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WO2017184751A1 - Compositions et méthodes pour traiter et prévenir l'hypertension - Google Patents

Compositions et méthodes pour traiter et prévenir l'hypertension Download PDF

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WO2017184751A1
WO2017184751A1 PCT/US2017/028389 US2017028389W WO2017184751A1 WO 2017184751 A1 WO2017184751 A1 WO 2017184751A1 US 2017028389 W US2017028389 W US 2017028389W WO 2017184751 A1 WO2017184751 A1 WO 2017184751A1
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uchll
subject
pah
gene
inhibitor
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Ankit Desai
Joe G. N. Garcia
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University of Arizona
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University of Arizona
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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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • 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
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • compositions and method for treating or preventing pulmonary hypertension and cancer are provided herein.
  • Pulmonary hypertension including pulmonary arterial hypertension (PAH) is an increase of blood pressure in the pulmonary artery, pulmonary vein, or pulmonary capillaries, together known as the lung vasculature, leading to shortness of breath, dizziness, fainting, leg swelling and other symptoms. Pulmonary hypertension can be a severe disease with a markedly decreased exercise tolerance.
  • the signs/symptoms of pulmonary hypertension are consistent with the following: shortness of breath, chest pain, heartbeat (increased), pain (right side of the chest/abdomen), poor appetite, lightheadedness, swelling (legs/ankles), and cyanosis.
  • Treatment of pulmonary hypertension is determined by whether the etiology is arterial, venous, hypoxic, thromboembolic, or miscellaneous. The treatment is dependent on the etiology of PH. Current conservative treatment for PAH (Group I defined by World
  • pulmonary vasoactive agents such as prostacyclin therapy, phosphodiesterase 5 inhibitors, and endothelin receptor antagonists are FDA-approved to treat patients with Group I and in some cases, Group IV
  • thromoboembolic thromoboembolic
  • Patients with left heart failure or hypoxemic lung diseases should not routinely be treated with vasoactive agents including prostanoids, phosphodiesterase inhibitors, or endothelin receptor antagonists.
  • vasoactive agents including prostanoids, phosphodiesterase inhibitors, or endothelin receptor antagonists.
  • doctors at a minimum will conduct cardiac catheterization of the right heart, echocardiography, chest CT, a six-minute walk test, and pulmonary function testing.
  • Using Group I-specific PAH treatments for other kinds of pulmonary hypertension in patients with these conditions can potentially harm the patient and waste substantial medical resources.
  • High dose calcium channel blockers are useful in less than 5% of IP AH patients who are vasoreactive by Swan-Ganz catheter.
  • calcium channel blockers have been largely misused, being prescribed to many patients with non-vasoreactive PAH, and furthermore, contributing to excess morbidity and mortality.
  • the criteria for vasoreactivity have changed. Only those patients whose mean pulmonary artery pressure falls by more than 10 mm Hg to less than 40 mm Hg with an unchanged or increased cardiac output when challenged with adenosine, epoprostenol, or nitric oxide are considered vasoreactive. Of these, only half of the patients are responsive to calcium channel blockers in the long term.
  • compositions and method for treating or preventing pulmonary hypertension and cancer are provided herein.
  • AKT1 plays a critical role in uncontrolled cancer cell proliferation as well as the hyper-proliferative state observed in ECs and smooth muscle cells of the lung vasculature in pulmonary arterial hypertension (Tang et al, Am J Physiol Lung Cell Mol Physiol, 308(2), L208-220 2015). While development of therapies have been ongoing that target inhibition of AKT expression and activation, emerging investigations now have also focused therapeutic discovery in the form of promoting AKT degradation (Chan et al, Cell Biosci, 4(1), 59 2014; Yang, Wu, Wu, & Lin, Cell Cycle, 9(3), 487-497 2010).
  • GADD45a loss of GADD45a, in particular, significantly reduced UCHLl expression associated with site-specific UCHLl promoter methylation, resulting in increased levels of site-specific Akt ubiquitination and reduced Akt levels.
  • UCHLl deficiency would promote protection from the development of chronic forms of pulmonary vascular disease via sustained reductions in AKT activity.
  • Gadd45a knockout mice (“epigenetic” model of UCHLl deficiency) and WT mice administered a UCHLl -specific inhibitor, demonstrated reduced right ventricular systolic pressures (RVSP), RV hypertrophy, and pathological and cancerous -like lung vascular remodeling when exposed to chronic hypoxia (a conventional murine model of pulmonary hypertension) compared to their controls. All three of these findings are considered surrogates for severity of PH. Both treatment-exposed conditions also revealed reduced vascular-specific UCHLl expression (predominantly in the endothelial cells, considered the initiating tissue source for the development of PAH) and decreased activated AKTl levels in the lungs.
  • UCHLl inhibitor exposure in vitro in lung vascular endothelial cells lead to reduced proliferation and angiogenesis after exposure to VEGF (a proliferative factor and known PH stimuli).
  • VEGF a proliferative factor and known PH stimuli.
  • UCHLl -Aktl is, therefore, a new candidate pathway in hypoxic PH and UCHLl inhibition represents a therapeutic target in PAH and broadly in hyper-proliferative and pro-angiogenic states such as cancers.
  • a method of treating or preventing pulmonary hypertension (PAH) in a subject comprising: administering a UCHLl inhibitor to the subject.
  • the UCHLl inhibitor is selected from, for example, a nucleic acid, a small molecule, a peptide, or an antibody.
  • the small molecule is LDN-57444.
  • the subject exhibits symptoms of PAH, does not exhibit symptoms of PAH, or is at increased risk for developing PAH.
  • the method further comprises the step of identifying the presence of a rs5030732 variant in a UCHLl gene in a sample from the subject.
  • the presence of the mutation is indicative of PAH in the subject.
  • Yet other embodiments provide a method of treating or preventing cancer in a subject, comprising: administering a UCHLl inhibitor to the subject.
  • the cancer is an endothelial cancer (e.g., breast or renal cancer).
  • Still other embodiments provide the use of a UCHLl inhibitor to treat or prevent cancer in a subject in need thereof.
  • Certain embodiments provide a method of characterizing a sample from a subject, comprising: detecting the presence of mutant UCHLl gene in the sample using a reagent that specifically binds to the mutant UCHLl gene but not a wild type UCHLl gene, wherein the mutant UCHLl gene comprises a rs5030732 mutation.
  • the reagent is one or more of one or more probes that specifically bind to the mutant UCHLl gene but not the wild type UCHLl gene and one or more primers that specifically bind to the mutant
  • the sample is tissue (e.g., lung tissue, cells, blood, blood products, or urine).
  • hypertension or cancer in a subject, comprising: administering a HIF-2a or PDGF inhibitor to the subject.
  • Also provided herein is a method of preventing UCHLl ubiquitination of downstream targets in a subject (e.g., HIF- ⁇ , HIF-2a, or PDGF), comprising administering an inhibitor of UCHLl, HIF- ⁇ , HIF-2a, or PDGF to the subject.
  • a subject e.g., HIF- ⁇ , HIF-2a, or PDGF
  • Figure 1 shows increased UCHL1 expression in PAH.
  • Figure 2 shows that UCHL1 inhibition reduces VEGF-induced cell proliferation in vitro.
  • BrdU incorporation assay reveals reduced cell proliferation in vitro with co-administration of LDN57444 in HPAECs exposed to VEGF.
  • C Reduced tube formation was observed with HPACEs after 20h treatment with LDN57444 at 5 ⁇ with in presence of VEGF compared to VEGF alone.
  • D LDN57444 exposure with VEGF results in significantly lower numbers of node, junction, branching intervals, meshes area and segment compared to VEGF treatment alone. * or p ⁇ 0.05, **p ⁇ 0.01, **** pO.0001
  • FIG. 3 shows that UCHL1 inhibition attenuates murine hypoxic PH.
  • WT mice exposed to LDN57444 for two weeks after 3 weeks of exposure to hypoxia reveal reduced (A. and B) RVSP and (C.) RVH compared to WT mice, which received vehicle.
  • Data presented as mean ⁇ SE. Normoxia (n 6-8) for all groups. **** pO.001, .***** pO.00001
  • Figure 4 shows that UCHL1 inhibition attenuates PH in a rodent PH models.
  • Figure 5 shows that UCHL1 inhibition reduces Akt activation and increases Relinked Akt ubiquitination in murine hypoxic PH.
  • Immunohistochemistry reveals increased UCHL1 staining in WT mice exposed to chronic hypoxia compared to normoxia mice, which appears to be predominantly within the endothelium.
  • FIG. 7 shows that Gadd45a deletion attenuates murine hypoxic PH. While both WT and Gadd45 1' mice exhibit elevated RVSP, Gadd45 1' mice demonstrate reduced (A.) RVSP and (B.) RVH compared to WT mice when exposed to hypoxia. C. While both WT and Gadd45a l ⁇ mice develop vascular remodeling after exposure to hypoxic PH, Gadd45a l ⁇ mice demonstrate reduced pulmonary artery medial thickness index compared to WT mice hypoxic lungs. .**p ⁇ 0.01, .**p ⁇ 0.01, **** pO.0001.
  • Figure 9 shows that UCHL1 inhibition reduces the levels of PAH mediators HIF-2a and PDGF in murine PH.
  • Both LDN5744 and chronic hypoxia (4 weeks, 10% Fi02) resulted in increased ubiquintanted Akt levels in whole lungs in mice.
  • FIG. 10 shows that TGF- ⁇ induces Snail (A) and Vimentin (B) gene expression levels, both associated with endothelial to mesenchymal transition (EndMT)- a cellular process contributing to the vascular remodeling in PAH, in human lung ECs while
  • Figure 11 shows genotype/phenotype with UCHL1 SNP in PAH.
  • the term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment.
  • the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.
  • the term "subject suspected of having a disease” refers to a subject that presents one or more symptoms indicative of a disease (e.g., pulmonary hypertension or cancer). A subject suspected of having a disease may also have one or more risk factors. A subject suspected of having disease has generally not been tested for the disease. However, a "subject suspected of having disease” encompasses an individual who has received a preliminary diagnosis but for whom a confirmatory test has not been done or for whom the level or severity of metabolic disease is not known.
  • the term "subject diagnosed with a disease” refers to a subject who has been tested and found to have a disease (e.g., pulmonary hypertension or cancer).
  • a disease e.g., pulmonary hypertension or cancer.
  • initial diagnosis refers to a test result of initial disease that reveals the presence or absence of disease.
  • the term "subject at risk for disease” refers to a subject with one or more risk factors for developing a specific disease (e.g., pulmonary hypertension or cancer).
  • Risk factors include, but are not limited to, gender, age, genetic predisposition, environmental exposure, and previous incidents of disease, preexisting diseases, and lifestyle.
  • non-human animals refers to all non-human animals including, but not limited to, vertebrates such as rodents, non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc.
  • cell culture refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, transformed cell lines, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro.
  • eukaryote refers to organisms distinguishable from
  • prokaryotes it is intended that the term encompass all organisms with cells that exhibit the usual characteristics of eukaryotes, such as the presence of a true nucleus bounded by a nuclear membrane, within which lie the chromosomes, the presence of membrane-bound organelles, and other characteristics commonly observed in eukaryotic organisms. Thus, the term includes, but is not limited to such organisms as fungi, protozoa, and animals (e.g., humans).
  • in vitro refers to an artificial environment and to processes or reactions that occur within an artificial environment.
  • in vitro environments can consist of, but are not limited to, test tubes and cell culture.
  • in vivo refers to the natural environment (e.g., an animal or a cell) and to processes or reaction that occur within a natural environment.
  • test compound and “candidate compound” refer to any chemical entity, pharmaceutical, drug, and the like that is a candidate for use to treat or prevent a disease, illness, sickness, or disorder of bodily function (e.g., hypertension or cancer).
  • Test compounds comprise both known and potential therapeutic compounds.
  • a test compound can be determined to be therapeutic by screening using the screening methods of the present disclosure.
  • sample is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, serum and the like. Environmental samples include environmental material such as surface matter, soil, water, and industrial samples. Such examples are not however to be construed as limiting the sample types applicable to the present disclosure.
  • an effective amount refers to the amount of a compound (e.g. , a compound described herein) sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations, applications or dosages and is not limited to or intended to be limited to a particular formulation or administration route.
  • co-administration refers to the administration of at least two agent(s) (e.g. , UCHL1 inhibitor compound having a structure presented above or elsewhere described herein) or therapies to a subject.
  • agent(s) e.g. , UCHL1 inhibitor compound having a structure presented above or elsewhere described herein
  • the coadministration of two or more agents/therapies is concurrent.
  • a first agent/therapy is administered prior to a second agent/therapy.
  • agents/therapies used may vary.
  • the appropriate dosage for co-administration can be readily determined by one skilled in the art.
  • the respective agents/therapies are administered at lower dosages than appropriate for their administration alone.
  • co-administration is especially desirable in embodiments where the co-administration of the agents/therapies lowers the requisite dosage of a known potentially harmful (e.g. , toxic) agent(s).
  • composition refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo, or ex vivo.
  • the term "toxic” refers to any detrimental or harmful effects on a cell or tissue as compared to the same cell or tissue prior to the administration of the toxicant.
  • compositions and method for treating or preventing pulmonary hypertension and cancer are provided herein.
  • Pulmonary arterial hypertension is a rare and fatal condition characterized by gradual occlusion of the small pulmonary arterioles leading to progressively increased pulmonary vascular resistance (PVR) with worsening right heart failure and death.
  • PVR pulmonary vascular resistance
  • data described herein reveal novel roles for the enzyme ubiquitin carboxy-terminal hydrolase LI (UCHL1) in AKT signaling, a well-known mediator of pulmonary vascular remodeling in PAH.
  • the studies provide further insight into UCHL1 genetics which may contribute to these roles, providing a new therapeutic target in PAH.
  • PAH is characterized by lung vascular remodeling, sustained vasoconstriction, in situ thrombosis and increased lung vascular wall stiffness, all which directly result in elevated PVR.
  • Histopathology reveals uncontrolled lung vascular smooth muscle (SMC) and endothelial cell (EC) hyperplasia with endothelial-to-mesenchymal transition (EndMT), a process that transforms fully differentiated lung EC to highly proliferative myofibroblasts (myoFB).
  • EndMT remodeling contributes to plexiform lesions, pathognomonic for PAH.
  • hypoxia-inducible factor-2a was reported to be a canonical genetic mediator of obliterative intimal lesions via both EC transcriptomic re- programming and paracrine effects on SMC proliferation.
  • Akt/mTOR signaling is well-known to regulate HIF-2a downstream.
  • Data described herein connects UCHLl activity to both the development of EndMT and HIF-2a protein regulation, linking its lung EC- specific expression to paracrine SMC pathology.
  • Chain-specific ubiquitin linkage of proteins regulate a host of cellular processes In general, lysine K48-linked polyubiquitin chains target proteins for proteolytic destruction. In contrast to proteasomal degradation, K63 -linked chains typically coordinate protein activation and trafficking as demonstrated by Akt in cancer. Linkage-specific Aktl ubiquitination in lung ECs has been shown; whereby Akt K63- linked ubiquitination was associated with its activation while K48-linked ubiquitination was associated with its degradation. In contrast to the paradigm where an individual enzyme has a single function, UCHLl manifests a dual and opposing role as a ligase increasing K63-linked ubiquitination in addition to its known DUB activity.
  • This ligase activity is enhanced by its concentration-dependent dimerization. Based on this linkage-specificity, it is contemplated that both UCHLl -mediated activities (DUB function and ligase ubiquitination) potentiate Akt signaling While dimer-associated K63-linked ubiquitination can further activate Akt, increased monomer associated DUB activity can reduce Akt degradation increasing total Akt protein levels. Based on these additive functions in Akt signaling, increased UCHLl dimer and monomer expression was shown in PAH samples (Example 1).
  • UCHLl inhibition resulted in increased total Akt ubiquitination, reduced EC/SMC proliferation, and attenuation in rodent PH models.
  • a novel role for growth arrest and DNA-damage-inducible, alpha has been reported in the regulation of site-specific AKT1 ubiquitination and activation via ubiquitin carboxyl-terminal esterase LI (UCHLl), a deubiquitinase.
  • Aktl is an established pro-survival candidate gene in the development of pulmonary arterial hypertension (PAH)
  • PAH pulmonary arterial hypertension
  • Gadd45a deficiency may also lead to protection from murine models of hypoxic pulmonary hypertension (PH) via UCHLl - mediated reductions in AKT1 activation.
  • UCHLl is a member of a gene family whose products hydrolyze small C-terminal adducts of ubiquitin to generate the ubiquitin monomer. Expression of UCHLl was thought to be highly specific to neurons and to cells of the diffuse neuroendocrine system and their tumors. It is abundantly present in all neurons (accounts for 1-2% of total brain protein), expressed specifically in neurons and testis/ovary. But, data as detailed above, also show significant pulmonary endothelial expression of UCHLl, which is a new finding opening the door to novel frontiers in pulmonary research.
  • the catalytic triad of UCH-Ll contains a cysteine at position 90, an aspartate at position 176, and a histidine at position 161 that are responsible for its hydrolase activity.
  • UCHLl inhibitors or inhibitor of downstream targets of UCHLl e.g., HIF-2a or PDGF
  • UCHLl e.g., HIF-2a or PDGF
  • methods and compositions for identifying variants (e.g., mutants, e.g., rs5030732 mutations) in UCHLl genes are provided herein.
  • UCHLl inhibitors include, but are not limited to, a nucleic acid, a small molecule, peptide, or an antibody.
  • the UCHLl inhibitor is a small molecule (e.g., LDN-57444; See e.g., Liu et al. (2003), Discovery of inhibitors that elucidate the role of UCH-Ll activity in the H1299 lung cancer cell line; Chem. Biol, 10 837; Tan et al. (2008), Endoplasmic reticulum stress contributes to the cell death induced by UCH-Ll inhibitor; Mol. Cell.
  • LDN-57444 small molecule
  • LDN-57444 has the structure
  • Small molecules inhibitors of downstream targets of UCHLl are (e.g., Roxadustat (FG-4592) and those described in Mohammad Ali Sadiq et al, Saudi J Ophthalmol. 2015 Oct-Dec; 29(4): 287- 291.
  • the UCHLl or UCHLl target inhibitor is a nucleic acid.
  • nucleic acids suitable for inhibiting UCHLl include, but are not limited to, antisense nucleic acids, miRNAs, and shRNAs.
  • nucleic acid therapies are complementary to and hybridize to at least a portion (e.g., at least 5, 8, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides) of SEQ ID NO: l (UCHLl mRNA; accession No. KR709885).
  • compositions comprising oligomeric antisense compounds, particularly oligonucleotides are used to modulate the function of nucleic acid molecules encoding UCHLl , ultimately modulating the amount of UCHLl expressed. This is accomplished by providing antisense compounds that specifically hybridize with one or more nucleic acids encoding UCHLl .
  • the specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid.
  • This modulation of function of a target nucleic acid by compounds that specifically hybridize to it is generally referred to as "antisense.
  • the functions of DNA to be interfered with include replication and transcription.
  • RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity that may be engaged in or facilitated by the RNA.
  • the overall effect of such interference with target nucleic acid function is modulation of UCHLl .
  • modulation means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. For example, expression may be inhibited to treat or prevent a metabolic disorder.
  • nucleic acids are siRNAs.
  • RNA interference is the process of sequence-specific, post-transcriptional gene silencing initiated by a small interfering RNA (siRNA). During RNAi, siRNA induces degradation of target mRNA with consequent sequence-specific inhibition of gene expression.
  • RNA interference is a RNA duplex of nucleotides that is targeted to a nucleic acid sequence of interest, for example, SIN3A.
  • siRNA is a generic term that encompasses all possible RNAi triggers.
  • RNA duplex refers to the structure formed by the complementary pairing between two regions of a RNA molecule.
  • siRNA is "targeted" to a gene in that the nucleotide sequence of the duplex portion of the siRNA is complementary to a nucleotide sequence of the targeted gene.
  • the siRNAs are targeted to the sequence encoding SIN3A.
  • the length of the duplex of siRNAs is less than 30 base pairs.
  • the duplex can be 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 or 10 base pairs in length.
  • the length of the duplex is 19 to 32 base pairs in length.
  • the length of the duplex is 19 or 21 base pairs in length.
  • the RNA duplex portion of the siRNA can be part of a hairpin structure.
  • the hairpin structure may contain a loop portion positioned between the two sequences that form the duplex.
  • the loop can vary in length. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26 or 27 nucleotides in length. In certain embodiments, the loop is 18 nucleotides in length.
  • the hairpin structure can also contain 3' and/or 5' overhang portions. In some embodiments, the overhang is a 3' and/or a 5' overhang 0, 1 , 2, 3, 4 or 5 nucleotides in length.
  • Dicer-substrate RNAs are chemically synthesized asymmetric 25-mer/27-mer duplex RNAs that have increased potency in RNA interference compared to traditional siRNAs.
  • Traditional 21-mer siRNAs are designed to mimic Dicer products and therefore bypass interaction with the enzyme Dicer.
  • Dicer has been recently shown to be a component of RISC and involved with entry of the siRNA duplex into RISC.
  • Dicer-substrate siRNAs are designed to be optimally processed by Dicer and show increased potency by engaging this natural processing pathway. Using this approach, sustained knockdown has been regularly achieved using sub-nanomolar concentrations. (U.S. Pat. No. 8,084,599; Kim et al., Nature Biotechnology 23:222 2005; Rose et al., Nucleic Acids Res., 33:4140 2005).
  • shRNA The transcriptional unit of a "shRNA” is comprised of sense and antisense sequences connected by a loop of unpaired nucleotides.
  • shRNAs are exported from the nucleus by Exportin-5, and once in the cytoplasm, are processed by Dicer to generate functional siRNAs.
  • miRNAs stem-loops are comprised of sense and antisense sequences connected by a loop of unpaired nucleotides typically expressed as part of larger primary transcripts (pri- miRNAs), which are excised by the Drosha-DGCR8 complex generating intermediates known as pre-miRNAs, which are subsequently exported from the nucleus by Exportin-5, and once in the cytoplasm, are processed by Dicer to generate functional miRNAs or siRNAs.
  • the term “artificial” arises from the fact the flanking sequences (.about.35 nucleotides upstream and .about.40 nucleotides downstream) arise from restriction enzyme sites within the multiple cloning site of the siRNA.
  • miRNA encompasses both the naturally occurring miRNA sequences as well as artificially generated miRNA shuttle vectors.
  • the siRNA can be encoded by a nucleic acid sequence, and the nucleic acid sequence can also include a promoter.
  • the nucleic acid sequence can also include a polyadenylation signal.
  • the polyadenylation signal is a synthetic minimal
  • polyadenylation signal or a sequence of six Ts.
  • the present disclosure contemplates the use of any genetic manipulation for use in modulating the expression of UCHL1 or a downstream target.
  • genetic manipulation include, but are not limited to, gene knockout (e.g., removing the UCHL1 gene from the chromosome using, for example, recombination), expression of antisense constructs with or without inducible promoters, and the like.
  • Delivery of nucleic acid construct to cells in vitro or in vivo may be conducted using any suitable method.
  • a suitable method is one that introduces the nucleic acid construct into the cell such that the desired event occurs (e.g., expression of an antisense construct).
  • Plasmids carrying genetic information into cells are achieved by any of various methods including, but not limited to, directed injection of naked DNA constructs, bombardment with gold particles loaded with said constructs, and macromolecule mediated gene transfer using, for example, liposomes, biopolymers, and the like.
  • Preferred methods use gene delivery vehicles derived from viruses, including, but not limited to, adenoviruses, retroviruses, vaccinia viruses, and adeno-associated viruses. Because of the higher efficiency as compared to retroviruses, vectors derived from adenoviruses are the preferred gene delivery vehicles for transferring nucleic acid molecules into host cells in vivo.
  • Adenoviral vectors have been shown to provide very efficient in vivo gene transfer into a variety of solid tumors in animal models and into human solid tumor xenografts in immune-deficient mice. Examples of adenoviral vectors and methods for gene transfer are described in PCT publications WO 00/12738 and WO 00/09675 and U. S. Pat. Appl. Nos. 6,033,908, 6,019,978, 6,001,557, 5,994, 132, 5,994, 128, 5,994,106, 5,981,225, 5,885,808, 5,872,154, 5,830,730, and 5,824,544, each of which is herein incorporated by reference in its entirety.
  • Vectors may be administered to subject in a variety of ways.
  • vectors are administered into tumors or tissue associated with tumors using direct injection.
  • administration is via the blood or lymphatic circulation (See e.g. , PCT publication 99/02685 herein incorporated by reference in its entirety).
  • Exemplary dose levels of adenoviral vector are preferably 108 to 1011 vector particles added to the perfusate.
  • the present disclosure provides antibodies that inhibit UCHL1.
  • Any suitable antibody e.g. , monoclonal, polyclonal, or synthetic
  • the antibodies are humanized antibodies. Methods for humanizing antibodies are well known in the art (See e.g. , U.S. Patents 6,180,370, 5,585,089, 6,054,297, and 5,565,332; each of which is herein incorporated by reference).
  • candidate UCHL1 inhibitors are screened for activity (e.g., using the methods described in Example 1 below or another suitable assay).
  • compositions comprising the compounds described above.
  • the pharmaceutical compositions of the present disclosure may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary (e.g. , by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.
  • compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets.
  • Thickeners flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
  • compositions of the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.
  • the pharmaceutical formulations of the present disclosure may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • compositions of the present disclosure may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas.
  • the compositions of the present disclosure may also be formulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • the pharmaceutical compositions may be formulated and used as foams.
  • Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product.
  • cationic lipids such as lipofectin (U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and poly cationic molecules, such as polylysine (WO 97/30731), also enhance the cellular uptake of oligonucleotides.
  • compositions of the present disclosure may additionally contain other adjunct components conventionally found in pharmaceutical compositions.
  • the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • such materials when added, should not unduly interfere with the biological activities of the components of the compositions of the present disclosure.
  • the formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved.
  • Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models or based on the examples described herein.
  • dosage is from 0.01 ⁇ g to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly.
  • the treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues.
  • UCHL1 inhibitors are administered to a subject found to be at risk for a disorder (e.g., a subject exhibiting one or more markers or symptoms of cancer and/or PAH but not meeting the diagnostic criteria for diagnosis of a disorder).
  • the compounds and pharmaceutical compositions described herein are administered in combination with one or more additional agents, treatment, or interventions (e.g., agents, treatments, or interventions useful in the treatment of cancer and/or PAH).
  • additional agents, treatment, or interventions e.g., agents, treatments, or interventions useful in the treatment of cancer and/or PAH.
  • agents useful in the treatment of PAH include, but are not limited to, bosetan, diuretics, digoxins, blood thinners, prostanoids, phosphodiesterase inhibitors, endothelin antagonists, high dose calcium channel blockers, or surgery to repair/replace the mitral valve or aortic valve.
  • UCHL1 inhibitors are used to treat or prevent cancer.
  • a non- limiting exemplary list of these diseases and conditions includes, but is not limited to, glioblastoma, pancreatic cancer, breast cancer, prostate cancer, lymphoma, skin cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain carcinoma, head-neck cancer, glioma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma, small- cell lung carcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortex carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma,
  • choriocarcinoma mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential thrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma, soft-tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma, and the like.
  • Some embodiments of the present invention provide methods for administering an effective amount of a compound of the invention and at least one additional therapeutic agent (including, but not limited to, chemotherapeutic antineoplastics, apoptosis-modulating agents, antimicrobials, antivirals, antifungals, and anti-inflammatory agents) and/or therapeutic technique (e.g. , surgical intervention, and/or radiotherapies).
  • the additional therapeutic agent(s) is a anticancer agent.
  • the additional therapeutic agent(s) is a radiation therapy.
  • anticancer agents are contemplated for use in the methods of the present invention. Indeed, the present invention contemplates, but is not limited to, administration of numerous anticancer agents such as: agents that induce apoptosis;
  • polynucleotides e.g., anti-sense, ribozymes, siRNA
  • polypeptides e.g. , enzymes and antibodies
  • biological mimetics alkaloids; alkylating agents; antitumor antibiotics;
  • antimetabolites include hormones; platinum compounds; monoclonal or polyclonal antibodies (e.g., antibodies conjugated with anticancer drugs, toxins, defensins), toxins; radionuclides;
  • biological response modifiers e.g., interferons (e.g. , IFN-a) and interleukins (e.g. , IL-2)
  • adoptive immunotherapy agents hematopoietic growth factors; agents that induce tumor cell differentiation (e.g. , all-trans-retinoic acid); gene therapy reagents (e.g. , antisense therapy reagents and nucleotides); tumor vaccines; angiogenesis inhibitors; proteosome inhibitors: NF-KB modulators; anti-CDK compounds; HDAC inhibitors; and the like.
  • chemotherapeutic compounds and anticancer therapies suitable for coadministration with the disclosed compounds are known to those skilled in the art.
  • anticancer agents comprise agents that induce or stimulate apoptosis.
  • Agents that induce apoptosis include, but are not limited to, radiation (e.g. , X- rays, gamma rays, UV); tumor necrosis factor (TNF)-related factors (e.g., TNF family receptor proteins, TNF family ligands, TRAIL, antibodies to TRAIL-R1 or TRAIL-R2); kinase inhibitors (e.g.
  • epidermal growth factor receptor (EGFR) kinase inhibitor vascular growth factor receptor (VGFR) kinase inhibitor, fibroblast growth factor receptor (FGFR) kinase inhibitor, platelet-derived growth factor receptor (PDGFR) kinase inhibitor, and Bcr- Abl kinase inhibitors (such as GLEEVEC)); antisense molecules; antibodies (e.g. ,
  • HERCEPTIN, RITUXAN, ZEVALIN, and AVASTIN anti-estrogens (e.g. , raloxifene and tamoxifen); anti-androgens (e.g. , flutamide, bicalutamide, finasteride, aminoglutethamide, ketoconazole, and corticosteroids); cyclooxygenase 2 (COX-2) inhibitors (e.g.
  • NSAIDs non-steroidal anti -inflammatory drugs
  • antiinflammatory drugs e.g., butazolidin, DECADRON, DELTASONE, dexamethasone, dexamethasone intensol, DEXONE, HEXADROL, hydroxychloroquine, METICORTEN, ORADEXON, ORASONE, oxyphenbutazone, PEDIAPRED, phenylbutazone, PLAQUENIL, prednisolone, prednisone, PRELONE, and TANDEARIL); and cancer chemotherapeutic drugs (e.g.
  • irinotecan CAMPTOSAR
  • CPT-11 CPT-11
  • fludarabine FLUDARA
  • dacarbazine DTIC
  • dexamethasone mitoxantrone
  • MYLOTARG VP- 16
  • cisplatin carboplatin, oxaliplatin, 5-FU, doxorubicin, gemcitabine, bortezomib, gefitinib, bevacizumab,
  • TAXOTERE or TAXOL cellular signaling molecules
  • ceramides and cytokines cellular signaling molecules
  • compositions and methods of the present invention provide a compound of the invention and at least one anti-hyperproliferative or antineoplastic agent selected from alkylating agents, antimetabolites, and natural products (e.g., herbs and other plant and/or animal derived compounds).
  • at least one anti-hyperproliferative or antineoplastic agent selected from alkylating agents, antimetabolites, and natural products (e.g., herbs and other plant and/or animal derived compounds).
  • Alkylating agents suitable for use in the present compositions and methods include, but are not limited to: 1) nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, ifosfamide, melphalan (L-sarcolysin); and chlorambucil); 2) ethylenimines and
  • methylmelamines e.g. , hexamethylmelamine and thiotepa
  • alkyl sulfonates e.g., busulfan
  • nitrosoureas e.g. , carmustine (BCNU); lomustine (CCNU); semustine (methyl- CCNU); and streptozocin (streptozotocin)
  • triazenes e.g., dacarbazine (DTIC;
  • antimetabolites suitable for use in the present compositions and methods include, but are not limited to: 1) folic acid analogs (e.g., methotrexate
  • pyrimidine analogs e.g. , fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorode-oxyuridine; FudR), and cytarabine (cytosine arabinoside)
  • purine analogs e.g. , mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG), and pentostatin (2'-deoxycoformycin)).
  • chemotherapeutic agents suitable for use in the compositions and methods of the present invention include, but are not limited to: 1) vinca alkaloids (e.g., vinblastine (VLB), vincristine); 2) epipodophyllotoxins (e.g., etoposide and teniposide); 3) antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin
  • vinca alkaloids e.g., vinblastine (VLB), vincristine
  • epipodophyllotoxins e.g., etoposide and teniposide
  • antibiotics e.g., dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin
  • mitomycin C (mitomycin C)); 4) enzymes (e.g. , L-asparaginase); 5) biological response modifiers (e.g., interferon-alfa); 6) platinum coordinating complexes (e.g., cisplatin (cis-DDP) and carboplatin); 7) anthracenediones (e.g., mitoxantrone); 8) substituted ureas (e.g. ,
  • methylhydrazine derivatives e.g. , procarbazine (N-methylhydrazine;
  • adrenocortical suppressants e.g., mitotane ( ⁇ , ⁇ '-DDD) and aminoglutethimide
  • 11 adrenocorticosteroids
  • 12 progestins (e.g. , hydroxy progesterone caproate, medroxyprogesterone acetate, and megestrol acetate)
  • 13 estrogens (e.g. , diethylstilbestrol and ethinyl estradiol)
  • antiestrogens e.g. , tamoxifen
  • 15) androgens e.g. , testosterone propionate and fluoxymesterone
  • 16 antiandrogens (e.g., flutamide): and 17) gonadotropin-releasing hormone analogs (e.g. , leuprolide).
  • any oncolytic agent that is routinely used in a cancer therapy context finds use in the compositions and methods of the present invention.
  • the U.S. Food and Drug Administration maintains a formulary of oncolytic agents approved for use in the United States. International counterpart agencies to the U.S.F.D.A. maintain similar formularies.
  • Table 1 provides a list of exemplary antineoplastic agents approved for use in the U.S. Those skilled in the art will appreciate that the "product labels" required on all U.S. approved chemotherapeutics describe approved indications, dosing information, toxicity data, and the like, for the exemplary agents.
  • Cisplatin Platinol Bristol-Myers Squibb PtCl 2 H 6 N 2
  • Daunorubicin HC1 daunomycin Cerubidin Wyeth Ayerst, Madison, ((1 S ,3 S )-3-Acetyl-l ,2,3,4,6, l l-hexahydro- e NJ
  • Tositumomab I 131 Tositumomab Bexxar Corixa Corp., Seattle, WA (recombinant murine immunotherapeutic
  • Anticancer agents further include compounds which have been identified to have anticancer activity. Examples include, but are not limited to, 3-AP, 12-0- tetradecanoylphorbol- 13 -acetate, 17AAG, 852A, ABI-007, ABR-217620, ABT-751, ADI- PEG 20, AE-941, AG-013736, AGRO100, alanosine, AMG 706, antibody G250, antineoplastons, AP23573, apaziquone, APC8015, atiprimod, ATN-161, atrasenten, azacitidine, BB-10901, BCX-1777, bevacizumab, BG00001, bicalutamide, BMS 247550, bortezomib, bryostatin-1, buserelin, calcitriol, CCI-779, CDB-2914, cefixime, cetuximab, CG0070, cilengitide, clofarabine, combreta
  • VNP40101M volociximab
  • vorinostat VX-680, ZD1839, ZD6474, zileuton, and zosuquidar trihydrochloride.
  • anticancer agents and other therapeutic agents those skilled in the art are referred to any number of instructive manuals including, but not limited to, the Physician's Desk Reference and to Goodman and Gilman's "Pharmaceutical Basis of Therapeutics" tenth edition, Eds. Hardman et al , 2002.
  • the present invention provides methods for administering a compound of the invention with radiation therapy.
  • the invention is not limited by the types, amounts, or delivery and administration systems used to deliver the therapeutic dose of radiation to an animal.
  • the animal may receive photon radiotherapy, particle beam radiation therapy, other types of radiotherapies, and combinations thereof.
  • the radiation is delivered to the animal using a linear accelerator.
  • the radiation is delivered using a gamma knife.
  • the source of radiation can be external or internal to the animal.
  • External radiation therapy is most common and involves directing a beam of high-energy radiation to a tumor site through the skin using, for instance, a linear accelerator. While the beam of radiation is localized to the tumor site, it is nearly impossible to avoid exposure of normal, healthy tissue. However, external radiation is usually well tolerated by animals.
  • Internal radiation therapy involves implanting a radiation-emitting source, such as beads, wires, pellets, capsules, particles, and the like, inside the body at or near the tumor site including the use of delivery systems that specifically target cancer cells (e.g., using particles attached to cancer cell binding ligands). Such implants can be removed following treatment, or left in the body inactive.
  • Types of internal radiation therapy include, but are not limited to, brachy therapy, interstitial irradiation, intracavity irradiation, radioimmunotherapy, and the like.
  • the animal may optionally receive radiosensitizers (e.g. , metronidazole,
  • misonidazole intra-arterial Budr, intravenous iododeoxyuridine (IudR), nitroimidazole, 5- substituted-4-nitroimidazoles, 2H-isoindolediones, [ [(2 -bromoethyl)-amino] methyl] -nitro- lH-imidazole-l-ethanol, nitroaniline derivatives, DNA-affinic hypoxia selective cytotoxins, halogenated DNA ligand, 1,2,4 benzotriazine oxides, 2-nitroimidazole derivatives, fluorine- containing nitroazole derivatives, benzamide, nicotinamide, acridine-intercalator, 5- thiotretrazole derivative, 3-nitro-l,2,4-triazole, 4,5-dinitroimidazole derivative, hydroxylated texaphrins, cisplatin, mitomycin, tiripazamine, nitrosourea,
  • Radiosensitizers enhance the killing of tumor cells. Radioprotectors protect healthy tissue from the harmful effects of radiation.
  • Radiotherapy any type of radiation can be administered to an animal, so long as the dose of radiation is tolerated by the animal without unacceptable negative side-effects.
  • Suitable types of radiotherapy include, for example, ionizing (electromagnetic) radiotherapy (e.g., X-rays or gamma rays) or particle beam radiation therapy (e.g. , high linear energy radiation).
  • Ionizing radiation is defined as radiation comprising particles or photons that have sufficient energy to produce ionization, i.e. , gain or loss of electrons (as described in, for example, U.S.
  • the effects of radiation can be at least partially controlled by the clinician.
  • the dose of radiation is fractionated for maximal target cell exposure and reduced toxicity.
  • the total dose of radiation administered to an animal is about .01 Gray (Gy) to about 100 Gy.
  • about 10 Gy to about 65 Gy e.g. , about 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, 50 Gy, 55 Gy, or 60 Gy
  • a complete dose of radiation can be administered over the course of one day
  • the total dose is ideally fractionated and administered over several days.
  • radiotherapy is administered over the course of at least about 3 days, e.g. , at least 5, 7, 10, 14, 17, 21, 25, 28, 32, 35, 38, 42, 46, 52, or 56 days (about 1-8 weeks).
  • a daily dose of radiation will comprise approximately 1-5 Gy (e.g., about 1 Gy, 1.5 Gy, 1.8 Gy, 2 Gy, 2.5 Gy, 2.8 Gy, 3 Gy, 3.2 Gy, 3.5 Gy, 3.8 Gy, 4 Gy, 4.2 Gy, or 4.5 Gy), or 1-2 Gy (e.g. , 1.5-2 Gy).
  • the daily dose of radiation should be sufficient to induce destruction of the targeted cells.
  • radiation is not administered every day, thereby allowing the animal to rest and the effects of the therapy to be realized.
  • radiation desirably is administered on 5 consecutive days, and not administered on 2 days, for each week of treatment, thereby allowing 2 days of rest per week.
  • radiation can be administered 1 day/week, 2 days/week, 3 days/week, 4 days/week, 5 days/week, 6 days/week, or all 7 days/week, depending on the animal's responsiveness and any potential side effects.
  • Radiation therapy can be initiated at any time in the therapeutic period. In one embodiment, radiation is initiated in week 1 or week 2, and is administered for the remaining duration of the therapeutic period. For example, radiation is administered in weeks 1-6 or in weeks 2-6 of a therapeutic period comprising 6 weeks for treating, for instance, a solid tumor. Altematively, radiation is administered in weeks 1-5 or weeks 2-5 of a therapeutic period comprising 5 weeks.
  • a compound of the invention and one or more therapeutic agents or anticancer agents are administered to an animal under one or more of the following conditions: at different periodicities, at different durations, at different concentrations, by different administration routes, etc.
  • the compound is administered prior to the therapeutic or anticancer agent (e.g., radiation therapy), e.g. , 0.5, 1, 2, 3, 4, 5, 10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks prior to the administration of the therapeutic or anticancer agent.
  • the compound is administered after the therapeutic or anticancer agent, e.g.
  • the compound and the therapeutic or anticancer agent are administered concurrently but on different schedules, e.g., the compound is administered daily while the therapeutic or anticancer agent is administered once a week, once every two weeks, once every three weeks, or once every four weeks. In other embodiments, the compound is administered once a week while the therapeutic or anticancer agent is administered daily, once a week, once every two weeks, once every three weeks, or once every four weeks.
  • the present invention provides a method of characterizing a sample from a subject, comprising: detecting the presence of mutant UCHL1 gene in the sample using a reagent that specifically binds to the mutant UCHL1 gene but not a wild type UCHL1 gene.
  • the mutant UCHL1 gene comprises a rs5030732 mutation.
  • the presence of the mutant UCHL1 gene is indicative of a diagnosis or prognosis of PAH (e.g., diagnosis of PAH, an increased risk of dying from PAH, or an increased severity of PAH).
  • the sample is tissue (e.g., lung tissue, cells, blood, blood products, or urine).
  • the presence of a mutation (e.g., rs5030732 mutation) in a UCHL1 gene is used to determine a treatment course of action (e.g., provide more aggressive therapy when the mutation is present).
  • a treatment course of action e.g., provide more aggressive therapy when the mutation is present.
  • the presence of UCHL1 mutations described herein is detected using a variety of nucleic acid techniques, including but not limited to: nucleic acid sequencing; nucleic acid hybridization; and, nucleic acid amplification.
  • nucleic acid sequencing methods are contemplated for use in the methods of the present disclosure including, for example, chain terminator (Sanger) sequencing, dye terminator sequencing, and high-throughput sequencing methods. Many of these sequencing methods are well known in the art. See, e.g., Sanger et al, Proc. Natl. Acad. Sci. USA 74:5463-5467 (1997); Maxam et al, Proc. Natl. Acad. Sci. USA 74:560-564 (1977);
  • NGS Next-generation sequencing
  • Amplification-requiring methods include pyrosequencing commercialized by Roche as the 454 technology platforms (e.g., GS 20 and GS FLX), the Solexa platform commercialized by Illumina, and the Supported Oligonucleotide Ligation and Detection (SOLiD) platform commercialized by Applied Biosystems.
  • Non-amplification approaches also known as single-molecule sequencing, are exemplified by the HeliScope platform commercialized by Helicos Biosciences, and emerging platforms commercialized by VisiGen, Oxford Nanopore Technologies Ltd., Life Technologies/Ion Torrent, and Pacific Biosciences, respectively.
  • nucleic acid hybridization techniques include, but are not limited to, in situ hybridization (ISH), microarray, and Southern or Northern blot.
  • In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA or RNA strand as a probe to localize a specific DNA or RNA sequence in a portion or section of tissue (in situ), or, if the tissue is small enough, the entire tissue (whole mount ISH).
  • DNA ISH can be used to determine the structure of chromosomes.
  • RNA ISH is used to measure and localize mRNAs and other transcripts within tissue sections or whole mounts. Sample cells and tissues are usually treated to fix the target transcripts in place and to increase access of the probe. The probe hybridizes to the target sequence at elevated temperature, and then the excess probe is washed away.
  • the probe that was labeled with either radio-, fluorescent- or antigen-labeled bases is localized and quantitated in the tissue using either
  • ISH fluorescence microscopy or immunohistochemistry, respectively.
  • ISH can also use two or more probes, labeled with radioactivity or the other non-radioactive labels, to simultaneously detect two or more transcripts.
  • FISH assays utilize bacterial artificial chromosomes (BACs). These have been used extensively in the human genome sequencing project (see Nature 409: 953-958 (2001)) and clones containing specific BACs are available through distributors that can be located through many sources, e.g. , NCBI. Each BAC clone from the human genome has been given a reference name that unambiguously identifies it. These names can be used to find a corresponding GenBank sequence and to order copies of the clone from a distributor.
  • BACs bacterial artificial chromosomes
  • the present invention further provides a method of performing a FISH assay on human cells (e.g., lung cells).
  • human cells e.g., lung cells.
  • Guidance regarding methodology may be obtained from many references including: In situ Hybridization: Medical Applications (eds. G. R. Coulton and J. de
  • kits that are commercially available and that provide protocols for performing FISH assays (available from e.g., Oncor, Inc., Gaithersburg, MD).
  • Patents providing guidance on methodology include U.S. 5,225,326; 5,545,524; 6,121,489 and 6,573,043. All of these references are hereby incorporated by reference in their entirety and may be used along with similar references in the art and with the information provided in the Examples section herein to establish procedural steps convenient for a particular laboratory.
  • DNA microarrays e.g., cDNA microarrays and oligonucleotide microarrays
  • protein microarrays e.g., cDNA microarrays and oligonucleotide microarrays
  • tissue microarrays e.g., tissue microarrays
  • transfection or cell microarrays e.g., cell microarrays
  • chemical compound microarrays e.g., antibody microarrays.
  • a DNA microarray commonly known as gene chip, DNA chip, or biochip, is a collection of microscopic DNA spots attached to a solid surface (e.g. , glass, plastic or silicon chip) forming an array for the purpose of expression profiling or monitoring expression levels for thousands of genes simultaneously.
  • the affixed DNA segments are known as probes, thousands of which can be used in a single DNA microarray.
  • Microarrays can be used to identify mutant UCHL1 genes.
  • Microarrays can be fabricated using a variety of technologies, including but not limiting: printing with fine-pointed pins onto glass slides; photolithography using pre-made masks; photolithography using dynamic micromirror devices; ink-jet printing; or, electrochemistry on microelectrode arrays.
  • Southern and Northern blotting is used to detect specific DNA or RNA sequences, respectively.
  • DNA or RNA extracted from a sample is fragmented, electrophoretically separated on a matrix gel, and transferred to a membrane filter.
  • the filter bound DNA or RNA is subject to hybridization with a labeled probe complementary to the sequence of interest. Hybridized probe bound to the filter is detected.
  • a variant of the procedure is the reverse Northern blot, in which the substrate nucleic acid that is affixed to the membrane is a collection of isolated DNA fragments and the probe is RNA extracted from a tissue and labeled.
  • Nucleic acids may be amplified prior to or simultaneous with detection.
  • Illustrative non-limiting examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), transcription-mediated amplification (TMA), ligase chain reaction (LCR), strand
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription polymerase chain reaction
  • TMA transcription-mediated amplification
  • LCR ligase chain reaction
  • RNA displacement amplification SDA
  • NASBA nucleic acid sequence based amplification
  • a computer-based analysis program is used to translate the raw data generated by the detection assay (e.g. , the presence or absence of a UCHLlmutation into data of predictive value for a clinician.
  • the clinician can access the predictive data using any suitable means.
  • the present invention provides the further benefit that the clinician, who is not likely to be trained in genetics or molecular biology, need not understand the raw data.
  • the data is presented directly to the clinician in its most useful form. The clinician is then able to immediately utilize the information in order to optimize the care of the subject.
  • a sample e.g. , a biopsy or a serum sample
  • a profiling service e.g., clinical lab at a medical facility, genomic profiling business, etc.
  • the subject may visit a medical center to have the sample obtained and sent to the profiling center, or subjects may collect the sample themselves (e.g.
  • the sample comprises previously determined biological information
  • the information may be directly sent to the profiling service by the subject (e.g. , an information card containing the information may be scanned by a computer and the data transmitted to a computer of the profiling center using an electronic communication systems).
  • the profiling service Once received by the profiling service, the sample is processed and a profile is produced (i.e. , mutation data), specific for the diagnostic or prognostic information desired for the subject.
  • the profile data is then prepared in a format suitable for interpretation by a treating clinician.
  • the prepared format may represent a diagnosis or risk assessment (e.g., presence or absence of UCHLl mutations) for the subject, along with recommendations for particular treatment options.
  • the data may be displayed to the clinician by any suitable method.
  • the profiling service generates a report that can be printed for the clinician (e.g., at the point of care) or displayed to the clinician on a computer monitor.
  • the information is first analyzed at the point of care or at a regional facility.
  • the raw data is then sent to a central processing facility for further analysis and/or to convert the raw data to information useful for a clinician or patient.
  • the central processing facility provides the advantage of privacy (all data is stored in a central facility with uniform security protocols), speed, and uniformity of data analysis.
  • the central processing facility can then control the fate of the data following treatment of the subject. For example, using an electronic communication system, the central facility can provide data to the clinician, the subject, or researchers.
  • the subj ect is able to directly access the data using the electronic communication system.
  • the subject may chose further intervention or counseling based on the results.
  • the data is used for research use.
  • the data may be used to further optimize the inclusion or elimination of markers as useful indicators of a particular condition or stage of disease or as a companion diagnostic to determine a treatment course of action.
  • compositions for use in the diagnostic methods described herein include, but are not limited to, probes, amplification oligonucleotides, and the like.
  • kits include all components necessary, sufficient or useful for detecting the markers described herein (e.g., reagents, controls, instructions, etc.). The kits described herein find use in research, therapeutic, screening, and clinical applications.
  • the present invention provides one or more nucleic acid probes or primers having 8 or more (e.g., 10 or more, 12 or more, 15 or more, 18 or more, etc.) nucleotides, and that specifically bind to mutant UCHL1 but not wild type UCHL1
  • Reagents Unless otherwise specified, all reagents were obtained from Sigma- Aldrich (St. Louis, MO). Reagents for SDS-PAGE electrophoresis and western blots supplies were purchased from Thermo Fischer Scientific (Waltham, MA) and Immobilon-P transfer membranes from Millipore (Bedford, MA). Mouse anti-ki-67, anti-UCHLl, anti-Aktl, anti- phospho-Akt (ser473), anti-Ubiquitin rabbit monoclonal antibodies and secondary anti-rabbit horseradish peroxidase (Klionsky DJ, et dk., Autophagy. 2016; 12(l): l-222)-labeled antibody for western blots were all purchased from Cell Signaling Technologies (Boston, MA).
  • VEGF Recombinant VEGF was purchased from Cell Signaling Technologies.
  • LDN57444 was purchased from Calbiochem EMD Millipore (Billercia, MA) and prepared into stock solution in DMSO.
  • iBlot2 premade transfer stack and iBlot2 membrane protein transfer instrument were purchased from Thermo Fischer Scientific.
  • Murine hypoxic PH model All experimental protocols were approved by the Animal Care and Use Committee of the University of Arizona.
  • LDN57444 injections were administered daily starting at week 3 to only WT mice (0.5mg/kg/day, IP) during exposure to both normoxia and hypoxia.
  • mice were housed in a chamber (Plexiglas) open to room air (normoxia) or to 10% inspired O 2 fraction for the 5-week duration (for the hypoxia-exposed conditions).
  • Chronic hypoxia was validated via a Pro:Ox sensor (BioSpherix, Redfield, NY) attached to the chamber after administration of nitrogen to reduce the fractional concentration of O 2 in the inspired gas.
  • Pro:Ox sensor BioSpherix, Redfield, NY
  • MCT Rodent monocrotaline
  • RVSP right ventricular systolic pressures
  • RVSP RVSP was recorded and analyzed using the AcgKnowledge software (Biopac Systems, Aero Camino Goleta, CA). RVSP was used as a surrogate for pulmonary arterial systolic pressure. After pressures were recorded, animals were euthanized by exsanguination, and the heart and lungs were removed en bloc.
  • RV hypertrophy was determined by the ratio of the weight of the RV wall divided by the sum of the weights of the left ventricle and the septum as previously reported (Tang et al, supra; Moreno-Vinasco L, et al., Physiol Genomics. 2008;33(2):278-91). Lungs were perfused with PBS, removed, and frozen in liquid nitrogen for Western blot and real-time RT-PCR analysis, as well as fixed in a 10% normalized formalin solution overnight for morphometric analysis.
  • Paraffin-embedded sections of lung tissue from patients with IP AH and control lungs were obtained from the University of Arizona Pathology Department after approval by the University of Arizona Institutional Review Board.
  • HPAEC human pulmonary artery endothelial cell
  • peripheral lung tissue which were lysed in 1 ⁇ RIPA buffer (Bio- Rad, Hercules, CA). Protein was loaded on to pre-made 4-12% NuPage or Bolt acrylamide gels from Thermo Fischer scientific, and separated at 150 volts and later transferred to a 0.2 ⁇ PVDF membrane from iBlot2 PVDF semi-dry transfer stack using iBlot2 protein transfer equipment and immunoblotted with anti-UCHLl (1 : 1000), anti-phospho-Akt (1 : 1000), and anti-Aktl monoclonal antibody (1 : 1000). Signals were detected using Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). The protein levels were normalized to ⁇ -Actin and expressed in arbitrary units.
  • Co-IP Co-immunoprecipitation
  • HPAECs Human pulmonary artery endothelial cells
  • HPAECs were cultured for 48h to 70% confluency in 100mm cell culture dish in EBM2 -Basal medium with EGMTM 2 Bullet Kit. After mixing an equal volume of basal medium with Gel trex@LDEV -free reduced growth factor basement membrane matrix (Thermo Fischer Scientific), 250 ⁇ of this mixture was used to coat the surface of 24 well cell culture plates (Thermo Fischer scientific). The matrix was allowed to gel at 37 C for 15 min at in a humidified atmosphere at 37°C and 5% C02.
  • HPAECs were then washed in calcium and magnesium-free IX PBS twice and were split into single cell suspensions in VEGF-free EBM2 basal medium and mixed with an equal volume of medium containing either vehicle or VEGF [20ng/ml] or VEGF [20ng/ml] plus LDN57444 [ ⁇ ] .
  • Tube formation was visualized and captured within 20 hours by EVOS FL cell imaging system (Thermo Fischer Scientific).
  • Image J (NIH) with Angiogenesis Analyzer was used to analyze a total 6 images from four different wells (replicates) to determine number of nodes, junctions, branching intervals, meshes, meshes area and segments.
  • RNA from ECs isolated from WT murine lungs exposed to 5 weeks normoxia and hypoxia were extracted using the RNeasy Plus Mini Kit (Qiagen, Germantown, MD).
  • RNA was reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA).
  • Real-time polymerase chain reaction (RT-qPCR) was performed using Taqman assays on a CFX384 Real-Time PCR Detection System (Bio-Rad).
  • Relative mRNA expression levels oiA tl and Gadd45a were normalized to 18S ribosomal RNA and determined by calculating the delta-delta-Ct value, according to manufacturer's guidelines. Differences in gene expression between hypoxic PH and normoxic tissue were calculated using Students t-test, with a -value ⁇ 0.05 considered significant.
  • Gadd45a a well-known demethylating protein
  • FIG. 9 shows that UCHLl inhibition reduces HIF-2a and PDGF levels in murine PH.
  • Both LDN5744 and chronic hypoxia (4 weeks, 10% Fi0 2 ) resulted in increased ubiquintanted Akt levels in whole lungs in mice.
  • HIF-2a up-regulation of HIF-2a in lung ECs ( Figure 9D), a downstream mediator of Akt/mTOR signaling, contributes to severe obliterative intimal remodeling and SMC proliferation in PAH via increased PDGF expression.
  • the genes that actively induce both HIF activities and consequently promote development of PAH remain largely unknown.
  • UCHLl abrogates Von Hippel-Lindau-mediated HIF- ⁇ ⁇ ubiquitination, the regulatory subunit of HIF- ⁇ ⁇ , consequently promoting cancer.
  • HIF-2a protein levels were observed in normoxic and hypoxic (5 wks, 10%FiO 2 ) murine lungs ( Figure 9B and 9C) after UCHLl inhibition (LDN57444-0.5mg/kg/day, daily for last 2 wks, IP).
  • UCHLl inhibition LDN57444-0.5mg/kg/day, daily for last 2 wks, IP.
  • a similar partem of expression was observed in PDGF AA and BB levels.
  • UCHLl mediates increased availability of HIF-2a and PDGF connecting EC-specific UCHLl activities to paracrine mediated SMC processes involved in vascular remodeling (e.g., proliferation).
  • FIG. 10 shows that TGF- ⁇ induces Snail (A) and Vimentin (B) gene expression levels in human lung ECs while LDN57444 reduces these levels. This indicates that reduced EndMT (endothelial to mesenchymal transition) with UCHLl inhibition. EndMT
  • Akt is a canonical mediator of cell survival and proliferation; furthermore, recently Aktl knockout mice have demonstrated reduced PH in a chronic hypoxia model with reduced SMC proliferation (Tang et al, Am J Physiol Lung Cell Mol Physiol, 308(2), L208-220 2015). While most cancer studies have evaluated inhibition of Akt via reductions in its expression or inhibition of its activation (via phosphorylation), there are a paucity of investigations into the degradation of Akt (Chan et al, Cell Biosci, 4(1), 59 2014;
  • This example demonstrates a therapeutic mediator of PH targeting Akt degradation via UCHLl inhibition, an established deubiquitinase in the murine model and in cells from patients with PAH.
  • Gadd45a knockout mice which have reduced UCHLl and Aktl levels, as well as WT mice, which were administered a targeted UCHLl inhibitor, demonstrated reduced RVSP, RVH, and pulmonary artery smooth muscle cell (PASMC) proliferation compared to their respective controls after exposure to chronic hypoxia.
  • UCHLl inhibition led to reductions in angiogenesis and EC proliferation in vitro.
  • Gadd45a knockout mice demonstrated reduced RVSP, RVH, and PASMC proliferation compared to WT mice after exposure to chronic hypoxia. These protective findings were associated with reduced levels of lung-specific expression of Akt in Gadd45a knockout mice compared to WT mice.
  • UCHL1 inhibitor LDN-57444
  • WT mice exposed to chronic hypoxia alleviated RVSP, RVH, and PASMC proliferation.
  • LDN-57444 also reduced VEGF-induced angiogenesis and HPAEC proliferation in vitro.
  • Histology demonstrates a striking predominance of reduced vascular-specific expression profiles of UCHL1 and Aktl levels in knockout mice compared to WT mice and increased EC-specific expression in human PAH samples compared to control samples. Additionally, ECs isolated from WT mice exposed to chronic hypoxia exhibited increases in GADD45a wAAKTl.
  • Akt expression and phosphorylation has been a major focus in several studies; however, substantial gaps remain in elucidating Aktl degradation. It has been established that deubiquitination (DUB) by the DUB enzyme, ubiquitin carboxy -terminal hydrolase LI (UCHL1), is important to Akt degradation in lung pathologies including acute lung injury, lung fibrosis, and most recently, in pulmonary arterial hypertension (PAH) with increased PA vascular remodeling.
  • UCHL1 ubiquitin carboxy -terminal hydrolase LI
  • PAH associations between genotype and indices of PAH were tested using linear regression, adjusting for age, race, sex, indexed pulmonary vascular resistance (PVR), and PAH medication use. PA elastance remained significantly associated with rs5030732 with increases observed in AA/CA genotypes (13% +0.06 mm Hg/mL,
  • Figure 11 shows genotype/phenotype with UCHL1 SNP in PAH.
  • PAH associations between genotype and indices of PAH were tested using linear regression, adjusting for age, race, sex, indexed PVR, and PAH medication use in case only analysis in 67 cases (subset analysis).
  • rs5030732 is a novel missense UCHL1 SNP more prevalent in PAH cases than healthy subjects. As this SNP is known to increase UCHL1 DUB activity, it is contemplated that the observed increased PA elastance in PAH patients is due, at least in part, to rs5030732-mediatd increased Akt signaling.

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Abstract

L'invention concerne des compositions et une méthode de traitement ou de prévention de l'hypertension pulmonaire et du cancer. En particulier, l'invention concerne des compositions, des méthodes et des utilisations de l'inhibition/de l'antagonisme/de la réduction de l'UCHL1 pour le traitement et la prévention de l'hypertension pulmonaire et du cancer.
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WO2024091607A1 (fr) * 2022-10-27 2024-05-02 The Regents Of The University Of Michigan Compositions et méthodes de traitement du cancer du rein

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11559545B2 (en) * 2019-01-29 2023-01-24 The Trustees Of Indiana University Inhibition of UCHL1 in high-grade serous ovarian cancer
WO2024091607A1 (fr) * 2022-10-27 2024-05-02 The Regents Of The University Of Michigan Compositions et méthodes de traitement du cancer du rein

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