WO2020006336A1 - Treatment of cardiovascular calcification using nuclear export inhibitors - Google Patents

Abstract

A method of treating or preventing cardiovascular calcification in a subject is described that includes administering to a subject in need thereof a therapeutically effective amount of a nuclear export inhibitor. In particular, the method is useful for treating or preventing calcific aortic valve disease by inhibiting activated canonical Wnt signaling.

Description

TREATMENT OF CARDIOVASCULAR CALCIFICATION USING NUCLEAR

EXPORT INHIBITORS

STATEMENT ON FEDERALLY FUNDED RESEARCH

[0001] This invention was made with government support under grant number R01 HL0127033 awarded by the National Institutes of Health. The Government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62/692,083, filed June 29, 2018, which is incorporated herein by reference in its entirety.

BACKGROUND

[0003] Heart valve disease results in over 23,000 annual deaths in the United States with calcific aortic valve disease (CAVD) being the most prevalent. There is no pharmacological treatment to prevent or reverse CAVD and therefore surgical intervention remains the only effective option which comes with insuperable complications and no guarantee of long-term success. Unfortunately, the development of alternative, mechanistic-based pharmacological therapies for CAVD has been hindered by our limited understanding of disease processes.

[0004] In healthy individuals, valve leaflets maintain unidirectional blood flow by sustaining a highly organized extracellular matrix (ECM) structure. Collagen, proteoglycan and elastin fiber layers confer distinct biomechanical properties that allow the leaflets to open and close. The major resident cell type responsible for ECM homeostasis is the valve interstitial cell (VIC) that resembles a fibroblast in healthy individuals. Rabkin-Aikawa et al ., J Heart Valve Dis., 13 :841-7 (2004). In CAVD, VICs transition towards an osteoblast-like cell as indicated by Runx2 expression. Rajamannan et al., Circulation, 124: 1783-91 (2011) This phenotypic change is associated with perturbations in ECM organization including calcific nodule formation on the aortic surface leading to stenosis.

[0005] At present there are no effective treatments other than surgical repair and replacement, and unfortunately replacement valves are often temporary due to their inability to grow, remodel, or withstand life-long mechanical demands in vivo , and therefore alternative approaches are required. The development of non-invasive approaches would be significantly improved by a more complete understanding of the molecular and cellular mechanisms that promote onset and progression of CAVD in vivo.

SUMMARY OF THE INVENTION

[0006] Calcific Aortic Valve Disease (CAVD) affects more than 5.2 million people living in the US and prevalence is increasing. There is no pharmacological therapy available to treat calcification and the beneficial effects of statins remains unsubstantiated and the field continues to explore better alternatives. However, to date none have been found and surgical repair or replacement remains the most effective treatment. Unfortunately, this is often impermanent and poses additional health risks particularly in the affected aging population. The inventors therefore sought to determine if the pharmacological XPOl (or CRM1) nuclear export inhibitor cancer drug (KPT-330, Selinexor) is beneficial in the treatment of CAVD.

[0007] The inventors demonstrate that KPT-330 has beneficial effects in preventing, attenuating and treating calcific nodule formation in porcine and human aortic valve interstitial cells cultured under osteogenic stimulus. In addition, KPT-330 is sufficient to prevent calcification of the aortic valve annulus in an established mouse model ( Klotho ^_/ ) in vivo. Previous studies have shown that increased canonical Wnt signaling is characteristic of CAVD. The inventors therefore have used a combination of RNA-sequencing and Mass Spectrometry to show that KPT- 330’ s prevents CAVD by repressing activated Wnt signaling and subsequently decreasing expression of pro-osteogenic markers and cell proliferation, potentially via the transcription factor C/EBRb

[0008] These findings have met the critical need to discover alterative, pharmacological-based therapies in the treatment of CAVD and demonstrated that targeting Wnt inhibition has beneficial effects in the development and progression of calcific nodule formation. BRIEF DESCRIPTION OF THE FIGURES

[0009] The present invention may be more readily understood by reference to the following drawings, wherein:

[0010] Figure 1 provides a scheme showing the effect of KPT-330 in preventing cardiovascular calcification.

[0011] Figures 2A-2X provide graphs and images showing an In vitro model of VIC calcific nodule formation. ( A-F) pAVICs were plated on glass in conditioned media. Bright field (A-C) and Alizarin Red stained images (D-F) of pAVICs at Stage I (~5 days), Stage II (~8-9 days), Stage III (1-0-12 days). Arrows depict quiescent (Stage I), pre-calcific (Stage II), and calcific nodules (Stage III) (n>3). (G-L) Immunofluorescence images to detect Runx2 (green), Osteopontin (red), or Cadherin-l 1 (red) and DAPI (blue) at Stage I and III. DAPI highlight nuclei in blue (n=3). (M) Cell proliferation assay of pAVICs harvested at Stage I and III (n=3) *, indicates P<0.0l compared to Stage I. (V) Heat map and ( O ) Venn diagram of the RNA-Seq analysis (n=3). (P-U) Immunofluorescence images to detect Osteomodulin (red) and Sclerostin (green) in pAVICs fixed at Stages I, II and III. Pentachrome (V), Alizarin red (W), and Immunofluorescence of Sclerostin (red) and Runx2 (green) (X) in a human aortic valve isolated from a rheumatic disease patient with aortic stenosis. Black arrow in W indicates calcific lesions and white arrow in X indicates co localization of Runx2 and Sclerostin.

[0012] Figures 3A-3W provide graphs and images showing KPT-330 prevents calcific nodule formation and reduces proliferation. (A) Schematic of the experimental timeline. ( B-E ) Bright field ( B , C) and Alizarin Red (D, E) images from DMSO or KPT-330 treated cells plated on glass. Arrows indicate Alizarin Red positive nodules (n>3). (F) Quantification of Alizarin Red positive nodules in pAVICs treated with increasing doses of KPT-330 (n=3), *p>0.05 compared to vehicle. (_' G-J) Immunofluorescence images of pAVICs to detect Sclerostin (green) or Osteomodulin (red) and DAPI (blue) (arrows showing calcified nodules) after DMSO or KPT-330 treatment stained with (n=3). (K-N) Alizarin Red staining of healthy and aortic stenosis (AS) human aortic VICs treated with DMSO or KPT-330 in OM (n=3). (0-R) Alizarin Red staining of pAVICs treated with DMSO or KPT-330 in OM ( O , P ) or inorganic phosphate ( Q , R ) (n=3). ( S) Luminescence from Caspase-glo 3/7 assay in pAVICs treated with DMSO or KPT-330 plated on glass (n=3). (7) Cell proliferation assay performed in in pAVICs treated with DMSO or KPT-330 plated on glass (n=3), *p>0.05 compared to DMSO. (U, V) Immunofluorescence images of pAVICs fixed after DMSO or KPT-330 treatment in OM, stained with pHH3 (red) and DAPI (blue) (n=3). (W) Quantification of data presented in U, V. *p>0.05 compared to DMSO.

[0013] Figures 4A-40 provide schemes and images showing KPT-330 attenuates and rescues nodule formation in vitro. (A) Schematic of the experimental timeline utilized in B-E. ( B-E ) Bright field ( B , C) and Alizarin Red (D, E) images from DMSO or KPT-330 treated cells at Stage II, plated on glass. Arrows indicate Alizarin Red positive nodules (n>3). (F) Schematic of the experimental timeline utilized in G-J. (G-J) Bright field (G, H) and Alizarin Red (I, ./) images from DMSO or KPT-330 treated cells at Stage III, plated on glass. Arrows indicate Alizarin Red positive nodules (n>3). ( K) Quantification of Alizarin Red positive nodules in pAVICs before and after KPT-330 treatment (n=3), *p>0.05 compared to before KPT-330 treatment. (L) Schematic of the experimental timeline utilized in M-O. ( M-0 ) pAVICs were treated with KPT-330 after calcific nodule formation for 72 hours and then re-plated with DMSO or KPT-330 treatment long term (14 days) to observe any re-appearance of nodules. Alizarin Red stained images are shown.

[0014] Figures 5A-5D provide schemes and images showing KPT-330 prevents aortic valve calcification in Klotho mice. (A) Schematic showing treatment paradigm in Klotho mice (B, B’) Low and high magnification Alizarin Red images of Klotho mice treated with vehicle. Arrows show presence of annular calcification and arrow heads show vascular calcification. (C, C’) Low and high magnification alizarin red images of Klotho mice treated with 30 mg/kg KPT-330. Arrows show absence of annular calcification and arrow heads show vascular calcification (n=6). ( D ) Quantification of Alizarin Red intensity within the aortic valve annular region of vehicle and KPT-330 treated Klotho mice (n=6).

[0015] Figures 6A-6G provide graphs and images showing KPT-330 retains C/EBRb in the nuclei of VICs. (A) Graph showing C/EBRb protein intensity from Mass Spec in Stages I and III (comparing non-calcified v/s calcified). ( B ) Graph showing C/EBRb protein intensity from Mass Spec in Stage III and Stage I+KPT-330 (comparing calcified v/s prevention i.e KPT-330 added at Stage I). (C) Graph showing C/EBRb protein intensity from Mass Spec in Stage III and Stage III+KPT-330 (comparing calcified v/s rescue i.e KPT-330 added at Stage III), p>0.05 compared to Stage I (A) or Stage III ( B , C). (D) Western Blot analysis and quantitation (E) of C/EBRb in cytoplasmic and nuclear lysates from pAVICs cultured in conditioned media (CM), osteogenic media (OM) or OM + KPT-330. (F) (H) Alizarin Red staining and quantitation (G) of pAVICs cultured in CM, OM alone, or OM + 400ng C/EBRb.

[0016] Figures 7A-7K provide graphs and images showing KPT-330 represses canonical Wnt signaling in pAVICs. (A) Western Blot analysis and quantitation (B) of active b-catenin expression in pAVICs cultured in OM alone, or OM + 200ng C/EBRb. (C) RPKM values obtained from RNA- Seq data for Wnt signaling mediator and regulatory genes, *p>0.05 compared to previous time point, #p>0.05 compared to Stage III. (D) Western Blot from nuclear and cytoplasmic fractions of pAVICs harvested at Stage I, Stage III, or KPT-330 treated at Stage I (n=3). (E) Quantitation of Western Blot shown in ( D ), *p>0.05 compared to Stage I. (F) Western Blot from whole cell lysates of pAVICs cultured in complete media (CM), osteogenic media (OM) or OM with KPT-330 (n=3). (G) Quantitation of Western Blot shown in ( D ), *p>0.05 compared to CM. ( H) Luminescence as %RLU/mg (relative light units normalized to protein concentration calculated via BCA assay) in pAVICs transfected with TOP/FOP Flash plasmids and Renilla plasmid treated with DMSO or KPT-330 in OM. LiCh treatment was performed for 24 hours to activate Wnt signaling (n=3), *p>0.05 compared to TOP -Flash, #>0.05 compared to DMSO. (I) Quantification of Alizarin Red intensity in pAVICs cultured in OM or OM with XAV-939 (n=3), *p>0.05 compared to OM. (./) Quantification of pHH3 positive pAVICs cultured in OM or OM with XAV-939 (n=3), *p>0.05 compared to OM. ( K) Quantification of the number of Alizarin Red positive nodules from pAVICs plated on glass and treated with various LiCh concentrations (n=3).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0017] The terminology as set forth herein is for description of the embodiments only and should not be construed as limiting of the invention as a whole. As used in the description of the invention and the appended claims, the singular forms“a”,“an”, and“the” are inclusive of their plural forms, unless contraindicated by the context surrounding such.

[0018] “Treat”,“treating”, and“treatment”, etc ., as used herein, refer to any action providing a benefit to a subject at risk for or afflicted with a condition or disease such as calcific aortic valve disease, including improvement in the condition through lessening or suppression of at least one symptom, delay in progression of the disease, prevention or delay in the onset of the disease, etc. The subject may be at risk due to the presence of a risk factor such as being over 65, male gender, being a smoker, being genetically predisposed to calcific aortic valve disease, and so on.

[0019] The term“in need of treatment” as used herein refers to a j udgment made by a caregiver that a patient requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a caregiver’s expertise, but that includes the knowledge that the patient is ill, or will be ill, as the result of a disease or condition that is treatable by a method or compound of the disclosure.

[0020] As used herein, the term“prevention” includes either preventing or decreasing the likelihood or severity of the onset of overgrowth by cardiovascular calcification altogether or preventing or decreasing the likelihood or severity of the onset of cardiovascular calcification in a subject. This includes prophylactic treatment of those having an increased risk of developing cardiovascular calcification. An increased risk represents an above-average risk that a subject will develop cardiovascular calcification, which can be determined, for example, through family history, detection of genes causing a predisposition to cardiovascular calcification, or through the presence of other risk factors such as diabetes or hypertension.

[0021] Within the present invention, a“therapeutically effective amount” of a composition is that amount which is sufficient to show a benefit (e.g., a reduction in a symptom associated with the disorder, disease, or condition being treated) while avoiding adverse side effects such as those typically associated with alternative therapies. When being used prophylactically, a therapeutically effective amount is an amount effective to decrease the likelihood that the subject will develop cardiovascular calcification. The therapeutically effective amount may be administered in one or more doses.

[0022] As used herein, the term“pharmaceutically acceptable carrier” refers to carriers that do not negatively affect the biological activity of the therapeutic molecule or compound to be placed therein. The characteristics of the delivery vehicle will depend on the route of administration. Therapeutic compositions may contain, in addition to the active compound, diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. A pharmaceutically acceptable carrier can deliver an active agent such as a nuclear transport inhibitor without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.

[0023] A subject, as defined herein, is an animal, preferably a mammal such as a domesticated farm animal ( e.g ., cow, horse, pig) or a pet (e.g, dog, cat). More preferably, the subject is a human. The subject may also be a subject having an increased risk of developing cardiovascular calcification, or being in need of treatment of calcific aortic valve disease.

Treatment or Prevention of Cardiovascular Calcification

[0024] In one aspect, the invention provides a method of treating or decreasing the risk of cardiovascular calcification in a subject that includes administering to a subject in need thereof a therapeutically effective amount of a nuclear export inhibitor. In some embodiments, the method is used to treat or decrease the risk of cardiovascular calcification in the cardiovascular interstitial cells in the heart of the subject. In a further embodiment, the cardiovascular calcification is calcific aortic valve disease and the cardiovascular interstitial cells are in the heart valve.

[0025] The method of treating or decreasing the risk of cardiovascular calcification includes administering a nuclear export inhibitor to the subject. Nuclear export, mediated mainly by the nuclear export factor exportin-l (Xpol), also known as chromosomal region maintenance 1 (CRM1), is an essential function in all eukaryote that transport nuclear export signal (NES) containing cargoes from the nucleus to the cytoplasm. Nuclear export inhibitors are compounds that inhibit this process, and include SINE and CRM1 nuclear export inhibitors. See Jans et al ., Curr Opin Cell Biol., 58:50-60 (2019). In some embodiments, the nuclear export inhibitor is a small molecule selective inhibitor of nuclear export (SINE). Examples of SINE compounds are described by Das et al. (Experimental Hematology & Oncology, 4, 7 (2015) and Parikh et al. (J Hematol Oncol., 7, 78 (2014)), and include the compounds KPT-185, KPT-276, KPT-251, and KPT-330. In some embodiments, the nuclear export inhibitor is a CRM1 inhibitor, also known as an Exportin 1 inhibitor. Examples of CRM1 inhibitors include CRM1 inhibitors available through Karyopharm Therapeutics Inc., such as KPT-185 and KPT-276, and leptomycin B. Additional CRM1 inhibitors are described by Gravina et al. , J Hematol Oncol., 7, 85 (2014) and Hill et al. , Oncotarget, 5, 11-28 (2014), both of which are incorporated by reference herein. A preferred compound, which is both a SINE compound and a CRM1 inhibitor, is the compound KPT-330. See Gravina et al. , BMC Cancer, 15, 941 (2015). The structure of KPT-330 is shown below:

In some embodiments, the method includes administering a therapeutically effective amount of KPT-330, or a pharmaceutically acceptable salt thereof, to a subject.

[0026] In some embodiments, the method is used specifically for decreasing the risk cardiovascular calcification, while in other embodiments the method is used specifically for treating cardiovascular calcification in a subject. When treating a subject, the method can further include the step of evaluating the need for continued treatment by imaging the level of cardiovascular calcification in the subject. Imaging can be used to revise the treatment being provided to the subject. For example, if imaging indicates that cardiovascular calcification is increasing, a higher dose of the nuclear export inhibitor can be administered to the subject. Alternately, if cardiovascular calcification has significantly decreased, it can indicate that treatment can be ceased, or a lower dose can be administered.

[0027] Cardiovascular calcification can be evaluated using a number of imaging techniques, such as magnetic resonance imaging, computed tomography, intravascular ultrasound, and positron emission tomography. See Wang et al ., J Am Heart Assoc., 28, 7(13) (2018). Imaging can identify microcalcification, a clinically significant manifestation of vascular mineralization which represents the early stages of intimal calcium formation, as well as macrocalcification, which represents a more advanced stage of cardiovascular calcification. Cardiovascular calcification is also associated with the development of increased vascular stiffness, which can be evaluated using brachial-ankle pulse wave velocity. See Vishnu et al. , Int J Cardiol., 189: 67-72 (2015).

[0028] In some embodiments, the method of the invention is used to prevent the development or decrease the risk of cardiovascular calcification in a subject. “Prevention” includes either preventing or decreasing the likelihood or severity of the onset of overgrowth by cardiovascular calcification altogether or preventing or decreasing the likelihood or severity of the onset of cardiovascular calcification in a subject. Decreasing the likelihood that a subject will develop cardiovascular calcification means that the subject has a decreased change of developing cardiovascular calcification in comparison with the rate for an untreated subject having similar risk factors. For example, the likelihood can be decrease by 5%, 10%, 20%, 30%, 40%, or by 50% or more. Treatment to prevent or decrease the risk that a subject will develop cardiovascular calcification will more likely be provided when a subject has been identified as having an increased risk of developing cardiovascular calcification. An increased risk represents an above-average risk that a subject will develop cardiovascular calcification as a result of having one or more risk factors for cardiovascular calcification.

[0029] Risk factors for cardiovascular calcification include a family history of cardiovascular calcification or detection of genes causing a predisposition to cardiovascular calcification. Other risk factors indicating an increased likelihood that a subject will develop cardiovascular calcification include atherosclerosis, diabetes, chronic kidney disease, hypertension, and age. See Krajnc et al ., J Int Med Res., 47(2):846-858 (2019). Low estrogen levels in postmenopausal women are also a risk factor for both loss of bone mass and accumulation of calcium salts in the vasculature. Fetuin or Fetuin-A levels have also been shown to be associated with the risk of developing or having cardiovascular calcification. See US Patent Publication No. 2005/0186647.

Cardiovascular Calcification

[0030] Cardiovascular calcification is a calcification of blood vessels that is known to be associated with features of metabolic syndrome, such as obesity, diabetes mellitus, chronic kidney disease, hypertension, and chronic inflammation. Calcification is the deposit of calcium salts, primarily calcium phosphate, in body tissues. While calcium deposition is important in the bone to insure strength and stability, cardiovascular calcification is an example of dystrophic calcification, which is the deposition of calcium in abnormal tissue sites. Examples of cardiovascular calcification include calcific aortic valve disease, calcific aortic valve stenosis, and calcification within various other regions of the vasculature, and particularly heart vasculature.

[0031] Cardiovascular calcification can occur as a result of atherosclerosis. However, while there are some overall similarities, a number of discrepancies exist between cardiovascular calcification and atherosclerosis. For example, whereas smooth muscle cells are prominently involved in atherosclerosis, typical smooth muscle cells are not seen in diseased aortic valve leaflets, where fibroblasts and myofibroblasts, a subset of differentiated fibroblasts, are more prominent. Also, although calcific changes can be seen in atherosclerotic plaques, calcification occurs earlier and is a more prominent feature of calcific aortic valve disease, particularly in the end stages of the disease process.

[0032] In some embodiments, the method is used to treat or prevent cardiovascular calcification in the cardiovascular interstitial cells in the heart of the subject. Interstitial cells are those situated between parts of a tissue. The heart valve includes valve interstitial cell that resembles a fibroblast in healthy individuals, but transition towards an osteoblast-like cell as indicated by Runx2 expression as a result of cardiovascular calcification.

[0033] In some embodiments, the present invention provides a method of treating or preventing calcific aortic valve disease. Calcific aortic valve disease is a slowly progressive disorder with a disease continuum that ranges from mild valve thickening without obstruction of blood flow, termed aortic sclerosis, to severe calcification with impaired leaflet motion, or aortic stenosis. Freeman et al, Circulation, 111, 3316-3326 (2005). Active calcification is prominent early in the disease process and is a major factor in the leaflet stiffness of severe stenosis. Microscopic areas of calcification colocalize in areas of lipoprotein accumulation and inflammatory cell infiltration. As the disease progresses, active bone formation may occur.

[0034] Diagnostic evaluation of calcific aortic valve disease includes assessment of leaflet anatomy and the extent of valvular calcification by echocardiography. The severity of aortic stenosis resulting from calcific aortic valve disease can be measured accurately and reliably on the basis of antegrade velocity, mean pressure gradient, and continuity equation valve area echocardiography provides an assessment of left ventricular hypertrophy, diastolic dysfunction, and regional and global systolic function with calculation of ejection fraction. Other associated abnormalities also are evaluated, including aortic dilation, coexisting mitral valve disease, and pulmonary hypertension. Serial echocardiography in patients with aortic stenosis provides valuable interval information, with the timing of examination determined by the stenosis severity and any changes in physical examination or clinical status. Although the cardinal symptoms of severe aortic stenosis are angina, congestive heart failure, and syncope, more subtle symptoms, such as a decrease in exercise tolerance or exertional dyspnea can also be present.

Administration and Formulation

[0035] The pharmaceutical compositions of the present invention comprise an activate agent (e.g., a nuclear export inhibitor), or a pharmaceutically acceptable salt thereof, and may also contain a pharmaceutically acceptable carrier and optionally other therapeutic ingredients. The term“pharmaceutically acceptable salts” refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic bases or acids and organic bases or acids.

[0036] The term“composition,” as in pharmaceutical composition, is intended to encompass a product comprising the active ingredient(s), and the inert ingredient(s) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound of the present invention and a pharmaceutically acceptable carrier.

[0037] The compositions include compositions suitable for oral, rectal, topical, parenteral (including subcutaneous, intramuscular, and intravenous), ocular (ophthalmic), pulmonary (nasal or buccal inhalation), or nasal administration, although the most suitable route in any given case will depend on the nature and severity of the conditions being treated and on the nature of the active ingredient. They may be conveniently presented in unit dosage form and prepared by any of the methods well-known in the art of pharmacy.

[0038] In practical use, the present compounds can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). In preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, hard and soft capsules and tablets, with the solid oral preparations being preferred over the liquid preparations.

[0039] Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be coated by standard aqueous or nonaqueous techniques. Such compositions and preparations should contain at least 0.1 percent of active compound. The percentage of active compound in these compositions may, of course, be varied and may conveniently be between about 2 percent to about 60 percent of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that an effective dosage will be obtained. The active compounds can also be administered intranasally as, for example, liquid drops or spray.

[0040] Tablets and capsules for oral administration are usually presented in a unit dose, and will typically contain conventional excipients such as a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as com starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin. When a dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil.

[0041] Various other materials may be present as coatings or to modify the physical form of the dosage unit. For instance, tablets may be coated with shellac, sugar or both. A syrup or elixir may contain, in addition to the active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and a flavoring such as cherry or orange flavor. [0042] The present compounds may also be administered parenterally. Solutions or suspensions of these active compounds can be prepared in water suitably mixed with a surfactant such as hydroxy-propylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0043] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

[0044] The term “pharmaceutically acceptable salts” refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts. Salts in the solid form may exist in more than one crystal structure, and may also be in the form of hydrates. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N’-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl- morpholine, N-ethypiperideine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

[0045] When the compound of the present invention is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like. Particularly preferred are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids.

[0046] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage may vary depending upon the dosage form employed and the route of administration. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the ICso (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0047] As defined herein, a therapeutically effective amount of an active compound (i.e., an effective dosage) ranges from 0.001 to 30 mg/kg body weight, preferably 0.01 to 25 mg/kg body weight, more preferably 0.1 to 20 mg/kg body weight, and even more preferably 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The active compounds can be administered one time per week for between 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between 3 to 7 weeks, and even more preferably for 4, 5, or 6 weeks. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a mammal including, but not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the mammal, and other diseases present. Moreover, treatment of a mammal with a therapeutically effective amount of an active compound can include a single treatment or, preferably, can include a series of treatments.

[0048] The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein. EXAMPLES

Example 1 : The pharmacological XPol antagonist KPT-330 prevents heart valve calcification via a novel C/EBRb signaling pathway

[0049] The pathogenesis of CAVD is not fully understood and therefore, the development of mechanistic-based therapies, beyond surgery has been hindered. Studies have shown histological and molecular similarities between calcified aortic valves and endochondral (and heterotopic) ossification. This includes formation of mineralized nodules closely associated with ectopic expression of osteogenic markers including Runx2, by valve interstitial cells. Dutta P, Lincoln T, Curr Cardiol Rep., 20(4) :2l (2018). Therefore, more recent studies have taken the approach of targeting these osteogenic signaling pathways in animal models to halt calcific valve pathology. However to date, no suitable therapeutic targets has been identified with clinical potential.

[0050] The inventors previously showed that treatment of porcine aortic VICs (pAVICs) with the nuclear export inhibitor Leptomycin B (LMB) prevents calcification under osteogenic stimulus in vitro. Huk et al ., Arterioscler Thromb Vase Biol., 36(2):328-38 (2016). However, due to the cytotoxicity of LMB, the therapeutic application in vivo is limited. Here, this is addressed by repurposing the XPOl (CRM1) antagonist drug KPT-330 (Selinexor) currently in Phase II/III clinical trials for treating solid tumor malignancies, and demonstrate its therapeutic potential in preventing CAVD in vivo and in vitro by retaining C/EBPB in the nuclei of VICs and inhibiting pro-osteogenic signaling and cell proliferation, via Wnt repression. The overall effect of KPT-330 as demonstrated by this work is shown in Figure 1.

Results

In vitro model of VIC-mediated calcific nodule formation.

[0051] Upon culturing porcine aortic valve interstitial cells (pAVICs) on glass in conditioned media, three stages of distinct cell morphology were observed. At Stage I (-day 5), pAVICs are elongated with cytoplasmic extensions (arrows, Figure 2A) and undetectable calcium deposition (Figure 2D). At Stage II (-day 8), cell confluency increases and‘swirling’ patterns of pAVICs form foci (arrows, Figure 18B) that are Alizarin Red negative (Figure 2E). By Stage III (days 12- 14), lesions referred to as calcific nodules are present and positive for Alizarin Red (arrows, Figures 2C, F). Immunohistochemistry detected expression of known pro-osteogenic markers including Runx2 (Figures 2G, J), Osteopontin (Figures 2H, K) and Cadherin-l 1 (Figures 21, L) in calcific nodules at Stage III, but not Stage I. Simultaneously, cell proliferation assay revealed increased cell growth rates during calcific nodule formation between Stages I and III (Figure 2M).

[0052] To examine differential transcriptomes at Stages I, II and III, RNA-sequencing was performed. Heat map hierarchical clustering analyses molecularly distinguishes the three stages (Figure 2N), and the Venn diagram (Figure 20) indicates common and unique mRNAs at each Stage. Pathway analysis between Stages I and II reveals enrichment of biological processes including‘osteogenic differentiation’, ‘cell adhesion’ and‘cell proliferation’ (Supplementary Figures 2B-D). Similar analysis comparing Stages II and III also identifies ‘osteogenic differentiation’ -related processes. RNA-seq analysis identified significant increases in the osteoblast marker Osteomodulin (Ninomiya et al ., 362(2):460-6 (2007)) at Stage II vs. Stage I (2.17-fold), and Stage III vs. Stage II (3.45-fold) and this is confirmed by immunohistochemistry (Figures 2P-R). Significant increases were also observed in the CAVD biomarker Sclerostin (Koos et al. , The J. Heart Valve Dis., 22(3):3 l7-25 (2013)) (3. l3-fold Stage II vs. Stage I, 9.09- fold Stage III vs. Stage II), and protein expression was found to be enriched in calcific nodules (Figure 2U). To further validate the significance of in vitro Sclerostin expression at Stage III, localization was examined in calcified valve tissue from a 49-year old female patient diagnosed with aortic stenosis. Pentachrome and Alizarin Red staining in Figure 2V indicates the calcific lesion (arrow, Figure 2W) with associated Runx2 and Sclerostin expression (Figure 2X). These studies highlight the molecular and cellular profiles of calcific nodule formation in pAVICs in vitro and identify similarities with human pathology.

The pharmacological XPOl inhibitor. KPT-330 prevents attenuates and rescues calcific nodule formation in vitro.

[0053] Leptomycin B prevents calcific nodule formation in pAVICs, however the known cytotoxicity hinders its pharmacological potential in humans. Therefore, we used the FDA- approved XPOl antagonist drug KPT-330. To determine the ability of KPT-330 to prevent calcific nodule formation, KPT-330 was added to pAVICs plated on glass at Stage I and harvested when calcific nodules were observed in controls (Figure 3A). Formation of Alizarin Red-positive nodules (Figures 3B-E), and expression of Sclerostin and Osteomodulin (Figures 3G-J), were reduced in pAVICs treated with KPT-330 in a dose-dependent manner (Figure 3F). KPT-330 also reduced Alizarin Red staining in VICs obtained from healthy donors (Figure 3K) and aortic stenosis (AS) patients (Figure 3M) cultured in osteogenic media (OM) (Figures 3K-N). In addition, treatment prevented Alizarin Red staining in osteogenic-stimulated pAVICs cultured in OM (Figures 30, P) and 6mM inorganic phosphate (Figures 3Q, R). KPT-330 did not significantly affect apoptosis of pAVICs by Stage III (Figure 3S), but significantly reduced proliferation by Stage II (Figures 3T-W). These data show that KPT-330 is sufficient to prevent calcific nodule formation, and this is associated with decreased cell proliferation and expression of pro-osteogenic markers.

[0054] In addition to preventing calcific nodule formation, treatment with KPT-330 at Stage II attenuates progression of calcific nodule formation (Figures 4A-E) and mitigates existing calcific nodules 72 hours later at Stage III (Figures 4F-K). RNA-seq analysis in pAVICs treated with KPT-330 at Stage III (‘rescue’) detected mRNAs were compared to those expressed at Stages I, II and III (with DMSO). Heat map analysis clustered biological replicates and showed further variance in the transcriptome of KPT-330 treated cells. Biological processes associated with differentially expressed mRNAs in Stage III+KPT-330 samples compared to Stage III+DMSO, ‘cell adhesion’,‘regulation of cell cycle’ and‘cell division’. In addition, pathways related to‘in utero embryonic development’ ‘heart development’ and ‘outflow tract morphogenesis’ were apparent following KPT-330 treatment. To determine the potency of KPT-330 in preventing the return of calcific nodules, pAVICs treated at Stage III were re-plated after 72 hours following the mitigation of calcific nodules, and exposed to DMSO or KPT-330 for an additional 14 days (Figure 4L). Calcific nodules did not return in DMSO-treated cells previously exposed to KPT-330 (Figure 4N), while pAVICs did not survive the continued KPT-330 treatment (Figure 40). These data suggest that short term exposure to KPT-330 prevents the recurrence of calcific nodule formation.

KPT-330 treatment prevents aortic valve calcification in Klotho ¹ mice

[0055] There are few suitable mouse models of aortic valve calcification in the field. Many rely on complex genetics, excess aging and the addition of Western diet; making cause and effect indistinguishable. Here, we utilize the established aging Klotho mouse model that develops calcification of the vessels and aortic valve annulus similar to humans by 6 weeks, (Cheek et al ., J Mol Cell Cardiol., 52(3):689-700 (2012)) and die prematurely at 8 weeks. While these mice do not exhibit significant valve dysfunction prior to premature death, they consistently develop calcific nodules at the base on the aortic valve annulus, similar to aging humans without the addition of other risk factors, making them suitable for prevention studies. To examine the therapeutic potential of KPT-330 in vivo, Klotho and Klotho^+/+ mice were treated with vehicle, or 30 mg/kg KPT-330 during weeks 4-5, weeks 5-6 and early week 7 (Figure 5A). Alizarin Red staining of cardiac tissue after 7 weeks did not detect calcification in DMSO or KPT-330-treated Klotho^+/+ littermates. As expected, the aortic valve annulus (arrows, Figures 5B, B’, D) and aorta (arrowhead, Figures 5B, B’) were calcified in Klotho mice treated with DMSO (n=6). However, annular calcification was not observed in 100% of Klotho mice (n=6) administered with KPT- 330 (Figures 5C, C’, D), while vascular calcification persisted. These observations suggest that KPT-330 can prevent calcification of the aortic valve annulus in Klotho mice.

KPT-330 retains C/EBRb in the nuclei of valve interstitial cells

[0056] KPT-330 is a nuclear export inhibitor drug and therefore, to determine the target protein(s) in preventing calcific nodule formation, mass spectrometry was performed on nuclear lysates isolated from pAVICs plated on glass at Stages I and III, and pAVICs treated with KPT- 330 at Stage I (prevention) and Stage III (rescue). Analysis demonstrates clustering of whole cell lysates samples within each biological replicate, and highlights homogeneity of replicates, and heterogeneity of experimental groups. Potential target proteins were considered if they were shown to i) leave the nuclei of pAVICs during calcific nodule formation (Stages I vs. Ill), and ii) be retained in the nuclei of pAVICs by KPT-330. The transcription factor C/EBRb fit these criteria (Figures 6A-C). Independent studies validate mass spec findings and show decreased C/EBRb in the nuclei of pAVICs cultured in OM, while KPT-330 treatment at Stage I prevented nuclear export of C/EBRb (Figures 6D, E). To determine if C/EBRb is sufficient to prevent calcific nodule formation, similar to KPT-330 treatment, pAVICs were treated with recombinant protein in the presence of osteogenic media. As shown in Figure 6F-G, Alizarin Red staining was significantly reduced with C/EBRb treatment compared to osteogenic media and vehicle controls. Together, these data suggest that KPT-330’ s beneficial mechanism of action is in part via preventing nuclear export of C/EBRb. KPT-330 repressed activated canonical Wnt signaling in pAVICs

[0057] Previous studies have shown that C/EBRb is a negative regulator of Wnt signaling (Park, S. et al ., Cell Death Dis 9, 1023 (2018)), and activated Wnt signaling has previously been shown to be present in excised calcified aortic valves. Gu et al., J Huazhong Univ Sci Technolog Med Sci 34, 33-36 (2014). Consistently, the inventors show that C/EBRb treatment in the presence of osteogenic media reduces active b-catenin (Figures 7A, B) associated with prevention of calcific nodule formation (Figure 6F, G). In addition, RNA-seq analysis identified a significant increase in Wnt-associated mRNAs between Stages I-III (Figure 7C). While treatment with KPT-330 decreased their expression (Lrp5, WntlOB, Lefl, Wispl, Wisp2) and increased mRNA levels of Wnt inhibitors (Wifl) (Figure 7C). In support of KPT-330 inhibiting Wnt signaling, treatment at Stage I (‘prevention’) decreases nuclear and cytoplasmic b-catenin (Figures 7D, E, lanes 3, 6), and active b-catenin expression in whole cell lysates (Figures 7F, G). This attenuation of active Wnt signaling by KPT-330 is associated with downregulation of the calcification marker, Osteomodulin (Figure 7F). LiCh is a Wnt agonist, and activity can be measured using the TOP- FLASH plasmid containing three copies of TCF binding sites. Here the inventors show that KPT- 330 treatment reduces LiCb-induced TOP -Flash activity in the presence of OM (Figure 7H), which is abolished when TCF sites are mutated (FOP -FLASH). Direct repression of Wnt signaling using the antagonist XAV939 (XAV) at Stage I prevented calcific nodule formation in OM (Figure 7G) and reduced cell proliferation (Figure 7H). Interestingly, direct activation of Wnt using LiCh (Figure 71) or Wnt3a was not sufficient to cause calcific nodule formation suggesting that inhibiting Wnt signaling prevents calcification, but activating the pathway is not sufficient to cause the process. These data show that like KPT-330-mediated retention of C/EBRb prevents calcification by repressing Wnt signaling to reduce pro-osteogenic signaling and cell proliferation.

Discussion

[0058] KPT-330 or Selinexor, is an XPOl antagonist currently in clinical trials as a promising anti-cancer drug causing cell cycle arrest resulting in apoptosis and cell death by retaining tumor suppressor and growth regulator proteins in the nucleus. Mahipal A, Malafa M., Pharmacol Then, 164: 135-43 (2016). The inventors demonstrate herein the therapeutic value of administrating KPT- 330 to treat CAVD, and use high throughput approaches to define its beneficial mechanism of action in VICs. The inventors’ data suggests that KPT-330 retains several proteins in the nucleus of VICs that rely on XPOl as a mechanism of nuclear export. In particular, they show that retaining nuclear C/EBRb upon KPT-330 treatment reduces canonical Wnt signaling and attenuates pro- osteogenic phenotypes as noted by decreased expression of markers such as osteomodulin. However, the phenotype of KPT-330-treated cells expressing nuclear C/EBRb has not yet been fully characterized, and it is anticipated that the forced localization of additional proteins may also contribute to the beneficial effect and phenotypic change. RNA-seq analysis demonstrates that KPT-330-treated VICs express a unique mRNA profile, dissimilar to that of untreated VICs at Stages I, II and III, suggesting that KPT-330 is not re-directing osteogenic VICs back to their quiescent/healthy or pre-calcific phenotypes, at least short-term (72 hours post treatment). Interestingly pathway analysis does reveal molecular similarities of KPT-330-treated pAVICs with ‘heart development’ and‘outflow tract morphogenesis’ and includes GATA4 , GATA5 , GATA6 , MEF2C , HEY2 that suggests a more mesoderm-like cell lineage.

[0059] Similar to cancer cells, KPT-330 has negative effects on VIC proliferation (Figure 3) and mRNA pathway analysis reveals that‘cell division’ and‘regulation of cell cycle’ are over represented following KPT-330 treatment. Increased VIC proliferation precedes differentiation to an osteoblast phenotype and calcification, and in vitro treatment of ERK pathway inhibitors or HMG-CoA reductase inhibitors (statins) reduce cell proliferation and calcification. Rajamannan el al., Circulation, 124(16): 1783 91 (2011) However, it is not known if cell proliferation, and potentially subsequent increased cell adhesion and subsequent strain is required for calcific nodule formation. Nonetheless, this biological process was significantly enriched during calcific nodule formation and repressed with KPT-330. It remains unknown if KPT-330 directly regulates the nuclear export of proteins related to cell proliferation, or if alterations in this biological process are secondary to repressed Wnt activity. Similarly, it is also possible that the anti-osteogenic effects of KPT-330 are also due to reduced Wnt as previously suggested in other systems. Cai et al., Experimental cell research, 345(2):206-l7 (2016) However unappreciated roles for nuclear C/EBPB are also considered and will be examined in the future.

[0060] Using mass spectrometry approaches, the inventors have shown a role for the XPOl- dependent transcription factor C/EBPB (Etchin et al., Leukemia, 30(1): 190-9 (2016)) in aortic valve calcification. C/EBPB overexpression is sufficient to prevent calcific nodule formation, and as calcific nodule formation develops, nuclear C/EBPB is reduced in VICs, however they have yet to determine if the loss of C/EBPB induces calcification. C/EBPB has been linked to the inflammatory response and in a recent study, pro-inflammatory signaling was shown to promote bone regeneration during fracture repair via C/EBPB, suggesting pro-bone in this system. Conversely, nuclear C/EBPB is highly expressed in‘quiescent’ VICs in conditioned media (Figure 6D) suggesting an anti-osteogenic role and may be required to maintain homeostasis of healthy valves.

[0061] In VICs treated with KPT-330, nuclear retention of C/EBPB is associated with decreased canonical Wnt activity. In addition, direct repression of Wnt by XAV had similar beneficial effects (Figure 7G, H). This is particularly relevant, as increased canonical Wnt signaling has been consistently reported to be associated with calcified valves from human patients, mouse models and cultured VICs, Albanese et al ., Arterioscler Thromb Vase Biol., 37(3):543-552 (2017). However, worthy of interest, in the inventors’ hands and in line with other studies, direct activation of Wnt by Li Cl 2 or Wnt3a treatment is not sufficient to promote calcification of VICs (Figure 7K), although this has been demonstrated in vascular smooth muscle cells (VSMCs). Kratchmarova et al. , Science 308, 1472-1477 (2005). This suggests divergent mechanisms of calcification between these two cell types as previously suggested (Ferdous, Z., Jo, H. & Nerem, R. M. Biomaterials 32, 2885-2893 (2011)), and this is further supported by the inability of KPT-330 to attenuate vascular calcification in Klotho^{~ ~} mice, despite 100% prevention in the aortic valves. In addition, it is considered that the effects of KPT-330 are likely cell dependent based on the pool of XPOl -dependent cargo proteins located in the nuclei at the time of treatment (Etchin et al ., Leukemia, 30, 190-199 (2016)), and these are likely different between VSMCs and VICs in Klotho^{~ ~} mice.

[0062] In other systems, C/EBPB represses Wnt in a number of ways including direct repression of the Wnt ligand, WntlOb, incidentally found to decrease in VICs following KPT-330 treatment (Figure 7A). In addition, C/EBPB binds and transcriptionally activates the Wnt antagonist Axinl to downregulate B-catenin in hepatic cancer cells. The inventors have yet to elucidate the mechanisms of C/EBPB-mediated Wnt repression, but several Wnt-related transcriptional targets have been identified by RNA-seq analysis and these are worthy of future studies. [0063] This is the first study to report inhibition of aortic valve calcification using an FDA- approved pharmacological reagent. In addition to demonstrating the ability of KPT-330 to prevent, attenuate and treat calcific nodule formation, the inventors have identified the mechanism of action in VICs via nuclear retention of C/EBPB and Wnt repression, leading to reduced pro-osteogenic signaling and cell proliferation (Figure 1). Findings from this study have clinical implications that hold promise for the development of non-surgical approaches in the treatment of CAVD.

Materials and Methods:

Cell Culture and Treatments

[0064] Porcine aortic valve interstitial cells (pAVICs) were isolated from porcine aortic valves and maintained by plating 250,000/well (of 6-well plate) in conditioned media defined as MEM media (containing L-glutamine) supplemented with l0%FBS and l%P/S. pAVICs were utilized from passage number 4 to 7 for all the experiments reported. Human VICs (hVICs) isolated from healthy and calcified aortic valve donors were a kind gift from Dr. Robert Hinton. Two normal and two diseased patient cell lines were utilized. The two normal VICs were of patient age-37 (sex-female, cause of death-cerebrovascular accident) and patient age-26 (sex-male, cause of death-anoxia). The two diseased cell lines were of patient age- 15 (sex-male, disease profile-BAV with severe aortic stenosis and aortic insufficiency) and patient age-23 (sex female, disease profile- BAV with aortic stenosis). pAVICs were maintained in DMEM media with L-glutamine supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). To stimulate calcification, pAVICs were cultured on glass cover slips in conditioned media, and monitored for different stages of calcification (Stages I-III). Alternatively, 250,000 pAVICs, or hVICs were cultured in osteogenic media (DMEM media with L-glutamine supplemented with 10% FBS and 1% P/S. Additionally, supplemented with Dexamethasone (100 nM), Ascorbic Acid (50 mM), and b-glycerophosphate (100 mM)) or conditioned media supplemented with 6 mM sodium phosphate dibasic.

[0065] KPT-330 was purchased from Apex Bio (B1464) and diluted in DMSO to a concentration of 100 nM (unless otherwise specified). KPT-330 treatment and cell harvesting was performed in cultured pAVICs as indicated in Figure 4 and 5. For hVICs, 20nM KPT-330 was added the day after plating the cells and cells harvested after 7-10 days. To inhibit canonical Wnt signaling, XAV-939 was purchased from Selleckchem, and diluted in lxPBS for use at a concentration of 10 mM. To activate canonical Wnt signaling, LiCl, or recombinant Wnt3a (R&D Systems) was diluted in distilled water and used at a concentration of 10 mM or 50ng/mL respectively. pAVICs were treated with XAV-939, LiCl or Wnt3a 24 hours after plating and harvested when calcific nodules were observed in vehicle controls.

Histological Staining.

[0066] Tissues: 4% paraformaldehyde (PFA)-fixed paraffin tissue sections (7pm thick) were deparafmized using xylene and ethyl alcohol (100%, 95%, 75%, 50%, 25%). They were then subjected to boiling for 10 minutes in antigen unmasking solution (Vector Laboratories) for antigenic retrieval. They were then blocked and primary antibody was applied overnight at 4°C. The following primary antibodies were utilized for the study: Sclerostin, ab85799 (Abeam), 1 :50; Runx2, SC-10758 (Santa Cruz), 1 :200. Following primary antibody incubation, appropriate secondary antibodies were applied (Alexa Fluor 488 or 586; Invitrogen; 1 :400). The sections were then mounted in Vectashield anti-fade medium with DAPI (Vector Laboratories) to detect cell nuclei. Pentachrome staining was performed on paraffin tissue sections of human aortic valve specimens according to the manufacturer’s instructions (Russel Movat, American MasterTech, #KTRMP) and then mounted using VectaMount Permanent Mounting Medium (Vector Laboratories, H-5000). All images were visualized using an Olympus BX51 microscope and captured using an Olympus DP71 camera and Cell Sens software. Image contrast and brightness were edited using Adobe Photoshop CC. Images were visualized using an Olympus BX51 microscope and captured using an Olympus DP71 camera and Cell Sens software. Image contrast and brightness were edited using Adobe Photoshop CC.

[0067] Cells: Media was removed from treated pAVICs and cells were rinsed in PBS, followed by fixation in 4% formaldehyde solution for 20 minutes at room temperature. Cells were then again rinsed in PBS and blocked for an hour at room temperature. Primary antibody was applied overnight at 4 °C. The following primary antibodies were utilized for the study: Sclerostin, ab85799 (Abeam), 1 :50; Runx2, SC-10758 (Santa Cruz), 1 :200; Osteomodulin/Osteoadherin, SC-271102 (Santa Cruz), 1 : 100; Osteopontin, SC-10593 (Santa Cruz), 1 : 100; Cadherin-l l/OB-Cadherin, #71-7600 (Thermo Scientific), 1 :400. The cells were then mounted in Vectashield anti-fade medium with DAPI (Vector Laboratories) to detect cell nuclei. Images were visualized using an Olympus BX51 microscope and captured using an Olympus DP71 camera and CellSens software. Image contrast and brightness were edited using Adobe Photoshop CC.

Alizarin Red Staining

[0068] Following treatments, cells and murine or human cardiac tissues were fixed in 4% PFA for 20 minutes at room temperature, then rinsed in 1 x PBS and water. Tissue sections (7pm thick) were deparafmized using xylene and ethyl alcohol (100%, 95%, 75%, 50%, 25%). Fixed tissue sections and cells were then stained with 2% alizarin red stain in water (pH-4.2) for 10 minutes and washed in water, mounted using xylene-based mounting media and imaged. For quantification, the number of alizarin red positive nodules were calculated and averaged for 3 independent biological replicates. Alternatively, alizarin red intensity was calculated using Image J analysis and averaged for 3 independent biological replicates. Images were visualized using an Olympus BX51 microscope and captured using an Olympus DP71 camera and CellSens software. Image contrast and brightness were edited using Adobe Photoshop CC.

Caspase-Glo 3/7 Assay

[0069] Caspase activity assay was performed as per the manufacturer’ s instructions (Promega, G8090). Briefly, 250,000 cells were grown and treated in 6 well plates. After appropriate treatments/time, cells were trypsinized and pelleted. Equal number of cells from each sample were counted and added to each well of a white opaque 96 well plate-a total volume of 100 mΐ. 100 mΐ of Caspase-glo 3/7 reagent was added to each well. Plate was incubated in the dark for an hour and luminescence was read. All values were normalized to media only control and averaged.

Cell Proliferation Assay

[0070] Cell proliferation assay was performed as per the manufacturer’ s instructions (Promega G400). Briefly, 250,000 cells were grown and treated in 6 well plates. After appropriate treatments/time, they were trypsinized and pelleted. Equal number of cells from each sample were counted and added to each well of a clear 96 well plate-a total volume of 100 mΐ. 15 mΐ of dye solution was added to each well. Plate was incubated at 37 °C for an hour, following which 100 mΐ of solubilization/stop solution was added to each well. Following overnight incubation, absorbance was read at 570 nm. All values were normalized to media only control and averaged.

Transfections and Dual-Luciferase Reporter Assay System (TOP/FOP Flash Assay)

[0071] Cells were grown in 6 well plates in osteogenic media with/without KPT (see above). After 5-6 days in culture (calcific nodules seen in osteogenic media wells), cells were trypsinized and re-plated in 12 well plates at a density of 125,000 cells/wells. Twenty-four hours post plating, cells were transfected with 500 ng of TOP/FOP Flash (Firefly) plasmids (M50 Super 8X TOP Flash plasmid; 12456 and M51 Super 8X FOP Flash plasmid; 12457) which were a gift from Randall Moon and 50ng of empty-Renilla plasmid (for transfection efficiency) using the Lipofectamine3000 transfection reagent kit from Invitrogen (L3000015) in serum free media. 24hours post transfection, cells were recovered in osteogenic media with/without KPT and LiCl (10 mM) was added to all the wells in order to induce the Wnt/[Symbol]-Catenin signaling response. Twenty-four hours post treatment, Dual-Luciferase Reporter Assay was performed according to the manufacturer’s instructions (Promega #E 1910) Luminescence Firefly values were normalized to Renilla and total protein (calculated via BCA assay). Values were averaged from three biological replicates.

Nuclear/Cvtoplasmic Lysate Extraction

[0072] Nuclear and cytoplasmic fractions were extracted based on the manufacture’ s protocol provided with the NE-PER Nuclear and Cytoplasmic Extraction Kit from Thermo Scientific (#78833). Briefly, cells were rinsed in PBS, trypsinized and pelleted via centrifugation. Cell pellets were washed in PBS substituted with Complete EDTA-free protease inhibitor cocktail (Sigma-Aldrich, 11836170001) in order to inactivate the proteases. Ice-cold CER-l buffer was added to the cell pellet. Cells were vigorously vortexed for 15 seconds to dissolve the cell pellet and incubated on ice for 10 minutes. Ice-cold CER-2 buffer was added to the samples and they were incubated on ice for a minute after vortexing for 5 seconds. Samples were spun down for 5 minutes at ~l6,000rcf. The supernatants (cytoplasmic fraction) were transferred to a fresh tube and stored. Ice-cold NER buffer was added to the pellets and incubated on ice for 40 minutes. Samples were vortexed every 10 minutes for 15 seconds. They were then centrifuged and supernatants (nuclear fraction) was stored for analysis. Whole Cell Protein Harvest and Western Blotting Analysis

[0073] Following treatments, protein from cells was harvested using IX Cell Lysis Buffer (Cell Signaling, #9803 S). Lysates were centrifuged for 10 minutes at 12,000 rpm at 4 °C to remove cell debris. Supernatants were collected and BCA assay was utilized to determine protein concentration to normalize samples. Samples were run on PAGE gel, transferred to a nitrocellulose membrane using the dry transfer apparatus from Invitrogen. Membrane was blocked for an hour before applying primary antibodies. The following primary antibodies were utilized for the study: GAPDH, #2118 S (Cell Signaling), 1 :2000; Osteomodulin/Osteoadherin, SC-271102 (Santa Cruz), 1 : 1000; active b-catenin, #05-665 (Millipore), 1 :500; b-catenin, #8480S (Cell Signaling), 1 :500; Lamin A/C, #4777S (Cell Signaling), 1 : 1000; b-tubulin, #2l28S (Cell Signaling), 1 : 1000.

Alkaline Phosphatase Assay

[0074] pAVICs were grown on glass and DMSO/KPT added to the wells -day 5. At day -12, cells were harvested, processed and alkaline phosphatase activity was calculated according to the manufacturer’s instructions provided with the kit (ab83369, Abeam).

Human AoV Specimens

[0075] Human aortic valve (AoV) specimens were a gift from Dr. Prasad Dasi, OSU. The valves were from a 49 year old women with rheumatic disease presenting aortic stenosis and valve calcification (IRB# is 2016H0280).

Animal Studies

[0076] The proposed research studies utilized mouse models. All mice for this study were housed in a USDA-certified, AALAC-accredited facility within Nationwide Children’s Hospital Research Institute. The vivarium was monitored daily by the investigator and animal care technicians. All animal protocols for the proposed studies have been approved by the Institutional Animal Care and Use Committee (IACUC #ARl 1-00026), and general animal training has been provided to all individuals involved in this study. Experimental mice were euthanized using CO2 anoxia, as recommended by the Panel of Euthanasia of the American Veterinary Medical Association, and great effort was made to ensure the mice do not suffer any unnecessary pain or discomfort.

[0077] Klotho heterozygous mice (B6; l29S5-Kltm lLex/Mmucd) were obtained from Mutant Mouse Resource and Research Centers supported by NIH (MMRCC) and bred to obtain Klotho wild-types, heterozygotes, and nulls. Klotho nulls were injected intraperitoneally with Vehicle (Pluronic F-68 and PVP K29) or KPT-330 (dissolved in Pluronic F-68 and PVP K29) at a dosage of 30 mg/kg. Injections began at 3-4 weeks of age and lasted until week 7-8 (a total of 6-7 injections/mouse). Mice were then euthanized and hearts were harvested for processing and analysis. For histological analysis, hearts were dissected and fixed in 4% PFA, paraffin-embedded and sectioned (7 pm).

RNA-Seq Analysis

[0078] pAVICs were plated as previously described and harvested either 5 days after plating (Day 5 or Stage I), ~8 days after plating (Day 8 or Stage II)(when pre-calcification was observed) or DMSO/KPT added when nodules observed and harvested ~72 hours post KPT-330 treatment (Stage 11 I/Stage III+KPT). Total RNA was extracted using the RNeasy Mini Kit from Qiagen (#74104).

[0079] Differential expression analysis: Gene-specific reads were calculated in the form of RPKM (Reads Per Kilobase of gene per Million fragments mapped). The RPKM of 10 reads was defined as the Detection Threshold, which was calculated from the formula: (10 reads/2kb)/(total mapped reads/lM). Values that were smaller than detection threshold were replaced with the average value of detection threshold across all samples. The Reliable Quantification Threshold (RQT) was defined as an RPKM equivalent to 50 reads and was calculated from the formula: (50 reads/2kb)/(total mapped reads/lM). Only the 13,934 porcine genes showing counts above RQT in one or more samples were analyzed for differential expression analysis. A l-way ANOVA was performed on the log2 RPKM data for the 13,934 detectable porcine genes to examine the effect of group (Day5, Day8, Nod+DMSO and Nod+KPT). Tukey tests were performed as a post-hoc test to determine the effect of treatment within each group (e.g. Day5 vs Day8). Likewise, fold changes were calculated for the same comparisons by taking the ratio of the mean values from both groups in the comparison. For example, when comparing Stage I vs. Stage II a fold change of 2.5 would mean that the gene expressed at Stage I was 2.5 times higher than at Stage II, or expression was higher in Stage I than Stage II. This logic was applied for all stage comparisons. ANOVA and Tukey P values were adjusted to control the False Discovery Rate according to the method of Benjamini and Hochberg (1995) J Roy Stat Soc B. 57:289-300. All statistical analysis was performed using R version 3.2.2 statistical computing software. Differential gene expression between stages was visualized utilizing volcano plots, which were generated with the R ggplot 2 package version 2.2.1.

[0080] Functional Annotation: To examine the differential gene expression between different stages, we used an FDR cut off of 0.05 for each comparison. This threshold identified the 1944 differentially expressed genes between Stage I and II, 2262 differentially expression genes between Stage II and III, and 3652 differentially expressed genes between Stage III and KPT treated cells. Functional annotation was then performed on each of these sets of differentially expressed genes using the Database for Annotation, Visualization and Integrated Discovery (DAVID) version 6.8. Gene Ontology (GO) Direct terms were utilized, which provide GO mappings directly annotated by the source database and have been filtered to remove broad parent terms. Enriched GO biological processes were then analyzed using the R package GOplot version 1.0.2. Bubble plots were generated using the GoBubble function to visualize the top 15 GO biological processes. The bubble plots represent each GO term as a circle, and these size of each circle correlates to the number of genes within the term, and is plotted as-log (FDR) vs. zscore. The zscore is a crude measurement, and predicts if a term will be up or downregulated. It is calculated by taking the number of upregulated genes and subtracting the number of downregulated genes and then dividing this number by the square root of the number of genes in each pathway. The top 12 GO biological processes were also visualized as a circle plot through the GoCircle function. The circle plots are able to show overall gene expression changes associated with each GO term, visualized by the red (upregulated genes) and blue (downregulated genes) dots. The circle plot also highlights the terms association to the zscore, visualized by color of the inner circles, where red is associated with terms that are predicted to have an overall increase, while blue is associated with an overall decrease. Chord plots where also generated, using the GoChord function, to highlight important GO terms and their association with statistically significant gene expression changes in genes associated with valve development and disease. [0081] The inventors also aimed to identify and functionally annotate the genes expressed explicitly during specific stages. Detectable genes with an average RPKM above the reliable quantification threshold were assessed that have ANOVA P values by group < 0.05 and a fold change > 2 in any of the 6 pairwise comparisons among the four groups. A gene was considered detectable in a group if the gene’s average RPKM value was above the average Reliable Quantification Threshold (RQT) value for that group. VennDiagram was used to generate overlapping data sets and to generate Venn Diagrams. Genes in the different overlapping datasets were analyzed using DAVID version 6.8 and were utilized the GO Direct terminology for functional annotation.

Mass Spectrometry/Proteomic Analysis

[0082] pAVICs were plated on glass coverslips and harvested either 5 days after plating (Day 5 or Stage I), or DMSO/KPT added on day 5 (prevention group) (Stage Ill/Stage III+KPT @Stage I) or when nodules observed and (rescue group) harvested ~72 hours post KPT-330 treatment (Stage Ill/Stage III+KPT @Stage III). Whole cell pellets were isolated via trypsinization, washed with PBS. Nuclear pellets were extracted as described above. Samples were sent to Duke Proteomic Core, Duke University, Durham, North Carolina for analysis.

[0083] Sample Preparation: Frozen samples were thawed to room temperature and 100 pL of 0.5% RapiGest in 50 mM ammonium bicarbonate (AmBic) was added to each. The samples were probe sonicated three times at power level 3 and each pulse was three seconds long. The samples were cooled on ice between pulses. A Bradford assay (Thermo) was performed with 5pL of each sample diluted with AmBic to 0.25% RapiGest to minimize interference in the measurement.

[0084] Sample Normalization and Digestion: Following the Bradford assay, 20 pg of each sample were normalized to an equivalent concentration (w/v) for the digestion using 0.5% RapiGest in AmBic was added to bring the total sample volume to 100 pL. Cysteine reduction was performed with 10 mM DTT, at 80 °C for 15 minutes with shaking at 750 rpm. Alkylation was performed with 2 mM iodoacetamide at room temperature in the dark for 30 minutes. Sequencing grade trypsin (Promega) at 100 ng/pL in AmBic was added at a ratio of 1 :50 (w/w) trypsimprotein. The samples digested at 37 °C overnight with shaking at 750 rpm. After digestion, each sample was acidified with 500 fmol ADH1 YEAST to a final concentration of 1% trifluoroacetic acid (TFA), 2% acetonitrile (MeCN) in water. The samples were heated to 60 °C for two hours with shaking at 750 rpm and then cooled to 4 °C for one hour. The samples were centrifuged at 15000 rpm for 5 minutes to pellet any undigested protein. 100 mΐ of the resulting supernatant was pipetted into an autosampler vial for analysis by LC-MS/MS. A Study Pool QC (SPQC) was made by combining 3 pL from each of the samples.

[0085] Quantitative Analysis of Whole Cell Lysate Proteins: Quantitative one-dimensional LC-MS/MS was performed once per sample using 2 pL (-250 ng) of the protein digest. Samples were analyzed using a nanoAcquity UPLC system (Waters) coupled to a Q Exactive Plus Orbitrap high resolution accurate mass tandem mass spectrometer (Thermo) via a nanoelectrospray ionization source. The sample was first trapped on a Symmetry C18 300pm x l80mm trapping column (5 pL/min at 99.9 0.1 v/v LLO/MeCN). Next, the analytical separation was performed using a 1.7 um Acquity HSS T3 Cl 8 75pm x 250mm column (Waters) with a 90 minute gradient of 5 to 40% MeCN in 0.1% formic acid at a flow rate of 400nL/min and a column temperature of 55 °C. Data collection on the Q Exactive Plus mass spectrometer was performed in a data- dependent MS/MS manner, using a 70,000 resolution precursor ion (MS1) scan followed by MS/MS (MS2) of the top 10 most abundant ions at 17,500 resolution. MS1 was accomplished using an automatic gain control (AGC) target of le6 ions and mass accumulation of 60 msec. MS2 used AGC target of 5e4 ions, 60msec mass accumulation, 2.0 m/z isolation window, 27V normalized collision energy, and 20s dynamic exclusion. The total analysis cycle time for each sample injection was approximately 2 hours, and the experiment totaled 21 injections (19 for quantitative analysis).

[0086] The SPQC was analyzed twice to condition the column and was analyzed four additional times (at the beginning and after every 5 samples). Following the analyses, the data was imported into Rosetta Elucidator v4.0 (Rosetta Biosoftware, Inc.), and all LC-MS files were aligned based on the accurate mass and retention time of detection ion (“features”) using a PeakTeller algorithm (Elucidator). The relative peptide abundance was calculated based on area-under-the-curve (AUC) of aligned features across all runs. The dataset had 835,562 MS/MS spectra for sequencing by database searching. This MS/MS data was searched against a custom RefSeq NCBI database with Sits scrofa taxonomy (downloaded on 12/19/2016) with additional and standard and contaminant proteins, including yeast ADH1 and bovine serum albumin, as well as an equal number of reversed-sequence“decoys” for false discovery rate (FDR) determination. Amino acid modifications allowed in database searching included fixed carbamidomethyl on Cys (+57), oxidation on Met (+16), deamidation on Asn and Gln (+1). The data was searched with 5 ppm precursor, 0.02Da product ion tolerance, and tryptic enzyme specificity, allowing up to two missed cleavages. The data was annotated at 1.2% peptide FDR using the PeptideTeller scoring algorithm.

[0087] During quantitative processing, the data was first curated to contain only high-quality peptides with appropriate chromatographic peak shape. To obtain the most robust protein quantification results, peptides with %CV > 50% were removed from the dataset prior to summing to the protein level, to yield a‘robust’ protein quantification dataset. The tool db2db on BiodbNet was used to convert the RefSeq protein identifier to a Gene Symbol to improve ease of interpretation.

Example 2: KPT-330 as a novel drug for the treatment of CAVD

[0088] Calcific aortic valve disease (CAVD) is a common disorder characterized by progressive buildup of calcium-rich nodules on the valve surface leading to stenosis. This process is mediated by dysregulation of valve interstitial cells (VIC) that abnormally differentiate towards an osteoblast-like lineage and promote matrix mineralization, similar to bone. Despite widespread prevalence, effective pharmaceutical treatments are lacking and surgical intervention remains standard treatment with no guarantee of long-term success. Therefore, there is a critical need to develop effective therapeutic alternatives.

[0089] KPT-330 or selinexor is a nuclear export inhibitor of XPOl -dependent proteins which is in Phase III clinical trials for the treatment of cancer. Its therapeutic potential for CAVD has not been examined. Using an established mouse model of CAVD ( Klothor' ), the inventors administered KPT-330 during early stages of annular valve calcification in an established mouse model (Klothcf ) and show that early KPT-330 treatment of Klotho prevents annular calcification in vivo (n=6). Similarly, treatment of human and porcine aortic valve interstitial cells (pAVICs) cultured under osteogenic stimulus at later stages is sufficient to attenuate and mitigate calcific nodule formation in vitro. RNA Seq analysis demonstrated that KPT-330 reduced the expression of osteogenic markers and mediators of pro-osteogenic Wnt signaling; a pathway previously shown to be activated in human calcific aortic valve disease. Specifically, KPT-330 reduced nuclear b-catenin, impeding its activity and thus inhibited Wnt signaling. To determine the proteins directly targeted by KPT-330 treatment, Mass Spectrometry was performed and suggests that KPT-330 retains C/EBRb in the nucleus of VICs to inhibit Wnt^-catenin signaling which in turn prevents pro-osteogenic signaling and cell proliferation of VICs, thereby preventing calcification.

[0090] The Wnt signaling pathway is regulated by a‘destruction complex’ and when Wnt is inactive, the inhibitory protein AXIN1 molecularly interacts with the signaling mediator, b-catenin in a complex to prevent b-catenin translocating to the nucleus to transcriptionally regulate target genes. However, when Wnt signaling is active, the molecular interaction between AXIN1 and b- catenin is reduced. In the inventors recent work, they show that that control conditions AXIN1 and b-catenin interact (suggesting inactive Wnt), however under osteogenic conditions this interaction is significantly reduced (activating Wnt). Furthermore, the inventors show that KPT-330 treatment restores the molecular interaction thereby decreasing Wnt activity to prevent calcification. These studies for the first time have identified KPT-330 as a novel therapeutic drug in the treatment of CAVD by targeting the C/EBP b/Axin/Wnt axis. Similar to pharmacological inhibition, genetic knockdown of XPOl in vitro also prevented calcification, indicating that nuclear shuttling of proteins is indeed crucial in CAVD pathogenesis. Collectively, the studies show that KPT-330 prevents calcific aortic valve disease both in vitro and in vivo.

[0091] The complete disclosure of all patents, patent applications, and publications, and electronically available materials cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Claims

CLAIMS What is claimed is:

1. A method of treating or decreasing the risk of cardiovascular calcification in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a nuclear export inhibitor.

2. The method of claim 1, wherein the cardiovascular calcification is calcific aortic valve disease.

3. The method of claim 1, wherein the nuclear export inhibitor is a small molecule selective inhibitor of nuclear export.

4. The method of claim 1, wherein the compound is a CRM1 inhibitor.

5. The method of claim 3, wherein the compound is KPT-330 or a pharmaceutically acceptable salt thereof.

6. The method of claim 1, wherein the method decreases the risk of cardiovascular calcification in a subject that has not been diagnosed as having cardiovascular calcification.

7. The method of claim 6, wherein the subject has one or more risk factors for cardiovascular calcification.

8. The method of claim 1, wherein the method treats cardiovascular calcification in a subject.

9. The method of claim 8, wherein the method further comprises evaluating the need for continued treatment by imaging the level of cardiovascular calcification in the subject.

10. The method of claim 1, wherein the compound is administered in a pharmaceutically acceptable carrier.

11. The method of claim 1, wherein the nuclear export inhibitor is orally administered.

12. The method of claim 1, wherein the subject is a human.