WO2018112409A1 - Methods of treating ataxia-telangiectasia - Google Patents

Methods of treating ataxia-telangiectasia Download PDF

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WO2018112409A1
WO2018112409A1 PCT/US2017/066818 US2017066818W WO2018112409A1 WO 2018112409 A1 WO2018112409 A1 WO 2018112409A1 US 2017066818 W US2017066818 W US 2017066818W WO 2018112409 A1 WO2018112409 A1 WO 2018112409A1
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administering
mometasone
loteprednol
cells
tyrphostin
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PCT/US2017/066818
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French (fr)
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Chris C. GIBSON
Blake BORGESON
Mason VICTORS
Gaelle MERCENNE
Timothy CONSIDINE
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Recursion Pharmaceuticals, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/275—Nitriles; Isonitriles
    • A61K31/277—Nitriles; Isonitriles having a ring, e.g. verapamil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Abstract

Methods are provided of treating neurodegenerative diseases such as ataxia-telangiectasia (A-T) by the administration of loteprednol, mometasone, tyrphostin AG 879, pharmaceutically-acceptable salts and/or esters of loteprednol, mometasone, or tyrphostin AG 879, or combinations thereof. Methods of treating cells with reduced expression of the ATM serine/threonine kinase (ATM) protein, reduced expression of an A-T gene, or both, are also provided. Such methods may include modulating a phenotypic profile of the cells by administering an effective amount of loteprednol, mometasone, tyrphostin AG 879, pharmaceutically-acceptable salts and/or esters of loteprednol, mometasone, or tyrphostin AG 879, or combinations thereof.

Description

METHODS OF TREATING ATAXIA-TELANGIECTASIA
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. provisional application No. 62/443, 144 filed January 6, 2017 and U.S. provisional application No. 62/434,790, filed December 15, 2016, both of which are incorporated by reference as if fully set forth herein.
TECHNICAL FIELD
[0002] The present disclosure relates to methods of treating diseases. More particularly, the disclosure relates to methods of treating neurodegenerative diseases such as ataxia-telangiectasia (A-T).
BACKGROUND
[0003] Classic A-T is an autosomal recessive disorder characterized by progressive cerebellar ataxia beginning between ages one and four years, oculomotor apraxia, choreoathetosis, telangiectasias of the conjunctivae, immunodeficiency, frequent infections, and an increased risk for malignancy, particularly leukemia and lymphoma.
[0004] Mutations in the ATM serine/threonine kinase (ATM) gene have been shown to be associated with A-T. The prevalence of A-T in the United States is between 1 :40,000-1 : 100,000 live births. A-T is the most common cause of progressive cerebellar ataxia in childhood in most countries; ataxia with oculomotor apraxia (AOA) may be more prevalent in Portugal and perhaps Japan. Prevalence varies with the degree of consanguinity in a country.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] The embodiments disclosed herein will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings.
[0006] FIG. 1 is a Phenotype Impact Plot depicting a subset of specific morphological features related to ATM-depleted cells. The degree to which each feature is changed compared to negative control cells (magnitude of bar) and the variability of each feature (darkness is inversely proportional to variability) is depicted. [0007] FIG. 2 is a Phenotype Impact Plot depicting the effect of administration of loteprednol etabonate on ATM-depleted cells for each of the features identified in FIG. 1 . The degree to which each feature is changed compared to ATM-depleted cells (magnitude of narrow bar) and the variability of each feature (darkness is inversely proportional to variability) is depicted.
[0008] FIG. 3 is a Phenotype Impact Plot depicting the effect of administration of mometasone furoate on ATM-depleted cells for each of the features identified in FIG. 1 . The degree to which each feature is changed compared to ATM-depleted cells (magnitude of narrow bar) and the variability of each feature (darkness is inversely proportional to variability) is depicted.
[0009] FIG. 4 is a Phenotype Impact Plot depicting the effect of administration of betamethasone on ATM-depleted cells for each of the features identified in FIG. 1 . The degree to which each feature is changed compared to ATM-depleted cells (magnitude of narrow bar) and the variability of each feature (darkness is inversely proportional to variability) is depicted.
[0010] FIG. 5 is a Phenotype Impact Plot depicting the effect of administration of tyrphostin AG 879 on ATM-depleted cells for each of the features identified in FIG. 1 . The degree to which each feature is changed compared to ATM-depleted cells (magnitude of narrow bar) and the variability of each feature (darkness is inversely proportional to variability) is depicted.
[0011] FIG. 6A is a series of western blots depicting protein expression in the absence of hydrogen peroxide (H2O2) treatment.
[0012] FIG. 6B is a series of western blots depicting protein expression in the presence of H202 treatment.
[0013] FIG. 7 is a graph quantifying the expression of phosphorylated Checkpoint kinase 2 (CHK2) protein in FIG. 6B.
[0014] FIG. 8 is a graph depicting the results of a propagation assay.
[0015] FIG. 9 is a graph depicting the results of another propagation assay.
[0016] FIG. 10 is a graph depicting the results of yet another propagation assay.
[0017] FIG. 1 1A is a series of representative western blots showing the expression of different proteins in the presence of H2O2 to induce oxidative stress and visualize the ATM response. The upper gel corresponds to ATM and shows that silencing is very efficient. The middle gels correspond to ataxia telangiectasia and Rad3-related (ATR) and DNA-dependent protein kinase, catalytic subunit (DNA- PKc), two proteins involved in the DNA repair pathway. The bottom gel shows the phosphorylated form of CHK2 protein and its activation in the presence of H2O2 and ATM. n=3.
[0018] FIG. 1 1 B is a graph showing quantification of the phosphorylated form of the CHK2 protein from FIG. 1 1A. n=3.
[0019] FIG. 12 is a graph showing the results of a proliferation assay. In the absence of ATM, cell number is increased by «50% compared to control. This number is rescued by addition of tyrphostin AG 879 (AG879).
DETAILED DESCRIPTION
[0020] The present disclosure provides methods of treating neurodegenerative diseases including, but not limited to, ataxia-telangiectasia (A-T).
[0021] It will be readily understood that the embodiments, as generally described herein, are exemplary. The following more detailed description of various embodiments is not intended to limit the scope of the present disclosure, but is merely representative of various embodiments. Moreover, the order of the steps or actions of the methods disclosed herein may be changed by those skilled in the art without departing from the scope of the present disclosure. In other words, unless a specific order of steps or actions is required for proper operation of the embodiment, the order or use of specific steps or actions may be modified.
[0022] A first aspect of the disclosure relates to methods of treating A-T. In some embodiments, the methods may include administering a therapeutically effective amount of loteprednol, mometasone, tyrphostin AG 879, an analog or derivative thereof, a pharmaceutically acceptable salt or ester of the foregoing, or combinations thereof. In certain embodiments, the loteprednol may include loteprednol etabonate. In various embodiments, the mometasone may include mometasone furoate.
[0023] The chemical structure of t rphostin AG 879 is as depicted below.
Figure imgf000005_0001
Tyrphostin AG 879 Tyrphostin AG 879 can also be referred to as a-cyano-(3,5-di-t-butyl-4- hydroxy)thiocinnamide (CAS Number, 148741 -30-4; empirical formula (Hill notation) C18H24N2OS; molecular weight, 316.46; MDL number, MFCD00236450; PubChem Substance ID, 24278728).
[0024] In some embodiments, administering a therapeutically effective amount of loteprednol, mometasone, tyrphostin AG 879, an analog or derivative thereof, a pharmaceutically acceptable salt or ester of the foregoing, or combinations thereof may include administering a composition or formulation consisting essentially of loteprednol, mometasone, tyrphostin AG 879, an analog or derivative thereof, a pharmaceutically acceptable salt or ester of the foregoing, or combinations thereof.
[0025] The administering may include orally administering. When prepared for oral administration, the compositions or formulations including loteprednol, mometasone, and/or tyrphostin AG 879 described herein may be prepared, for example, in capsules, tablets, caplets, lozenges, aqueous suspensions or solutions, and oral sprays.
[0026] Another aspect of the disclosure relates to methods of treating cells with reduced expression of the ATM protein, reduced expression of an A-T gene (e.g., a gene associated with A-T), or both. In some embodiments, the methods may include modulating a phenotypic profile of the cells by administering an effective amount of loteprednol, mometasone, tyrphostin AG 879, an analog or derivative thereof, a pharmaceutically acceptable salt or ester of the foregoing, or combinations thereof. In various embodiments, the cells may be mammalian cells. For example, the cells may be in a mammal such as a human.
[0027] In certain embodiments, the A-T gene may include the ATM gene. In various embodiments, the phenotypic profile may be generated from a profiling process including metabolomic profiling, proteomic profiling, gene expression profiling, morphological profiling, image-based morphological profiling, or combinations thereof.
[0028] In some embodiments, image-based morphological profiling may include tracking staining intensities in one or more imaging channels, correlations between imaging channels, textural patterns, size and shape of cellular structures, geometric relationships between adjacent cells, and/or geometric relationships between intracellular structures. For example, cells may be probed with Hoechst (DNA/nuclei); concanavalin A (endoplasmic reticulum); phalloidin (actin); wheat germ agglutinin (WGA; membranes and Golgi apparatus); SYTO 14 (nucleoli and cytosolic RNA); and/or MITOTRACKER® (mitochondria). The probed cells may then be imaged and/or analyzed.
[0029] In certain embodiments, the profiling process may include tracking 100 or more cellular features, 500 or more cellular features, or 1000 or more cellular features. In various embodiments, the profiling process may include generating a negative control phenotypic profile; profiling the cells with reduced expression of the ATM protein, reduced expression of an A-T gene, or both, to generate an A-T phenotypic profile; and/or determining phenotypic differences unique to the A-T phenotypic profile, as compared to the negative control phenotypic profile. In some embodiments, generating the negative control phenotypic profile may include generating a random composite phenotypic profile of numerous disease models unrelated to A-T (e.g., but not limited to, about 30 disease models). As used herein, "unrelated" refers to diseases or disease models not known to be associated with A- T.
[0030] In some embodiments, determining phenotypic differences unique to the A-T phenotypic profile may include determining statistically significant phenotypic features associated with the reduction of expression or function of the ATM gene. In certain embodiments, modulating the phenotypic profile may include normalizing the phenotypic profile so as to minimize phenotypic differences unique to the A-T phenotypic profile.
[0031] In certain embodiments, the methods may further include identifying a mammal as having cells with reduced expression of the ATM protein, reduced expression of an A-T gene, or both. In certain other embodiments, the methods may further include identifying the mammal as in need of modulation of the phenotypic profile of the cells of the mammal.
[0032] In some embodiments, administering a therapeutically effective amount of loteprednol, mometasone, tyrphostin AG 879, an analog or derivative thereof, a pharmaceutically acceptable salt or ester of the foregoing, or combinations thereof may include administering a composition or formulation consisting essentially of loteprednol, mometasone, tyrphostin AG 879, an analog or derivative thereof, a pharmaceutically acceptable salt or ester of the foregoing, or combinations thereof. The administering may include orally administering. [0033] Another aspect of the disclosure relates to methods of activating a pathway. In some embodiments, the methods may include activating phosphorylation of CHK2 protein by administering a therapeutically effective amount of loteprednol, mometasone, tyrphostin AG 879, an analog or derivative thereof, a pharmaceutically acceptable salt or ester of the foregoing, or combinations thereof to cells of a mammal with reduced expression of the ATM protein, reduced expression of an A-T gene, or both.
[0034] In various embodiments, administering a therapeutically effective amount of loteprednol, mometasone, an analog or derivative thereof, a pharmaceutically acceptable salt or ester of the foregoing, or combinations thereof may result in at least about 10% greater, at least about 15% greater, at least about 20% greater, or at least about 25% greater phosphorylation of the CHK2 protein than an equimolar dose of betamethasone dipropionate or dexamethasone sodium phosphate.
[0035] Another aspect of the disclosure relates to methods of initiating double- stranded deoxyribonucleic acid (dsDNA) repair. In certain embodiments, the methods may include administering a therapeutically effective amount of loteprednol, mometasone, tyrphostin AG 879, an analog or derivative thereof, a pharmaceutically acceptable salt or ester of the foregoing, or combinations thereof to cells of a mammal with reduced expression of the ATM protein, reduced expression of an A-T gene, or both.
[0036] Another aspect of the disclosure relates to methods of controlling cellular proliferation. In some embodiments, the methods may include administering a therapeutically effective amount of mometasone, tyrphostin AG 879, an analog or derivative thereof, a pharmaceutically acceptable salt or ester of the foregoing, or combinations thereof to cells of a mammal, wherein the cells have reduced expression of the ATM protein, reduced expression of an A-T gene, or both.
[0037] In certain embodiments, the methods may further include identifying the mammal as having cells with reduced expression of the ATM protein, reduced expression of an A-T gene, or both. In various embodiments, healthy cellular proliferation levels of the cells may be promoted by the administration step.
EXAMPLES
[0038] To further illustrate these embodiments, the following examples are provided. These examples are not intended to limit the scope of the claimed invention, which should be determined solely on the basis of the attached claims. Example 1 - In vitro analysis of ATM gene knockdown models
[0039] An A-T model was developed based on ATM protein knockdown. Image- based, morphological profiling methods were used to characterize and/or determine the cellular state from relevant cellular morphology and to analyze the effect of dosages of compounds described herein.
[0040] Measured features in the morphological profiling included staining intensities, textural patterns, size and shape of cellular structures, as well as correlation between stains across channels and neighborhood relationships between cells and among intracellular structures. This technique enabled single-cell resolution and enabled detection of perturbations in subsets of cells.
[0041] FIG. 1 is a Phenotype Impact Plot, which displays a selection of many of the top differences for each morphological feature measured. Approximately 800 features were quantified for each cell using analytical software (CELL-PROFILER™, discussed more below). Subsequent analysis of these data identified separate complex morphological phenotypes. Some of the phenotypes included many different features. Together, these features constitute a phenotypic signature or phenotypic profile for the A-T disease model (i.e. , an A-T phenotypic profile).
[0042] The Phenotype Impact Plot of FIG. 1 displays some of the phenotypic features that are most contributory to the ATM gene's unique disease signature. Feature ranking depended on both the magnitude of the effect of ATM depletion (i.e. , bar length) as well as the consistency or variability of the effect of ATM depletion (i.e. , darkness). The magnitude of each bar represents the average magnitude of the change for that feature, with the horizontal axis at the bottom oriented to display decreases in features toward the left and increases in features toward the right. The units on the x-axis are standard deviations from the non-disease state as determined by the negative control composite (discussed in more detail below). As mentioned previously, the darkness of each bar represents variability of the effect of ATM depletion for a given feature (e.g. , across wells, across siRNAs, and across experiments). For example, the darker the intensity of the bar the more consistent the effect of ATM depletion is for the indicated feature.
[0043] A negative control phenotypic profile was also generated. The negative control phenotypic profile was generated based on a random composite phenotypic profile of 30 disease models not known to be associated with A-T. Phenotypic differences unique to the A-T phenotypic profile were then determined by comparing the A-T phenotypic profile to the negative control phenotypic profile. Thus, features unique to the A-T disease that are not shared with other diseases were identified.
[0044] Analysis of the unique features of the A-T phenotypic profile revealed the following trends, among others:
• Increase in the "Texture_Entropy" of the "OrigBlue" channel within the nucleus.
• Decreases in the "TextureJnverseDifferenceMoment" of the "OrigBlue" channel within the nucleus, cytoplasm, and cell.
• Decrease in the "Texture_SumVariance" of the OrigRed" channel within the nucleus.
• Decrease in the "Texture_Sum Entropy" of the OrigRed" channel within the cell.
• Increase in the intensity of the "OrigGreen" channel within the cytoplasm.
• Increase in the intensity of the "OrigBlue" channel within the cell.
• Increases in the "Texture_Contrast" of the "OrigRed" and "OrigBlue" channels within the cytoplasm.
• Increase in the "Texture_DifferenceVariance" of the "OrigGreen" channel within the cell.
[0045] Images of stained cells presented various disease-specific features. Disease-specific phenotypes were constructed from the features extracted by CELL-PROFILER™ image processing software across multiple functional cellular compartments. Images of stained cells also presented various disease-non-specific phenotypes. Disease-non-specific phenotypes were also constructed, taking as an input the hundreds of features extracted by CELLPROFILER™ image processing software across multiple functional cellular compartments. Furthermore, the features were extracted from cells probed with Hoechst (DNA/nuclei), concanavalin A (endoplasmic reticulum), phalloidin (actin) plus WGA (membranes and Golgi apparatus), SYTO® 14 (nucleoli and cytosolic RNA), and MITOTRACKER® (mitochondria) using an IMAGEXPRESS® MICRO XLS epifluorescent microscope (MOLECULAR DEVICES™).
[0046] FIG. 2 is a Phenotype Impact Plot depicting the effect of administration of 1 μΜ of loteprednol etabonate to ATM-depleted cells for each of the features identified in FIG. 1 . FIG. 3 is a Phenotype Impact Plot depicting the effect of administration of 1 μΜ of mometasone furoate to ATM-depleted cells for each of the features identified in FIG. 1 . FIG. 4 is a Phenotype Impact Plot depicting the effect of administration of 1 μΜ of betamethasone to ATM-depleted cells for each of the features identified in FIG. 1 . FIG. 5 is a Phenotype Impact Plot depicting the effect of administration of 1 μΜ of tyrphostin AG 879 to ATM-depleted cells for each of the features identified in FIG. 1 . In FIGS. 2-5, the degree to which each feature is changed compared to ATM-depleted cells (magnitude of narrow bar) and the variability of each feature (darkness is inversely proportional to variability) is depicted.
[0047] Analysis of the results depicted in FIGS. 2 and 3 revealed the following trends (i.e., in ATM-depleted cells treated with loteprednol etabonate or mometasone furoate versus untreated ATM-depleted cells):
• Increases in the "TextureJnverseDifferenceMoment" of the OrigBlue" channel within the nucleus and cytoplasm.
• Decrease in the "Texture_Entropy" of the OrigBlue" channel within the nucleus.
• Decreases in the "Texture_Contrast" of the "OrigBlue" and "OrigRed" channels within the cytoplasm.
• Decrease in the intensity of the OrigBlue" channel within the cell.
[0048] Analysis of the results depicted in FIG. 5 revealed the following trends (i.e. , in ATM-depleted cells treated with tyrphostin AG 879 versus untreated ATM- depleted cells):
• Decrease in the "Texture_Entropy" of the OrigBlue" channel within the nucleus.
• Increases in the "TextureJnverseDifferenceMoment" of the "OrigBlue" channel within the nucleus, cytoplasm, and cell.
• Increase in the "Texture_SumVariance" of the "OrigRed" channel within the nucleus.
• Increase in the "Texture_Sum Entropy" of the "OrigRed" channel within the cell.
• Decrease in the intensity of the "OrigGreen" channel within the cytoplasm.
• Decrease in the intensity of the "OrigBlue" channel within the cell. • Decreases in the "Texture_Contrast" of the "OrigRed" and "OrigBlue" channels within the cytoplasm.
• Decrease in the "Texture_DifferenceVariance" of the "OrigGreen" channel within the cell.
[0049] The methods used in this example were based in part on the methods described in Nature Protocols 1 1 , 1757-1774 (2016), which is hereby incorporated herein by reference in its entirety.
Example 2 - Glucocorticoid treatment in ATM-depleted cells
[0050] ATM protein was depleted in A549 cells using siRNA directed to ATM mRNA. ATM-depleted cells were treated with one of seven compounds, including: betamethasone dipropionate (Drug 1 ), dexamethasone sodium phosphate (Drug 2), fluocinolone acetonide (Drug 3), fluocinonide (Drug 4), fluticasone propionate (Drug 5), loteprednol etabonate (Drug 6), and mometasone furoate (Drug 7). The compounds are also referred to herein as the glucocorticoids (GCs). A positive control cell sample was not treated with any one of the seven compounds, but was treated with a non-targeting siRNA and dimethyl sulfoxide (DMSO). A negative control cell sample was treated with the ATM siRNA and DMSO, but was not treated with any one of the seven compounds.
[0051] The serine/threonine kinase activity of ATM protein was assessed using immunoblotting of cell lysates to phosphorylated ATM target substrates. Cells treated as outlined above were first stressed with H202 to create double-strand DNA breaks (DSB) to activate ATM serine/threonine kinase. H2O2 treatment induces oxidative stress such that the ATM response can be visualized. In parallel, a second set of cells were also treated as outlined above but without being stressed with H2O2. If evaluated under stress, ATM serine/threonine kinase activity is non-detectable in situations in which ATM protein is absent.
[0052] The CHK2 protein is a major downstream signaling target of ATM. It has been shown to orchestrate the ATM cascade (e.g. , DNA repair, cell cycle block, etc.). Cell lysates were collected from cells treated as described above and immunoblotting of the cell lysates was performed.
[0053] Western blots were generated to show the expression patterns of the following proteins: ATM; ataxia telangiectasia and Rad3-related protein (ATR); DNA- dependent protein kinase, catalytic subunit (DNA-PKC); and phosphorylated CHK2. As depicted, expression patterns of these proteins were analyzed in the absence (see FIG. 6A) or presence (see FIG. 6B) of H2O2 treatment.
[0054] As shown, CHK2 was not phosphorylated in the non-H202-treated cell lysates, while CHK2 was phosphorylated in the H202-treated cell lysates. FIG. 7 depicts quantification of the phosphorylated CHK2 signal (n=3) in the samples from FIG. 6B.
[0055] It was observed that the siRNA efficiently silenced ATM in the cells. Further, ATM expression was not rescued in the presence of any of the tested compounds (i.e. , Drugs 1 -7). ATR and DNA-PKC, in addition to ATM, are kinases that have been shown to be part of the same DSB response. Without being bound by any particular theory, ATR and DNA-PKC do not appear to be stimulated by the GCs to compensate for the loss of ATM as there does not appear to be a change of expression for ATR or DNA-PKC in the absence or presence of the GCs. As depicted, each of the GCs rescued the phosphorylation of CHK2, which is the main effector of the ATM response.
[0056] As depicted in FIG. 7, CHK2 phosphorylation was enhanced by about 20% in ATM-depleted cells treated with loteprednol etabonate (ATM-6) in comparison to the ATM-depleted cells treated with betamethasone dipropionate (ATM-1 ) and CHK2 phosphorylation was enhanced by about 25% in ATM-depleted cells treated with loteprednol etabonate (ATM-6) in comparison to the ATM-depleted cells treated with dexamethasone sodium phosphate (ATM-2). Likewise, CHK2 phosphorylation was enhanced by about 15% in ATM-depleted cells treated with mometasone furoate (ATM-7) in comparison to the ATM-depleted cells treated with betamethasone dipropionate and CHK2 phosphorylation was enhanced by about 20% in ATM- depleted cells treated with mometasone furoate (ATM-7) in comparison to the ATM- depleted cells treated with dexamethasone sodium phosphate.
Example 3 - Glucocorticoid treatment and cell proliferation measurements
[0057] Cell proliferation under different conditions was also analyzed. As depicted in FIG. 8, ATM depletion increased cell propagation (i.e. , cell number increased by about 50%). This was at least partially rescued by administration of mometasone furoate.
Example 4 - Propagation assay with H?0^and glucocorticoid treatment
[0058] A propagation assay was conducted in ATM-depleted cells using multiple concentrations of loteprednol etabonate (1 μΜ, 0.1 μΜ, and 0.01 μΜ) and mometasone furoate (1 μΜ, 0.1 μΜ, and 0.01 μΜ), in parallel with a betamethasone control (1 μΜ and 0.1 μΜ). The propagation assay was conducted after testing the ATM response in cells treated with 250 μΜ H202 for one hour. FIG. 9 depicts the results of this assay (n=1 ; 3 technical replicates). As shown, the cell number was similar to control in the 1 μΜ and 0.1 μΜ mometasone furoate samples, and even in the 0.01 μΜ mometasone furoate samples to at least some extent.
Example 5 - Propagation assay with bleomycin treatment
[0059] A propagation assay was conducted in ATM-depleted cells using multiple concentrations of loteprednol etabonate (1 μΜ, 0.1 μΜ, and 0.01 μΜ) and mometasone furoate (1 μΜ, 0.1 μΜ, and 0.01 μΜ), in parallel with a betamethasone control (1 μΜ and 0.1 μΜ). The propagation assay was conducted after testing the ATM response in cells treated with bleomycin. Like H2O2, bleomycin induces DSB; however, bleomycin is not specific to the ATM response. Bleomycin has been shown to activate ATR. FIG. 10 depicts the results of this assay (n=1 ). As shown, ATM-depleted cells treated with bleomycin continued to propagate. In the presence of the GCs, cell growth appeared to be more controlled.
Example 6 - Tyrphostin AG 879 treatment in ATM-depleted cells
[0060] ATM protein was depleted in A549 cells using siRNA directed to ATM mRNA. ATM-depleted cells were treated with tyrphostin AG 879 (AG879) (see FIG. 1 1 A). A positive control cell sample was not treated with tyrphostin AG 879, but was treated with a non-targeting siRNA and dimethyl sulfoxide (DMSO). A negative control cell sample was treated with the ATM siRNA and DMSO, but was not treated with tyrphostin AG 879.
[0061] The serine/threonine kinase activity of ATM protein was assessed using immunoblotting of cell lysates to phosphorylated ATM target substrates. Cells treated as outlined above were first stressed with H2O2 to create double-strand DNA breaks (DSB) to activate ATM serine/threonine kinase. H2O2 treatment induces oxidative stress such that the ATM response can be visualized. In parallel, a second set of cells were also treated as outlined above but without being stressed with H2O2. In the non-H2O2-treated cells the results were substantially the same as the H2O2- treated cells except that CHK2 was not phosphorylated. If evaluated under stress, ATM serine/threonine kinase activity is non-detectable in situations in which ATM protein is absent. [0062] The CHK2 protein is a major downstream signaling target of ATM. It has been shown to orchestrate the ATM cascade {e.g. , DNA repair, cell cycle block, etc.). Cell lysates were collected from cells treated as described above and immunoblotting of the cell lysates was performed.
[0063] Western blots were generated to show the expression patterns of the following proteins: ATM, ATR, DNA-dependent protein kinase, catalytic subunit (DNA-PKc), and phosphorylated CHK2.
[0064] CHK2 was not phosphorylated in the non-H202-treated cell lysates, while, as shown in FIG. 1 1 A, CHK2 was phosphorylated in the H202-treated cell lysates. FIG. 1 1 B depicts quantification of the phosphorylated CHK2 signal (n=3) in the samples from FIG. 1 1 A.
[0065] It was observed that the siRNA efficiently silenced ATM in the cells. Further, ATM expression was not rescued in the presence of tyrphostin AG 879. ATR and DNA-PKc, in addition to ATM, are kinases that have been shown to be part of the same DSB response.
Example 7 - Tyrphostin AG 879 treatment and cell proliferation measurements
[0066] Cell proliferation under different conditions was also analyzed. As depicted in FIG. 12, ATM depletion increased cell propagation (i.e. , cell number increased by about 50%). This was at least partially rescued by administration of tyrphostin AG 879.
[0067] It will be apparent to those having skill in the art that many changes may be made to the details of the above-described embodiments without departing from the underlying principles of the invention.

Claims

Claims:
1 . A method of treating ataxia-telangiectasia, the method comprising administering a therapeutically effective amount of loteprednol, mometasone, tyrphostin AG 879, an analog or derivative thereof, a pharmaceutically acceptable salt or ester of the foregoing, or combinations thereof.
2. The method of claim 1 , wherein the loteprednol comprises loteprednol etabonate and the mometasone comprises mometasone furoate.
3. The method of claim 1 or claim 2, wherein administering a therapeutically effective amount of loteprednol, mometasone, tyrphostin AG 879, an analog or derivative thereof, a pharmaceutically acceptable salt or ester of the foregoing, or combinations thereof comprises administering a composition or formulation consisting essentially of loteprednol, mometasone, tyrphostin AG 879, an analog or derivative thereof, a pharmaceutically acceptable salt or ester of the foregoing, or combinations thereof.
4. The method of any one of claims 1 -3, wherein administering comprises orally administering.
5. A method of treating cells with reduced expression of the ATM protein, reduced expression of an ataxia-telangiectasia gene, or both, the method comprising modulating a phenotypic profile of the cells by administering an effective amount of loteprednol, mometasone, tyrphostin AG 879, an analog or derivative thereof, a pharmaceutically acceptable salt or ester of the foregoing, or combinations thereof.
6. The method of claim 5, wherein the ataxia-telangiectasia gene comprises the ATM gene.
7. The method of claim 5 or claim 6, wherein the phenotypic profile is generated from a profiling process comprising metabolomic profiling, proteomic profiling, gene expression profiling, morphological profiling, image-based morphological profiling, or combinations thereof.
8. The method of claim 7, wherein image-based morphological profiling comprises tracking staining intensities in one or more imaging channels, correlations between imaging channels, textural patterns, size and shape of cellular structures, geometric relationships between adjacent cells, and geometric relationships between intracellular structures.
9. The method of claim 7 or claim 8, wherein the profiling process comprises tracking 100 or more cellular features, 500 or more cellular features, or 1000 or more cellular features.
10. The method of any one of claims 7-9, wherein the profiling process comprises:
generating a negative control phenotypic profile;
profiling the cells with reduced expression of the ATM protein, reduced expression of an ataxia-telangiectasia gene, or both, to generate an ataxia- telangiectasia phenotypic profile; and
determining phenotypic differences unique to the ataxia-telangiectasia phenotypic profile, as compared to the negative control phenotypic profile.
1 1 . The method of claim 10, wherein generating the negative control phenotypic profile comprises generating a random composite phenotypic profile of numerous disease models unrelated to ataxia-telangiectasia.
12. The method of claim 10 or claim 1 1 , wherein determining phenotypic differences unique to the ataxia-telangiectasia phenotypic profile comprises determining statistically significant phenotypic features associated with the presence of the ATM gene.
13. The method of claim 12, wherein modulating the phenotypic profile comprises normalizing the phenotypic profile so as to minimize phenotypic differences unique to the ataxia-telangiectasia phenotypic profile.
14. The method of any one of claims 5-13, wherein the cells are in a mammal.
15. The method of claim 14, further comprising identifying the mammal as having cells with reduced expression of the ATM protein, reduced expression of an ataxia- telangiectasia gene, or both.
16. The method of claim 14, further comprising identifying the mammal as in need of modulation of the phenotypic profile of the cells.
17. The method of any one of claims 5-16, wherein administering a therapeutically effective amount of loteprednol, mometasone, tyrphostin AG 879, an analog or derivative thereof, a pharmaceutically acceptable salt or ester of the foregoing, or combinations thereof comprises administering a composition or formulation consisting essentially of loteprednol, mometasone, tyrphostin AG 879, an analog or derivative thereof, a pharmaceutically acceptable salt or ester of the foregoing, or combinations thereof.
18. The method of any one of claims 5-17, wherein administering comprises orally administering.
19. A method of activating a pathway, the method comprising activating phosphorylation of CHK2 protein by administering a therapeutically effective amount of loteprednol, mometasone, tyrphostin AG 879, an analog or derivative thereof, a pharmaceutically acceptable salt or ester of the foregoing, or combinations thereof to cells of a mammal with reduced expression of an ATM protein, reduced expression of an ataxia-telangiectasia gene, or both.
20. The method of claim 19, comprising administering a therapeutically effective amount of loteprednol, mometasone, an analog or derivative thereof, a pharmaceutically acceptable salt or ester of the foregoing, or combinations thereof, wherein the administering step results in at least about 10% greater, at least about 15% greater, at least about 20% greater, or at least about 25% greater phosphorylation of the CHK2 protein than an equimolar dose of betamethasone dipropionate or dexamethasone sodium phosphate.
21 . The method of claim 19 or claim 20, wherein the loteprednol comprises loteprednol etabonate and the mometasone comprises mometasone furoate.
22. The method of any one of claims 19-21 , wherein administering a therapeutically effective amount of loteprednol, mometasone, tyrphostin AG 879, an analog or derivative thereof, a pharmaceutically acceptable salt or ester of the foregoing, or combinations thereof comprises administering a composition or formulation consisting essentially of loteprednol, mometasone, tyrphostin AG 879, an analog or derivative thereof, a pharmaceutically acceptable salt or ester of the foregoing, or combinations thereof.
23. The method of any one of claims 19-22, wherein administering comprises orally administering to the mammal.
24. The method of any one of claims 19-23, further comprising identifying the mammal as having cells with reduced expression of an ATM protein, reduced expression of an ataxia-telangiectasia gene, or both.
25. A method of initiating double-stranded deoxyribonucleic acid repair, the method comprising administering a therapeutically effective amount of loteprednol, mometasone, tyrphostin AG 879, an analog or derivative thereof, a pharmaceutically acceptable salt or ester of the foregoing, or combinations thereof to cells of a mammal with reduced expression of an ATM protein, reduced expression of an ataxia-telangiectasia gene, or both.
26. The method of claim 25, comprising administering a therapeutically effective amount of loteprednol, mometasone, an analog or derivative thereof, a pharmaceutically acceptable salt or ester of the foregoing, or combinations thereof, wherein the administering step results in at least about 10% greater, at least about 15% greater, at least about 20% greater, or at least about 25% greater initiation of DNA repair than an equimolar dose of betamethasone dipropionate or dexamethasone sodium phosphate.
27. The method of claim 25 or claim 26, wherein the loteprednol comprises loteprednol etabonate and the mometasone comprises mometasone furoate.
28. The method of any one of claims 25-27, wherein administering a therapeutically effective amount of loteprednol, mometasone, tyrphostin AG 879, an analog or derivative thereof, a pharmaceutically acceptable salt or ester of the foregoing, or combinations thereof comprises administering a composition or formulation consisting essentially of loteprednol, mometasone, tyrphostin AG 879, an analog or derivative thereof, a pharmaceutically acceptable salt or ester of the foregoing, or combinations thereof.
29. The method of any one of claims 25-28, wherein administering comprises orally administering to the mammal.
30. The method of any one of claims 25-29, further comprising identifying the mammal as having cells with reduced expression of the ATM protein, reduced expression of an ataxia-telangiectasia gene, or both.
31 . A method of controlling cellular proliferation, the method comprising administering a therapeutically effective amount of mometasone, tyrphostin AG 879, an analog or derivative thereof, a pharmaceutically acceptable salt or ester of the foregoing, or combinations thereof to cells of a mammal, wherein the cells have reduced expression of the ATM protein, reduced expression of an ataxia- telangiectasia gene, or both.
32. The method of claim 31 , further comprising identifying the mammal as having cells with reduced expression of an ATM protein, reduced expression of an ataxia- telangiectasia gene, or both.
33. The method of claim 31 or claim 32, wherein healthy cellular proliferation levels of the cells are promoted by the administration step.
34. The method of any one of claims 31 -33, wherein the mometasone comprises mometasone furoate.
35. The method of any one of claims 31 -34, wherein administering a therapeutically effective amount of mometasone, tyrphostin AG 879, an analog or derivative thereof, a pharmaceutically acceptable salt or ester of the foregoing, or combinations thereof comprises administering a composition or formulation consisting essentially of mometasone, tyrphostin AG 879, an analog or derivative thereof, a pharmaceutically acceptable salt or ester of the foregoing, or combinations thereof.
36. The method of any one of claims 31 -35, wherein administering comprises orally administering to the mammal.
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