WO2019246572A1 - Compositions et méthodes de traitement d'un aldostéronisme primaire - Google Patents

Compositions et méthodes de traitement d'un aldostéronisme primaire Download PDF

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WO2019246572A1
WO2019246572A1 PCT/US2019/038564 US2019038564W WO2019246572A1 WO 2019246572 A1 WO2019246572 A1 WO 2019246572A1 US 2019038564 W US2019038564 W US 2019038564W WO 2019246572 A1 WO2019246572 A1 WO 2019246572A1
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fgfr2
subject
agent
inhibitor
gof
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PCT/US2019/038564
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David T. BREAULT
Leng SINING
Diana L. CARLONE
Emanuele PIGNATTI
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Children's Medical Center Corporation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • PA Primary aldosteronism
  • the invention provides compositions for ameliorating the symptoms of PA and methods for treating PA.
  • PA is the most common form of secondary hypertension and is often associated with hypokalemia and low plasma renin activity. Delays in diagnosis and treatment result in a higher risk of stroke, cardiac failure, and renal damage. Thus, understanding the mechanisms underlying the pathogenesis of PA is crucial to facilitate early diagnosis and to develop alternative treatment strategies.
  • the present inventors generated a mouse model with zG-specific beta-catenin gain-of- function ( CatGOF), which develops the hallmarks of PA, including zG hyperplasia, hyperaldosteronism, and high blood pressure. Using RNA sequencing, the present inventors have identified 790 transcripts that are differentially expressed in CatGOF adrenals, including increased levels of Fibroblast Growth Factor Receptor 2 (Fgfr2).
  • CatGOF zG-specific beta-catenin gain-of- function
  • Results presented herein show that Fgfr2 deletion in the adult adrenal results in disrupted zG morphology and impaired aldosterone production, suggesting an important role for FGFR2 in regulating zG function. These findings suggested that FGFR2 is an important mediator of PA progression driven by WNT/beta-catenin activation, and that its inhibition will effectively block the progression of PA. To test these hypotheses, the present inventors have investigated the mechanism by which FGFR2 regulates aldosterone production and the impact of FGFR2 inhibition on PA progression in CatGOF mice, using genetic ablation and pharmacological inhibition strategies. Results presented herein provide the first molecular insights into how WNT/beta-catenin signaling drives PA progression in vivo. Accordingly, results presented herein provide critical proof-of-principle evidence in support of a novel non-invasive therapeutic strategy for the treatment of hypertension resulting from PA.
  • a method of inhibiting cell proliferation in the zona granulosa comprising contacting a cell of the zona granulosa with an agent that inhibits Fibroblast Growth Factor Receptor 2 (FGFR2) activity.
  • FGFR2 Fibroblast Growth Factor Receptor 2
  • a method of reducing aldosterone secretion in a subject comprising administering to the subject a therapeutically effective amount of an agent that inhibits Fibroblast Growth Factor Receptor 2 (FGFR2) activity.
  • FGFR2 Fibroblast Growth Factor Receptor 2
  • a method for treating primary aldosteronism (PA) in a subject in need thereof comprising administering to the subject a
  • FGFR2 Fibroblast Growth Factor Receptor 2
  • a method for treating primary aldosteronism (PA) in a subject in need thereof comprising treating a subject selected for having elevated aldosterone levels and administering to the subject a therapeutically effective amount of an agent that inhibits Fibroblast Growth Factor Receptor 2 (FGFR2) activity.
  • PA primary aldosteronism
  • the agent is a small molecule, protein, or polynucleotide.
  • the agent binds to FGFR2 and inhibits ligand binding.
  • the agent is a tyrosine kinase inhibitor.
  • the agent is a small molecule inhibitor, an antibody specific for fgfr2, or an antigen-binding fragment of an antibody specific for FGFR2.
  • the small molecule inhibitor is selected from the group consisting of AZD4547, BGJ398 (infigratinib), Alofanib (RPT835), and SSR128129E; and the protein is Bemarituzumab (FPA144).
  • the agent inhibits FGFR2 expression.
  • the agent is a polynucleotide.
  • the agent is an inhibitory nucleic acid molecule.
  • Exemplary inhibitory nucleic acid molecules are oligonucleotides comprising: single and double stranded nucleic acid molecules (e.g., DNA, RNA, and analogs thereof) that bind a nucleic acid molecule that encodes an FGFR2 polypeptide (e.g., antisense molecules, siRNA, shRNA) as well as nucleic acid molecules that bind directly to the polypeptide to modulate its biological activity (e.g., aptamers).
  • the inhibitory nucleic acid molecule is an antisense nucleic acid molecule, siRNA or a vector encoding an inhibitory nucleic acid molecule.
  • the subject is a mammal.
  • the mammal is a human.
  • the agent or composition comprising same is administered intravenously, subcutaneously, intraperitoneally, orally, via inhalation, or locally.
  • the administering involves direct administration into and around the adrenal gland.
  • the direct administration into and around the adrenal gland is achieved via injection or implantation of a drug-delivery device.
  • the subject has at least one of expansion of a zona glomerulosa (zG) expansion in an adrenal gland, hyperaldosteronism, and hypertension.
  • zG zona glomerulosa
  • the subject has resistant hypertension.
  • Resistant hypertension may be used to refer to high blood pressure that requires several medications to control.
  • the method further comprises determining at least one of the subject’s blood pressure, aldosterone levels, and serum renin levels.
  • the aldosterone levels are determined in at least one of the subject’s serum and urine.
  • the method further comprises administering a therapeutically effective amount of at least one additional therapeutic agent.
  • the at least one additional therapeutic agent is an aldosterone blocking drug.
  • the aldosterone-blocking drug is a mineralocorticoid receptor antagonist.
  • FGFR2 fibroblast growth factor receptor 2
  • PA primary aldosteronism
  • fgfr2 fibroblast growth factor receptor 2
  • PA primary aldosteronism
  • fgfr2 fibroblast growth factor receptor 2
  • PA primary aldosteronism
  • composition comprising a therapeutically effective amount of at least one inhibitor of fibroblast growth factor receptor 2 (fgfr2) and a therapeutically effective amount of at least one inhibitor of WNT/beta-catenin signaling pathway and a pharmaceutically acceptable carrier.
  • fgfr2 fibroblast growth factor receptor 2
  • compositions for ameliorating the symptoms of PA and methods for treating PA.
  • compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.
  • ameliorate is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
  • agent any small molecule chemical compound, antibody or functional fragment thereof, nucleic acid molecule, peptide, or polypeptide.
  • alteration is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein.
  • an alteration includes a 10% change in expression levels, more particularly a 25% change, more particularly a 40% change, and most particularly a 50% or greater change in expression levels.
  • an analog is meant a molecule that is not identical, but has analogous functional or structural features.
  • a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding.
  • An analog may include an unnatural amino acid.
  • Detect refers to identifying the presence, absence or amount of the analyte to be detected.
  • disease is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.
  • diseases include PA, both familial forms and non-familial forms of the disease.
  • non-familial forms of PA are most commonly caused by unilateral aldosterone producing adenoma (APA) or bilateral adrenal hyperplasia (BAH), it is envisioned that therapeutic methods described herein may be used to advantage to treat APA and/or BAH.
  • a patient with PA does not have a detectable cancer.
  • an effective amount is meant the amount required to ameliorate the symptoms of a disease relative to an untreated patient.
  • the effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject.
  • the dosage regimen calls for twice daily, once daily, twice per week, or once per week.
  • the duration of treatment may extend from 7-28 days.
  • the duration of treatment may be extended for longer periods of time for the purposes of chronic treatment of, for example, hypertension.
  • a dosage regimen is intermittent, wherein a period during which time an effective amount of active compound(s) is administered is followed by a period of time wherein no active compound is administered.
  • Such an intermittent regimen may be used when the active compound(s) is associated with deleterious side effects.
  • subjects/patients may respond differently to various active compound(s) and thus, sensitivities may also vary in a subject/patient dependent manner.
  • a skilled practitioner may employ an intermittent dosage regimen under circumstances wherein a subject/patient exhibits side effects to an extent unacceptable to the subject/patient.
  • the invention provides a number of targets that are useful for the development of highly specific drugs to treat a disease or a disorder characterized by the methods delineated herein.
  • the methods of the invention provide a facile means to identify therapies that are safe for use in subjects.
  • the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high-volume throughput, high sensitivity, and low complexity.
  • fragment is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide.
  • a fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
  • “Hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.
  • inhibitory nucleic acid is meant a double-stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that when administered to a mammalian cell results in a decrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a target gene.
  • a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule, or an ortholog thereof, or comprises at least a portion of the complementary strand of a target nucleic acid molecule.
  • an inhibitory nucleic acid molecule comprises at least a portion of any or all of the nucleic acids delineated herein.
  • isolated refers to material that is free to varying degrees from components which normally accompany it as found in its native state.
  • Isolate denotes a degree of separation from original source or surroundings.
  • Purify denotes a degree of separation that is higher than isolation.
  • a “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography.
  • the term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel.
  • modifications for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
  • isolated polynucleotide is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene.
  • the term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences.
  • the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
  • an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it.
  • the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.
  • the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention.
  • An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
  • marker is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.
  • “obtaining” as in“obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.
  • Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having“substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity.
  • Polynucleotides having“substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule.
  • hybridize is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency.
  • complementary polynucleotide sequences e.g., a gene described herein
  • stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide.
  • Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C.
  • hybridization time the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA
  • concentration of detergent e.g., sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • Various levels of stringency are accomplished by combining these various conditions as needed.
  • hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS.
  • hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 .mu.g/ml denatured salmon sperm DNA (ssDNA).
  • hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate,
  • wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature.
  • stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.
  • Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C.
  • wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.
  • Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
  • substantially identical is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein).
  • a reference amino acid sequence for example, any one of the amino acid sequences described herein
  • nucleic acid sequence for example, any one of the nucleic acid sequences described herein.
  • such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
  • Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705,
  • BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications.
  • Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine;
  • a BLAST program may be used, with a probability score between e 3 and e 100 indicating a closely related sequence.
  • subject is meant a mammal, including, but not limited to, a human or non human mammal, such as a rodent (e.g., a mouse) bovine, equine, canine, ovine, or feline.
  • rodent e.g., a mouse
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40
  • the terms“treat,” treating,”“treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
  • the term“about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
  • compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
  • FGFR2 Fibroblast Growth Factor Receptor 2
  • FGFR2 polypeptide a cell surface receptor for fibroblast growth factor or a fragment thereof having tyrosine kinase activity and having at least about 85% or greater amino acid sequence identity to Uniprot Identifier P21802-1.
  • An exemplary FGFR2 amino acid sequence is provided below.
  • FGFR2 Fibroblast Growth Factor Receptor 2
  • FGFR2 polypeptide or fragment thereof.
  • An exemplary FGFR2 nucleic acid sequence is provided at NM_000l4l.4, which is reproduced below.
  • gagatttagc agccagaaat gttttggtaa cagaaacaa tgtgatgaaa atagcagact
  • Fig. 1A-1F depicts (A) a schematic of the conditional deletion of Ctnnbl exon 3 following Cre-mediated recombination; (B) a histogram depicting results comparing b wildtype (b WT) and b gain of function (b GOF); and (C)-(F) immunohistochemistry results comparing b WT and b GOF adrenals.
  • Fig. 2A-2F depicts (A) immunohistochemistry results comparing b WT and b GOF adrenal staining patterns; (B) a schematic of functional lineage-tracing experiments using the R26R mTmG reporter allele; (C)-(F) immunohistochemistry results comparing b WT and b GOF adrenals.
  • Fig. 3A-3H depicts (A) - (D) histograms depicting results comparing b WT and b GOF biological read outs; (E) immunohistochemistry results comparing b WT and b GOF adrenals; (F) histograms depicting results comparing b WT and b GOF adult and aged mice; (G) a plot depicting plasma aldosterone levels in b WT and b GOF mice; and (H) histogram plots demonstrating that b-catenin gain-of-function (Bcat-GOF) increases the adrenal NADPH/NADP+ ratio.
  • Fig. 3A-3H depicts (A) - (D) histograms depicting results comparing b WT and b GOF biological read outs; (E) immunohistochemistry results comparing b WT and b GOF adrenals; (F) histograms depicting results comparing b WT and b GOF adult and aged mice;
  • 4A-4F depicts (A) a schematic depicting the generation of Aff re/Cre :: Ctnnb l FloxedEx3 (AS(KO)-Pcat-GOF) mice; (B) a cartoon illustrating RAAS activation; (C) immunohistochemistry results comparing b WT and b GOF adrenals; (D) a histogram depicting results comparing b WT and b GOF biological parameters; (E) immunohistochemistry results comparing b WT and b GOF adrenals; and (F) a histogram depicting results comparing b WT and b GOF.
  • Fig. 5A-5F depicts (A) a cartoon illustrating the aldosterone/Renin/Angll pathway
  • FIG. 1 A cartoon illustrating the experimental design for evaluating the impact of spironolactone on b WT and b GOF mice;
  • C immunohistochemistry results comparing b WT and b GOF adrenals;
  • D a histogram depicting results comparing b WT and b GOF biological parameters;
  • E immunohistochemistry results comparing b WT and b GOF adrenals;
  • F a histogram depicting results comparing b WT and b GOF.
  • Fig. 6A-6D depicts (A) a histogram depicting results comparing b WT and b GOF in young adult, adult, and aged mice; (B) - (C) immunohistochemistry results comparing b WT and b GOF adrenals; and (D) ) a histogram depicting results comparing b WT and b GOF adrenals.
  • Fig. 7A-7D depicts (A) and (C) immunohistochemistry results comparing b WT and b GOF adrenals; and (B) and (D) histograms depicting results comparing b WT and b GOF adrenals.
  • Fig. 8A-8E depicts (A) and (B) histograms depicting results comparing b WT and b GOF mice biological parameters; and (C) - (E) immunohistochemistry results comparing b WT and b GOF adrenals.
  • Fig. 9A-9E depicts (A) and (B) histograms depicting results comparing b WT and b GOF mice adrenal glands; and (C) - (E) immunohistochemistry results comparing b WT and b GOF adrenals.
  • Fig. 10A-10E depicts (A), (C), and (E) immunohistochemistry results comparing b WT and b GOF adrenals; and (B) and (D) histograms depicting results comparing b WT and b GOF mice biological parameters and adrenal gland weight.
  • Fig. 11A-11C depicts transcriptome analysis of b Cat-GOF adrenals.
  • B Functional annotation enrichment analysis using DAVID (v6.8).
  • C RPKM fold change of Fgfr2 and FGF target genes, *, ***, ****, adjusted p-value ⁇ 0.05, 0.001, 0.0001. Error bar, SEM.
  • Fig. 12 depicts a schematic of Fgfr2 cKO mouse model. Cre mediates deletion of exon 8-10, resulting in loss of function of Fgfr2.
  • Fig. 13 depicts zG morphology is disrupted in Fgfr2 cKO adrenals. Immunostaining of laminin bl (Lambl), outlining glomerulus structures in control and Fgfr2 cKO adrenals. Bar, 50pm.
  • Fig. 14A- 14B Fgfr2 deletion causes impaired aldosterone production.
  • Fig. 15 presents histogram plots depicting results comparing aldosterone production and aldosterone to renin ratios in fgfr2 WT and fgfr2 knock out (KO) mice which show that FGFR2 is required for normal aldosterone production.
  • Fig. 16 presents photographs of AS-/- Control and AS-/- cKO mice and a histogram plot depicting results comparing weight gain in AS-/- Control and AS-/- cKO mice.
  • Fig. 17 presents a schematic of the conditional mouse alleles used to generate b Cat- GOF and fgfr2 loss of function (LOF)/KO mice.
  • Fig. 18 presents immunohistochemistry results comparing b GOF and b GOF :: fgfr2c LOF mice adrenals which shows that fgfr2 deletion rescues the b catenin GOF phenotype.
  • Fig. 19 presents immunohistochemistry results comparing b GOF adrenals with and without treatment with the indicated FGFR2 inhibitor and histogram plots depicting these results which show that FGFR2 inhibitor treatment impairs zG proliferation, a hallmark of the b catenin GOF phenotype.
  • FIGs. 20A and 20B illustrate that FGFR inhibitors impair zG function.
  • FIG. 20 A is a plot showing urinary aldosterone levels over time in adult mice treated with AZD4547. The arrow denotes the start of treatments. denotes p ⁇ 0.01 and“*” denotes p ⁇ 0.05.
  • FIG. 20B is a graph showing the fold change in Cypl lb2 and Cyclin Dl transcript levels in adrenals from untreated mice and mice treated with BGJ398 for 7 days. “*” denotes p ⁇ 0.05. Error bars, denote the mean ⁇ SEM.
  • FIG. 21 provides a series of images illustratrating that Fgfr2 deletion prevents zG hyperplasia driven by b-catenin gain-of-function.
  • Representative images of GFP and Dab2 co-immunostaining of control (ASCre/+; mTmG), bCat GOF (ASCre/+; mTmG; b-catenin fl(ex3)/+), and bCat GOF; Fgfr2 cKO (ASCre/+; mTmG; b-catenin fl(ex3)/+; Fgfr2 fl/fl) adult mice adrenals. Bars denote 50 pm DETAILED DESCRIPTION OF THE INVENTION
  • the invention provides compositions for ameliorating the symptoms of PA and methods for treating PA.
  • PA is markedly underdiagnosed and managed with limited medical and surgical options. Apart from familial forms, PA is most commonly caused by unilateral aldosterone producing adenoma (APA) or bilateral adrenal hyperplasia (BAH).
  • APA aldosterone producing adenoma
  • BAH bilateral adrenal hyperplasia
  • the WNT/b- catenin signaling pathway is a well-known regulator of adrenal homeostasis and is found activated in -70% of aldosterone-producing Conn’s adenomas.
  • the present inventors have generated a mouse model with b-catenin gain-of-function ⁇ Cat-GOF) in the adult zG, which develops the hallmarks of PA, including zG expansion, increased plasma aldosterone/renin ratio, and high blood pressure.
  • this model offers a unique opportunity to study the downstream cellular and molecular mechanisms by which WNT ⁇ -catenin signaling leads to PA, and to develop targeted therapies for its treatment.
  • Fgfr2 Fibroblast growth factor receptor 2
  • bCat-GOF adrenals a Fibroblast growth factor receptor 2
  • Fgfr2 has previously been shown to regulate adrenal development and in the adult its expression is restricted to the zG. Genetic deletion of Fgfr2 in the adult adrenal results in disrupted zG morphology and impaired aldosterone production.
  • treatment of mice with pharmacological antagonists of FGFR signaling results in a marked decrease in zG proliferation.
  • FGFR2 is a highly targetable receptor tyrosine kinase. Results from studies presented herein provide important insights into the mechanisms of PA pathogenesis and guidance regarding the development of novel, non- invasive therapeutic strategies for the treatment of PA using FGF inhibitors, including those currently under development for the treatment of cancer.
  • the invention features compositions and methods that are useful for treating PA and ameliorating symptoms thereof, including, without limitation, hypertension, zG hyperplasia, and hyperaldosteronism.
  • the present invention provides methods of treating disease and/or disorders or symptoms thereof which comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the formulae herein to a subject (e.g., a mammal such as a human).
  • a subject e.g., a mammal such as a human
  • a subject e.g., a mammal such as a human
  • a method of treating a subject suffering from or susceptible to a PA or a symptom thereof includes the step of administering to the mammal a therapeutic amount of an amount of a compound herein sufficient to treat the disease or disorder or symptom thereof or a composition comprising same, under conditions such that the disease or disorder is treated.
  • the methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a compound described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).
  • the terms“treat,” treating,”“treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
  • the terms“prevent,”“preventing,”“prevention,”“prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
  • the therapeutic methods of the invention in general comprise administration of a therapeutically effective amount of the compounds described herein, such as, for example a small molecule inhibitor of Fgfr2 to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human.
  • a subject e.g., animal, human
  • Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects "at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like).
  • Inhibitors of Fgfr2 activity are known in the art and include small molecule inhibitors and monoclonal antibodies specific for Fgfr2.
  • Inhibitors of Fgfr2 activity include, without limitation, AZD4547, a tyrosine kinase inhibitor which targets FGFR1-3 (available from AstraZeneca); BGJ398 (infigratinib), a Pan-FGF receptor kinase inhibitor (available from Novartis); Bemarituzumab (FPA144), a monoclonal antibody that binds to FGFR2b preventing binding of certain FGFs (available from Five Prime); Alofanib (RPT835), a novel first-in-class allosteric small-molecular inhibitor of FGFR2 (available from Ruspharmtech); and SSR128129E, an allosteric inhibitor of FGF receptor signaling (available from Sanofi Aventis).
  • AZD4547 may be dosed at l2.5mg/kg once daily, 24d (Gavine et al, 2012, Cancer Res. 72:2045-2056); or in accordance with the following clinical trials: PHASE 1/2, 40, 60, 80mg, 2lday, lowest does resulted in dose limiting toxicity (NCT01824901); PHASE 2, 80mg, 2wk on lwk off (NCT01457846); or phase 1/2, 40, 80mg, tablet twice daily
  • BGJ398 may be dosed at 30mg/kg/day (Guagnano et al., 2011, J Med Chem
  • phase 2 l25mg flat, 3wk on lwk off (NCT02160041) ); the entire content of each of which is incorporated herein by reference.
  • FPAl44/bemarituzumab may be dosed at 5mg/kg bidaily (Gemo et al. 2014); or in accordance with the following clinical trials: phase 1 (NCT03343301); the entire content of each of which is incorporated herein by reference.
  • RPT835/Alofanib may be dosed at 50mg/kg oral daily, l-3wk.
  • RPT835/Alofanib may be dosed at 50mg/kg oral daily, l-3wk.
  • a therapeutically effective amount of at least one inhibitor of Fgfr2 activity or expression is administered to a subject in need thereof.
  • a therapeutically effective amount of at least one inhibitor of Fgfr2 activity is administered to a subject in need thereof in conjunction with a therapeutically effective amount of at least one inhibitor of WNT/beta-catenin signaling.
  • Such a combination may be administered concomitantly or in succession.
  • the invention provides a method of monitoring treatment progress.
  • the method includes the step of determining a level of diagnostic marker (Marker) (e.g., any target delineated herein modulated by a compound herein, a protein or indicator thereof, etc.) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof associated with PA (e.g., hypertension), in which the subject has been administered a therapeutic amount of a compound herein sufficient to treat the disease or symptoms thereof.
  • the level of Marker determined in the method can be compared to known levels of Marker in either healthy normal controls or in other afflicted patients to establish the subject’s disease status.
  • a second level of Marker in the subject is determined at a time point later than the
  • a pre-treatment level of Marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of Marker can then be compared to the level of Marker in the subject after the treatment commences, to determine the efficacy of the treatment.
  • a compound or agent e.g., small molecule inhibitor of Fgfr2
  • a pharmaceutically acceptable salt thereof is formulated with a carrier that is pharmaceutically acceptable and is appropriate for delivering the compound or agent by the chosen route of administration.
  • Suitable pharmaceutically acceptable carriers are those used conventionally with small molecules, such as diluents, excipients and the like. See, for example, "Remington s Pharmaceutical Sciences", l7th Ed., Mack Publishing Company, Easton, Pa., 1995, for guidance on drug formulations.
  • the compounds are formulated for administration by infusion or by injection, either sub-cutaneously or intravenously, and are accordingly utilized as aqueous solutions in sterile and pyrogen-free form and optionally buffered to a slightly acidic or physiological pH.
  • the compounds/agents may be administered in distilled water, saline, buffered saline or 5% dextrose solution. Water solubility of compositions comprising a compound or agent may be enhanced by
  • solubility enhancer such as acetic acid
  • compositions comprising same may be administered via a variety of methods.
  • Such methods include, without limitation, intravesicular, intralesional (in and around an adrenal gland), oral, intravenous (iv), subcutaneous (sc or sq), intraperitoneal, intramuscular intradermal, rectal, nasal, or topical administration, or inhalation via nebulizer or inhaler, to a subject (e.g., a mammal) in need thereof.
  • the therapeutic dosing and regimen best suited for treatment of a subject vary with the disorder or condition to be treated, and according to the patient's weight and other parameters.
  • a dose of at least one compound/agent described herein may, for example, be administered at about 2.5 mg/kg, administered twice daily over 10 days. Smaller doses, e.g., in the pg/kg range, and shorter or longer duration or frequency of treatment, are also envisioned to produce therapeutically useful results, i.e., a statistically significant decrease in hypertension.
  • localized administration to, e.g., at least one adrenal gland may be optimized based on the response of adrenal cells therein.
  • An effective dosage and treatment protocol may be determined by conventional means, starting with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Numerous factors may be taken into consideration by a clinician when determining an optimal dosage for a given subject, including the size, age, and general condition of the patient, the particular disorder being treated, the severity of the disorder, and the presence of other drugs in the patient. Trial dosages may be chosen after consideration of the results of animal studies and the clinical literature.
  • a typical human dose of a compound/agent may be from about 10 pg/kg body weight/day to about 10 mg/kg/day, more particularly from about 50 pg/kg/day to about 5 mg/kg/day, and even more particularly about 100 pg/kg/day to 1 mg/kg/day.
  • Therapeutic efficacy of a compound/agent and/or compositions comprising same may be determined by evaluating and comparing patient symptoms and quality of life pre- and post-administration. Such methods apply irrespective of the mode of administration.
  • pre-administration refers to evaluating patient symptoms and quality of life prior to onset of therapy and post-administration refers to evaluating patient symptoms and quality of life at least 2-8 weeks after onset of therapy.
  • the post-administration evaluating is performed about 2-8, 2-6, 4-6, or 4 weeks after onset of therapy.
  • patient symptoms (e.g., hypertension) and quality of life pre- and post-administration are evaluated via questionnaire assessment.
  • the formulation comprising a compound/agent comprises one or more additional components, wherein the additional component is at least one of an osmolar component that provides an isotonic, or near isotonic solution compatible with human cells or blood, and a preservative.
  • the osmolar component is a salt, such as sodium chloride, or a sugar or a combination of two or more of these components.
  • the sugar may be a monosaccharide such as dextrose, a disaccharide such as sucrose or lactose, a polysaccharide such as dextran 40, dextran 60, or starch, or a sugar alcohol such as mannitol.
  • the osmolar component is readily selected by those skilled in the art.
  • the preservative is at least one of parabens, chlorobutanol, phenol, sorbic acid, and thimerosal.
  • the formulation comprising a compound/agent is in the form of a sustained release formulation and further comprises one or more additional components, wherein the additional component is at least one of an anti-inflammatory agent; and a preservative.
  • the sustained release formulation is administered as a suppository.
  • the sustained release formulation is administered in an implant designed for subcutaneous (sc or sq) implantation.
  • sc or sq subcutaneous
  • Exemplary sc implants are known to those of skill in the art and may involve a port or catheter or the like.
  • the port or catheter is implanted in or near an adrenal gland.
  • Inhibitory nucleic acid molecules are those oligonucleotides that inhibit the expression or activity of an FGFR2 polypeptide.
  • Such oligonucleotides include single and double stranded nucleic acid molecules (e.g., DNA, RNA, and analogs thereof) that bind a nucleic acid molecule that encodes an FGFR2 polypeptide (e.g., antisense molecules, siRNA, shRNA) as well as nucleic acid molecules that bind directly to the polypeptide to modulate its biological activity (e.g., aptamers).
  • Short twenty-one to twenty-five nucleotide double-stranded RNAs are effective at down-regulating gene expression (Zamore et al, Cell 101 : 25-33; Elbashir et al., Nature 411: 494-498, 2001, hereby incorporated by reference).
  • the therapeutic effectiveness of an siRNA approach in mammals was demonstrated in vivo by McCaffrey et al. (Nature 418: 38- 39.2002).
  • siRNAs may be designed to inactivate that gene. Such siRNAs, for example, could be administered directly to an affected tissue, or administered systemically.
  • the nucleic acid sequence of a gene can be used to design small interfering RNAs (siRNAs).
  • the 21 to 25 nucleotide siRNAs may be used, for example, as therapeutics to treat FSHD.
  • the inhibitory nucleic acid molecules of the present invention may be employed as double-stranded RNAs for RNA interference (RNAi)-mediated knock-down of expression.
  • RNAi is a method for decreasing the cellular expression of specific proteins of interest (reviewed in Tuschl, Chembiochem 2:239-245, 2001; Sharp, Genes & Devel.
  • siRNAs introduction of siRNAs into cells either by transfection of dsRNAs or through expression of siRNAs using a plasmid-based expression system is increasingly being used to create loss-of-function phenotypes in mammalian cells.
  • a double-stranded RNA (dsRNA) molecule is made that includes between eight and nineteen consecutive nucleobases of a nucleobase oligomer of the invention.
  • the dsRNA can be two distinct strands of RNA that have duplexed, or a single RNA strand that has self-duplexed (small hairpin (sh)RNA).
  • small hairpin (sh)RNA small hairpin
  • dsRNAs are about 21 or 22 base pairs, but may be shorter or longer (up to about 29 nucleobases) if desired.
  • dsRNA can be made using standard techniques (e.g., chemical synthesis or in vitro transcription).
  • Kits are available, for example, from Ambion (Austin, Tex.) and Epicentre (Madison, Wis.). Methods for expressing dsRNA in mammalian cells are described in Brummelkamp et al. Science 296:550-553, 2002; Paddison et al. Genes & Devel. 16:948-958, 2002. Paul et al. Nature Biotechnol. 20:505-508, 2002; Sui et al. Proc. Natl. Acad. Sci. USA 99:5515-5520, 2002; Yu et al. Proc. Natl. Acad. Sci. USA 99:6047- 6052, 2002; Miyagishi et al. Nature Biotechnol. 20:497-500, 2002; and Lee et al. Nature Biotechnol. 20:500-505 2002, each of which is hereby incorporated by reference.
  • Small hairpin RNAs comprise an RNA sequence having a stem-loop structure.
  • a "stem-loop structure” refers to a nucleic acid having a secondary structure that includes a region of nucleotides which are known or predicted to form a double strand or duplex (stem portion) that is linked on one side by a region of predominantly single-stranded nucleotides (loop portion).
  • the term “hairpin” is also used herein to refer to stem-loop structures. Such structures are well known in the art and the term is used consistently with its known meaning in the art.
  • the secondary structure does not require exact base-pairing.
  • the stem can include one or more base mismatches or bulges.
  • the base-pairing can be exact, i.e. not include any mismatches.
  • the multiple stem-loop structures can be linked to one another through a linker, such as, for example, a nucleic acid linker, a miRNA flanking sequence, other molecule, or some combination thereof.
  • small hairpin RNA includes a conventional stem-loop shRNA, which forms a precursor miRNA (pre-miRNA). While there may be some variation in range, a conventional stem-loop shRNA can comprise a stem ranging from 19 to 29 bp, and a loop ranging from 4 to 30 bp. "shRNA” also includes micro-RNA embedded shRNAs (miRNA-based shRNAs), wherein the guide strand and the passenger strand of the miRNA duplex are incorporated into an existing (or natural) miRNA or into a modified or synthetic (designed) miRNA. In some instances the precursor miRNA molecule can include more than one stem-loop structure.
  • MicroRNAs are endogenously encoded RNA molecules that are about 22 -nucleotides long and generally expressed in a highly tissue- or developmental- stage-specific fashion and that post-transcriptionally regulate target genes. More than 200 distinct miRNAs have been identified in plants and animals. These small regulatory RNAs are believed to serve important biological functions by two prevailing modes of action: (1) by repressing the translation of target mRNAs, and (2) through RNA interference (RNAi), that is, cleavage and degradation of mRNAs. In the latter case, miRNAs function analogously to small interfering RNAs (siRNAs). Thus, one can design and express artificial miRNAs based on the features of existing miRNA genes.
  • RNAi RNA interference
  • shRNAs can be expressed from DNA vectors to provide sustained silencing and high yield delivery into almost any cell type.
  • the vector is a viral vector.
  • Exemplary viral vectors include retroviral, including lentiviral, adenoviral, baculoviral and avian viral vectors, and including such vectors allowing for stable, single-copy genomic integrations.
  • Retroviruses from which the retroviral plasmid vectors can be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
  • a retroviral plasmid vector can be employed to transduce packaging cell lines to form producer cell lines.
  • packaging cells which can be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14c, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy 1 :5-14 (1990), which is incorporated herein by reference in its entirety.
  • the vector can transduce the packaging cells through any means known in the art.
  • a producer cell line generates infectious retroviral vector particles which include polynucleotide encoding a DNA replication protein. Such retroviral vector particles then can be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express a DNA replication protein.
  • Catalytic RNA molecules or ribozymes that include an antisense sequence of the present invention can be used to inhibit expression of a nucleic acid molecule in vivo (e.g., a nucleic acid molecule encoding an FGFR2 polypeptide).
  • a nucleic acid molecule e.g., a nucleic acid molecule encoding an FGFR2 polypeptide.
  • the inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs.
  • the design and use of target RNA-specific ribozymes is described in Haseloff et al, Nature 334:585-591. 1988, and U.S. Patent Application Publication No. 2003/0003469 Al, each of which is incorporated by reference.
  • the invention also features a catalytic RNA molecule that includes, in the binding arm, an antisense RNA having between eight and nineteen consecutive nucleobases.
  • the catalytic nucleic acid molecule is formed in a hammerhead or hairpin motif. Examples of such hammerhead motifs are described by Rossi et al., Aids Research and Human Retroviruses, 8: 183, 1992. Example of hairpin motifs are described by Hampel et al, "RNA Catalyst for Cleaving Specific RNA Sequences," filed Sep. 20, 1989, which is a continuation-in-part of U.S. Ser. No. 07/247,100 filed Sep.
  • any method for introducing a nucleic acid construct into cells can be employed.
  • Physical methods of introducing nucleic acids include injection of a solution containing the construct, bombardment by particles covered by the construct, soaking a cell, tissue sample or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the construct.
  • a viral construct packaged into a viral particle can be used to accomplish both efficient introduction of an expression construct into the cell and transcription of the encoded shRNA.
  • Other methods known in the art for introducing nucleic acids to cells can be used, such as lipid-mediated carrier transport, chemical mediated transport, such as calcium phosphate, and the like.
  • shRNA-encoding nucleic acid construct can be introduced along with components that perform one or more of the following activities: enhance RNA uptake by the cell, promote annealing of the duplex strands, stabilize the annealed strands, or otherwise increase inhibition of the target gene.
  • DNA vectors for example plasmid vectors comprising either an RNA polymerase II or RNA polymerase III promoter can be employed.
  • Expression of endogenous miRNAs is controlled by RNA polymerase II (Pol II) promoters and in some cases, shRNAs are most efficiently driven by Pol II promoters, as compared to RNA polymerase III promoters (Dickins et al, 2005, Nat. Genet. 39: 914-921).
  • expression of the shRNA can be controlled by an inducible promoter or a conditional expression system, including, without limitation, RNA polymerase type II promoters.
  • promoters in the context of the invention are tetracycline- inducible promoters (including TRE-tight), IPTG-inducible promoters, tetracycline transactivator systems, and reverse tetracycline trans activator (rtTA) systems.
  • Constitutive promoters can also be used, as can cell- or tissue-specific promoters. Many promoters will be ubiquitous, such that they are expressed in all cell and tissue types.
  • a certain embodiment uses tetracycline-responsive promoters, one of the most effective conditional gene expression systems in in vitro and in vivo studies. See International Patent Application
  • Naked polynucleotides, or analogs thereof, are capable of entering mammalian cells and inhibiting expression of a gene of interest. Nonetheless, it may be desirable to utilize a formulation that aids in the delivery of oligonucleotides or other nucleobase oligomers to cells (see, e.g., U.S. Pat. Nos. 5,656,611, 5,753,613, 5,785,992, 6,120,798, 6,221,959, 6,346,613, and 6,353,055, each of which is hereby incorporated by reference).
  • oligonucleotides induce the cleavage of RNA by RNase H: polydeoxynucleotides with phosphodi ester (PO) or phosphorothioate (PS) linkages.
  • PO phosphodi ester
  • PS phosphorothioate
  • 2'-OMe-RNA sequences exhibit a high affinity for RNA targets, these sequences are not substrates for RNase H.
  • a desirable oligonucleotide is one based on 2'-modified oligonucleotides containing oligodeoxynucleotide gaps with some or all intemucleotide linkages modified to phosphorothioates for nuclease resistance. The presence of
  • methylphosphonate modifications increases the affinity of the oligonucleotide for its target RNA and thus reduces the IC50. This modification also increases the nuclease resistance of the modified oligonucleotide.
  • CMAS covalently-closed multiple antisense
  • SiAS ribbon-type antisense
  • nucleoside is a nucleobase-sugar combination.
  • the base portion of the nucleoside is normally a heterocyclic base.
  • the two most common classes of such heterocyclic bases are the purines and the pyrimidines.
  • Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.
  • the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar.
  • the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound.
  • the respective ends of this linear polymeric structure can be further joined to form a circular structure; open linear structures are generally preferred.
  • the phosphate groups are commonly referred to as forming the backbone of the oligonucleotide.
  • the normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.
  • nucleobase oligomers useful in this invention include oligonucleotides containing modified backbones or non-natural intemucleoside linkages.
  • nucleobase oligomers having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
  • modified oligonucleotides that do not have a phosphorus atom in their intemucleoside backbone are also considered to be nucleobase oligomers.
  • Nucleobase oligomers that have modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,
  • thionoalkylphosphonates thionoalkylphosphotriest- ers, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity, wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.
  • Various salts, mixed salts and free acid forms are also included.
  • Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos.
  • Nucleobase oligomers having modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages.
  • oligonucleotides include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH.sub.2 component parts.
  • Representative United States patents that teach the preparation of the above oligonucleotides include, but are not limited to, U.S. Pat. Nos.
  • nucleobase oligomers In other nucleobase oligomers, both the sugar and the intemucleoside linkage, i.e., the backbone, are replaced with novel groups.
  • the nucleobase units are maintained for hybridization with a gene encoding an FGFR2 polypeptide.
  • One such nucleobase oligomer is referred to as a Peptide Nucleic Acid (PNA).
  • PNA Peptide Nucleic Acid
  • the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an
  • nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Methods for making and using these nucleobase oligomers are described, for example, in "Peptide Nucleic Acids: Protocols and Applications” Ed. P. E. Nielsen, Horizon Press, Norfolk, United Kingdom, 1999. Representative United States patents that teach the preparation of PNAs include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497-1500.
  • the nucleobase oligomers have phosphorothioate backbones and nucleosides with heteroatom backbones, and in particular - CH2-NH-O-CH2-, -CH2-N(CH3)-0-CH2- (known as a methylene (methylimino) or MMI backbone), -CH 2 -0-N(CH 3 )-CH 2 -, -CH 2 -N(CH 3 )-N(CH 3 )-CH 2 -, and -0-N(CH 3 )-CH 2 -CH 2 -.
  • the oligonucleotides have morpholino backbone structures described in U.S. Pat. No. 5,034,506.
  • Nucleobase oligomers may also contain one or more substituted sugar moieties. Nucleobase oligomers comprise one of the following at the 2' position: OH; F; O-, S-, or N- alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl may be substituted or unsubstituted Ci to C10 alkyl or C2 to C10 alkenyl and alkynyl.
  • n and m are from 1 to about 10.
  • nucleobase oligomers include one of the following at the 2' position: Ci to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, or O- aralkyl, SH, SOU, OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCTU, S0 2 CH 3 , ONO2, NO2, NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the following at the 2' position: Ci to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, or O- aralkyl, SH, SOU, OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCTU, S0 2 CH 3 , ONO2, NO2,
  • modifications are 2'-0-methyl and 2'-methoxyethoxy (2'-0- CH2CH20CH 3 , also known as 2'-0-(2-methoxyethyl) or 2'-MOE).
  • Another desirable modification is 2'-dimethylaminooxyethoxy (i.e., 0(CH2) 20N(CH 3 ) 2), also known as 2'- DMAOE.
  • Other modifications include, 2'-aminopropoxy (2'-OCH2CH2CH2NH2) and 2'- fluoro (2'-F). Similar modifications may also be made at other positions on an
  • nucleobase oligomers may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos.
  • Nucleobase oligomers may also include nucleobase modifications or substitutions.
  • "unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me- C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine and 2-thiocytosine; 5-halouracil and cytosine; 5- propynyl uracil and cytosine; 6-azo uracil, cytosine and thymine; 5-uracil (pseudouracil); 4- thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines; 5-halo (e.g., 5-bromo), 5-trifluoromethyl
  • nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al, Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of an antisense oligonucleotide of the invention.
  • nucleobase oligomer of the invention involves chemically linking to the nucleobase oligomer one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide.
  • moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al, Proc. Natl. Acad. Sci. USA, 86:6553-6556, 1989), cholic acid (Manoharan et al, Bioorg. Med. Chem.
  • a thioether e.g., hexyl-S-tritylthiol
  • Manoharan et al Ann. N.Y. Acad. Sci., 660:306-309, 1992; Manoharan et al., Bioorg. Med. Chem. Let., 3:2765-2770, 1993
  • a thiocholesterol Olet al, Nucl.
  • the present invention also includes nucleobase oligomers that are chimeric compounds.
  • "Chimeric" nucleobase oligomers are nucleobase oligomers, particularly oligonucleotides, that contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide.
  • These nucleobase oligomers typically contain at least one region where the nucleobase oligomer is modified to confer, upon the nucleobase oligomer, increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid.
  • An additional region of the nucleobase oligomer may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
  • RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of nucleobase oligomer inhibition of gene expression. Consequently, comparable results can often be obtained with shorter nucleobase oligomers when chimeric nucleobase oligomers are used, compared to phosphorothioate deoxy oligonucleotides hybridizing to the same target region.
  • Chimeric nucleobase oligomers of the invention may be formed as composite structures of two or more nucleobase oligomers as described above. Such nucleobase oligomers, when oligonucleotides, have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference in its entirety.
  • nucleobase oligomers used in accordance with this invention may be any nucleobase oligomers.
  • nucleobase oligomers of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption.
  • Results presented herein show that Fgfr2 deletion in the adult adrenal results in disrupted zG morphology and impaired aldosterone production, suggesting an important role for FGFR2 in regulating zG function. These findings suggested that FGFR2 is an important mediator of PA progression driven by WNT/beta-catenin activation, and that its inhibition will effectively block the progression of PA. To test these hypotheses, the present inventors have investigated the mechanism by which FGFR2 regulates aldosterone production and the impact of FGFR2 inhibition on PA progression in CatGOF mice, using genetic ablation and pharmacological inhibition strategies. Results presented herein provide the first molecular insights into how WNT/beta- catenin signaling drives PA progression in vivo.
  • results presented herein provide critical proof-of-principle evidence in support of a novel non-invasive therapeutic strategy for the treatment of hypertension resulting from PA.
  • Example 1 Stabilization of b-catenin in zG cells results in ectopic accumulation of the zG.
  • mice with zG-specific stabilization of b- catenin [Cypl lb2(AS) Cre/+ :: Ctnnb l FloxedEx3 ) (pcat-GOF] ( Figure 1A ⁇ .
  • conditional deletion of Ctnnbl exon 3 following Cre-mediated recombination results in stabilization of b- catenin and constitutive activation of the canonical (c)WNT pathway) specifically within zG cells ( Figure 1A).
  • Increased expression of Axin2 and Lefl, well-established targets of cWNT signaling, confirmed activation of this pathway in Pcat-GOF adrenals ( Figure IB).
  • the present inventors examined the expression pattern of Aldose reductase-related protein 1 (Akrlb7), a zF cell marker, which revealed mutually exclusive expression of b-catenin and Akrlb7 in both Pcat- WT and Pcat-GOF adrenals ( Figure IF). Finally, since the histomorphometry of the orthotopic zG and zF are notably distinct, the present inventors analyzed the cellular density of the ectopic domain by assessing the number of DAPI-positive nuclei per unit area, which was
  • Example 2 Stabilization of b-catenin in zG cells blocks zG-to-zF cell transdifferentiation.
  • the present inventors To understand the cellular mechanisms underlying the ectopic accumulation of zG cells in Pcat-GOF mice, the present inventors first tested the hypothesis that accumulation of zG cells resulted from increased proliferation of zG cells in response to the oncogenic activity of stabilized b-catenin. To address this, the present inventors determined the proliferation index in bq3R ⁇ UT and bq8uOOR adrenals by scoring the number of Ki67-positive cells per zG area, defined by the b-catenin expression domain. Surprisingly, the proliferation index in bq8uOOR adrenals was reduced compared to bq8RnnT controls ( Figure 2A), ruling out increased cell division as the explanation for the expanded zG.
  • Example 3 Stabilization of b-catenin in zG cells results in increased aldosterone and high blood pressure.
  • Example 4 Stabilization of b-catenin in zG cells does not lead to autonomous aldosterone production.
  • Example 5 Stabilization of b-catenin in zG cells is not associated with increased CYP11B2 expression.
  • Example 6 Stabilization of b-catenin does not change zG responsiveness to Angll.
  • Pcat-GOF might lead to an increase in Angll responsiveness of zG cells leading to an increase in aldosterone production on a per cell basis.
  • Pcat-WT and Pcat-GOF mice were subjected to intravascular volume depletion (using either a low salt diet or acute water restriction) to activate the renin- angiotensin-aldosterone system (RAAS).
  • RAAS renin- angiotensin-aldosterone system
  • Immunostaining for CYPllB2-expressing cells revealed the expected overall increase in the number of cells in response to volume depletion, including in the expanded zG domain, but no difference in the absolute number of CYP11B2- expressing cells between Pcat-WT and Pcat-GOF mice (Figure 8D-E).
  • Example 7 Stabilization of b-catenin in zG cells leads to increased NADPH and
  • Example 8 Chronic activation of RAAS in ficat-GOF mice leads to accelerated zG
  • AS(KO) AS Cre / Cre mice
  • CYP11B2 deficiency results in a ⁇ 3 fold increase in plasma renin activity and generated A ⁇ f re/Cre : : ( 'innh 1 ' AS ( K 0 ) - b ca t- G 0 F) mice
  • spironolactone a mineralocorticoid receptor antagonist
  • a low-salt diet two treatments commonly prescribed for patients with hyperaldosteronism.
  • spironolactone a mineralocorticoid receptor antagonist
  • a low-salt diet two treatments commonly prescribed for patients with hyperaldosteronism.
  • bcat-WT and bq8 ⁇ -OOR mice were treated with spironolactone for 50 days ( Figure 5, A, B).
  • Cypllb2 transcript levels and the overall number of CYPllB2-expressing cells were increased in both bcat-WT and bq8 ⁇ -OOR mice, compared to non-treated controls ( Figure 10A and 10B).
  • spironolactone-treated bq8 ⁇ -OOR mice demonstrated increased adrenal mass compared with age-matched
  • Example 9 Transcriptome analysis of ⁇ Cat-GOF adrenals revealed upregulation of FGF Signaling.
  • the present inventors performed RNA sequencing analysis on whole adrenal mRNA from the present inventors pCat-GOF and control animals. Differential expression analysis revealed 790 unique genes significantly changed in pCat-GOF adrenals compared to controls ( Figure 11 A). The present inventors further analyzed the data for enrichment of Gene Ontology and KEGG pathway annotations and found significant enrichment in genes associated with pathways such as WNT signaling, MAPK signaling, as well as biological processes such as cell adhesion, migration, and morphogenesis (Figure 11B).
  • Fibroblast growth factor receptor 2 (Fgfr2) expression is significantly upregulated in pCat-GOF adrenals ( Figure 11C), while the expression of other FGFRs and FGF ligands are unchanged. Further to this point, FGF-activated ETV transcription factors, Etv4 and Etv5, were both significantly upregulated in pCat-GOF adrenals, along with other FGF-regulated target
  • Example 10 FGFR2 regulates aldosterone production in adult mice.
  • FGFR2 signaling in mediating PA progression in pCat-GOF mice, the present inventors first sought to determine its function in wild type animals.
  • the function of FGFR2 in adult adrenals was evaluated by generating a zG-specific knockout mouse model (ASCre/+ : : Fgfr2flox/flox mice, hereafter referred to as Fgfr2 cKO).
  • Fgfr2 cKO zG-specific knockout mouse model
  • Example 11 Evaluation of the effects of inhibiting FGFR2 signaling on PA progression in Cat-GOF mice.
  • Results presented herein indicate that activation of Wnt/p-catenin signaling in zG cells, which leads to PA in mice, may result from activation of FGFR2 and its downstream signaling. Targeting the FGFR2 signaling pathway, therefore, represents a robust and non- invasive therapeutic strategy for the treatment of patients with PA.
  • the present inventors investigated this therapeutic avenue by experimentally testing whether inhibition of FGFR2 signaling prevents and/or reverses the progression of PA in pCat-GOF animals.
  • the present inventors employed two complementary strategies to inhibit FGFR2 signaling in pCat-GOF mice: (1) genetic ablation using th c Fgfr2flox conditional allele and (2) pharmacological inhibition using selective FGFR small molecule inhibitors.
  • the first strategy of using genetic ablation the present inventors assessed whether concomitant Fgfr2 deletion in pCat-GOF animals can prevent the onset of PA including zG expansion, hyperaldosteronism, and high blood pressure.
  • the conditional mouse alleles and crossing to achieve this plan experimentally are shown in schematic in Figure 17.
  • Fgfr2 cKO mice Male ririCre/Cre :: Fgfr2+/flox mice were bred with female Fgfr2flox/flox : : fi-catenin(Ex3)flox/flox mice to generate ASCre/+ ::
  • Fgfr2flox/flox ficatenin (Ex3)flox/+ double mutant mice (pCat-FKO) and ASCre/+ ::
  • Fgfr2+/flox fi-catenin(Ex3)flox/+ single mutant mice (pCat-GOF).
  • Results presented in Figure 18 show that Fgfr2 deletion in pCat-GOF rescues the pcatenin GOF phenotype.
  • the present inventors used pCat-GOF and ASCre/+ (pCat-Ctrl) mice.
  • FGFR inhibitors are widely studied chemotherapeutic agents
  • Both small molecule agents are currently in phase II clinical trials for the treatment of various malignancies and are second generation FGFR inhibitors that target FGFR1/2/3 with high selectivity and minimum off- target effects.
  • both compounds demonstrate strong antitumor effects at a dose of lOmg/kg/day with minimum toxicity for a maximum period of 28 days.
  • Age and sex-matched adult pCat-Ctrl mice were treated with vehicle or lOmg/kg inhibitor (AZD4547 or BGJ398) daily by oral gavage for 12 days.
  • Figure 19 depicts the results of experiments wherein the effects of AZD4547 and BGJ398 were evaluated in the context of pCat-Ctrl adrenals.
  • Aldosterone levels for the treatment mice and control mice were compared using two-way ANOVA analysis, followed by Bonferroni’s multiple comparison correction test. Decreased aldosterone levels were observed in treated mice compared to untreated mice (FIG. 20A).
  • An additional cohort of mice were treated with 30 mg/kg/day p. o. of BGJ398 for 7 days.
  • plasma aldosterone levels and blood pressure in pCat-GOF animals can be determined before and after administration of at least one FGFR inhibitor.
  • both female and male mice are studied at 12-15 weeks of age.
  • Adrenal collection and statistical analyses is performed as described herein above.
  • blood is collected retro-orbitally from conscious mice using EDTA- containing capillary tubes and centrifuged to obtain plasma.
  • Aldosterone levels are determined using a radioimmunoassay (RIA; IBL International). Renin activity is determined by RIA (IBL International) for Angiotensin I generated by incubating plasma with excess angiotensinogen.
  • RIA radioimmunoassay

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Abstract

L'invention concerne des méthodes de traitement d'un aldostéronisme primaire (PA) par l'administration d'une quantité thérapeutiquement efficace d'au moins un inhibiteur du récepteur 2 du facteur de croissance des fibroblastes ou d'une composition associée. L'invention concerne également des compositions comprenant une quantité thérapeutiquement efficace d'au moins un inhibiteur du récepteur 2 du facteur de croissance des fibroblastes, une quantité thérapeutiquement efficace d'au moins un inhibiteur de la signalisation WNT/β-caténine, et un excipient pharmaceutiquement acceptable. De telles compositions sont utilisées pour atténuer les symptômes de PA, ce qui permet de traiter la maladie.
PCT/US2019/038564 2018-06-22 2019-06-21 Compositions et méthodes de traitement d'un aldostéronisme primaire WO2019246572A1 (fr)

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