HDAC6 inhibition in preventing cyst formation. Immunocytochemistry was used to assess presence or absence of primary cilia in renal tubules following HDAC6 inhibition. All animal studies were conducted in compliance with Institutional IACUC approved protocols.
  • TABLE 1 illustrates the incidence of renal cystogenesis observed in the Arm 3 mice after 4 months of continuous daily treatment with ACY-1215. Incidence of renal cystogenesis was calculated as a percentage of mice that developed cysts. A significantly reduced incidence of cystogenesis was observed in Tsc2-null mice that were treated with ACY-1215. Specifically, the incidence reduced from 78% in the vehicle treated cohort to 31% in the ACY-1215 treated cohort.
  • HDAC6 inhibitor ACY-1215 HDAC6 inhibitor ACY-1215.
  • HDAC6 inhibitor ACY-1215 shows promise for significantly reducing incidence of cystogenesisis in vivo, and restoring ciliary defects in vitro.
  • this compound is already in clinical trials for distinct and unrelated diseases, such as myeloma and lymphomas, and is thus has been shown to be well -tolerated for in vivo administration in humans.
  • Hippel-Lindau tumor suppressor protein mediates ubiquitination of activated atypical protein kinase C. The Journal of biological chemistry 2001; 276(47): 43611-7.
  • Thoma CR, Frew IJ, Hoerner CR, Montani M, Moch H, Krek W., pVHL and GSK3beta are components of a primary cilium-maintenance signalling network. Nat Cell Biol 2007; 9(5):588-95.
  • Boutet SC Biressi S, Iori K, Natu V, Rando TA., Tafl regulates Pax3 protein by monoubiquitination in skeletal muscle progenitors. Mol Cell 2010; 40(5):749-61.
  • Ferchichi I, Kourda N, Sassi S, Romdhane KB, Balatgi S, Cremet JY, et al., Aurora A overexpression and pVHL reduced expression are correlated with a bad kidney cancer prognosis. Dis Markers 2012; 33(6):333-40.

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Abstract

La présente invention concerne l'utilisation d'inhibiteurs de HDAC6 pour le traitement de maladies et troubles rénaux, comprenant une ciliopathie, la maladie de Von Hippel-Lindau (VHL), la cystogenèse rénale, un carcinome à cellules rénales et le complexe de la sclérose tubéreuse (CST), et similaire. Dans certains modes de réalisation, l'inhibiteur de HDAC6 est un inhibiteur direct de HDAC6. Dans un autre mode de réalisation, l'inhibiteur de signalisation HDAC6 est ACY-1215 ou un sel pharmaceutiquement acceptable de celui-ci. Dans certains modes de réalisation, l'inhibiteur de HDAC6 est combiné avec un inhibiteur d'AURKA.
PCT/US2017/048646 2016-08-26 2017-08-25 Utilisation d'inhibiteurs de hdac6 pour la prévention et le traitement de la cystogenèse rénale, d'un carcinome à cellules rénales et de ciliopathies rénales Ceased WO2018039581A1 (fr)

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CN115074429A (zh) * 2021-03-11 2022-09-20 首都医科大学附属北京胸科医院 一个结核易感性筛查的生物标志物及其应用
US20220372491A1 (en) * 2019-10-28 2022-11-24 Monash University Treatment of renal cystic disease
CN116492466A (zh) * 2023-04-17 2023-07-28 中国人民解放军总医院第三医学中心 Hedgehog通路抑制剂在治疗肾透明细胞癌中的应用

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
US20220372491A1 (en) * 2019-10-28 2022-11-24 Monash University Treatment of renal cystic disease
EP4051273A4 (fr) * 2019-10-28 2023-11-08 Monash University Traitement de la maladie kystique rénale
CN115074429A (zh) * 2021-03-11 2022-09-20 首都医科大学附属北京胸科医院 一个结核易感性筛查的生物标志物及其应用
CN116492466A (zh) * 2023-04-17 2023-07-28 中国人民解放军总医院第三医学中心 Hedgehog通路抑制剂在治疗肾透明细胞癌中的应用

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